Replication Timing Research Articles

KMT2C/KMT2D-dependent H3K4me1 mediates changes in DNA replication timing and origin activity during a cell fate transition.

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.

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy

Development of a Novel Single Cell Multiomics Approach for Simultaneous Analysis of Replication Timing and Gene Expression.

Replication timing (RT) allows us to analyze temporal patterns of genome-wide replication, i.e., if genes replicate early or late during the S-phase of the cell cycle. RT has been linked to gene expression in normal and diseased acute and chronic states such as cancer. However, studies done to date focused on bulk cell populations that required tens of thousands of cells for RT analysis. Here, we developed an affordable novel single cell (sc)-multiomics approach to simultaneously analyze RT and gene expression from cells or nuclei. We used this approach to generate sc-RT profiles and sc-gene expression data from the well-established human liver cancer cell line, HepG2. We demonstrated that as few as 17 mid S-phase cells were sufficient to produce cell-type specific pseudo bulk RT profiles that had a high correlation to previously published HepG2 bulk RT profiles. The sc-RT profiles allowed us to visualize how individual cells progressed through genome replication. We were also able to demonstrate high-resolution correlations between RT and gene expression within each individual cell, which to our knowledge, has not been reported. We observed trends that were conserved between individual cells, as well as cell-to-cell variations, which were not possible to detect with the bulk RT studies.

Nucleosome wrapping states encode principles of 3D genome organization

Nucleosome is the basic structural unit of the genome. During processes like DNA replication and gene transcription, the conformation of nucleosomes undergoes dynamic changes, including DNA unwrapping and rewrapping, as well as histone disassembly and assembly. However, the wrapping characteristics of nucleosomes across the entire genome, including region-specificity and their correlation with higher-order chromatin organization, remains to be studied. In this study, we investigate the wrapping length of DNA on nucleosomes across the whole genome using wrapping-seq. We discover that the chromatin of mouse ES cells forms Nucleosome Wrapping Domains (NRDs), which can also be observed in yeast and fly genomes. We find that the degree of nucleosome wrapping decreases after DNA replication and is promoted by transcription. Furthermore, we observe that nucleosome wrapping domains delineate Hi-C compartments and replication timing domains. In conclusion, we have unveiled a previously unrecognized domainization principle of the chromatin, encoded by nucleosome wrapping states.

Multi-omics approaches reveal that diffuse midline gliomas present altered DNA replication and are susceptible to replication stress therapy

BackgroundThe fatal diffuse midline gliomas (DMG) are characterized by an undruggable H3K27M mutation in H3.1 or H3.3. K27M impairs normal development by stalling differentiation. The identification of targetable pathways remains very poorly explored. Toward this goal, we undertake a multi-omics approach to evaluate replication timing profiles, transcriptomics, and cell cycle features in DMG cells from both H3.1K27M and H3.3K27M subgroups and perform a comparative, integrative data analysis with healthy brain tissue.ResultsDMG cells present differential replication timing in each subgroup, which, in turn, correlates with significant differential gene expression. Differentially expressed genes in S phase are involved in various pathways related to DNA replication. We detect increased expression of DNA replication genes earlier in the cell cycle in DMG cell lines compared to normal brain cells. Furthermore, the distance between origins of replication in DMG cells is smaller than in normal brain cells and their fork speed is slower, a read-out of replication stress. Consistent with these findings, DMG tumors present high replication stress signatures in comparison to normal brain cells. Finally, DMG cells are specifically sensitive to replication stress therapy.ConclusionsThis whole genome multi-omics approach provides insights into the cell cycle regulation of DMG via the H3K27M mutations and establishes a pharmacologic vulnerability in DNA replication, which resolves a potentially novel therapeutic strategy for this non-curable disease.

