1
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Li Y, Meng Z, Fan C, Rong H, Xi Y, Liao Q. Identification and multi-omics analysis of essential coding and long non-coding genes in colorectal cancer. Biochem Biophys Rep 2025; 41:101938. [PMID: 40034256 PMCID: PMC11874739 DOI: 10.1016/j.bbrep.2025.101938] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2024] [Revised: 01/19/2025] [Accepted: 01/28/2025] [Indexed: 03/05/2025] Open
Abstract
Essential genes are indispensable for the survival of cancer cell. CRISPR/Cas9-based pooled genetic screens have distinguished the essential genes and their functions in distinct cellular processes. Nevertheless, the landscape of essential genes at the single cell levels and the effect on the tumor microenvironment (TME) remains limited. Here, we identified 396 essential protein-coding genes (ESPs) by integration of 8 genome-wide CRISPR loss-of-function screen datasets of colorectal cancer (CRC) cell lines and single-cell RNA sequencing (scRNA-seq) data of CRC tissues. Then, 29 essential long non-coding genes (ESLs) were predicted using Hypergeometric Test (HT) and Personalized PageRank (PPR) algorithms based on ESPs and co-expressed network constructed from scRNA-seq. CRISPR/Cas9 knockout experiment verified the effect of several ESPs and ESLs on the survival of CRC cell line. Furthermore, multi-omics features of ESPs and ESLs were illustrated by examining their expression patterns and transcription factor (TF) regulatory network at the single cell level, as well as DNA mutation and DNA methylation events at bulk level. Finally, through integrating multiple intracellular regulatory networks with cell-cell communication network (CCN), we elucidated that CD47 and MIF are regulated by multiple CRC essential genes, and the anti-cancer drugs sunitinib can interfere the expression of them potentially. Our findings provide a comprehensive asset of CRC ESPs and ESLs, sheding light on the mining of potential therapy targets for CRC.
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Affiliation(s)
- Yanguo Li
- Institute of Drug Discovery Technology, Ningbo University, Ningbo, Zhejiang, China
| | - Zixing Meng
- Department of Biochemistry and Molecular Biology and Zhejiang Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang, China
| | - Chengjiang Fan
- Department of Biochemistry and Molecular Biology and Zhejiang Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang, China
| | - Hao Rong
- Department of Biochemistry and Molecular Biology and Zhejiang Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang, China
| | - Yang Xi
- Department of Biochemistry and Molecular Biology and Zhejiang Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang, China
| | - Qi Liao
- Department of Biochemistry and Molecular Biology and Zhejiang Key Laboratory of Pathophysiology, Health Science Center, Ningbo University, Ningbo, Zhejiang, China
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2
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Hogrebe NJ, Schmidt MD, Augsornworawat P, Gale SE, Shunkarova M, Millman JR. Depolymerizing F-actin accelerates the exit from pluripotency to enhance stem cell-derived islet differentiation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2024.10.21.618465. [PMID: 39484596 PMCID: PMC11526947 DOI: 10.1101/2024.10.21.618465] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/03/2024]
Abstract
In this study, we demonstrate that cytoskeletal state at the onset of directed differentiation is critical for the specification of human pluripotent stem cells (hPSCs) to all three germ layers. In particular, a polymerized actin cytoskeleton facilitates directed ectoderm differentiation, while depolymerizing F-actin promotes mesendoderm lineages. Applying this concept to a stem cell-derived islet (SC-islet) differentiation protocol, we show that depolymerizing F-actin with latrunculin A (latA) during the first 24 hours of definitive endoderm formation facilitates rapid exit from pluripotency and alters Activin/Nodal, BMP, JNK-JUN, and WNT pathway signaling dynamics. These signaling changes influence downstream patterning of the gut tube, leading to improved pancreatic progenitor identity and decreased expression of markers associated with other endodermal lineages. Continued differentiation generates islets containing a higher percentage of β cells that exhibit improved maturation, insulin secretion, and ability to reverse hyperglycemia. Furthermore, this latA treatment reduces enterochromaffin cells in the final cell population and corrects differentiations from hPSC lines that otherwise fail to consistently produce pancreatic islets, highlighting the importance of cytoskeletal signaling at the onset of directed differentiation.
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Affiliation(s)
- Nathaniel J. Hogrebe
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, MSC 8127-057-08, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Mason D. Schmidt
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, MSC 8127-057-08, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Punn Augsornworawat
- Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
| | - Sarah E. Gale
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, MSC 8127-057-08, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Mira Shunkarova
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, MSC 8127-057-08, 660 South Euclid Avenue, St. Louis, MO 63110, USA
| | - Jeffrey R. Millman
- Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, MSC 8127-057-08, 660 South Euclid Avenue, St. Louis, MO 63110, USA
- Department of Biomedical Engineering, Washington University in St. Louis, 1 Brookings Drive, St. Louis, MO 63130, USA
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3
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Su C, Pastor WA, Emad A. Deciphering lineage-relevant gene regulatory networks during endoderm formation by InPheRNo-ChIP. Brief Bioinform 2024; 25:bbae592. [PMID: 39535258 PMCID: PMC11558691 DOI: 10.1093/bib/bbae592] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2024] [Revised: 10/09/2024] [Accepted: 11/01/2024] [Indexed: 11/16/2024] Open
Abstract
Deciphering the underlying gene regulatory networks (GRNs) that govern early human embryogenesis is critical for understanding developmental mechanisms yet remains challenging due to limited sample availability and the inherent complexity of the biological processes involved. To address this, we developed InPheRNo-ChIP, a computational framework that integrates multimodal data, including RNA-seq, transcription factor (TF)-specific ChIP-seq, and phenotypic labels, to reconstruct phenotype-relevant GRNs associated with endoderm development. The core of this method is a probabilistic graphical model that models the simultaneous effect of TFs on their putative target genes to influence a particular phenotypic outcome. Unlike the majority of existing GRN inference methods that are agnostic to the phenotypic outcomes, InPheRNo-ChIP directly incorporates phenotypic information during GRN inference, enabling the distinction between lineage-specific and general regulatory interactions. We integrated data from three experimental studies and applied InPheRNo-ChIP to infer the GRN governing the differentiation of human embryonic stem cells into definitive endoderm. Benchmarking against a scRNA-seq CRISPRi study demonstrated InPheRNo-ChIP's ability to identify regulatory interactions involving endoderm markers FOXA2, SMAD2, and SOX17, outperforming other methods. This highlights the importance of incorporating the phenotypic context during network inference. Furthermore, an ablation study confirms the synergistic contribution of ChIP-seq, RNA-seq, and phenotypic data, highlighting the value of multimodal integration for accurate phenotype-relevant GRN reconstruction.
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Affiliation(s)
- Chen Su
- Department of Electrical and Computer Engineering, McGill University, 845 Sherbrooke Street West, Montreal, Quebec H3A 0G4, Canada
| | - William A Pastor
- Department of Biochemistry, McGill University, 845 Sherbrooke Street West, Montreal, Quebec H3A 0G4, Canada
- The Rosalind and Morris Goodman Cancer Institute, 1160 Pine Avenue, Montreal, Quebec H3A 1A3, Canada
| | - Amin Emad
- Department of Electrical and Computer Engineering, McGill University, 845 Sherbrooke Street West, Montreal, Quebec H3A 0G4, Canada
- The Rosalind and Morris Goodman Cancer Institute, 1160 Pine Avenue, Montreal, Quebec H3A 1A3, Canada
- Mila, Quebec AI Institute, 6666 St-Urbain Street #200, Montreal, Quebec H2S 3H1, Canada
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4
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Barrero M, Lazarenkov A, Blanco E, Palma LG, López-Rubio AV, Bauer M, Bigas A, Di Croce L, Sardina JL, Payer B. The interferon γ pathway enhances pluripotency and X-chromosome reactivation in iPSC reprogramming. SCIENCE ADVANCES 2024; 10:eadj8862. [PMID: 39110794 PMCID: PMC11305397 DOI: 10.1126/sciadv.adj8862] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 07/21/2023] [Accepted: 06/28/2024] [Indexed: 08/10/2024]
Abstract
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here, we reveal important pathways for pluripotency acquisition and X-reactivation using a genome-wide CRISPR screen during neural precursor to iPSC reprogramming. In particular, we discover that activation of the interferon γ (IFNγ) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFNγ stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFNγ in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes.
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Affiliation(s)
- Mercedes Barrero
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain
| | | | - Enrique Blanco
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain
| | - Luis G. Palma
- Josep Carreras Leukemia Research Institute (IJC), Badalona 08916, Spain
- Institut Hospital del Mar d’Investigacions Mèdiques, CIBERONC, Barcelona 08003, Spain
| | | | - Moritz Bauer
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain
| | - Anna Bigas
- Josep Carreras Leukemia Research Institute (IJC), Badalona 08916, Spain
- Institut Hospital del Mar d’Investigacions Mèdiques, CIBERONC, Barcelona 08003, Spain
| | - Luciano Di Croce
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain
- Universitat Pompeu Fabra (UPF), Barcelona 08003, Spain
- ICREA, Passeig Lluís Companys 23, Barcelona 08010, Spain
| | - José Luis Sardina
- Josep Carreras Leukemia Research Institute (IJC), Badalona 08916, Spain
| | - Bernhard Payer
- Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain
- Universitat Pompeu Fabra (UPF), Barcelona 08003, Spain
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5
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Madrigal A, Lu T, Soto LM, Najafabadi HS. A unified model for interpretable latent embedding of multi-sample, multi-condition single-cell data. Nat Commun 2024; 15:6573. [PMID: 39097589 PMCID: PMC11298001 DOI: 10.1038/s41467-024-50963-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2023] [Accepted: 07/23/2024] [Indexed: 08/05/2024] Open
Abstract
Single-cell analysis across multiple samples and conditions requires quantitative modeling of the interplay between the continuum of cell states and the technical and biological sources of sample-to-sample variability. We introduce GEDI, a generative model that identifies latent space variations in multi-sample, multi-condition single-cell datasets and attributes them to sample-level covariates. GEDI enables cross-sample cell state mapping on par with state-of-the-art integration methods, cluster-free differential gene expression analysis along the continuum of cell states, and machine learning-based prediction of sample characteristics from single-cell data. GEDI can also incorporate gene-level prior knowledge to infer pathway and regulatory network activities in single cells. Finally, GEDI extends all these concepts to previously unexplored modalities that require joint consideration of dual measurements, such as the joint analysis of exon inclusion/exclusion reads to model alternative cassette exon splicing, or spliced/unspliced reads to model the mRNA stability landscapes of single cells.
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Affiliation(s)
- Ariel Madrigal
- Department of Human Genetics, McGill University, Montreal, QC, H3A 0C7, Canada
- Victor P. Dahdaleh Institute of Genomic Medicine, Montreal, QC, H3A 0G1, Canada
| | - Tianyuan Lu
- Lady Davis Institute for Medical Research, Montreal, QC, H3T 1E2, Canada
- Department of Statistical Sciences, University of Toronto, Toronto, ON, M5S 1A1, Canada
- Department of Population Health Sciences, University of Wisconsin-Madison, Madison, WI, 53726, USA
| | - Larisa M Soto
- Department of Human Genetics, McGill University, Montreal, QC, H3A 0C7, Canada
- Victor P. Dahdaleh Institute of Genomic Medicine, Montreal, QC, H3A 0G1, Canada
| | - Hamed S Najafabadi
- Department of Human Genetics, McGill University, Montreal, QC, H3A 0C7, Canada.
- Victor P. Dahdaleh Institute of Genomic Medicine, Montreal, QC, H3A 0G1, Canada.
- McGill Centre for RNA Sciences, McGill University, Montreal, Canada.
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6
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Zhou H, Ye P, Xiong W, Duan X, Jing S, He Y, Zeng Z, Wei Y, Ye Q. Genome-scale CRISPR-Cas9 screening in stem cells: theories, applications and challenges. Stem Cell Res Ther 2024; 15:218. [PMID: 39026343 PMCID: PMC11264826 DOI: 10.1186/s13287-024-03831-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2024] [Accepted: 07/02/2024] [Indexed: 07/20/2024] Open
Abstract
Due to the rapid development of stem cell technology, there have been tremendous advances in molecular biological and pathological research, cell therapy as well as organoid technologies over the past decades. Advances in genome editing technology, particularly the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related protein 9 (Cas9), have further facilitated the rapid development of stem cell researches. The CRISPR-Cas9 technology now goes beyond creating single gene editing to enable the inhibition or activation of endogenous gene loci by fusing inhibitory (CRISPRi) or activating (CRISPRa) domains with deactivated Cas9 proteins (dCas9). These tools have been utilized in genome-scale CRISPRi/a screen to recognize hereditary modifiers that are synergistic or opposing to malady mutations in an orderly and fair manner, thereby identifying illness mechanisms and discovering novel restorative targets to accelerate medicinal discovery investigation. However, the application of this technique is still relatively rare in stem cell research. There are numerous specialized challenges in applying large-scale useful genomics approaches to differentiated stem cell populations. Here, we present the first comprehensive review on CRISPR-based functional genomics screening in the field of stem cells, as well as practical considerations implemented in a range of scenarios, and exploration of the insights of CRISPR-based screen into cell fates, disease mechanisms and cell treatments in stem cell models. This review will broadly benefit scientists, engineers and medical practitioners in the areas of stem cell research.
