Publications by authors named "David M Gilbert"

124 Publications

Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.

Mol Cell 2021 Jul 21;81(14):2975-2988.e6. Epub 2021 Jun 21.

University of Massachusetts Medical School, Department of Biochemistry and Molecular Pharmacology, Worcester, MA 01605, USA. Electronic address:

The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging ∼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.
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http://dx.doi.org/10.1016/j.molcel.2021.05.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8286344PMC
July 2021

Nuclear organisation and replication timing are coupled through RIF1-PP1 interaction.

Nat Commun 2021 05 18;12(1):2910. Epub 2021 May 18.

Epigenetics & Neurobiology Unit, European Molecular Biology Laboratory (EMBL Rome), Monterotondo, Italy.

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture.
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http://dx.doi.org/10.1038/s41467-021-22899-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131703PMC
May 2021

Replication timing maintains the global epigenetic state in human cells.

Science 2021 04 22;372(6540):371-378. Epub 2021 Apr 22.

Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.

The temporal order of DNA replication [replication timing (RT)] is correlated with chromatin modifications and three-dimensional genome architecture; however, causal links have not been established, largely because of an inability to manipulate the global RT program. We show that loss of RIF1 causes near-complete elimination of the RT program by increasing heterogeneity between individual cells. RT changes are coupled with widespread alterations in chromatin modifications and genome compartmentalization. Conditional depletion of RIF1 causes replication-dependent disruption of histone modifications and alterations in genome architecture. These effects were magnified with successive cycles of altered RT. These results support models in which the timing of chromatin replication and thus assembly plays a key role in maintaining the global epigenetic state.
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http://dx.doi.org/10.1126/science.aba5545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173839PMC
April 2021

Catalytically Enhanced Cas9 through Directed Protein Evolution.

CRISPR J 2021 Apr;4(2):223-232

Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, USA; Florida State University, Tallahassee, Florida, USA.

Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the catalytic efficiency by AceCECas9. We further evolved the more widely used Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.
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http://dx.doi.org/10.1089/crispr.2020.0092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8182482PMC
April 2021

Mammalian DNA Replication Timing.

Cold Spring Harb Perspect Biol 2021 Jul 1;13(7). Epub 2021 Jul 1.

Department of Biological Science, Florida State University, Tallahassee, Florida 32306, USA.

Immediately following the discovery of the structure of DNA and the semi-conservative replication of the parental DNA sequence into two new DNA strands, it became apparent that DNA replication is organized in a temporal and spatial fashion during the S phase of the cell cycle, correlated with the large-scale organization of chromatin in the nucleus. After many decades of limited progress, technological advances in genomics, genome engineering, and imaging have finally positioned the field to tackle mechanisms underpinning the temporal and spatial regulation of DNA replication and the causal relationships between DNA replication and other features of large-scale chromosome structure and function. In this review, we discuss these major recent discoveries as well as expectations for the coming decade.
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http://dx.doi.org/10.1101/cshperspect.a040162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8247564PMC
July 2021

The Tiger Rattlesnake genome reveals a complex genotype underlying a simple venom phenotype.

Proc Natl Acad Sci U S A 2021 01;118(4)

Department of Biological Sciences, Clemson University, Clemson, SC 29634;

Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.
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http://dx.doi.org/10.1073/pnas.2014634118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848695PMC
January 2021

SPIN reveals genome-wide landscape of nuclear compartmentalization.

Genome Biol 2021 Jan 14;22(1):36. Epub 2021 Jan 14.

Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, 15213, PA, USA.

We report SPIN, an integrative computational method to reveal genome-wide intranuclear chromosome positioning and nuclear compartmentalization relative to multiple nuclear structures, which are pivotal for modulating genome function. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and putative associations with nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to other 3D genome features and genome function (transcription and replication timing). SPIN provides critical insights into nuclear spatial and functional compartmentalization.
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http://dx.doi.org/10.1186/s13059-020-02253-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809771PMC
January 2021

Cohesin depleted cells rebuild functional nuclear compartments after endomitosis.

Nat Commun 2020 12 1;11(1):6146. Epub 2020 Dec 1.

Anthropology and Human Genomics, Department Biology II, Ludwig-Maximilians-Universität München, München, Germany.

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.
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http://dx.doi.org/10.1038/s41467-020-19876-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708632PMC
December 2020

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

Nature 2020 07 29;583(7818):699-710. Epub 2020 Jul 29.

