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1
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Song S, Fan Y, Zou G, Huo L, Kumar J, Li Y, Wang R, Dai E, Jin J, Scott AW, Shao S, Pizzi MP, Vykoukal JV, Katayama H, Hanash S, Calin GA, Zhang X, Lee MG, Wang Z, Lo YH, Gan Q, Waters RE, Yin F, Wang L, Cheng X, Ajani JA, Dhar SS. KAP1 promotes gastric adenocarcinoma progression by activating Hippo/YAP1 signaling via binding to HNRNPAB. Cancer Lett 2025; 621:217695. [PMID: 40189014 PMCID: PMC12165730 DOI: 10.1016/j.canlet.2025.217695] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2024] [Revised: 03/31/2025] [Accepted: 04/03/2025] [Indexed: 04/15/2025]
Abstract
Gastric adenocarcinoma (GAC) remains a significant global health challenge, with over a million new cases annually. Peritoneal carcinomatosis (PC), detected in ∼20 % of cases at diagnosis and ∼45 % later, is uniformly fatal, with limited treatment options. This study investigated the role of KAP1 in GAC progression, focusing on its interaction with YAP1 and cancer stemness traits. Analysis of over 596 primary GACs and 72 PC samples revealed that high nuclear KAP1 expression correlates with poor prognosis. KAP1 knockdown reduced oncogenic activity and stemness traits in GAC cells. Mechanistically, KAP1 positively regulates YAP1 transcription by binding to its promoter and reducing H3K27ac levels. Mass spectrometry identified an interaction between KAP1 and HNRNPAB, further modulating YAP1 signaling. Expression of the KRAB domain of ZFP568 without its DNA-binding zinc fingers inhibited both KAP1 and YAP1 expression, significantly reducing colony formation and tumor growth in vivo. Additionally, emerging antisense oligonucleotides (ASOs) targeting KAP1 or YAP1 effectively suppressed mouse tumor progression. These findings establish KAP1 as a critical driver of tumor progression in GAC through YAP1 regulation and HNRNPAB interaction, highlighting its potential therapeutic target. This study advances our understanding and offers a preclinical framework to improve outcomes for GAC.
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Affiliation(s)
- Shumei Song
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Yibo Fan
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Gengyi Zou
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Longfei Huo
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Janani Kumar
- Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Yuan Li
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Surgical Oncology and General Surgery, First Hospital of China Medical University, Shenyang, 110001, PR China
| | - Ruiping Wang
- Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Enyu Dai
- Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Jiankang Jin
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Ailing W Scott
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Shan Shao
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Melissa Pool Pizzi
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Jody V Vykoukal
- Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Hiroyuki Katayama
- Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Samir Hanash
- Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - George A Calin
- Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Xing Zhang
- Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Min Gyu Lee
- Molecular & Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Zhenning Wang
- Department of Surgical Oncology and General Surgery, First Hospital of China Medical University, Shenyang, 110001, PR China
| | - Yuan-Hung Lo
- Molecular & Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Qiong Gan
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Rebecca E Waters
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Feng Yin
- Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Linghua Wang
- Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Xiaodong Cheng
- Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Jaffer A Ajani
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
| | - Shilpa S Dhar
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
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Leach DA, Chatterjee N, Spahr K, de Almeida GS, Varela-Carver A, Shah TT, Winkler M, Ahmed HU, Bevan CL. Simultaneous inhibition of TRIM24 and TRIM28 sensitises prostate cancer cells to antiandrogen therapy, decreasing VEGF signalling and angiogenesis. Mol Oncol 2025. [PMID: 40411304 DOI: 10.1002/1878-0261.70065] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2024] [Revised: 03/20/2025] [Accepted: 05/13/2025] [Indexed: 05/26/2025] Open
Abstract
Castrate-resistant prostate cancer (CRPC) is a likely outcome of hormone treatment for advanced prostate cancer. Although no longer dependent on androgen levels, CRPC remains driven by the androgen receptor (AR). One proposed progression mechanism is altered repertoires of coregulator proteins possessing the ability to alter AR activity. Increased expression of tripartite motif-containing 24 (TRIM24) and TRIM28-two members of a distinct bromodomain-containing subfamily of Tripartite motif (TRIM) coregulators-occurs in CRPC. Endogenous TRIM24 and TRIM28 interact with each other and AR, bind to chromatin and regulate genes such as the angiogenic factor vascular endothelial growth factor A (VEGFA) and oncogene MYC. Silencing of TRIM24 and TRIM28 simultaneously, but not either alone, sensitised CRPC model cell lines to the antiandrogen enzalutamide and bicalutamide. This re-sensitisation to antiandrogen therapeutics could then be reversed by addition of VEGF. Furthermore, both TRIM24 and TRIM28 expression associated with angiogenesis signatures in tumour samples, and conditioned media from TRIM24 and TRIM28-silenced cancer cells inhibited endothelial cell proliferation and formation of vascular tube structures. Our data suggest that TRIM24 and TRIM28 proteins interact, in gene-specific manners, to regulate AR activity, increase VEGF signalling and angiogenesis, and that targeting these coregulators may increase the effectiveness of antiandrogen therapy.
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Affiliation(s)
- Damien A Leach
- Division of Cancer, Imperial Centre for Translational & Experimental Medicine, Imperial College London, UK
| | - Nilesh Chatterjee
- Division of Cancer, Imperial Centre for Translational & Experimental Medicine, Imperial College London, UK
- St George's University of London, UK
| | - Kellie Spahr
- University of Michigan, Ann Arbor, MI, USA
- University of Pittsburgh, PA, USA
| | | | - Anabel Varela-Carver
- Division of Cancer, Imperial Centre for Translational & Experimental Medicine, Imperial College London, UK
| | - Taimur T Shah
- Imperial Urology, Division of Surgery, Imperial College Healthcare NHS Trust, London, UK
- Imperial Prostate, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, UK
| | - Mathias Winkler
- Imperial Urology, Division of Surgery, Imperial College Healthcare NHS Trust, London, UK
- Imperial Prostate, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, UK
| | - Hashim U Ahmed
- Imperial Urology, Division of Surgery, Imperial College Healthcare NHS Trust, London, UK
- Imperial Prostate, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, UK
| | - Charlotte L Bevan
- Division of Cancer, Imperial Centre for Translational & Experimental Medicine, Imperial College London, UK
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3
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Schmidleithner L, Stüve P, Feuerer M. Transposable elements as instructors of the immune system. Nat Rev Immunol 2025:10.1038/s41577-025-01172-3. [PMID: 40301669 DOI: 10.1038/s41577-025-01172-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/27/2025] [Indexed: 05/01/2025]
Abstract
Transposable elements (TEs) are mobile repetitive nucleic acid sequences that have been incorporated into the genome through spontaneous integration, accounting for almost 50% of human DNA. Even though most TEs are no longer mobile today, studies have demonstrated that they have important roles in different biological processes, such as ageing, embryonic development, and cancer. TEs influence these processes through various mechanisms, including active transposition of TEs contributing to ongoing evolution, transposon transcription generating RNA or protein, and by influencing gene regulation as enhancers. However, how TEs interact with the immune system remains a largely unexplored field. In this Perspective, we describe how TEs might influence different aspects of the immune system, such as innate immune responses, T cell activation and differentiation, and tissue adaptation. Furthermore, TEs can serve as a source of neoantigens for T cells in antitumour immunity. We suggest that TE biology is an important emerging field of immunology and discuss the potential to harness the TE network therapeutically, for example, to improve immunotherapies for cancer and autoimmune and inflammatory diseases.
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Affiliation(s)
| | - Philipp Stüve
- Leibniz Institute for Immunotherapy, Regensburg, Germany
| | - Markus Feuerer
- Leibniz Institute for Immunotherapy, Regensburg, Germany.
- Chair for Immunology, University Regensburg, Regensburg, Germany.
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Li BI, Alvarez MJ, Zhao H, Chirathivat N, Califano A, Shen MM. The regulatory architecture of the primed pluripotent cell state. Nat Commun 2025; 16:3351. [PMID: 40204698 PMCID: PMC11982361 DOI: 10.1038/s41467-025-57894-4] [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: 04/14/2024] [Accepted: 03/06/2025] [Indexed: 04/11/2025] Open
Abstract
Despite extensive research, the gene regulatory architecture governing mammalian cell states remains poorly understood. Here we present an integrative systems biology approach to elucidate the network architecture of primed state pluripotency. Using an unbiased methodology, we identified and experimentally confirmed 132 transcription factors as master regulators (MRs) of mouse epiblast stem cell (EpiSC) pluripotency, many of which were further validated by CRISPR-mediated functional assays. To assemble a comprehensive regulatory network, we silenced each of the 132 MRs to assess their effects on the other MRs and their transcriptional targets, yielding a network of 1273 MR → MR interactions. Network architecture analyses revealed four functionally distinct MR modules (communities), and identified key Speaker and Mediator MRs based on their hierarchical rank and centrality. Our findings elucidate the de-centralized logic of a "communal interaction" model in which the balanced activities of four MR communities maintain primed state pluripotency.
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Affiliation(s)
- Bo I Li
- Department of Medicine, New York, NY, USA
- Systems Biology, New York, NY, USA
- Genetics and Development, New York, NY, USA
- Urology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA
- Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Mariano J Alvarez
- Systems Biology, New York, NY, USA
- DarwinHealth, Inc., New York, NY, USA
| | - Hui Zhao
- Department of Medicine, New York, NY, USA
- Systems Biology, New York, NY, USA
- Genetics and Development, New York, NY, USA
- Urology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA
- Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Napon Chirathivat
- Department of Medicine, New York, NY, USA
- Systems Biology, New York, NY, USA
- Genetics and Development, New York, NY, USA
- Urology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA
- Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA
| | - Andrea Califano
- Department of Medicine, New York, NY, USA.
- Systems Biology, New York, NY, USA.
- Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA.
- DarwinHealth, Inc., New York, NY, USA.
- Department of Biochemistry and Molecular Biophysics, New York, NY, USA.
- Biomedical Informatics, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA.
- Chan Zuckerberg Biohub, New York, NY, USA.
| | - Michael M Shen
- Department of Medicine, New York, NY, USA.
- Systems Biology, New York, NY, USA.
- Genetics and Development, New York, NY, USA.
- Urology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA.
- Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, NY, USA.
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5
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Kim YK, Ramalho-Santos M. 20 years of stemness: From stem cells to hypertranscription and back. Stem Cell Reports 2025; 20:102406. [PMID: 39919752 PMCID: PMC11960510 DOI: 10.1016/j.stemcr.2025.102406] [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: 11/01/2024] [Revised: 01/08/2025] [Accepted: 01/09/2025] [Indexed: 02/09/2025] Open
Abstract
Transcriptional profiling of stem cells came of age at the beginning of the century with the use of microarrays to analyze cell populations in bulk. Since then, stem cell transcriptomics has become increasingly sophisticated, notably with the recent widespread use of single-cell RNA sequencing. Here, we provide a perspective on how an early signature of genes upregulated in embryonic and adult stem cells, identified using microarrays over 20 years ago, serendipitously led to the recent discovery that stem/progenitor cells across organs are in a state of hypertranscription, a global elevation of the transcriptome. Looking back, we find that the 2002 stemness signature is a robust marker of stem cell hypertranscription, even though it was developed well before it was known what hypertranscription meant or how to detect it. We anticipate that studies of stem cell hypertranscription will be rich in novel insights in physiological and disease contexts for years to come.
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Affiliation(s)
- Yun-Kyo Kim
- Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto ON M5G 0A4, Canada; Department of Molecular Genetics, University of Toronto, Toronto ON M5G 1X5, Canada.
| | - Miguel Ramalho-Santos
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto ON M5T 3L9, Canada; Department of Molecular Genetics, University of Toronto, Toronto ON M5G 1X5, Canada.
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6
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Bhaduri-McIntosh S, Rousseau BA. KAP1/TRIM28 - antiviral and proviral protagonist of herpesvirus biology. Trends Microbiol 2024; 32:1179-1189. [PMID: 38871562 PMCID: PMC11620967 DOI: 10.1016/j.tim.2024.05.007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2024] [Revised: 05/19/2024] [Accepted: 05/21/2024] [Indexed: 06/15/2024]
Abstract
Dysregulation of the constitutive heterochromatin machinery (HCM) that silences pericentromeric regions and endogenous retroviral elements in the human genome has consequences for aging and cancer. By recruiting epigenetic regulators, Krüppel-associated box (KRAB)-associated protein 1 (KAP1/TRIM28/TIF1β) is integral to the function of the HCM. Epigenetically silencing DNA genomes of incoming herpesviruses to enforce latency, KAP1 and HCM also serve in an antiviral capacity. In addition to gene silencing, newer reports highlight KAP1's ability to directly activate cellular gene transcription. Here, we discuss the many facets of KAP1, including recent findings that unexpectedly connect KAP1 to the inflammasome, reveal KAP1 cleavage as a novel mode of regulation, and argue for a pro-herpesviral KAP1 function that ensures transition from transcription to replication of the herpesvirus genome.