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Affiliation(s)
- Heng Zhou
- Center of Regenerative Medicine and Department of Stomatology, Renmin Hospital of Wuhan University, Wuhan, 430060, People's Republic of China
| | - Peng Ye
- Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan, 430060, People's Republic of China
| | - Wei Xiong
- Center of Regenerative Medicine and Department of Stomatology, Renmin Hospital of Wuhan University, Wuhan, 430060, People's Republic of China
| | - Xingxiang Duan
- Center of Regenerative Medicine and Department of Stomatology, Renmin Hospital of Wuhan University, Wuhan, 430060, People's Republic of China
| | - Shuili Jing
- Center of Regenerative Medicine and Department of Stomatology, Renmin Hospital of Wuhan University, Wuhan, 430060, People's Republic of China
| | - Yan He
- Institute of Regenerative and Translational Medicine, Tianyou Hospital of Wuhan University of Science and Technology, Wuhan, 430064, Hubei, People's Republic of China
- Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
| | - Zhi Zeng
- Department of Pathology, Renmin Hospital of Wuhan University, Wuhan, 430060, People's Republic of China.
| | - Yen Wei
- The Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing, 100084, People's Republic of China.
| | - Qingsong Ye
- Center of Regenerative Medicine and Department of Stomatology, Renmin Hospital of Wuhan University, Wuhan, 430060, People's Republic of China.
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7
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Edenhofer FC, Térmeg A, Ohnuki M, Jocher J, Kliesmete Z, Briem E, Hellmann I, Enard W. Generation and characterization of inducible KRAB-dCas9 iPSCs from primates for cross-species CRISPRi. iScience 2024; 27:110090. [PMID: 38947524 PMCID: PMC11214527 DOI: 10.1016/j.isci.2024.110090] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2023] [Revised: 03/28/2024] [Accepted: 05/21/2024] [Indexed: 07/02/2024] Open
Abstract
Comparisons of molecular phenotypes across primates provide unique information to understand human biology and evolution, and single-cell RNA-seq CRISPR interference (CRISPRi) screens are a powerful approach to analyze them. Here, we generate and validate three human, three gorilla, and two cynomolgus iPS cell lines that carry a dox-inducible KRAB-dCas9 construct at the AAVS1 locus. We show that despite variable expression levels of KRAB-dCas9 among lines, comparable downregulation of target genes and comparable phenotypic effects are observed in a single-cell RNA-seq CRISPRi screen. Hence, we provide valuable resources for performing and further extending CRISPRi in human and non-human primates.
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Affiliation(s)
- Fiona C. Edenhofer
- Anthropology and Human Genomics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany
| | - Anita Térmeg
- Anthropology and Human Genomics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany
| | - Mari Ohnuki
- Anthropology and Human Genomics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany
- Institute for the Advanced Study of Human Biology, Kyoto University, Kyoto 606-8501, Japan
- Hakubi Center, Kyoto University, Kyoto 606-8501, Japan
| | - Jessica Jocher
- Anthropology and Human Genomics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany
| | - Zane Kliesmete
- Anthropology and Human Genomics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany
| | - Eva Briem
- Anthropology and Human Genomics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany
| | - Ines Hellmann
- Anthropology and Human Genomics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany
| | - Wolfgang Enard
- Anthropology and Human Genomics, Faculty of Biology, Ludwig-Maximilians-Universität München, 82152 Planegg, Germany
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8
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Barry T, Mason K, Roeder K, Katsevich E. Robust differential expression testing for single-cell CRISPR screens at low multiplicity of infection. Genome Biol 2024; 25:124. [PMID: 38760839 PMCID: PMC11100084 DOI: 10.1186/s13059-024-03254-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Accepted: 04/19/2024] [Indexed: 05/19/2024] Open
Abstract
Single-cell CRISPR screens (perturb-seq) link genetic perturbations to phenotypic changes in individual cells. The most fundamental task in perturb-seq analysis is to test for association between a perturbation and a count outcome, such as gene expression. We conduct the first-ever comprehensive benchmarking study of association testing methods for low multiplicity-of-infection (MOI) perturb-seq data, finding that existing methods produce excess false positives. We conduct an extensive empirical investigation of the data, identifying three core analysis challenges: sparsity, confounding, and model misspecification. Finally, we develop an association testing method - SCEPTRE low-MOI - that resolves these analysis challenges and demonstrates improved calibration and power.
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Affiliation(s)
- Timothy Barry
- Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, USA.
| | - Kaishu Mason
- Department of Statistics and Data Science, Wharton School, University of Pennsylvania, Philadelphia, USA
| | - Kathryn Roeder
- Department of Statistics and Data Science, Carnegie Mellon University, Pittsburgh, USA
- Computational Biology Department, Carnegie Mellon University, Pittsburgh, USA
| | - Eugene Katsevich
- Department of Statistics and Data Science, Wharton School, University of Pennsylvania, Philadelphia, USA.
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9
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Barry T, Mason K, Roeder K, Katsevich E. Robust differential expression testing for single-cell CRISPR screens at low multiplicity of infection. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.05.15.540875. [PMID: 38659821 PMCID: PMC11042176 DOI: 10.1101/2023.05.15.540875] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/26/2024]
Abstract
Single-cell CRISPR screens (perturb-seq) link genetic perturbations to phenotypic changes in individual cells. The most fundamental task in perturb-seq analysis is to test for association between a perturbation and a count outcome, such as gene expression. We conduct the first-ever comprehensive benchmarking study of association testing methods for low multiplicity-of-infection (MOI) perturb-seq data, finding that existing methods produce excess false positives. We conduct an extensive empirical investigation of the data, identifying three core analysis challenges: sparsity, confounding, and model misspecification. Finally, we develop an association testing method - SCEPTRE low-MOI - that resolves these analysis challenges and demonstrates improved calibration and power.
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10
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Xing M, Yao B, Xu J, Lu P, Li Q, Wu D, Chen B, Wei J, Su L, Zhao Q. NatD epigenetically activates FOXA2 expression to promote breast cancer progression by facilitating MMP14 expression. iScience 2024; 27:108840. [PMID: 38303717 PMCID: PMC10830889 DOI: 10.1016/j.isci.2024.108840] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2023] [Revised: 12/09/2023] [Accepted: 01/03/2024] [Indexed: 02/03/2024] Open
Abstract
N-α-acetyltransferase D (NatD) mediates N-α-terminal acetylation of histone H4 (Nt-Ac-H4), but its role in breast cancer metastasis remains unknown. Here, we show that depletion of NatD directly represses the expression of FOXA2, and is accompanied by a significant reduction in Nt-Ac-H4 enrichment at the FOXA2 promoter. We show that NatD is commonly upregulated in primary breast cancer tissues, where its expression level correlates with FOXA2 expression, enhanced invasiveness, and poor clinical outcomes. Furthermore, we show that FOXA2 promotes the migration and invasion of breast cancer cells by activating MMP14 expression. MMP14 is also upregulated in breast cancer tissues, where its expression level correlates with FOXA2 expression and poor clinical prognosis. Our study shows that the NatD-FOXA2-MMP14 axis functions as a key signaling pathway to promote the migratory and invasive capabilities of breast cancer cells, suggesting that NatD is a critical epigenetic modulator of cell invasion during breast cancer progression.
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Affiliation(s)
- Mengying Xing
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and General Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing 210046, China
| | - Bing Yao
- National Experimental Teaching Center of Basic Medical Science, Nanjing Medical University, Nanjing, China
| | - Jiaxuan Xu
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and General Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing 210046, China
| | - Peifen Lu
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and General Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing 210046, China
| | - Qixiang Li
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and General Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing 210046, China
| | - Dongliang Wu
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and General Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing 210046, China
| | - Bing Chen
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and General Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing 210046, China
| | - Jiwu Wei
- Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, China
| | - Lei Su
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and General Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing 210046, China
| | - Quan Zhao
- The State Key Laboratory of Pharmaceutical Biotechnology, Department of Hematology and General Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, China-Australia Institute of Translational Medicine, School of Life Sciences, Nanjing University, Nanjing 210046, China
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11
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Matsui S, Granitto M, Buckley M, Ludwig K, Koigi S, Shiley J, Zacharias WJ, Mayhew CN, Lim HW, Iwafuchi M. Pioneer and PRDM transcription factors coordinate bivalent epigenetic states to safeguard cell fate. Mol Cell 2024; 84:476-489.e10. [PMID: 38211589 PMCID: PMC10872272 DOI: 10.1016/j.molcel.2023.12.007] [Citation(s) in RCA: 15] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2023] [Revised: 10/30/2023] [Accepted: 12/08/2023] [Indexed: 01/13/2024]
Abstract
Pioneer transcription factors (TFs) regulate cell fate by establishing transcriptionally primed and active states. However, cell fate control requires the coordination of both lineage-specific gene activation and repression of alternative-lineage programs, a process that is poorly understood. Here, we demonstrate that the pioneer TF FOXA coordinates with PRDM1 TF to recruit nucleosome remodeling and deacetylation (NuRD) complexes and Polycomb repressive complexes (PRCs), which establish highly occupied, accessible nucleosome conformation with bivalent epigenetic states, thereby preventing precocious and alternative-lineage gene expression during human endoderm differentiation. Similarly, the pioneer TF OCT4 coordinates with PRDM14 to form bivalent enhancers and repress cell differentiation programs in human pluripotent stem cells, suggesting that this may be a common and critical function of pioneer TFs. We propose that pioneer and PRDM TFs coordinate to safeguard cell fate through epigenetic repression mechanisms.
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Affiliation(s)
- Satoshi Matsui
- Division of Developmental Biology, Center for Stem Cell & Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
| | - Marissa Granitto
- Division of Developmental Biology, Center for Stem Cell & Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
| | - Morgan Buckley
- Division of Developmental Biology, Center for Stem Cell & Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
| | - Katie Ludwig
- Division of Developmental Biology, Center for Stem Cell & Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
| | - Sandra Koigi
- Division of Developmental Biology, Center for Stem Cell & Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
| | - Joseph Shiley
- Division of Developmental Biology, Center for Stem Cell & Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
| | - William J Zacharias
- Division of Pulmonary Biology and Pulmonary and Critical Care Medicine, Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
| | - Christopher N Mayhew
- Division of Developmental Biology, Center for Stem Cell & Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
| | - Hee-Woong Lim
- Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
| | - Makiko Iwafuchi
- Division of Developmental Biology, Center for Stem Cell & Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
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12
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Yang C, Lei Y, Ren T, Yao M. The Current Situation and Development Prospect of Whole-Genome Screening. Int J Mol Sci 2024; 25:658. [PMID: 38203828 PMCID: PMC10779205 DOI: 10.3390/ijms25010658] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2023] [Revised: 12/22/2023] [Accepted: 12/29/2023] [Indexed: 01/12/2024] Open
Abstract
High-throughput genetic screening is useful for discovering critical genes or gene sequences that trigger specific cell functions and/or phenotypes. Loss-of-function genetic screening is mainly achieved through RNA interference (RNAi), CRISPR knock-out (CRISPRko), and CRISPR interference (CRISPRi) technologies. Gain-of-function genetic screening mainly depends on the overexpression of a cDNA library and CRISPR activation (CRISPRa). Base editing can perform both gain- and loss-of-function genetic screening. This review discusses genetic screening techniques based on Cas9 nuclease, including Cas9-mediated genome knock-out and dCas9-based gene activation and interference. We compare these methods with previous genetic screening techniques based on RNAi and cDNA library overexpression and propose future prospects and applications for CRISPR screening.
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Affiliation(s)
| | | | | | - Mingze Yao
- Shanxi Provincial Key Laboratory for Medical Molecular Cell Biology, Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education and Institute of Biomedical Sciences, Shanxi University, Taiyuan 030006, China; (C.Y.); (Y.L.); (T.R.)
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13
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Li Q, Li J, Wang P, He X, Hong M, Liu F. A Comparative Study of Endoderm Differentiation Between Activin A and Small Molecules. Exp Clin Endocrinol Diabetes 2023; 131:667-675. [PMID: 38056491 DOI: 10.1055/a-2182-8936] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/08/2023]
Abstract
Small molecules such as ROCK inhibitors (Fasudil) and inducer of definitive endoderm 1 (IDE1) can promote differentiation of definitive endoderm, but their effects remain controversial. Therefore, we attempted to verify the effect of these small molecules on promoting definitive endoderm differentiation and found that Fasudil or IDE1 alone could not achieve a similar effect as activin A. On the contrary, CHIR99021 could efficiently promote definitive endoderm differentiation. Nearly 43.4% of experimental cells were SRY-box transcription factor 17 (SOX17)-positive under the synergistic effect of IDE1 and CHIR99021, but its ability to differentiate towards definitive endoderm was still insufficient. Transcriptional analysis and comparison of IDE1 and CHIR99021 synergistic groups (IC) and activin A and CHIR99021 synergistic groups (AC) showed significantly down-regulated definitive endoderm markers in the IC group compared with those in the AC group and the differences between the two groups were mainly due to bone morphogenetic proteins (BMP4) and fibroblast growth factor 17 (FGF17). Further single-cell transcriptome analysis revealed lower expression of BMP4 in SOX17-positive populations, while mothers against decapentaplegic homolog (SMAD) protein translation signal and FGF17 in the AC group were higher than that in the IC group. Western blot analysis showed a significant difference in levels of p-SMAD2/3 between AC and IC groups, which suggests that regulating p-SMAD2/3 may provide a reference to improve the differentiation of definitive endoderm.