Department of Biological Science, Florida State University, Tallahassee, FL, USA.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE and Roadmap Epigenomics data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.
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http://dx.doi.org/10.1038/s41586-020-2493-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410828PMC
July 2020

An integrative ENCODE resource for cancer genomics.

Nat Commun 2020 07 29;11(1):3696. Epub 2020 Jul 29.

Program in Computational Biology & Bioinformatics, Yale University, New Haven, CT, 06520, USA.

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.
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http://dx.doi.org/10.1038/s41467-020-14743-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391744PMC
July 2020

3D genome organization contributes to genome instability at fragile sites.

Nat Commun 2020 07 17;11(1):3613. Epub 2020 Jul 17.

Department of Genetics, The Life Sciences Institute, Hebrew University, Jerusalem, 9190401, Israel.

Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer.
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http://dx.doi.org/10.1038/s41467-020-17448-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367836PMC
July 2020

4D Genome Rewiring during Oncogene-Induced and Replicative Senescence.

Mol Cell 2020 05 26;78(3):522-538.e9. Epub 2020 Mar 26.

Institute of Human Genetics, UMR 9002, CNRS and University of Montpellier, Montpellier, France. Electronic address:

To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.
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http://dx.doi.org/10.1016/j.molcel.2020.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7208559PMC
May 2020

High-resolution Repli-Seq defines the temporal choreography of initiation, elongation and termination of replication in mammalian cells.

Genome Biol 2020 03 24;21(1):76. Epub 2020 Mar 24.

Department of Biological Science, Florida State University, 319 Stadium Drive, Tallahassee, FL, 32306, USA.

Background: DNA replication in mammalian cells occurs in a defined temporal order during S phase, known as the replication timing (RT) programme. Replication timing is developmentally regulated and correlated with chromatin conformation and local transcriptional potential. Here, we present RT profiles of unprecedented temporal resolution in two human embryonic stem cell lines, human colon carcinoma line HCT116, and mouse embryonic stem cells and their neural progenitor derivatives.

Results: Fine temporal windows revealed a remarkable degree of cell-to-cell conservation in RT, particularly at the very beginning and ends of S phase, and identified 5 temporal patterns of replication in all cell types, consistent with varying degrees of initiation efficiency. Zones of replication initiation (IZs) were detected throughout S phase and interacted in 3D space preferentially with other IZs of similar firing time. Temporal transition regions were resolved into segments of uni-directional replication punctuated at specific sites by small, inefficient IZs. Sites of convergent replication were divided into sites of termination or large constant timing regions consisting of many synchronous IZs in tandem. Developmental transitions in RT occured mainly by activating or inactivating individual IZs or occasionally by altering IZ firing time, demonstrating that IZs, rather than individual origins, are the units of developmental regulation. Finally, haplotype phasing revealed numerous regions of allele-specific and allele-independent asynchronous replication. Allele-independent asynchronous replication was correlated with the presence of previously mapped common fragile sites.

Conclusions: Altogether, these data provide a detailed temporal choreography of DNA replication in mammalian cells.
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http://dx.doi.org/10.1186/s13059-020-01983-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092589PMC
March 2020

Local rewiring of genome-nuclear lamina interactions by transcription.

EMBO J 2020 03 21;39(6):e103159. Epub 2020 Feb 21.

Division of Gene Regulation and Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands.

Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). Activation of such genes is often accompanied by repositioning toward the nuclear interior. How this process works and how it impacts flanking chromosomal regions are poorly understood. We addressed these questions by systematic activation or inactivation of individual genes, followed by detailed genome-wide analysis of NL interactions, replication timing, and transcription patterns. Gene activation inside LADs typically causes NL detachment of the entire transcription unit, but rarely more than 50-100 kb of flanking DNA, even when multiple neighboring genes are activated. The degree of detachment depends on the expression level and the length of the activated gene. Loss of NL interactions coincides with a switch from late to early replication timing, but the latter can involve longer stretches of DNA. Inactivation of active genes can lead to increased NL contacts. These extensive datasets are a resource for the analysis of LAD rewiring by transcription and reveal a remarkable flexibility of interphase chromosomes.
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http://dx.doi.org/10.15252/embj.2019103159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073462PMC
March 2020

Replication timing alterations in leukemia affect clinically relevant chromosome domains.

Blood Adv 2019 11;3(21):3201-3213

Department of Biological Science, Florida State University, Tallahassee, FL.

