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1
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Xiao Y, Jin W, Ju L, Fu J, Wang G, Yu M, Chen F, Qian K, Wang X, Zhang Y. Tracking single-cell evolution using clock-like chromatin accessibility loci. Nat Biotechnol 2025; 43:784-798. [PMID: 38724668 DOI: 10.1038/s41587-024-02241-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2022] [Accepted: 04/10/2024] [Indexed: 05/18/2025]
Abstract
Single-cell chromatin accessibility sequencing (scATAC-seq) reconstructs developmental trajectory by phenotypic similarity. However, inferring the exact developmental trajectory is challenging. Previous studies showed age-associated DNA methylation (DNAm) changes in specific genomic regions, termed clock-like differential methylation loci (ClockDML). Age-associated DNAm could either result from or result in chromatin accessibility changes at ClockDML. As cells undergo mitosis, the heterogeneity of chromatin accessibility on clock-like loci is reduced, providing a measure of mitotic age. In this study, we developed a method, called EpiTrace, that counts the fraction of opened clock-like loci from scATAC-seq data to determine cell age and perform lineage tracing in various cell lineages and animal species. It shows concordance with known developmental hierarchies, correlates well with DNAm-based clocks and is complementary with mutation-based lineage tracing, RNA velocity and stemness predictions. Applying EpiTrace to scATAC-seq data reveals biological insights with clinically relevant implications, ranging from hematopoiesis, organ development, tumor biology and immunity to cortical gyrification.
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Affiliation(s)
- Yu Xiao
- Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Wan Jin
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
- Euler Technology, ZGC Life Sciences Park, Beijing, China
| | - Lingao Ju
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Jie Fu
- Hong Kong University of Science and Technology, Hong Kong, China
| | - Gang Wang
- Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Mengxue Yu
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
| | - Fangjin Chen
- High Performance Computing Center, Peking-Tsinghua College of Life Sciences, Peking University, Beijing, China
| | - Kaiyu Qian
- Department of Biological Repositories, Human Genetic Resources Preservation Center of Hubei Province, Hubei Key Laboratory of Urological Diseases, Zhongnan Hospital of Wuhan University, Wuhan, China
- Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, China
| | - Xinghuan Wang
- Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan, China.
- Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, China.
- Wuhan Research Center for Infectious Diseases and Cancer, Chinese Academy of Medical Sciences, Wuhan, China.
| | - Yi Zhang
- Euler Technology, ZGC Life Sciences Park, Beijing, China.
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2
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Deshmukh MG, Brooks VT, Roy SF, Milette S, Bosenberg M, Micevic G. DNA methylation in melanoma immunotherapy: mechanisms and therapeutic opportunities. Clin Epigenetics 2025; 17:71. [PMID: 40307913 PMCID: PMC12044936 DOI: 10.1186/s13148-025-01865-5] [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: 10/24/2024] [Accepted: 03/26/2025] [Indexed: 05/02/2025] Open
Abstract
Abnormal DNA methylation is a hallmark of cancer and a nearly universal feature of melanoma. DNA methylation plays well-appreciated melanoma cell-intrinsic roles, including silencing tumor-suppressor genes, regulating genomic stability, deregulating expression of oncogenes to potentiate proliferative signaling and tumor migration. With the recent success of immunological therapies for melanoma, important roles for DNA methylation are also emerging at the interface between melanoma and immune cells with the potential to regulate the anti-tumor immune response. These newly recognized roles for DNA methylation in controlling melanoma cell immunogenicity, expression of MHC and immune checkpoint molecules as well as T cell phenotypes in the tumor microenvironment raise the possibility of using DNA methylation to develop improved therapies and methylation-based biomarkers. In addition to reviewing the "immune dimension" of DNA methylation, we summarize recent developments with potential clinical applications in melanoma, such as targeted DNA methylation editing, single-cell methylation approaches, and measurement of circulating methylated DNA. An improved understanding of the immune roles of DNA methylation presents an exciting opportunity for continued improvement of care and outcomes for patients with melanoma.
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Affiliation(s)
- Maya G Deshmukh
- Medical Scientist Training Program (MD-PhD), Yale School of Medicine, New Haven, CT, 06520, USA
- Department of Immunobiology, Yale School of Medicine, New Haven, CT, 06520, USA
- Department of Dermatology, Yale School of Medicine, New Haven, CT, 06520, USA
| | - Veronica T Brooks
- Department of Dermatology, Yale School of Medicine, New Haven, CT, 06520, USA
| | - Simon F Roy
- Department of Dermatology, Yale School of Medicine, New Haven, CT, 06520, USA
| | - Simon Milette
- Department of Immunobiology, Yale School of Medicine, New Haven, CT, 06520, USA
- Department of Dermatology, Yale School of Medicine, New Haven, CT, 06520, USA
| | - Marcus Bosenberg
- Department of Immunobiology, Yale School of Medicine, New Haven, CT, 06520, USA
- Department of Pathology, Yale School of Medicine, New Haven, CT, 06520, USA
- Department of Dermatology, Yale School of Medicine, New Haven, CT, 06520, USA
- Yale Cancer Center, Yale School of Medicine, New Haven, CT, 06520, USA
- Yale Center for Immuno-Oncology, Yale School of Medicine, New Haven, CT, 06520, USA
| | - Goran Micevic
- Department of Immunobiology, Yale School of Medicine, New Haven, CT, 06520, USA.
- Department of Pathology, Yale School of Medicine, New Haven, CT, 06520, USA.
- Department of Dermatology, Yale School of Medicine, New Haven, CT, 06520, USA.
- Yale Cancer Center, Yale School of Medicine, New Haven, CT, 06520, USA.
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3
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Tobias IC, Moorthy SD, Shchuka VM, Langroudi L, Cherednychenko M, Gillespie ZE, Duncan AG, Tian R, Gajewska NA, Di Roberto RB, Mitchell JA. A Sox2 enhancer cluster regulates region-specific neural fates from mouse embryonic stem cells. G3 (BETHESDA, MD.) 2025; 15:jkaf012. [PMID: 39849901 PMCID: PMC12005160 DOI: 10.1093/g3journal/jkaf012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/28/2024] [Revised: 01/14/2025] [Accepted: 01/19/2025] [Indexed: 01/25/2025]
Abstract
Sex-determining region Y box 2 (Sox2) is a critical transcription factor for embryogenesis and neural stem and progenitor cell (NSPC) maintenance. While distal enhancers control Sox2 in embryonic stem cells (ESCs), enhancers closer to the gene are implicated in Sox2 transcriptional regulation in neural development. We hypothesize that a downstream enhancer cluster, termed Sox2 regulatory regions 2-18 (SRR2-18), regulates Sox2 transcription in neural stem cells and we investigate this in NSPCs derived from mouse ESCs. Using functional genomics and CRISPR-Cas9-mediated deletion analyses, we investigate the role of SRR2-18 in Sox2 regulation during neural differentiation. Transcriptome analyses demonstrate that the loss of even 1 copy of SRR2-18 disrupts the region-specific identity of NSPCs, reducing the expression of genes associated with more anterior regions of the embryonic nervous system. Homozygous deletion of this Sox2 neural enhancer cluster causes reduced SOX2 protein, less frequent interaction with transcriptional machinery, and leads to perturbed chromatin accessibility genome-wide further affecting the expression of neurodevelopmental and anterior-posterior regionalization genes. Furthermore, homozygous NSPC deletants exhibit self-renewal defects and impaired differentiation into cell types found in the brain. Altogether, our data define a cis-regulatory enhancer cluster controlling Sox2 transcription in NSPCs and highlight the sensitivity of neural differentiation processes to decreased Sox2 transcription, which causes differentiation into posterior neural fates, specifically the caudal neural tube. This study highlights the importance of precise Sox2 regulation by SRR2-18 in neural differentiation.
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Affiliation(s)
- Ian C Tobias
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Sakthi D Moorthy
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Virlana M Shchuka
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Lida Langroudi
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Mariia Cherednychenko
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Zoe E Gillespie
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Andrew G Duncan
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Ruxiao Tian
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Natalia A Gajewska
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Raphaël B Di Roberto
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
| | - Jennifer A Mitchell
- Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada
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4
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Chen S, He Y, Lv L, Liu B, Li C, Deng H, Xu J. Transient chemical-mediated epigenetic modulation confers unrestricted lineage potential on human primed pluripotent stem cells. SCIENCE CHINA. LIFE SCIENCES 2025; 68:1084-1101. [PMID: 39825205 DOI: 10.1007/s11427-024-2660-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Accepted: 06/19/2024] [Indexed: 01/20/2025]
Abstract
Human primed pluripotent stem cells are capable of generating all the embryonic lineages. However, their extraembryonic trophectoderm potentials are limited. It remains unclear how to expand their developmental potential to trophectoderm lineages. Here we show that transient treatment with a cocktail of small molecule epigenetic modulators imparts trophectoderm lineage potentials to human primed pluripotent stem cells while preserving their embryonic potential. These chemically treated cells can generate trophectoderm-like cells and downstream trophoblast stem cells, diverging into syncytiotrophoblast and extravillous trophoblast lineages. Transcriptomic and CUT&Tag analyses reveal that these induced cells share transcriptional profiles with in vivo trophectoderm and cytotrophoblast, and exhibit reduced H3K27me3 modification at gene loci specific to trophoblast lineages compared with primed pluripotent cells. Mechanistic exploration highlighted the critical roles of epigenetic modulators HDAC2, EZH1/2, and KDM5s in the activation of trophoblast lineage potential. Our findings demonstrate that transient epigenetic resetting activates unrestricted lineage potential in human primed pluripotent stem cells, and offer new mechanistic insights into human trophoblast lineage specification and in vitro models for studying placental development and related disorders.
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Affiliation(s)
- Shi Chen
- Department of Cell Biology, School of Basic Medical Sciences, Peking University Stem Cell Research Center, Peking University Health Science Center, Peking University, Beijing, 100191, China
| | - Yuanyuan He
- Academy of Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China
| | - Lejun Lv
- BeiCell Therapeutics, Beijing, 100094, China
| | - Bei Liu
- BeiCell Therapeutics, Beijing, 100094, China.
| | - Cheng Li
- School of Life Sciences, Center for Bioinformatics, Center for Statistical Science, Peking University, Beijing, 100871, China.
| | - Hongkui Deng
- MOE Engineering Research Center of Regenerative Medicine, School of Basic Medical Sciences, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center and the MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China.
| | - Jun Xu
- Department of Cell Biology, School of Basic Medical Sciences, Peking University Stem Cell Research Center, Peking University Health Science Center, Peking University, Beijing, 100191, China.
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5
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Schmidt T, Wiesbeck M, Egert L, Truong TT, Danese A, Voshagen L, Imhof S, Iraci Borgia M, Deeksha, Neuner A, Köferle A, Geerlof A, Santos Dias Mourão A, Stricker S. Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP) complexes. Nucleic Acids Res 2025; 53:gkaf235. [PMID: 40156858 PMCID: PMC11952960 DOI: 10.1093/nar/gkaf235] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2024] [Revised: 03/03/2025] [Accepted: 03/17/2025] [Indexed: 04/01/2025] Open
Abstract
For genome editing, the use of CRISPR ribonucleoprotein (RNP) complexes is well established and often the superior choice over plasmid-based or viral strategies. RNPs containing dCas9 fusion proteins, which enable the targeted manipulation of transcriptomes and epigenomes, remain significantly less accessible. Here, we describe the production, delivery, and optimization of second generation CRISPRa RNPs (dRNPs). We characterize the transcriptional and cellular consequences of dRNP treatments in a variety of human target cells and show that the uptake is very efficient. The targeted activation of genes demonstrates remarkable potency, even for genes that are strongly silenced, such as developmental master transcription factors. In contrast to DNA-based CRISPRa strategies, gene activation is immediate and characterized by a sharp temporal precision. We also show that dRNPs allow very high-target multiplexing, enabling undiminished gene activation of multiple genes simultaneously. Applying these insights, we find that intensive target multiplexing at single promoters synergistically elevates gene transcription. Finally, we demonstrate in human stem and differentiated cells that the preferable features of dRNPs allow to instruct and convert cell fates efficiently without the need for DNA delivery or viral vectors.