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Affiliation(s)
- Qiang Li
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
| | - Jin Li
- Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha 410078, Hunan, PR China
| | - Ping Wang
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
| | - Xiaoqun He
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
| | - Mingzhao Hong
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
| | - Feng Liu
- Department of Endocrinology, University of Chinese Academy of Sciences Shenzhen Hospital, Shenzhen 518106, Guangdong Province, P.R. China
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14
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Rummel CK, Gagliardi M, Ahmad R, Herholt A, Jimenez-Barron L, Murek V, Weigert L, Hausruckinger A, Maidl S, Hauger B, Raabe FJ, Fürle C, Trastulla L, Turecki G, Eder M, Rossner MJ, Ziller MJ. Massively parallel functional dissection of schizophrenia-associated noncoding genetic variants. Cell 2023; 186:5165-5182.e33. [PMID: 37852259 DOI: 10.1016/j.cell.2023.09.015] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 06/12/2023] [Accepted: 09/14/2023] [Indexed: 10/20/2023]
Abstract
Schizophrenia (SCZ) is a highly heritable mental disorder with thousands of associated genetic variants located mostly in the noncoding space of the genome. Translating these associations into insights regarding the underlying pathomechanisms has been challenging because the causal variants, their mechanisms of action, and their target genes remain largely unknown. We implemented a massively parallel variant annotation pipeline (MVAP) to perform SCZ variant-to-function mapping at scale in disease-relevant neural cell types. This approach identified 620 functional variants (1.7%) that operate in a highly developmental context and neuronal-activity-dependent manner. Multimodal integration of epigenomic and CRISPRi screening data enabled us to link these functional variants to target genes, biological processes, and ultimately alterations of neuronal physiology. These results provide a multistage prioritization strategy to map functional single-nucleotide polymorphism (SNP)-to-gene-to-endophenotype relations and offer biological insights into the context-dependent molecular processes modulated by SCZ-associated genetic variation.
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Affiliation(s)
- Christine K Rummel
- Max Planck Institute of Psychiatry, Munich 80804, Germany; International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich 80804, Germany
| | - Miriam Gagliardi
- Department of Psychiatry, University of Münster, Münster 48149, Germany
| | - Ruhel Ahmad
- Max Planck Institute of Psychiatry, Munich 80804, Germany
| | - Alexander Herholt
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU, Munich 80336, Germany; Systasy Bioscience GmbH, Munich 81669, Germany
| | - Laura Jimenez-Barron
- Max Planck Institute of Psychiatry, Munich 80804, Germany; International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich 80804, Germany
| | - Vanessa Murek
- Max Planck Institute of Psychiatry, Munich 80804, Germany
| | - Liesa Weigert
- Max Planck Institute of Psychiatry, Munich 80804, Germany
| | | | - Susanne Maidl
- Max Planck Institute of Psychiatry, Munich 80804, Germany
| | - Barbara Hauger
- Max Planck Institute of Psychiatry, Munich 80804, Germany
| | - Florian J Raabe
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU, Munich 80336, Germany
| | | | - Lucia Trastulla
- International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich 80804, Germany; Department of Psychiatry, University of Münster, Münster 48149, Germany; Technische Universität München Medical Graduate Center Experimental Medicine, Munich 80333, Germany
| | - Gustavo Turecki
- Douglas Mental Health University Institute, Department of Psychiatry, McGill University, Montreal, QC, Canada
| | - Matthias Eder
- Max Planck Institute of Psychiatry, Munich 80804, Germany
| | - Moritz J Rossner
- Department of Psychiatry and Psychotherapy, LMU University Hospital, LMU, Munich 80336, Germany; Systasy Bioscience GmbH, Munich 81669, Germany
| | - Michael J Ziller
- Max Planck Institute of Psychiatry, Munich 80804, Germany; Department of Psychiatry, University of Münster, Münster 48149, Germany; Center for Soft Nanoscience, University of Münster, Münster 48149, Germany.
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15
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Kolvenbach CM, Shril S, Hildebrandt F. The genetics and pathogenesis of CAKUT. Nat Rev Nephrol 2023; 19:709-720. [PMID: 37524861 DOI: 10.1038/s41581-023-00742-9] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/29/2023] [Indexed: 08/02/2023]
Abstract
Congenital anomalies of the kidney and urinary tract (CAKUT) comprise a large variety of malformations that arise from defective kidney or urinary tract development and frequently lead to kidney failure. The clinical spectrum ranges from severe malformations, such as renal agenesis, to potentially milder manifestations, such as vesicoureteral reflux. Almost 50% of cases of chronic kidney disease that manifest within the first three decades of life are caused by CAKUT. Evidence suggests that a large number of CAKUT are genetic in origin. To date, mutations in ~54 genes have been identified as monogenic causes of CAKUT, contributing to 12-20% of the aetiology of the disease. Pathogenic copy number variants have also been shown to cause CAKUT and can be detected in 4-11% of patients. Furthermore, environmental and epigenetic factors can increase the risk of CAKUT. The discovery of novel CAKUT-causing genes is challenging owing to variable expressivity, incomplete penetrance and variable genotype-phenotype correlation. However, such a discovery could ultimately lead to improvements in the accurate molecular genetic diagnosis, assessment of prognosis and multidisciplinary clinical management of patients with CAKUT, potentially including personalized therapeutic approaches.
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Affiliation(s)
- Caroline M Kolvenbach
- Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Shirlee Shril
- Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Friedhelm Hildebrandt
- Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
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16
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Meyers S, Demeyer S, Cools J. CRISPR screening in hematology research: from bulk to single-cell level. J Hematol Oncol 2023; 16:107. [PMID: 37875911 PMCID: PMC10594891 DOI: 10.1186/s13045-023-01495-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2023] [Accepted: 08/21/2023] [Indexed: 10/26/2023] Open
Abstract
The CRISPR genome editing technology has revolutionized the way gene function is studied. Genome editing can be achieved in single genes or for thousands of genes simultaneously in sensitive genetic screens. While conventional genetic screens are limited to bulk measurements of cell behavior, recent developments in single-cell technologies make it possible to combine CRISPR screening with single-cell profiling. In this way, cell behavior and gene expression can be monitored simultaneously, with the additional possibility of including data on chromatin accessibility and protein levels. Moreover, the availability of various Cas proteins leading to inactivation, activation, or other effects on gene function further broadens the scope of such screens. The integration of single-cell multi-omics approaches with CRISPR screening open the path to high-content information on the impact of genetic perturbations at single-cell resolution. Current limitations in cell throughput and data density need to be taken into consideration, but new technologies are rapidly evolving and are likely to easily overcome these limitations. In this review, we discuss the use of bulk CRISPR screening in hematology research, as well as the emergence of single-cell CRISPR screening and its added value to the field.
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Affiliation(s)
- Sarah Meyers
- Center for Human Genetics, KU Leuven, Leuven, Belgium
- Center for Cancer Biology, VIB, Leuven, Belgium
- Leuvens Kanker Instituut (LKI), KU Leuven - UZ Leuven, Leuven, Belgium
| | - Sofie Demeyer
- Center for Human Genetics, KU Leuven, Leuven, Belgium
- Center for Cancer Biology, VIB, Leuven, Belgium
- Leuvens Kanker Instituut (LKI), KU Leuven - UZ Leuven, Leuven, Belgium
| | - Jan Cools
- Center for Human Genetics, KU Leuven, Leuven, Belgium.
- Center for Cancer Biology, VIB, Leuven, Belgium.
- Leuvens Kanker Instituut (LKI), KU Leuven - UZ Leuven, Leuven, Belgium.
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17
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Calandrelli R, Wen X, Charles Richard JL, Luo Z, Nguyen TC, Chen CJ, Qi Z, Xue S, Chen W, Yan Z, Wu W, Zaleta-Rivera K, Hu R, Yu M, Wang Y, Li W, Ma J, Ren B, Zhong S. Genome-wide analysis of the interplay between chromatin-associated RNA and 3D genome organization in human cells. Nat Commun 2023; 14:6519. [PMID: 37845234 PMCID: PMC10579264 DOI: 10.1038/s41467-023-42274-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2023] [Accepted: 10/05/2023] [Indexed: 10/18/2023] Open
Abstract
The interphase genome is dynamically organized in the nucleus and decorated with chromatin-associated RNA (caRNA). It remains unclear whether the genome architecture modulates the spatial distribution of caRNA and vice versa. Here, we generate a resource of genome-wide RNA-DNA and DNA-DNA contact maps in human cells. These maps reveal the chromosomal domains demarcated by locally transcribed RNA, hereafter termed RNA-defined chromosomal domains. Further, the spreading of caRNA is constrained by the boundaries of topologically associating domains (TADs), demonstrating the role of the 3D genome structure in modulating the spatial distribution of RNA. Conversely, stopping transcription or acute depletion of RNA induces thousands of chromatin loops genome-wide. Activation or suppression of the transcription of specific genes suppresses or creates chromatin loops straddling these genes. Deletion of a specific caRNA-producing genomic sequence promotes chromatin loops that straddle the interchromosomal target sequences of this caRNA. These data suggest a feedback loop where the 3D genome modulates the spatial distribution of RNA, which in turn affects the dynamic 3D genome organization.
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Affiliation(s)
- Riccardo Calandrelli
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Xingzhao Wen
- Bioinformatics and Systems Biology Program, University of California San Diego, La Jolla, CA, USA
| | | | - Zhifei Luo
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Tri C Nguyen
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Chien-Ju Chen
- Bioinformatics and Systems Biology Program, University of California San Diego, La Jolla, CA, USA
| | - Zhijie Qi
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Shuanghong Xue
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Weizhong Chen
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Zhangming Yan
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Weixin Wu
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Kathia Zaleta-Rivera
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA
| | - Rong Hu
- Department of Cellular and Molecular Medicine, Center for Epigenomics, University of California San Diego, La Jolla, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
| | - Miao Yu
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
| | - Yuchuan Wang
- Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA
| | - Wenbo Li
- Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center, Houston, TX, USA
| | - Jian Ma
- Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA
| | - Bing Ren
- Department of Cellular and Molecular Medicine, Center for Epigenomics, University of California San Diego, La Jolla, CA, USA
- Ludwig Institute for Cancer Research, La Jolla, CA, USA
| | - Sheng Zhong
- Shu Chien-Gene Lay Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
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18
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Kearns NA, Lobo M, Genga RMJ, Abramowitz RG, Parsi KM, Min J, Kernfeld EM, Huey JD, Kady J, Hennessy E, Brehm MA, Ziller MJ, Maehr R. Generation and molecular characterization of human pluripotent stem cell-derived pharyngeal foregut endoderm. Dev Cell 2023; 58:1801-1818.e15. [PMID: 37751684 PMCID: PMC10637111 DOI: 10.1016/j.devcel.2023.08.024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2023] [Revised: 05/15/2023] [Accepted: 08/18/2023] [Indexed: 09/28/2023]
Abstract
Approaches to study human pharyngeal foregut endoderm-a developmental intermediate that is linked to various human syndromes involving pharynx development and organogenesis of tissues such as thymus, parathyroid, and thyroid-have been hampered by scarcity of tissue access and cellular models. We present an efficient stepwise differentiation method to generate human pharyngeal foregut endoderm from pluripotent stem cells. We determine dose and temporal requirements of signaling pathway engagement for optimized differentiation and characterize the differentiation products on cellular and integrated molecular level. We present a computational classification tool, "CellMatch," and transcriptomic classification of differentiation products on an integrated mouse scRNA-seq developmental roadmap confirms cellular maturation. Integrated transcriptomic and chromatin analyses infer differentiation stage-specific gene regulatory networks. Our work provides the method and integrated multiomic resource for the investigation of disease-relevant loci and gene regulatory networks and their role in developmental defects affecting the pharyngeal endoderm and its derivatives.
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Affiliation(s)
- Nicola A Kearns
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Macrina Lobo
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Ryan M J Genga
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Ryan G Abramowitz
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Krishna M Parsi
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Jiang Min
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Eric M Kernfeld
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Jack D Huey
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Jamie Kady
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Erica Hennessy
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Michael A Brehm
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Michael J Ziller
- Department of Psychiatry, University of Münster, Münster, Germany
| | - René Maehr
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA; Diabetes Center of Excellence, University of Massachusetts Chan Medical School, Worcester, MA, USA.