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Affiliation(s)
- Sumita Bhaduri-McIntosh
- Division of Infectious Diseases, Department of Pediatrics, University of Florida, Gainesville, FL, USA; Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA.
| | - Beth A Rousseau
- Division of Infectious Diseases, Department of Pediatrics, University of Florida, Gainesville, FL, USA
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7
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Maghsoudloo M, Mokhtari K, Jamali B, Gholamzad A, Entezari M, Hashemi M, Fu J. Multifaceted role of TRIM28 in health and disease. MedComm (Beijing) 2024; 5:e790. [PMID: 39534556 PMCID: PMC11554878 DOI: 10.1002/mco2.790] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2024] [Revised: 09/28/2024] [Accepted: 09/28/2024] [Indexed: 11/16/2024] Open
Abstract
The TRIM (tripartite motif) family, with TRIM28 as a key member, plays a vital role in regulating health and disease. TRIM28 contains various functional domains essential for transcriptional regulation, primarily through its interaction with KRAB-ZNF proteins, which influence chromatin remodeling and gene expression. Despite extensive research, the precise mechanisms by which TRIM28 impacts health and disease remain elusive. This review delves into TRIM28's multifaceted roles in maintaining health, contributing to a variety of diseases, and influencing cancer progression. In cancers, TRIM28 exhibits a dual nature, functioning as both a tumor promoter and suppressor depending on the cellular context and cancer type. The review also explores its critical involvement in processes such as DNA repair, cell cycle regulation, epithelial-to-mesenchymal transition, and the maintenance of stem cell properties. By uncovering TRIM28's complex roles across different cancers and other diseases, this review underscores its potential as a therapeutic target. The significance of TRIM28 as a versatile regulator opens the door to innovative therapeutic strategies, particularly in cancer treatment and the management of other diseases. Ongoing research into TRIM28 may yield key insights into disease progression and novel treatment options.
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Affiliation(s)
- Mazaher Maghsoudloo
- Key Laboratory of Epigenetics and Oncologythe Research Center for Preclinical MedicineSouthwest Medical UniversityLuzhouSichuanChina
| | - Khatere Mokhtari
- Department of Cellular and Molecular Biology and MicrobiologyFaculty of Biological Science and TechnologyUniversity of IsfahanIsfahanIran
| | - Behdokht Jamali
- Department of Microbiology and GeneticKherad Institute of Higher EducationBusheherIran
| | - Amir Gholamzad
- Farhikhtegan Medical Convergence Sciences Research CenterFarhikhtegan Hospital Tehran Medical SciencesIslamic Azad UniversityTehranIran
| | - Maliheh Entezari
- Farhikhtegan Medical Convergence Sciences Research CenterFarhikhtegan Hospital Tehran Medical SciencesIslamic Azad UniversityTehranIran
- Department of GeneticsFaculty of Advanced Science and TechnologyTehran Medical SciencesIslamic Azad UniversityTehranIran
| | - Mehrdad Hashemi
- Farhikhtegan Medical Convergence Sciences Research CenterFarhikhtegan Hospital Tehran Medical SciencesIslamic Azad UniversityTehranIran
- Department of GeneticsFaculty of Advanced Science and TechnologyTehran Medical SciencesIslamic Azad UniversityTehranIran
| | - Junjiang Fu
- Key Laboratory of Epigenetics and Oncologythe Research Center for Preclinical MedicineSouthwest Medical UniversityLuzhouSichuanChina
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8
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Mote RD, Tiwari M, Yadavalli N, Rajan R, Subramanyam D. A high-throughput screen in mESCs to identify the cross-talk between signaling, endocytosis, and pluripotency. Cell Biol Int 2024. [PMID: 38706123 DOI: 10.1002/cbin.12168] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2023] [Revised: 03/23/2024] [Accepted: 04/14/2024] [Indexed: 05/07/2024]
Abstract
Embryonic stem cell fate is regulated by various cellular processes. Recently, the process of endocytosis has been implicated in playing a role in the maintenance of self-renewal and pluripotency of mouse embryonic stem cells. A previous siRNA-based screen interrogated the function of core components of the endocytic machinery in maintaining the pluripotency of embryonic stem cells, revealing a crucial role for clathrin mediated endocytosis. A number of other proteins involved in key signaling pathways have also been shown to both regulate and be regulated by endocytosis. We collated a list of 1141 genes in connection to the term "endocytosis" from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), excluding those previously interrogated, and examined the effect of their knockdown on the pluripotency of mouse embryonic stem cells (mESCs) using levels of green fluorescent protein driven by the Oct4 promoter. We used high-throughput screening followed by an automated MATrix LABoratory (MATLAB)-based analysis pipeline and assessed changes in GFP fluorescence as a readout for ESC pluripotency. Through this screen we identified a number of genes, many hitherto not associated with stem cell pluripotency, which upon knockdown either resulted in a significant increase or decrease of GFP fluorescence. We further present validation for some of these hits. We present a workflow aimed to identify genes involved in signaling pathways which can be regulated by endocytosis, and that affect the pluripotency of ESCs.
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Affiliation(s)
- Ridim D Mote
- Stem Cell Biology Lab, National Centre for Cell Science, SP Pune University Campus, Pune, India
| | - Mahak Tiwari
- Stem Cell Biology Lab, National Centre for Cell Science, SP Pune University Campus, Pune, India
- SP Pune University, Pune, India
| | - Narayana Yadavalli
- Stem Cell Biology Lab, National Centre for Cell Science, SP Pune University Campus, Pune, India
| | - Raghav Rajan
- Indian Institute of Science Education and Research, Pune, India
| | - Deepa Subramanyam
- Stem Cell Biology Lab, National Centre for Cell Science, SP Pune University Campus, Pune, India
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9
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Amiri M, Kiniry SJ, Possemato AP, Mahmood N, Basiri T, Dufour CR, Tabatabaei N, Deng Q, Bellucci MA, Harwalkar K, Stokes MP, Giguère V, Kaufman RJ, Yamanaka Y, Baranov PV, Tahmasebi S, Sonenberg N. Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells. Cell Rep 2024; 43:113615. [PMID: 38159280 PMCID: PMC10962698 DOI: 10.1016/j.celrep.2023.113615] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2023] [Revised: 10/24/2023] [Accepted: 12/06/2023] [Indexed: 01/03/2024] Open
Abstract
The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α+/+) and phosphorylation-deficient mutant eIF2α (eIF2αA/A) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/β) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network.
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Affiliation(s)
- Mehdi Amiri
- Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada
| | - Stephen J Kiniry
- School of Biochemistry and Cell Biology, University College Cork, T12 XF62 Cork, Ireland
| | | | - Niaz Mahmood
- Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada
| | - Tayebeh Basiri
- Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada
| | - Catherine R Dufour
- Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada
| | - Negar Tabatabaei
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL 60612, USA
| | - Qiyun Deng
- Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada
| | - Michael A Bellucci
- Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada
| | - Keerthana Harwalkar
- Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada; Department of Human Genetics, McGill University, Montreal, QC H3A 0C7, Canada
| | - Matthew P Stokes
- Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923, USA
| | - Vincent Giguère
- Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada
| | - Randal J Kaufman
- Degenerative Diseases Program, Center for Genetic Disorders and Aging Research, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA
| | - Yojiro Yamanaka
- Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada; Department of Human Genetics, McGill University, Montreal, QC H3A 0C7, Canada
| | - Pavel V Baranov
- School of Biochemistry and Cell Biology, University College Cork, T12 XF62 Cork, Ireland
| | - Soroush Tahmasebi
- Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL 60612, USA.
| | - Nahum Sonenberg
- Department of Biochemistry, McGill University, Montreal, QC H3A 1A3, Canada; Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC H3A 1A3, Canada.
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10
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Ning T, Zhao M, Zhang N, Wang Z, Zhang S, Liu M, Zhu S. TRIM28 suppresses cancer stem-like characteristics in gastric cancer cells through Wnt/β-catenin signaling pathways. Exp Biol Med (Maywood) 2023; 248:2210-2218. [PMID: 38058023 PMCID: PMC10903244 DOI: 10.1177/15353702231211970] [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: 04/27/2023] [Accepted: 08/23/2023] [Indexed: 12/08/2023] Open
Abstract
The influences of TRIM28 on the gastric tumorigenesis together with potential molecular mechanisms remain to be studied. We aimed at exploring the important effects of TRIM28 on gastric cancer (GC) and uncovering underling molecular mechanisms. Through immunohistochemistry analysis of 20 pairs of GC and the peritumoral tissues, the expression level of TRIM28 was determined. A variety of assays were applied to explore the important roles of TRIM28 in GC. Western blotting and qRT-PCR analyses were used to analyze the association between TRIM28 and the Wnt/β-catenin signaling pathway. TRIM28 was highly expressed in GC tissues than peritumoral tissues. And high expression level of TRIM28 in GC was associated with good prognostic effects. In vitro functional assays suggested TRIM28 knockdown enhanced the proliferation and clone formation of GC cell. Moreover, TRIM28 knockdown enhanced the expression level of stemness markers, strengthened sphere-forming and drug-resistance properties of GC cells, suggesting important effect on GC cell stemness. Besides, our analysis showed that the Wnt/β-catenin signaling was involved in the effect of TRIM28 on GC cell stemness property, and blocking Wnt/β-catenin signaling pathway obviously rescued the promotion influence of TRIM28 knockdown. Overall, TRIM28 has an important influence on regulating the stem-like property of GC cell via Wnt/β-catenin signaling, suggesting TRIM28 a promising drug target and a potential predictor of prognosis.
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Affiliation(s)
- Tingting Ning
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, China
| | - Mengran Zhao
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, China
| | - Nan Zhang
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, China
| | - Zhaoqing Wang
- Department of Pathology, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China
| | - Shutian Zhang
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, China
| | - Mo Liu
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, China
| | - Shengtao Zhu
- Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, National Clinical Research Center for Digestive Disease, Beijing Digestive Disease Center, Beijing Key Laboratory for Precancerous Lesion of Digestive Disease, Beijing, 100050, China
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11
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Talukdar PD, Chatterji U. Transcriptional co-activators: emerging roles in signaling pathways and potential therapeutic targets for diseases. Signal Transduct Target Ther 2023; 8:427. [PMID: 37953273 PMCID: PMC10641101 DOI: 10.1038/s41392-023-01651-w] [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: 04/18/2023] [Revised: 08/27/2023] [Accepted: 09/10/2023] [Indexed: 11/14/2023] Open
Abstract
Specific cell states in metazoans are established by the symphony of gene expression programs that necessitate intricate synergic interactions between transcription factors and the co-activators. Deregulation of these regulatory molecules is associated with cell state transitions, which in turn is accountable for diverse maladies, including developmental disorders, metabolic disorders, and most significantly, cancer. A decade back most transcription factors, the key enablers of disease development, were historically viewed as 'undruggable'; however, in the intervening years, a wealth of literature validated that they can be targeted indirectly through transcriptional co-activators, their confederates in various physiological and molecular processes. These co-activators, along with transcription factors, have the ability to initiate and modulate transcription of diverse genes necessary for normal physiological functions, whereby, deregulation of such interactions may foster tissue-specific disease phenotype. Hence, it is essential to analyze how these co-activators modulate specific multilateral processes in coordination with other factors. The proposed review attempts to elaborate an in-depth account of the transcription co-activators, their involvement in transcription regulation, and context-specific contributions to pathophysiological conditions. This review also addresses an issue that has not been dealt with in a comprehensive manner and hopes to direct attention towards future research that will encompass patient-friendly therapeutic strategies, where drugs targeting co-activators will have enhanced benefits and reduced side effects. Additional insights into currently available therapeutic interventions and the associated constraints will eventually reveal multitudes of advanced therapeutic targets aiming for disease amelioration and good patient prognosis.
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Affiliation(s)
- Priyanka Dey Talukdar
- Cancer Research Laboratory, Department of Zoology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, West Bengal, India
| | - Urmi Chatterji
- Cancer Research Laboratory, Department of Zoology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, West Bengal, India.
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12
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Zhang X, Wang Y, Lu J, Xiao L, Chen H, Li Q, Li YY, Xu P, Ruan C, Zhou H, Zhao Y. A conserved ZFX/WNT3 axis modulates the growth and imatinib response of chronic myeloid leukemia stem/progenitor cells. Cell Mol Biol Lett 2023; 28:83. [PMID: 37864206 PMCID: PMC10589942 DOI: 10.1186/s11658-023-00496-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 10/03/2023] [Indexed: 10/22/2023] Open
Abstract
BACKGROUND Zinc finger protein X-linked (ZFX) has been shown to promote the growth of tumor cells, including leukemic cells. However, the role of ZFX in the growth and drug response of chronic myeloid leukemia (CML) stem/progenitor cells remains unclear. METHODS Real-time quantitative PCR (RT-qPCR) and immunofluorescence were used to analyze the expression of ZFX and WNT3 in CML CD34+ cells compared with normal control cells. Short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) technologies were used to study the role of ZFX in growth and drug response of CML cells. Microarray data were generated to compare ZFX-silenced CML CD34+ cells with their controls. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to study the molecular mechanisms of ZFX to regulate WNT3 expression. RT-qPCR and western blotting were used to study the effect of ZFX on β-catenin signaling. RESULTS We showed that ZFX expression was significantly higher in CML CD34+ cells than in control cells. Overexpression and gene silencing experiments indicated that ZFX promoted the in vitro growth of CML cells, conferred imatinib mesylate (IM) resistance to these cells, and enhanced BCR/ABL-induced malignant transformation. Microarray data and subsequent validation revealed that WNT3 transcription was conservatively regulated by ZFX. WNT3 was highly expressed in CML CD34+ cells, and WNT3 regulated the growth and IM response of these cells similarly to ZFX. Moreover, WNT3 overexpression partially rescued ZFX silencing-induced growth inhibition and IM hypersensitivity. ZFX silencing decreased WNT3/β-catenin signaling, including c-MYC and CCND1 expression. CONCLUSION The present study identified a novel ZFX/WNT3 axis that modulates the growth and IM response of CML stem/progenitor cells.
