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1
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Liu Y, Zhangding Z, Liu X, Hu J. Chromatin-centric insights into DNA replication. Trends Genet 2025; 41:412-424. [PMID: 39765445 DOI: 10.1016/j.tig.2024.12.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2024] [Revised: 11/28/2024] [Accepted: 12/06/2024] [Indexed: 05/08/2025]
Abstract
DNA replication ensures the precise transmission of genetic information from parent to daughter cells. In eukaryotes, this process involves the replication of every base pair within a highly complex chromatin environment, encompassing multiple levels of chromatin structure and various chromatin metabolic processes. Recent evidence has demonstrated that DNA replication is strictly regulated in both temporal and spatial dimensions by factors such as 3D genome structure and transcription, which is crucial for maintaining genomic stability in each cell cycle. In this review, we discuss the diverse mechanisms that govern eukaryotic DNA replication, emphasizing the roles of chromatin architecture and transcriptional activity within the mammalian chromatin landscape. These insights provide a foundation for future investigations in this field.
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Affiliation(s)
- Yang Liu
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, PKU-THU Center for Life Sciences, Peking University, Beijing 100871, China; Department of Medical Genetics, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China
| | - Zhengrong Zhangding
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, PKU-THU Center for Life Sciences, Peking University, Beijing 100871, China
| | - Xuhao Liu
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, PKU-THU Center for Life Sciences, Peking University, Beijing 100871, China
| | - Jiazhi Hu
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, PKU-THU Center for Life Sciences, Peking University, Beijing 100871, China; Peking University Chengdu Academy for Advanced Interdisciplinary Biotechnologies, Chengdu, Sichuan 610213, China.
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2
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Shibata Y, Peycheva M, Shibata E, Malzl D, Pavri R, Dutta A. Specific origin selection and excess functional MCM2-7 loading in ORC-deficient cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2024.10.30.621095. [PMID: 39554186 PMCID: PMC11565923 DOI: 10.1101/2024.10.30.621095] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2024]
Abstract
The six subunit Origin Recognition Complex (ORC) loads excess MCM2-7 on chromosomes to promote initiation of DNA replication and is believed to be important for origin specification. Mapping of origins in cancer cell lines engineered to delete three of the subunits, ORC1 , ORC2 or ORC5 shows that specific origins are still used and are mostly at the same sites in the genome as in wild type cells. The few thousand origins that were up-regulated in the absence of ORC suggest that GC/TA skewness and simple repeat sequences facilitate, but are not essential for, origin selection in the absence of the six-subunit ORC. Despite the lack of ORC, excess MCM2-7 is still loaded at comparable rates in G1 phase to license dormant origins and is also repeatedly loaded in the same S phase to permit re-replication. Thus, origin specification and excess MCM2-7 loading on origins do not require the six-subunit ORC in human cancer cell lines.
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3
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Krude T, Bi J, Doran R, Jones R, Smith J. Human DNA replication initiation sites are specified epigenetically by oxidation of 5-methyl-deoxycytidine. Nucleic Acids Res 2025; 53:gkaf362. [PMID: 40323014 PMCID: PMC12051107 DOI: 10.1093/nar/gkaf362] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2024] [Revised: 04/03/2025] [Accepted: 04/17/2025] [Indexed: 05/08/2025] Open
Abstract
DNA replication initiates at tens of thousands of sites on the human genome during each S phase. However, no consensus DNA sequence has been found that specifies the locations of these replication origins. Here, we investigate modifications of human genomic DNA by density equilibrium centrifugation and DNA sequencing. We identified short discrete sites with increased density during quiescence and G1 phase that overlap with DNA replication origins before their activation in S phase. The increased density is due to the oxidation of 5-methyl-deoxycytidines by ten-eleven-translocation DNA dioxygenase (TET) enzymes at GC-rich domains. Reversible inhibition of de novo methylation and of subsequent oxidation of deoxycytidines results in a reversible inhibition of DNA replication and of cell proliferation. Our findings suggest a mechanism for the epigenetic specification and semiconservative inheritance of DNA replication origin sites in human cells that also provides a stable integral DNA replication licence to support once-per-cell cycle control of origin activation.
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Affiliation(s)
- Torsten Krude
- Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
| | - Jiaming Bi
- Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
| | - Rachel Doran
- Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
| | - Rebecca A Jones
- Developmental Biology Laboratory, Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | - James C Smith
- Developmental Biology Laboratory, Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
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4
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Matsumoto A, Daigaku Y, Tsubouchi T. Polymerase-usage sequencing identifies initiation zones with less bias across S phase in mouse embryonic stem cells. J Biochem 2025; 177:213-223. [PMID: 39745849 PMCID: PMC11879308 DOI: 10.1093/jb/mvae097] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2024] [Revised: 12/19/2024] [Accepted: 12/21/2024] [Indexed: 01/04/2025] Open
Abstract
Various methods have been developed to map replication initiation zones (IZs) genome-wide, often finding far fewer IZs than expected. In particular, IZs corresponding to later stages of S phase are under-represented. Here, we reanalysed IZs with respect to replication timing in mouse ES cells. These datasets identified over five times as many early IZs compared to late IZs. In addition, we have set up a polymerase-usage sequencing (Pu-seq) system in mouse ES cells to map IZs genome-wide. Pu-seq showed less bias towards early IZs, potentially indicating better sensitivity for identifying IZs in late S phase.
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Affiliation(s)
- Akino Matsumoto
- Laboratory of Stem Cell Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan
- Department of Basic Biology, the Graduate University for Advanced Studies, SOKENDAI, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan
| | - Yasukazu Daigaku
- Cancer Genome Dynamics Project, Cancer Institute, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan
| | - Tomomi Tsubouchi
- Laboratory of Stem Cell Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan
- Department of Basic Biology, the Graduate University for Advanced Studies, SOKENDAI, 38 Nishigonaka, Myodaiji, Okazaki, Aichi 444-8585, Japan
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5
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Hyrien O, Guilbaud G, Krude T. The double life of mammalian DNA replication origins. Genes Dev 2025; 39:304-324. [PMID: 39904559 PMCID: PMC11874978 DOI: 10.1101/gad.352227.124] [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] [Indexed: 02/06/2025]
Abstract
Mammalian DNA replication origins have been historically difficult to identify and their determinants are still unresolved. Here, we first review methods developed over the last decades to map replication initiation sites either directly via initiation intermediates or indirectly via determining replication fork directionality profiles. We also discuss the factors that may specify these sites as replication initiation sites. Second, we address the controversy that has emerged from these results over whether origins are narrowly defined and localized to specific sites or are more dispersed and organized into broad zones. Ample evidence in favor of both scenarios currently creates an impression of unresolved confusion in the field. We attempt to formulate a synthesis of both models and to reconcile discrepant findings. It is evident that not only one approach is sufficient in isolation but that the combination of several is instrumental toward understanding initiation sites in mammalian genomes. We argue that an aggregation of several individual and often inefficient initiation sites into larger initiation zones and the existence of efficient unidirectional initiation sites and fork stalling at the borders of initiation zones can reconcile the different observations.
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Affiliation(s)
- Olivier Hyrien
- Département de Biologie, École Normale Supérieure, Université Paris Science and Letters, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Institut de Biologie de l'Ecole Normale Superieure, 75005 Paris, France
| | - Guillaume Guilbaud
- Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom
| | - Torsten Krude
- Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom
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6
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Gökbuget D, Goehring L, Boileau RM, Lenshoek K, Huang TT, Blelloch R. KMT2C/KMT2D-dependent H3K4me1 mediates changes in DNA replication timing and origin activity during a cell fate transition. Cell Rep 2025; 44:115272. [PMID: 39908143 DOI: 10.1016/j.celrep.2025.115272] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2023] [Revised: 10/10/2024] [Accepted: 01/15/2025] [Indexed: 02/07/2025] Open
Abstract
Mammalian genomes replicate in a cell-type-specific order during the S phase, correlated to transcriptional activity, histone modifications, and chromatin structure. The causal relationships between these features and DNA replication timing (RT), especially during cell fate changes, are largely unknown. Using machine learning, we quantify 21 chromatin features predicting local RT and RT changes during differentiation in embryonic stem cells (ESCs). About one-third of the genome shows RT changes during differentiation. Chromatin features accurately predict both steady-state RT and RT changes. Histone H3 lysine 4 monomethylation (H3K4me1), catalyzed by KMT2C and KMT2D (KMT2C/D), emerges as a top predictor. Loss of KMT2C/D or their enzymatic activities impairs RT changes during differentiation. This correlates with local H3K4me1 loss and reduced replication origin firing, while transcription remains largely unaffected. Our findings reveal KMT2C/D-dependent H3K4me1 as a key regulator of RT and replication initiation, a role that likely impacts diseases associated with KMT2C/D mutations.
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Affiliation(s)
- Deniz Gökbuget
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA; Department of Urology, University of California, San Francisco, San Francisco, CA, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
| | - Liana Goehring
- Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA
| | - Ryan M Boileau
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA; Department of Urology, University of California, San Francisco, San Francisco, CA, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA
| | - Kayla Lenshoek
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA; Department of Urology, University of California, San Francisco, San Francisco, CA, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA
| | - Tony T Huang
- Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA
| | - Robert Blelloch
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA; Department of Urology, University of California, San Francisco, San Francisco, CA, USA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
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7
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Turner JL, Hinojosa-Gonzalez L, Sasaki T, Uchino S, Vouzas A, Soto MS, Chakraborty A, Alexander KE, Fitch CA, Brown AN, Ay F, Gilbert DM. Master transcription factor binding sites constitute the core of early replication control elements. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2023.10.22.563497. [PMID: 39990485 PMCID: PMC11844392 DOI: 10.1101/2023.10.22.563497] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/25/2025]
Abstract
Eukaryotic genomes replicate in a defined temporal order called the replication timing (RT) program. RT is developmentally regulated with potential to drive cell fate transitions, but mechanisms controlling RT remain elusive. We previously identified "Early Replication Control Elements" (ERCEs) necessary for early RT, domain-wide transcription, 3D chromatin architecture and compartmentalization in mouse embryonic stem cells (mESCs) but, deletions identifying ERCEs were large and encompassed many putative regulatory elements. Here, we show that ERCEs are compound elements whose RT activity can largely be accounted for by multiple sites of diverse master transcription factor binding (subERCEs), distinguished from other such sites by their long-range interactions. While deletion of subERCEs had large effects on both transcription and RT, deleting transcription start sites eliminated nearly all transcription with moderate effects on RT. Our results suggest a model in which subERCEs respond to diverse master transcription factors by functioning both as transcription enhancers and as elements that organize chromatin domains structurally and support early RT, potentially providing a feed-forward loop to drive robust epigenomic change during cell fate transitions.