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19
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Yi Y, Lan X, Li Y, Yan C, Lv J, Zhang T, Jiang W. Fatty acid synthesis and oxidation regulate human endoderm differentiation by mediating SMAD3 nuclear localization via acetylation. Dev Cell 2023; 58:1670-1687.e4. [PMID: 37516106 DOI: 10.1016/j.devcel.2023.07.005] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2022] [Revised: 05/02/2023] [Accepted: 07/07/2023] [Indexed: 07/31/2023]
Abstract
Metabolic remodeling is one of the earliest events that occur during cell differentiation. Here, we define fatty acid metabolism as a key player in definitive endoderm differentiation from human embryonic stem cells. Fatty acid β-oxidation is enhanced while lipogenesis is decreased, and this is due to the phosphorylation of lipogenic enzyme acetyl-CoA carboxylase by AMPK. More importantly, inhibition of fatty acid synthesis by either its inhibitors or AMPK agonist significantly promotes human endoderm differentiation, while blockade of fatty acid oxidation impairs differentiation. Mechanistically, reduced de novo fatty acid synthesis and enhanced fatty acid β-oxidation both contribute to the accumulation of intracellular acetyl-CoA, which guarantees the acetylation of SMAD3 and further causes nuclear localization to promote endoderm differentiation. Thus, our current study identifies a fatty acid synthesis/oxidation shift during early differentiation and presents an instructive role for fatty acid metabolism in regulating human endoderm differentiation.
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Affiliation(s)
- Ying Yi
- Department of Biological Repositories, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Xianchun Lan
- Department of Biological Repositories, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Yinglei Li
- Department of Biological Repositories, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Chenchao Yan
- Department of Biological Repositories, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Jing Lv
- Department of Biological Repositories, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China; College of Life Science, Cangzhou Normal University, Cangzhou 061000, China
| | - Tianzhe Zhang
- Department of Biological Repositories, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Wei Jiang
- Department of Biological Repositories, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China; Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan 430071, China.
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20
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Yu Q, Van Minsel P, Galle E, Thienpont B. GiRAFR improves gRNA detection and annotation in single-cell CRISPR screens. Commun Biol 2023; 6:975. [PMID: 37741886 PMCID: PMC10518011 DOI: 10.1038/s42003-023-05351-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2023] [Accepted: 09/12/2023] [Indexed: 09/25/2023] Open
Abstract
Novel methods that combine single cell RNA-seq with CRISPR screens enable high-throughput characterization of transcriptional changes caused by genetic perturbations. Dedicated software is however lacking to annotate CRISPR guide RNA (gRNA) libraries and associate them with single cell transcriptomes. Here, we describe a CRISPR droplet sequencing (CROP-seq) dataset. During analysis, we observed that the most commonly used method fails to detect mutant gRNAs. We therefore developed a python tool to identify and characterize intact and mutant gRNAs, called GiRAFR. We show that mutant gRNAs are dysfunctional, and failure to detect and annotate them leads to an inflated estimate of the number of untransformed cells, attenuated downregulation of target genes, as well as an underestimated multiplet frequency. These findings are mirrored in publicly available datasets, where we find that up to 35% of cells are transduced with a mutant gRNA. Applying GiRAFR hence stands to improve the annotation and quality of single cell CRISPR screens.
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Affiliation(s)
- Qian Yu
- Laboratory for Functional Epigenetics, Department of Human Genetics, KU Leuven, 3000, Leuven, Belgium
| | - Paulien Van Minsel
- Laboratory for Functional Epigenetics, Department of Human Genetics, KU Leuven, 3000, Leuven, Belgium
| | - Eva Galle
- Laboratory for Functional Epigenetics, Department of Human Genetics, KU Leuven, 3000, Leuven, Belgium
| | - Bernard Thienpont
- Laboratory for Functional Epigenetics, Department of Human Genetics, KU Leuven, 3000, Leuven, Belgium.
- Leuven Institute for Single Cell Omics, KU Leuven, 3000, Leuven, Belgium.
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21
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Balmas E, Sozza F, Bottini S, Ratto ML, Savorè G, Becca S, Snijders KE, Bertero A. Manipulating and studying gene function in human pluripotent stem cell models. FEBS Lett 2023; 597:2250-2287. [PMID: 37519013 DOI: 10.1002/1873-3468.14709] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 07/04/2023] [Accepted: 07/05/2023] [Indexed: 08/01/2023]
Abstract
Human pluripotent stem cells (hPSCs) are uniquely suited to study human development and disease and promise to revolutionize regenerative medicine. These applications rely on robust methods to manipulate gene function in hPSC models. This comprehensive review aims to both empower scientists approaching the field and update experienced stem cell biologists. We begin by highlighting challenges with manipulating gene expression in hPSCs and their differentiated derivatives, and relevant solutions (transfection, transduction, transposition, and genomic safe harbor editing). We then outline how to perform robust constitutive or inducible loss-, gain-, and change-of-function experiments in hPSCs models, both using historical methods (RNA interference, transgenesis, and homologous recombination) and modern programmable nucleases (particularly CRISPR/Cas9 and its derivatives, i.e., CRISPR interference, activation, base editing, and prime editing). We further describe extension of these approaches for arrayed or pooled functional studies, including emerging single-cell genomic methods, and the related design and analytical bioinformatic tools. Finally, we suggest some directions for future advancements in all of these areas. Mastering the combination of these transformative technologies will empower unprecedented advances in human biology and medicine.
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Affiliation(s)
- Elisa Balmas
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Federica Sozza
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Sveva Bottini
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Maria Luisa Ratto
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Giulia Savorè
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Silvia Becca
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Kirsten Esmee Snijders
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
| | - Alessandro Bertero
- Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center "Guido Tarone", University of Turin, Torino, Italy
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22
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Armendariz DA, Sundarrajan A, Hon GC. Breaking enhancers to gain insights into developmental defects. eLife 2023; 12:e88187. [PMID: 37497775 PMCID: PMC10374278 DOI: 10.7554/elife.88187] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2023] [Accepted: 07/19/2023] [Indexed: 07/28/2023] Open
Abstract
Despite ground-breaking genetic studies that have identified thousands of risk variants for developmental diseases, how these variants lead to molecular and cellular phenotypes remains a gap in knowledge. Many of these variants are non-coding and occur at enhancers, which orchestrate key regulatory programs during development. The prevailing paradigm is that non-coding variants alter the activity of enhancers, impacting gene expression programs, and ultimately contributing to disease risk. A key obstacle to progress is the systematic functional characterization of non-coding variants at scale, especially since enhancer activity is highly specific to cell type and developmental stage. Here, we review the foundational studies of enhancers in developmental disease and current genomic approaches to functionally characterize developmental enhancers and their variants at scale. In the coming decade, we anticipate systematic enhancer perturbation studies to link non-coding variants to molecular mechanisms, changes in cell state, and disease phenotypes.
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Affiliation(s)
- Daniel A Armendariz
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, United States
| | - Anjana Sundarrajan
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, United States
| | - Gary C Hon
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, United States
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, United States
- Lyda Hill Department of Bioinformatics, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, United States
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23
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Rummel CK, Gagliardi M, Herholt A, Ahmad R, Murek V, Weigert L, Hausruckinger A, Maidl S, Jimenez-Barron L, Trastulla L, Eder M, Rossner M, Ziller MJ. Cell type and condition specific functional annotation of schizophrenia associated non-coding genetic variants. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.06.27.545266. [PMID: 37425902 PMCID: PMC10326990 DOI: 10.1101/2023.06.27.545266] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/11/2023]
Abstract
Schizophrenia (SCZ) is a highly polygenic disease and genome wide association studies have identified thousands of genetic variants that are statistically associated with this psychiatric disorder. However, our ability to translate these associations into insights on the disease mechanisms has been challenging since the causal genetic variants, their molecular function and their target genes remain largely unknown. In order to address these questions, we established a functional genomics pipeline in combination with induced pluripotent stem cell technology to functionally characterize ~35,000 non-coding genetic variants associated with schizophrenia along with their target genes. This analysis identified a set of 620 (1.7%) single nucleotide polymorphisms as functional on a molecular level in a highly cell type and condition specific fashion. These results provide a high-resolution map of functional variant-gene combinations and offer comprehensive biological insights into the developmental context and stimulation dependent molecular processes modulated by SCZ associated genetic variation.
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Affiliation(s)
- Christine K. Rummel
- Max Planck Institute of Psychiatry, Munich, Germany
- International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich, Germany
| | - Miriam Gagliardi
- Department of Psychiatry, University of Münster, Münster, Germany
| | - Alexander Herholt
- Department of Psychiatry and Psychotherapy, University Hospital, LMU Munich, Munich, Germany
| | - Ruhel Ahmad
- Max Planck Institute of Psychiatry, Munich, Germany
| | | | | | | | | | - Laura Jimenez-Barron
- Max Planck Institute of Psychiatry, Munich, Germany
- International Max Planck Research School for Translational Psychiatry (IMPRS-TP), Munich, Germany
| | - Lucia Trastulla
- Department of Psychiatry, University of Münster, Münster, Germany
| | - Mathias Eder
- Max Planck Institute of Psychiatry, Munich, Germany
| | - Moritz Rossner
- Department of Psychiatry and Psychotherapy, University Hospital, LMU Munich, Munich, Germany
| | - Michael J. Ziller
- Max Planck Institute of Psychiatry, Munich, Germany
- Department of Psychiatry, University of Münster, Münster, Germany
- Center for Soft Nanoscience, University of Münster, Münster, Germany
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24
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Armendariz DA, Goetsch SC, Sundarrajan A, Sivakumar S, Wang Y, Xie S, Munshi NV, Hon GC. CHD-associated enhancers shape human cardiomyocyte lineage commitment. eLife 2023; 12:e86206. [PMID: 37096669 PMCID: PMC10156167 DOI: 10.7554/elife.86206] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2023] [Accepted: 03/10/2023] [Indexed: 04/26/2023] Open
Abstract
Enhancers orchestrate gene expression programs that drive multicellular development and lineage commitment. Thus, genetic variants at enhancers are thought to contribute to developmental diseases by altering cell fate commitment. However, while many variant-containing enhancers have been identified, studies to endogenously test the impact of these enhancers on lineage commitment have been lacking. We perform a single-cell CRISPRi screen to assess the endogenous roles of 25 enhancers and putative cardiac target genes implicated in genetic studies of congenital heart defects (CHDs). We identify 16 enhancers whose repression leads to deficient differentiation of human cardiomyocytes (CMs). A focused CRISPRi validation screen shows that repression of TBX5 enhancers delays the transcriptional switch from mid- to late-stage CM states. Endogenous genetic deletions of two TBX5 enhancers phenocopy epigenetic perturbations. Together, these results identify critical enhancers of cardiac development and suggest that misregulation of these enhancers could contribute to cardiac defects in human patients.
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Affiliation(s)
- Daniel A Armendariz
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical CenterDallasUnited States
| | - Sean C Goetsch
- Department of Internal Medicine, University of Texas Southwestern Medical CenterDallasUnited States
| | - Anjana Sundarrajan
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical CenterDallasUnited States
| | - Sushama Sivakumar
- Department of Internal Medicine, University of Texas Southwestern Medical CenterDallasUnited States
| | - Yihan Wang
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical CenterDallasUnited States
| | - Shiqi Xie
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical CenterDallasUnited States
| | - Nikhil V Munshi
- Department of Internal Medicine, University of Texas Southwestern Medical CenterDallasUnited States
- Division of Cardiology, Department of Molecular Biology, McDermott Center for Human Growth and Development, University of Texas Southwestern Medical CenterDallasUnited States
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical CenterDallasUnited States
| | - Gary C Hon
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical CenterDallasUnited States
- Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical CenterDallasUnited States
- Lyda Hill Department of Bioinformatics, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical CenterDallasUnited States
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25
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Landshammer A, Bolondi A, Kretzmer H, Much C, Buschow R, Rose A, Wu HJ, Mackowiak SD, Braendl B, Giesselmann P, Tornisiello R, Parsi KM, Huey J, Mielke T, Meierhofer D, Maehr R, Hnisz D, Michor F, Rinn JL, Meissner A. T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation. eLife 2023; 12:e83077. [PMID: 36719724 PMCID: PMC9889090 DOI: 10.7554/elife.83077] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2022] [Accepted: 01/06/2023] [Indexed: 02/01/2023] Open
Abstract
Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation.