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MESH Headings
- Humans
- Imatinib Mesylate/pharmacology
- Imatinib Mesylate/metabolism
- beta Catenin/metabolism
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism
- Stem Cells/metabolism
- Signal Transduction
- Drug Resistance, Neoplasm/genetics
- Neoplastic Stem Cells/metabolism
- Wnt3 Protein/metabolism
- Wnt3 Protein/pharmacology
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Affiliation(s)
- Xiuyan Zhang
- Cyrus Tang Medical Institute, Soochow University, Suzhou, 215123, China.
- Jiangsu Institute of Hematology, NHC Key Laboratory of Thrombosis and Hemostasis, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China.
| | - Yu Wang
- Cyrus Tang Medical Institute, Soochow University, Suzhou, 215123, China
| | - Jinchang Lu
- Cyrus Tang Medical Institute, Soochow University, Suzhou, 215123, China
| | - Lun Xiao
- Department of Vascular Surgery, The Affiliated Drum Tower Hospital, Nanjing University Medical School, Nanjing, 210008, China
| | - Hui Chen
- Cyrus Tang Medical Institute, Soochow University, Suzhou, 215123, China
| | - Quanxue Li
- Shanghai-MOST Key Laboratory of Health and Disease Genomics, Shanghai Institute for Biomedical and Pharmaceutical Technologies, Shanghai, 200237, China
| | - Yuan-Yuan Li
- Shanghai-MOST Key Laboratory of Health and Disease Genomics, Shanghai Institute for Biomedical and Pharmaceutical Technologies, Shanghai, 200237, China
| | - Peng Xu
- Cyrus Tang Medical Institute, Soochow University, Suzhou, 215123, China
| | - Changgeng Ruan
- Jiangsu Institute of Hematology, NHC Key Laboratory of Thrombosis and Hemostasis, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China
- National Clinical Research Center for Hematologic Diseases, Suzhou, 215006, China
- Collaborative Innovation Center of Hematology, Soochow University, Suzhou, 215006, China
- MOE Engineering Center of Hematological Disease, Soochow University, Suzhou, 21513, China
| | - Haixia Zhou
- Jiangsu Institute of Hematology, NHC Key Laboratory of Thrombosis and Hemostasis, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China.
- National Clinical Research Center for Hematologic Diseases, Suzhou, 215006, China.
- MOE Engineering Center of Hematological Disease, Soochow University, Suzhou, 21513, China.
| | - Yun Zhao
- Cyrus Tang Medical Institute, Soochow University, Suzhou, 215123, China.
- National Clinical Research Center for Hematologic Diseases, Suzhou, 215006, China.
- Collaborative Innovation Center of Hematology, Soochow University, Suzhou, 215006, China.
- MOE Engineering Center of Hematological Disease, Soochow University, Suzhou, 21513, China.
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13
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Chen Q, Malki S, Xu X, Bennett B, Lackford BL, Kirsanov O, Geyer CB, Hu G. Cnot3 is required for male germ cell development and spermatogonial stem cell maintenance. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.13.562256. [PMID: 37873304 PMCID: PMC10592795 DOI: 10.1101/2023.10.13.562256] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2023]
Abstract
The foundation of spermatogenesis and lifelong fertility is provided by spermatogonial stem cells (SSCs). SSCs divide asymmetrically to either replenish their numbers (self-renewal) or produce undifferentiated progenitors that proliferate before committing to differentiation. However, regulatory mechanisms governing SSC maintenance are poorly understood. Here, we show that the CCR4-NOT mRNA deadenylase complex subunit CNOT3 plays a critical role in maintaining spermatogonial populations in mice. Cnot3 is highly expressed in undifferentiated spermatogonia, and its deletion in spermatogonia resulted in germ cell loss and infertility. Single cell analyses revealed that Cnot3 deletion led to the de-repression of transcripts encoding factors involved in spermatogonial differentiation, including those in the glutathione redox pathway that are critical for SSC maintenance. Together, our study reveals that CNOT3 - likely via the CCR4-NOT complex - actively degrades transcripts encoding differentiation factors to sustain the spermatogonial pool and ensure the progression of spermatogenesis, highlighting the importance of CCR4-NOT-mediated post-transcriptional gene regulation during male germ cell development.
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Affiliation(s)
- Qing Chen
- Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
- Present address: Clinical Microbiome Unit (CMU), Laboratory of Host Immunity and Microbiome (LHIM), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD 20892, USA
| | - Safia Malki
- Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
| | - Xiaojiang Xu
- Integrative Bioinformatics Support Group, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
- Present address: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, LA 70112
| | - Brian Bennett
- Integrative Bioinformatics Support Group, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
| | - Brad L. Lackford
- Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
| | - Oleksandr Kirsanov
- Department of Anatomy & Cell Biology, Brody School of Medicine at East Carolina University, Greenville, NC, USA
| | - Christopher B. Geyer
- Department of Anatomy & Cell Biology, Brody School of Medicine at East Carolina University, Greenville, NC, USA
- East Carolina Diabetes and Obesity Institute East Carolina University, Greenville, NC, USA
| | - Guang Hu
- Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
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14
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Shaba E, Landi C, Marzocchi C, Vantaggiato L, Bini L, Ricci C, Cantara S. Proteomics Reveals How the Tardigrade Damage Suppressor Protein Teaches Transfected Human Cells to Survive UV-C Stress. Int J Mol Sci 2023; 24:11463. [PMID: 37511223 PMCID: PMC10380570 DOI: 10.3390/ijms241411463] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Revised: 06/12/2023] [Accepted: 07/12/2023] [Indexed: 07/30/2023] Open
Abstract
The genome sequencing of the tardigrade Ramazzottius varieornatus revealed a unique nucleosome-binding protein named damage suppressor (Dsup), which was discovered to be crucial for the extraordinary abilities of tardigrades in surviving extreme stresses, such as UV. Evidence in Dsup-transfected human cells suggests that Dsup mediates an overall response in DNA damage signaling, DNA repair, and cell cycle regulation, resulting in an acquired resistance to stress. Given these promising outcomes, our study attempts to provide a wider comprehension of the molecular mechanisms modulated by Dsup in human cells and to explore the Dsup-activated molecular pathways under stress. We performed a differential proteomic analysis of Dsup-transfected and control human cells under basal conditions and at 24 h recovery after exposure to UV-C. We demonstrate via enrichment and network analyses, for the first time, that even in the absence of external stimuli, and more significantly, after stress, Dsup activates mechanisms involved with the unfolded protein response, the mRNA processing and stability, cytoplasmic stress granules, the DNA damage response, and the telomere maintenance. In conclusion, our results shed new light on Dsup-mediated protective mechanisms and increases our knowledge of the molecular machineries of extraordinary protection against UV-C stress.
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Affiliation(s)
- Enxhi Shaba
- Functional Proteomics Lab, Department of Life Sciences, University of Siena, 53100 Siena, Italy
| | - Claudia Landi
- Functional Proteomics Lab, Department of Life Sciences, University of Siena, 53100 Siena, Italy
| | - Carlotta Marzocchi
- Department of Medical, Surgical and Neurological Sciences, University of Siena, 53100 Siena, Italy
| | - Lorenza Vantaggiato
- Functional Proteomics Lab, Department of Life Sciences, University of Siena, 53100 Siena, Italy
| | - Luca Bini
- Functional Proteomics Lab, Department of Life Sciences, University of Siena, 53100 Siena, Italy
| | - Claudia Ricci
- Department of Medical, Surgical and Neurological Sciences, University of Siena, 53100 Siena, Italy
| | - Silvia Cantara
- Department of Medical, Surgical and Neurological Sciences, University of Siena, 53100 Siena, Italy
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15
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Liu C, Yu P, Ren Z, Yao F, Wang L, Hu G, Li P, Zhao Q. Rif1 Regulates Self-Renewal and Impedes Mesendodermal Differentiation of Mouse Embryonic Stem Cells. Stem Cell Rev Rep 2023:10.1007/s12015-023-10525-1. [PMID: 36971904 PMCID: PMC10366267 DOI: 10.1007/s12015-023-10525-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/02/2023] [Indexed: 03/29/2023]
Abstract
Abstract
Background
RAP1 interacting factor 1 (Rif1) is highly expressed in mice embryos and mouse embryonic stem cells (mESCs). It plays critical roles in telomere length homeostasis, DNA damage, DNA replication timing and ERV silencing. However, whether Rif1 regulates early differentiation of mESC is still unclear.
Methods
In this study, we generated a Rif1 conditional knockout mouse embryonic stem (ES) cell line based on Cre-loxP system. Western blot, flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), RNA high-throughput sequencing (RNA-Seq), chromatin immunoprecipitation followed high-throughput sequencing (ChIP-Seq), chromatin immunoprecipitation quantitative PCR (ChIP-qPCR), immunofluorescence, and immunoprecipitation were employed for phenotype and molecular mechanism assessment.
Results
Rif1 plays important roles in self-renewal and pluripotency of mESCs and loss of Rif1 promotes mESC differentiation toward the mesendodermal germ layers. We further show that Rif1 interacts with histone H3K27 methyltransferase EZH2, a subunit of PRC2, and regulates the expression of developmental genes by directly binding to their promoters. Rif1 deficiency reduces the occupancy of EZH2 and H3K27me3 on mesendodermal gene promoters and activates ERK1/2 activities.
Conclusion
Rif1 is a key factor in regulating the pluripotency, self-renewal, and lineage specification of mESCs. Our research provides new insights into the key roles of Rif1 in connecting epigenetic regulations and signaling pathways for cell fate determination and lineage specification of mESCs.
Graphical abstract
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16
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Yang Y, Tan S, Han Y, Huang L, Yang R, Hu Z, Tao Y, Oyang L, Lin J, Peng Q, Jiang X, Xu X, Xia L, Peng M, Wu N, Tang Y, Li X, Liao Q, Zhou Y. The role of tripartite motif-containing 28 in cancer progression and its therapeutic potentials. Front Oncol 2023; 13:1100134. [PMID: 36756159 PMCID: PMC9899900 DOI: 10.3389/fonc.2023.1100134] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Accepted: 01/04/2023] [Indexed: 01/24/2023] Open
Abstract
Tripartite motif-containing 28 (TRIM28) belongs to tripartite motif (TRIM) family. TRIM28 not only binds and degrades its downstream target, but also acts as a transcription co-factor to inhibit gene expression. More and more studies have shown that TRIM28 plays a vital role in tumor genesis and progression. Here, we reviewed the role of TRIM28 in tumor proliferation, migration, invasion and cell death. Moreover, we also summarized the important role of TRIM28 in tumor stemness sustainability and immune regulation. Because of the importance of TRIM28 in tumors, TIRM28 may be a candidate target for anti-tumor therapy and play an important role in tumor diagnosis and treatment in the future.
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Affiliation(s)
- Yiqing Yang
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Shiming Tan
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Yaqian Han
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Lisheng Huang
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China,University of South China, Hengyang, Hunan, China
| | - Ruiqian Yang
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China,University of South China, Hengyang, Hunan, China
| | - Zifan Hu
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China,University of South China, Hengyang, Hunan, China
| | - Yi Tao
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China,University of South China, Hengyang, Hunan, China
| | - Linda Oyang
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Jinguan Lin
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Qiu Peng
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Xianjie Jiang
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Xuemeng Xu
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Longzheng Xia
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Mingjing Peng
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Nayiyuan Wu
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Yanyan Tang
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China
| | - Xiaoling Li
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China,*Correspondence: Yujuan Zhou, ; Qianjin Liao, ; Xiaoling Li,
| | - Qianjin Liao
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China,Hunan Key Laboratory of Translational Radiation Oncology, Changsha, Hunan, China,*Correspondence: Yujuan Zhou, ; Qianjin Liao, ; Xiaoling Li,
| | - Yujuan Zhou
- Hunan Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China,Hunan Key Laboratory of Translational Radiation Oncology, Changsha, Hunan, China,*Correspondence: Yujuan Zhou, ; Qianjin Liao, ; Xiaoling Li,
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17
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Burramsetty AK, Nishimura K, Kishimoto T, Hamzah M, Kuno A, Fukuda A, Hisatake K. Locus-Specific Isolation of the Nanog Chromatin Identifies Regulators Relevant to Pluripotency of Mouse Embryonic Stem Cells and Reprogramming of Somatic Cells. Int J Mol Sci 2022; 23:ijms232315242. [PMID: 36499566 PMCID: PMC9740452 DOI: 10.3390/ijms232315242] [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: 11/14/2022] [Revised: 11/28/2022] [Accepted: 11/30/2022] [Indexed: 12/11/2022] Open
Abstract
Pluripotency is a crucial feature of pluripotent stem cells, which are regulated by the core pluripotency network consisting of key transcription factors and signaling molecules. However, relatively less is known about the molecular mechanisms that modify the core pluripotency network. Here we used the CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) to unbiasedly isolate proteins assembled on the Nanog promoter in mouse embryonic stem cells (mESCs), and then tested their functional relevance to the maintenance of mESCs and reprogramming of somatic cells. Gene ontology analysis revealed that the identified proteins, including many RNA-binding proteins (RBPs), are enriched in RNA-related functions and gene expression. ChIP-qPCR experiments confirmed that BCLAF1, FUBP1, MSH6, PARK7, PSIP1, and THRAP3 occupy the Nanog promoter region in mESCs. Knockdown experiments of these factors show that they play varying roles in self-renewal, pluripotency gene expression, and differentiation of mESCs as well as in the reprogramming of somatic cells. Our results show the utility of unbiased identification of chromatin-associated proteins on a pluripotency gene in mESCs and reveal the functional relevance of RBPs in ESC differentiation and somatic cell reprogramming.