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8
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Wong LH, Tremethick DJ. Multifunctional histone variants in genome function. Nat Rev Genet 2025; 26:82-104. [PMID: 39138293 DOI: 10.1038/s41576-024-00759-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/18/2024] [Indexed: 08/15/2024]
Abstract
Histones are integral components of eukaryotic chromatin that have a pivotal role in the organization and function of the genome. The dynamic regulation of chromatin involves the incorporation of histone variants, which can dramatically alter its structural and functional properties. Contrary to an earlier view that limited individual histone variants to specific genomic functions, new insights have revealed that histone variants exert multifaceted roles involving all aspects of genome function, from governing patterns of gene expression at precise genomic loci to participating in genome replication, repair and maintenance. This conceptual change has led to a new understanding of the intricate interplay between chromatin and DNA-dependent processes and how this connection translates into normal and abnormal cellular functions.
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Affiliation(s)
- Lee H Wong
- Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
| | - David J Tremethick
- The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capial Territory, Australia.
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9
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Arroyo M, Casas-Delucchi C, Pabba M, Prorok P, Pradhan S, Rausch C, Lehmkuhl A, Maiser A, Buschbeck M, Pasque V, Bernstein E, Luck K, Cardoso M. Histone variant macroH2A1 regulates synchronous firing of replication origins in the inactive X chromosome. Nucleic Acids Res 2024; 52:11659-11688. [PMID: 39189450 PMCID: PMC11514477 DOI: 10.1093/nar/gkae734] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2024] [Revised: 08/07/2024] [Accepted: 08/14/2024] [Indexed: 08/28/2024] Open
Abstract
MacroH2A has been linked to transcriptional silencing, cell identity, and is a hallmark of the inactive X chromosome (Xi). However, it remains unclear whether macroH2A plays a role in DNA replication. Using knockdown/knockout cells for each macroH2A isoform, we show that macroH2A-containing nucleosomes slow down replication progression rate in the Xi reflecting the higher nucleosome stability. Moreover, macroH2A1, but not macroH2A2, regulates the number of nano replication foci in the Xi, and macroH2A1 downregulation increases DNA loop sizes corresponding to replicons. This relates to macroH2A1 regulating replicative helicase loading during G1 by interacting with it. We mapped this interaction to a phenylalanine in macroH2A1 that is not conserved in macroH2A2 and the C-terminus of Mcm3 helicase subunit. We propose that macroH2A1 enhances the licensing of pre-replication complexes via DNA helicase interaction and loading onto the Xi.
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Affiliation(s)
- Maria Arroyo
- Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
| | - Corella S Casas-Delucchi
- Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
| | - Maruthi K Pabba
- Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
| | - Paulina Prorok
- Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
| | - Sunil K Pradhan
- Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
| | - Cathia Rausch
- Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
| | - Anne Lehmkuhl
- Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
| | - Andreas Maiser
- Faculty of Biology and Center for Molecular Biosystems (BioSysM), Human Biology and BioImaging, LMU Munich, Munich 81377, Germany
| | - Marcus Buschbeck
- Program of Myeloid Neoplasms, Program of Applied Epigenetics, Josep Carreras Leukaemia Research Institute (IJC), Germans Trias i Pujol Research Institute (IGTP), Campus Can Ruti, Camí de les Escoles, 08916 Badalona, Barcelona, Spain
| | - Vincent Pasque
- Department of Development and Regeneration, Leuven Stem Cell Institute, Leuven Institute for Single-Cell Omics (LISCO), KU Leuven-University of Leuven, 3000 Leuven, Belgium
| | - Emily Bernstein
- Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, Tisch Cancer Institute, NY, NY 10029, USA
| | - Katja Luck
- Institute of Molecular Biology (IMB) gGmbH, 55128 Mainz, Germany
| | - M Cristina Cardoso
- Cell Biology and Epigenetics, Department of Biology, Technical University of Darmstadt, 64287 Darmstadt, Germany
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10
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Xu S, Egli D. Genome organization and stability in mammalian pre-implantation development. DNA Repair (Amst) 2024; 144:103780. [PMID: 39504608 DOI: 10.1016/j.dnarep.2024.103780] [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: 05/25/2024] [Revised: 10/14/2024] [Accepted: 10/22/2024] [Indexed: 11/08/2024]
Abstract
A largely stable genome is required for normal development, even as genetic change is an integral aspect of reproduction, genetic adaptation and evolution. Recent studies highlight a critical window of mammalian development with intrinsic DNA replication stress and genome instability in the first cell divisions after fertilization. Patterns of DNA replication and genome stability are established very early in mammals, alongside patterns of nuclear organization, and before the emergence of gene expression patterns, and prior to cell specification and germline formation. The study of DNA replication and genome stability in the mammalian embryo provides a unique cellular system due to the resetting of the epigenome to a totipotent state, and the de novo establishment of the patterns of nuclear organization, gene expression, DNA methylation, histone modifications and DNA replication. Studies on DNA replication and genome stability in the early mammalian embryo is relevant for understanding both normal and disease-causing genetic variation, and to uncover basic principles of genome regulation.
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Affiliation(s)
- Shuangyi Xu
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA
| | - Dieter Egli
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Obstetrics and Gynecology, Columbia University, New York, NY 10032, USA.
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11
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Zhu X, Kanemaki MT. Replication initiation sites and zones in the mammalian genome: Where are they located and how are they defined? DNA Repair (Amst) 2024; 141:103713. [PMID: 38959715 DOI: 10.1016/j.dnarep.2024.103713] [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: 04/19/2024] [Revised: 06/14/2024] [Accepted: 06/15/2024] [Indexed: 07/05/2024]
Abstract
Eukaryotic DNA replication is a tightly controlled process that occurs in two main steps, i.e., licensing and firing, which take place in the G1 and S phases of the cell cycle, respectively. In Saccharomyces cerevisiae, the budding yeast, replication origins contain consensus sequences that are recognized and bound by the licensing factor Orc1-6, which then recruits the replicative Mcm2-7 helicase. By contrast, mammalian initiation sites lack such consensus sequences, and the mammalian ORC does not exhibit sequence specificity. Studies performed over the past decades have identified replication initiation sites in the mammalian genome using sequencing-based assays, raising the question of whether replication initiation occurs at confined sites or in broad zones across the genome. Although recent reports have shown that the licensed MCMs in mammalian cells are broadly distributed, suggesting that ORC-dependent licensing may not determine the initiation sites/zones, they are predominantly located upstream of actively transcribed genes. This review compares the mechanism of replication initiation in yeast and mammalian cells, summarizes the sequencing-based technologies used for the identification of initiation sites/zones, and proposes a possible mechanism of initiation-site/zone selection in mammalian cells. Future directions and challenges in this field are also discussed.
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Affiliation(s)
- Xiaoxuan Zhu
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Shizuoka, Mishima 411-8540, Japan.
| | - Masato T Kanemaki
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Shizuoka, Mishima 411-8540, Japan; Graduate Institute for Advanced Studies, SOKENDAI, Yata 1111, Shizuoka, Mishima 411-8540, Japan; Department of Biological Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
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12
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Park D, Chung WC, Gong S, Ravichandran S, Lee GM, Han M, Kim KK, Ahn JH. G-quadruplex as an essential structural element in cytomegalovirus replication origin. Nat Commun 2024; 15:7353. [PMID: 39191758 PMCID: PMC11350156 DOI: 10.1038/s41467-024-51797-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2024] [Accepted: 08/16/2024] [Indexed: 08/29/2024] Open
Abstract
G-quadruplex (G4) structures are found in eukaryotic cell replication origins, but their role in origin function remains unclear. In this study G4 motifs are found in the lytic DNA replication origin (oriLyt) of human cytomegalovirus (HCMV) and recombinant viruses show that a G4 motif in oriLyt essential region I (ER-I) is necessary for viral growth. Replication assays of oriLyt-containing plasmids and biochemical/biophysical analyses show that G4 formation in ER-I is crucial for viral DNA replication. G4 pull-down analysis identifies viral DNA replication factors, such as IE2, UL84, and UL44, as G4-binding proteins. In enzyme-linked immunosorbent assays, specific G4-binding ligands inhibit G4 binding by the viral proteins. The Epstein-Barr virus oriLyt core element also forms a stable G4 that could substitute for the oriLyt ER-I G4 in HCMV. These results demonstrate that viral G4s in replication origins represent an essential structural element in recruiting replication factors and might be a therapeutic target against viral infections.
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Affiliation(s)
- Daegyu Park
- Department of Microbiology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea
| | - Woo-Chang Chung
- Department of Microbiology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea
| | - Shuang Gong
- Department of Microbiology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea
| | | | - Gwang Myeong Lee
- Department of Microbiology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea
| | - Minji Han
- Department of Microbiology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea
| | - Kyeong Kyu Kim
- Department of Precision Medicine, Institute for Antimicrobial Resistance Research and Therapeutics, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea
- Biomedical Research Institute, Samsung Medical Center, Seoul, Republic of Korea
| | - Jin-Hyun Ahn
- Department of Microbiology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea.
- Biomedical Research Institute, Samsung Medical Center, Seoul, Republic of Korea.