Human B-cell precursor acute lymphoid leukemias (BCP-ALLs) comprise a group of genetically and clinically distinct disease entities with features of differentiation arrest at known stages of normal B-lineage differentiation. We previously showed that BCP-ALL cells display unique and clonally heritable, stable DNA replication timing (RT) programs (ie, programs describing the variable order of replication and subnuclear 3D architecture of megabase-scale chromosomal units of DNA in different cell types). To determine the extent to which BCP-ALL RT programs mirror or deviate from specific stages of normal human B-cell differentiation, we transplanted immunodeficient mice with quiescent normal human CD34+ cord blood cells and obtained RT signatures of the regenerating B-lineage populations. We then compared these with RT signatures for leukemic cells from a large cohort of BCP-ALL patients with varied genetic subtypes and outcomes. The results identify BCP-ALL subtype-specific features that resemble specific stages of B-cell differentiation and features that seem to be associated with relapse. These results suggest that the genesis of BCP-ALL involves alterations in RT that reflect biologically significant and potentially clinically relevant leukemia-specific epigenetic changes.
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http://dx.doi.org/10.1182/bloodadvances.2019000641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855107PMC
November 2019

Control of DNA replication timing in the 3D genome.

Nat Rev Mol Cell Biol 2019 12 2;20(12):721-737. Epub 2019 Sep 2.

Department of Biological Science, Florida State University, Tallahassee, FL, USA.

The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later. During the past decade, this spatiotemporal DNA replication programme has been mapped along the genome and found to correlate with epigenetic marks, transcriptional activity and features of 3D genome architecture such as chromosome compartments and topologically associated domains. But the causal relationship between these features and DNA replication timing and the regulatory mechanisms involved have remained an enigma. The recent identification of cis-acting elements regulating the replication time and 3D architecture of individual replication domains and of long non-coding RNAs that coordinate whole chromosome replication provide insights into such mechanisms.
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http://dx.doi.org/10.1038/s41580-019-0162-yDOI Listing
December 2019

Replication timing networks reveal a link between transcription regulatory circuits and replication timing control.

Genome Res 2019 09 21;29(9):1415-1428. Epub 2019 Aug 21.

Department of Biological Science, Florida State University, Tallahassee, Florida, 32306-4295, USA.

DNA replication occurs in a defined temporal order known as the replication timing (RT) program and is regulated during development, coordinated with 3D genome organization and transcriptional activity. However, transcription and RT are not sufficiently coordinated to predict each other, suggesting an indirect relationship. Here, we exploit genome-wide RT profiles from 15 human cell types and intermediate differentiation stages derived from human embryonic stem cells to construct different types of RT regulatory networks. First, we constructed networks based on the coordinated RT changes during cell fate commitment to create highly complex RT networks composed of thousands of interactions that form specific functional subnetwork communities. We also constructed directional regulatory networks based on the order of RT changes within cell lineages, and identified master regulators of differentiation pathways. Finally, we explored relationships between RT networks and transcriptional regulatory networks (TRNs) by combining them into more complex circuitries of composite and bipartite networks. Results identified novel interactions linking transcription factors that are core to the regulatory circuitry of each cell type to RT changes occurring in those cell types. These core transcription factors were found to bind cooperatively to sites in the affected replication domains, providing provocative evidence that they constitute biologically significant directional interactions. Our findings suggest a regulatory link between the establishment of cell-type-specific TRNs and RT control during lineage specification.
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http://dx.doi.org/10.1101/gr.247049.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724675PMC
September 2019

Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication.

J Virol 2019 10 30;93(20). Epub 2019 Sep 30.

Department of Biological Science, Florida State University, Tallahassee, Florida, USA

Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication. Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.
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http://dx.doi.org/10.1128/JVI.00638-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798117PMC
October 2019

Rapid Irreversible Transcriptional Reprogramming in Human Stem Cells Accompanied by Discordance between Replication Timing and Chromatin Compartment.

Stem Cell Reports 2019 07 20;13(1):193-206. Epub 2019 Jun 20.

Department of Biological Science, Florida State University, 319 Stadium Drive, Tallahassee, FL 32306, USA. Electronic address:

The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties.
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http://dx.doi.org/10.1016/j.stemcr.2019.05.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627004PMC
July 2019

Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication.

Cell 2019 02 27;176(4):816-830.e18. Epub 2018 Dec 27.

Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA. Electronic address:

The temporal order of DNA replication (replication timing [RT]) is highly coupled with genome architecture, but cis-elements regulating either remain elusive. We created a series of CRISPR-mediated deletions and inversions of a pluripotency-associated topologically associating domain (TAD) in mouse ESCs. CTCF-associated domain boundaries were dispensable for RT. CTCF protein depletion weakened most TAD boundaries but had no effect on RT or A/B compartmentalization genome-wide. By contrast, deletion of three intra-TAD CTCF-independent 3D contact sites caused a domain-wide early-to-late RT shift, an A-to-B compartment switch, weakening of TAD architecture, and loss of transcription. The dispensability of TAD boundaries and the necessity of these "early replication control elements" (ERCEs) was validated by deletions and inversions at additional domains. Our results demonstrate that discrete cis-regulatory elements orchestrate domain-wide RT, A/B compartmentalization, TAD architecture, and transcription, revealing fundamental principles linking genome structure and function.
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http://dx.doi.org/10.1016/j.cell.2018.11.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546437PMC
February 2019

RT States: systematic annotation of the human genome using cell type-specific replication timing programs.

Bioinformatics 2019 07;35(13):2167-2176

Department of Biostatistics and Bioinformatics, Rollins School of Public Health, Emory University, Atlanta, GA, USA.

Motivation: The replication timing (RT) program has been linked to many key biological processes including cell fate commitment, 3D chromatin organization and transcription regulation. Significant technology progress now allows to characterize the RT program in the entire human genome in a high-throughput and high-resolution fashion. These experiments suggest that RT changes dynamically during development in coordination with gene activity. Since RT is such a fundamental biological process, we believe that an effective quantitative profile of the local RT program from a diverse set of cell types in various developmental stages and lineages can provide crucial biological insights for a genomic locus.

Results: In this study, we explored recurrent and spatially coherent combinatorial profiles from 42 RT programs collected from multiple lineages at diverse differentiation states. We found that a Hidden Markov Model with 15 hidden states provide a good model to describe these genome-wide RT profiling data. Each of the hidden state represents a unique combination of RT profiles across different cell types which we refer to as 'RT states'. To understand the biological properties of these RT states, we inspected their relationship with chromatin states, gene expression, functional annotation and 3D chromosomal organization. We found that the newly defined RT states possess interesting genome-wide functional properties that add complementary information to the existing annotation of the human genome.

Availability And Implementation: R scripts for inferring HMM models and Perl scripts for further analysis are available https://github.com/PouletAxel/script_HMM_Replication_timing.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681175PMC
July 2019

Integrative detection and analysis of structural variation in cancer genomes.

Nat Genet 2018 10 10;50(10):1388-1398. Epub 2018 Sep 10.

Altius institute for Biomedical Sciences, Seattle, WA, USA.

Structural variants (SVs) can contribute to oncogenesis through a variety of mechanisms. Despite their importance, the identification of SVs in cancer genomes remains challenging. Here, we present a framework that integrates optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole-genome sequencing to systematically detect SVs in a variety of normal or cancer samples and cell lines. We identify the unique strengths of each method and demonstrate that only integrative approaches can comprehensively identify SVs in the genome. By combining Hi-C and optical mapping, we resolve complex SVs and phase multiple SV events to a single haplotype. Furthermore, we observe widespread structural variation events affecting the functions of noncoding sequences, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel three-dimensional chromatin structural domains. Our results indicate that noncoding SVs may be underappreciated mutational drivers in cancer genomes.
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http://dx.doi.org/10.1038/s41588-018-0195-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301019PMC
October 2018

Cellular senescence induces replication stress with almost no affect on DNA replication timing.

Cell Cycle 2018 21;17(13):1667-1681. Epub 2018 Aug 21.

b Laboratory of Genome and Stem Cell Plasticity in Development and Aging , Institute of Regenerative Medicine, U1183, Université de Montpellier , Montpellier Cedex , France.

Organismal aging entails a gradual decline of normal physiological functions and a major contributor to this decline is withdrawal of the cell cycle, known as senescence. Senescence can result from telomere diminution leading to a finite number of population doublings, known as replicative senescence (RS), or from oncogene overexpression, as a protective mechanism against cancer. Senescence is associated with large-scale chromatin re-organization and changes in gene expression. Replication stress is a complex phenomenon, defined as the slowing or stalling of replication fork progression and/or DNA synthesis, which has serious implications for genome stability, and consequently in human diseases. Aberrant replication fork structures activate the replication stress response leading to the activation of dormant origins, which is thought to be a safeguard mechanism to complete DNA replication on time. However, the relationship between replicative stress and the changes in the spatiotemporal program of DNA replication in senescence progression remains unclear. Here, we studied the DNA replication program during senescence progression in proliferative and pre-senescent cells from donors of various ages by single DNA fiber combing of replicated DNA, origin mapping by sequencing short nascent strands and genome-wide profiling of replication timing (TRT). We demonstrate that, progression into RS leads to reduced replication fork rates and activation of dormant origins, which are the hallmarks of replication stress. However, with the exception of a delay in RT of the CREB5 gene in all pre-senescent cells, RT was globally unaffected by replication stress during entry into either oncogene-induced or RS. Consequently, we conclude that RT alterations associated with physiological and accelerated aging, do not result from senescence progression. Our results clarify the interplay between senescence, aging and replication programs and demonstrate that RT is largely resistant to replication stress.
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http://dx.doi.org/10.1080/15384101.2018.1491235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133336PMC
November 2019

