Neural network and kinetic modelling of human genome replication reveal replication origin locations and strengths.

In human and other metazoans, the determinants of replication origin location and strength are still elusive. Origins are licensed in G1 phase and fired in S phase of the cell cycle, respectively. It is debated which of these two temporally separate steps determines origin efficiency. Experiments can independently profile mean replication timing (MRT) and replication fork directionality (RFD) genome-wide. Such profiles contain information on multiple origins' properties and on fork speed. Due to possible origin inactivation by passive replication, however, observed and intrinsic origin efficiencies can markedly differ. Thus, there is a need for methods to infer intrinsic from observed origin efficiency, which is context-dependent. Here, we show that MRT and RFD data are highly consistent with each other but contain information at different spatial scales. Using neural networks, we infer an origin licensing landscape that, when inserted in an appropriate simulation framework, jointly predicts MRT and RFD data with unprecedented precision and underlies the importance of dispersive origin firing. We furthermore uncover an analytical formula that predicts intrinsic from observed origin efficiency combined with MRT data. Comparison of inferred intrinsic origin efficiencies with experimental profiles of licensed origins (ORC, MCM) and actual initiation events (Bubble-seq, SNS-seq, OK-seq, ORM) show that intrinsic origin efficiency is not solely determined by licensing efficiency. Thus, human replication origin efficiency is set at both the origin licensing and firing steps.

Developmental and cancer-associated plasticity of DNA replication preferentially targets GC-poor, lowly expressed and late-replicating regions.

The spatiotemporal program of metazoan DNA replication is regulated during development and altered in cancers. We have generated novel OK-seq, Repli-seq and RNA-seq data to compare the DNA replication and gene expression programs of twelve cancer and non-cancer human cell types. Changes in replication fork directionality (RFD) determined by OK-seq are widespread but more frequent within GC-poor isochores and largely disconnected from transcription changes. Cancer cell RFD profiles cluster with non-cancer cells of similar developmental origin but not with different cancer types. Importantly, recurrent RFD changes are detected in specific tumour progression pathways. Using a model for establishment and early progression of chronic myeloid leukemia (CML), we identify 1027 replication initiation zones (IZs) that progressively change efficiency during long-term expression of the BCR-ABL1 oncogene, being twice more often downregulated than upregulated. Prolonged expression of BCR-ABL1 results in targeting of new IZs and accentuation of previous efficiency changes. Targeted IZs are predominantly located in GC-poor, late replicating gene deserts and frequently silenced in late CML. Prolonged expression of BCR-ABL1 results in massive deletion of GC-poor, late replicating DNA sequences enriched in origin silencing events. We conclude that BCR-ABL1 expression progressively affects replication and stability of GC-poor, late-replicating regions during CML progression.

Evidence for DNA Sequence Encoding of an Accessible Nucleosomal Array across Vertebrates.

Nucleosome-depleted regions around which nucleosomes order following the "statistical" positioning scenario were recently shown to be encoded in the DNA sequence in human. This intrinsic nucleosomal ordering strongly correlates with oscillations in the local GC content as well as with the interspecies and intraspecies mutation profiles, revealing the existence of both positive and negative selection. In this letter, we show that these predicted nucleosome inhibitory energy barriers (NIEBs) with compacted neighboring nucleosomes are indeed ubiquitous to all vertebrates tested. These 1 kb-sized chromatin patterns are widely distributed along vertebrate chromosomes, overall covering more than a third of the genome. We have previously observed in human deviations from neutral evolution at these genome-wide distributed regions, which we interpreted as a possible indication of the selection of an open, accessible, and dynamic nucleosomal array to constitutively facilitate the epigenetic regulation of nuclear functions in a cell-type-specific manner. As a first, very appealing observation supporting this hypothesis, we report evidence of a strong association between NIEB borders and the poly(A) tails of Alu sequences in human. These results suggest that NIEBs provide adequate chromatin patterns favorable to the integration of Alu retrotransposons and, more generally to various transposable elements in the genomes of primates and other vertebrates.

Multi-scale structural community organisation of the human genome.