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13
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Feng J, Chuah Y, Liang Y, Cipta N, Zeng Y, Warrier T, Elfar G, Yoon J, Grinchuk O, Tay E, Lok KZ, Zheng ZQ, Khong Z, Chong ZS, Teo J, Sanford E, Neo C, Chiu H, Leung J, Wang L, Lim Y, Zhao T, Sobota R, Crasta K, Tergaonkar V, Taneja R, Ng SY, Cheok C, Ling SC, Loh YH, Ong D. PHF2 regulates genome topology and DNA replication in neural stem cells via cohesin. Nucleic Acids Res 2024; 52:7063-7080. [PMID: 38808662 PMCID: PMC11229317 DOI: 10.1093/nar/gkae457] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2023] [Revised: 04/15/2024] [Accepted: 05/15/2024] [Indexed: 05/30/2024] Open
Abstract
Cohesin plays a crucial role in the organization of topologically-associated domains (TADs), which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here, we discover that the histone demethylase PHF2 associates with RAD21, a core subunit of cohesin, to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably, the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient, active replication origins, and can act as boundaries to separate adjacent domains. Accordingly, PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore, we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation, and that PHF2's histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops, where cohesin represses dormant replication origins directly or indirectly, to sustain DNA replication in NSC.
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Affiliation(s)
- Jia Feng
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
| | - You Heng Chuah
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- NUS Centre for Cancer Research Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Yajing Liang
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
| | - Nadia Omega Cipta
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Yingying Zeng
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Tushar Warrier
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Gamal Ahmed Rashed Elsayed Elfar
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
- Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, National University Hospital, Singapore 119074, Singapore
| | - Jeehyun Yoon
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- NUS Centre for Cancer Research Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Oleg V Grinchuk
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- NUS Centre for Cancer Research Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Emmy Xue Yun Tay
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
| | - Ker-Zhing Lok
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
| | - Zong-Qing Zheng
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
| | - Zi Jian Khong
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Zheng-Shan Chong
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Jackie Teo
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Emma May Sanford
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- Healthy Longevity Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Cheryl Jia Yi Neo
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- Healthy Longevity Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Hsin Yao Chiu
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
| | - Jia Yu Leung
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
- Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, National University Hospital, Singapore 119074, Singapore
| | - Loo Chien Wang
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Yan Ting Lim
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Tianyun Zhao
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Radoslaw M Sobota
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
| | - Karen Carmelina Crasta
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- NUS Centre for Cancer Research Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
- Healthy Longevity Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Vinay Tergaonkar
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, MD7, Singapore 117596, Singapore
- Laboratory of NFκB Signalling, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore
| | - Reshma Taneja
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- NUS Centre for Cancer Research Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Healthy Longevity Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Shi-Yan Ng
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
- National Neuroscience Institute, 308433, Singapore
| | - Chit Fang Cheok
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
- Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, National University Hospital, Singapore 119074, Singapore
| | - Shuo-Chien Ling
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- Healthy Longevity Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Yuin-Han Loh
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
| | - Derrick Sek Tong Ong
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore
- NUS Centre for Cancer Research Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore 138673, Singapore
- Healthy Longevity Translation Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- National Neuroscience Institute, 308433, Singapore
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14
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Xu S, Wang N, Zuccaro MV, Gerhardt J, Iyyappan R, Scatolin GN, Jiang Z, Baslan T, Koren A, Egli D. DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility. Nat Commun 2024; 15:5247. [PMID: 38898078 PMCID: PMC11187207 DOI: 10.1038/s41467-024-49565-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2023] [Accepted: 06/10/2024] [Indexed: 06/21/2024] Open
Abstract
DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.
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Affiliation(s)
- Shuangyi Xu
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY, 10032, USA
| | - Ning Wang
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY, 10032, USA
| | - Michael V Zuccaro
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY, 10032, USA
- Graduate Program, Department of Cellular Physiology and Biophysics, Columbia University, New York, NY, USA
| | - Jeannine Gerhardt
- The Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical School, New York, NY, USA
| | - Rajan Iyyappan
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL, USA
| | | | - Zongliang Jiang
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL, USA
| | - Timour Baslan
- Department of Biomedical Sciences, School of Veterinary Medicine, The University of Pennsylvania, Philadelphia, PA, USA
| | - Amnon Koren
- Department of Molecular and Cellular Biology, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Dieter Egli
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY, 10032, USA.
- Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Columbia University, New York, NY, USA.
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15
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Rojas P, Wang J, Guglielmi G, Sadurnì MM, Pavlou L, Leung GHD, Rajagopal V, Spill F, Saponaro M. Genome-wide identification of replication fork stalling/pausing sites and the interplay between RNA Pol II transcription and DNA replication progression. Genome Biol 2024; 25:126. [PMID: 38773641 PMCID: PMC11106976 DOI: 10.1186/s13059-024-03278-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2023] [Accepted: 05/14/2024] [Indexed: 05/24/2024] Open
Abstract
BACKGROUND DNA replication progression can be affected by the presence of physical barriers like the RNA polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know how transcription influences overall DNA replication progression. RESULTS To characterize sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase to identify replication fork/stalling hotspots as replication progresses through the genome. These sites are typically associated with increased DNA damage, overlapped with fragile sites and with breakpoints of rearrangements identified in cancers but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. Indeed, we find that slowing down transcription elongation slows down directly replication progression through genes. This indicates that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. At the same time, we find instances where transcription activity favors replication progression because it reduces histone density. CONCLUSIONS Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.
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Affiliation(s)
- Patricia Rojas
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Jianming Wang
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Giovanni Guglielmi
- School of Mathematics, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
- Department of Biomedical Engineering, University of Melbourne, Melbourne, VIC, 3010, Australia
| | - Martina Mustè Sadurnì
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Lucas Pavlou
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Geoffrey Ho Duen Leung
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, B15 2TT, UK
| | - Vijay Rajagopal
- Department of Biomedical Engineering, University of Melbourne, Melbourne, VIC, 3010, Australia
| | - Fabian Spill
- School of Mathematics, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
| | - Marco Saponaro
- Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, B15 2TT, UK.
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16
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Tian M, Wang Z, Su Z, Shibata E, Shibata Y, Dutta A, Zang C. Integrative analysis of DNA replication origins and ORC-/MCM-binding sites in human cells reveals a lack of overlap. eLife 2024; 12:RP89548. [PMID: 38567819 PMCID: PMC10990492 DOI: 10.7554/elife.89548] [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] [Indexed: 04/05/2024] Open
Abstract
Based on experimentally determined average inter-origin distances of ~100 kb, DNA replication initiates from ~50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the origin recognition complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and five ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of ~7.5 million union origins identified by all datasets, only 0.27% (20,250 shared origins) were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques, suggesting extensive variability in origin usage and identification. Also, 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF-binding sites, G-quadruplex sites, and activating histone marks, these overlaps are comparable or less than that of known transcription start sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ~13,000 reproducible ORC-binding sites in human cancer cells, and only 4.5% were within 1 kb of the ~11,000 union MCM2-7-binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast, Saccharomyces cerevisiae. Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites.
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Affiliation(s)
- Mengxue Tian
- Center for Public Health Genomics, University of Virginia School of MedicineCharlottesvilleUnited States
- Department of Biochemistry and Molecular Genetics, University of Virginia School of MedicineCharlottesvilleUnited States
| | - Zhenjia Wang
- Center for Public Health Genomics, University of Virginia School of MedicineCharlottesvilleUnited States
| | - Zhangli Su
- Department of Biochemistry and Molecular Genetics, University of Virginia School of MedicineCharlottesvilleUnited States
- Department of Genetics, University of Alabama at BirminghamBirminghamUnited States
| | - Etsuko Shibata
- Department of Biochemistry and Molecular Genetics, University of Virginia School of MedicineCharlottesvilleUnited States
- Department of Genetics, University of Alabama at BirminghamBirminghamUnited States
| | - Yoshiyuki Shibata
- Department of Biochemistry and Molecular Genetics, University of Virginia School of MedicineCharlottesvilleUnited States
- Department of Genetics, University of Alabama at BirminghamBirminghamUnited States
| | - Anindya Dutta
- Department of Biochemistry and Molecular Genetics, University of Virginia School of MedicineCharlottesvilleUnited States
- Department of Genetics, University of Alabama at BirminghamBirminghamUnited States
| | - Chongzhi Zang
- Center for Public Health Genomics, University of Virginia School of MedicineCharlottesvilleUnited States
- Department of Biochemistry and Molecular Genetics, University of Virginia School of MedicineCharlottesvilleUnited States
- Department of Public Health Sciences, University of VirginiaCharlottesvilleUnited States
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17
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Lai PM, Chan KM. Roles of Histone H2A Variants in Cancer Development, Prognosis, and Treatment. Int J Mol Sci 2024; 25:3144. [PMID: 38542118 PMCID: PMC10969971 DOI: 10.3390/ijms25063144] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Revised: 02/09/2024] [Accepted: 02/09/2024] [Indexed: 07/16/2024] Open
Abstract
Histones are nuclear proteins essential for packaging genomic DNA and epigenetic gene regulation. Paralogs that can substitute core histones (H2A, H2B, H3, and H4), named histone variants, are constitutively expressed in a replication-independent manner throughout the cell cycle. With specific chaperones, they can be incorporated to chromatin to modify nucleosome stability by modulating interactions with nucleosomal DNA. This allows the regulation of essential fundamental cellular processes for instance, DNA damage repair, chromosomal segregation, and transcriptional regulation. Among all the histone families, histone H2A family has the largest number of histone variants reported to date. Each H2A variant has multiple functions apart from their primary role and some, even be further specialized to perform additional tasks in distinct lineages, such as testis specific shortH2A (sH2A). In the past decades, the discoveries of genetic alterations and mutations in genes encoding H2A variants in cancer had revealed variants' potentiality in driving carcinogenesis. In addition, there is growing evidence that H2A variants may act as novel prognostic indicators or biomarkers for both early cancer detection and therapeutic treatments. Nevertheless, no studies have ever concluded all identified variants in a single report. Here, in this review, we summarize the respective functions for all the 19 mammalian H2A variants and their roles in cancer biology whilst potentiality being used in clinical setting.