Characterizing the evolutionary dynamics of cancer proliferation in single-cell clones with SPRINTER

Proliferation is a key hallmark of cancer, but whether it differs between evolutionarily distinct clones co-existing within a tumor is unknown. We introduce the Single-cell Proliferation Rate Inference in Non-homogeneous Tumors through Evolutionary Routes (SPRINTER) algorithm that uses single-cell whole-genome DNA sequencing data to enable accurate identification and clone assignment of S- and G2-phase cells, as assessed by generating accurate ground truth data. Applied to a newly generated longitudinal, primary-metastasis-matched dataset of 14,994 non-small cell lung cancer cells, SPRINTER revealed widespread clone proliferation heterogeneity, orthogonally supported by Ki-67 staining, nuclei imaging and clinical imaging. We further demonstrated that high-proliferation clones have increased metastatic seeding potential, increased circulating tumor DNA shedding and clone-specific altered replication timing in proliferation- or metastasis-related genes associated with expression changes. Applied to previously generated datasets of 61,914 breast and ovarian cancer cells, SPRINTER revealed increased single-cell rates of different genomic variants and enrichment of proliferation-related gene amplifications in high-proliferation clones.

ChromEMT: visualizing and reconstructing chromatin ultrastructure and 3D organization in situ.

Structure determines function. The discovery of the DNA double-helix structure revealed how genetic information is stored and copied. In the mammalian cell nucleus, up to two meters of DNA is compacted by histones to form nucleosome/DNA particle chains that form euchromatin and heterochromatin domains, chromosome territories and mitotic chromosomes upon cell division. A critical question is what are the structures, interactions and 3D organization of DNA as chromatin in the nucleus and how do they determine DNA replication timing, gene expression and ultimately cell fate. To visualize genomic DNA across these different length scales in the nucleus, we developed ChromEMT, a method that selectively enhances the electron density and contrast of DNA and interacting nucleosome particles, which enables nucleosome chains, chromatin domains, chromatin ultrastructure and 3D organization to be imaged and reconstructed by using multi-tilt electron microscopy tomography (EMT). ChromEMT exploits a membrane-permeable, fluorescent DNA-binding dye, DRAQ5, which upon excitation drives the photo-oxidation and precipitation of diaminobenzidine polymers on the surface of DNA/nucleosome particles that are visible in the electron microscope when stained with osmium. Here, we describe a detailed protocol for ChromEMT, including DRAQ5 staining, photo-oxidation, sample preparation and multi-tilt EMT that can be applied broadly to reconstruct genomic DNA structure and 3D interactions in cells and tissues and different kingdoms of life. The entire procedure takes ~9 days and requires expertise in electron microscopy sample sectioning and acquisition of multi-tilt EMT data sets.

Phage cocktail amikacin combination as a potential therapy for bacteremia associated with carbapenemase producing colistin resistant Klebsiella pneumoniae

The increasing occurrence of hospital-associated infections, particularly bacteremia, caused by extensively drug-resistant (XDR) carbapenemase-producing colistin-resistant Klebsiella pneumoniae highlights a critical requirement to discover new therapeutic alternatives. Bacteriophages having host-specific bacteriolytic effects are promising alternatives for combating these pathogens. Among 12 phages isolated from public wastewater in Thailand, two phages-vB_kpnM_05 (myovirus) and vB_kpnP_08 (podovirus) showed broad-host range, producing bacteriolytic activities against 81.3% (n = 26) and 78.1% (n = 25) of 32 XDR carbapenemase-producing colistin-resistant K. pneumoniae, with capsular types—K15, K17, K50, K51, K52/wzi-50 and K2/wzi-2. Both phages showed short replication times, large burst sizes with rapid adsorptions. They exhibited significant stability under various environmental conditions. Genomic analysis revealed that both phages are genetically distinct phages from Myoviridae and Podoviridae family, with the lack of toxin, virulence, lysogeny and antibiotic resistance genes. These characteristics highlighted their promising potential for utilizing in phage therapy for combating XDR K. pneumoniae. Although phage cocktail combining vB_kpnM_05 and vB_kpnP_08 provided significant bacteriolysis for longer duration (8 h) than its monophage (6 h), bacterial regrowth was observed which suggested an evitable development of phage resistance under phages’ selection pressures. Future study will be undertaken to elucidate the precise mechanisms by which these XDR K. pneumoniae developed phage resistance and their associated fitness cost. Remarkably, combining phage cocktail with amikacin at their sub-inhibitory concentrations produced potent synergy by completely suppressing bacterial regrowth in vitro. Our study demonstrated the significant therapeutic and prophylactic effectiveness of a phage cocktail-amikacin combination as a promising alternative strategy for overcoming bacteremia associated with XDR K. pneumoniae having carbapenemase and colistin resistance in vivo.