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Affiliation(s)
- Arun Kumar Burramsetty
- Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan
| | - Ken Nishimura
- Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan
- Correspondence: (K.N.); (K.H.)
| | - Takumi Kishimoto
- Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan
| | - Muhammad Hamzah
- Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan
| | - Akihiro Kuno
- Laboratory of Animal Resource Center, Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan
- Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba, Tsukuba 305-8575, Japan
| | - Aya Fukuda
- Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan
| | - Koji Hisatake
- Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan
- Correspondence: (K.N.); (K.H.)
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18
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Nyberg WA, Velasquez‐Pulgarin DA, He T, Sjöstrand M, Pellé L, Covacu R, Espinosa A. The bromodomain protein TRIM28 controls the balance between growth and invasiveness in melanoma. EMBO Rep 2022; 24:e54944. [PMID: 36341538 PMCID: PMC9827549 DOI: 10.15252/embr.202254944] [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: 02/28/2022] [Revised: 10/18/2022] [Accepted: 10/20/2022] [Indexed: 11/09/2022] Open
Abstract
Melanoma tumors are highly metastatic partly due to the ability of melanoma cells to transition between invasive and proliferative states. However, the mechanisms underlying this plasticity are still not fully understood. To identify new epigenetic regulators of melanoma plasticity, we combined data mining, tumor models, proximity proteomics, and CUT&RUN sequencing. We focus on the druggable family of bromodomain epigenetic readers and identify TRIM28 as a new regulator of melanoma plasticity. We find that TRIM28 promotes the expression of pro-invasive genes and that TRIM28 controls the balance between invasiveness and growth of melanoma cells. We demonstrate that TRIM28 acts via the transcription factor JUNB that directly regulates the expression of pro-invasive and pro-growth genes. Mechanistically, TRIM28 controls the expression of JUNB by negatively regulating its transcriptional elongation by RNA polymerase II. In conclusion, our results demonstrate that a TRIM28-JUNB axis controls the balance between invasiveness and growth in melanoma tumors and suggest that the bromodomain protein TRIM28 could be targeted to reduce tumor spread.
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Affiliation(s)
- William A Nyberg
- Department of Medicine, Center for Molecular Medicine, Karolinska InstitutetKarolinska University HospitalStockholmSweden,Present address:
Department of MedicineUniversity of California San FranciscoSan FranciscoCAUSA
| | - Diego A Velasquez‐Pulgarin
- Department of Medicine, Center for Molecular Medicine, Karolinska InstitutetKarolinska University HospitalStockholmSweden,Department of Biomedical EngineeringUniversity of MemphisMemphisTNUSA
| | - Tianlin He
- Department of Medicine, Center for Molecular Medicine, Karolinska InstitutetKarolinska University HospitalStockholmSweden
| | - Maria Sjöstrand
- Department of Medicine, Center for Molecular Medicine, Karolinska InstitutetKarolinska University HospitalStockholmSweden,Present address:
Department of Medicine, Center for Cell EngineeringMemorial Sloan‐Kettering Cancer CenterNew YorkNYUSA
| | - Lucia Pellé
- Department of Medicine, Center for Molecular Medicine, Karolinska InstitutetKarolinska University HospitalStockholmSweden
| | - Ruxandra Covacu
- Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska InstitutetKarolinska University HospitalStockholmSweden
| | - Alexander Espinosa
- Department of Medicine, Center for Molecular Medicine, Karolinska InstitutetKarolinska University HospitalStockholmSweden
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19
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Kim YS, Potashnikova DM, Gisina AM, Kholodenko IV, Kopylov AT, Tikhonova OV, Kurbatov LK, Saidova AA, Tvorogova AV, Kholodenko RV, Belousov PV, Vorobjev IA, Zgoda VG, Yarygin KN, Lupatov AY. TRIM28 Is a Novel Regulator of CD133 Expression Associated with Cancer Stem Cell Phenotype. Int J Mol Sci 2022; 23:9874. [PMID: 36077272 PMCID: PMC9456468 DOI: 10.3390/ijms23179874] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 08/24/2022] [Accepted: 08/27/2022] [Indexed: 11/16/2022] Open
Abstract
CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines. Despite a substantial difference in the proliferative activities of cell populations with different levels of CD133 expression, transcriptomic and proteomic profiling revealed only minor distinctions between them. Nonetheless, a further in silico assessment of the differentially expressed transcripts and proteins revealed 16 proteins that could be involved in the regulation of CD133 expression; these were assigned ranks reflecting the apparent extent of their involvement. Among them, the TRIM28 transcription factor had the highest rank. The prominent role of TRIM28 in CD133 expression modulation was confirmed experimentally in the Caco2 cell line clones: the knockout, though not the knockdown, of the TRIM28 gene downregulated CD133. These results for the first time highlight an important role of the TRIM28 transcription factor in the regulation of CD133-associated cancer cell heterogeneity.
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Affiliation(s)
- Yan S. Kim
- Laboratory of Cell Biology, V.N. Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia
| | - Daria M. Potashnikova
- Cell Biology and Histology Department, School of Biology, M.V. Lomonosov Moscow State University, 119234 Moscow, Russia
| | - Alisa M. Gisina
- Laboratory of Cell Biology, V.N. Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia
| | - Irina V. Kholodenko
- Laboratory of Cell Biology, V.N. Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia
| | - Arthur T. Kopylov
- Laboratory of Systems Biology, V.N. Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia
| | - Olga V. Tikhonova
- Laboratory of Systems Biology, V.N. Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia
| | - Leonid K. Kurbatov
- Transcriptome Analysis Group, Analytical Branch Department, V.N. Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia
| | - Aleena A. Saidova
- Cell Biology and Histology Department, School of Biology, M.V. Lomonosov Moscow State University, 119234 Moscow, Russia
- Department of Transcription Factors, V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Anna V. Tvorogova
- Laboratory of Cell Motility, A.N. Belozersky Research Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119992 Moscow, Russia
| | - Roman V. Kholodenko
- Laboratory of Molecular Immunology, M.M. Shemyakin–Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 117997 Moscow, Russia
| | - Pavel V. Belousov
- Endocrinology Research Centre, 117292 Moscow, Russia
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, V.A. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Ivan A. Vorobjev
- Laboratory of Cell Motility, A.N. Belozersky Research Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119992 Moscow, Russia
- Department of Biology, School of Sciences and Humanities, Nazarbayev University, Nur-Sultan 010000, Kazakhstan
- Laboratory of Biophotonics and Imaging, National Laboratory Astana, Nazarbayev University, Nur-Sultan 010000, Kazakhstan
| | - Victor G. Zgoda
- Laboratory of Systems Biology, V.N. Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia
| | - Konstantin N. Yarygin
- Laboratory of Cell Biology, V.N. Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia
| | - Alexey Yu. Lupatov
- Laboratory of Cell Biology, V.N. Orekhovich Institute of Biomedical Chemistry, 119121 Moscow, Russia
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20
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Gao C, Qi X, Gao X, Li J, Qin Y, Yin Y, Gao F, Feng T, Wu S, Du X. A Genome-Wide CRISPR Screen Identifies Factors Regulating Pluripotency Exit in Mouse Embryonic Stem Cells. Cells 2022; 11:cells11152289. [PMID: 35892587 PMCID: PMC9331787 DOI: 10.3390/cells11152289] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2022] [Revised: 07/22/2022] [Accepted: 07/22/2022] [Indexed: 02/05/2023] Open
Abstract
Pluripotency maintenance and exit in embryonic stem cells is a focal topic in stem cell biology. However, the effects of screening under very stringent culture conditions (e.g., differentiation medium, no leukemia inhibitory factor, no chemical inhibitors such as PD0325901 and CHIR99021, and no feeder cells) and of prolonging culture for key factors that regulate pluripotency exit, have not yet been reported. Here, we used a genome-wide CRISPR library to perform such a screen in mouse embryonic stem cells. Naïve NANOG-GFP mESCs were first transfected with a mouse genome-wide CRISPR knockout library to obtain a mutant mESCs library, followed by screening for two months in a strict N2B27 differentiation medium. The clones that survived our stringent screening were analyzed to identify the inserted sgRNAs. In addition to identifying the enriched genes that were reported in previous studies (Socs3, Tsc1, Trp53, Nf2, Tcf7l1, Csnk1a1, and Dhx30), we found 17 unreported genes, among which Zfp771 and Olfr769 appeared to be involved in pluripotency exit. Furthermore, Zfp771 knockout ESCs showed a differentiation delay in embryonic chimera experiments, indicating Zfp771 played an important role in pluripotency exit. Our results show that stringent screening with the CRISPR library can reveal key regulators of pluripotency exit.
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Affiliation(s)
- Chen Gao
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; (C.G.); (X.G.); (J.L.); (Y.Q.); (Y.Y.); (F.G.)
| | - Xiaolan Qi
- State Key Laboratory of Veterinary Etiological Biology, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
| | - Xin Gao
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; (C.G.); (X.G.); (J.L.); (Y.Q.); (Y.Y.); (F.G.)
| | - Jin Li
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; (C.G.); (X.G.); (J.L.); (Y.Q.); (Y.Y.); (F.G.)
| | - Yumin Qin
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; (C.G.); (X.G.); (J.L.); (Y.Q.); (Y.Y.); (F.G.)
| | - Yunjun Yin
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; (C.G.); (X.G.); (J.L.); (Y.Q.); (Y.Y.); (F.G.)
| | - Fei Gao
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; (C.G.); (X.G.); (J.L.); (Y.Q.); (Y.Y.); (F.G.)
| | - Tao Feng
- Sanya Institute of China Agricultural University, Sanya 572000, China;
| | - Sen Wu
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; (C.G.); (X.G.); (J.L.); (Y.Q.); (Y.Y.); (F.G.)
- Sanya Institute of China Agricultural University, Sanya 572000, China;
- Correspondence: (S.W.); (X.D.); Tel.: +86-10-62733075 (S.W.)
| | - Xuguang Du
- State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; (C.G.); (X.G.); (J.L.); (Y.Q.); (Y.Y.); (F.G.)
- Sanya Institute of China Agricultural University, Sanya 572000, China;
- Correspondence: (S.W.); (X.D.); Tel.: +86-10-62733075 (S.W.)
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21
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Hijacking of transcriptional condensates by endogenous retroviruses. Nat Genet 2022; 54:1238-1247. [PMID: 35864192 PMCID: PMC9355880 DOI: 10.1038/s41588-022-01132-w] [Citation(s) in RCA: 52] [Impact Index Per Article: 17.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2021] [Accepted: 05/26/2022] [Indexed: 12/20/2022]
Abstract
Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA polymerase II and the Mediator coactivator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and noncoding nascent RNAs, including those produced by retrotransposons, may facilitate ‘hijacking’ of transcriptional condensates in various developmental and disease contexts. TRIM28 depletion in embryonic stem cells disconnects transcriptional condensates from super-enhancers, which is rescued by knockdown of endogenous retroviruses.
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22
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Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits. Int J Mol Sci 2022; 23:ijms23126839. [PMID: 35743282 PMCID: PMC9224613 DOI: 10.3390/ijms23126839] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2022] [Revised: 06/14/2022] [Accepted: 06/16/2022] [Indexed: 12/10/2022] Open
Abstract
TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.
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23
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Quan Y, Wang M, Xu C, Wang X, Wu Y, Qin D, Lin Y, Lu X, Lu F, Li L. Cnot8 eliminates naïve regulation networks and is essential for naïve-to-formative pluripotency transition. Nucleic Acids Res 2022; 50:4414-4435. [PMID: 35390160 PMCID: PMC9071485 DOI: 10.1093/nar/gkac236] [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: 06/01/2021] [Revised: 03/11/2022] [Accepted: 03/26/2022] [Indexed: 11/14/2022] Open
Abstract
Mammalian early epiblasts at different phases are characterized by naïve, formative, and primed pluripotency states, involving extensive transcriptome changes. Here, we report that deadenylase Cnot8 of Ccr4-Not complex plays essential roles during the transition from naïve to formative state. Knock out (KO) Cnot8 resulted in early embryonic lethality in mice, but Cnot8 KO embryonic stem cells (ESCs) could be established. Compared with the cells differentiated from normal ESCs, Cnot8 KO cells highly expressed a great many genes during their differentiation into the formative state, including several hundred naïve-like genes enriched in lipid metabolic process and gene expression regulation that may form the naïve regulation networks. Knockdown expression of the selected genes of naïve regulation networks partially rescued the differentiation defects of Cnot8 KO ESCs. Cnot8 depletion led to the deadenylation defects of its targets, increasing their poly(A) tail lengths and half-life, eventually elevating their expression levels. We further found that Cnot8 was involved in the clearance of targets through its deadenylase activity and the binding of Ccr4-Not complex, as well as the interacting with Tob1 and Pabpc1. Our results suggest that Cnot8 eliminates naïve regulation networks through mRNA clearance, and is essential for naïve-to-formative pluripotency transition.