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Affiliation(s)
| | - Kui Ming Chan
- Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China;
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18
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Ahmad H, Chetlangia N, Prasanth SG. Chromatin's Influence on Pre-Replication Complex Assembly and Function. BIOLOGY 2024; 13:152. [PMID: 38534422 PMCID: PMC10968542 DOI: 10.3390/biology13030152] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/10/2024] [Revised: 02/19/2024] [Accepted: 02/19/2024] [Indexed: 03/28/2024]
Abstract
In all eukaryotes, the initiation of DNA replication requires a stepwise assembly of factors onto the origins of DNA replication. This is pioneered by the Origin Recognition Complex, which recruits Cdc6. Together, they bring Cdt1, which shepherds MCM2-7 to form the OCCM complex. Sequentially, a second Cdt1-bound hexamer of MCM2-7 is recruited by ORC-Cdc6 to form an MCM double hexamer, which forms a part of the pre-RC. Although the mechanism of ORC binding to DNA varies across eukaryotes, how ORC is recruited to replication origins in human cells remains an area of intense investigation. This review discusses how the chromatin environment influences pre-RC assembly, function, and, eventually, origin activity.
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Affiliation(s)
- Hina Ahmad
- Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601S Goodwin Avenue, Urbana, IL 61801, USA; (H.A.); (N.C.)
| | - Neha Chetlangia
- Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601S Goodwin Avenue, Urbana, IL 61801, USA; (H.A.); (N.C.)
| | - Supriya G. Prasanth
- Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601S Goodwin Avenue, Urbana, IL 61801, USA; (H.A.); (N.C.)
- Cancer Center at Illinois, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
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19
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González-Acosta D, Lopes M. DNA replication and replication stress response in the context of nuclear architecture. Chromosoma 2024; 133:57-75. [PMID: 38055079 PMCID: PMC10904558 DOI: 10.1007/s00412-023-00813-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2023] [Revised: 10/23/2023] [Accepted: 10/24/2023] [Indexed: 12/07/2023]
Abstract
The DNA replication process needs to be coordinated with other DNA metabolism transactions and must eventually extend to the full genome, regardless of chromatin status, gene expression, secondary structures and DNA lesions. Completeness and accuracy of DNA replication are crucial to maintain genome integrity, limiting transformation in normal cells and offering targeting opportunities for proliferating cancer cells. DNA replication is thus tightly coordinated with chromatin dynamics and 3D genome architecture, and we are only beginning to understand the underlying molecular mechanisms. While much has recently been discovered on how DNA replication initiation is organised and modulated in different genomic regions and nuclear territories-the so-called "DNA replication program"-we know much less on how the elongation of ongoing replication forks and particularly the response to replication obstacles is affected by the local nuclear organisation. Also, it is still elusive how specific components of nuclear architecture participate in the replication stress response. Here, we review known mechanisms and factors orchestrating replication initiation, and replication fork progression upon stress, focusing on recent evidence linking genome organisation and nuclear architecture with the cellular responses to replication interference, and highlighting open questions and future challenges to explore this exciting new avenue of research.
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Affiliation(s)
| | - Massimo Lopes
- Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.
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20
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Xu S, Wang N, Zuccaro MV, Gerhardt J, Baslan T, Koren A, Egli D. DNA replication in early mammalian embryos is patterned, predisposing lamina-associated regions to fragility. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.25.573304. [PMID: 38234839 PMCID: PMC10793403 DOI: 10.1101/2023.12.25.573304] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2024]
Abstract
DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that both bovine and mouse cleavage stage embryos progress through S-phase in a defined pattern. Late replicating regions are associated with the nuclear lamina from the first cell cycle after fertilization, and contain few active origins, and few but long genes. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to segregation of soma and germ line. Our studies show that the formation of early and late replicating regions is among the first layers of epigenetic regulation established on the mammalian genome after fertilization.
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Affiliation(s)
- Shuangyi Xu
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY, 10032, USA
| | - Ning Wang
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY, 10032, USA
| | - Michael V Zuccaro
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY, 10032, USA
- Graduate Program, Department of Cellular Physiology and Biophysics, Columbia University, New York
| | | | - Timour Baslan
- Department of Biomedical Sciences, The University of Pennsylvania, Philadelphia, PA, 19104
| | - Amnon Koren
- Department of Molecular Biology and Genetics, Cornell University, Ithaca NY, 14853, USA
| | - Dieter Egli
- Division of Molecular Genetics, Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia Stem Cell Initiative, Columbia University, New York, NY, 10032, USA
- Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Columbia University, New York, NY, 10032, USA
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21
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Lee CSK, Weiβ M, Hamperl S. Where and when to start: Regulating DNA replication origin activity in eukaryotic genomes. Nucleus 2023; 14:2229642. [PMID: 37469113 PMCID: PMC10361152 DOI: 10.1080/19491034.2023.2229642] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Revised: 06/20/2023] [Accepted: 06/21/2023] [Indexed: 07/21/2023] Open
Abstract
In eukaryotic genomes, hundreds to thousands of potential start sites of DNA replication named origins are dispersed across each of the linear chromosomes. During S-phase, only a subset of origins is selected in a stochastic manner to assemble bidirectional replication forks and initiate DNA synthesis. Despite substantial progress in our understanding of this complex process, a comprehensive 'identity code' that defines origins based on specific nucleotide sequences, DNA structural features, the local chromatin environment, or 3D genome architecture is still missing. In this article, we review the genetic and epigenetic features of replication origins in yeast and metazoan chromosomes and highlight recent insights into how this flexibility in origin usage contributes to nuclear organization, cell growth, differentiation, and genome stability.
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Affiliation(s)
- Clare S K Lee
- Chromosome Dynamics and Genome Stability, Institute of Epigenetics and Stem Cells, Helmholtz Zentrum München, Munich, Germany
| | - Matthias Weiβ
- Chromosome Dynamics and Genome Stability, Institute of Epigenetics and Stem Cells, Helmholtz Zentrum München, Munich, Germany
| | - Stephan Hamperl
- Chromosome Dynamics and Genome Stability, Institute of Epigenetics and Stem Cells, Helmholtz Zentrum München, Munich, Germany
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22
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Bellani MA, Shaik A, Majumdar I, Ling C, Seidman MM. The Response of the Replication Apparatus to Leading Template Strand Blocks. Cells 2023; 12:2607. [PMID: 37998342 PMCID: PMC10670059 DOI: 10.3390/cells12222607] [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: 10/23/2023] [Revised: 11/07/2023] [Accepted: 11/08/2023] [Indexed: 11/25/2023] Open
Abstract
Duplication of the genome requires the replication apparatus to overcome a variety of impediments, including covalent DNA adducts, the most challenging of which is on the leading template strand. Replisomes consist of two functional units, a helicase to unwind DNA and polymerases to synthesize it. The helicase is a multi-protein complex that encircles the leading template strand and makes the first contact with a leading strand adduct. The size of the channel in the helicase would appear to preclude transit by large adducts such as DNA: protein complexes (DPC). Here we discuss some of the extensively studied pathways that support replication restart after replisome encounters with leading template strand adducts. We also call attention to recent work that highlights the tolerance of the helicase for adducts ostensibly too large to pass through the central channel.
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Affiliation(s)
| | | | | | | | - Michael M. Seidman
- Laboratory of Molecular Biology and Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA; (M.A.B.)
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23
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Poulet-Benedetti J, Tonnerre-Doncarli C, Valton AL, Laurent M, Gérard M, Barinova N, Parisis N, Massip F, Picard F, Prioleau MN. Dimeric G-quadruplex motifs-induced NFRs determine strong replication origins in vertebrates. Nat Commun 2023; 14:4843. [PMID: 37563125 PMCID: PMC10415359 DOI: 10.1038/s41467-023-40441-4] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Accepted: 07/28/2023] [Indexed: 08/12/2023] Open
Abstract
Replication of vertebrate genomes is tightly regulated to ensure accurate duplication, but our understanding of the interplay between genetic and epigenetic factors in this regulation remains incomplete. Here, we investigated the involvement of three elements enriched at gene promoters and replication origins: guanine-rich motifs potentially forming G-quadruplexes (pG4s), nucleosome-free regions (NFRs), and the histone variant H2A.Z, in the firing of origins of replication in vertebrates. We show that two pG4s on the same DNA strand (dimeric pG4s) are sufficient to induce the assembly of an efficient minimal replication origin without inducing transcription in avian DT40 cells. Dimeric pG4s in replication origins are associated with formation of an NFR next to precisely-positioned nucleosomes enriched in H2A.Z on this minimal origin and genome-wide. Thus, our data suggest that dimeric pG4s are important for the organization and duplication of vertebrate genomes. It supports the hypothesis that a nucleosome close to an NFR is a shared signal for the formation of replication origins in eukaryotes.
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Affiliation(s)
| | | | - Anne-Laure Valton
- Université Paris Cité, CNRS, Institut Jacques Monod, F-75013, Paris, France
- Department of Systems Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA
- Howard Hughes Medical Institute, Chevy Chase, MD, USA
| | - Marc Laurent
- Université Paris Cité, CNRS, Institut Jacques Monod, F-75013, Paris, France
| | - Marie Gérard
- Université Paris Cité, CNRS, Institut Jacques Monod, F-75013, Paris, France
| | - Natalja Barinova
- Université Paris Cité, CNRS, Institut Jacques Monod, F-75013, Paris, France
| | - Nikolaos Parisis
- Université Paris Cité, CNRS, Institut Jacques Monod, F-75013, Paris, France
| | - Florian Massip
- MINES ParisTech, PSL-Research University, CBIO-Centre for Computational Biology, 75006, Paris, France
- Institut Curie, Paris, Cedex, France
- INSERM, U900, Paris, Cedex, France
| | - Franck Picard
- Laboratory of Biology and Modelling of the Cell, Université de Lyon, Ecole Normale Supérieure de Lyon, CNRS, UMR5239, Université Claude Bernard Lyon 1, Lyon, France.