EPCO-06. DIFFUSE MIDLINE GLIOMAS: FROM CELL CYCLE TO THERAPEUTIC OPPORTUNITIES

Abstract The fatal Diffuse Midline Gliomas (DMG) are characterized by an undruggable H3K27M mutation in H3.1 or H3.3. K27M impairs normal development by stalling of differentiation. As replication timing influences gene expression, cell fate, and cellular response to therapeutics, we undertook a multi-omics approach (Repli-seq, cell cycle RNA-seq, single cell RNA-seq) in DMG cells (H3.1K27M and H3.3K27M subgroups) and tumors in comparison to normal brain to gain an appreciation for targetable pathways in DMG. DMG cells presented differential replication timing in each subgroup, which, in turn, correlated with significant differential gene expression including the cholesterol biosynthesis pathway. Consistent with these findings, DMG tumors presented high replication stress and cholesterol biosynthesis pathway signatures. Moreover, DMG cells were specifically vulnerable to an inhibitor of the cholesterol pathway and to a replication stress therapy, singly and in combination. In conclusion, this combinatorial genomics approach revealed insights into therapeutic opportunities for this incurable disease.

NARA: Network-Aware Resource Allocation mechanism for minimizing quality-of-service impact while dealing with energy consumption in volunteer networks

A large-scale volunteer computing system is a type of distributed system in which contributors volunteer their computing resources, such as personal computers or mobile devices, to contribute to a larger computing effort. Volunteer resources are connected over the Internet and together form a powerful computing system capable of providing a service without depending on a service provider. Volunteer network resource allocation is the process of assigning computing tasks or services to a network of volunteer resources. The allocation process includes identifying the needed resources, selecting appropriate volunteers, and assigning tasks or services based on their capabilities. Volunteer computing systems consist of a large number of heterogeneous resources - in terms of processing power, storage, and availability - belonging to different authorities - users or organizations - and exhibiting uncertain behavior in terms of connection, disconnection, capacity, and failure. All of this makes resource allocation a challenging task in terms of ensuring a minimum quality of service, requiring complex algorithms and optimization techniques to ensure that services are efficiently allocated while respecting the constraints of the available resource. This paper introduces the Network-Aware Resource Allocation mechanism, which leverages the location, connectivity, and network latency of volunteer nodes to minimize the time a service runs with degraded quality of service and aims to deal with the energy consumption resulting from data replication requirements. This resource allocation mechanism applies to both the initial deployment of the service in the network and to the reallocation of nodes in the event that one of the allocated nodes fails or becomes unavailable. Our method has been validated in a simulation environment of a realistic volunteer system. The analysis of the results shows how our mechanism meets the quality requirements of users while minimizing the synchronization and replication times of service data replicas, as well as the time that services run with degraded quality of service, reducing the times by more than 70% in the service deployment phase and by more than 60% in the service execution phase. It also helps to reduce overall energy consumption.

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae , the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2 , a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30 . Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

Reducing the replication time for structural estimations: A successful replication of “An Anatomy of International Trade” using GPU computing

AbstractEaton, Kortum, and Kramarz (2011) (EKK) discovered empirical patterns from French manufacturing firms that a baseline firm heterogeneity model could not explain. The authors proposed and estimated a model that closely matches the patterns observed in French data. This paper successfully replicates their findings using author‐provided data, re‐implementing their algorithms in Python and leveraging graphics processing unit computing to significantly boost computational speed. Applying the model to Chinese manufacturing data, despite differences in context, showed that the model explains most observed patterns well.