BACKGROUND: Structural interaction frequency matrices between all genome loci are now experimentally achievable thanks to high-throughput chromosome conformation capture technologies. This ensues a new methodological challenge for computational biology which consists in objectively extracting from these data the structural motifs characteristic of genome organisation. RESULTS: We deployed the fast multi-scale community mining algorithm based on spectral graph wavelets to characterise the networks of intra-chromosomal interactions in human cell lines. We observed that there exist structural domains of all sizes up to chromosome length and demonstrated that the set of structural communities forms a hierarchy of chromosome segments. Hence, at all scales, chromosome folding predominantly involves interactions between neighbouring sites rather than the formation of links between distant loci. CONCLUSIONS: Multi-scale structural decomposition of human chromosomes provides an original framework to question structural organisation and its relationship to functional regulation across the scales. By construction the proposed methodology is independent of the precise assembly of the reference genome and is thus directly applicable to genomes whose assembly is not fully determined.

The spatiotemporal program of DNA replication is associated with specific combinations of chromatin marks in human cells.

The duplication of mammalian genomes is under the control of a spatiotemporal program that orchestrates the positioning and the timing of firing of replication origins. The molecular mechanisms coordinating the activation of about [Formula: see text] predicted origins remain poorly understood, partly due to the intrinsic rarity of replication bubbles, making it difficult to purify short nascent strands (SNS). The precise identification of origins based on the high-throughput sequencing of SNS constitutes a new methodological challenge. We propose a new statistical method with a controlled resolution, adapted to the detection of replication origins from SNS data. We detected an average of 80,000 replication origins in different cell lines. To evaluate the consistency between different protocols, we compared SNS detections with bubble trapping detections. This comparison demonstrated a good agreement between genome-wide methods, with 65% of SNS-detected origins validated by bubble trapping, and 44% of bubble trapping origins validated by SNS origins, when compared at the same resolution. We investigated the interplay between the spatial and the temporal programs of replication at fine scales. We show that most of the origins detected in regions replicated in early S phase are shared by all the cell lines investigated whereas cell-type-specific origins tend to be replicated in late S phase. We shed a new light on the key role of CpG islands, by showing that 80% of the origins associated with CGIs are constitutive. Our results further show that at least 76% of CGIs are origins of replication. The analysis of associations with chromatin marks at different timing of cell division revealed new potential epigenetic regulators driving the spatiotemporal activity of replication origins. We highlight the potential role of H4K20me1 and H3K27me3, the coupling of which is correlated with increased efficiency of replication origins, clearly identifying those marks as potential key regulators of replication origins.

Evidence of selection for an accessible nucleosomal array in human.

BACKGROUND: Recently, a physical model of nucleosome formation based on sequence-dependent bending properties of the DNA double-helix has been used to reveal some enrichment of nucleosome-inhibiting energy barriers (NIEBs) nearby ubiquitous human "master" replication origins. Here we use this model to predict the existence of about 1.6 millions NIEBs over the 22 human autosomes. RESULTS: We show that these high energy barriers of mean size 153 bp correspond to nucleosome-depleted regions (NDRs) in vitro, as expected, but also in vivo. On either side of these NIEBs, we observe, in vivo and in vitro, a similar compacted nucleosome ordering, suggesting an absence of chromatin remodeling. This nucleosomal ordering strongly correlates with oscillations of the GC content as well as with the interspecies and intraspecies mutation profiles along these regions. Comparison of these divergence rates reveals the existence of both positive and negative selections linked to nucleosome positioning around these intrinsic NDRs. Overall, these NIEBs and neighboring nucleosomes cover 37.5 % of the human genome where nucleosome occupancy is stably encoded in the DNA sequence. These 1 kb-sized regions of intrinsic nucleosome positioning are equally found in GC-rich and GC-poor isochores, in early and late replicating regions, in intergenic and genic regions but not at gene promoters. CONCLUSION: The source of selection pressure on the NIEBs has yet to be resolved in future work. One possible scenario is that these widely distributed chromatin patterns have been selected in human to impair the condensation of the nucleosomal array into the 30 nm chromatin fiber, so as to facilitate the epigenetic regulation of nuclear functions in a cell-type-specific manner.

Embryonic stem cell specific "master" replication origins at the heart of the loss of pluripotency.