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24
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Han ZQ, Wen LN. Application of G-quadruplex targets in gastrointestinal cancers: Advancements, challenges and prospects. World J Gastrointest Oncol 2023; 15:1149-1173. [PMID: 37546556 PMCID: PMC10401460 DOI: 10.4251/wjgo.v15.i7.1149] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/11/2023] [Revised: 04/11/2023] [Accepted: 05/08/2023] [Indexed: 07/12/2023] Open
Abstract
Genomic instability and inflammation are considered to be two enabling characteristics that support cancer development and progression. G-quadruplex structure is a key element that contributes to genomic instability and inflammation. G-quadruplexes were once regarded as simply an obstacle that can block the transcription of oncogenes. A ligand targeting G-quadruplexes was found to have anticancer activity, making G-quadruplexes potential anticancer targets. However, further investigation has revealed that G-quadruplexes are widely distributed throughout the human genome and have many functions, such as regulating DNA replication, DNA repair, transcription, translation, epigenetics, and inflammatory response. G-quadruplexes play double regulatory roles in transcription and translation. In this review, we focus on G-quadruplexes as novel targets for the treatment of gastrointestinal cancers. We summarize the application basis of G-quadruplexes in gastrointestinal cancers, including their distribution sites, structural characteristics, and physiological functions. We describe the current status of applications for the treatment of esophageal cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, colorectal cancer, and gastrointestinal stromal tumors, as well as the associated challenges. Finally, we review the prospective clinical applications of G-quadruplex targets, providing references for targeted treatment strategies in gastrointestinal cancers.
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Affiliation(s)
- Zong-Qiang Han
- Department of Laboratory Medicine, Beijing Xiaotangshan Hospital, Beijing 102211, China
| | - Li-Na Wen
- Department of Clinical Nutrition, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China
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25
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Zou Y, Pei J, Long H, Lan L, Dong K, Wang T, Li M, Zhao Z, Zhu L, Zhang G, Jin X, Wang Y, Wen Z, Wei M, Feng Y. H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins. Nat Struct Mol Biol 2023:10.1038/s41594-023-00998-6. [PMID: 37202474 DOI: 10.1038/s41594-023-00998-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2022] [Accepted: 04/12/2023] [Indexed: 05/20/2023]
Abstract
The transmission and maintenance of genetic information in eukaryotic cells relies on the faithful duplication of the entire genome. In each round of division, excessive replication origins are licensed, with only a fraction activated to give rise to bi-directional replication forks in the context of chromatin. However, it remains elusive how eukaryotic replication origins are selectively activated. Here we demonstrate that O-GlcNAc transferase (OGT) enhances replication initiation by catalyzing H4S47 O-GlcNAcylation. Mutation of H4S47 impairs DBF4-dependent protein kinase (DDK) recruitment on chromatin, causing reduced phosphorylation of the replicative helicase mini-chromosome maintenance (MCM) complex and compromised DNA unwinding. Our short nascent-strand sequencing results further confirm the importance of H4S47 O-GlcNAcylation in origin activation. We propose that H4S47 O-GlcNAcylation directs origin activation through facilitating MCM phosphorylation, and this may shed light on the control of replication efficiency by chromatin environment.
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Affiliation(s)
- Yingying Zou
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Jiayao Pei
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Haizhen Long
- Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China
| | - Liting Lan
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Kejian Dong
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Tingting Wang
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Ming Li
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Zhexuan Zhao
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Lirun Zhu
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Gangxuan Zhang
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Xin Jin
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Yang Wang
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Zengqi Wen
- School of Medicine, Sun Yat-sen University, Shenzhen, China
| | - Min Wei
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China
| | - Yunpeng Feng
- Key Laboratory of Molecular Epigenetics of the Ministry of Education (MOE), Northeast Normal University, Changchun, Jilin, China.
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26
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Prorok P, Forouzanfar F, Murugarren N, Peiffer I, Charton R, Akerman I, Méchali M. Loss of Ezh2 function remodels the DNA replication initiation landscape. Cell Rep 2023; 42:112280. [PMID: 36995935 DOI: 10.1016/j.celrep.2023.112280] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 12/13/2022] [Accepted: 03/03/2023] [Indexed: 03/31/2023] Open
Abstract
In metazoan cells, DNA replication initiates from thousands of genomic loci scattered throughout the genome called DNA replication origins. Origins are strongly associated with euchromatin, particularly open genomic regions such as promoters and enhancers. However, over a third of transcriptionally silent genes are associated with DNA replication initiation. Most of these genes are bound and repressed by the Polycomb repressive complex-2 (PRC2) through the repressive H3K27me3 mark. This is the strongest overlap observed for a chromatin regulator with replication origin activity. Here, we asked whether Polycomb-mediated gene repression is functionally involved in recruiting DNA replication origins to transcriptionally silent genes. We show that the absence of EZH2, the catalytic subunit of PRC2, results in increased DNA replication initiation, specifically in the vicinity of EZH2 binding sites. The increase in DNA replication initiation does not correlate with transcriptional de-repression or the acquisition of activating histone marks but does correlate with loss of H3K27me3 from bivalent promoters.
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Affiliation(s)
- Paulina Prorok
- Institute of Human Genetics, CNRS-University of Montpellier, Montpellier 34090, France.
| | - Faezeh Forouzanfar
- Institute of Human Genetics, CNRS-University of Montpellier, Montpellier 34090, France
| | - Nerea Murugarren
- Institute of Metabolism and Systems Research (IMSR), University of Birmingham, Birmingham B152TT, UK; Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham B152TT, UK
| | - Isabelle Peiffer
- Institute of Human Genetics, CNRS-University of Montpellier, Montpellier 34090, France
| | - Romain Charton
- Institute of Human Genetics, CNRS-University of Montpellier, Montpellier 34090, France
| | - Ildem Akerman
- Institute of Metabolism and Systems Research (IMSR), University of Birmingham, Birmingham B152TT, UK; Centre for Endocrinology, Diabetes and Metabolism, Birmingham Health Partners, Birmingham B152TT, UK.
| | - Marcel Méchali
- Institute of Human Genetics, CNRS-University of Montpellier, Montpellier 34090, France.
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27
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Liu D, Sonalkar J, Prasanth SG. ORChestra coordinates the replication and repair music. Bioessays 2023; 45:e2200229. [PMID: 36811379 PMCID: PMC10023367 DOI: 10.1002/bies.202200229] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Revised: 02/03/2023] [Accepted: 02/06/2023] [Indexed: 02/24/2023]
Abstract
Error-free genome duplication and accurate cell division are critical for cell survival. In all three domains of life, bacteria, archaea, and eukaryotes, initiator proteins bind replication origins in an ATP-dependent manner, play critical roles in replisome assembly, and coordinate cell-cycle regulation. We discuss how the eukaryotic initiator, Origin recognition complex (ORC), coordinates different events during the cell cycle. We propose that ORC is the maestro driving the orchestra to coordinately perform the musical pieces of replication, chromatin organization, and repair.
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Affiliation(s)
- Dazhen Liu
- Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601S Goodwin Avenue, Urbana, IL 61801 USA
| | - Jay Sonalkar
- Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601S Goodwin Avenue, Urbana, IL 61801 USA
| | - Supriya G. Prasanth
- Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, 601S Goodwin Avenue, Urbana, IL 61801 USA
- Cancer center at Illinois, UIUC
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28
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Oberdoerffer P, Miller KM. Histone H2A variants: Diversifying chromatin to ensure genome integrity. Semin Cell Dev Biol 2023; 135:59-72. [PMID: 35331626 PMCID: PMC9489817 DOI: 10.1016/j.semcdb.2022.03.011] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2021] [Revised: 03/07/2022] [Accepted: 03/08/2022] [Indexed: 12/12/2022]
Abstract
Histone variants represent chromatin components that diversify the structure and function of the genome. The variants of H2A, primarily H2A.X, H2A.Z and macroH2A, are well-established participants in DNA damage response (DDR) pathways, which function to protect the integrity of the genome. Through their deposition, post-translational modifications and unique protein interaction networks, these variants guard DNA from endogenous threats including replication stress and genome fragility as well as from DNA lesions inflicted by exogenous sources. A growing body of work is now providing a clearer picture on the involvement and mechanistic basis of H2A variant contribution to genome integrity. Beyond their well-documented role in gene regulation, we review here how histone H2A variants promote genome stability and how alterations in these pathways contribute to human diseases including cancer.
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Affiliation(s)
- Philipp Oberdoerffer
- Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21287 USA.
| | - Kyle M Miller
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA; Livestrong Cancer Institutes, Dell Medical School, The University of Texas at Austin, Austin, TX 78712, USA.
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29
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Hu Y, Stillman B. Origins of DNA replication in eukaryotes. Mol Cell 2023; 83:352-372. [PMID: 36640769 PMCID: PMC9898300 DOI: 10.1016/j.molcel.2022.12.024] [Citation(s) in RCA: 66] [Impact Index Per Article: 33.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2022] [Revised: 12/19/2022] [Accepted: 12/21/2022] [Indexed: 01/15/2023]
Abstract
Errors occurring during DNA replication can result in inaccurate replication, incomplete replication, or re-replication, resulting in genome instability that can lead to diseases such as cancer or disorders such as autism. A great deal of progress has been made toward understanding the entire process of DNA replication in eukaryotes, including the mechanism of initiation and its control. This review focuses on the current understanding of how the origin recognition complex (ORC) contributes to determining the location of replication initiation in the multiple chromosomes within eukaryotic cells, as well as methods for mapping the location and temporal patterning of DNA replication. Origin specification and configuration vary substantially between eukaryotic species and in some cases co-evolved with gene-silencing mechanisms. We discuss the possibility that centromeres and origins of DNA replication were originally derived from a common element and later separated during evolution.
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Affiliation(s)
- Yixin Hu
- Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA; Program in Molecular and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA
| | - Bruce Stillman
- Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
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30
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Stem cell plasticity, acetylation of H3K14, and de novo gene activation rely on KAT7. Cell Rep 2023; 42:111980. [PMID: 36641753 DOI: 10.1016/j.celrep.2022.111980] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2019] [Revised: 09/30/2022] [Accepted: 12/23/2022] [Indexed: 01/16/2023] Open
Abstract
In the conventional model of transcriptional activation, transcription factors bind to response elements and recruit co-factors, including histone acetyltransferases. Contrary to this model, we show that the histone acetyltransferase KAT7 (HBO1/MYST2) is required genome wide for histone H3 lysine 14 acetylation (H3K14ac). Examining neural stem cells, we find that KAT7 and H3K14ac are present not only at transcribed genes but also at inactive genes, intergenic regions, and in heterochromatin. KAT7 and H3K14ac were not required for the continued transcription of genes that were actively transcribed at the time of loss of KAT7 but indispensable for the activation of repressed genes. The absence of KAT7 abrogates neural stem cell plasticity, diverse differentiation pathways, and cerebral cortex development. Re-expression of KAT7 restored stem cell developmental potential. Overexpression of KAT7 enhanced neuron and oligodendrocyte differentiation. Our data suggest that KAT7 prepares chromatin for transcriptional activation and is a prerequisite for gene activation.