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Affiliation(s)
- Alexandro Landshammer
- Department of Genome Regulation, Max Planck Institute for Molecular GeneticsBerlinGermany
- Institute of Chemistry and Biochemistry, Freie Universität BerlinBerlinGermany
| | - Adriano Bolondi
- Department of Genome Regulation, Max Planck Institute for Molecular GeneticsBerlinGermany
- Institute of Chemistry and Biochemistry, Freie Universität BerlinBerlinGermany
| | - Helene Kretzmer
- Department of Genome Regulation, Max Planck Institute for Molecular GeneticsBerlinGermany
| | - Christian Much
- Department of Biochemistry, University of Colorado Boulder and BioFrontiers InstituteBoulderUnited States
| | - René Buschow
- Max Planck Institute for Molecular Genetics, Microscopy Core FacilityBerlinGermany
| | - Alina Rose
- Helmholtz Institute for Metabolic, Obesity and Vascular ResearchLeipzigGermany
| | - Hua-Jun Wu
- Department of Data Science, Dana-Farber Cancer Institute, Department of Biostatistics, Harvard T. H. Chan School of Public HealthBostonUnited States
- Center for Precision Medicine Multi-Omics Research, School of Basic Medical Sciences, Peking University Health Science Center and Peking University Cancer Hospital and InstituteBeijingChina
| | - Sebastian D Mackowiak
- Department of Genome Regulation, Max Planck Institute for Molecular GeneticsBerlinGermany
| | - Bjoern Braendl
- Department of Genome Regulation, Max Planck Institute for Molecular GeneticsBerlinGermany
| | - Pay Giesselmann
- Department of Genome Regulation, Max Planck Institute for Molecular GeneticsBerlinGermany
| | - Rosaria Tornisiello
- Department of Genome Regulation, Max Planck Institute for Molecular GeneticsBerlinGermany
| | - Krishna Mohan Parsi
- Program in Molecular Medicine, University of Massachusetts Medical SchoolWorcesterUnited States
| | - Jack Huey
- Program in Molecular Medicine, University of Massachusetts Medical SchoolWorcesterUnited States
| | - Thorsten Mielke
- Max Planck Institute for Molecular Genetics, Microscopy Core FacilityBerlinGermany
| | - David Meierhofer
- Max Planck Institute for Molecular Genetics, Mass Spectrometry Core FacilityBerlinGermany
| | - René Maehr
- Center for Precision Medicine Multi-Omics Research, School of Basic Medical Sciences, Peking University Health Science Center and Peking University Cancer Hospital and InstituteBeijingChina
- Diabetes Center of Excellence, University of Massachusetts Medical SchoolWorcesterUnited States
| | - Denes Hnisz
- Department of Genome Regulation, Max Planck Institute for Molecular GeneticsBerlinGermany
| | - Franziska Michor
- Department of Stem Cell and Regenerative Biology, Harvard UniversityCambridgeUnited States
- Broad Institute of MIT and HarvardCambridgeUnited States
- Department of Data Science, Dana-Farber Cancer Institute, and Department of Biostatistics, Harvard T. H. Chan School of Public HealthBostonUnited States
- The Ludwig Center at Harvard, Boston, MA 02215, USA, and Center for Cancer Evolution, Dana-Farber Cancer InstituteBostonUnited States
| | - John L Rinn
- Department of Biochemistry, University of Colorado Boulder and BioFrontiers InstituteBoulderUnited States
| | - Alexander Meissner
- Department of Genome Regulation, Max Planck Institute for Molecular GeneticsBerlinGermany
- Institute of Chemistry and Biochemistry, Freie Universität BerlinBerlinGermany
- Department of Stem Cell and Regenerative Biology, Harvard UniversityCambridgeUnited States
- Broad Institute of MIT and HarvardCambridgeUnited States
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26
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Madrigal P, Deng S, Feng Y, Militi S, Goh KJ, Nibhani R, Grandy R, Osnato A, Ortmann D, Brown S, Pauklin S. Epigenetic and transcriptional regulations prime cell fate before division during human pluripotent stem cell differentiation. Nat Commun 2023; 14:405. [PMID: 36697417 PMCID: PMC9876972 DOI: 10.1038/s41467-023-36116-9] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Accepted: 01/17/2023] [Indexed: 01/26/2023] Open
Abstract
Stem cells undergo cellular division during their differentiation to produce daughter cells with a new cellular identity. However, the epigenetic events and molecular mechanisms occurring between consecutive cell divisions have been insufficiently studied due to technical limitations. Here, using the FUCCI reporter we developed a cell-cycle synchronised human pluripotent stem cell (hPSC) differentiation system for uncovering epigenome and transcriptome dynamics during the first two divisions leading to definitive endoderm. We observed that transcription of key differentiation markers occurs before cell division, while chromatin accessibility analyses revealed the early inhibition of alternative cell fates. We found that Activator protein-1 members controlled by p38/MAPK signalling are necessary for inducing endoderm while blocking cell fate shifting toward mesoderm, and that enhancers are rapidly established and decommissioned between different cell divisions. Our study has practical biomedical utility for producing hPSC-derived patient-specific cell types since p38/MAPK induction increased the differentiation efficiency of insulin-producing pancreatic beta-cells.
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Affiliation(s)
- Pedro Madrigal
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, CB10 1SA, UK
- Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, CB2 0SZ, UK
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, CB10 1SD, UK
| | - Siwei Deng
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Yuliang Feng
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Stefania Militi
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Kim Jee Goh
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
- The Francis Crick Institute, London, NW1 1AT, UK
| | - Reshma Nibhani
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK
| | - Rodrigo Grandy
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Anna Osnato
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Daniel Ortmann
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Stephanie Brown
- Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK
| | - Siim Pauklin
- Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Old Road, University of Oxford, Headington, Oxford, OX3 7LD, UK.
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27
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Yu C, Li X, Zhao Y, Hu Y. The role of FOXA family transcription factors in glucolipid metabolism and NAFLD. Front Endocrinol (Lausanne) 2023; 14:1081500. [PMID: 36798663 PMCID: PMC9927216 DOI: 10.3389/fendo.2023.1081500] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Accepted: 01/17/2023] [Indexed: 02/04/2023] Open
Abstract
Abnormal glucose metabolism and lipid metabolism are common pathological processes in many metabolic diseases, such as nonalcoholic fatty liver disease (NAFLD). Many studies have shown that the forkhead box (FOX) protein subfamily FOXA has a role in regulating glucolipid metabolism and is closely related to hepatic steatosis and NAFLD. FOXA exhibits a wide range of functions ranging from the initiation steps of metabolism such as the development of the corresponding metabolic organs and the differentiation of cells, to multiple pathways of glucolipid metabolism, to end-of-life problems of metabolism such as age-related obesity. The purpose of this article is to review and discuss the currently known targets and signal transduction pathways of FOXA in glucolipid metabolism. To provide more experimental evidence and basis for further research and clinical application of FOXA in the regulation of glucolipid metabolism and the prevention and treatment of NAFLD.
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Affiliation(s)
- Chuchu Yu
- Key Laboratory of Liver and Kidney Diseases (Ministry of Education), Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Institute of Liver Diseases, Shuguang Hospital Affifiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Xiaojing Li
- Key Laboratory of Liver and Kidney Diseases (Ministry of Education), Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Institute of Liver Diseases, Shuguang Hospital Affifiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Yu Zhao
- Key Laboratory of Liver and Kidney Diseases (Ministry of Education), Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Institute of Liver Diseases, Shuguang Hospital Affifiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
- *Correspondence: Yu Zhao, ; Yiyang Hu,
| | - Yiyang Hu
- Key Laboratory of Liver and Kidney Diseases (Ministry of Education), Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Institute of Liver Diseases, Shuguang Hospital Affifiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
- Institute of Clinical Pharmacology, Shuguang Hospital Affifiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, China
- *Correspondence: Yu Zhao, ; Yiyang Hu,
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28
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Warren I, Moeller MM, Guiggey D, Chiang A, Maloy M, Ogoke O, Groth T, Mon T, Meamardoost S, Liu X, Thompson S, Szeglowski A, Thompson R, Chen P, Paulmurugan R, Yarmush ML, Kidambi S, Parashurama N. FOXA1/2 depletion drives global reprogramming of differentiation state and metabolism in a human liver cell line and inhibits differentiation of human stem cell-derived hepatic progenitor cells. FASEB J 2023; 37:e22652. [PMID: 36515690 DOI: 10.1096/fj.202101506rrr] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2021] [Revised: 10/28/2022] [Accepted: 10/31/2022] [Indexed: 12/15/2022]
Abstract
FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.
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Affiliation(s)
- Iyan Warren
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Michael M Moeller
- Department of Chemical and Biomolecular Engineering, University of Nebraska- Lincoln, Lincoln, Nebraska, USA
| | - Daniel Guiggey
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Alexander Chiang
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Mitchell Maloy
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Ogechi Ogoke
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Theodore Groth
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Tala Mon
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Saber Meamardoost
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Xiaojun Liu
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Sarah Thompson
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Antoni Szeglowski
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Ryan Thompson
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Peter Chen
- Department of Biomedical Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA
| | - Ramasamy Paulmurugan
- Department of Radiology, Canary Center for Early Cancer Detection and the Molecular Imaging Program at Stanford, Stanford University, Palo Alto, California, USA
| | - Martin L Yarmush
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.,Department of Biomedical Engineering, Rutgers University, Piscataway, New Jersey, USA
| | - Srivatsan Kidambi
- Department of Chemical and Biomolecular Engineering, University of Nebraska- Lincoln, Lincoln, Nebraska, USA
| | - Natesh Parashurama
- Department of Chemical and Biological Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA.,Department of Biomedical Engineering, University at Buffalo (State University of New York), Buffalo, New York, USA.,Clinical and Translation Research Center (CTRC), University at Buffalo (State University of New York), Buffalo, New York, USA
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29
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Yamatani Y, Nakai K. Comprehensive comparison of gene expression diversity among a variety of human stem cells. NAR Genom Bioinform 2022; 4:lqac087. [PMID: 36458020 PMCID: PMC9706419 DOI: 10.1093/nargab/lqac087] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Revised: 10/26/2022] [Accepted: 11/08/2022] [Indexed: 12/02/2022] Open
Abstract
Several factors, including tissue origins and culture conditions, affect the gene expression of undifferentiated stem cells. However, understanding the basic identity across different stem cells has not been pursued well despite its importance in stem cell biology. Thus, we aimed to rank the relative importance of multiple factors to gene expression profile among undifferentiated human stem cells by analyzing publicly available RNA-seq datasets. We first conducted batch effect correction to avoid undefined variance in the dataset as possible. Then, we highlighted the relative impact of biological and technical factors among undifferentiated stem cell types: a more influence on tissue origins in induced pluripotent stem cells than in other stem cell types; a stronger impact of culture condition in embryonic stem cells and somatic stem cell types, including mesenchymal stem cells and hematopoietic stem cells. In addition, we found that a characteristic gene module, enriched in histones, exhibits higher expression across different stem cell types that were annotated by specific culture conditions. This tendency was also observed in mouse stem cell RNA-seq data. Our findings would help to obtain general insights into stem cell quality, such as the balance of differentiation potentials that undifferentiated stem cells possess.
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Affiliation(s)
- Yukiyo Yamatani
- Department of Computational Biology and Medical Sciences, the University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan
| | - Kenta Nakai
- Department of Computational Biology and Medical Sciences, the University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan
- Human Genome Center, the Institute of Medical Science, the University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan
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30
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Medina-Cano D, Corrigan EK, Glenn RA, Islam MT, Lin Y, Kim J, Cho H, Vierbuchen T. Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids. Development 2022; 149:dev200561. [PMID: 35899604 PMCID: PMC10655922 DOI: 10.1242/dev.200561] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2022] [Accepted: 07/04/2022] [Indexed: 11/20/2022]
Abstract
Directed differentiation of pluripotent stem cells (PSCs) is a powerful model system for deconstructing embryonic development. Although mice are the most advanced mammalian model system for genetic studies of embryonic development, state-of-the-art protocols for directed differentiation of mouse PSCs into defined lineages require additional steps and generates target cell types with lower purity than analogous protocols for human PSCs, limiting their application as models for mechanistic studies of development. Here, we examine the potential of mouse epiblast stem cells cultured in media containing Wnt pathway inhibitors as a starting point for directed differentiation. As a proof of concept, we focused our efforts on two specific cell/tissue types that have proven difficult to generate efficiently and reproducibly from mouse embryonic stem cells: definitive endoderm and neural organoids. We present new protocols for rapid generation of nearly pure definitive endoderm and forebrain-patterned neural organoids that model the development of prethalamic and hippocampal neurons. These differentiation models present new possibilities for combining mouse genetic tools with in vitro differentiation to characterize molecular and cellular mechanisms of embryonic development.
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Affiliation(s)
- Daniel Medina-Cano
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Emily K. Corrigan
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Rachel A. Glenn
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Cell and Developmental Biology Program, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA
| | - Mohammed T. Islam
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Yuan Lin
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Juliet Kim
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Hyunwoo Cho
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
| | - Thomas Vierbuchen
- Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
- Center for Stem Cell Biology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA
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31
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An artificial LAMA2-GelMA hydrogel microenvironment for the development of pancreatic endocrine progenitors. Biomaterials 2022; 291:121882. [DOI: 10.1016/j.biomaterials.2022.121882] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2022] [Revised: 10/15/2022] [Accepted: 10/23/2022] [Indexed: 11/21/2022]
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32
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Zheng B, Seltzsam S, Wang C, Schierbaum L, Schneider S, Wu CHW, Dai R, Connaughton DM, Nakayama M, Mann N, Stajic N, Mane S, Bauer SB, Tasic V, Nam HJ, Shril S, Hildebrandt F. Whole-exome sequencing identifies FOXL2, FOXA2 and FOXA3 as candidate genes for monogenic congenital anomalies of the kidneys and urinary tract. Nephrol Dial Transplant 2022; 37:1833-1843. [PMID: 34473308 PMCID: PMC9755999 DOI: 10.1093/ndt/gfab253] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2021] [Indexed: 11/14/2022] Open
Abstract
BACKGROUND Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the most common cause of chronic kidney disease in the first three decades of life. Variants in four Forkhead box (FOX) transcription factors have been associated with CAKUT. We hypothesized that other FOX genes, if highly expressed in developing kidneys, may also represent monogenic causes of CAKUT. METHODS We here performed whole-exome sequencing (WES) in 541 families with CAKUT and generated four lists of CAKUT candidate genes: (A) 36 FOX genes showing high expression during renal development, (B) 4 FOX genes known to cause CAKUT to validate list A, (C) 80 genes that we identified as unique potential novel CAKUT candidate genes when performing WES in 541 CAKUT families and (D) 175 genes identified from WES as multiple potential novel CAKUT candidate genes. RESULTS To prioritize potential novel CAKUT candidates in the FOX gene family, we overlapped 36 FOX genes (list A) with lists C and D of WES-derived CAKUT candidates. Intersection with list C identified a de novo FOXL2 in-frame deletion in a patient with eyelid abnormalities and ureteropelvic junction obstruction, and a homozygous FOXA2 missense variant in a patient with horseshoe kidney. Intersection with list D identified a heterozygous FOXA3 missense variant in a CAKUT family with multiple affected individuals. CONCLUSIONS We hereby identified FOXL2, FOXA2 and FOXA3 as novel monogenic candidate genes of CAKUT, supporting the utility of a paralog-based approach to discover mutated genes associated with human disease.