Continuous-Trait Probabilistic Model for Comparing Multi-species Functional Genomic Data.

Cell Syst 2018 08 20;7(2):208-218.e11. Epub 2018 Jun 20.

Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA 15213, USA. Electronic address:

A large amount of multi-species functional genomic data from high-throughput assays are becoming available to help understand the molecular mechanisms for phenotypic diversity across species. However, continuous-trait probabilistic models, which are key to such comparative analysis, remain under-explored. Here we develop a new model, called phylogenetic hidden Markov Gaussian processes (Phylo-HMGP), to simultaneously infer heterogeneous evolutionary states of functional genomic features in a genome-wide manner. Both simulation studies and real data application demonstrate the effectiveness of Phylo-HMGP. Importantly, we applied Phylo-HMGP to analyze a new cross-species DNA replication timing (RT) dataset from the same cell type in five primate species (human, chimpanzee, orangutan, gibbon, and green monkey). We demonstrate that our Phylo-HMGP model enables discovery of genomic regions with distinct evolutionary patterns of RT. Our method provides a generic framework for comparative analysis of multi-species continuous functional genomic signals to help reveal regions with conserved or lineage-specific regulatory roles.
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http://dx.doi.org/10.1016/j.cels.2018.05.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107375PMC
August 2018

Allele-specific control of replication timing and genome organization during development.

Genome Res 2018 06 7;28(6):800-811. Epub 2018 May 7.

Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4295, USA.

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid × mouse crosses and exploited the high single-nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (total nuclear RNA-seq), and chromatin accessibility (ATAC-seq). We also present HARP, a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of six different hybrid mESC clones with different genomes (C57BL/6, 129/sv, and CAST/Ei), parental configurations, and gender revealed significant RT asynchrony between alleles across ∼12% of the autosomal genome linked to subspecies genomes but not to parental origin, growth conditions, or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not as strongly with SNP density, gene expression, imprinting, or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types, including extraembryonic endoderm stem (XEN) cells, four male and female primary mouse embryonic fibroblasts (MEFs), and neural precursor cells (NPCs) differentiated in vitro from mESCs with opposite parental configurations. We found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs were largely lost in all differentiated cell types, accompanied by novel sites of allelic asynchrony at a considerably smaller proportion of the genome, suggesting that genome organization of homologs converges to similar folding patterns during cell fate commitment.
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http://dx.doi.org/10.1101/gr.232561.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991511PMC
June 2018

Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq.

Nat Protoc 2018 05 29;13(5):819-839. Epub 2018 Mar 29.

Department of Biological Science, Florida State University, Tallahassee, Florida, USA.

This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and subnuclear position. Moreover, RT is regulated during development and is altered in diseases. Here, we describe E/L Repli-seq, an extension of our Repli-chip protocol. E/L Repli-seq is a rapid, robust and relatively inexpensive protocol for analyzing RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse-labeled with BrdU, and early and late S-phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S-phase fractions. The results are comparable to those of Repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single-nucleotide polymorphisms (SNPs) to analyze RT allelic asynchrony, and for direct comparison to Repli-chip data. This protocol can be performed in up to 3 d before sequencing, and requires basic cellular and molecular biology skills, as well as a basic understanding of Unix and R.
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http://dx.doi.org/10.1038/nprot.2017.148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044726PMC
May 2018

Single-cell replication profiling to measure stochastic variation in mammalian replication timing.

Nat Commun 2018 01 30;9(1):427. Epub 2018 Jan 30.

Department of Biological Science, Florida State University, 319 Stadium Drive, Tallahassee, FL, 32306, USA.