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31
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Jodkowska K, Pancaldi V, Rigau M, Almeida R, Fernández-Justel J, Graña-Castro O, Rodríguez-Acebes S, Rubio-Camarillo M, Carrillo-de Santa Pau E, Pisano D, Al-Shahrour F, Valencia A, Gómez M, Méndez J. 3D chromatin connectivity underlies replication origin efficiency in mouse embryonic stem cells. Nucleic Acids Res 2022; 50:12149-12165. [PMID: 36453993 PMCID: PMC9757045 DOI: 10.1093/nar/gkac1111] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Revised: 10/31/2022] [Accepted: 11/25/2022] [Indexed: 12/03/2022] Open
Abstract
In mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.
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Affiliation(s)
| | | | | | | | - José M Fernández-Justel
- Functional Organization of the Mammalian Genome Group, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Madrid, Spain
| | - Osvaldo Graña-Castro
- Bioinformatics Unit, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain,Institute of Applied Molecular Medicine (IMMA-Nemesio Díez), San Pablo-CEU University, Boadilla del Monte, Madrid, Spain
| | - Sara Rodríguez-Acebes
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Miriam Rubio-Camarillo
- Bioinformatics Unit, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | | | - David Pisano
- Bioinformatics Unit, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Fátima Al-Shahrour
- Bioinformatics Unit, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
| | - Alfonso Valencia
- Computational Biology Life Sciences Group, Barcelona Supercomputing Center (BSC), Barcelona, Spain
| | - María Gómez
- Correspondence may also be addressed to María Gómez. Tel: +34 911964724; Fax: +34 911964420;
| | - Juan Méndez
- To whom correspondence should be addressed. Tel: +34 917328000; Fax: +34 917328033;
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32
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Masai H. Replicon hypothesis revisited. Biochem Biophys Res Commun 2022; 633:77-80. [DOI: 10.1016/j.bbrc.2022.09.060] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2022] [Accepted: 09/14/2022] [Indexed: 11/06/2022]
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33
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Rhind N. DNA replication timing: Biochemical mechanisms and biological significance. Bioessays 2022; 44:e2200097. [PMID: 36125226 PMCID: PMC9783711 DOI: 10.1002/bies.202200097] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Revised: 08/31/2022] [Accepted: 09/02/2022] [Indexed: 12/27/2022]
Abstract
The regulation of DNA replication is a fascinating biological problem both from a mechanistic angle-How is replication timing regulated?-and from an evolutionary one-Why is replication timing regulated? Recent work has provided significant insight into the first question. Detailed biochemical understanding of the mechanism and regulation of replication initiation has made possible robust hypotheses for how replication timing is regulated. Moreover, technical progress, including high-throughput, single-molecule mapping of replication initiation and single-cell assays of replication timing, has allowed for direct testing of these hypotheses in mammalian cells. This work has consolidated the conclusion that differential replication timing is a consequence of the varying probability of replication origin initiation. The second question is more difficult to directly address experimentally. Nonetheless, plausible hypotheses can be made and one-that replication timing contributes to the regulation of chromatin structure-has received new experimental support.
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Affiliation(s)
- Nicholas Rhind
- Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, Massachusetts, USA
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34
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Dao FY, Lv H, Fullwood MJ, Lin H. Accurate Identification of DNA Replication Origin by Fusing Epigenomics and Chromatin Interaction Information. RESEARCH (WASHINGTON, D.C.) 2022; 2022:9780293. [PMID: 36405252 PMCID: PMC9667886 DOI: 10.34133/2022/9780293] [Citation(s) in RCA: 24] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Accepted: 09/30/2022] [Indexed: 07/29/2023]
Abstract
DNA replication initiation is a complex process involving various genetic and epigenomic signatures. The correct identification of replication origins (ORIs) could provide important clues for the study of a variety of diseases caused by replication. Here, we design a computational approach named iORI-Epi to recognize ORIs by incorporating epigenome-based features, sequence-based features, and 3D genome-based features. The iORI-Epi displays excellent robustness and generalization ability on both training datasets and independent datasets of K562 cell line. Further experiments confirm that iORI-Epi is highly scalable in other cell lines (MCF7 and HCT116). We also analyze and clarify the regulatory role of epigenomic marks, DNA motifs, and chromatin interaction in DNA replication initiation of eukaryotic genomes. Finally, we discuss gene enrichment pathways from the perspective of ORIs in different replication timing states and heuristically dissect the effect of promoters on replication initiation. Our computational methodology is worth extending to ORI identification in other eukaryotic species.
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Affiliation(s)
- Fu-Ying Dao
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China
- School of Biological Sciences, Nanyang Technological University, Singapore 639798, Singapore
- Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Dr, Singapore 117599, Singapore
| | - Hao Lv
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China
- Department of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Melissa J. Fullwood
- School of Biological Sciences, Nanyang Technological University, Singapore 639798, Singapore
- Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Dr, Singapore 117599, Singapore
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A∗STAR), Singapore 138673, Singapore
| | - Hao Lin
- Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China
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35
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Episomes and Transposases-Utilities to Maintain Transgene Expression from Nonviral Vectors. Genes (Basel) 2022; 13:genes13101872. [PMID: 36292757 PMCID: PMC9601623 DOI: 10.3390/genes13101872] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Revised: 10/07/2022] [Accepted: 10/14/2022] [Indexed: 11/04/2022] Open
Abstract
The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and adverse immunological reactions. Nonviral vector systems have been extensively studied and refined during the last decades. Emerging knowledge of the epigenetic regulation of replication and spatial chromatin organisation, as well as new technologies, such as Crispr/Cas, were employed to enhance the performance of different nonviral vector systems. Thus, nonviral vectors are in focus and hold some promising perspectives for future applications in gene therapy. This review addresses three prominent nonviral vector systems: the Sleeping Beauty transposase, S/MAR-based episomes, and viral plasmid replicon-based EBV vectors. Exemplarily, we review different utilities, modifications, and new concepts that were pursued to overcome limitations regarding stable transgene expression and mitotic stability. New insights into the nuclear localisation of nonviral vector molecules and the potential consequences thereof are highlighted. Finally, we discuss the remaining limitations and provide an outlook on possible future developments in nonviral vector technology.
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36
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Peycheva M, Neumann T, Malzl D, Nazarova M, Schoeberl UE, Pavri R. DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations. Science 2022; 377:eabj5502. [DOI: 10.1126/science.abj5502] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Chromosomal translocations result from the joining of DNA double-strand breaks (DSBs) and frequently cause cancer. However, the steps linking DSB formation to DSB ligation remain undeciphered. We report that DNA replication timing (RT) directly regulates lymphomagenic
Myc
translocations during antibody maturation in B cells downstream of DSBs and independently of DSB frequency. Depletion of minichromosome maintenance complexes alters replication origin activity, decreases translocations, and deregulates global RT. Ablating a single origin at
Myc
causes an early-to-late RT switch, loss of translocations, and reduced proximity with the immunoglobulin heavy chain (
Igh
) gene, its major translocation partner. These phenotypes were reversed by restoring early RT. Disruption of early RT also reduced tumorigenic translocations in human leukemic cells. Thus, RT constitutes a general mechanism in translocation biogenesis linking DSB formation to DSB ligation.
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Affiliation(s)
- Mihaela Peycheva
- Research Institute of Molecular Pathology (IMP), Vienna Biocenter, 1030 Vienna, Austria
| | - Tobias Neumann
- Research Institute of Molecular Pathology (IMP), Vienna Biocenter, 1030 Vienna, Austria
- Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna Biocenter, 1030 Vienna, Austria
| | - Daniel Malzl
- Research Institute of Molecular Pathology (IMP), Vienna Biocenter, 1030 Vienna, Austria
| | - Mariia Nazarova
- Research Institute of Molecular Pathology (IMP), Vienna Biocenter, 1030 Vienna, Austria
| | - Ursula E. Schoeberl
- Research Institute of Molecular Pathology (IMP), Vienna Biocenter, 1030 Vienna, Austria
| | - Rushad Pavri
- Research Institute of Molecular Pathology (IMP), Vienna Biocenter, 1030 Vienna, Austria
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37
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Seddon AR, Das AB, Hampton MB, Stevens AJ. Site-specific decreases in DNA methylation in replicating cells following exposure to oxidative stress. Hum Mol Genet 2022; 32:632-648. [PMID: 36106794 PMCID: PMC9896486 DOI: 10.1093/hmg/ddac232] [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: 05/18/2022] [Revised: 09/01/2022] [Accepted: 09/03/2022] [Indexed: 02/07/2023] Open
Abstract
Oxidative stress is a common feature of inflammation-driven cancers, and it promotes genomic instability and aggressive tumour phenotypes. It is known that oxidative stress transiently modulates gene expression through the oxidation of transcription factors and associated regulatory proteins. Neutrophils are our most abundant white blood cells and accumulate at sites of infection and inflammation. Activated neutrophils produce hypochlorous acid and chloramines, which can disrupt DNA methylation by oxidizing methionine. The goal of the current study was to determine whether chloramine exposure results in sequence-specific modifications in DNA methylation that enable long-term alterations in transcriptional output. Proliferating Jurkat T-lymphoma cells were exposed to sublethal doses of glycine chloramine and differential methylation patterns were compared using Illumina EPIC 850 K bead chip arrays. There was a substantial genome-wide decrease in methylation 4 h after exposure that correlated with altered RNA expression for 24 and 48 h, indicating sustained impacts on exposed cells. A large proportion of the most significant differentially methylated CpG sites were situated towards chromosomal ends, suggesting that these regions are most susceptible to inhibition of maintenance DNA methylation. This may contribute to epigenetic instability of chromosomal ends in rapidly dividing cells, with potential implications for the regulation of telomere length and cellular longevity.