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Affiliation(s)
- Tobias Schmidt
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
- Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Maximilian Wiesbeck
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
- Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Luisa Egert
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
- Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Thi-Tram Truong
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
- Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Anna Danese
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Lukas Voshagen
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Simon Imhof
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Matilde Iraci Borgia
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Deeksha
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
- Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Andrea M Neuner
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
- Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Anna Köferle
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
- Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
| | - Arie Geerlof
- Institute of Structural Biology, Helmholtz Centre Munich, German Research Center for Environmental Health, Neuherberg, 85764, Germany
| | - André Santos Dias Mourão
- Institute of Structural Biology, Helmholtz Centre Munich, German Research Center for Environmental Health, Neuherberg, 85764, Germany
| | - Stefan H Stricker
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
- Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, Grosshaderner Strasse 9, Planegg-Martinsried, 82152, Germany
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6
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Fu Y, Yang X, Li S, Ma C, An Y, Cheng T, Liang Y, Sun S, Cheng T, Zhao Y, Wang J, Wang X, Xu P, Yin Y, Liang H, Liu N, Zou W, Chen B. Dynamic properties of transcriptional condensates modulate CRISPRa-mediated gene activation. Nat Commun 2025; 16:1640. [PMID: 39952932 PMCID: PMC11828908 DOI: 10.1038/s41467-025-56735-8] [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: 04/23/2024] [Accepted: 01/28/2025] [Indexed: 02/17/2025] Open
Abstract
CRISPR activation (CRISPRa) is a powerful tool for endogenous gene activation, yet the mechanisms underlying its optimal transcriptional activation remain unclear. By monitoring real-time transcriptional bursts, we find that CRISPRa modulates both burst duration and amplitude. Our quantitative imaging reveals that CRISPR-SunTag activators, with three tandem VP64-p65-Rta (VPR), form liquid-like transcriptional condensates and exhibit high activation potency. Although visible CRISPRa condensates are associated with some RNA bursts, the overall levels of phase separation do not correlate with transcriptional bursting or activation strength in individual cells. When the number of SunTag scaffolds is increased to 10 or more, solid-like condensates form, sequestering co-activators such as p300 and MED1. These condensates display low dynamicity and liquidity, resulting in ineffective gene activation. Overall, our studies characterize various phase-separated CRISPRa systems for gene activation, highlighting the foundational principles for engineering CRISPR-based programmable synthetic condensates with appropriate properties to effectively modulate gene expression.
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Affiliation(s)
- Yujuan Fu
- Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China
- Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, China
- Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, China
| | - Xiaoxuan Yang
- Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China
- Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, China
| | - Sihui Li
- Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China
- Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, China
| | - Chenyang Ma
- Women's Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Yao An
- Center of Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, China
| | - Tao Cheng
- Women's Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Ying Liang
- Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China
- Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, China
| | - Shengbai Sun
- Center of Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, China
| | - Tianyi Cheng
- Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China
- Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, China
| | - Yongyang Zhao
- Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China
- Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, China
| | - Jianghu Wang
- Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China
- The State Key Laboratory of Southwest Karst Mountain Biodiversity Conservation of Forestry Administration, School of Life Science, Guizhou Normal University, Guiyang, China
| | - Xiaoyue Wang
- The State Key Laboratory of Southwest Karst Mountain Biodiversity Conservation of Forestry Administration, School of Life Science, Guizhou Normal University, Guiyang, China
| | - Pengfei Xu
- Women's Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Yafei Yin
- Center of Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, China
| | - Hongqing Liang
- Women's Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Nan Liu
- Liangzhu Laboratory, Zhejiang University Medical Center, Hangzhou, China.
- Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, China.
| | - Wei Zou
- The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu, China.
- Insititute of Translational Medicine, Zhejiang University, Hangzhou, China.
| | - Baohui Chen
- Bone Marrow Transplantation Center of the First Affiliated Hospital and Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China.
- Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou, China.
- Zhejiang Provincial Key Laboratory of Genetic & Developmental Disorders, Hangzhou, China.
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7
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Mengistu DY, Terribili M, Pellacani C, Ciapponi L, Marzullo M. Epigenetic regulation of TDP-43: potential implications for amyotrophic lateral sclerosis. FRONTIERS IN MOLECULAR MEDICINE 2025; 5:1530719. [PMID: 40017539 PMCID: PMC11865237 DOI: 10.3389/fmmed.2025.1530719] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/19/2024] [Accepted: 01/22/2025] [Indexed: 03/01/2025]
Abstract
Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterized by the progressive degeneration of motor neurons. One of the key pathogenic factors implicated in ALS is TDP-43 (TAR DNA-binding protein 43), an RNA-binding protein encoded by the TARDBP gene. Under normal physiological conditions, TDP-43 predominantly resides in the nucleus, where it plays a critical role in regulating gene expression, alternative splicing, RNA transport, and stability. In ALS, TDP-43 undergoes pathological mislocalization from the nucleus to the cytoplasm, disrupting its normal function and contributing to disease progression. The nuclear loss of TDP-43 leads to widespread dysregulation of RNA metabolism. Moreover, mislocalized TDP-43 aggregates in the cytoplasm, acquires toxic properties that sequester essential RNA molecules and proteins. Importantly, deviations in TDP-43 levels, whether excessive or reduced, can lead to cellular dysfunction, and contribute to disease progression, highlighting the delicate balance required for neuronal health. Emerging evidence suggests that epigenetic mechanisms may play a crucial role in regulating TARDBP expression and, consequently, TDP-43 cellular levels. Epigenetic modifications such as DNA methylation, histone modifications, and non-coding RNAs are increasingly recognized as modulators of gene expression and cellular function in neurodegenerative diseases, including ALS. Dysregulation of these processes could contribute to aberrant TARDBP expression, amplifying TDP-43-associated pathologies. This review explores and summarizes the recent findings on how specific epigenetic modifications influence TDP-43 expression and discusses their possible implications for disease progression.
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Affiliation(s)
- D. Y. Mengistu
- Department of Biology and Biotechnologies, Sapienza University of Rome, Rome, Italy
| | - M. Terribili
- Department of Biology and Biotechnologies, Sapienza University of Rome, Rome, Italy
| | - C. Pellacani
- Istituto Di Biologia e Patologia Molecolari, CNR, Sapienza Università Di Roma, Rome, Italy
| | - L. Ciapponi
- Department of Biology and Biotechnologies, Sapienza University of Rome, Rome, Italy
| | - M. Marzullo
- Department of Biology and Biotechnologies, Sapienza University of Rome, Rome, Italy
- Istituto Di Biologia e Patologia Molecolari, CNR, Sapienza Università Di Roma, Rome, Italy
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8
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Guo X, Li X, Wang S, Shi Y, Huang J, Liu X, Lu Y, Zhang J, Luo L, You J. Optimizing Adoptive Cell Therapy for Solid Tumors via Epigenetic Regulation of T-cell Destiny. Adv Healthc Mater 2024; 13:e2402209. [PMID: 39301920 DOI: 10.1002/adhm.202402209] [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: 06/17/2024] [Revised: 09/03/2024] [Indexed: 09/22/2024]
Abstract
Adoptive cell therapy (ACT) emerged as a promising approach for cancer treatment, yet its application in solid tumors faced challenges such as inadequate tumor infiltration and cellular dysfunction. Histone acetylation is reported to play a crucial role in restoring T-cell function within tumor tissues. Building upon previous research, a novel strategy involving the co-loading of two drugs, G3C12 and vorinostat (SAHA), into PLGA microspheres to form G3C12+SAHA@PLGA is developed for intratumoral injection. The G3C12 peptide enhances adoptive T-cell recruitment to the tumor site by modulating the binding state of IFN-γ. While SAHA, a histone deacetylase inhibitor, promotes memory phenotypes of infiltrating T-cells and prevents their transition to an exhausted state. This synergistic approach effectively augmentes the efficacy of ACT in the "cold" tumor model (4T1) or the "hot" tumor model (CT26). These findings highlight the potential of combining epigenetic regulation with recruitment signaling as a means to enhance the therapeutic impact of ACT in treating solid tumors.
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Affiliation(s)
- Xuemeng Guo
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
| | - Xiang Li
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
| | - Sijie Wang
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
| | - Yingying Shi
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
| | - Jiaxin Huang
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
| | - Xu Liu
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
| | - Yichao Lu
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
| | - Junlei Zhang
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
| | - Lihua Luo
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
| | - Jian You
- College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, P. R. China
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, 79 Qingchun Road, Shangcheng District, Hangzhou, Zhejiang, 310006, P. R. China
- The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 QingChun Road, Hangzhou, Zhejiang, 310000, P. R. China
- Jinhua Institute of Zhejiang University, 498 Yiwu Street, Jinhua, Zhejiang, 321299, P. R. China
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9
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Sun Y, Wang HY, Liu B, Yue B, Liu Q, Liu Y, Rosa IF, Doretto LB, Han S, Lin L, Gong X, Shao C. CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole ( Cynoglossus semilaevis) Testis Cells. Int J Mol Sci 2024; 25:7637. [PMID: 39062879 PMCID: PMC11277268 DOI: 10.3390/ijms25147637] [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: 05/27/2024] [Revised: 07/03/2024] [Accepted: 07/09/2024] [Indexed: 07/28/2024] Open
Abstract
DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.
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Affiliation(s)
- Yanxu Sun
- College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China; (Y.S.); (B.Y.); (X.G.)
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
| | - Hong-Yan Wang
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
| | - Binghua Liu
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
| | - Bowen Yue
- College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China; (Y.S.); (B.Y.); (X.G.)
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
| | - Qian Liu
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
| | - Yuyan Liu
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
| | - Ivana F. Rosa
- Department of Structural and Functional Biology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu 01049-010, Brazil;
| | - Lucas B. Doretto
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
| | - Shenglei Han
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
| | - Lei Lin
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
| | - Xiaoling Gong
- College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China; (Y.S.); (B.Y.); (X.G.)
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources (Shanghai Ocean University), Ministry of Education, Shanghai 201306, China
- National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
| | - Changwei Shao
- State Key Laboratory of Mariculture Biobreeding and Sustainable Goods, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China; (H.-Y.W.); (B.L.); (Q.L.); (Y.L.); (L.B.D.); (S.H.); (L.L.)
- Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao 266237, China
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10
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Corveleyn L, Sen P, Adams P, Sidoli S. Linking Aging to Cancer: The Role of Chromatin Biology. J Gerontol A Biol Sci Med Sci 2024; 79:glae133. [PMID: 38761362 PMCID: PMC11170291 DOI: 10.1093/gerona/glae133] [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: 02/20/2024] [Indexed: 05/20/2024] Open
Abstract
Epigenetic changes have been established to be a hallmark of aging, which implies that aging science requires collaborating with the field of chromatin biology. DNA methylation patterns, changes in relative abundance of histone post-translational modifications, and chromatin remodeling are the central players in modifying chromatin structure. Aging is commonly associated with an overall increase in chromatin instability, loss of homeostasis, and decondensation. However, numerous publications have highlighted that the link between aging and chromatin changes is not nearly as linear as previously expected. This complex interplay of these epigenetic elements during the lifetime of an organism likely contributes to cellular senescence, genomic instability, and disease susceptibility. Yet, the causal links between these phenomena still need to be fully unraveled. In this perspective article, we discuss potential future directions of aging chromatin biology.
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Affiliation(s)
- Laura Corveleyn
- Laboratory of Pharmaceutical Biotechnology, Department of Pharmaceutics, Ghent University, Ghent, Belgium
| | - Payel Sen
- Laboratory of Genetics and Genomics, National Institute on Aging, Baltimore, Maryland, USA
| | - Peter Adams
- Sanford Burnham Prebys Medical Discovery Institute, San Diego, California, USA
| | - Simone Sidoli
- Department of Biochemistry, Albert Einstein College of Medicine, New York, New York, USA
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11
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Pereira A, Diwakar J, Masserdotti G, Beşkardeş S, Simon T, So Y, Martín-Loarte L, Bergemann F, Vasan L, Schauer T, Danese A, Bocchi R, Colomé-Tatché M, Schuurmans C, Philpott A, Straub T, Bonev B, Götz M. Direct neuronal reprogramming of mouse astrocytes is associated with multiscale epigenome remodeling and requires Yy1. Nat Neurosci 2024; 27:1260-1273. [PMID: 38956165 PMCID: PMC11239498 DOI: 10.1038/s41593-024-01677-5] [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: 09/16/2022] [Accepted: 05/10/2024] [Indexed: 07/04/2024]
Abstract
Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.