Mammalian DNA replication is regulated via multi-replicon segments that replicate in a defined temporal order during S-phase. Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments. Although replication origins are selected stochastically, variation in replication timing is poorly understood. Here we devise a strategy to measure variation in replication timing using DNA copy number in single mouse embryonic stem cells. We find that borders between replicated and unreplicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus. Fifty percent of replication events deviated from their average replication time by ± 15% of S phase. This degree of variation is similar between cells, between homologs within cells and between all domains genomewide, regardless of their replication timing. These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.
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http://dx.doi.org/10.1038/s41467-017-02800-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789892PMC
January 2018

Replication Domains: Genome Compartmentalization into Functional Replication Units.

Adv Exp Med Biol 2017 ;1042:229-257

Department of Biological Science, Florida State University, Tallahassee, FL, USA.

DNA replication occurs in a defined temporal order during S phase, known as the replication timing programme, which is regulated not only during the cell cycle but also during the process of development and differentiation. The units of replication timing regulation, known as replication domains (RDs), frequently comprise several nearly synchronously firing replication origins. Replication domains correspond to topologically associating domains (TADs) mapped by chromatin conformation capture methods and are likely to be the molecular equivalents of replication foci observed using cytogenetic methods. Both TAD and replication foci are considered to be stable structural units of chromosomes, conserved through the cell cycle and development, and accordingly, the boundaries of RDs also appear to be stable in different cell types. During both normal development and progression of disease, distinct cell states are characterized by unique replication timing signatures, with approximately half of genomic RDs switching replication timing between these cell states. Advances in functional genomics provide hope that we can soon gain an understanding of the cause and consequence of the replication timing programme and its myriad correlations with chromatin context and gene regulation.
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http://dx.doi.org/10.1007/978-981-10-6955-0_11DOI Listing
July 2018

Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors.

Nucleic Acids Res 2018 02;46(4):1810-1820

Department of Biological Science, 319 Stadium Drive, Florida State University, Tallahassee, FL 32306, USA.

The role of DNA sequence in determining replication timing (RT) and chromatin higher order organization remains elusive. To address this question, we have developed an extra-chromosomal replication system (E-BACs) consisting of ∼200 kb human bacterial artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) stable segregation elements. E-BACs were stably maintained as autonomous mini-chromosomes in EBNA1-expressing HeLa or human induced pluripotent stem cells (hiPSCs) and established distinct RT patterns. An E-BAC harboring an early replicating chromosomal region replicated early during S phase, while E-BACs derived from RT transition regions (TTRs) and late replicating regions replicated in mid to late S phase. Analysis of E-BAC interactions with cellular chromatin (4C-seq) revealed that the early replicating E-BAC interacted broadly throughout the genome and preferentially with the early replicating compartment of the nucleus. In contrast, mid- to late-replicating E-BACs interacted with more specific late replicating chromosomal segments, some of which were shared between different E-BACs. Together, we describe a versatile system in which to study the structure and function of chromosomal segments that are stably maintained separately from the influence of cellular chromosome context.
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http://dx.doi.org/10.1093/nar/gkx1265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829748PMC
February 2018

DNA replication timing alterations identify common markers between distinct progeroid diseases.

Proc Natl Acad Sci U S A 2017 12 1;114(51):E10972-E10980. Epub 2017 Dec 1.

Department of Biological Science, Florida State University, Tallahassee, FL 32306;

Progeroid syndromes are rare genetic disorders that phenotypically resemble natural aging. Different causal mutations have been identified, but no molecular alterations have been identified that are in common to these diseases. DNA replication timing (RT) is a robust cell type-specific epigenetic feature highly conserved in the same cell types from different individuals but altered in disease. Here, we characterized DNA RT program alterations in Hutchinson-Gilford progeria syndrome (HGPS) and Rothmund-Thomson syndrome (RTS) patients compared with natural aging and cellular senescence. Our results identified a progeroid-specific RT signature that is common to cells from three HGPS and three RTS patients and distinguishes them from healthy individuals across a wide range of ages. Among the RT abnormalities, we identified the tumor protein p63 gene () as a gene marker for progeroid syndromes. By using the redifferentiation of four patient-derived induced pluripotent stem cells as a model for the onset of progeroid syndromes, we tracked the progression of RT abnormalities during development, revealing altered RT of the gene as an early event in disease progression of both HGPS and RTS. Moreover, the RT abnormalities in progeroid patients were associated with altered isoform expression of Our findings demonstrate the value of RT studies to identify biomarkers not detected by other methods, reveal abnormal RT as an early event in progeroid disease progression, and suggest gene regulation as a potential therapeutic target.
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http://dx.doi.org/10.1073/pnas.1711613114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754778PMC
December 2017
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