Biology subjects

Gilbert, D. M.

Publications and source records attributed to Gilbert, D. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Decoding single-cell replication states from imaging data reveals locus-specific subnuclear position dependencies of replication initiation

Replication timing (RT) is a fundamental feature of genome regulation, tightly linked to chromatin state and three-dimensional (3D) nuclear organization. Yet how the reproducible population-level timing program emerges from stochastic replication decisions in individual cells remains unclear, because it requires measuring RT and 3D genome organization jointly in the same cell. Here, we introduce RepTile, the first systematic framework that infers allele-resolved, single-cell replication states directly from multiplexed FISH and chromosome tracing experiments. By extracting replication information from spot-count distributions while correcting for genomic and spatial detection biases, RepTile infers cell-cycle progression, locus-specific replication timing, cell-to-cell heterogeneity in replication timing, and replication states of individual allele-resolved locus copies within their native three-dimensional nuclear context. Using RepTile on mouse embryonic stem cells, we find that the association between replication timing and subnuclear position observed in population-level genomic experiments conceals fundamentally different locus-specific behaviors. We found two classes of chromatin regions: "position-sensitive" and "position-insensitive". Position-sensitive regions replicate at different times depending on their subnuclear location, typically firing earliest when positioned in their canonical locale. Strikingly, this holds even when the canonical locale is repressive, such as the nuclear envelope. On the other hand, position-insensitive loci replicate at their characteristic times regardless of subnuclear position. These classes map onto distinct regulatory regimes, which are particularly pronounced at replication initiation zones (IZ): position-sensitive IZs are enriched in domains that replicate constitutively early or late across cell types, whereas position-insensitive IZs are enriched in domains that switch RT developmentally and contain early replication control elements (ERCEs), cis-regulatory elements known to govern IZ firing times. Together, these results challenge the view that subnuclear location broadly defines replication timing, indicating that positional dependence is specific to constitutive domains, while developmental domains carry intrinsic cis-acting programs that decouple their replication from subnuclear location.

genomics↗

bioRxiv2026

Microhomology-mediated end joining acts directly on replication forks to repair single-ended double strand breaks

Replication stress, intrinsic to oncogenesis, often leads to fork breakage and double-strand break (DSB) formation. Conventionally, break-induced replication (BIR) is considered the primary mechanism for repairing replication-associated single-ended DSBs (seDSBs). Here, we demonstrate that microhomology-mediated end joining (MMEJ) acts directly to repair seDSBs at broken replication forks (fork-MMEJ), preferentially on the leading strands, and functions cooperatively with BIR. We also showed that while fork-MMEJ is promoted by Pol{theta}, it operates independently of MRE11/CtIP-mediated end resection, relies on RPA, and produces asymmetric deletion patterns, which is distinct from canonical MMEJ (cMMEJ) defined at replication-independent double-ended DSBs (deDSBs). ATR, activated as end resection proceeds, serves as a pivotal switch to suppress fork-MMEJ while promoting BIR. Combined inactivation of ATR and Pol{theta} synergistically kills cancer cells under high replication stress with minimal toxicity to normal cells. Together, our study provides fundamental insights into the MMEJ mechanism and offers new strategies for cancer treatment.

molecular biology↗

bioRxiv2025

Autosomal Allelic Inactivation: Variable Replication and Dosage Sensitivity

Autosomal monoallelic gene expression and asynchronous replication between alleles are established features of imprinted genes and genes regulated by allelic exclusion. Inactivation/Stability Centers (I/SCs) are recently described autosomal loci that exhibit epigenetic regulation of allelic expression and replication timing, with differences that can be comparable to those observed between the active and inactive X chromosomes1. Here we characterize >100 autosomal loci with allele-specific epigenetic regulation of replication timing and gene expression, defining them as I/SCs. I/SCs are approximately 1 megabase in size and can contain both protein-coding and noncoding genes. In different single cell derived clones, these genes may be expressed from a single allele, the opposite allele, both alleles, or not expressed at all. This stochastic, yet mitotically stable, pattern indicates that the choice of which allele is expressed is independent of parent of origin and independent of the expression status of the other allele. Similarly, alleles within I/SCs show varying replication timing, either earlier or later, that is also independent of the other allele. Additionally, we identify syntenic loci in the mouse genome that display epigenetic regulation of allelic replication timing, highlighting the genomic organization and conservation of I/SC-associated regulation between human and mouse genomes. The allele-restricted regulation described here creates extensive cellular mosaicism through a stable epigenetic mechanism. This mosaicism impacts numerous dosage-sensitive genes associated with human diseases such as Alzheimer, Parkinson, epilepsy, deafness, and impaired intellectual development.