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Affiliation(s)
- Allwyn Pereira
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Institute of Stem Cell Research, Helmholtz Center Munich, BMC LMU Munich, Planegg, Germany
- Nantes Université, CHU Nantes, INSERM, TaRGeT - Translational Research in Gene Therapy, UMR 1089, Nantes, France
| | - Jeisimhan Diwakar
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Helmholtz Pioneer Campus, Helmholtz Center Munich, Neuherberg, Germany
| | - Giacomo Masserdotti
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Institute of Stem Cell Research, Helmholtz Center Munich, BMC LMU Munich, Planegg, Germany
| | - Sude Beşkardeş
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Helmholtz Pioneer Campus, Helmholtz Center Munich, Neuherberg, Germany
| | - Tatiana Simon
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Institute of Stem Cell Research, Helmholtz Center Munich, BMC LMU Munich, Planegg, Germany
| | - Younju So
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Institute of Stem Cell Research, Helmholtz Center Munich, BMC LMU Munich, Planegg, Germany
| | - Lucía Martín-Loarte
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Institute of Stem Cell Research, Helmholtz Center Munich, BMC LMU Munich, Planegg, Germany
| | - Franziska Bergemann
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Institute of Stem Cell Research, Helmholtz Center Munich, BMC LMU Munich, Planegg, Germany
| | - Lakshmy Vasan
- Biological Science Platform, Sunnybrook Research Institute; Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | - Tamas Schauer
- Biomedical Center Munich (BMC), Bioinformatic Core Facility, Faculty of Medicine, LMU Munich, Planegg, Germany
- Institute of Stem Cells and Epigenetics, Helmholtz Center Munich, Neuherberg, Germany
| | - Anna Danese
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Institute of Stem Cell Research, Helmholtz Center Munich, BMC LMU Munich, Planegg, Germany
| | - Riccardo Bocchi
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany
- Institute of Stem Cell Research, Helmholtz Center Munich, BMC LMU Munich, Planegg, Germany
- Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland
| | - Maria Colomé-Tatché
- Institute of Computational Biology, Helmholtz Center Munich, Neuherberg, Germany
- Biomedical Center Munich (BMC), Physiological Chemistry, Faculty of Medicine, LMU Munich, Planegg, Germany
| | - Carol Schuurmans
- Biological Science Platform, Sunnybrook Research Institute; Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | - Anna Philpott
- Wellcome-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK
| | - Tobias Straub
- Biological Science Platform, Sunnybrook Research Institute; Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | - Boyan Bonev
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany.
- Helmholtz Pioneer Campus, Helmholtz Center Munich, Neuherberg, Germany.
| | - Magdalena Götz
- Biomedical Center Munich (BMC), Physiological Genomics, LMU Munich, Planegg, Germany.
- Institute of Stem Cell Research, Helmholtz Center Munich, BMC LMU Munich, Planegg, Germany.
- Excellence Cluster of Systems Neurology (SYNERGY), Munich, Germany.
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12
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Cerneckis J, Cai H, Shi Y. Induced pluripotent stem cells (iPSCs): molecular mechanisms of induction and applications. Signal Transduct Target Ther 2024; 9:112. [PMID: 38670977 PMCID: PMC11053163 DOI: 10.1038/s41392-024-01809-0] [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: 07/28/2023] [Revised: 03/09/2024] [Accepted: 03/17/2024] [Indexed: 04/28/2024] Open
Abstract
The induced pluripotent stem cell (iPSC) technology has transformed in vitro research and holds great promise to advance regenerative medicine. iPSCs have the capacity for an almost unlimited expansion, are amenable to genetic engineering, and can be differentiated into most somatic cell types. iPSCs have been widely applied to model human development and diseases, perform drug screening, and develop cell therapies. In this review, we outline key developments in the iPSC field and highlight the immense versatility of the iPSC technology for in vitro modeling and therapeutic applications. We begin by discussing the pivotal discoveries that revealed the potential of a somatic cell nucleus for reprogramming and led to successful generation of iPSCs. We consider the molecular mechanisms and dynamics of somatic cell reprogramming as well as the numerous methods available to induce pluripotency. Subsequently, we discuss various iPSC-based cellular models, from mono-cultures of a single cell type to complex three-dimensional organoids, and how these models can be applied to elucidate the mechanisms of human development and diseases. We use examples of neurological disorders, coronavirus disease 2019 (COVID-19), and cancer to highlight the diversity of disease-specific phenotypes that can be modeled using iPSC-derived cells. We also consider how iPSC-derived cellular models can be used in high-throughput drug screening and drug toxicity studies. Finally, we discuss the process of developing autologous and allogeneic iPSC-based cell therapies and their potential to alleviate human diseases.
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Affiliation(s)
- Jonas Cerneckis
- Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA
- Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA
| | - Hongxia Cai
- Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA
| | - Yanhong Shi
- Department of Neurodegenerative Diseases, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA.
- Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA, 91010, USA.
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13
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Stricker SH, Pereira CF. Reprogramming Stars #15: Colliding Cellular Reprogramming Paths- An Interview with Dr. Stefan Stricker. Cell Reprogram 2024; 26:37-42. [PMID: 38635923 DOI: 10.1089/cell.2024.29115.shs] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/20/2024] Open
Affiliation(s)
- Stefan H Stricker
- Reprogramming and Regeneration, Biomedical Center (BMC), Physiological Genomics, Faculty of Medicine, LMU Munich. Epigenetic Engineering, Institute of Stem Cell Research, Helmholtz Zentrum, German Research Center for Environmental Health, Planegg-Martinsried, Germany
| | - Carlos-Filipe Pereira
- Molecular Medicine and Gene Therapy, Lund Stem Cell Centre, Wallenberg Centre for Molecular Medicine, Lund University, Lund, Sweden
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14
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Seem K, Kaur S, Kumar S, Mohapatra T. Epigenome editing for targeted DNA (de)methylation: a new perspective in modulating gene expression. Crit Rev Biochem Mol Biol 2024; 59:69-98. [PMID: 38440883 DOI: 10.1080/10409238.2024.2320659] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2023] [Accepted: 02/15/2024] [Indexed: 03/06/2024]
Abstract
Traditionally, it has been believed that inheritance is driven as phenotypic variations resulting from changes in DNA sequence. However, this paradigm has been challenged and redefined in the contemporary era of epigenetics. The changes in DNA methylation, histone modification, non-coding RNA biogenesis, and chromatin remodeling play crucial roles in genomic functions and regulation of gene expression. More importantly, some of these changes are inherited to the next generations as a part of epigenetic memory and play significant roles in gene expression. The sum total of all changes in DNA bases, histone proteins, and ncRNA biogenesis constitutes the epigenome. Continuous progress in deciphering epigenetic regulations and the existence of heritable epigenetic/epiallelic variations associated with trait of interest enables to deploy epigenome editing tools to modulate gene expression. DNA methylation marks can be utilized in epigenome editing for the manipulation of gene expression. Initially, genome/epigenome editing technologies relied on zinc-finger protein or transcriptional activator-like effector protein. However, the discovery of clustered regulatory interspaced short palindromic repeats CRISPR)/deadCRISPR-associated protein 9 (dCas9) enabled epigenome editing to be more specific/efficient for targeted DNA (de)methylation. One of the major concerns has been the off-target effects, wherein epigenome editing may unintentionally modify gene/regulatory element which may cause unintended change/harmful effects. Moreover, epigenome editing of germline cell raises several ethical/safety issues. This review focuses on the recent developments in epigenome editing tools/techniques, technological limitations, and future perspectives of this emerging technology in therapeutics for human diseases as well as plant improvement to achieve sustainable developmental goals.
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Affiliation(s)
- Karishma Seem
- Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi, India
| | - Simardeep Kaur
- Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi, India
| | - Suresh Kumar
- Division of Biochemistry, ICAR-Indian Agricultural Research Institute, New Delhi, India
| | - Trilochan Mohapatra
- Protection of Plant Varieties and Farmers' Rights Authority, New Delhi, India
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15
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Fatima N, Saif Ur Rahman M, Qasim M, Ali Ashfaq U, Ahmed U, Masoud MS. Transcriptional Factors Mediated Reprogramming to Pluripotency. Curr Stem Cell Res Ther 2024; 19:367-388. [PMID: 37073151 DOI: 10.2174/1574888x18666230417084518] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2022] [Revised: 02/01/2023] [Accepted: 02/06/2023] [Indexed: 04/20/2023]
Abstract
A unique kind of pluripotent cell, i.e., Induced pluripotent stem cells (iPSCs), now being targeted for iPSC synthesis, are produced by reprogramming animal and human differentiated cells (with no change in genetic makeup for the sake of high efficacy iPSCs formation). The conversion of specific cells to iPSCs has revolutionized stem cell research by making pluripotent cells more controllable for regenerative therapy. For the past 15 years, somatic cell reprogramming to pluripotency with force expression of specified factors has been a fascinating field of biomedical study. For that technological primary viewpoint reprogramming method, a cocktail of four transcription factors (TF) has required: Kruppel-like factor 4 (KLF4), four-octamer binding protein 34 (OCT3/4), MYC and SOX2 (together referred to as OSKM) and host cells. IPS cells have great potential for future tissue replacement treatments because of their ability to self-renew and specialize in all adult cell types, although factor-mediated reprogramming mechanisms are still poorly understood medically. This technique has dramatically improved performance and efficiency, making it more useful in drug discovery, disease remodeling, and regenerative medicine. Moreover, in these four TF cocktails, more than 30 reprogramming combinations were proposed, but for reprogramming effectiveness, only a few numbers have been demonstrated for the somatic cells of humans and mice. Stoichiometry, a combination of reprogramming agents and chromatin remodeling compounds, impacts kinetics, quality, and efficiency in stem cell research.
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Affiliation(s)
- Nazira Fatima
- Laboratory Animal Center, Xi'an Jiaotong University Health Science Center, Xi'an, Shaanxi, 710061, China
| | - Muhammad Saif Ur Rahman
- Institute of Advanced Studies, Shenzhen University, Shenzhen, 518060, China
- Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, 518060, China
| | - Muhammad Qasim
- Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad, 38000, Pakistan
| | - Usman Ali Ashfaq
- Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad, 38000, Pakistan
| | - Uzair Ahmed
- EMBL Partnership Institute for Genome Editing Technologies, Vilnius University, Vilnius, 10257, Lithuania
| | - Muhammad Shareef Masoud
- Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad, 38000, Pakistan
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16
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Péron S, Miyakoshi LM, Brill MS, Manzano-Franco D, Serrano-López J, Fan W, Marichal N, Ghanem A, Conzelmann KK, Karow M, Ortega F, Gascón S, Berninger B. Programming of neural progenitors of the adult subependymal zone towards a glutamatergic neuron lineage by neurogenin 2. Stem Cell Reports 2023; 18:2418-2433. [PMID: 37995703 PMCID: PMC10724369 DOI: 10.1016/j.stemcr.2023.10.019] [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: 08/21/2021] [Revised: 10/26/2023] [Accepted: 10/27/2023] [Indexed: 11/25/2023] Open
Abstract
Although adult subependymal zone (SEZ) neural stem cells mostly generate GABAergic interneurons, a small progenitor population expresses the proneural gene Neurog2 and produces glutamatergic neurons. Here, we determined whether Neurog2 could respecify SEZ neural stem cells and their progeny toward a glutamatergic fate. Retrovirus-mediated expression of Neurog2 induced the glutamatergic lineage markers TBR2 and TBR1 in cultured SEZ progenitors, which differentiated into functional glutamatergic neurons. Likewise, Neurog2-transduced SEZ progenitors acquired glutamatergic neuron hallmarks in vivo. Intriguingly, they failed to migrate toward the olfactory bulb and instead differentiated within the SEZ or the adjacent striatum, where they received connections from local neurons, as indicated by rabies virus-mediated monosynaptic tracing. In contrast, lentivirus-mediated expression of Neurog2 failed to reprogram early SEZ neurons, which maintained GABAergic identity and migrated to the olfactory bulb. Our data show that NEUROG2 can program SEZ progenitors toward a glutamatergic identity but fails to reprogram their neuronal progeny.
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Affiliation(s)
- Sophie Péron
- Research Group "Adult Neurogenesis and Cellular Reprogramming", Institute of Physiological Chemistry, University Medical Center Johannes Gutenberg University, Mainz, Germany; Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK
| | - Leo M Miyakoshi
- Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, Munich, Germany
| | - Monika S Brill
- Institute of Neuronal Cell Biology, Technical University Munich, Munich, Germany; Munich Cluster of Systems Neurology (SyNergy), Munich, Germany
| | - Diana Manzano-Franco
- Department of Molecular, Cellular and Developmental Neurobiology, Cajal Institute - CSIC, Madrid, Spain
| | - Julia Serrano-López
- Department of Biochemistry and Molecular Biology, Faculty of Veterinary, Universidad Complutense de Madrid (UCM), Madrid, Spain; Instituto Universitario de Investigación en Neuroquímica (IUIN), Madrid, Spain; Instituto de Investigación Sanitaria San Carlos (IdISSC), Madrid, Spain
| | - Wenqiang Fan
- Research Group "Adult Neurogenesis and Cellular Reprogramming", Institute of Physiological Chemistry, University Medical Center Johannes Gutenberg University, Mainz, Germany
| | - Nicolás Marichal
- Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK
| | - Alexander Ghanem
- Max von Pettenkofer Institute and Gene Center, Ludwig Maximilians-University Munich, Munich, Germany
| | - Karl-Klaus Conzelmann
- Max von Pettenkofer Institute and Gene Center, Ludwig Maximilians-University Munich, Munich, Germany
| | - Marisa Karow
- Institute of Biochemistry, Friedrich-Alexander Universität Nürnberg-Erlangen, Erlangen, Germany
| | - Felipe Ortega
- Department of Biochemistry and Molecular Biology, Faculty of Veterinary, Universidad Complutense de Madrid (UCM), Madrid, Spain; Instituto Universitario de Investigación en Neuroquímica (IUIN), Madrid, Spain; Instituto de Investigación Sanitaria San Carlos (IdISSC), Madrid, Spain
| | - Sergio Gascón
- Department of Molecular, Cellular and Developmental Neurobiology, Cajal Institute - CSIC, Madrid, Spain.
| | - Benedikt Berninger
- Research Group "Adult Neurogenesis and Cellular Reprogramming", Institute of Physiological Chemistry, University Medical Center Johannes Gutenberg University, Mainz, Germany; Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK; MRC Centre for Neurodevelopmental Disorders, Institute of Psychiatry, Psychology & Neuroscience, King's College London, London, UK; Focus Program Translational Neurosciences, Johannes Gutenberg University, Mainz, Germany.