Epigenetic regulation of the replication program during mammalian cell differentiation remains poorly understood. We performed an integrative analysis of eleven genome-wide epigenetic profiles at 100 kb resolution of Mean Replication Timing (MRT) data in six human cell lines. Compared to the organization in four chromatin states shared by the five somatic cell lines, embryonic stem cell (ESC) line H1 displays (i) a gene-poor but highly dynamic chromatin state (EC4) associated to histone variant H2AZ rather than a HP1-associated heterochromatin state (C4) and (ii) a mid-S accessible chromatin state with bivalent gene marks instead of a polycomb-repressed heterochromatin state. Plastic MRT regions (≲ 20% of the genome) are predominantly localized at the borders of U-shaped timing domains. Whereas somatic-specific U-domain borders are gene-dense GC-rich regions, 31.6% of H1-specific U-domain borders are early EC4 regions enriched in pluripotency transcription factors NANOG and OCT4 despite being GC poor and gene deserts. Silencing of these ESC-specific "master" replication initiation zones during differentiation corresponds to a loss of H2AZ and an enrichment in H3K9me3 mark characteristic of late replicating C4 heterochromatin. These results shed a new light on the epigenetically regulated global chromatin reorganization that underlies the loss of pluripotency and lineage commitment.

Human genome replication proceeds through four chromatin states.

Advances in genomic studies have led to significant progress in understanding the epigenetically controlled interplay between chromatin structure and nuclear functions. Epigenetic modifications were shown to play a key role in transcription regulation and genome activity during development and differentiation or in response to the environment. Paradoxically, the molecular mechanisms that regulate the initiation and the maintenance of the spatio-temporal replication program in higher eukaryotes, and in particular their links to epigenetic modifications, still remain elusive. By integrative analysis of the genome-wide distributions of thirteen epigenetic marks in the human cell line K562, at the 100 kb resolution of corresponding mean replication timing (MRT) data, we identify four major groups of chromatin marks with shared features. These states have different MRT, namely from early to late replicating, replication proceeds though a transcriptionally active euchromatin state (C1), a repressive type of chromatin (C2) associated with polycomb complexes, a silent state (C3) not enriched in any available marks, and a gene poor HP1-associated heterochromatin state (C4). When mapping these chromatin states inside the megabase-sized U-domains (U-shaped MRT profile) covering about 50% of the human genome, we reveal that the associated replication fork polarity gradient corresponds to a directional path across the four chromatin states, from C1 at U-domains borders followed by C2, C3 and C4 at centers. Analysis of the other genome half is consistent with early and late replication loci occurring in separate compartments, the former correspond to gene-rich, high-GC domains of intermingled chromatin states C1 and C2, whereas the latter correspond to gene-poor, low-GC domains of alternating chromatin states C3 and C4 or long C4 domains. This new segmentation sheds a new light on the epigenetic regulation of the spatio-temporal replication program in human and provides a framework for further studies in different cell types, in both health and disease.

3D chromatin conformation correlates with replication timing and is conserved in resting cells.

Although chromatin folding is known to be of functional importance to control the gene expression program, less is known regarding its interplay with DNA replication. Here, using Circular Chromatin Conformation Capture combined with high-throughput sequencing, we identified megabase-sized self-interacting domains in the nucleus of a human lymphoblastoid cell line, as well as in cycling and resting peripheral blood mononuclear cells (PBMC). Strikingly, the boundaries of those domains coincide with early-initiation zones in every cell types. Preferential interactions have been observed between the consecutive early-initiation zones, but also between those separated by several tens of megabases. Thus, the 3D conformation of chromatin is strongly correlated with the replication timing along the whole chromosome. We furthermore provide direct clues that, in addition to the timing value per se, the shape of the timing profile at a given locus defines its set of genomic contacts. As this timing-related scheme of chromatin organization exists in lymphoblastoid cells, resting and cycling PBMC, this indicates that it is maintained several weeks or months after the previous S-phase. Lastly, our work highlights that the major chromatin changes accompanying PBMC entry into cell cycle occur while keeping largely unchanged the long-range chromatin contacts.

Megabase replication domains along the human genome: relation to chromatin structure and genome organisation.

In higher eukaryotes, the absence of specific sequence motifs, marking the origins of replication has been a serious hindrance to the understanding of (i) the mechanisms that regulate the spatio-temporal replication program, and (ii) the links between origins activation, chromatin structure and transcription. In this chapter, we review the partitioning of the human genome into megabased-size replication domains delineated as N-shaped motifs in the strand compositional asymmetry profiles. They collectively span 28.3% of the genome and are bordered by more than 1,000 putative replication origins. We recapitulate the comparison of this partition of the human genome with high-resolution experimental data that confirms that replication domain borders are likely to be preferential replication initiation zones in the germline. In addition, we highlight the specific distribution of experimental and numerical chromatin marks along replication domains. Domain borders correspond to particular open chromatin regions, possibly encoded in the DNA sequence, and around which replication and transcription are highly coordinated. These regions also present a high evolutionary breakpoint density, suggesting that susceptibility to breakage might be linked to local open chromatin fiber state. Altogether, this chapter presents a compartmentalization of the human genome into replication domains that are landmarks of the human genome organization and are likely to play a key role in genome dynamics during evolution and in pathological situations.