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Affiliation(s)
- Bixia Zheng
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
- Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China
| | - Steve Seltzsam
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Chunyan Wang
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Luca Schierbaum
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Sophia Schneider
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Chen-Han Wilfred Wu
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Rufeng Dai
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Dervla M Connaughton
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Makiko Nakayama
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Nina Mann
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Natasa Stajic
- Department of Pediatric Nephrology, Institute for Mother and Child Health Care, Belgrade, Serbia
| | - Shrikant Mane
- Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
| | - Stuart B Bauer
- Department of Urology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Velibor Tasic
- Medical Faculty of Skopje, University Children's Hospital, Skopje, Macedonia
| | - Hyun Joo Nam
- Department of Biological and Environmental Science, Texas A&M University at Commerce, Commerce, TX, USA
| | - Shirlee Shril
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
| | - Friedhelm Hildebrandt
- Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA
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33
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Loss of FOXA2 induces ER stress and hepatic steatosis and alters developmental gene expression in human iPSC-derived hepatocytes. Cell Death Dis 2022; 13:713. [PMID: 35973994 PMCID: PMC9381545 DOI: 10.1038/s41419-022-05158-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Revised: 07/29/2022] [Accepted: 08/03/2022] [Indexed: 01/21/2023]
Abstract
FOXA2 has been known to play important roles in liver functions in rodents. However, its role in human hepatocytes is not fully understood. Recently, we generated FOXA2 mutant induced pluripotent stem cell (FOXA2-/-iPSC) lines and illustrated that loss of FOXA2 results in developmental defects in pancreatic islet cells. Here, we used FOXA2-/-iPSC lines to understand the role of FOXA2 on the development and function of human hepatocytes. Lack of FOXA2 resulted in significant alterations in the expression of key developmental and functional genes in hepatic progenitors (HP) and mature hepatocytes (MH) as well as an increase in the expression of ER stress markers. Functional assays demonstrated an increase in lipid accumulation, bile acid synthesis and glycerol production, while a decrease in glucose uptake, glycogen storage, and Albumin secretion. RNA-sequencing analysis further validated the findings by showing a significant increase in genes associated with lipid metabolism, bile acid secretion, and suggested the activation of hepatic stellate cells and hepatic fibrosis in MH lacking FOXA2. Overexpression of FOXA2 reversed the defective phenotypes and improved hepatocyte functionality in iPSC-derived hepatic cells lacking FOXA2. These results highlight a potential role of FOXA2 in regulating human hepatic development and function and provide a human hepatocyte model, which can be used to identify novel therapeutic targets for FOXA2-associated liver disorders.
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Application of Gene Editing Technology in Resistance Breeding of Livestock. LIFE (BASEL, SWITZERLAND) 2022; 12:life12071070. [PMID: 35888158 PMCID: PMC9325061 DOI: 10.3390/life12071070] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/17/2022] [Revised: 06/27/2022] [Accepted: 07/06/2022] [Indexed: 02/06/2023]
Abstract
As a new genetic engineering technology, gene editing can precisely modify the specific gene sequence of the organism’s genome. In the last 10 years, with the rapid development of gene editing technology, zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR/Cas9 systems have been applied to modify endogenous genes in organisms accurately. Now, gene editing technology has been used in mice, zebrafish, pigs, cattle, goats, sheep, rabbits, monkeys, and other species. Breeding for disease-resistance in agricultural animals tends to be a difficult task for traditional breeding, but gene editing technology has made this easier. In this work, we overview the development and application of gene editing technology in the resistance breeding of livestock. Also, we further discuss the prospects and outlooks of gene editing technology in disease-resistance breeding.
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35
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Hammelman J, Patel T, Closser M, Wichterle H, Gifford D. Ranking reprogramming factors for cell differentiation. Nat Methods 2022; 19:812-822. [PMID: 35710610 PMCID: PMC10460539 DOI: 10.1038/s41592-022-01522-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Accepted: 05/13/2022] [Indexed: 12/16/2022]
Abstract
Transcription factor over-expression is a proven method for reprogramming cells to a desired cell type for regenerative medicine and therapeutic discovery. However, a general method for the identification of reprogramming factors to create an arbitrary cell type is an open problem. Here we examine the success rate of methods and data for differentiation by testing the ability of nine computational methods (CellNet, GarNet, EBseq, AME, DREME, HOMER, KMAC, diffTF and DeepAccess) to discover and rank candidate factors for eight target cell types with known reprogramming solutions. We compare methods that use gene expression, biological networks and chromatin accessibility data, and comprehensively test parameter and preprocessing of input data to optimize performance. We find the best factor identification methods can identify an average of 50-60% of reprogramming factors within the top ten candidates, and methods that use chromatin accessibility perform the best. Among the chromatin accessibility methods, complex methods DeepAccess and diffTF have higher correlation with the ranked significance of transcription factor candidates within reprogramming protocols for differentiation. We provide evidence that AME and diffTF are optimal methods for transcription factor recovery that will allow for systematic prioritization of transcription factor candidates to aid in the design of new reprogramming protocols.
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Affiliation(s)
- Jennifer Hammelman
- Computational and Systems Biology, MIT, Cambridge, MA, USA
- Computer Science and Artificial Intelligence Laboratory, MIT, Cambridge, MA, USA
| | - Tulsi Patel
- Departments of Pathology and Cell Biology, Neuroscience, Rehabilitation and Regenerative Medicine (in Neurology), Columbia University Irving Medical Center, New York, NY, USA
- Center for Motor Neuron Biology and Disease, Columbia University Irving Medical Center, New York, NY, USA
- Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY, USA
| | - Michael Closser
- Departments of Pathology and Cell Biology, Neuroscience, Rehabilitation and Regenerative Medicine (in Neurology), Columbia University Irving Medical Center, New York, NY, USA
- Center for Motor Neuron Biology and Disease, Columbia University Irving Medical Center, New York, NY, USA
- Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY, USA
| | - Hynek Wichterle
- Departments of Pathology and Cell Biology, Neuroscience, Rehabilitation and Regenerative Medicine (in Neurology), Columbia University Irving Medical Center, New York, NY, USA
- Center for Motor Neuron Biology and Disease, Columbia University Irving Medical Center, New York, NY, USA
- Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY, USA
| | - David Gifford
- Computational and Systems Biology, MIT, Cambridge, MA, USA.
- Computer Science and Artificial Intelligence Laboratory, MIT, Cambridge, MA, USA.
- Department of Biological Engineering, MIT, Cambridge, MA, USA.
- Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA, USA.
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36
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Yang D, Cho H, Tayyebi Z, Shukla A, Luo R, Dixon G, Ursu V, Stransky S, Tremmel DM, Sackett SD, Koche R, Kaplan SJ, Li QV, Park J, Zhu Z, Rosen BP, Pulecio J, Shi ZD, Bram Y, Schwartz RE, Odorico JS, Sidoli S, Wright CV, Leslie CS, Huangfu D. CRISPR screening uncovers a central requirement for HHEX in pancreatic lineage commitment and plasticity restriction. Nat Cell Biol 2022; 24:1064-1076. [PMID: 35787684 PMCID: PMC9283336 DOI: 10.1038/s41556-022-00946-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2021] [Accepted: 05/25/2022] [Indexed: 01/07/2023]
Abstract
The pancreas and liver arise from a common pool of progenitors. However, the underlying mechanisms that drive their lineage diversification from the foregut endoderm are not fully understood. To tackle this question, we undertook a multifactorial approach that integrated human pluripotent-stem-cell-guided differentiation, genome-scale CRISPR-Cas9 screening, single-cell analysis, genomics and proteomics. We discovered that HHEX, a transcription factor (TF) widely recognized as a key regulator of liver development, acts as a gatekeeper of pancreatic lineage specification. HHEX deletion impaired pancreatic commitment and unleashed an unexpected degree of cellular plasticity towards the liver and duodenum fates. Mechanistically, HHEX cooperates with the pioneer TFs FOXA1, FOXA2 and GATA4, shared by both pancreas and liver differentiation programmes, to promote pancreas commitment, and this cooperation restrains the shared TFs from activating alternative lineages. These findings provide a generalizable model for how gatekeeper TFs like HHEX orchestrate lineage commitment and plasticity restriction in broad developmental contexts.
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Affiliation(s)
- Dapeng Yang
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
| | - Hyunwoo Cho
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
| | - Zakieh Tayyebi
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA,Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
| | - Abhijit Shukla
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
| | - Renhe Luo
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA,Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA
| | - Gary Dixon
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA,Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA,Present address: Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA
| | - Valeria Ursu
- Vanderbilt University Program in Developmental Biology and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 37203, USA
| | - Stephanie Stransky
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | | | | | - Richard Koche
- Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
| | - Samuel J. Kaplan
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA,Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
| | - Qing V. Li
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA,Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA
| | - Jiwoon Park
- Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA,Division of Gastroenterology and Hepatology, Department of Medicine, Weill Medical College of Cornell University, New York, NY, 10065, USA
| | - Zengrong Zhu
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
| | - Bess P. Rosen
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA,Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
| | - Julian Pulecio
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
| | - Zhong-Dong Shi
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA
| | - Yaron Bram
- Division of Gastroenterology and Hepatology, Department of Medicine, Weill Medical College of Cornell University, New York, NY, 10065, USA
| | - Robert E. Schwartz
- Division of Gastroenterology and Hepatology, Department of Medicine, Weill Medical College of Cornell University, New York, NY, 10065, USA
| | | | - Simone Sidoli
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA
| | - Christopher V. Wright
- Vanderbilt University Program in Developmental Biology and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 37203, USA
| | - Christina S. Leslie
- Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA,Correspondence to: (DH), (CSL)
| | - Danwei Huangfu
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA,Correspondence to: (DH), (CSL)
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37
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Zhang SY, Zhao J, Ni JJ, Li H, Quan ZZ, Qing H. Application and prospects of high-throughput screening for in vitro neurogenesis. World J Stem Cells 2022; 14:393-419. [PMID: 35949394 PMCID: PMC9244953 DOI: 10.4252/wjsc.v14.i6.393] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2021] [Revised: 04/07/2022] [Accepted: 05/28/2022] [Indexed: 02/06/2023] Open
Abstract
Over the past few decades, high-throughput screening (HTS) has made great contributions to new drug discovery. HTS technology is equipped with higher throughput, minimized platforms, more automated and computerized operating systems, more efficient and sensitive detection devices, and rapid data processing systems. At the same time, in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes. However, challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional (3D) in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions. Here, we review the applications of HTS technologies on in vitro neurogenesis, especially aiming at identifying the essential genes, chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids. We also discuss the developmental tendency of HTS technology, e.g., so-called next-generation screening, which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.
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Affiliation(s)
- Shu-Yuan Zhang
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Juan Zhao
- Aerospace Medical Center, Aerospace Center Hospital, Beijing 100049, China
| | - Jun-Jun Ni
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Hui Li
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Zhen-Zhen Quan
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Hong Qing
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
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38
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Haellman V, Pirkl M, Akmammedov A, Saxena P, Beerenwinkel N, Paro R, Teixeira AP, Fussenegger M. dCas9-mediated dysregulation of gene expression in human induced pluripotent stem cells during primitive streak differentiation. Metab Eng 2022; 73:70-81. [PMID: 35724832 DOI: 10.1016/j.ymben.2022.06.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Revised: 06/14/2022] [Accepted: 06/14/2022] [Indexed: 11/30/2022]
Abstract
CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation. We observed, however, that the dCas9 protein itself exerts a potential non-specific effect in hiPSCs, affecting the cell's phenotype and gene expression patterns during subsequent directed differentiation. We show that this effect is specific to the condition when cells are cultured in medium that does not actively maintain the pluripotency network, and that the sgRNA-free apo-dCas9 protein itself influences endogenous gene expression. Transcriptomics analysis revealed that a significant number of genes involved in developmental processes and various other genes with non-overlapping biological functions are affected by dCas9 overexpression. This suggests a potential adverse phenotypic effect of dCas9 itself in hiPSCs, which could have implications for when and how CRISPR/Cas9-based tools can be used reliably and safely in pluripotent stem cells.