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17
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Cai R, Lv R, Shi X, Yang G, Jin J. CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation. Int J Mol Sci 2023; 24:14865. [PMID: 37834313 PMCID: PMC10573330 DOI: 10.3390/ijms241914865] [Citation(s) in RCA: 29] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2023] [Revised: 09/29/2023] [Accepted: 10/01/2023] [Indexed: 10/15/2023] Open
Abstract
CRISPR/Cas9-mediated cleavage of DNA, which depends on the endonuclease activity of Cas9, has been widely used for gene editing due to its excellent programmability and specificity. However, the changes to the DNA sequence that are mediated by CRISPR/Cas9 affect the structures and stability of the genome, which may affect the accuracy of results. Mutations in the RuvC and HNH regions of the Cas9 protein lead to the inactivation of Cas9 into dCas9 with no endonuclease activity. Despite the loss of endonuclease activity, dCas9 can still bind the DNA strand using guide RNA. Recently, proteins with active/inhibitory effects have been linked to the end of the dCas9 protein to form fusion proteins with transcriptional active/inhibitory effects, named CRISPRa and CRISPRi, respectively. These CRISPR tools mediate the transcription activity of protein-coding and non-coding genes by regulating the chromosomal modification states of target gene promoters, enhancers, and other functional elements. Here, we highlight the epigenetic mechanisms and applications of the common CRISPR/dCas9 tools, by which we hope to provide a reference for future related gene regulation, gene function, high-throughput target gene screening, and disease treatment.
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Affiliation(s)
- Ruijie Cai
- Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Runyu Lv
- Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Xin'e Shi
- Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Gongshe Yang
- Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Jianjun Jin
- Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
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18
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Zhang X, Zhang Y, Wang C, Wang X. TET (Ten-eleven translocation) family proteins: structure, biological functions and applications. Signal Transduct Target Ther 2023; 8:297. [PMID: 37563110 PMCID: PMC10415333 DOI: 10.1038/s41392-023-01537-x] [Citation(s) in RCA: 50] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2022] [Revised: 05/24/2023] [Accepted: 06/05/2023] [Indexed: 08/12/2023] Open
Abstract
Ten-eleven translocation (TET) family proteins (TETs), specifically, TET1, TET2 and TET3, can modify DNA by oxidizing 5-methylcytosine (5mC) iteratively to yield 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC), and then two of these intermediates (5fC and 5caC) can be excised and return to unmethylated cytosines by thymine-DNA glycosylase (TDG)-mediated base excision repair. Because DNA methylation and demethylation play an important role in numerous biological processes, including zygote formation, embryogenesis, spatial learning and immune homeostasis, the regulation of TETs functions is complicated, and dysregulation of their functions is implicated in many diseases such as myeloid malignancies. In addition, recent studies have demonstrated that TET2 is able to catalyze the hydroxymethylation of RNA to perform post-transcriptional regulation. Notably, catalytic-independent functions of TETs in certain biological contexts have been identified, further highlighting their multifunctional roles. Interestingly, by reactivating the expression of selected target genes, accumulated evidences support the potential therapeutic use of TETs-based DNA methylation editing tools in disorders associated with epigenetic silencing. In this review, we summarize recent key findings in TETs functions, activity regulators at various levels, technological advances in the detection of 5hmC, the main TETs oxidative product, and TETs emerging applications in epigenetic editing. Furthermore, we discuss existing challenges and future directions in this field.
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Affiliation(s)
- Xinchao Zhang
- Department of Pathology, Ruijin Hospital and College of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
| | - Yue Zhang
- Department of Pathology, Ruijin Hospital and College of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
| | - Chaofu Wang
- Department of Pathology, Ruijin Hospital and College of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
| | - Xu Wang
- Department of Pathology, Ruijin Hospital and College of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
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19
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Wang SE, Jiang YH. Novel epigenetic molecular therapies for imprinting disorders. Mol Psychiatry 2023; 28:3182-3193. [PMID: 37626134 PMCID: PMC10618104 DOI: 10.1038/s41380-023-02208-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/12/2023] [Revised: 07/21/2023] [Accepted: 07/27/2023] [Indexed: 08/27/2023]
Abstract
Genomic imprinting disorders are caused by the disruption of genomic imprinting processes leading to a deficit or increase of an active allele. Their unique molecular mechanisms underlying imprinted genes offer an opportunity to investigate epigenetic-based therapy for reactivation of an inactive allele or reduction of an active allele. Current treatments are based on managing symptoms, not targeting the molecular mechanisms underlying imprinting disorders. Here, we highlight molecular approaches of therapeutic candidates in preclinical and clinical studies for individual imprinting disorders. These include the significant progress of discovery and testing of small molecules, antisense oligonucleotides, and CRISPR mediated genome editing approaches as new therapeutic strategies. We discuss the significant challenges of translating these promising therapies from the preclinical stage to the clinic, especially for genome editing based approaches.
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Affiliation(s)
- Sung Eun Wang
- Department of Genetics, Yale University School of Medicine, 333 Cedar street, New Haven, CT, 06520, USA
| | - Yong-Hui Jiang
- Department of Genetics, Yale University School of Medicine, 333 Cedar street, New Haven, CT, 06520, USA.
- Department of Neuroscience, Yale University School of Medicine, 333 Cedar street, New Haven, CT, 06520, USA.
- Department of Pediatrics, Yale University School of Medicine, 333 Cedar street, New Haven, CT, 06520, USA.
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20
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Abstract
DNA-editing enzymes perform chemical reactions on DNA nucleobases. These reactions can change the genetic identity of the modified base or modulate gene expression. Interest in DNA-editing enzymes has burgeoned in recent years due to the advent of clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) systems, which can be used to direct their DNA-editing activity to specific genomic loci of interest. In this review, we showcase DNA-editing enzymes that have been repurposed or redesigned and developed into programmable base editors. These include deaminases, glycosylases, methyltransferases, and demethylases. We highlight the astounding degree to which these enzymes have been redesigned, evolved, and refined and present these collective engineering efforts as a paragon for future efforts to repurpose and engineer other families of enzymes. Collectively, base editors derived from these DNA-editing enzymes facilitate programmable point mutation introduction and gene expression modulation by targeted chemical modification of nucleobases.
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Affiliation(s)
- Kartik L Rallapalli
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA;
| | - Alexis C Komor
- Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA;
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21
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Damle EB, Morrison VE, Cioma J, Volic M, Bix GJ. Co-administration of extracellular matrix-based biomaterials with neural stem cell transplantation for treatment of central nervous system injury. Front Neurosci 2023; 17:1177040. [PMID: 37255752 PMCID: PMC10225608 DOI: 10.3389/fnins.2023.1177040] [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: 03/01/2023] [Accepted: 04/28/2023] [Indexed: 06/01/2023] Open
Abstract
Injuries and disorders of the central nervous system (CNS) present a particularly difficult challenge for modern medicine to address, given the complex nature of the tissues, obstacles in researching and implementing therapies, and barriers to translating efficacious treatments into human patients. Recent advancements in neural stem cell (NSC) transplantation, endogenous neurogenesis, and in vivo reprogramming of non-neural cells into the neuronal lineage represent multiple approaches to resolving CNS injury. However, we propose that one practice that must be incorporated universally in neuroregeneration studies is the use of extracellular matrix (ECM)-mimicking biomaterials to supply the architectural support and cellular microenvironment necessary for partial or complete restoration of function. Through consideration of developmental processes including neurogenesis, cellular migration, and establishment of functional connectivity, as well as evaluation of process-specific interactions between cells and ECM components, insights can be gained to harness and modulate native and induced neurobiological processes to promote CNS tissue repair. Further, evaluation of the current landscape of regenerative medicine and tissue engineering techniques external to the neurosciences provides key perspectives into the role of the ECM in the use of stem cell-based therapies, and the potential directions future neuroregenerative approaches may take. If the most successful of these approaches achieve wide-spread adoption, innovative paired NSC-ECM strategies for neuroregeneration may become prominent in the near future, and with the rapid advances these techniques are poised to herald, a new era of treatment for CNS injury may dawn.
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Affiliation(s)
- Eshan B. Damle
- Clinical Neuroscience Research Center, Tulane University School of Medicine, New Orleans, LA, United States
- Tulane Brain Institute, Tulane University, New Orleans, LA, United States
- Department of Cell and Molecular Biology, Tulane University, New Orleans, LA, United States
| | - Vivianne E. Morrison
- Clinical Neuroscience Research Center, Tulane University School of Medicine, New Orleans, LA, United States
| | - Jozef Cioma
- Faculty of Biology, Medicine, and Health, School of Biological Sciences, University of Manchester, Manchester, United Kingdom
| | - Milla Volic
- Faculty of Biology, Medicine, and Health, School of Biological Sciences, University of Manchester, Manchester, United Kingdom
| | - Gregory J. Bix
- Clinical Neuroscience Research Center, Tulane University School of Medicine, New Orleans, LA, United States
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22
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Murtaj V, Butti E, Martino G, Panina-Bordignon P. Endogenous neural stem cells characterization using omics approaches: Current knowledge in health and disease. Front Cell Neurosci 2023; 17:1125785. [PMID: 37091923 PMCID: PMC10113633 DOI: 10.3389/fncel.2023.1125785] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Accepted: 03/03/2023] [Indexed: 04/08/2023] Open
Abstract
Neural stem cells (NSCs), an invaluable source of neuronal and glial progeny, have been widely interrogated in the last twenty years, mainly to understand their therapeutic potential. Most of the studies were performed with cells derived from pluripotent stem cells of either rodents or humans, and have mainly focused on their potential in regenerative medicine. High-throughput omics technologies, such as transcriptomics, epigenetics, proteomics, and metabolomics, which exploded in the past decade, represent a powerful tool to investigate the molecular mechanisms characterizing the heterogeneity of endogenous NSCs. The transition from bulk studies to single cell approaches brought significant insights by revealing complex system phenotypes, from the molecular to the organism level. Here, we will discuss the current literature that has been greatly enriched in the “omics era”, successfully exploring the nature and function of endogenous NSCs and the process of neurogenesis. Overall, the information obtained from omics studies of endogenous NSCs provides a sharper picture of NSCs function during neurodevelopment in healthy and in perturbed environments.
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Affiliation(s)
- Valentina Murtaj
- Division of Neuroscience, San Raffaele Vita-Salute University, Milan, Italy
- Neuroimmunology, Division of Neuroscience, Institute of Experimental Neurology, IRCCS Ospedale San Raffaele, Milan, Italy
| | - Erica Butti
- Neuroimmunology, Division of Neuroscience, Institute of Experimental Neurology, IRCCS Ospedale San Raffaele, Milan, Italy
| | - Gianvito Martino
- Division of Neuroscience, San Raffaele Vita-Salute University, Milan, Italy
- Neuroimmunology, Division of Neuroscience, Institute of Experimental Neurology, IRCCS Ospedale San Raffaele, Milan, Italy
| | - Paola Panina-Bordignon
- Division of Neuroscience, San Raffaele Vita-Salute University, Milan, Italy
- Neuroimmunology, Division of Neuroscience, Institute of Experimental Neurology, IRCCS Ospedale San Raffaele, Milan, Italy
- *Correspondence: Paola Panina-Bordignon
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23
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Toward the Development of Epigenome Editing-Based Therapeutics: Potentials and Challenges. Int J Mol Sci 2023; 24:ijms24054778. [PMID: 36902207 PMCID: PMC10003136 DOI: 10.3390/ijms24054778] [Citation(s) in RCA: 22] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 02/27/2023] [Accepted: 02/28/2023] [Indexed: 03/06/2023] Open
Abstract
The advancement in epigenetics research over the past several decades has led to the potential application of epigenome-editing technologies for the treatment of various diseases. In particular, epigenome editing is potentially useful in the treatment of genetic and other related diseases, including rare imprinted diseases, as it can regulate the expression of the epigenome of the target region, and thereby the causative gene, with minimal or no modification of the genomic DNA. Various efforts are underway to successfully apply epigenome editing in vivo, such as improving target specificity, enzymatic activity, and drug delivery for the development of reliable therapeutics. In this review, we introduce the latest findings, summarize the current limitations and future challenges in the practical application of epigenome editing for disease therapy, and introduce important factors to consider, such as chromatin plasticity, for a more effective epigenome editing-based therapy.