genomics↗

bioRxiv2025

RIF1 orchestrates a multi-step restoration of post-replicative H3K9me3

DNA replication causes the dilution of parental histones along with their specific post-translational modifications. The kinetics of restoring these marks on newly incorporated histones dictate how quickly genomic domains regain their epigenetic identity after replication. H3K9me3 is restored extremely slowly; the process of reconstitution, to achieve the pre-replication levels, continues throughout the following G1 phase. The molecular mechanisms behind this slow reconstitution are unknown. We show here that RIF1s reassociation with heterochromatin during mitotic exit is required to set up a chromatin environment permissive for histone methyltransferases to resume H3K9me3 deposition. RIF1 facilitates the recruitment of SUV39H1, HP1 and HP1{beta} and is required for the increased tri-methylation of H3K9 that occurs during G1 phase. RIF1 is also indispensable for recruiting Protein Phosphatase 1 (PP1) to heterochromatin, and the interaction between RIF1 and PP1 is essential for the maintenance of H3K9me3 levels. In addition, RIF1-PP1 complex temporally restrains the activity of Aurora kinase at heterochromatin, ensuring that phosphorylation of H3S10 does not precede replication. This creates a time- window permissive for SUV39Hs to initiate the reinstatement of H3K9me3.

cell biology↗

bioRxiv2025

Transcription can be sufficient, but is not necessary, to advance replication timing

DNA replication timing (RT) is correlated with transcription during cell fate changes but there are many exceptions and our understanding of this relationship suffers from a paucity of reductionist approaches. Here, we manipulated length and strength of transcription in hybrid-genome mouse embryonic stem cells (mESCs) at a single locus upstream of the silent, late replicating, Pleiotrophin (Ptn) gene, directly comparing RT to nascent transcription rates at engineered vs. wild-type alleles. First, we inserted four reporter genes that differ only in their promoter. Two promoters transcribed the reporter gene at high rates and advanced RT. The other two transcribed at lower rates and did not advance RT. Since these promoters may prove useful in applications where effects on RT are undesirable, we confirmed the inability of one of them to advance RT at numerous ectopic sites. We next juxtaposed these same four promoters upstream of the Ptn transcription start site where they all transcribed the 96kb Ptn gene and advanced RT to different extents correlated with transcription rates. Indeed, a doxycycline-responsive promoter, which could not advance RT when induced as a small reporter gene, elicited a rapid and reversible RT advance proportional to the rate of transcription, providing direct evidence that transcription itself can advance RT. However, deletion of the Ptn promoter and enhancer, followed by directed differentiation to neural precursors, eliminated induction of transcription throughout the entire Ptn replication domain, without preventing the switch to early replication. Our results provide a solid empirical base with which to re-evaluate many decades of literature, demonstrating that length and strength of transcription is sufficient but not necessary to advance RT. Our results also provide a robust system in which to rapidly effect an RT change, permitting mechanistic studies of the role of transcription in RT and the consequences of RT changes to epigenomic remodeling.

genomics↗

bioRxiv2024

Centromeric transposable elements and epigenetic status drive karyotypic variation in the eastern hoolock gibbon