A novel strategy of transcription regulation by intragenic nucleosome ordering.

Numerous studies of chromatin structure showed that nucleosome free regions (NFRs) located at 5' gene ends contribute to transcription initiation regulation. Here, we determine the role of intragenic chromatin structure on gene expression regulation. We show that, along Saccharomyces cerevisiae genes, nucleosomes are highly organized following two types of architecture that depend only on the distance between the NFRs located at the 5' and 3' gene ends. In the first type, this distance constrains in vivo the positioning of n nucleosomes regularly organized in a "crystal-like" array. In the second type, this distance is such that the corresponding genes can accommodate either n or (n + 1) nucleosomes, thereby displaying two possible crystal-like arrays of n weakly compacted or n + 1 highly compacted nucleosomes. This adaptability confers "bi-stable" properties to chromatin and is a key to its dynamics. Compared to crystal-like genes, bi-stable genes present higher transcriptional plasticity, higher sensitivity to chromatin regulators, higher H3 turnover rate, and lower H2A.Z enrichment. The results strongly suggest that transcription elongation is facilitated by higher chromatin compaction. The data allow us to propose a new paradigm of transcriptional control mediated by the stability and the level of compaction of the intragenic chromatin architecture and open new ways for investigating eukaryotic gene expression regulation.

Impact of replication timing on non-CpG and CpG substitution rates in mammalian genomes.

Neutral nucleotide substitutions occur at varying rates along genomes, and it remains a major issue to unravel the mechanisms that cause these variations and to analyze their evolutionary consequences. Here, we study the role of replication in the neutral substitution pattern. We obtained a high-resolution replication timing profile of the whole human genome by massively parallel sequencing of nascent BrdU-labeled replicating DNA. These data were compared to the neutral substitution rates along the human genome, obtained by aligning human and chimpanzee genomes using macaque and orangutan as outgroups. All substitution rates increase monotonously with replication timing even after controlling for local or regional nucleotide composition, crossover rate, distance to telomeres, and chromatin compaction. The increase in non-CpG substitution rates might result from several mechanisms including the increase in mutation-prone activities or the decrease in efficiency of DNA repair during the S phase. In contrast, the rate of C --> T transitions in CpG dinucleotides increases in later-replicating regions due to increasing DNA methylation level that reflects a negative correlation between timing and gene expression. Similar results are observed in the mouse, which indicates that replication timing is a main factor affecting nucleotide substitution dynamics at non-CpG sites and constitutes a major neutral process driving mammalian genome evolution.

Replication fork polarity gradients revealed by megabase-sized U-shaped replication timing domains in human cell lines.

In higher eukaryotes, replication program specification in different cell types remains to be fully understood. We show for seven human cell lines that about half of the genome is divided in domains that display a characteristic U-shaped replication timing profile with early initiation zones at borders and late replication at centers. Significant overlap is observed between U-domains of different cell lines and also with germline replication domains exhibiting a N-shaped nucleotide compositional skew. From the demonstration that the average fork polarity is directly reflected by both the compositional skew and the derivative of the replication timing profile, we argue that the fact that this derivative displays a N-shape in U-domains sustains the existence of large-scale gradients of replication fork polarity in somatic and germline cells. Analysis of chromatin interaction (Hi-C) and chromatin marker data reveals that U-domains correspond to high-order chromatin structural units. We discuss possible models for replication origin activation within U/N-domains. The compartmentalization of the genome into replication U/N-domains provides new insights on the organization of the replication program in the human genome.

Replication-associated mutational asymmetry in the human genome.