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24
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Dennison R, Usuga E, Chen H, Paul JZ, Arbelaez CA, Teng YD. Direct Cell Reprogramming and Phenotypic Conversion: An Analysis of Experimental Attempts to Transform Astrocytes into Neurons in Adult Animals. Cells 2023; 12:618. [PMID: 36831283 PMCID: PMC9954435 DOI: 10.3390/cells12040618] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Revised: 02/06/2023] [Accepted: 02/09/2023] [Indexed: 02/17/2023] Open
Abstract
Central nervous system (CNS) repair after injury or disease remains an unresolved problem in neurobiology research and an unmet medical need. Directly reprogramming or converting astrocytes to neurons (AtN) in adult animals has been investigated as a potential strategy to facilitate brain and spinal cord recovery and advance fundamental biology. Conceptually, AtN strategies rely on forced expression or repression of lineage-specific transcription factors to make endogenous astrocytes become "induced neurons" (iNs), presumably without re-entering any pluripotent or multipotent states. The AtN-derived cells have been reported to manifest certain neuronal functions in vivo. However, this approach has raised many new questions and alternative explanations regarding the biological features of the end products (e.g., iNs versus neuron-like cells, neural functional changes, etc.), developmental biology underpinnings, and neurobiological essentials. For this paper per se, we proposed to draw an unconventional distinction between direct cell conversion and direct cell reprogramming, relative to somatic nuclear transfer, based on the experimental methods utilized to initiate the transformation process, aiming to promote a more in-depth mechanistic exploration. Moreover, we have summarized the current tactics employed for AtN induction, comparisons between the bench endeavors concerning outcome tangibility, and discussion of the issues of published AtN protocols. Lastly, the urgency to clearly define/devise the theoretical frameworks, cell biological bases, and bench specifics to experimentally validate primary data of AtN studies was highlighted.
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Affiliation(s)
- Rachel Dennison
- Department of Physical Medicine and Rehabilitation, Harvard Medical School, Boston, MA 02129, USA
- Laboratory of SCI, Stem Cell and Recovery Neurobiology Research, Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital Network, Mass General Brigham, and Harvard Medical School, Boston, MA 02115, USA
| | - Esteban Usuga
- Department of Physical Medicine and Rehabilitation, Harvard Medical School, Boston, MA 02129, USA
- Laboratory of SCI, Stem Cell and Recovery Neurobiology Research, Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital Network, Mass General Brigham, and Harvard Medical School, Boston, MA 02115, USA
| | - Harriet Chen
- Department of Physical Medicine and Rehabilitation, Harvard Medical School, Boston, MA 02129, USA
- Laboratory of SCI, Stem Cell and Recovery Neurobiology Research, Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital Network, Mass General Brigham, and Harvard Medical School, Boston, MA 02115, USA
| | - Jacob Z. Paul
- Department of Physical Medicine and Rehabilitation, Harvard Medical School, Boston, MA 02129, USA
- Laboratory of SCI, Stem Cell and Recovery Neurobiology Research, Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital Network, Mass General Brigham, and Harvard Medical School, Boston, MA 02115, USA
| | - Christian A. Arbelaez
- Department of Physical Medicine and Rehabilitation, Harvard Medical School, Boston, MA 02129, USA
- Laboratory of SCI, Stem Cell and Recovery Neurobiology Research, Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital Network, Mass General Brigham, and Harvard Medical School, Boston, MA 02115, USA
| | - Yang D. Teng
- Department of Physical Medicine and Rehabilitation, Harvard Medical School, Boston, MA 02129, USA
- Laboratory of SCI, Stem Cell and Recovery Neurobiology Research, Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital Network, Mass General Brigham, and Harvard Medical School, Boston, MA 02115, USA
- Neurotrauma Recovery Research, Department of Physical Medicine and Rehabilitation, Spaulding Rehabilitation Hospital Network, Mass General Brigham, and Harvard Medical School, Boston, MA 02115, USA
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25
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Sapozhnikov DM, Szyf M. Enzyme-free targeted DNA demethylation using CRISPR-dCas9-based steric hindrance to identify DNA methylation marks causal to altered gene expression. Nat Protoc 2022; 17:2840-2881. [PMID: 36207463 DOI: 10.1038/s41596-022-00741-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2022] [Accepted: 06/22/2022] [Indexed: 11/09/2022]
Abstract
DNA methylation involves the enzymatic addition of a methyl group primarily to cytosine residues in DNA. This protocol describes how to produce complete and minimally confounded DNA demethylation of specific sites in the genome of cultured cells by clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and without the involvement of an epigenetic-modifying enzyme, the purpose of which is the evaluation of the functional (i.e., gene expression or phenotypic) consequences of DNA demethylation of specific sites that have been previously implicated in particular pathological or physiological contexts. This protocol maximizes the ability of the easily reprogrammable CRISPR-dCas9 system to assess the impact of DNA methylation from a causal rather than correlational perspective: alternative protocols for CRISPR-dCas9-based site-specific DNA methylation or demethylation rely on the recruitment of epigenetic enzymes that exhibit additional nonspecific activities at both the targeted site and throughout the genome, confounding conclusions of causality of DNA methylation. Inhibition or loss of DNA methylation is accomplished by three consecutive lentiviral transductions. The first two lentiviruses establish stable expression of dCas9 and a guide RNA, which will physically obstruct either maintenance or de novo DNA methyltransferase activity at the guide RNA target site. A third lentivirus introduces Cre recombinase to delete the dCas9 transgene, which leads to loss of dCas9 from the target site, allowing transcription factors and/or the transcription machinery to interact with the demethylated target site. This protocol requires 3-8 months to complete owing to prolonged cell passaging times, but there is little hands-on time, and no specific skills beyond basic molecular biology techniques are necessary.
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Affiliation(s)
- Daniel M Sapozhnikov
- Department of Pharmacology and Therapeutics, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
| | - Moshe Szyf
- Department of Pharmacology and Therapeutics, Faculty of Medicine, McGill University, Montreal, Quebec, Canada.
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26
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Stolz P, Mantero AS, Tvardovskiy A, Ugur E, Wange LE, Mulholland CB, Cheng Y, Wierer M, Enard W, Schneider R, Bartke T, Leonhardt H, Elsässer SJ, Bultmann S. TET1 regulates gene expression and repression of endogenous retroviruses independent of DNA demethylation. Nucleic Acids Res 2022; 50:8491-8511. [PMID: 35904814 PMCID: PMC9410877 DOI: 10.1093/nar/gkac642] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Revised: 04/25/2022] [Accepted: 07/20/2022] [Indexed: 11/13/2022] Open
Abstract
DNA methylation (5-methylcytosine (5mC)) is critical for genome stability and transcriptional regulation in mammals. The discovery that ten-eleven translocation (TET) proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) revolutionized our perspective on the complexity and regulation of DNA modifications. However, to what extent the regulatory functions of TET1 can be attributed to its catalytic activity remains unclear. Here, we use genome engineering and quantitative multi-omics approaches to dissect the precise catalytic vs. non-catalytic functions of TET1 in murine embryonic stem cells (mESCs). Our study identifies TET1 as an essential interaction hub for multiple chromatin modifying complexes and a global regulator of histone modifications. Strikingly, we find that the majority of transcriptional regulation depends on non-catalytic functions of TET1. In particular, we show that TET1 is critical for the establishment of H3K9me3 and H4K20me3 at endogenous retroviral elements (ERVs) and their silencing that is independent of its canonical role in DNA demethylation. Furthermore, we provide evidence that this repression of ERVs depends on the interaction between TET1 and SIN3A. In summary, we demonstrate that the non-catalytic functions of TET1 are critical for regulation of gene expression and the silencing of endogenous retroviruses in mESCs.
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Affiliation(s)
- Paul Stolz
- Faculty of Biology and Center for Molecular Biosystems (BioSysM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Munich 81377, Germany
| | - Angelo Salazar Mantero
- Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet 17165 Stockholm, Sweden, Ming Wai Lau Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet 17177 Stockholm, Sweden
| | - Andrey Tvardovskiy
- Institute of Functional Epigenetics (IFE), Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Enes Ugur
- Faculty of Biology and Center for Molecular Biosystems (BioSysM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Munich 81377, Germany.,Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried 82152, Germany
| | - Lucas E Wange
- Faculty of Biology, Anthropology and Human Genomics, Ludwig-Maximilians-Universität München 82152, Planegg-Martinsried, Germany
| | - Christopher B Mulholland
- Faculty of Biology and Center for Molecular Biosystems (BioSysM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Munich 81377, Germany
| | - Yuying Cheng
- Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet 17165 Stockholm, Sweden, Ming Wai Lau Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet 17177 Stockholm, Sweden
| | - Michael Wierer
- Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried 82152, Germany
| | - Wolfgang Enard
- Faculty of Biology, Anthropology and Human Genomics, Ludwig-Maximilians-Universität München 82152, Planegg-Martinsried, Germany
| | - Robert Schneider
- Institute of Functional Epigenetics (IFE), Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Till Bartke
- Institute of Functional Epigenetics (IFE), Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Heinrich Leonhardt
- Faculty of Biology and Center for Molecular Biosystems (BioSysM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Munich 81377, Germany
| | - Simon J Elsässer
- Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet 17165 Stockholm, Sweden, Ming Wai Lau Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet 17177 Stockholm, Sweden
| | - Sebastian Bultmann
- Faculty of Biology and Center for Molecular Biosystems (BioSysM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Munich 81377, Germany
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27
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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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28
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Oberkofler V, Bäurle I. Inducible epigenome editing probes for the role of histone H3K4 methylation in Arabidopsis heat stress memory. PLANT PHYSIOLOGY 2022; 189:703-714. [PMID: 35285498 PMCID: PMC9157090 DOI: 10.1093/plphys/kiac113] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Accepted: 02/15/2022] [Indexed: 05/26/2023]
Abstract
Histone modifications play a crucial role in the integration of environmental signals to mediate gene expression outcomes. However, genetic and pharmacological interference often causes pleiotropic effects, creating the urgent need for methods that allow locus-specific manipulation of histone modifications, preferably in an inducible manner. Here, we report an inducible system for epigenome editing in Arabidopsis (Arabidopsis thaliana) using a heat-inducible dCas9 to target a JUMONJI (JMJ) histone H3 lysine 4 (H3K4) demethylase domain to a locus of interest. As a model locus, we target the ASCORBATE PEROXIDASE2 (APX2) gene that shows transcriptional memory after heat stress (HS), correlating with H3K4 hyper-methylation. We show that dCas9-JMJ is targeted in a HS-dependent manner to APX2 and that the HS-induced overaccumulation of H3K4 trimethylation (H3K4me3) decreases when dCas9-JMJ binds to the locus. This results in reduced HS-mediated transcriptional memory at the APX2 locus. Targeting an enzymatically inactive JMJ protein in an analogous manner affected transcriptional memory less than the active JMJ protein; however, we still observed a decrease in H3K4 methylation levels. Thus, the inducible targeting of dCas9-JMJ to APX2 was effective in reducing H3K4 methylation levels. As the effect was not fully dependent on enzyme activity of the eraser domain, the dCas9-JMJ fusion protein may act in part independently of its demethylase activity. This underlines the need for caution in the design and interpretation of epigenome editing studies. We expect our versatile inducible epigenome editing system to be especially useful for studying temporal dynamics of chromatin modifications.