Great apes have maintained a stable karyotype with few large-scale rearrangements; in contrast, gibbons have undergone a high rate of chromosomal rearrangements coincident with rapid centromere turnover. Here we characterize assembled centromeres in the Eastern hoolock gibbon, Hoolock leuconedys (HLE), finding a diverse group of transposable elements (TEs) that differ from the canonical alpha satellites found across centromeres of other apes. We find that HLE centromeres contain a CpG methylation centromere dip region, providing evidence this epigenetic feature is conserved in the absence of satellite arrays; nevertheless, we report a variety of atypical centromeric features, including protein-coding genes and mismatched replication timing. Further, large structural variations define HLE centromeres and distinguish them from other gibbons. Combined with differentially methylated TEs, topologically associated domain boundaries, and segmental duplications at chromosomal breakpoints, we propose that a "perfect storm" of multiple genomic attributes with propensities for chromosome instability shaped gibbon centromere evolution.

genomics↗

bioRxiv2024

Beyond A and B Compartments: how major nuclear locales define nuclear genome organization and function

Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Whereas gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.

genomics↗

bioRxiv2023

Brd2 is dispensable for genome compartmentalization and replication timing

Replication Timing (RT) refers to the temporal order in which the genome is replicated during S phase. Early replicating regions correlate with the transcriptionally active, accessible euchromatin (A) compartment, while late replicating regions correlate with the heterochromatin (B) compartment and repressive histone marks. Previously, widespread A/B genome compartmentalization changes were reported following Brd2 depletion. Since RT and A/B compartmentalization are two of the most highly correlated chromosome properties, we evaluated the effects of Brd2 depletion on RT. We performed E/L Repli-Seq following Brd2 depletion in the previously described Brd2 conditional degron cell line and found no significant alterations in RT after Brd2 KD. This finding prompted us to re-analyze the Micro-C data from the previous publication. We report that we were unable to detect any compartmentalization changes in Brd2 depleted cells compared to DMSO control using the same data. Taken together, our findings demonstrate that Brd2 depletion alone does not affect A/B compartmentalization or RT in mouse embryonic stem cells.

genomics↗

bioRxiv2023

Nucleolus and centromere TSA-Seq reveals variable localization of heterochromatin in different cell types

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology.

cell biology↗

bioRxiv2023

Master transcription factor binding sites are necessary for early replication control element activity

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.

molecular biology↗

bioRxiv2023

Transcription-Replication Conflict Shapes DNA Break Dynamics

Recurrent DNA break clusters (RDCs) are replication-transcription collision hotspots; many are unique to neural progenitor cells. Through high-resolution replication sequencing and a capture-ligation assay in mouse neural progenitor cells experiencing replication stress, we unraveled the replication features dictating RDC location and orientation. Most RDCs occur at the replication forks traversing timing transition regions (TTRs), where sparse replication origins connect unidirectional forks. Leftward-moving forks generate telomere-connected DNA double-strand breaks (DSBs), while rightward-moving forks lead to centromere-connected DSBs. Strand-specific mapping for DNA-bound RNA revealed co-transcriptional dual-strand DNA:RNA hybrids present at a higher density in RDC than in other actively transcribed long genes. In addition, mapping RNA polymerase activity revealed that head-to-head interactions between replication and transcription machinery resulted in 60% DSB contribution to the head-on compared to 40% for co-directional. Our findings revealed TTR as a novel fragile class and highlighted how the linear interaction between transcription and replication impacts genome stability.

genomics↗

bioRxiv2023

RIF1 regulates replication origin activity and early replication timing in B cells

The mammalian DNA replication timing (RT) program is crucial for the proper functioning and integrity of the genome. The best-known mechanism for controlling RT is the suppression of late origins of replication in heterochromatin by RIF1. Here, we report that in antigen-activated B lymphocytes, RIF1 binds predominantly to early-replicating active chromatin, regulates early origin firing and promotes early replication. RIF1 has a minor role in gene expression and genome organization in B cells. Furthermore, we find that RIF1 functions in a complementary and non-epistatic manner with minichromosome maintenance (MCM) proteins to establish early RT signatures genome-wide and, specifically, to ensure the early replication of highly transcribed genes. These findings reveal new layers of regulation within the B cell RT program, driven by the coordinated activity of RIF1 and MCM proteins.