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Affiliation(s)
- Viktor Haellman
- Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland
| | - Martin Pirkl
- Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland; SIB Swiss Institute of Bioinformatics, Mattenstrasse 26, CH, 4058, Basel, Switzerland
| | - Arslan Akmammedov
- Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland
| | - Pratik Saxena
- Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland
| | - Niko Beerenwinkel
- Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland; SIB Swiss Institute of Bioinformatics, Mattenstrasse 26, CH, 4058, Basel, Switzerland
| | - Renato Paro
- Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland; Faculty of Science, University of Basel, Mattenstrasse 26, CH, 4058, Basel, Switzerland
| | - Ana Palma Teixeira
- Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland.
| | - Martin Fussenegger
- Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH, 4058, Basel, Switzerland; Faculty of Science, University of Basel, Mattenstrasse 26, CH, 4058, Basel, Switzerland.
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39
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Fang F, Iaquinta PJ, Xia N, Liu L, Diao L, Reijo Pera RA. Transcriptional control of human gametogenesis. Hum Reprod Update 2022; 28:313-345. [PMID: 35297982 PMCID: PMC9071081 DOI: 10.1093/humupd/dmac002] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2021] [Revised: 11/22/2021] [Indexed: 11/14/2022] Open
Abstract
The pathways of gametogenesis encompass elaborate cellular specialization accompanied by precise partitioning of the genome content in order to produce fully matured spermatozoa and oocytes. Transcription factors are an important class of molecules that function in gametogenesis to regulate intrinsic gene expression programs, play essential roles in specifying (or determining) germ cell fate and assist in guiding full maturation of germ cells and maintenance of their populations. Moreover, in order to reinforce or redirect cell fate in vitro, it is transcription factors that are most frequently induced, over-expressed or activated. Many reviews have focused on the molecular development and genetics of gametogenesis, in vivo and in vitro, in model organisms and in humans, including several recent comprehensive reviews: here, we focus specifically on the role of transcription factors. Recent advances in stem cell biology and multi-omic studies have enabled deeper investigation into the unique transcriptional mechanisms of human reproductive development. Moreover, as methods continually improve, in vitro differentiation of germ cells can provide the platform for robust gain- and loss-of-function genetic analyses. These analyses are delineating unique and shared human germ cell transcriptional network components that, together with somatic lineage specifiers and pluripotency transcription factors, function in transitions from pluripotent stem cells to gametes. This grand theme review offers additional insight into human infertility and reproductive disorders that are linked predominantly to defects in the transcription factor networks and thus may potentially contribute to the development of novel treatments for infertility.
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Affiliation(s)
- Fang Fang
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
| | - Phillip J Iaquinta
- Division of Research, Economic Development, and Graduate Education, California Polytechnic State University, San Luis Obispo, CA, USA
| | - Ninuo Xia
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
| | - Lei Liu
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
| | - Lei Diao
- The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China
| | - Renee A Reijo Pera
- Division of Research, Economic Development, and Graduate Education, California Polytechnic State University, San Luis Obispo, CA, USA
- McLaughlin Research Institute, Great Falls, MT, USA
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40
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Jovic D, Liang X, Zeng H, Lin L, Xu F, Luo Y. Single-cell RNA sequencing technologies and applications: A brief overview. Clin Transl Med 2022; 12:e694. [PMID: 35352511 PMCID: PMC8964935 DOI: 10.1002/ctm2.694] [Citation(s) in RCA: 514] [Impact Index Per Article: 171.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Revised: 12/09/2021] [Accepted: 12/20/2021] [Indexed: 12/19/2022] Open
Abstract
Single-cell RNA sequencing (scRNA-seq) technology has become the state-of-the-art approach for unravelling the heterogeneity and complexity of RNA transcripts within individual cells, as well as revealing the composition of different cell types and functions within highly organized tissues/organs/organisms. Since its first discovery in 2009, studies based on scRNA-seq provide massive information across different fields making exciting new discoveries in better understanding the composition and interaction of cells within humans, model animals and plants. In this review, we provide a concise overview about the scRNA-seq technology, experimental and computational procedures for transforming the biological and molecular processes into computational and statistical data. We also provide an explanation of the key technological steps in implementing the technology. We highlight a few examples on how scRNA-seq can provide unique information for better understanding health and diseases. One important application of the scRNA-seq technology is to build a better and high-resolution catalogue of cells in all living organism, commonly known as atlas, which is key resource to better understand and provide a solution in treating diseases. While great promises have been demonstrated with the technology in all areas, we further highlight a few remaining challenges to be overcome and its great potentials in transforming current protocols in disease diagnosis and treatment.
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Affiliation(s)
- Dragomirka Jovic
- Lars Bolund Institute of Regenerative MedicineQingdao‐Europe Advanced Institute for Life SciencesQingdaoChina
- BGI‐ShenzhenShenzhenChina
| | - Xue Liang
- Lars Bolund Institute of Regenerative MedicineQingdao‐Europe Advanced Institute for Life SciencesQingdaoChina
- BGI‐ShenzhenShenzhenChina
- Department of BiologyUniversity of CopenhagenCopenhagenDenmark
| | - Hua Zeng
- Nanjing University of Chinese MedicineNanjingChina
| | - Lin Lin
- Department of BiomedicineAarhus UniversityAarhusDenmark
- Steno Diabetes Center AarhusAarhus University HospitalAarhusDenmark
| | - Fengping Xu
- Lars Bolund Institute of Regenerative MedicineQingdao‐Europe Advanced Institute for Life SciencesQingdaoChina
- BGI‐ShenzhenShenzhenChina
| | - Yonglun Luo
- Lars Bolund Institute of Regenerative MedicineQingdao‐Europe Advanced Institute for Life SciencesQingdaoChina
- BGI‐ShenzhenShenzhenChina
- Department of BiomedicineAarhus UniversityAarhusDenmark
- Steno Diabetes Center AarhusAarhus University HospitalAarhusDenmark
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41
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Su EY, Spangler A, Bian Q, Kasamoto JY, Cahan P. Reconstruction of dynamic regulatory networks reveals signaling-induced topology changes associated with germ layer specification. Stem Cell Reports 2022; 17:427-442. [PMID: 35090587 PMCID: PMC8828556 DOI: 10.1016/j.stemcr.2021.12.018] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Revised: 12/21/2021] [Accepted: 12/26/2021] [Indexed: 11/17/2022] Open
Abstract
Elucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. Unfortunately, existing computational gene regulatory network (GRN) reconstruction methods are imprecise, computationally burdensome, and fail to reveal dynamic regulatory topologies. Here, we present Epoch, a reconstruction tool that uses single-cell transcriptomics to accurately infer dynamic networks. We apply Epoch to identify the dynamic networks underpinning directed differentiation of mouse embryonic stem cells (ESCs) guided by multiple signaling pathways, and we demonstrate that modulating these pathways drives topological changes that bias cell fate potential. We also find that Peg3 rewires the pluripotency network to favor mesoderm specification. By integrating signaling pathways with GRNs, we trace how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively. Finally, we identify regulatory circuits of patterning and axis formation that distinguish in vitro and in vivo mesoderm specification.
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Affiliation(s)
- Emily Y Su
- Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Abby Spangler
- Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Qin Bian
- Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Jessica Y Kasamoto
- Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Patrick Cahan
- Institute for Cell Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
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42
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Metabolic and epigenetic regulation of endoderm differentiation. Trends Cell Biol 2022; 32:151-164. [PMID: 34607773 PMCID: PMC8760149 DOI: 10.1016/j.tcb.2021.09.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2021] [Revised: 08/31/2021] [Accepted: 09/10/2021] [Indexed: 02/06/2023]
Abstract
The endoderm, one of the three primary germ layers, gives rise to lung, liver, stomach, intestine, colon, pancreas, bladder, and thyroid. These endoderm-originated organs are subject to many life-threatening diseases. However, primary cells/tissues from endodermal organs are often difficult to grow in vitro. Human pluripotent stem cells (hPSCs), therefore, hold great promise for generating endodermal cells and their derivatives for the development of new therapeutics against these human diseases. Although a wealth of research has provided crucial information on the mechanisms underlying endoderm differentiation from hPSCs, increasing evidence has shown that metabolism, in connection with epigenetics, actively regulates endoderm differentiation in addition to the conventional endoderm inducing signals. Here we review recent advances in metabolic and epigenetic regulation of endoderm differentiation.
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43
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Geusz RJ, Wang A, Lam DK, Vinckier NK, Alysandratos KD, Roberts DA, Wang J, Kefalopoulou S, Ramirez A, Qiu Y, Chiou J, Gaulton KJ, Ren B, Kotton DN, Sander M. Sequence logic at enhancers governs a dual mechanism of endodermal organ fate induction by FOXA pioneer factors. Nat Commun 2021; 12:6636. [PMID: 34789735 PMCID: PMC8599738 DOI: 10.1038/s41467-021-26950-0] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2020] [Accepted: 10/28/2021] [Indexed: 01/15/2023] Open
Abstract
FOXA pioneer transcription factors (TFs) associate with primed enhancers in endodermal organ precursors. Using a human stem cell model of pancreas differentiation, we here discover that only a subset of pancreatic enhancers is FOXA-primed, whereas the majority is unprimed and engages FOXA upon lineage induction. Primed enhancers are enriched for signal-dependent TF motifs and harbor abundant and strong FOXA motifs. Unprimed enhancers harbor fewer, more degenerate FOXA motifs, and FOXA recruitment to unprimed but not primed enhancers requires pancreatic TFs. Strengthening FOXA motifs at an unprimed enhancer near NKX6.1 renders FOXA recruitment pancreatic TF-independent, induces priming, and broadens the NKX6.1 expression domain. We make analogous observations about FOXA binding during hepatic and lung development. Our findings suggest a dual role for FOXA in endodermal organ development: first, FOXA facilitates signal-dependent lineage initiation via enhancer priming, and second, FOXA enforces organ cell type-specific gene expression via indirect recruitment by lineage-specific TFs.
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Affiliation(s)
- Ryan J. Geusz
- grid.266100.30000 0001 2107 4242Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA 92093 USA ,grid.266100.30000 0001 2107 4242Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA 92093 USA ,grid.468218.10000 0004 5913 3393Sanford Consortium for Regenerative Medicine, La Jolla, San Diego, CA 92093 USA ,grid.266100.30000 0001 2107 4242Biomedical Graduate Studies Program, University of California San Diego, La Jolla, San Diego, CA 92037 USA
| | - Allen Wang
- grid.266100.30000 0001 2107 4242Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA 92093 USA ,grid.266100.30000 0001 2107 4242Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA 92093 USA ,grid.468218.10000 0004 5913 3393Sanford Consortium for Regenerative Medicine, La Jolla, San Diego, CA 92093 USA
| | - Dieter K. Lam
- grid.266100.30000 0001 2107 4242Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA 92093 USA ,grid.266100.30000 0001 2107 4242Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA 92093 USA ,grid.468218.10000 0004 5913 3393Sanford Consortium for Regenerative Medicine, La Jolla, San Diego, CA 92093 USA
| | - Nicholas K. Vinckier
- grid.266100.30000 0001 2107 4242Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA 92093 USA ,grid.266100.30000 0001 2107 4242Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA 92093 USA ,grid.468218.10000 0004 5913 3393Sanford Consortium for Regenerative Medicine, La Jolla, San Diego, CA 92093 USA
| | - Konstantinos-Dionysios Alysandratos
- grid.239424.a0000 0001 2183 6745Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118 USA ,grid.189504.10000 0004 1936 7558The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA 02118 USA
| | - David A. Roberts
- grid.239424.a0000 0001 2183 6745Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118 USA
| | - Jinzhao Wang
- grid.266100.30000 0001 2107 4242Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA 92093 USA ,grid.266100.30000 0001 2107 4242Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA 92093 USA ,grid.468218.10000 0004 5913 3393Sanford Consortium for Regenerative Medicine, La Jolla, San Diego, CA 92093 USA
| | - Samy Kefalopoulou
- grid.266100.30000 0001 2107 4242Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA 92093 USA ,grid.266100.30000 0001 2107 4242Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA 92093 USA ,grid.468218.10000 0004 5913 3393Sanford Consortium for Regenerative Medicine, La Jolla, San Diego, CA 92093 USA
| | - Araceli Ramirez
- grid.266100.30000 0001 2107 4242Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA 92093 USA ,grid.266100.30000 0001 2107 4242Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA 92093 USA ,grid.468218.10000 0004 5913 3393Sanford Consortium for Regenerative Medicine, La Jolla, San Diego, CA 92093 USA
| | - Yunjiang Qiu
- grid.266100.30000 0001 2107 4242Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA 92093 USA
| | - Joshua Chiou
- grid.266100.30000 0001 2107 4242Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA 92093 USA ,grid.266100.30000 0001 2107 4242Biomedical Graduate Studies Program, University of California San Diego, La Jolla, San Diego, CA 92037 USA
| | - Kyle J. Gaulton
- grid.266100.30000 0001 2107 4242Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA 92093 USA
| | - Bing Ren
- grid.266100.30000 0001 2107 4242Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA 92093 USA ,grid.1052.60000000097371625Ludwig Institute for Cancer Research, La Jolla, San Diego, CA 92093-0653 USA
| | - Darrell N. Kotton
- grid.239424.a0000 0001 2183 6745Center for Regenerative Medicine of Boston University and Boston Medical Center, Boston, MA 02118 USA ,grid.189504.10000 0004 1936 7558The Pulmonary Center and Department of Medicine, Boston University School of Medicine, Boston, MA 02118 USA
| | - Maike Sander
- Department of Pediatrics, Pediatric Diabetes Research Center, University of California, La Jolla, San Diego, CA, 92093, USA. .,Department of Cellular & Molecular Medicine, University of California, La Jolla, San Diego, CA, 92093, USA. .,Sanford Consortium for Regenerative Medicine, La Jolla, San Diego, CA, 92093, USA.