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Affiliation(s)
- Vicky Oberkofler
- Institute for Biochemistry and Biology, University of Potsdam, Potsdam, 14476, Germany
| | - Isabel Bäurle
- Institute for Biochemistry and Biology, University of Potsdam, Potsdam, 14476, Germany
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29
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Sano T, Ito T, Ishigami S, Bandaru S, Sano S. Intrinsic activation of cardiosphere-derived cells enhances myocardial repair. J Thorac Cardiovasc Surg 2022; 163:1479-1490.e5. [PMID: 32682583 DOI: 10.1016/j.jtcvs.2020.05.040] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/19/2019] [Revised: 05/01/2020] [Accepted: 05/01/2020] [Indexed: 12/11/2022]
Abstract
OBJECTIVE Permanent loss of cardiomyocytes after myocardial infarction results in irreversible damage to cardiac function. The present study aims to enhance the cardiomyogenic efficiency of cardiosphere-derived cells (CDCs) to develop into large populations of cardiomyocytes by intrinsic activation of cardio-specific differentiation factors (Gata4, Mef2c, Nkx2-5, Hand2, and Tnnt2) by a CRISPR/dCas9 assisted transcriptional enhancement system. METHODS Exhaustive screening was performed to identify the specific sequences in endogenous regulatory regions (enhancers and promoters) responsible for transcriptional activation of the target genes, via a CRISPR/dCas9 system fused with transcriptional activator VP64 (CRISPR-dCas9-VP64). In a rat model of acute myocardial infarction, we compared the regenerative potential and functional benefits of CDCs with or without transcriptional activation. RESULTS We identified a panel of specific CRISPR RNA targeting the enhancers and promoters, which demonstrated significantly higher expression of differentiation factors of Gata4, Hand2, and Tnnt2. The group of CDCs with transcriptional activator VP64 (CDC with VP64) showed significant improvement in the left ventricular ejection fraction (61.9% vs 52.5% and 44.1% in the CDC without transcriptional activation group and control) and decreased scar area in the heart. CONCLUSIONS We have identified endogenous regulatory regions responsible for an intrinsic activation of cardio-specific differentiation factors assisted via a CRISPR/dCas9 gene transcriptional system. The CRISPR/dCas9 system may provide an efficient and effective means of regulating Tnnt2 gene activation within stem cells. Subsequently, this system can be used to enhance transplanted CDCs differentiation potential within ischemic myocardia to better therapeutic outcomes of patients with ischemic heart disease.
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Affiliation(s)
- Toshikazu Sano
- Division of Pediatric Cardiothoracic Surgery, Department of Surgery, University of California at San Francisco, San Francisco, Calif
| | - Tatsuo Ito
- Department of Hygiene, Kawasaki Medical University, Okayama, Japan
| | - Shuta Ishigami
- Division of Pediatric Cardiothoracic Surgery, Department of Surgery, University of California at San Francisco, San Francisco, Calif
| | - Srinivas Bandaru
- Department of Hygiene, Kawasaki Medical University, Okayama, Japan
| | - Shunji Sano
- Division of Pediatric Cardiothoracic Surgery, Department of Surgery, University of California at San Francisco, San Francisco, Calif.
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Duddu AS, Majumdar SS, Sahoo S, Jhunjhunwala S, Jolly MK. Emergent dynamics of a three-node regulatory network explain phenotypic switching and heterogeneity: a case study of Th1/Th2/Th17 cell differentiation. Mol Biol Cell 2022; 33:ar46. [PMID: 35353012 PMCID: PMC9265159 DOI: 10.1091/mbc.e21-10-0521] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Naïve helper (CD4+) T-cells can differentiate into distinct functional subsets including Th1, Th2, and Th17 phenotypes. Each of these phenotypes has a 'master regulator' - T-bet (Th1), GATA3 (Th2) and RORγT (Th17) - that inhibits the other two master regulators. Such mutual repression among them at a transcriptional level can enable multistability, giving rise to six experimentally observed phenotypes - Th1, Th2, Th17, hybrid Th/Th2, hybrid Th2/Th17 and hybrid Th1/Th17. However, the dynamics of switching among these phenotypes, particularly in the case of epigenetic influence, remains unclear. Here, through mathematical modeling, we investigated the coupled transcription-epigenetic dynamics in a three-node mutually repressing network to elucidate how epigenetic changes mediated by any 'master regulator' can influence the transition rates among different cellular phenotypes. We show that the degree of plasticity exhibited by one phenotype depends on relative strength and duration of mutual epigenetic repression mediated among the master regulators in a three-node network. Further, our model predictions can offer putative mechanisms underlying relatively higher plasticity of Th17 phenotype as observed in vitro and in vivo. Together, our modeling framework characterizes phenotypic plasticity and heterogeneity as an outcome of emergent dynamics of a three-node regulatory network, such as the one mediated by T-bet/GATA3/RORγT.
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Affiliation(s)
- Atchuta Srinivas Duddu
- Centre for BioSystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India
| | - Sauma Suvra Majumdar
- epartment of Biotechnology, National Institute of Technology, Durgapur 713216, India
| | - Sarthak Sahoo
- Centre for BioSystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India
| | - Siddharth Jhunjhunwala
- Centre for BioSystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India
| | - Mohit Kumar Jolly
- Centre for BioSystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India
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31
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Xi J, Xu Y, Guo Z, Li J, Wu Y, Sun Q, Wang Y, Chen M, Zhu S, Bian S, Kang J. LncRNA SOX1-OT V1 acts as a decoy of HDAC10 to promote SOX1-dependent hESC neuronal differentiation. EMBO Rep 2022; 23:e53015. [PMID: 34927789 PMCID: PMC8811645 DOI: 10.15252/embr.202153015] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2021] [Revised: 11/15/2021] [Accepted: 11/17/2021] [Indexed: 02/05/2023] Open
Abstract
Long noncoding RNAs (lncRNAs) are abundantly expressed in the nervous system, but their regulatory roles in neuronal differentiation are poorly understood. Using a human embryonic stem cell (hESC)-based 2D neural differentiation approach and a 3D cerebral organoid system, we show that SOX1-OT variant 1 (SOX1-OT V1), a SOX1 overlapping noncoding RNA, plays essential roles in both dorsal cortical neuron differentiation and ventral GABAergic neuron differentiation by facilitating SOX1 expression. SOX1-OT V1 physically interacts with HDAC10 through its 5' region, acts as a decoy to block HDAC10 binding to the SOX1 promoter, and thus maintains histone acetylation levels at the SOX1 promoter. SOX1 in turn activates ASCL1 expression and promotes neuronal differentiation. Taken together, we identify a SOX1-OT V1/HDAC10-SOX1-ASCL1 axis, which promotes neurogenesis, highlighting a role for lncRNAs in hESC neuronal differentiation.
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Affiliation(s)
- Jiajie Xi
- Clinical and Translational Research Center of Shanghai First Maternity and Infant HospitalShanghai Key Laboratory of Maternal Fetal MedicineShanghai Key Laboratory of Signaling and Disease ResearchFrontier Science Center for Stem Cell ResearchNational Stem Cell Translational Resource CenterSchool of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Yanxin Xu
- Clinical and Translational Research Center of Shanghai First Maternity and Infant HospitalShanghai Key Laboratory of Maternal Fetal MedicineShanghai Key Laboratory of Signaling and Disease ResearchFrontier Science Center for Stem Cell ResearchNational Stem Cell Translational Resource CenterSchool of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Zhenming Guo
- Institute for Regenerative MedicineShanghai East HospitalSchool of Life Sciences and TechnologyFrontier Science Center for Stem Cell ResearchTongji UniversityShanghaiChina
| | - Jianguo Li
- Clinical and Translational Research Center of Shanghai First Maternity and Infant HospitalShanghai Key Laboratory of Maternal Fetal MedicineShanghai Key Laboratory of Signaling and Disease ResearchFrontier Science Center for Stem Cell ResearchNational Stem Cell Translational Resource CenterSchool of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Yukang Wu
- Clinical and Translational Research Center of Shanghai First Maternity and Infant HospitalShanghai Key Laboratory of Maternal Fetal MedicineShanghai Key Laboratory of Signaling and Disease ResearchFrontier Science Center for Stem Cell ResearchNational Stem Cell Translational Resource CenterSchool of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Qiaoyi Sun
- Clinical and Translational Research Center of Shanghai First Maternity and Infant HospitalShanghai Key Laboratory of Maternal Fetal MedicineShanghai Key Laboratory of Signaling and Disease ResearchFrontier Science Center for Stem Cell ResearchNational Stem Cell Translational Resource CenterSchool of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Yuxi Wang
- Clinical and Translational Research Center of Shanghai First Maternity and Infant HospitalShanghai Key Laboratory of Maternal Fetal MedicineShanghai Key Laboratory of Signaling and Disease ResearchFrontier Science Center for Stem Cell ResearchNational Stem Cell Translational Resource CenterSchool of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Mengxia Chen
- Institute for Regenerative MedicineShanghai East HospitalSchool of Life Sciences and TechnologyFrontier Science Center for Stem Cell ResearchTongji UniversityShanghaiChina
| | - Songcheng Zhu
- Clinical and Translational Research Center of Shanghai First Maternity and Infant HospitalShanghai Key Laboratory of Maternal Fetal MedicineShanghai Key Laboratory of Signaling and Disease ResearchFrontier Science Center for Stem Cell ResearchNational Stem Cell Translational Resource CenterSchool of Life Sciences and TechnologyTongji UniversityShanghaiChina
| | - Shan Bian
- Institute for Regenerative MedicineShanghai East HospitalSchool of Life Sciences and TechnologyFrontier Science Center for Stem Cell ResearchTongji UniversityShanghaiChina
| | - Jiuhong Kang
- Clinical and Translational Research Center of Shanghai First Maternity and Infant HospitalShanghai Key Laboratory of Maternal Fetal MedicineShanghai Key Laboratory of Signaling and Disease ResearchFrontier Science Center for Stem Cell ResearchNational Stem Cell Translational Resource CenterSchool of Life Sciences and TechnologyTongji UniversityShanghaiChina
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Epigenetic modifications of histones during osteoblast differentiation. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2022; 1865:194780. [PMID: 34968769 DOI: 10.1016/j.bbagrm.2021.194780] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/29/2021] [Revised: 10/30/2021] [Accepted: 12/08/2021] [Indexed: 12/20/2022]
Abstract
In bone biology, epigenetics plays a key role in mesenchymal stem cells' (MSCs) commitment towards osteoblasts. It involves gene regulatory mechanisms governed by chromatin modulators. Predominant epigenetic mechanisms for efficient osteogenic differentiation include DNA methylation, histone modifications, and non-coding RNAs. Among these mechanisms, histone modifications critically contribute to altering chromatin configuration. Histone based epigenetic mechanisms are an essential mediator of gene expression during osteoblast differentiation as it directs the bivalency of the genome. Investigating the importance of histone modifications in osteogenesis may lead to the development of epigenetic-based remedies for genetic disorders of bone. Hence, in this review, we have highlighted the importance of epigenetic modifications such as post-translational modifications of histones, including methylation, acetylation, phosphorylation, ubiquitination, and their role in the activation or suppression of gene expression during osteoblast differentiation. Further, we have emphasized the future advancements in the field of epigenetics towards orthopaedical therapeutics.
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33
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Yildiz CB, Zimmer-Bensch G. Role of DNMTs in the Brain. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2022; 1389:363-394. [DOI: 10.1007/978-3-031-11454-0_15] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
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34
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Direct neuronal reprogramming: Fast forward from new concepts toward therapeutic approaches. Neuron 2021; 110:366-393. [PMID: 34921778 DOI: 10.1016/j.neuron.2021.11.023] [Citation(s) in RCA: 68] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2021] [Revised: 10/25/2021] [Accepted: 11/19/2021] [Indexed: 12/21/2022]
Abstract
Differentiated cells have long been considered fixed in their identity. However, about 20 years ago, the first direct conversion of glial cells into neurons in vitro opened the field of "direct neuronal reprogramming." Since then, neuronal reprogramming has achieved the generation of fully functional, mature neurons with remarkable efficiency, even in diseased brain environments. Beyond their clinical implications, these discoveries provided basic insights into crucial mechanisms underlying conversion of specific cell types into neurons and maintenance of neuronal identity. Here we discuss such principles, including the importance of the starter cell for shaping the outcome of neuronal reprogramming. We further highlight technical concerns for in vivo reprogramming and propose a code of conduct to avoid artifacts and pitfalls. We end by pointing out next challenges for development of less invasive cell replacement therapies for humans.
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35
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Deciphering Plant Chromatin Regulation via CRISPR/dCas9-Based Epigenome Engineering. EPIGENOMES 2021; 5:epigenomes5030017. [PMID: 34968366 PMCID: PMC8594717 DOI: 10.3390/epigenomes5030017] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2021] [Revised: 08/11/2021] [Accepted: 08/18/2021] [Indexed: 01/23/2023] Open
Abstract
CRISPR-based epigenome editing uses dCas9 as a platform to recruit transcription or chromatin regulators at chosen loci. Despite recent and ongoing advances, the full potential of these approaches to studying chromatin functions in vivo remains challenging to exploit. In this review we discuss how recent progress in plants and animals provides new routes to investigate the function of chromatin regulators and address the complexity of associated regulations that are often interconnected. While efficient transcriptional engineering methodologies have been developed and can be used as tools to alter the chromatin state of a locus, examples of direct manipulation of chromatin regulators remain scarce in plants. These reports also reveal pitfalls and limitations of epigenome engineering approaches that are nevertheless informative as they are often associated with locus- and context-dependent features, which include DNA accessibility, initial chromatin and transcriptional state or cellular dynamics. Strategies implemented in different organisms to overcome and even take advantage of these limitations are highlighted, which will further improve our ability to establish the causality and hierarchy of chromatin dynamics on genome regulation.