molecular biology↗

bioRxiv2022

Epigenetic Control of Hundreds of Chromosome-Associated lncRNA Genes Essential for Replication and Stability

ASARs are long noncoding RNA genes that control replication timing of entire human chromosomes in cis. The three known ASAR genes are located on human chromosomes 6 and 15, and are essential for chromosome integrity. To identify ASARs on all human chromosomes we utilized a set of distinctive ASAR characteristics that allowed for the identification of hundreds of autosomal loci with epigenetically controlled, allele-restricted behavior in expression and replication timing of coding and noncoding genes, and is distinct from genomic imprinting. Disruption of noncoding RNA genes at five of five tested loci resulted in chromosome-wide delayed replication and chromosomal instability, validating their ASAR activity. In addition to the three known essential cis-acting chromosomal loci, origins, centromeres, and telomeres, we propose that all mammalian chromosomes also contain "Inactivation/Stability Centers" that display allele-restricted epigenetic regulation of protein coding and noncoding ASAR genes that are essential for replication and stability of each chromosome.

genetics↗

bioRxiv2021

Dynamic chromosomal interactions and control of heterochromatin positioning by Ki67

Ki-67 is a chromatin-associated protein with a dynamic distribution pattern throughout the cell cycle, and is thought to be involved in chromatin organization. Lack of genomic interaction maps has hampered a detailed understanding of its roles, particularly during interphase. By pA-DamID mapping in human cell lines we found that Ki-67 associates with large genomic domains that overlap mostly with late-replicating regions. Early in interphase, when Ki-67 is present in pre-nucleolar bodies, it interacts with these domains on all chromosomes. However, later in interphase, when Ki-67 is confined to nucleoli, it shows a striking shift towards small chromosomes. Nucleolar perturbations indicate that these cell cycle dynamics correspond to nucleolar maturation during interphase, and suggest that nucleolar sequestration of Ki-67 limits its interactions with larger chromosomes. Furthermore, we demonstrate that Ki-67 does not detectably control chromatin-chromatin interactions during interphase, but it competes with the nuclear lamina for interaction with late-replicating DNA, and it controls replication timing of (peri)centromeric regions. Together, these results reveal a highly dynamic choreography of genome interactions and roles for Ki-67 in heterochromatin organization.

genomics↗

bioRxiv2021

High throughput genome-wide single cell protein:DNA binding site mapping by targeted insertion of promoters (TIP-seq)

Assessing cell to cell, and importantly, chromosome to chromosome, heterogeneity in cellular phenotypes is a central goal of modern cell biology. However, chromatin profiling in single cells has been extremely challenging, and single chromosome profiling has not been achieved. In cases where single cell methods have shown promise, success has been mainly limited to histone proteins and/or require highly specialized equipment or cell type specific protocols and are relatively low throughput. Here, we have combined the advantages of tagmentation, linear amplification and combinatorial indexing to produce a high throughput single cell DNA binding site mapping method that does not require specialized equipment and is capable of multiplexing several samples/ target proteins in one experiment. Targeted Insertion of Promoters (TIP-seq) uses Tn5 fused to protein A (as with CUT&Tag) to insert a T7 RNA polymerase promoter into sites adjacent to an antibody bound to a chromatin protein of interest, followed by linear amplification of flanking DNA with T7 polymerase, cDNA preparation and PCR indexing. Tip-seq provides [~]10-fold higher unique reads and thus higher coverage per single cell compared to state-of-the-art methods. We apply TIP-seq to map histone modifications, RNA PolII and CTCF binding sites in single human and mouse cells. TIP-seq will also be adaptable for other platforms, such as 10X genomics and ICELL8. In summary, TIP-seq provides a high-throughput, low-cost method for single cell protein mapping, that yields substantially higher coverage per cell and signal to noise than existing methods.