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Affiliation(s)
- Yujun Quan
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Meijiao Wang
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing 100101, China
| | - Chengpeng Xu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiaoxiao Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Yu Wu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Dandan Qin
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Yuxuan Lin
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Xukun Lu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Falong Lu
- State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Lei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Stem Cell and Regeneration, Beijing Institute of Stem Cell and Regenerative Medicine, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
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24
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Olechnowicz A, Oleksiewicz U, Machnik M. KRAB-ZFPs and cancer stem cells identity. Genes Dis 2022. [PMID: 37492743 PMCID: PMC10363567 DOI: 10.1016/j.gendis.2022.03.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
Studies on carcinogenesis continue to provide new information about different disease-related processes. Among others, much research has focused on the involvement of cancer stem cells (CSCs) in tumor initiation and progression. Studying the similarities and differences between CSCs and physiological stem cells (SCs) allows for a better understanding of cancer biology. Recently, it was shown that stem cell identity is partially governed by the Krϋppel-associated box domain zinc finger proteins (KRAB-ZFPs), the biggest family of transcription regulators. Several KRAB-ZFP factors exert a known effect in tumor cells, acting as tumor suppressor genes (TSGs) or oncogenes, yet their role in CSCs is still poorly characterized. Here, we review recent studies regarding the influence of KRAB-ZFPs and their cofactor protein TRIM28 on CSCs phenotype, stemness features, migration and invasion potential, metastasis, and expression of parental markers.
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25
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Moon HJ, Lee NY, Do EK, Lee SY, Park GT, Lim JK, Seo JK, Kim JH. Kap1 Regulates the Stability of Lin28A in Embryonic Stem Cells. Stem Cells 2022; 40:385-396. [PMID: 35262736 DOI: 10.1093/stmcls/sxac010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Accepted: 01/24/2022] [Indexed: 11/14/2022]
Abstract
Lin28A is an RNA-binding protein that controls mammalian development and maintenance of the pluripotency of embryonic stem cells (ESCs) via regulating the processing of the microRNA let-7. Lin28A is highly expressed in ESCs, and ectopic expression of this protein facilitates reprogramming of somatic cells to induced pluripotent stem cells. However, the mechanisms underlying the post-translational regulation of Lin28A protein stability in ESCs remain unclear. In the present study, we identified Kap1 (KRAB-associated protein 1) as a novel Lin28A-binding protein using affinity purification and mass spectrometry. Kap1 specifically interacted with the N-terminal region of Lin28A through its coiled-coil domain. Kap1 overexpression significantly attenuated Lin28A ubiquitination and increased its stability. However, small interfering RNA-mediated knockdown of Kap1 promoted the ubiquitination of Lin28A, leading to its proteasomal degradation. Trim71, an E3 ubiquitin ligase, induced Lin28A degradation and Kap1 knockdown accelerated the Trim71-dependent degradation of Lin28A. Mutation of the lysine 177 residue of Lin28A to arginine abrogated the ubiquitination and degradation of Lin28A which were accelerated by Kap1 silencing. Moreover, Kap1 overexpression led to the accumulation of Lin28A in the cytoplasm, but not in the nucleus, and reduced the levels of let-7 subtypes. These results suggest that Kap1 plays a key role in regulation of the stability of Lin28A by modulating the Trim71-mediated ubiquitination and subsequent degradation of Lin28A, thus playing a pivotal role in the regulation of ESC self-renewal and pluripotency.
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Affiliation(s)
- Hye Ji Moon
- Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan, Republic of Korea.,Department of Physiology, School of Medicine, Pusan National University, Yangsan, Republic of Korea
| | - Na Yeon Lee
- Department of Physiology, School of Medicine, Pusan National University, Yangsan, Republic of Korea
| | - Eun Kyoung Do
- Department of Physiology, School of Medicine, Pusan National University, Yangsan, Republic of Korea
| | - Seo Yul Lee
- Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan, Republic of Korea.,Department of Physiology, School of Medicine, Pusan National University, Yangsan, Republic of Korea
| | - Gyu Tae Park
- Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan, Republic of Korea.,Department of Physiology, School of Medicine, Pusan National University, Yangsan, Republic of Korea
| | - Jae Kyong Lim
- Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan, Republic of Korea.,Department of Physiology, School of Medicine, Pusan National University, Yangsan, Republic of Korea
| | - Jeong Kon Seo
- School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea
| | - Jae Ho Kim
- Gene & Cell Therapy Research Center for Vessel-associated Diseases, Pusan National University, Yangsan, Republic of Korea.,Department of Physiology, School of Medicine, Pusan National University, Yangsan, Republic of Korea
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26
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Randolph K, Hyder U, D’Orso I. KAP1/TRIM28: Transcriptional Activator and/or Repressor of Viral and Cellular Programs? Front Cell Infect Microbiol 2022; 12:834636. [PMID: 35281453 PMCID: PMC8904932 DOI: 10.3389/fcimb.2022.834636] [Citation(s) in RCA: 40] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2021] [Accepted: 02/03/2022] [Indexed: 01/01/2023] Open
Abstract
Several transcriptional and epigenetic regulators have been functionally linked to the control of viral and cellular gene expression programs. One such regulator is Krüppel-associated box (KRAB)-associated protein 1 (KAP1: also named TRIM28 or TIF1β), which has been extensively studied in the past three decades. Here we offer an up-to date review of its various functions in a diversity of contexts. We first summarize the discovery of KAP1 repression of endogenous retroviruses during development. We then deliberate evidence in the literature suggesting KAP1 is both an activator and repressor of HIV-1 transcription and discuss experimental differences and limitations of previous studies. Finally, we discuss KAP1 regulation of DNA and RNA viruses, and then expand on KAP1 control of cellular responses and immune functions. While KAP1 positive and negative regulation of viral and cellular transcriptional programs is vastly documented, our mechanistic understanding remains narrow. We thus propose that precision genetic tools to reveal direct KAP1 functions in gene regulation will be required to not only illuminate new biology but also provide the foundation to translate the basic discoveries from the bench to the clinics.
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27
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He QL, Yuan P, Yang L, Yan ZQ, Chen W, Chen YD, Kong SM, Tang FC, Qiao J, Yan LY. Single-cell RNA sequencing reveals abnormal fluctuations in human eight-cell embryos associated with blastocyst formation failure. Mol Hum Reprod 2022; 28:6460826. [PMID: 34904654 DOI: 10.1093/molehr/gaab069] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2021] [Revised: 12/01/2021] [Accepted: 12/09/2021] [Indexed: 12/22/2022] Open
Abstract
Infertility has become a global health issue, with the number of people suffering from the disease increasing year by year, and ART offering great promise for infertility treatment. However, the regulation of early embryonic development is complicated and a series of processes takes place, including the maternal-to-zygotic transition. In addition, developmental arrest is frequently observed during human early embryonic development. In this study, we performed single-cell RNA sequencing on a biopsied blastomere from human eight-cell embryos and tracked the developmental potential of the remaining cells. To compare the sequencing results between different eight-cell embryos, we have combined the research data of this project with the data previously shared in the database and found that cells from the same embryo showed a higher correlation. Additionally, the transcriptome of embryos with blastocyst formation failure was significantly different from developed embryos, and the gene expression as well as cell signaling pathways related to embryonic development were also altered. In particular, the expression of some maternal and zygotic genes in the failed blastocyst formation group was significantly altered: the overall expression level of maternal genes was significantly higher in the failed blastocyst than the developed blastocyst group. In general, these findings provide clues for the causes of human embryonic arrest after the eight-cell stage, and they also provide new ideas for improving the success rate of ART in clinical practice.
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Affiliation(s)
- Qi-Long He
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.,Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
| | - Peng Yuan
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.,Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
| | - Lu Yang
- Beijing Advanced Innovation Center for Genomics, Peking University, Beijing, China.,Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Zhi-Qiang Yan
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.,Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
| | - Wei Chen
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.,Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China.,Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Yi-Dong Chen
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.,Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China
| | - Si-Ming Kong
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.,Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China.,Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Fu-Chou Tang
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China.,Beijing Advanced Innovation Center for Genomics, Peking University, Beijing, China.,Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Jie Qiao
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.,Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China.,Beijing Advanced Innovation Center for Genomics, Peking University, Beijing, China.,Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
| | - Li-Ying Yan
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing, China.,National Clinical Research Center for Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China.,Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China.,Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China.,Beijing Advanced Innovation Center for Genomics, Peking University, Beijing, China.,Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
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28
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Yoshizawa-Sugata N, Yamazaki S, Mita-Yoshida K, Ono T, Nishito Y, Masai H. Loss of full-length DNA replication regulator Rif1 in two-cell embryos is associated with zygotic transcriptional activation. J Biol Chem 2021; 297:101367. [PMID: 34736895 PMCID: PMC8686075 DOI: 10.1016/j.jbc.2021.101367] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Revised: 10/17/2021] [Accepted: 10/20/2021] [Indexed: 11/21/2022] Open
Abstract
Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of two-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing, whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2C embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction and are likely nonfunctional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides may be involved in 2C-specific transcription program.
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Key Words
- 2c, two-cell (embryo)
- 4-oht, 4-hydroxytamoxifen
- dox, doxycycline
- erv, endogenous retrovirus
- es, embryonic stem
- hpf, hours post fertilization
- idr, intrinsic disordered region
- ivf, in vitro fertilization
- kd, knockdown
- ko, knockout
- rt, room temperature
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Affiliation(s)
| | - Satoshi Yamazaki
- Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Kaoru Mita-Yoshida
- Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Tomio Ono
- Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Yasumasa Nishito
- Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
| | - Hisao Masai
- Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
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29
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Sarkar M, Martufi M, Roman-Trufero M, Wang YF, Whilding C, Dormann D, Sabbattini P, Dillon N. CNOT3 interacts with the Aurora B and MAPK/ERK kinases to promote survival of differentiating mesendodermal progenitor cells. Mol Biol Cell 2021; 32:ar40. [PMID: 34613789 PMCID: PMC8694085 DOI: 10.1091/mbc.e21-02-0089] [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: 02/26/2021] [Revised: 09/23/2021] [Accepted: 10/01/2021] [Indexed: 11/18/2022] Open
Abstract
Mesendoderm cells are key intermediate progenitors that form at the early primitive streak (PrS) and give rise to mesoderm and endoderm in the gastrulating embryo. We have identified an interaction between CNOT3 and the cell cycle kinase Aurora B that requires sequences in the NOT box domain of CNOT3 and regulates MAPK/ERK signaling during mesendoderm differentiation. Aurora B phosphorylates CNOT3 at two sites located close to a nuclear localization signal and promotes localization of CNOT3 to the nuclei of mouse embryonic stem cells (ESCs) and metastatic lung cancer cells. ESCs that have both sites mutated give rise to embryoid bodies that are largely devoid of mesoderm and endoderm and are composed mainly of cells with ectodermal characteristics. The mutant ESCs are also compromised in their ability to differentiate into mesendoderm in response to FGF2, BMP4, and Wnt3 due to reduced survival and proliferation of differentiating mesendoderm cells. We also show that the double mutation alters the balance of interaction of CNOT3 with Aurora B and with ERK and reduces phosphorylation of ERK in response to FGF2. Our results identify a potential adaptor function for CNOT3 that regulates the Ras/MEK/ERK pathway during embryogenesis.
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Affiliation(s)
- Moumita Sarkar
- Gene Regulation and Chromatin Group, Imperial College London, London W12 0NN, UK
| | - Matteo Martufi
- Gene Regulation and Chromatin Group, Imperial College London, London W12 0NN, UK
| | - Monica Roman-Trufero
- Gene Regulation and Chromatin Group, Imperial College London, London W12 0NN, UK
| | - Yi-Fang Wang
- Bioinformatics and Computing, Imperial College London, London W12 0NN, UK
| | - Chad Whilding
- Microscopy Facility, MRC London Institute of Medical Sciences, Imperial College London, London W12 0NN, UK
| | - Dirk Dormann
- Microscopy Facility, MRC London Institute of Medical Sciences, Imperial College London, London W12 0NN, UK
| | | | - Niall Dillon
- Gene Regulation and Chromatin Group, Imperial College London, London W12 0NN, UK
- Institute of Clinical Sciences, Imperial College London, London W12 0NN, UK
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30
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Naxerova K, Di Stefano B, Makofske JL, Watson EV, de Kort MA, Martin TD, Dezfulian M, Ricken D, Wooten EC, Kuroda MI, Hochedlinger K, Elledge SJ. Integrated loss- and gain-of-function screens define a core network governing human embryonic stem cell behavior. Genes Dev 2021; 35:1527-1547. [PMID: 34711655 PMCID: PMC8559676 DOI: 10.1101/gad.349048.121] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2021] [Accepted: 09/22/2021] [Indexed: 12/13/2022]
Abstract
In this Resource/Methodology, Naxerova et al. describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. They identify a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance, and their results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks. Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.
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Affiliation(s)
- Kamila Naxerova
- Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.,Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.,Center for Systems Biology, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA
| | - Bruno Di Stefano
- Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA
| | - Jessica L Makofske
- Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA
| | - Emma V Watson
- Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.,Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Marit A de Kort
- Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA
| | - Timothy D Martin
- Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.,Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Mohammed Dezfulian
- Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.,Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Dominik Ricken
- Center for Systems Biology, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA
| | - Eric C Wooten
- Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.,Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Mitzi I Kuroda
- Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA
| | - Konrad Hochedlinger
- Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA
| | - Stephen J Elledge
- Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.,Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA
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31
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Transposable Element Dynamics and Regulation during Zygotic Genome Activation in Mammalian Embryos and Embryonic Stem Cell Model Systems. Stem Cells Int 2021; 2021:1624669. [PMID: 34691189 PMCID: PMC8536462 DOI: 10.1155/2021/1624669] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2021] [Revised: 08/31/2021] [Accepted: 09/08/2021] [Indexed: 12/25/2022] Open
Abstract
Transposable elements (TEs) are mobile genetic sequences capable of duplicating and reintegrating at new regions within the genome. A growing body of evidence has demonstrated that these elements play important roles in host genome evolution, despite being traditionally viewed as parasitic elements. To prevent ectopic activation of TE transposition and transcription, they are epigenetically silenced in most somatic tissues. Intriguingly, a specific class of TEs-retrotransposons-is transiently expressed at discrete phases during mammalian development and has been linked to the establishment of totipotency during zygotic genome activation (ZGA). While mechanisms controlling TE regulation in somatic tissues have been extensively studied, the significance underlying the unique transcriptional reactivation of retrotransposons during ZGA is only beginning to be uncovered. In this review, we summarize the expression dynamics of key retrotransposons during ZGA, focusing on findings from in vivo totipotent embryos and in vitro totipotent-like embryonic stem cells (ESCs). We then dissect the functions of retrotransposons and discuss how their transcriptional activities are finetuned during early stages of mammalian development.