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36
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Basu A, Tiwari VK. Epigenetic reprogramming of cell identity: lessons from development for regenerative medicine. Clin Epigenetics 2021; 13:144. [PMID: 34301318 PMCID: PMC8305869 DOI: 10.1186/s13148-021-01131-4] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2021] [Accepted: 07/13/2021] [Indexed: 12/17/2022] Open
Abstract
Epigenetic mechanisms are known to define cell-type identity and function. Hence, reprogramming of one cell type into another essentially requires a rewiring of the underlying epigenome. Cellular reprogramming can convert somatic cells to induced pluripotent stem cells (iPSCs) that can be directed to differentiate to specific cell types. Trans-differentiation or direct reprogramming, on the other hand, involves the direct conversion of one cell type into another. In this review, we highlight how gene regulatory mechanisms identified to be critical for developmental processes were successfully used for cellular reprogramming of various cell types. We also discuss how the therapeutic use of the reprogrammed cells is beginning to revolutionize the field of regenerative medicine particularly in the repair and regeneration of damaged tissue and organs arising from pathological conditions or accidents. Lastly, we highlight some key challenges hindering the application of cellular reprogramming for therapeutic purposes.
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Affiliation(s)
- Amitava Basu
- Institute of Molecular Biology (IMB), 55128, Mainz, Germany.
| | - Vijay K Tiwari
- Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Science, Queens University Belfast, Belfast, BT9 7BL, UK.
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37
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Brown KE, Fisher AG. Reprogramming lineage identity through cell-cell fusion. Curr Opin Genet Dev 2021; 70:15-23. [PMID: 34087754 DOI: 10.1016/j.gde.2021.04.004] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Revised: 04/12/2021] [Accepted: 04/23/2021] [Indexed: 12/23/2022]
Abstract
The conversion of differentiated cells to a pluripotent state through somatic cell nuclear transfer provided the first unequivocal evidence that differentiation was reversible. In more recent times, introducing a combination of key transcription factors into terminally differentiated mammalian cells was shown to drive their conversion to induced pluripotent stem cells (iPSCs). These discoveries were transformative, but the relatively slow speed (2-3 weeks) and low efficiency of reprogramming (0.1-1%) made deciphering the underlying molecular mechanisms difficult and complex. Cell fusion provides an alternative reprogramming approach that is both efficient and tractable, particularly when combined with modern multi-omics analysis of individual cells. Here we review the history and the recent advances in cell-cell fusion that are enabling a better understanding cell fate conversion, and we discuss how this knowledge could be used to shape improved strategies for regenerative medicine.
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Affiliation(s)
- Karen E Brown
- Epigenetic Memory Group, MRC London Institute of Medical Sciences (LMS), Imperial College London, Du Cane Road, London W12 0NN, UK.
| | - Amanda G Fisher
- Epigenetic Memory Group, MRC London Institute of Medical Sciences (LMS), Imperial College London, Du Cane Road, London W12 0NN, UK
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38
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Epigenetic memory in reprogramming. Curr Opin Genet Dev 2021; 70:24-31. [PMID: 34058535 DOI: 10.1016/j.gde.2021.04.007] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Revised: 04/20/2021] [Accepted: 04/27/2021] [Indexed: 01/23/2023]
Abstract
A central question of biology is the basis of stable cell fates. Cell fates are formed during development, where the zygote progresses from totipotency to terminal differentiation. Each step of lineage commitment involves establishment of stable states encoding-specific developmental commitments that can be faithfully transmitted to daughter cells - a 'memory' of cell fate is acquired. However, this cell-fate memory is reversible and can be changed when experimental reprogramming procedures such as nuclear transfer to eggs or transcription factor overexpression are used. The ability to reprogram cell fates impacts regenerative medicine, as progress in understanding underlying molecular mechanisms of cell-fate changes can allow the generation of any cell type needed for cell replacement therapies. Given its potential, studies are currently aiming at improving the low efficiency of cell-fate conversion. In recent years, epigenetic mechanisms suggested to promote stable cell-fate memory emerged as factors that cause resistance to cell-fate conversions during nuclear reprogramming. In this review, we highlight the latest work that has characterised epigenetic barriers to reprogramming which, during normal development, help to maintain the stable differentiation status of cells.
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39
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Bragg-Gonzalo L, De León Reyes NS, Nieto M. Genetic and activity dependent-mechanisms wiring the cortex: Two sides of the same coin. Semin Cell Dev Biol 2021; 118:24-34. [PMID: 34030948 DOI: 10.1016/j.semcdb.2021.05.011] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Revised: 04/27/2021] [Accepted: 05/08/2021] [Indexed: 01/17/2023]
Abstract
The cerebral cortex is responsible for the higher-order functions of the brain such as planning, cognition, or social behaviour. It provides us with the capacity to interact with and transform our world. The substrates of cortical functions are complex neural circuits that arise during development from the dynamic remodelling and progressive specialization of immature undefined networks. Here, we review the genetic and activity-dependent mechanisms of cortical wiring focussing on the importance of their interaction. Cortical circuits emerge from an initial set of neuronal types that engage in sequential forms of embryonic and postnatal activity. Such activities further complement the cells' genetic programs, increasing neuronal diversity and modifying the electrical properties while promoting selective connectivity. After a temporal window of enhanced plasticity, the main features of mature circuits are established. Failures in these processes can lead to neurodevelopmental disorders whose treatment remains elusive. However, a deeper dissection of cortical wiring will pave the way for innovative therapies.
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Affiliation(s)
- L Bragg-Gonzalo
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, (CNB-CSIC) Campus de Cantoblanco, Darwin 3, 28049 Madrid, Spain
| | - N S De León Reyes
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, (CNB-CSIC) Campus de Cantoblanco, Darwin 3, 28049 Madrid, Spain; Instituto de Neurociencias de Alicante, CSIC-UMH, 03550 San Juan de Alicante, Spain
| | - M Nieto
- Department of Cellular and Molecular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, (CNB-CSIC) Campus de Cantoblanco, Darwin 3, 28049 Madrid, Spain.
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40
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Goell JH, Hilton IB. CRISPR/Cas-Based Epigenome Editing: Advances, Applications, and Clinical Utility. Trends Biotechnol 2021; 39:678-691. [PMID: 33972106 DOI: 10.1016/j.tibtech.2020.10.012] [Citation(s) in RCA: 50] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2020] [Revised: 10/21/2020] [Accepted: 10/29/2020] [Indexed: 02/06/2023]
Abstract
The epigenome dynamically regulates gene expression and guides cellular differentiation throughout the lifespan of eukaryotic organisms. Recent advances in clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based epigenome editing technologies have enabled researchers to site-specifically program epigenetic modifications to endogenous DNA and histones and to manipulate the architecture of native chromatin. As a result, epigenome editing has helped to uncover the causal relationships between epigenetic marks and gene expression. As epigenome editing tools have continued to develop, researchers have applied them in new ways to explore the function of the epigenome in human health and disease. In this review, we discuss the recent technical improvements in CRISPR/Cas-based epigenome editing that have advanced clinical research and examine how these technologies could be improved for greater future utility.
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Affiliation(s)
- Jacob H Goell
- Department of Bioengineering, Rice University, Houston, TX, USA
| | - Isaac B Hilton
- Department of Bioengineering, Rice University, Houston, TX, USA; Department of BioSciences, Rice University, Houston, TX, USA.
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41
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de Lima Camillo LP, Quinlan RBA. A ride through the epigenetic landscape: aging reversal by reprogramming. GeroScience 2021; 43:463-485. [PMID: 33825176 PMCID: PMC8110674 DOI: 10.1007/s11357-021-00358-6] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2020] [Accepted: 02/08/2021] [Indexed: 02/06/2023] Open
Abstract
Aging has become one of the fastest-growing research topics in biology. However, exactly how the aging process occurs remains unknown. Epigenetics plays a significant role, and several epigenetic interventions can modulate lifespan. This review will explore the interplay between epigenetics and aging, and how epigenetic reprogramming can be harnessed for age reversal. In vivo partial reprogramming holds great promise as a possible therapy, but several limitations remain. Rejuvenation by reprogramming is a young but rapidly expanding subfield in the biology of aging.
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42
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MacArthur IC, Dawlaty MM. TET Enzymes and 5-Hydroxymethylcytosine in Neural Progenitor Cell Biology and Neurodevelopment. Front Cell Dev Biol 2021; 9:645335. [PMID: 33681230 PMCID: PMC7930563 DOI: 10.3389/fcell.2021.645335] [Citation(s) in RCA: 38] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2020] [Accepted: 02/01/2021] [Indexed: 12/19/2022] Open
Abstract
Studies of tissue-specific epigenomes have revealed 5-hydroxymethylcytosine (5hmC) to be a highly enriched and dynamic DNA modification in the metazoan nervous system, inspiring interest in the function of this epigenetic mark in neurodevelopment and brain function. 5hmC is generated by oxidation of 5-methylcytosine (5mC), a process catalyzed by the ten–eleven translocation (TET) enzymes. 5hmC serves not only as an intermediate in DNA demethylation but also as a stable epigenetic mark. Here, we review the known functions of 5hmC and TET enzymes in neural progenitor cell biology and embryonic and postnatal neurogenesis. We also discuss how TET enzymes and 5hmC regulate neuronal activity and brain function and highlight their implications in human neurodevelopmental and neurodegenerative disorders. Finally, we present outstanding questions in the field and envision new research directions into the roles of 5hmC and TET enzymes in neurodevelopment.
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Affiliation(s)
- Ian C MacArthur
- Department of Genetics, Albert Einstein College of Medicine, Bronx, NY, United States.,Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY, United States.,Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY, United States
| | - Meelad M Dawlaty
- Department of Genetics, Albert Einstein College of Medicine, Bronx, NY, United States.,Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY, United States.,Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY, United States
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43
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Kazi TA, Biswas SR. CRISPR/dCas system as the modulator of gene expression. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 178:99-122. [PMID: 33685602 DOI: 10.1016/bs.pmbts.2020.12.002] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
CRISPR/Cas has been a very exciting field of research because of its multifaceted applications in biological science for editing genome. This tool can be programmed to target any region of DNA of choice by designing gRNA. The potential of gRNA to recruit a CRISPR-associated protein at a specific genomic site allowed scientists to engineer genome of diverse species for research and development. The application of Cas9 has been further expanded with a recently developed catalytically inactive protein (dead Cas9). CRISPR/dCas system is widely used as a programmable vector to deliver functional cargo (transcriptional effectors) to the desired sites at the genome for targeted transcriptional repression (CRISPR interference, CRISPRi) or activation (CRISPR activation, CRISPRa). It is now possible to regulate gene expression in cells without altering the DNA sequence. These CRISPRi/a toolboxes have explored many unsolved biological issues. Further research on CRISPR system could help diagnose and treat various human diseases.
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Affiliation(s)
- Tawsif Ahmed Kazi
- Department of Botany, Visva-Bharati, Santiniketan, West Bengal, India
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Sgro A, Blancafort P. Epigenome engineering: new technologies for precision medicine. Nucleic Acids Res 2021; 48:12453-12482. [PMID: 33196851 PMCID: PMC7736826 DOI: 10.1093/nar/gkaa1000] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Revised: 10/10/2020] [Accepted: 10/16/2020] [Indexed: 02/07/2023] Open
Abstract
Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more 'normal-like state', having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.
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Affiliation(s)
- Agustin Sgro
- Cancer Epigenetics Laboratory, The Harry Perkins Institute of Medical Research, Nedlands, Western Australia 6009, Australia.,School of Human Sciences, The University of Western Australia, Crawley, Perth, Western Australia 6009, Australia
| | - Pilar Blancafort
- Cancer Epigenetics Laboratory, The Harry Perkins Institute of Medical Research, Nedlands, Western Australia 6009, Australia.,School of Human Sciences, The University of Western Australia, Crawley, Perth, Western Australia 6009, Australia.,The Greehey Children's Cancer Research Institute, The University of Texas Health Science Center, San Antonio, TX 78229, USA
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45
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Kressler C, Gasparoni G, Nordström K, Hamo D, Salhab A, Dimitropoulos C, Tierling S, Reinke P, Volk HD, Walter J, Hamann A, Polansky JK. Targeted De-Methylation of the FOXP3-TSDR Is Sufficient to Induce Physiological FOXP3 Expression but Not a Functional Treg Phenotype. Front Immunol 2021; 11:609891. [PMID: 33488615 PMCID: PMC7817622 DOI: 10.3389/fimmu.2020.609891] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Accepted: 11/20/2020] [Indexed: 12/22/2022] Open
Abstract
CD4+ regulatory T cells (Tregs) are key mediators of immunological tolerance and promising effector cells for immuno-suppressive adoptive cellular therapy to fight autoimmunity and chronic inflammation. Their functional stability is critical for their clinical utility and has been correlated to the demethylated state of the TSDR/CNS2 enhancer element in the Treg lineage transcription factor FOXP3. However, proof for a causal contribution of the TSDR de-methylation to FOXP3 stability and Treg induction is so far lacking. We here established a powerful transient-transfection CRISPR-Cas9-based epigenetic editing method for the selective de-methylation of the TSDR within the endogenous chromatin environment of a living cell. The induced de-methylated state was stable over weeks in clonal T cell proliferation cultures even after expression of the editing complex had ceased. Epigenetic editing of the TSDR resulted in FOXP3 expression, even in its physiological isoform distribution, proving a causal role for the de-methylated TSDR in FOXP3 regulation. However, successful FOXP3 induction was not associated with a switch towards a functional Treg phenotype, in contrast to what has been reported from FOXP3 overexpression approaches. Thus, TSDR de-methylation is required, but not sufficient for a stable Treg phenotype induction. Therefore, targeted demethylation of the TSDR may be a critical addition to published in vitro Treg induction protocols which so far lack FOXP3 stability.