During evolution, mutations occur at rates that can differ between the two DNA strands. In the human genome, nucleotide substitutions occur at different rates on the transcribed and non-transcribed strands that may result from transcription-coupled repair. These mutational asymmetries generate transcription-associated compositional skews. To date, the existence of such asymmetries associated with replication has not yet been established. Here, we compute the nucleotide substitution matrices around replication initiation zones identified as sharp peaks in replication timing profiles and associated with abrupt jumps in the compositional skew profile. We show that the substitution matrices computed in these regions fully explain the jumps in the compositional skew profile when crossing initiation zones. In intergenic regions, we observe mutational asymmetries measured as differences between complementary substitution rates; their sign changes when crossing initiation zones. These mutational asymmetries are unlikely to result from cryptic transcription but can be explained by a model based on replication errors and strand-biased repair. In transcribed regions, mutational asymmetries associated with replication superimpose on the previously described mutational asymmetries associated with transcription. We separate the substitution asymmetries associated with both mechanisms, which allows us to determine for the first time in eukaryotes, the mutational asymmetries associated with replication and to reevaluate those associated with transcription. Replication-associated mutational asymmetry may result from unequal rates of complementary base misincorporation by the DNA polymerases coupled with DNA mismatch repair (MMR) acting with different efficiencies on the leading and lagging strands. Replication, acting in germ line cells during long evolutionary times, contributed equally with transcription to produce the present abrupt jumps in the compositional skew. These results demonstrate that DNA replication is one of the major processes that shape human genome composition.

Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome.

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics.

Nucleosome positioning by genomic excluding-energy barriers.

Recent genome-wide nucleosome mappings along with bioinformatics studies have confirmed that the DNA sequence plays a more important role in the collective organization of nucleosomes in vivo than previously thought. Yet in living cells, this organization also results from the action of various external factors like DNA-binding proteins and chromatin remodelers. To decipher the code for intrinsic chromatin organization, there is thus a need for in vitro experiments to bridge the gap between computational models of nucleosome sequence preferences and in vivo nucleosome occupancy data. Here we combine atomic force microscopy in liquid and theoretical modeling to demonstrate that a major sequence signaling in vivo are high-energy barriers that locally inhibit nucleosome formation rather than favorable positioning motifs. We show that these genomic excluding-energy barriers condition the collective assembly of neighboring nucleosomes consistently with equilibrium statistical ordering principles. The analysis of two gene promoter regions in Saccharomyces cerevisiae and the human genome indicates that these genomic barriers direct the intrinsic nucleosome occupancy of regulatory sites, thereby contributing to gene expression regulation.

FORK-seq: Single-Molecule Profiling of DNA Replication.

Most genome replication mapping methods profile cell populations, masking cell-to-cell heterogeneity. Here, we describe FORK-seq, a nanopore sequencing method to map replication of single DNA molecules at 200 nucleotide resolution using a nanopore current interpretation tool allowing the quantification of BrdU incorporation. Along pulse-chased replication intermediates from Saccharomyces cerevisiae, we can orient replication tracks and reproduce population-based replication directionality profiles. Additionally, we can map individual initiation and termination events. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.

Genome-wide mapping of individual replication fork velocities using nanopore sequencing.

Little is known about replication fork velocity variations along eukaryotic genomes, since reference techniques to determine fork speed either provide no sequence information or suffer from low throughput. Here we present NanoForkSpeed, a nanopore sequencing-based method to map and extract the velocity of individual forks detected as tracks of the thymidine analogue bromodeoxyuridine incorporated during a brief pulse-labelling of asynchronously growing cells. NanoForkSpeed retrieves previous Saccharomyces cerevisiae mean fork speed estimates (≈2 kb/min) in the BT1 strain exhibiting highly efficient bromodeoxyuridine incorporation and wild-type growth, and precisely quantifies speed changes in cells with altered replisome progression or exposed to hydroxyurea. The positioning of >125,000 fork velocities provides a genome-wide map of fork progression based on individual fork rates, showing a uniform fork speed across yeast chromosomes except for a marked slowdown at known pausing sites.

Open chromatin encoded in DNA sequence is the signature of 'master' replication origins in human cells.

For years, progress in elucidating the mechanisms underlying replication initiation and its coupling to transcriptional activities and to local chromatin structure has been hampered by the small number (approximately 30) of well-established origins in the human genome and more generally in mammalian genomes. Recent in silico studies of compositional strand asymmetries revealed a high level of organization of human genes around 1000 putative replication origins. Here, by comparing with recently experimentally identified replication origins, we provide further support that these putative origins are active in vivo. We show that regions approximately 300-kb wide surrounding most of these putative replication origins that replicate early in the S phase are hypersensitive to DNase I cleavage, hypomethylated and present a significant enrichment in genomic energy barriers that impair nucleosome formation (nucleosome-free regions). This suggests that these putative replication origins are specified by an open chromatin structure favored by the DNA sequence. We discuss how this distinctive attribute makes these origins, further qualified as 'master' replication origins, priviledged loci for future research to decipher the human spatio-temporal replication program. Finally, we argue that these 'master' origins are likely to play a key role in genome dynamics during evolution and in pathological situations.