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32
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Vojtek M, Chambers I. Loss of Resf1 reduces the efficiency of embryonic stem cell self-renewal and germline entry. Life Sci Alliance 2021; 4:4/12/e202101190. [PMID: 34607919 PMCID: PMC8500223 DOI: 10.26508/lsa.202101190] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 09/23/2021] [Accepted: 09/23/2021] [Indexed: 11/25/2022] Open
Abstract
RESF1 supports ESC self-renewal by raising expression of transmembrane LIF receptor and key pluripotency transcription factors and increases in vitro primordial germ cell differentiation efficiency. Retroelement silencing factor 1 (RESF1) interacts with the key regulators of mouse embryonic stem cells (ESCs) OCT4 and NANOG, and its absence results in sterility of mice. However, the function of RESF1 in ESCs and germline specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESC self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential, whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces the efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification.
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Affiliation(s)
- Matúš Vojtek
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, Scotland.,Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland
| | - Ian Chambers
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, Scotland .,Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland
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33
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Zeng P, Tang X, Wu T, Tian Q, Li M, Ding J. [Identification of potential regulatory genes for embryonic stem cell self-renewal and pluripotency by random forest]. NAN FANG YI KE DA XUE XUE BAO = JOURNAL OF SOUTHERN MEDICAL UNIVERSITY 2021; 41:1234-1238. [PMID: 34549716 DOI: 10.12122/j.issn.1673-4254.2021.08.16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
OBJECTIVE To identify novel genes associated with self-renewal and pluripotency of mouse embryonic stem cells(mESCs)by integrating multiomics data based on machine learning methods. METHODS We integrated multiomics information of mESCs involving transcriptome, histone modifications, chromatin accessibility, transcription factor binding and architectural protein binding, and compared the signal differences between known stem cell self-renewal and pluripotency genes and other genes.By integrating these multiomics data, we established prediction models based on several machine learning classifiers including random forests and performed 5-fold cross validations.The model was trained using the training dataset containing two thirds of the input samples, and the remaining one third of the input samples were used as the test dataset to assess the performance of the model in independent tests.Finally, the results predicted by the model were validated through gene function annotation and cell function experiments including cell viability assay, colony formation assay and cell cycle analysis. RESULTS Compared with the random genes, the genes known to be associated with self-renewal and pluripotency of mESCs in the multiomics data showed significantly different features.Random forest outperformed the other machine learning algorithms tested on these multiomics data, with an area under the curve (AUC) of 0.883±0.018 for cross validation and an AUC of 0.880±0.028 for independent test.Based on this model, we identified 893 potential regulatory genes associated wwith self-renewal and pluripotency of mESCs, which were similar to the known genes in functional annotation.Known-down of the predicted novel regulator gene Cct6a resulted in significant decreases in the cell viability of mESCs (P < 0.0001) and the number of cell clones (P < 0.01), significantly increased the number of cells in G1 phase (P < 0.01) and decreasedthe number of S phase cells (P < 0.05).Knockdown of Cct6a also led to failure of positive alkaline phosphatase staining of the mESCs. CONCLUSION Machine learning model based on multiomics data can be used to predict potential self-renewal and pluripotency regulators with high performance.By using this model, we predicted potential self-renewal and pluripotency regulatory genes including Cct6a and applied experimental validation.This model provides new insights into the regulatory mechanism of mESCs and contribute to stem cell research.
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Affiliation(s)
- P Zeng
- School of Basic Medical Science, Southern Medical University, Guangzhou 510515, China
| | - X Tang
- Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
| | - T Wu
- Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
| | - Q Tian
- Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
| | - M Li
- School of Basic Medical Science, Southern Medical University, Guangzhou 510515, China
| | - J Ding
- Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
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34
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Qin W, Ugur E, Mulholland CB, Bultmann S, Solovei I, Modic M, Smets M, Wierer M, Forné I, Imhof A, Cardoso MC, Leonhardt H. Phosphorylation of the HP1β hinge region sequesters KAP1 in heterochromatin and promotes the exit from naïve pluripotency. Nucleic Acids Res 2021; 49:7406-7423. [PMID: 34214177 PMCID: PMC8287961 DOI: 10.1093/nar/gkab548] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Revised: 05/31/2021] [Accepted: 06/11/2021] [Indexed: 12/26/2022] Open
Abstract
Heterochromatin binding protein HP1β plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1β−/− embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1β is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1β−/− and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1−/− cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.
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Affiliation(s)
- Weihua Qin
- Faculty of Biology, Ludwig-Maximilians-Universität München, Butenandtstraße 1, D-81377 Munich, Germany
| | - Enes Ugur
- Faculty of Biology, Ludwig-Maximilians-Universität München, Butenandtstraße 1, D-81377 Munich, Germany.,Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
| | - Christopher B Mulholland
- Faculty of Biology, Ludwig-Maximilians-Universität München, Butenandtstraße 1, D-81377 Munich, Germany
| | - Sebastian Bultmann
- Faculty of Biology, Ludwig-Maximilians-Universität München, Butenandtstraße 1, D-81377 Munich, Germany
| | - Irina Solovei
- Faculty of Biology, Ludwig-Maximilians-Universität München, Butenandtstraße 1, D-81377 Munich, Germany
| | - Miha Modic
- The Francis Crick Institute and UCL Queen Square Institute of Neurology, London NW1 1AT, United Kingdom
| | - Martha Smets
- Faculty of Biology, Ludwig-Maximilians-Universität München, Butenandtstraße 1, D-81377 Munich, Germany
| | - Michael Wierer
- Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
| | - Ignasi Forné
- Biomedical Center Munich, Faculty of Medicine, Ludwig-Maximilians-Universität München, Großhaderner Str. 9, 82152 Planegg-Martinsried, Germany
| | - Axel Imhof
- Biomedical Center Munich, Faculty of Medicine, Ludwig-Maximilians-Universität München, Großhaderner Str. 9, 82152 Planegg-Martinsried, Germany
| | - M Cristina Cardoso
- Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
| | - Heinrich Leonhardt
- Faculty of Biology, Ludwig-Maximilians-Universität München, Butenandtstraße 1, D-81377 Munich, Germany
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35
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Wang C, Songyang Z, Huang Y. TRIM28 inhibits alternative lengthening of telomere phenotypes by protecting SETDB1 from degradation. Cell Biosci 2021; 11:149. [PMID: 34330324 PMCID: PMC8325274 DOI: 10.1186/s13578-021-00660-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2021] [Accepted: 07/15/2021] [Indexed: 01/04/2023] Open
Abstract
Background About 10–15% of tumor cells extend telomeres through the alternative lengthening of telomeres (ALT) mechanism, which is a recombination-dependent replication pathway. It is generally believed that ALT cells are related to the chromatin modification of telomeres. However, the mechanism of ALT needs to be further explored. Results Here we found that TRIM28/KAP1 is preferentially located on the telomeres of ALT cells and interacts with telomeric shelterin/telosome complex. Knocking down TRIM28 in ALT cells delayed cell growth, decreased the level of C-circle which is one kind of extrachromosomal circular telomeric DNA, increased the frequency of ALT-associated promyelocytic leukemia bodies (APBs), led to telomere prolongation and increased the telomere sister chromatid exchange in ALT cells. Mechanistically, TRIM28 protects telomere histone methyltransferase SETDB1 from degradation, thus maintaining the H3K9me3 heterochromatin state of telomere DNA. Conclusions Our work provides a model that TRIM28 inhibits alternative lengthening of telomere phenotypes by protecting SETDB1 from degradation. In general, our results reveal the mechanism of telomere heterochromatin maintenance and its effect on ALT, and TRIM28 may serve as a target for the treatment of ALT tumor cells. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-021-00660-y.
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Affiliation(s)
- Chuanle Wang
- MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research and SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, China
| | - Zhou Songyang
- MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research and SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, China.,Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 510120, China.,Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030, USA.,Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, 510005, China
| | - Yan Huang
- MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research and SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, China.
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36
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Disruption of RING and PHD Domains of TRIM28 Evokes Differentiation in Human iPSCs. Cells 2021; 10:cells10081933. [PMID: 34440702 PMCID: PMC8394524 DOI: 10.3390/cells10081933] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2021] [Revised: 07/18/2021] [Accepted: 07/26/2021] [Indexed: 12/31/2022] Open
Abstract
TRIM28, a multi-domain protein, is crucial in the development of mouse embryos and the maintenance of embryonic stem cells’ (ESC) self-renewal potential. As the epigenetic factor modulating chromatin structure, TRIM28 regulates the expression of numerous genes and is associated with progression and poor prognosis in many types of cancer. Because of many similarities between highly dedifferentiated cancer cells and normal pluripotent stem cells, we applied human induced pluripotent stem cells (hiPSC) as a model for stemness studies. For the first time in hiPSC, we analyzed the function of individual TRIM28 domains. Here we demonstrate the essential role of a really interesting new gene (RING) domain and plant homeodomain (PHD) in regulating pluripotency maintenance and self-renewal capacity of hiPSC. Our data indicate that mutation within the RING or PHD domain leads to the loss of stem cell phenotypes and downregulation of the FGF signaling. Moreover, impairment of RING or PHD domain results in decreased proliferation and impedes embryoid body formation. In opposition to previous data indicating the impact of phosphorylation on TRIM28 function, our data suggest that TRIM28 phosphorylation does not significantly affect the pluripotency and self-renewal maintenance of hiPSC. Of note, iPSC with disrupted RING and PHD functions display downregulation of genes associated with tumor metastasis, which are considered important targets in cancer treatment. Our data suggest the potential use of RING and PHD domains of TRIM28 as targets in cancer therapy.
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37
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Li B, Qiao C, Jin X, Chan HM. Characterizing the Low-Dose Effects of Methylmercury on the Early Stages of Embryo Development Using Cultured Human Embryonic Stem Cells. ENVIRONMENTAL HEALTH PERSPECTIVES 2021; 129:77007. [PMID: 34328791 PMCID: PMC8323991 DOI: 10.1289/ehp7349] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Revised: 06/18/2021] [Accepted: 07/02/2021] [Indexed: 06/13/2023]
Abstract
BACKGROUND Global concerns of methylmercury (MeHg) exposure have been raised, especially on its effects on pregnant women. Recent epidemiological studies have revealed associations between maternal blood/hair MeHg concentrations, adverse pregnancy outcomes, and developmental deficits. However, the underlying mechanisms remain unclear. OBJECTIVES In this study, we characterized the effects of MeHg exposure on undifferentiated human embryonic stem cells (hESCs) and extrapolated the effects to human embryonic development. METHODS hESCs were exposed to 0, 1, 5, 10, 50, 100 or 200nM MeHg for 24 h or 6 d. Cell adherence and colony formation and expansion were examined under the microscope. Cell attachment, viability/proliferation, apoptosis, stress response, cell cycle, autophagy, and expression of cell lineage marker genes and proteins were measured at the end of exposures. RESULTS Our results indicated that exposure to nanomolar concentrations of MeHg was associated with a) higher levels of reactive oxygen species (ROS) and hemeoxygenase-1 (HO-1), suggesting increased stress and adaptive responses; b) lower cellular adhesion, viability/proliferation, and colony formation and expansion; c) higher levels of apoptosis, reflected by higher cleaved caspase-3 expression and Annexin V binding; d) higher levels of cytoskeleton protein α-tubulin expression; e) higher rates of G1/S phase cell cycle arrest; and f) autophagy inhibition, as shown by a lower LC3BII/LC3BI ratio and accumulation of SQSTM1 (p62). These outcomes were accompanied by higher expressions of self-renewal genes or proteins or both, including OCT4, SOX2, NANOG, and cytokine receptor IL6ST, as well as pluripotency and the cell fate regulator cyclin D1. DISCUSSION These results revealed that under the selection pressure of exposure to low doses of MeHg, some hESCs underwent apoptosis, whereas others adapted and survived with enhanced self-renewal gene expression and specific morphological phenotypes. Findings from the present study provide in vitro evidence that low doses of MeHg adversely affect hESCs when exposed during a period of time that models embryonic pre-, during, and early postimplantation stages. https://doi.org/10.1289/EHP7349.