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Affiliation(s)
- Christopher Kressler
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.,Immuno-Epigenetics, German Rheumatism Research Centre (DRFZ), Berlin, Germany
| | | | - Karl Nordström
- Genetics/Epigenetics, Saarland University, Saarbrücken, Germany
| | - Dania Hamo
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.,Immuno-Epigenetics, German Rheumatism Research Centre (DRFZ), Berlin, Germany
| | | | | | - Sascha Tierling
- Genetics/Epigenetics, Saarland University, Saarbrücken, Germany
| | - Petra Reinke
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.,Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Hans-Dieter Volk
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.,Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Jörn Walter
- Genetics/Epigenetics, Saarland University, Saarbrücken, Germany
| | - Alf Hamann
- Immuno-Epigenetics, German Rheumatism Research Centre (DRFZ), Berlin, Germany
| | - Julia K Polansky
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.,Immuno-Epigenetics, German Rheumatism Research Centre (DRFZ), Berlin, Germany.,Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, Berlin, Germany
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46
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Nakamura M, Gao Y, Dominguez AA, Qi LS. CRISPR technologies for precise epigenome editing. Nat Cell Biol 2021; 23:11-22. [PMID: 33420494 DOI: 10.1038/s41556-020-00620-7] [Citation(s) in RCA: 217] [Impact Index Per Article: 54.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Accepted: 11/30/2020] [Indexed: 01/29/2023]
Abstract
The epigenome involves a complex set of cellular processes governing genomic activity. Dissecting this complexity necessitates the development of tools capable of specifically manipulating these processes. The repurposing of prokaryotic CRISPR systems has allowed for the development of diverse technologies for epigenome engineering. Here, we review the state of currently achievable epigenetic manipulations along with corresponding applications. With future optimization, CRISPR-based epigenomic editing stands as a set of powerful tools for understanding and controlling biological function.
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Affiliation(s)
- Muneaki Nakamura
- Department of Bioengineering, Stanford University, Stanford, CA, USA
| | - Yuchen Gao
- Department of Bioengineering, Stanford University, Stanford, CA, USA.,Cancer Biology Program, Stanford University, Stanford, CA, USA.,Mammoth Biosciences, South San Francisco, CA, USA
| | - Antonia A Dominguez
- Department of Bioengineering, Stanford University, Stanford, CA, USA.,Sana Biotechnology, South San Francisco, CA, USA
| | - Lei S Qi
- Department of Bioengineering, Stanford University, Stanford, CA, USA. .,Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA. .,Stanford ChEM-H Institute, Stanford University, Stanford, CA, USA.
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47
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Stricker SH, Götz M. Epigenetic regulation of neural lineage elaboration: Implications for therapeutic reprogramming. Neurobiol Dis 2020; 148:105174. [PMID: 33171228 DOI: 10.1016/j.nbd.2020.105174] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2020] [Revised: 10/19/2020] [Accepted: 11/06/2020] [Indexed: 01/14/2023] Open
Abstract
The vulnerability of the mammalian brain is mainly due to its limited ability to generate new neurons once fully matured. Direct conversion of non-neuronal cells to neurons opens up a new avenue for therapeutic intervention and has made great strides also for in vivo applications in the injured brain. These great achievements raise the issue of adequate identity and chromatin hallmarks of the induced neurons. This may be particularly important, as aberrant epigenetic settings may reveal their adverse effects only in certain brain activity states. Therefore, we review here the knowledge about epigenetic memory and partially resetting of chromatin hallmarks from other reprogramming fields, before moving to the knowledge in direct neuronal reprogramming, which is still limited. Most importantly, novel tools are available now to manipulate specific epigenetic marks at specific sites of the genome. Applying these will eventually allow erasing aberrant epigenetic memory and paving the way towards new therapeutic approaches for brain repair.
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Affiliation(s)
- Stefan H Stricker
- Institute for Stem Cell Research, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, 82152 Planegg, Germany; Physiological Genomics, Biomedical Center (BMC), Ludwig-Maximilians Universitaet Muenchen, 82152 Planegg, Munich, Germany; MCN Junior Research Group, Munich Center for Neurosciences, Ludwig-Maximilian-Universität, BioMedical Center, Grosshaderner Strasse 9, Planegg-Martinsried 82152, Germany.
| | - Magdalena Götz
- Institute for Stem Cell Research, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, 82152 Planegg, Germany; Physiological Genomics, Biomedical Center (BMC), Ludwig-Maximilians Universitaet Muenchen, 82152 Planegg, Munich, Germany; SYNERGY, Excellence Cluster of Systems Neurology, BioMedical Center (BMC), Ludwig-Maximilians-Universitaet Muenchen, 82152 Planegg, Munich, Germany.
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48
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Gupta✉ N, Dilmen E, Morizane R. 3D kidney organoids for bench-to-bedside translation. J Mol Med (Berl) 2020; 99:477-487. [PMID: 33034708 PMCID: PMC8026465 DOI: 10.1007/s00109-020-01983-y] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2020] [Revised: 06/30/2020] [Accepted: 09/22/2020] [Indexed: 12/23/2022]
Abstract
The kidneys are essential organs that filter the blood, removing urinary waste while maintaining fluid and electrolyte homeostasis. Current conventional research models such as static cell cultures and animal models are insufficient to grasp the complex human in vivo situation or lack translational value. To accelerate kidney research, novel research tools are required. Recent developments have allowed the directed differentiation of induced pluripotent stem cells to generate kidney organoids. Kidney organoids resemble the human kidney in vitro and can be applied in regenerative medicine and as developmental, toxicity, and disease models. Although current studies have shown great promise, challenges remain including the immaturity, limited reproducibility, and lack of perfusable vascular and collecting duct systems. This review gives an overview of our current understanding of nephrogenesis that enabled the generation of kidney organoids. Next, the potential applications of kidney organoids are discussed followed by future perspectives. This review proposes that advancement in kidney organoid research will be facilitated through our increasing knowledge on nephrogenesis and combining promising techniques such as organ-on-a-chip models.
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Affiliation(s)
- Navin Gupta✉
- Nephrology Division, Massachusetts General Hospital, Boston, MA USA
- Department of Medicine, Harvard Medical School, Boston, MA USA
- The Wyss Institute, Harvard University, Cambridge, MA USA
| | - Emre Dilmen
- Nephrology Division, Massachusetts General Hospital, Boston, MA USA
| | - Ryuji Morizane
- Nephrology Division, Massachusetts General Hospital, Boston, MA USA
- Department of Medicine, Harvard Medical School, Boston, MA USA
- The Wyss Institute, Harvard University, Cambridge, MA USA
- Harvard Stem Cell Institute, Cambridge, MA USA
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49
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Carter JL, Halmai JANM, Fink KD. The iNs and Outs of Direct Reprogramming to Induced Neurons. Front Genome Ed 2020; 2:7. [PMID: 34713216 PMCID: PMC8525349 DOI: 10.3389/fgeed.2020.00007] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Accepted: 07/24/2020] [Indexed: 12/12/2022] Open
Abstract
Understanding of cell-type specific transcription factors has promoted progress in methods for cellular reprogramming, such as directly reprogramming somatic cells to induced neurons (iN). Methods for direct reprogramming require neuronal-fate determining gene activation via neuron-specific microRNAs, chemical modulation of key neuronal signaling pathways or overexpression via viral vectors, with some reprogramming strategies requiring a combination of these methods to induce the neuronal-cell fate. These methods have been employed in a multitude of cell types, including fibroblasts, hepatocytes, peripheral blood mononuclear, and T cells. The ability to create iN from skin biopsies and blood samples coupled with recent advancements in artificially inducing age- and disease-associated phenotypes are accelerating the development of disease models for late-onset neurodegenerative disorders. Here, we review how activation of the neuronal transcriptome alters the epigenetic landscape of the donor cell to facilitate reprogramming to neurons. We also discuss the advantages of using DNA binding domains such as CRISPR/dCas9 to overcome epigenetic barriers to induce neuronal-cell fate by activating endogenous neuronal cell-fate determining genes.
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50
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Luz-Madrigal A, Grajales-Esquivel E, Tangeman J, Kosse S, Liu L, Wang K, Fausey A, Liang C, Tsonis PA, Del Rio-Tsonis K. DNA demethylation is a driver for chick retina regeneration. Epigenetics 2020; 15:998-1019. [PMID: 32290791 PMCID: PMC7518676 DOI: 10.1080/15592294.2020.1747742] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2019] [Revised: 02/26/2020] [Accepted: 03/09/2020] [Indexed: 01/01/2023] Open
Abstract
Cellular reprogramming resets the epigenetic landscape to drive shifts in transcriptional programmes and cell identity. The embryonic chick can regenerate a complete neural retina, after retinectomy, via retinal pigment epithelium (RPE) reprogramming in the presence of FGF2. In this study, we systematically analysed the reprogramming competent chick RPE prior to injury, and during different stages of reprogramming. In addition to changes in the expression of genes associated with epigenetic modifications during RPE reprogramming, we observed dynamic changes in histone marks associated with bivalent chromatin (H3K27me3/H3K4me3) and intermediates of the process of DNA demethylation including 5hmC and 5caC. Comprehensive analysis of the methylome by whole-genome bisulphite sequencing (WGBS) confirmed extensive rearrangements of DNA methylation patterns including differentially methylated regions (DMRs) found at promoters of genes associated with chromatin organization and fibroblast growth factor production. We also identified Tet methylcytosine dioxygenase 3 (TET3) as an important factor for DNA demethylation and retina regeneration, capable of reprogramming RPE in the absence of exogenous FGF2. In conclusion, we demonstrate that injury early in RPE reprogramming triggers genome-wide dynamic changes in chromatin, including bivalent chromatin and DNA methylation. In the presence of FGF2, these dynamic modifications are further sustained in the commitment to form a new retina. Our findings reveal active DNA demethylation as an important process that may be applied to remove the epigenetic barriers in order to regenerate retina in mammals. ABBREVIATIONS bp: Base pair; DMR: Differentially methylated region; DMC: Differentially methylated cytosines; GFP: Green fluorescent protein; PCR: Polymerase chain reaction. TET: Ten-eleven translocation; RPE: retinal pigment epithelium.
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Affiliation(s)
- Agustín Luz-Madrigal
- Department of Biology and Center for Visual Sciences at Miami University, Miami University, Oxford, OH, USA
- Department of Biology and Center for Stem Cell & Organoid Medicine (CuSTOM), Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
| | - Erika Grajales-Esquivel
- Department of Biology and Center for Visual Sciences at Miami University, Miami University, Oxford, OH, USA
| | - Jared Tangeman
- Department of Biology and Center for Visual Sciences at Miami University, Miami University, Oxford, OH, USA
| | - Sarah Kosse
- Department of Biology and Center for Visual Sciences at Miami University, Miami University, Oxford, OH, USA
| | - Lin Liu
- Department of Biology and Center for Visual Sciences at Miami University, Miami University, Oxford, OH, USA
| | - Kai Wang
- Department of Biology and Center for Visual Sciences at Miami University, Miami University, Oxford, OH, USA
| | - Andrew Fausey
- Department of Biology and Center for Visual Sciences at Miami University, Miami University, Oxford, OH, USA
| | - Chun Liang
- Department of Biology and Center for Visual Sciences at Miami University, Miami University, Oxford, OH, USA
- Department of Computer Science and Software Engineering, Miami University, Oxford, OH, USA
| | - Panagiotis A. Tsonis
- Department of Biology, University of Dayton and Center for Tissue Regeneration and Engineering at the University of Dayton (TREND), Dayton, OH, USA
| | - Katia Del Rio-Tsonis
- Department of Biology and Center for Visual Sciences at Miami University, Miami University, Oxford, OH, USA
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