genomics↗

bioRxiv2021

Cohesin-mediated loop anchors confine the location of human replication origins

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability across dividing cells in metazoans. It is currently unknown how the location of replication origins and the timing of their activation is determined in the human genome. Here, we dissect the role for G1 phase topologically associating domains (TADs), subTADs, and loops in the activation of replication initiation zones (IZs). We identify twelve subtypes of self-interacting chromatin domains distinguished by their degree of nesting, the presence of corner dot structures indicative of loops, and their co-localization with A/B compartments. Early replicating IZs localize to boundaries of nested corner-dot TAD/subTADs anchored by high density arrays of co-occupied CTCF+cohesin binding sites with divergently oriented motifs. By contrast, late replicating IZs localize to weak TADs/subTAD boundaries devoid of corner dots and most often anchored by singlet CTCF+cohesin sites. Upon global knock-down of cohesin-mediated loops in G1, early wave focal IZs replicate later in S phase and convert to diffuse placement along the genome. Moreover, IZs in mid-late S phase are delayed to the final minutes before entry into G2 when cohesin-mediated dot-less boundaries are ablated. We also delete a specific loop anchor and observe a sharp local delay of an early wave IZ to replication in late S phase. Our data demonstrate that cohesin-mediated loops at genetically-encoded TAD/subTAD boundaries in G1 phase are an essential determinant of the precise genomic placement of human replication origins in S phase.

genomics↗

bioRxiv2020

Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication Timing

DNA replication is regulated by the location and timing of replication initiation. Therefore, much effort has been invested in identifying and analyzing the sites of human replication initiation. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation site utilization in metazoans has made mapping the location and timing of replication initiation in human cells difficult. A potential solution to the problem of human replication mapping is single-molecule analysis. However, current approaches do not provide the throughput required for genome-wide experiments. To address this challenge, we have developed Optical Replication Mapping (ORM), a high-throughput single-molecule approach to map newly replicated DNA, and used it to map early initiation events in human cells. The single-molecule nature of our data, and a total of more than 2000-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. In particular, for the first time, we are able to measure genome-wide the absolute efficiency of human replication initiation. We find that the distribution of human replication initiation is consistent with inefficient, stochastic initiation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. In particular, we find sites of human replication initiation are not confined to well-defined replication origins but are instead distributed across broad initiation zones consisting of many initiation sites. Furthermore, we find no correlation of initiation events between neighboring initiation zones. Although most early initiation events occur in early-replicating regions of the genome, a significant number occur in late-replicating regions. The fact that initiation sites in typically late-replicating regions have some probability of firing in early S phase suggests that the major difference between initiation events in early and late replicating regions is their intrinsic probability of firing, as opposed to a qualitative difference in their firing-time distributions. Moreover, modeling of replication kinetics demonstrates that measuring the efficiency of initiation-zone firing in early S phase suffices to predict the average firing time of such initiation zones throughout S phase, further suggesting that the differences between the firing times of early and late initiation zones are quantitative, rather than qualitative. 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.

genomics↗

bioRxiv2020

Catalytically Enhanced Cas9 through Directed Protein Evolution

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 system has found widespread applications in genome manipulations due to its simplicity and effectiveness. Significant efforts in enzyme engineering have been made to improve the CRISPR-Cas9 systems beyond their natural power with additional functionalities such as DNA modification, transcriptional regulation, and high target selectivity1-10. Relatively less attention, however, has been paid to improving the catalytic efficiency of CRISPR-Cas9. Increased catalytic efficiency may be desired in applications where the currently available CRISPR-Cas9 tools are either ineffective4, 11-14 or of low efficiency such as with type II-C Cas915-18 or in non-mammals19, 20. We describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9). We demonstrate the effectiveness of this method with a previously characterized Type IIC Cas9 from Acidothermus cellulolyticus (AceCas9) with up to 4-fold improvement of in vitro catalytic efficiency, as well as the widely used Streptococcus pyogenes Cas9 (SpyCas9), which showed a 2-fold increase in homology directed repair (HDR)-based gene insertion in human colon cancer cells.

synthetic biology↗