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Affiliation(s)
- Bai Li
- Department of Biology, University of Ottawa, Ottawa, Ontario, Canada
| | - Cunye Qiao
- Biostatistics and Modeling Division, Bureau of Food Surveillance and Science Integration, Food Directorate, Health Products and Food Branch (HPFB), Health Canada, Ottawa, Ontario, Canada
| | - Xiaolei Jin
- Regulatory Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, HPFB, Health Canada, Ottawa, Ontario, Canada
| | - Hing Man Chan
- Department of Biology, University of Ottawa, Ottawa, Ontario, Canada
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38
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Phase separation of OCT4 controls TAD reorganization to promote cell fate transitions. Cell Stem Cell 2021; 28:1868-1883.e11. [PMID: 34038708 DOI: 10.1016/j.stem.2021.04.023] [Citation(s) in RCA: 88] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2019] [Revised: 08/06/2020] [Accepted: 04/19/2021] [Indexed: 02/05/2023]
Abstract
Topological-associated domains (TADs) are thought to be relatively stable across cell types, although some TAD reorganization has been observed during cellular differentiation. However, little is known about the mechanisms through which TAD reorganization affects cell fate or how master transcription factors affect TAD structures during cell fate transitions. Here, we show extensive TAD reorganization during somatic cell reprogramming, which is correlated with gene transcription and changes in cellular identity. Manipulating TAD reorganization promotes reprogramming, and the dynamics of concentrated chromatin loops in OCT4 phase separated condensates contribute to TAD reorganization. Disrupting OCT4 phase separation attenuates TAD reorganization and reprogramming, which can be rescued by fusing an intrinsically disordered region (IDR) to OCT4. We developed an approach termed TAD reorganization-based multiomics analysis (TADMAN), which identified reprogramming regulators. Together, these findings elucidate a role and mechanism of TAD reorganization, regulated by OCT4 phase separation, in cellular reprogramming.
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Akinci E, Hamilton MC, Khowpinitchai B, Sherwood RI. Using CRISPR to understand and manipulate gene regulation. Development 2021; 148:dev182667. [PMID: 33913466 PMCID: PMC8126405 DOI: 10.1242/dev.182667] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Understanding how genes are expressed in the correct cell types and at the correct level is a key goal of developmental biology research. Gene regulation has traditionally been approached largely through observational methods, whereas perturbational approaches have lacked precision. CRISPR-Cas9 has begun to transform the study of gene regulation, allowing for precise manipulation of genomic sequences, epigenetic functionalization and gene expression. CRISPR-Cas9 technology has already led to the discovery of new paradigms in gene regulation and, as new CRISPR-based tools and methods continue to be developed, promises to transform our knowledge of the gene regulatory code and our ability to manipulate cell fate. Here, we discuss the current and future application of the emerging CRISPR toolbox toward predicting gene regulatory network behavior, improving stem cell disease modeling, dissecting the epigenetic code, reprogramming cell fate and treating diseases of gene dysregulation.
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Affiliation(s)
- Ersin Akinci
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
- Department of Agricultural Biotechnology, Faculty of Agriculture, Akdeniz University, Antalya, 07070, Turkey
| | - Marisa C. Hamilton
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
| | - Benyapa Khowpinitchai
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
| | - Richard I. Sherwood
- Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA
- Hubrecht Institute, 3584 CT, Utrecht, The Netherlands
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Terzi Cizmecioglu N, Huang J, Keskin EG, Wang X, Esen I, Chen F, Orkin SH. ARID4B is critical for mouse embryonic stem cell differentiation towards mesoderm and endoderm, linking epigenetics to pluripotency exit. J Biol Chem 2021; 295:17738-17751. [PMID: 33454011 DOI: 10.1074/jbc.ra120.015534] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2020] [Revised: 10/13/2020] [Indexed: 11/06/2022] Open
Abstract
Distinct cell types emerge from embryonic stem cells through a precise and coordinated execution of gene expression programs during lineage commitment. This is established by the action of lineage specific transcription factors along with chromatin complexes. Numerous studies have focused on epigenetic factors that affect embryonic stem cells (ESC) self-renewal and pluripotency. However, the contribution of chromatin to lineage decisions at the exit from pluripotency has not been as extensively studied. Using a pooled epigenetic shRNA screen strategy, we identified chromatin-related factors critical for differentiation toward mesodermal and endodermal lineages. Here we reveal a critical role for the chromatin protein, ARID4B. Arid4b-deficient mESCs are similar to WT mESCs in the expression of pluripotency factors and their self-renewal. However, ARID4B loss results in defects in up-regulation of the meso/endodermal gene expression program. It was previously shown that Arid4b resides in a complex with SIN3A and HDACS 1 and 2. We identified a physical and functional interaction of ARID4B with HDAC1 rather than HDAC2, suggesting functionally distinct Sin3a subcomplexes might regulate cell fate decisions Finally, we observed that ARID4B deficiency leads to increased H3K27me3 and a reduced H3K27Ac level in key developmental gene loci, whereas a subset of genomic regions gain H3K27Ac marks. Our results demonstrate that epigenetic control through ARID4B plays a key role in the execution of lineage-specific gene expression programs at pluripotency exit.
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Affiliation(s)
- Nihal Terzi Cizmecioglu
- Department of Biological Sciences, Faculty of Arts and Sciences, Middle East Technical University, Ankara, Turkey.
| | - Jialiang Huang
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian China
| | - Ezgi G Keskin
- Department of Biological Sciences, Faculty of Arts and Sciences, Middle East Technical University, Ankara, Turkey
| | - Xiaofeng Wang
- Geisel School of Medicine, Dartmouth University, Hanover, New Hampshire USA
| | - Idil Esen
- Howard Hughes Medical Institute, Dana Farber/Boston Children's Cancer and Blood Disorders Center, Dept. of Pediatrics, Harvard Medical School, Boston, Massachusetts USA
| | - Fei Chen
- State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian China
| | - Stuart H Orkin
- Howard Hughes Medical Institute, Dana Farber/Boston Children's Cancer and Blood Disorders Center, Dept. of Pediatrics, Harvard Medical School, Boston, Massachusetts USA.
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Sohn EJ, Moon HJ, Lim JK, Kim DS, Kim JH. Regulation of the protein stability and transcriptional activity of OCT4 in stem cells. Adv Biol Regul 2020; 79:100777. [PMID: 33451972 DOI: 10.1016/j.jbior.2020.100777] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 12/15/2020] [Accepted: 12/21/2020] [Indexed: 12/31/2022]
Abstract
OCT4 (also known as Oct3 and Oct3/4), which is encoded by Pou5f1, is expressed in early embryonic cells and plays an important role in early development, pluripotency maintenance, and self-renewal of embryonic stem cells. It also regulates the reprogramming of somatic cells into induced pluripotent stem cells. Several OCT4-binding proteins, including SOX2 and NANOG, reportedly regulate gene transcription in stem cells. An increasing number of evidence suggests that not only gene transcription but also post-translational modifications of OCT4 play a pivotal role in regulating the expression and activity of OCT4. For instance, ubiquitination and sumoylation have been reported to regulate OCT4 protein stability. In addition, the phosphorylation of Ser347 in OCT4 also stabilizes the OCT4 protein level. Recently, we identified KAP1 as an OCT4-binding protein and reported the KAP1-mediated regulation of OCT4 protein stability. KAP1 overexpression led to an increased proliferation of mouse embryonic stem cells and promoted the reprogramming of somatic cells resulting in induced pluripotent stem cells. In this review, we discuss how the protein stability and function of OCT4 are regulated by protein-protein interaction in stem cells.
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Affiliation(s)
- Eun Jung Sohn
- Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan, 50612, Republic of Korea; Department of Physiology, School of Medicine, Pusan National University, Yangsan, 50612, Republic of Korea
| | - Hye Ji Moon
- Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan, 50612, Republic of Korea; Department of Physiology, School of Medicine, Pusan National University, Yangsan, 50612, Republic of Korea
| | - Jae Kyong Lim
- Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan, 50612, Republic of Korea; Department of Physiology, School of Medicine, Pusan National University, Yangsan, 50612, Republic of Korea
| | - Da Sol Kim
- Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan, 50612, Republic of Korea; Department of Physiology, School of Medicine, Pusan National University, Yangsan, 50612, Republic of Korea
| | - Jae Ho Kim
- Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Yangsan, 50612, Republic of Korea.
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No Easy Way Out for EZH2: Its Pleiotropic, Noncanonical Effects on Gene Regulation and Cellular Function. Int J Mol Sci 2020; 21:ijms21249501. [PMID: 33327550 PMCID: PMC7765048 DOI: 10.3390/ijms21249501] [Citation(s) in RCA: 58] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2020] [Revised: 12/04/2020] [Accepted: 12/08/2020] [Indexed: 12/14/2022] Open
Abstract
Enhancer of zeste homolog 2 (EZH2) plays critical roles in a range of biological processes including organ development and homeostasis, epigenomic and transcriptomic regulation, gene repression and imprinting, and DNA damage repair. A widely known function of EZH2 is to serve as an enzymatic subunit of Polycomb repressive complex 2 (PRC2) and catalyze trimethylation of histone H3 lysine 27 (H3K27me3) for repressing target gene expression. However, an increasing body of evidence demonstrates that EZH2 has many "non-conventional" functions that go beyond H3K27 methylation as a Polycomb factor. First, EZH2 can methylate a number of nonhistone proteins, thereby regulating cellular processes in an H3K27me3-independent fashion. Furthermore, EZH2 relies on both methyltransferase-dependent and methyltransferase-independent mechanisms for modulating gene-expression programs and/or epigenomic patterns of cells. Importantly, independent of PRC2, EZH2 also forms physical interactions with a number of DNA-binding factors and transcriptional coactivators to context-dependently influence gene expression. The purpose of this review is to detail the complex, noncanonical roles of EZH2, which are generally less appreciated in gene and (epi)genome regulation. Because EZH2 deregulation is prevalent in human diseases such as cancer, there is increased dependency on its noncanonical function, which shall have important implications in developing more effective therapeutics.
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The Regulatory Properties of the Ccr4-Not Complex. Cells 2020; 9:cells9112379. [PMID: 33138308 PMCID: PMC7692201 DOI: 10.3390/cells9112379] [Citation(s) in RCA: 41] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2020] [Revised: 10/21/2020] [Accepted: 10/26/2020] [Indexed: 12/12/2022] Open
Abstract
The mammalian Ccr4–Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. In the nucleus, it is involved in the regulation of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, nuclear RNA surveillance, and DNA damage repair. In the cytoplasm, the Ccr4–Not complex plays a central role in mRNA decay and affects protein quality control. Most of our original knowledge of the Ccr4–Not complex is derived, primarily, from studies in yeast. More recent studies have shown that the mammalian complex has a comparable structure and similar properties. In this review, we summarize the evidence for the multiple roles of both the yeast and mammalian Ccr4–Not complexes, highlighting their similarities.
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Czerwinska P, Jaworska AM, Wlodarczyk NA, Mackiewicz AA. Melanoma Stem Cell-Like Phenotype and Significant Suppression of Immune Response within a Tumor Are Regulated by TRIM28 Protein. Cancers (Basel) 2020; 12:E2998. [PMID: 33076560 PMCID: PMC7650661 DOI: 10.3390/cancers12102998] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2020] [Revised: 10/07/2020] [Accepted: 10/13/2020] [Indexed: 12/20/2022] Open
Abstract
TRIM28 emerged as a guard of the intrinsic "state of cell differentiation", facilitating self-renewal of pluripotent stem cells. Recent reports imply TRIM28 engagement in cancer stem cell (CSC) maintenance, although the exact mechanism remains unresolved. TRIM28 high expression is associated with worse melanoma patient outcomes. Here, we investigated the association between TRIM28 level and melanoma stemness, and aligned it with the antitumor immune response to find the mechanism of "stemness high/immune low" melanoma phenotype acquisition. Based on the SKCM TCGA data, the TRIM28 expression profile, clinicopathological features, expression of correlated genes, and the level of stemness and immune scores were analyzed in patient samples. The biological function for differentially expressed genes was annotated with GSEA. Results were validated with additional datasets from R2: Genomics Analysis and Visualization Platform and in vitro with a panel of seven melanoma cell lines. All statistical analyses were accomplished using GraphPad Prism 8. TRIM28HIGH-expressing melanoma patients are characterized by worse outcomes and significantly different gene expression profiles than the TRIM28NORM cohort. TRIM28 high level related to higher melanoma stemness as measured with several distinct scores and TRIM28HIGH-expressing melanoma cell lines possess the greater potential of melanosphere formation. Moreover, TRIM28HIGH melanoma tumors were significantly depleted with infiltrating immune cells, especially cytotoxic T cells, helper T cells, and B cells. Furthermore, TRIM28 emerged as a good predictor of "stemness high/immune low" melanoma phenotype. Our data indicate that TRIM28 might facilitate this phenotype by direct repression of interferon signaling. TRIM28 emerged as a direct link between stem cell-like phenotype and attenuated antitumor immune response in melanoma, although further studies are needed to evaluate the direct mechanism of TRIM28-mediated stem-like phenotype acquisition.
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Affiliation(s)
- Patrycja Czerwinska
- Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, 15 Garbary St., 61-866 Poznan, Poland; (A.M.J.); (N.A.W.)
- Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, 15 Garbary St., 61-866 Poznan, Poland
| | - Anna Maria Jaworska
- Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, 15 Garbary St., 61-866 Poznan, Poland; (A.M.J.); (N.A.W.)
| | - Nikola Agata Wlodarczyk
- Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, 15 Garbary St., 61-866 Poznan, Poland; (A.M.J.); (N.A.W.)
| | - Andrzej Adam Mackiewicz
- Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, 15 Garbary St., 61-866 Poznan, Poland; (A.M.J.); (N.A.W.)