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Affiliation(s)
- Annika R Seddon
- University of Otago, Christchurch, Department of Pathology and Biomedical Science, Christchurch, 8011, New Zealand
| | - Andrew B Das
- University of Otago, Christchurch, Department of Pathology and Biomedical Science, Christchurch, 8011, New Zealand,Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia,Sir Peter MacCallum Department of Oncology, The University of Melbourne, Victoria 3010, Australia
| | - Mark B Hampton
- University of Otago, Christchurch, Department of Pathology and Biomedical Science, Christchurch, 8011, New Zealand
| | - Aaron J Stevens
- To whom correspondence should be addressed at: Department of Pathology, University of Otago, Wellington, 23 Mein St, Newtown, Wellington 6021, New Zealand. Tel: +64 43855541; Fax: +64 4 389 5725;
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38
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Gutierrez C. A Journey to the Core of the Plant Cell Cycle. Int J Mol Sci 2022; 23:8154. [PMID: 35897730 PMCID: PMC9330084 DOI: 10.3390/ijms23158154] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Revised: 07/16/2022] [Accepted: 07/21/2022] [Indexed: 02/04/2023] Open
Abstract
Production of new cells as a result of progression through the cell division cycle is a fundamental biological process for the perpetuation of both unicellular and multicellular organisms. In the case of plants, their developmental strategies and their largely sessile nature has imposed a series of evolutionary trends. Studies of the plant cell division cycle began with cytological and physiological approaches in the 1950s and 1960s. The decade of 1990 marked a turn point with the increasing development of novel cellular and molecular protocols combined with advances in genetics and, later, genomics, leading to an exponential growth of the field. In this article, I review the current status of plant cell cycle studies but also discuss early studies and the relevance of a multidisciplinary background as a source of innovative questions and answers. In addition to advances in a deeper understanding of the plant cell cycle machinery, current studies focus on the intimate interaction of cell cycle components with almost every aspect of plant biology.
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Affiliation(s)
- Crisanto Gutierrez
- Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, Nicolas Cabrera 1, Cantoblanco, 28049 Madrid, Spain
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39
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Guilbaud G, Murat P, Wilkes HS, Lerner LK, Sale JE, Krude T. Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation. Nucleic Acids Res 2022; 50:7436-7450. [PMID: 35801867 PMCID: PMC9303276 DOI: 10.1093/nar/gkac555] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 06/14/2022] [Accepted: 06/20/2022] [Indexed: 12/16/2022] Open
Abstract
Replication of the human genome initiates within broad zones of ∼150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq), based on density substitution. Newly replicated DNA is rendered 'heavy-light' (HL) by incorporation of BrdUTP while unreplicated DNA remains 'light-light' (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within early initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.
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Affiliation(s)
- Guillaume Guilbaud
- Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
| | - Pierre Murat
- Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
| | - Helen S Wilkes
- Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK
| | - Leticia Koch Lerner
- Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
| | - Julian E Sale
- Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK
| | - Torsten Krude
- Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK
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40
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Impact of Chromosomal Context on Origin Selection and the Replication Program. Genes (Basel) 2022; 13:genes13071244. [PMID: 35886027 PMCID: PMC9318681 DOI: 10.3390/genes13071244] [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: 04/12/2022] [Revised: 06/28/2022] [Accepted: 07/08/2022] [Indexed: 02/01/2023] Open
Abstract
Eukaryotic DNA replication is regulated by conserved mechanisms that bring about a spatial and temporal organization in which distinct genomic domains are copied at characteristic times during S phase. Although this replication program has been closely linked with genome architecture, we still do not understand key aspects of how chromosomal context modulates the activity of replication origins. To address this question, we have exploited models that combine engineered genomic rearrangements with the unique replication programs of post-quiescence and pre-meiotic S phases. Our results demonstrate that large-scale inversions surprisingly do not affect cell proliferation and meiotic progression, despite inducing a restructuring of replication domains on each rearranged chromosome. Remarkably, these alterations in the organization of DNA replication are entirely due to changes in the positions of existing origins along the chromosome, as their efficiencies remain virtually unaffected genome wide. However, we identified striking alterations in origin firing proximal to the fusion points of each inversion, suggesting that the immediate chromosomal neighborhood of an origin is a crucial determinant of its activity. Interestingly, the impact of genome reorganization on replication initiation is highly comparable in the post-quiescent and pre-meiotic S phases, despite the differences in DNA metabolism in these two physiological states. Our findings therefore shed new light on how origin selection and the replication program are governed by chromosomal architecture.
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41
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Emerson DJ, Zhao PA, Cook AL, Barnett RJ, Klein KN, Saulebekova D, Ge C, Zhou L, Simandi Z, Minsk MK, Titus KR, Wang W, Gong W, Zhang D, Yang L, Venev SV, Gibcus JH, Yang H, Sasaki T, Kanemaki MT, Yue F, Dekker J, Chen CL, Gilbert DM, Phillips-Cremins JE. Cohesin-mediated loop anchors confine the locations of human replication origins. Nature 2022; 606:812-819. [PMID: 35676475 PMCID: PMC9217744 DOI: 10.1038/s41586-022-04803-0] [Citation(s) in RCA: 68] [Impact Index Per Article: 22.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2020] [Accepted: 04/26/2022] [Indexed: 12/18/2022]
Abstract
DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.
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Affiliation(s)
- Daniel J Emerson
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Peiyao A Zhao
- Department of Biological Science, Florida State University, Tallahassee, FL, USA
| | - Ashley L Cook
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - R Jordan Barnett
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Kyle N Klein
- Department of Biological Science, Florida State University, Tallahassee, FL, USA
| | - Dalila Saulebekova
- Institut Curie, PSL Research University, CNRS UMR3244, Dynamics of Genetic Information, Sorbonne Université, Paris, France
| | - Chunmin Ge
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Linda Zhou
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Zoltan Simandi
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Miriam K Minsk
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Katelyn R Titus
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Weitao Wang
- Institut Curie, PSL Research University, CNRS UMR3244, Dynamics of Genetic Information, Sorbonne Université, Paris, France
| | - Wanfeng Gong
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Di Zhang
- Children's Hospital of Pennsylvania, Philadelphia, PA, USA
| | - Liyan Yang
- University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Sergey V Venev
- University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Johan H Gibcus
- University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Hongbo Yang
- Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA
| | - Takayo Sasaki
- San Diego Biomedical Research Institute, San Diego, CA, USA
| | - Masato T Kanemaki
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Mishima, Japan
- Department of Genetics, The Graduate University for Advanced Studies (Sokendai), Mishima, Japan
| | - Feng Yue
- Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA
| | - Job Dekker
- University of Massachusetts Chan Medical School, Worcester, MA, USA
- Howard Hughes Medical Institute, Chevy Chase, MD, USA
| | - Chun-Long Chen
- Institut Curie, PSL Research University, CNRS UMR3244, Dynamics of Genetic Information, Sorbonne Université, Paris, France
| | - David M Gilbert
- Department of Biological Science, Florida State University, Tallahassee, FL, USA
- San Diego Biomedical Research Institute, San Diego, CA, USA
| | - Jennifer E Phillips-Cremins
- Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
- Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
- New York Stem Cell Foundation Robertson Investigator, New York, NY, USA.
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42
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Gatto A, Forest A, Quivy JP, Almouzni G. HIRA-dependent boundaries between H3 variants shape early replication in mammals. Mol Cell 2022; 82:1909-1923.e5. [PMID: 35381196 DOI: 10.1016/j.molcel.2022.03.017] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2021] [Revised: 12/16/2021] [Accepted: 03/09/2022] [Indexed: 10/18/2022]
Abstract
The lack of a consensus DNA sequence defining replication origins in mammals has led researchers to consider chromatin as a means to specify these regions. However, to date, there is no mechanistic understanding of how this could be achieved and maintained given that nucleosome disruption occurs with each fork passage and with transcription. Here, by genome-wide mapping of the de novo deposition of the histone variants H3.1 and H3.3 in human cells during S phase, we identified how their dual deposition mode ensures a stable marking with H3.3 flanked on both sides by H3.1. These H3.1/H3.3 boundaries correspond to the initiation zones of early origins. Loss of the H3.3 chaperone HIRA leads to the concomitant disruption of H3.1/H3.3 boundaries and initiation zones. We propose that the HIRA-dependent deposition of H3.3 preserves H3.1/H3.3 boundaries by protecting them from H3.1 invasion linked to fork progression, contributing to a chromatin-based definition of early replication zones.
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Affiliation(s)
- Alberto Gatto
- Institut Curie, PSL Research University, CNRS, Sorbonne Université, Nuclear Dynamics Unit, Equipe Labellisée Ligue contre le Cancer, 26 rue d'Ulm, 75005 Paris, France
| | - Audrey Forest
- Institut Curie, PSL Research University, CNRS, Sorbonne Université, Nuclear Dynamics Unit, Equipe Labellisée Ligue contre le Cancer, 26 rue d'Ulm, 75005 Paris, France
| | - Jean-Pierre Quivy
- Institut Curie, PSL Research University, CNRS, Sorbonne Université, Nuclear Dynamics Unit, Equipe Labellisée Ligue contre le Cancer, 26 rue d'Ulm, 75005 Paris, France.
| | - Geneviève Almouzni
- Institut Curie, PSL Research University, CNRS, Sorbonne Université, Nuclear Dynamics Unit, Equipe Labellisée Ligue contre le Cancer, 26 rue d'Ulm, 75005 Paris, France.
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43
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Mei L, Kedziora KM, Song EA, Purvis JE, Cook J. The consequences of differential origin licensing dynamics in distinct chromatin environments. Nucleic Acids Res 2022; 50:9601-9620. [PMID: 35079814 PMCID: PMC9508807 DOI: 10.1093/nar/gkac003] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Revised: 12/17/2021] [Accepted: 01/05/2022] [Indexed: 02/01/2023] Open
Abstract
Eukaryotic chromosomes contain regions of varying accessibility, yet DNA replication factors must access all regions. The first replication step is loading MCM complexes to license replication origins during the G1 cell cycle phase. It is not yet known how mammalian MCM complexes are adequately distributed to both accessible euchromatin regions and less accessible heterochromatin regions. To address this question, we combined time-lapse live-cell imaging with immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in euchromatin and heterochromatin throughout G1. We report here that MCM loading in euchromatin is faster than that in heterochromatin in early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. This differential acceleration allows both chromatin types to begin S phase with similar concentrations of loaded MCM. The different loading dynamics require ORCA-dependent differences in origin recognition complex distribution. A consequence of heterochromatin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with underlicensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus, G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.
