Our long-term goal is to understand the role of DNA replication in cellular epigenetic states. Chromatin is assembled at the replication fork and different types of chromatin are assembled at different times during S-phase. Moreover, many studies have correlated changes in replication timing to changes in gene expression in different cell lineages and in cancer but none have been able to address the intermediate states that accompany these changes. Mechanistic studies will require a system in which these changes can be elicited with sufficient synchrony and homogeneity as to permit biochemical and molecular analyses. We describe such a system in this proposal. We detect dynamic changes in replication timin within a single cell cycle and coincident with key cell fate changes during the differentiation of mouse ES cells to neural precursors. Early to late replication changes coincide with loss of pluripotence and irreversible down-regulation of ES-specific genes, while late to early changes coincide with commitment to neural lineages and up-regulation of neural specific genes. Since replication timing is regulated at the level of large chromosomal domains, our studies have the potential to open a novel chapter in gene regulation. Our working hypothesis is that changes in replication timing during differentiation reinforce the heritability of changes in chromatin structure across large chromosome domains that in turn modulate the responsiveness of genes during stem cell commitment.
In Aim 1 we will perform genome-wide analyses of replication timing, transcription and chromatin states at key stages during differentiation to identify biologically significant relationships. We demonstrate that ES cells lacking the G9a histone methyltransferase replicate a subset of neural-induced genes earlier during S-phase, suggesting a link between histone methylation and replication. One of these genes, the Pleiotrophin (Ptn) gene resides within a 500 kb chromatin domain that switches as a unit from late to early replicating within the same cell cycle in which transcription is induced. Intriguingly, a wave of non-coding transcription begins throughput this chromatin domain 1-2 cell cycles prior to the replication switch, during a definitive ectoderm-like stage. We propose a model in which non-coding transcription elicits changes in histone modifications that accumulate until they trigger a switch in replication timing that in turn transmits the chromatin state to the entire domain, committing the domain to a responsive chromatin state.
Aim 2 addresses the role of transcription in remodeling domain-wide chromatin structure while Aim 3 addresses the role of the G9a histone methyltransferase in regulating replication timing and chromatin structure at the level of large chromatin domains. Lay Relevance: All cells contain the same genetic information (DNA) but package it with proteins into """"""""chromatin"""""""" in characteristic ways that define each cell type. Chromatin is dismantled and re-assembled during each cell division, and we have discovered that the sequence in which segments of DNA are packaged into chromatin changes as stem cells turn into different cell types. Understanding how to manipulate this packaging process may help us engineer different cell types, a central goal in stem cell therapy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Neurogenesis and Cell Fate Study Section (NCF)
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Haynes, Susan R
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Florida State University
Schools of Arts and Sciences
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Rivera-Mulia, Juan Carlos; Dimond, Andrew; Vera, Daniel et al. (2018) Allele-specific control of replication timing and genome organization during development. Genome Res 28:800-811
Sima, Jiao; Bartlett, Daniel A; Gordon, Molly R et al. (2018) Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors. Nucleic Acids Res 46:1810-1820
Dixon, Jesse R; Xu, Jie; Dileep, Vishnu et al. (2018) Integrative detection and analysis of structural variation in cancer genomes. Nat Genet 50:1388-1398
Dileep, Vishnu; Gilbert, David M (2018) Single-cell replication profiling to measure stochastic variation in mammalian replication timing. Nat Commun 9:427
Yang, Yang; Gu, Quanquan; Zhang, Yang et al. (2018) Continuous-Trait Probabilistic Model for Comparing Multi-species Functional Genomic Data. Cell Syst 7:208-218.e11
Marchal, Claire; Sasaki, Takayo; Vera, Daniel et al. (2018) Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq. Nat Protoc 13:819-839
Rivera-Mulia, Juan Carlos; Schwerer, Hélène; Besnard, Emilie et al. (2018) Cellular senescence induces replication stress with almost no affect on DNA replication timing. Cell Cycle 17:1667-1681
Sasaki, Takayo; Rivera-Mulia, Juan Carlos; Vera, Daniel et al. (2017) Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia. Exp Hematol 51:71-82.e3
Rivera-Mulia, Juan Carlos; Desprat, Romain; Trevilla-Garcia, Claudia et al. (2017) DNA replication timing alterations identify common markers between distinct progeroid diseases. Proc Natl Acad Sci U S A 114:E10972-E10980
Sasaki, Takayo; Gilbert, David M (2017) Unearthing worm replication origins. Nat Struct Mol Biol 24:195-196

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