- Department of Diagnostics and Cancer Immunology, Greater Poland Cancer Centre, 15 Garbary St., 61-866 Poznan, Poland
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Lu HP, Lin CJ, Chen WC, Chang YJ, Lin SW, Wang HH, Chang CJ. TRIM28 Regulates Dlk1 Expression in Adipogenesis. Int J Mol Sci 2020; 21:ijms21197245. [PMID: 33008113 PMCID: PMC7582669 DOI: 10.3390/ijms21197245] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Revised: 09/20/2020] [Accepted: 09/27/2020] [Indexed: 12/23/2022] Open
Abstract
The tripartite motif-containing protein 28 (TRIM28) is a transcription corepressor, interacting with histone deacetylase and methyltransferase complexes. TRIM28 is a crucial regulator in development and differentiation. We would like to investigate its function and regulation in adipogenesis. Knockdown of Trim28 by transducing lentivirus-carrying shRNAs impairs the differentiation of 3T3-L1 preadipocytes, demonstrated by morphological observation and gene expression analysis. To understand the molecular mechanism of Trim28-mediated adipogenesis, the RNA-seq was performed to find out the possible Trim28-regulated genes. Dlk1 (delta-like homolog 1) was increased in Trim28 knockdown 3T3-L1 cells both untreated and induced to differentiation. Dlk1 is an imprinted gene and known as an inhibitor of adipogenesis. Further knockdown of Dlk1 in Trim28 knockdown 3T3-L1 would rescue cell differentiation. The epigenetic analysis showed that DNA methylation of Dlk1 promoter and differentially methylated regions (DMRs) was not altered significantly in Trim28 knockdown cells. However, compared to control cells, the histone methylation on the Dlk1 promoter was increased at H3K4 and decreased at H3K27 in Trim28 knockdown cells. Finally, we found Trim28 might be recruited by transcription factor E2f1 to regulate Dlk1 expression. The results imply Trim28-Dlk1 axis is critical for adipogenesis.
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Affiliation(s)
- Hsin-Pin Lu
- Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 10617, Taiwan; (H.-P.L.); (C.-J.L.); (W.-C.C.)
| | - Chieh-Ju Lin
- Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 10617, Taiwan; (H.-P.L.); (C.-J.L.); (W.-C.C.)
| | - Wen-Ching Chen
- Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 10617, Taiwan; (H.-P.L.); (C.-J.L.); (W.-C.C.)
| | - Yao-Jen Chang
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan; (Y.-J.C.); (S.-W.L.)
| | - Sheng-Wei Lin
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan; (Y.-J.C.); (S.-W.L.)
| | - Hsin-Hui Wang
- Department of Pediatrics, Division of Pediatric Immunology and Nephrology, Taipei Veterans General Hospital, Taipei 11217, Taiwan;
- Department of Pediatrics, Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei 11217, Taiwan
- Institute of Emergency and Critical Care Medicine, School of Medicine, National Yang-Ming University, Taipei 11217, Taiwan
| | - Ching-Jin Chang
- Graduate Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 10617, Taiwan; (H.-P.L.); (C.-J.L.); (W.-C.C.)
- Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan; (Y.-J.C.); (S.-W.L.)
- Correspondence:
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Liu M, Zhu Y, Xing F, Liu S, Xia Y, Jiang Q, Qin J. The polycomb group protein PCGF6 mediates germline gene silencing by recruiting histone-modifying proteins to target gene promoters. J Biol Chem 2020; 295:9712-9724. [PMID: 32482889 PMCID: PMC7363147 DOI: 10.1074/jbc.ra119.012121] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Revised: 05/27/2020] [Indexed: 01/18/2023] Open
Abstract
Polycomb group (PcG) proteins are essential for maintenance of lineage fidelity by coordinating developmental gene expression programs. Polycomb group ring finger 6 (PCGF6) has been previously reported to repress expression of lineage-specific genes, especially germ cell-related genes in mouse embryonic stem cells (ESCs) via the noncanonical polycomb repressive complex PRC1.6. However, the molecular mechanism of this repression remains largely unknown. Here, using RNA-Seq, real-time RT-PCR, immunohistochemistry, immunoprecipitation, and ChIP analyses, we demonstrate that PCGF6 plays an essential role in embryonic development, indicated by the partially penetrant embryonic lethality in homozygous PCGF6 (Pcgf6-/-)-deficient mice. We also found that surviving Pcgf6-deficient mice exhibit reduced fertility. Using the Pcgf6-deficient mice, we observed that ablation of Pcgf6 in somatic tissues robustly derepresses germ cell-related genes. We further provide evidence that these genes are direct targets of PCGF6 in ESCs and that endogenous PCGF6 co-localizes with the histone-modifying proteins G9A histone methyltransferase (G9A)/G9a-like protein (GLP) and histone deacetylase 1/2 (HDAC1/2) on the promoters of the germ cell-related genes. Moreover, the binding of these proteins to their target genes correlated with methylation of Lys-9 of histone 3 and with the status of histone acetylation at these genes. Moreover, the recruitment of G9A/GLP and HDAC1/2 to target promoters depended on the binding of PCGF6. Our findings indicate that PCGF6 has a critical role in safeguarding lineage decisions and in preventing aberrant expression of germ cell-related genes.
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Affiliation(s)
- Mengjie Liu
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, Nanjing University, Nanjing, China
| | - Yaru Zhu
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, Nanjing University, Nanjing, China
| | - Fei Xing
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China
| | - Shuang Liu
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, Nanjing University, Nanjing, China
| | - Yin Xia
- School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, China
| | - Qing Jiang
- Department of Sports Medicine and Adult Reconstructive Surgery, Nanjing Drum Tower Hospital Affiliated to Medical School of Nanjing University, Nanjing, China
| | - Jinzhong Qin
- State Key Laboratory of Pharmaceutical Biotechnology and MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center, Nanjing University, Nanjing, China
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Shanak S, Helms V. DNA methylation and the core pluripotency network. Dev Biol 2020; 464:145-160. [PMID: 32562758 DOI: 10.1016/j.ydbio.2020.06.001] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2020] [Revised: 05/01/2020] [Accepted: 06/04/2020] [Indexed: 01/06/2023]
Abstract
From the onset of fertilization, the genome undergoes cell division and differentiation. All of these developmental transitions and differentiation processes include cell-specific signatures and gradual changes of the epigenome. Understanding what keeps stem cells in the pluripotent state and what leads to differentiation are fascinating and biomedically highly important issues. Numerous studies have identified genes, proteins, microRNAs and small molecules that exert essential effects. Notably, there exists a core pluripotency network that consists of several transcription factors and accessory proteins. Three eminent transcription factors, OCT4, SOX2 and NANOG, serve as hubs in this core pluripotency network. They bind to the enhancer regions of their target genes and modulate, among others, the expression levels of genes that are associated with Gene Ontology terms related to differentiation and self-renewal. Also, much has been learned about the epigenetic rewiring processes during these changes of cell fate. For example, DNA methylation dynamics is pivotal during embryonic development. The main goal of this review is to highlight an intricate interplay of (a) DNA methyltransferases controlling the expression levels of core pluripotency factors by modulation of the DNA methylation levels in their enhancer regions, and of (b) the core pluripotency factors controlling the transcriptional regulation of DNA methyltransferases. We discuss these processes both at the global level and in atomistic detail based on information from structural studies and from computer simulations.
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Affiliation(s)
- Siba Shanak
- Faculty of Science, Arab-American University, Jenin, Palestine; Center for Bioinformatics, Saarland University, Saarbruecken, Germany
| | - Volkhard Helms
- Center for Bioinformatics, Saarland University, Saarbruecken, Germany.
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Justice M, Carico ZM, Stefan HC, Dowen JM. A WIZ/Cohesin/CTCF Complex Anchors DNA Loops to Define Gene Expression and Cell Identity. Cell Rep 2020; 31:107503. [PMID: 32294452 PMCID: PMC7212317 DOI: 10.1016/j.celrep.2020.03.067] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2019] [Revised: 01/14/2020] [Accepted: 03/20/2020] [Indexed: 12/31/2022] Open
Abstract
Chromosome structure is a key regulator of gene expression. CTCF and cohesin play critical roles in structuring chromosomes by mediating physical interactions between distant genomic sites. The resulting DNA loops often contain genes and their cis-regulatory elements. Despite the importance of DNA loops in maintaining proper transcriptional regulation and cell identity, there is limited understanding of the molecular mechanisms that regulate their dynamics and function. We report a previously unrecognized role for WIZ (widely interspaced zinc finger-containing protein) in DNA loop architecture and regulation of gene expression. WIZ forms a complex with cohesin and CTCF that occupies enhancers, promoters, insulators, and anchors of DNA loops. Aberrant WIZ function alters cohesin occupancy and increases the number of DNA loop structures in the genome. WIZ is required for proper gene expression and transcriptional insulation. Our results uncover an unexpected role for WIZ in DNA loop architecture, transcriptional control, and maintenance of cell identity.
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Affiliation(s)
- Megan Justice
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Zachary M Carico
- Cancer Epigenetics Training Program, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Holden C Stefan
- Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Jill M Dowen
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Cancer Epigenetics Training Program, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
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Takahashi A, Suzuki T, Soeda S, Takaoka S, Kobori S, Yamaguchi T, Mohamed HMA, Yanagiya A, Abe T, Shigeta M, Furuta Y, Kuba K, Yamamoto T. The CCR4-NOT complex maintains liver homeostasis through mRNA deadenylation. Life Sci Alliance 2020; 3:3/5/e201900494. [PMID: 32238456 PMCID: PMC7119370 DOI: 10.26508/lsa.201900494] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2019] [Revised: 03/11/2020] [Accepted: 03/14/2020] [Indexed: 12/12/2022] Open
Abstract
The biological significance of deadenylation in global gene expression is not fully understood. Here, we show that the CCR4-NOT deadenylase complex maintains expression of mRNAs, such as those encoding transcription factors, cell cycle regulators, DNA damage response-related proteins, and metabolic enzymes, at appropriate levels in the liver. Liver-specific disruption of Cnot1, encoding a scaffold subunit of the CCR4-NOT complex, leads to increased levels of mRNAs for transcription factors, cell cycle regulators, and DNA damage response-related proteins because of reduced deadenylation and stabilization of these mRNAs. CNOT1 suppression also results in an increase of immature, unspliced mRNAs (pre-mRNAs) for apoptosis-related and inflammation-related genes and promotes RNA polymerase II loading on their promoter regions. In contrast, mRNAs encoding metabolic enzymes become less abundant, concomitant with decreased levels of these pre-mRNAs. Lethal hepatitis develops concomitantly with abnormal mRNA expression. Mechanistically, the CCR4-NOT complex targets and destabilizes mRNAs mainly through its association with Argonaute 2 (AGO2) and butyrate response factor 1 (BRF1) in the liver. Therefore, the CCR4-NOT complex contributes to liver homeostasis by modulating the liver transcriptome through mRNA deadenylation.
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Affiliation(s)
- Akinori Takahashi
- Cell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan
| | - Toru Suzuki
- Laboratory for Immunogenetics, Center for Integrative Medical Sciences, RIKEN, Yokohama City, Kanagawa, Japan
| | - Shou Soeda
- Cell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan
| | - Shohei Takaoka
- Cell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan
| | - Shungo Kobori
- Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan
| | - Tomokazu Yamaguchi
- Department of Biochemistry and Metabolic Science, Graduate School of Medicine, Akita University, Akita, Japan
| | | | - Akiko Yanagiya
- Cell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan
| | - Takaya Abe
- Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
| | - Mayo Shigeta
- Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
| | - Yasuhide Furuta
- Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
| | - Keiji Kuba
- Department of Biochemistry and Metabolic Science, Graduate School of Medicine, Akita University, Akita, Japan
| | - Tadashi Yamamoto
- Cell Signal Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan .,Laboratory for Immunogenetics, Center for Integrative Medical Sciences, RIKEN, Yokohama City, Kanagawa, Japan
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Oh E, Mark KG, Mocciaro A, Watson ER, Prabu JR, Cha DD, Kampmann M, Gamarra N, Zhou CY, Rape M. Gene expression and cell identity controlled by anaphase-promoting complex. Nature 2020; 579:136-140. [PMID: 32076268 PMCID: PMC7402266 DOI: 10.1038/s41586-020-2034-1] [Citation(s) in RCA: 69] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2018] [Accepted: 01/01/2020] [Indexed: 01/08/2023]
Abstract
Metazoan development requires robust proliferation of progenitor cells, whose identities are established by tightly controlled transcriptional networks 1. As gene expression is globally inhibited during mitosis, the transcriptional programs defining cell identity must be restarted in each cell cycle 2-5, yet how this is accomplished is poorly understood. Here, we identified a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters 6,7, recruit the anaphase-promoting complex (APC/C) to specific transcription start sites (TSS) during mitosis. This allows APC/C to decorate histones with K11/K48-branched ubiquitin chains that recruit p97/VCP and the proteasome and ensure rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and transcription re-initiation are thus controlled by the same regulator, APC/C, which provides a robust mechanism to maintain cell identity through cell division.
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Affiliation(s)
- Eugene Oh
- Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, CA, USA.,Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA
| | - Kevin G Mark
- Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, CA, USA.,Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA
| | - Annamaria Mocciaro
- Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, CA, USA.,Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA.,Berkeley Lights, Emeryville, CA, USA
| | - Edmond R Watson
- Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - J Rajan Prabu
- Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany
| | - Denny D Cha
- Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, CA, USA.,Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA
| | - Martin Kampmann
- Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA.,Institute for Neurodegenerative Diseases, University of California at San Francisco, San Francisco, CA, USA.,Chan Zuckerberg Biohub, San Francisco, CA, USA
| | - Nathan Gamarra
- Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA
| | - Coral Y Zhou
- Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA
| | - Michael Rape
- Howard Hughes Medical Institute, University of California at Berkeley, Berkeley, CA, USA. .,Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA.
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