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44
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Haswell JR, Mattioli K, Gerhardinger C, Maass PG, Foster DJ, Peinado P, Wang X, Medina PP, Rinn JL, Slack FJ. Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency. PLoS One 2021; 16:e0252848. [PMID: 34731163 PMCID: PMC8565776 DOI: 10.1371/journal.pone.0252848] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2021] [Accepted: 08/26/2021] [Indexed: 12/26/2022] Open
Abstract
Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.
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Affiliation(s)
- Jeffrey R. Haswell
- Department of Pathology, HMS Initiative for RNA Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America
- Department of Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Kaia Mattioli
- Department of Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, United States of America
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, United States of America
| | - Chiara Gerhardinger
- Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, United States of America
- Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America
| | - Philipp G. Maass
- Genetics and Genome Biology Program, SickKids Research Institute, Toronto, Ontario, Canada
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Daniel J. Foster
- Department of Pathology, HMS Initiative for RNA Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America
- Department of Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Paola Peinado
- Department of Biochemistry and Molecular Biology, University of Granada, Centre for Genomics and Oncological Research (GENYO), Granada, Spain
| | - Xiaofeng Wang
- Department of Molecular and Systems Biology, Geisel School of Medicine, Dartmouth College, Hanover, New Hampshire, United States of America
| | - Pedro P. Medina
- Department of Biochemistry and Molecular Biology, University of Granada, Centre for Genomics and Oncological Research (GENYO), Granada, Spain
| | - John L. Rinn
- Department of Biochemistry, University of Colorado, BioFrontiers Institute, Boulder, Colorado, United States of America
| | - Frank J. Slack
- Department of Pathology, HMS Initiative for RNA Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America
- * E-mail:
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45
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Abstract
Transcription factors (TFs) are essential mediators of epigenetic regulation and modifiers of penetrance. Studies from the past decades have revealed a sub-class of TF that is capable of remodeling closed chromatin states through targeting nucleosomal motifs. This pioneer factor (PF) class of chromatin remodeler is ATP independent in its roles in epigenetic initiation, with nucleosome-motif recognition and association with repressive chromatin regions. Increasing evidence suggests that the fundamental properties of PFs can be coopted in human cancers. We explore the role of PFs in the larger context of tissue-specific epigenetic regulation. Moreover, we highlight an emerging class of chimeric PF derived from translocation partners in human disease and PFs associated with rare tumors. In the age of site-directed genome editing and targeted protein degradation, increasing our understanding of PFs will provide access to next-generation therapy for human disease driven from altered transcriptional circuitry.
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46
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Li B, Hon GC. Single-Cell Genomics: Catalyst for Cell Fate Engineering. Front Bioeng Biotechnol 2021; 9:748942. [PMID: 34733831 PMCID: PMC8558416 DOI: 10.3389/fbioe.2021.748942] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Accepted: 10/05/2021] [Indexed: 12/14/2022] Open
Abstract
As we near a complete catalog of mammalian cell types, the capability to engineer specific cell types on demand would transform biomedical research and regenerative medicine. However, the current pace of discovering new cell types far outstrips our ability to engineer them. One attractive strategy for cellular engineering is direct reprogramming, where induction of specific transcription factor (TF) cocktails orchestrates cell state transitions. Here, we review the foundational studies of TF-mediated reprogramming in the context of a general framework for cell fate engineering, which consists of: discovering new reprogramming cocktails, assessing engineered cells, and revealing molecular mechanisms. Traditional bulk reprogramming methods established a strong foundation for TF-mediated reprogramming, but were limited by their small scale and difficulty resolving cellular heterogeneity. Recently, single-cell technologies have overcome these challenges to rapidly accelerate progress in cell fate engineering. In the next decade, we anticipate that these tools will enable unprecedented control of cell state.
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Affiliation(s)
- Boxun Li
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, United States
| | - Gary C. Hon
- Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, United States
- Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, United States
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47
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Rankin SA, Steimle JD, Yang XH, Rydeen AB, Agarwal K, Chaturvedi P, Ikegami K, Herriges MJ, Moskowitz IP, Zorn AM. Tbx5 drives Aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development. eLife 2021; 10:69288. [PMID: 34643182 PMCID: PMC8555986 DOI: 10.7554/elife.69288] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2021] [Accepted: 09/23/2021] [Indexed: 12/14/2022] Open
Abstract
The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects.
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Affiliation(s)
- Scott A Rankin
- Center for Stem Cell and Organoid Medicine (CuSTOM), Division of Developmental Biology, Perinatal Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, United States
| | - Jeffrey D Steimle
- Department of Pediatrics, University of Chicago, Chicago, United States.,Department of Pathology, University of Chicago, Chicago, United States.,Department of Human Genetics, University of Chicago, Chicago, United States
| | - Xinan H Yang
- Department of Pediatrics, University of Chicago, Chicago, United States.,Department of Pathology, University of Chicago, Chicago, United States.,Department of Human Genetics, University of Chicago, Chicago, United States
| | - Ariel B Rydeen
- Department of Pediatrics, University of Chicago, Chicago, United States.,Department of Pathology, University of Chicago, Chicago, United States.,Department of Human Genetics, University of Chicago, Chicago, United States
| | - Kunal Agarwal
- Center for Stem Cell and Organoid Medicine (CuSTOM), Division of Developmental Biology, Perinatal Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, United States
| | - Praneet Chaturvedi
- Center for Stem Cell and Organoid Medicine (CuSTOM), Division of Developmental Biology, Perinatal Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, United States
| | - Kohta Ikegami
- Department of Pediatrics, University of Chicago, Chicago, United States
| | | | - Ivan P Moskowitz
- Department of Pediatrics, University of Chicago, Chicago, United States.,Department of Pathology, University of Chicago, Chicago, United States.,Department of Human Genetics, University of Chicago, Chicago, United States
| | - Aaron M Zorn
- Center for Stem Cell and Organoid Medicine (CuSTOM), Division of Developmental Biology, Perinatal Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, United States.,University of Cincinnati, College of Medicine, Department of Pediatrics, Chicago, United States
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48
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Akgol Oksuz B, Yang L, Abraham S, Venev SV, Krietenstein N, Parsi KM, Ozadam H, Oomen ME, Nand A, Mao H, Genga RMJ, Maehr R, Rando OJ, Mirny LA, Gibcus JH, Dekker J. Systematic evaluation of chromosome conformation capture assays. Nat Methods 2021; 18:1046-1055. [PMID: 34480151 PMCID: PMC8446342 DOI: 10.1038/s41592-021-01248-7] [Citation(s) in RCA: 127] [Impact Index Per Article: 31.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2020] [Accepted: 07/18/2021] [Indexed: 01/05/2023]
Abstract
Chromosome conformation capture (3C) assays are used to map chromatin interactions genome-wide. Chromatin interaction maps provide insights into the spatial organization of chromosomes and the mechanisms by which they fold. Hi-C and Micro-C are widely used 3C protocols that differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of 3C experimental parameters. We identified optimal protocol variants for either loop or compartment detection, optimizing fragment size and cross-linking chemistry. We used this knowledge to develop a greatly improved Hi-C protocol (Hi-C 3.0) that can detect both loops and compartments relatively effectively. In addition to providing benchmarked protocols, this work produced ultra-deep chromatin interaction maps using Micro-C, conventional Hi-C and Hi-C 3.0 for key cell lines used by the 4D Nucleome project. This analysis systematically evaluates cross-linking chemistry and chromatin fragmentation strategies commonly used in 3C assays and introduces an improved Hi-C protocol for detecting loops and compartments.
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Affiliation(s)
- Betul Akgol Oksuz
- Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA
| | - Liyan Yang
- Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA
| | - Sameer Abraham
- Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Sergey V Venev
- Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA
| | - Nils Krietenstein
- Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA
| | - Krishna Mohan Parsi
- Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA.,Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, USA
| | - Hakan Ozadam
- Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.,Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA
| | - Marlies E Oomen
- Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA
| | - Ankita Nand
- Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA
| | - Hui Mao
- Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA.,Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, USA
| | - Ryan M J Genga
- Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA.,Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, USA
| | - Rene Maehr
- Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA.,Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, USA
| | - Oliver J Rando
- Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA
| | - Leonid A Mirny
- Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA.,Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA.,Graduate Program in Biophysics, Harvard University, Cambridge, MA, USA
| | - Johan H Gibcus
- Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA.
| | - Job Dekker
- Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. .,Howard Hughes Medical Institute, Chevy Chase, MD, USA.
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49
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Hertzano R, Gwilliam K, Rose K, Milon B, Matern MS. Cell Type-Specific Expression Analysis of the Inner Ear: A Technical Report. Laryngoscope 2021; 131 Suppl 5:S1-S16. [PMID: 32579737 PMCID: PMC8996438 DOI: 10.1002/lary.28765] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2020] [Revised: 04/21/2020] [Accepted: 05/01/2020] [Indexed: 01/11/2023]
Abstract
OBJECTIVE The cellular diversity of the inner ear has presented a technical challenge in obtaining molecular insight into its development and function. The application of technological advancements in cell type-specific expression enable clinicians and researchers to leap forward from classic genetics to obtaining mechanistic understanding of congenital and acquired hearing loss. This understanding is essential for development of therapeutics to prevent and reverse diseases of the inner ear, including hearing loss. The objective of this study is to describe and compare the available tools for cell type-specific analysis of the ear, as a means to support decision making in study design. STUDY DESIGN Three major approaches for cell type-specific analysis of the ear including fluorescence-activated cell sorting (FACS), ribosomal and RNA pulldown techniques, and single cell RNA-seq (scRNA-seq) are compared and contrasted using both published and original data. RESULTS We demonstrate the strength and weaknesses of these approaches leading to the inevitable conclusion that to maximize the utility of these approaches, it is important to match the experimental approach with the tissue of origin, cell type of interest, and the biological question. Often, a combined approach (eg, cell sorting and scRNA-seq or expression analysis using 2 separate approaches) is required. Finally, new tools for visualization and analysis of complex expression data, such as the gEAR platform (umgear.org), collate cell type-specific gene expression from the ear field and provide unprecedented access to both clinicians and researchers. LEVEL OF EVIDENCE N/A Laryngoscope, 131:S1-S16, 2021.
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Affiliation(s)
- Ronna Hertzano
- Department of Otorhinolaryngology Head and Neck Surgery University of Maryland School of Medicine 16 S Eutaw St. Suite 500 Baltimore Maryland 21201 U.S.A
- Institute for Genome Sciences, University of Maryland School of Medicine Baltimore Maryland U.S.A
- Department of Anatomy and Neurobiology University of Maryland School of Medicine Baltimore Maryland U.S.A
| | - Kathleen Gwilliam
- Department of Otorhinolaryngology Head and Neck Surgery University of Maryland School of Medicine 16 S Eutaw St. Suite 500 Baltimore Maryland 21201 U.S.A
| | - Kevin Rose
- Department of Otorhinolaryngology Head and Neck Surgery University of Maryland School of Medicine 16 S Eutaw St. Suite 500 Baltimore Maryland 21201 U.S.A
| | - Beatrice Milon
- Department of Otorhinolaryngology Head and Neck Surgery University of Maryland School of Medicine 16 S Eutaw St. Suite 500 Baltimore Maryland 21201 U.S.A
| | - Maggie S. Matern
- Department of Otorhinolaryngology Head and Neck Surgery University of Maryland School of Medicine 16 S Eutaw St. Suite 500 Baltimore Maryland 21201 U.S.A
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50
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Hammelman J, Gifford DK. Discovering differential genome sequence activity with interpretable and efficient deep learning. PLoS Comput Biol 2021; 17:e1009282. [PMID: 34370721 PMCID: PMC8376110 DOI: 10.1371/journal.pcbi.1009282] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2021] [Revised: 08/19/2021] [Accepted: 07/16/2021] [Indexed: 11/23/2022] Open
Abstract
Discovering sequence features that differentially direct cells to alternate fates is key to understanding both cellular development and the consequences of disease related mutations. We introduce Expected Pattern Effect and Differential Expected Pattern Effect, two black-box methods that can interpret genome regulatory sequences for cell type-specific or condition specific patterns. We show that these methods identify relevant transcription factor motifs and spacings that are predictive of cell state-specific chromatin accessibility. Finally, we integrate these methods into framework that is readily accessible to non-experts and available for download as a binary or installed via PyPI or bioconda at https://cgs.csail.mit.edu/deepaccess-package/.
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Affiliation(s)
- Jennifer Hammelman
- Computational and Systems Biology, MIT, Cambridge, Massachusetts, United States of America
- Computer Science and Artificial Intelligence Laboratory, MIT, Cambridge, Massachusetts, United States of America
| | - David K. Gifford
- Computer Science and Artificial Intelligence Laboratory, MIT, Cambridge, Massachusetts, United States of America
- Department of Electrical Engineering & Computer Science, MIT, Cambridge, Massachusetts, United States of America
- Department of Biological Engineering, MIT, Cambridge, Massachusetts, United States of America
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