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Affiliation(s)
- Liu Mei
- Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Katarzyna M Kedziora
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
- Bioinformatics and Analytics Research Collaborative (BARC), University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Eun-Ah Song
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Jeremy E Purvis
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Jeanette Gowen Cook
- Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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44
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Zyner KG, Simeone A, Flynn SM, Doyle C, Marsico G, Adhikari S, Portella G, Tannahill D, Balasubramanian S. G-quadruplex DNA structures in human stem cells and differentiation. Nat Commun 2022; 13:142. [PMID: 35013231 PMCID: PMC8748810 DOI: 10.1038/s41467-021-27719-1] [Citation(s) in RCA: 60] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Accepted: 12/02/2021] [Indexed: 12/13/2022] Open
Abstract
The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.
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Affiliation(s)
- Katherine G Zyner
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK
| | - Angela Simeone
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK
| | - Sean M Flynn
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK
| | - Colm Doyle
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK
| | - Giovanni Marsico
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK
| | - Santosh Adhikari
- Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK
| | - Guillem Portella
- Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK
| | - David Tannahill
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK
| | - Shankar Balasubramanian
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK.
- Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
- School of Clinical Medicine, University of Cambridge, Cambridge, CB2 0SP, UK.
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45
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Li Y, Hartemink AJ, MacAlpine DM. Cell-Cycle-Dependent Chromatin Dynamics at Replication Origins. Genes (Basel) 2021; 12:genes12121998. [PMID: 34946946 PMCID: PMC8701747 DOI: 10.3390/genes12121998] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Revised: 12/02/2021] [Accepted: 12/08/2021] [Indexed: 01/20/2023] Open
Abstract
Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell-cycle–dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors, and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between chromatin occupancy at the ACS and origin efficiency occurred in early S phase, consistent with the rate-limiting formation of the Cdc45–Mcm2-7–GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell-cycle–regulated chromatin dynamics and how they relate to the regulation of origin activity.
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Affiliation(s)
- Yulong Li
- Department of Computer Science, Duke University, Durham, NC 27708, USA;
| | - Alexander J. Hartemink
- Department of Computer Science, Duke University, Durham, NC 27708, USA;
- Correspondence: (A.J.H.); (D.M.M.)
| | - David M. MacAlpine
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
- Correspondence: (A.J.H.); (D.M.M.)
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46
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The genetic architecture of DNA replication timing in human pluripotent stem cells. Nat Commun 2021; 12:6746. [PMID: 34799581 PMCID: PMC8604924 DOI: 10.1038/s41467-021-27115-9] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Accepted: 10/29/2021] [Indexed: 12/11/2022] Open
Abstract
DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome's replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) - sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.
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47
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Colino-Sanguino Y, Clark SJ, Valdes-Mora F. The H2A.Z-nuclesome code in mammals: emerging functions. Trends Genet 2021; 38:273-289. [PMID: 34702577 DOI: 10.1016/j.tig.2021.10.003] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Revised: 09/30/2021] [Accepted: 10/04/2021] [Indexed: 12/12/2022]
Abstract
H2A.Z is a histone variant that provides specific structural and docking-side properties to the nucleosome, resulting in diverse and specialised molecular and cellular functions. In this review, we discuss the latest studies uncovering new functional aspects of mammalian H2A.Z in gene transcription, including pausing and elongation of RNA polymerase II (RNAPII) and enhancer activity; DNA repair; DNA replication; and 3D chromatin structure. We also review the recently described role of H2A.Z in embryonic development, cell differentiation, neurodevelopment, and brain function. In conclusion, our cumulative knowledge of H2A.Z over the past 40 years, in combination with the implementation of novel molecular technologies, is unravelling an unexpected and complex role of histone variants in gene regulation and disease.
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Affiliation(s)
- Yolanda Colino-Sanguino
- Cancer Epigenetics Biology and Therapeutics, Precision Medicine Theme, Children's Cancer Institute, Sydney, NSW, Australia; School of Children and Women Health, University of NSW Sydney, Sydney, NSW, Australia.
| | - Susan J Clark
- Epigenetics Research Laboratory, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia; St. Vincent's Clinical School, University of NSW Sydney, Sydney, NSW, Australia
| | - Fatima Valdes-Mora
- Cancer Epigenetics Biology and Therapeutics, Precision Medicine Theme, Children's Cancer Institute, Sydney, NSW, Australia; School of Children and Women Health, University of NSW Sydney, Sydney, NSW, Australia.
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48
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Colonnetta MM, Abrahante JE, Schedl P, Gohl DM, Deshpande G. CLAMP regulates zygotic genome activation in Drosophila embryos. Genetics 2021; 219:iyab107. [PMID: 34849887 PMCID: PMC8633140 DOI: 10.1093/genetics/iyab107] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2020] [Accepted: 06/15/2020] [Indexed: 11/13/2022] Open
Abstract
Embryonic patterning is critically dependent on zygotic genome activation (ZGA). In Drosophila melanogaster embryos, the pioneer factor Zelda directs ZGA, possibly in conjunction with other factors. Here, we have explored the novel involvement of Chromatin-Linked Adapter for MSL Proteins (CLAMP) during ZGA. CLAMP binds thousands of sites genome-wide throughout early embryogenesis. Interestingly, CLAMP relocates to target promoter sequences across the genome when ZGA is initiated. Although there is a considerable overlap between CLAMP and Zelda binding sites, the proteins display distinct temporal dynamics. To assess whether CLAMP occupancy affects gene expression, we analyzed transcriptomes of embryos zygotically compromised for either clamp or zelda and found that transcript levels of many zygotically activated genes are similarly affected. Importantly, compromising either clamp or zelda disrupted the expression of critical segmentation and sex determination genes bound by CLAMP (and Zelda). Furthermore, clamp knockdown embryos recapitulate other phenotypes observed in Zelda-depleted embryos, including nuclear division defects, centrosome aberrations, and a disorganized actomyosin network. Based on these data, we propose that CLAMP acts in concert with Zelda to regulate early zygotic transcription.
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Affiliation(s)
- Megan M Colonnetta
- Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA
| | - Juan E Abrahante
- University of Minnesota Informatics Institute, Minneapolis, MN 55455, USA
| | - Paul Schedl
- Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA
| | - Daryl M Gohl
- University of Minnesota Genomics Center, Minneapolis, MN 55455, USA
| | - Girish Deshpande
- Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA
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49
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Brossas C, Duriez B, Valton AL, Prioleau MN. Promoters are key organizers of the duplication of vertebrate genomes. Bioessays 2021; 43:e2100141. [PMID: 34319621 DOI: 10.1002/bies.202100141] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2021] [Revised: 07/13/2021] [Accepted: 07/15/2021] [Indexed: 11/06/2022]
Abstract
In vertebrates, single cell analyses of replication timing patterns brought to light a very well controlled program suggesting a tight regulation on initiation sites. Mapping of replication origins with different methods has revealed discrete preferential sites, enriched in promoters and potential G-quadruplex motifs, which can aggregate into initiation zones spanning several tens of kilobases (kb). Another characteristic of replication origins is a nucleosome-free region (NFR). A modified yeast strain containing a humanized origin recognition complex (ORC) fires new origins at NFRs revealing their regulatory role. In cooperation with NFRs, the histone variant H2A.Z facilitates ORC loading through di-methylation of lysine 20 of histone H4. Recent studies using genome editing methods show that efficient initiation sites associated with transcriptional activity can synergize over several tens of kb by establishing physical contacts and lead to the formation of early domains of DNA replication demonstrating a co-regulation between replication initiation and transcription.
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Affiliation(s)
- Caroline Brossas
- Université de Paris, CNRS, Institut Jacques Monod, Paris, France
| | - Bénédicte Duriez
- IMRB, INSERM U955, Equipe GEIC2O, Faculté de Santé, Créteil, France
| | - Anne-Laure Valton
- Department of Biochemistry and Molecular Pharmacology, Program in Systems Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.,Howard Hughes Medical Institute, Chevy Chase, Maryland, USA
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Shi J, Zhang X, Li J, Huang W, Wang Y, Wang Y, Qin J. MTA2 sensitizes gastric cancer cells to PARP inhibition by induction of DNA replication stress. Transl Oncol 2021; 14:101167. [PMID: 34280886 PMCID: PMC8313750 DOI: 10.1016/j.tranon.2021.101167] [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: 06/24/2021] [Accepted: 06/28/2021] [Indexed: 12/24/2022] Open
Abstract
Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib selectively kills cancer cells with BRCA-deficiency and is approved for BRCA-mutated breast, ovarian and pancreatic cancers by FDA. However, phase III study of olaparib failed to show a significant improvement in overall survival in patients with gastric cancer (GC). To discover an effective biomarker for GC patient-selection in olaparib treatment, we analyzed proteomic profiling of 12 GC cell lines. MTA2 was identified to confer sensitivity to olaparib by aggravating olaparib-induced replication stress in cancer cells. Mechanistically, we applied Cleavage Under Targets and Tagmentation assay to find that MTA2 proteins preferentially bind regions of replication origin-associated DNA sequences, which could be enhanced by olaparib treatment. Furthermore, MTA2 was validated here to render cancer cells susceptible to combination of olaparib with ATR inhibitor AZD6738. In general, our study identified MTA2 as a potential biomarker for olaparib sensitivity by aggravating olaparib-induced replication stress.
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Affiliation(s)
- Jinwen Shi
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China
| | - Xiaofeng Zhang
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China
| | - Jin'e Li
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China
| | - Wenwen Huang
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Sun Yat-sen University, Guangzhou 510060, China
| | - Yini Wang
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China
| | - Yi Wang
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China
| | - Jun Qin
- State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing 102206, China.
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