Inferring replication timing and proliferation dynamics from single-cell DNA sequencing data

Dysregulated DNA replication is a cause and a consequence of aneuploidy in cancer, yet the interplay between copy number alterations (CNAs), replication timing (RT) and cell cycle dynamics remain understudied in aneuploid tumors. We developed a probabilistic method, PERT, for simultaneous inference of cell-specific replication and copy number states from single-cell whole genome sequencing (scWGS) data. We used PERT to investigate clone-specific RT and proliferation dynamics in >50,000 cells obtained from aneuploid and clonally heterogeneous cell lines, xenografts and primary cancers. We observed bidirectional relationships between RT and CNAs, with CNAs affecting X-inactivation producing the largest RT shifts. Additionally, we found that clone-specific S-phase enrichment positively correlated with ground-truth proliferation rates in genomically stable but not unstable cells. Together, these results demonstrate robust computational identification of S-phase cells from scWGS data, and highlight the importance of RT and cell cycle properties in studying the genomic evolution of aneuploid tumors.

Mathematical model for the distribution of DNA replication origins.

DNA replication in yeast and in many other organisms starts from well-defined locations on the DNA known as replication origins. The spatial distribution of these origins in the genome is particularly important in ensuring that replication is completed quickly. Cells are more vulnerable to DNA damage and other forms of stress while they are replicating their genome. This raises the possibility that the spatial distribution of origins is under selection pressure. In this paper we investigate the hypothesis that natural selection favors origin distributions leading to shorter replication times. Using a simple mathematical model, we show that this hypothesis leads to two main predictions about the origin distributions: that neighboring origins that are inefficient (less likely to fire) are more likely to be close to each other than efficient origins; and that neighboring origins with larger differences in firing times are more likely to be close to each other than origins with similar firing times. We test these predictions using next-generation sequencing data, and show that they are both supported by the data.

Nucleolus and centromere Tyramide Signal Amplification-Seq reveals variable localization of heterochromatin in different cell types

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated Tyramide Signal Amplification (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 human embryonic stem cells (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.

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.

Embryonic genome instability upon DNA replication timing program emergence

Faithful DNA replication is essential for genome integrity1–4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5–8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.

ERCC2 mutations alter the genomic distribution pattern of somatic mutations and are independently prognostic in bladder cancer

Excision repair cross-complementation group 2 (ERCC2) encodes the DNA helicase xeroderma pigmentosum group D, which functions in transcription and nucleotide excision repair. Point mutations in ERCC2 are putative drivers in around 10% of bladder cancers (BLCAs) and a potential positive biomarker for cisplatin therapy response. Nevertheless, the prognostic significance directly attributed to ERCC2 mutations and its pathogenic role in genome instability remain poorly understood. We first demonstrated that mutant ERCC2 is an independent predictor of prognosis in BLCA. We then examined its impact on the somatic mutational landscape using a cohort of ERCC2 wild-type (n= 343) and mutant (n= 39) BLCA whole genomes. The genome-wide distribution of somatic mutations is significantly altered in ERCC2 mutants, including T[C>T]N enrichment, altered replication time correlations, and CTCF-cohesin binding site mutation hotspots. We leverage these alterations to develop a machine learning model for predicting pathogenic ERCC2 mutations, which may be useful to inform treatment of patients with BLCA.

Sex-specific DNA-replication in the early mammalian embryo

The timing of DNA replication in mammals is crucial for minimizing errors and influenced by genome usage and chromatin states. Replication timing in the newly formed mammalian embryo remains poorly understood. Here, we have investigated replication timing in mouse zygotes and 2-cell embryos, revealing that zygotes lack a conventional replication timing program, which then emerges in 2-cell embryos. This program differs from embryonic stem cells and generally correlates with transcription and genome compartmentalization of both parental genomes. However, consistent and systematic differences existed between the replication timing of the two parental genomes, including considerably later replication of maternal pericentromeric regions compared to paternal counterparts. Moreover, maternal chromatin modified by Polycomb Repressive Complexes in the oocyte, undergoes early replication, despite belonging to the typically late-replicating B-compartment of the genome. This atypical and asynchronous replication of the two parental genomes may advance our understanding of replication stress in early human embryos and trigger strategies to reduce errors and aneuploidies.