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.

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.

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.

Organization of DNA Replication Origin Firing in Xenopus Egg Extracts: The Role of Intra-S Checkpoint.

During cell division, the duplication of the genome starts at multiple positions called replication origins. Origin firing requires the interaction of rate-limiting factors with potential origins during the S(ynthesis)-phase of the cell cycle. Origins fire as synchronous clusters which is proposed to be regulated by the intra-S checkpoint. By modelling the unchallenged, the checkpoint-inhibited and the checkpoint protein Chk1 over-expressed replication pattern of single DNA molecules from Xenopus sperm chromatin replicated in egg extracts, we demonstrate that the quantitative modelling of data requires: (1) a segmentation of the genome into regions of low and high probability of origin firing; (2) that regions with high probability of origin firing escape intra-S checkpoint regulation and (3) the variability of the rate of DNA synthesis close to replication forks is a necessary ingredient that should be taken in to account in order to describe the dynamic of replication origin firing. This model implies that the observed origin clustering emerges from the apparent synchrony of origin firing in regions with high probability of origin firing and challenge the assumption that the intra-S checkpoint is the main regulator of origin clustering.

Coupling between Sequence-Mediated Nucleosome Organization and Genome Evolution.

The nucleosome is a major modulator of DNA accessibility to other cellular factors. Nucleosome positioning has a critical importance in regulating cell processes such as transcription, replication, recombination or DNA repair. The DNA sequence has an influence on the position of nucleosomes on genomes, although other factors are also implicated, such as ATP-dependent remodelers or competition of the nucleosome with DNA binding proteins. Different sequence motifs can promote or inhibit the nucleosome formation, thus influencing the accessibility to the DNA. Sequence-encoded nucleosome positioning having functional consequences on cell processes can then be selected or counter-selected during evolution. We review the interplay between sequence evolution and nucleosome positioning evolution. We first focus on the different ways to encode nucleosome positions in the DNA sequence, and to which extent these mechanisms are responsible of genome-wide nucleosome positioning in vivo. Then, we discuss the findings about selection of sequences for their nucleosomal properties. Finally, we illustrate how the nucleosome can directly influence sequence evolution through its interactions with DNA damage and repair mechanisms. This review aims to provide an overview of the mutual influence of sequence evolution and nucleosome positioning evolution, possibly leading to complex evolutionary dynamics.

Human ORC/MCM density is low in active genes and correlates with replication time but does not delimit initiation zones.

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late-replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

FORK-seq: replication landscape of the Saccharomyces cerevisiae genome by nanopore sequencing.

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. By quantifying BrdU incorporation along pulse-chased replication intermediates from Saccharomyces cerevisiae, we orient 58,651 replication tracks reproducing population-based replication directionality profiles and map 4964 and 4485 individual initiation and termination events, respectively. Although most events cluster at known origins and fork merging zones, 9% and 18% of initiation and termination events, respectively, occur at many locations previously missed. Thus, FORK-seq reveals the full extent of cell-to-cell heterogeneity in DNA replication.

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.

The eukaryotic bell-shaped temporal rate of DNA replication origin firing emanates from a balance between origin activation and passivation.

The time-dependent rate [Formula: see text] of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here, we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the [Formula: see text] universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris. We predict the [Formula: see text] maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics.

Sensitive Hg2+ Ion Detection Using Metal Enhanced Fluorescence of Novel Polyvinyl Pyrrolidone (PVP)-Templated Gold Nanoparticles.

In this study, we report a straightforward strategy for Hg2+ ion detection. Fluorescent Au nanoparticles (NPs) were one-pot synthesized using a polymer (polyvinyl pyrrolidone [PVP]) as both capping and fluorescence agent. The as-synthesized PVP-Au NPs showed a remarkably rapid response selectively for Hg2+ ions compared to 14 other metal ions. The detection limit of Hg2+ was estimated at 100 nM. We discuss the emission and quenching mechanism of the PVP-Au NPs, the former being attributed to metal enhanced fluorescence and the latter being related to static quenching by Hg2+. The fluorescence of PVP-Au NPs offers an efficient and reliable strategy for Hg2+ ions detection. They therefore have a great potential for applications in health and environmental monitoring.

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.

Numerical Study of Novel Ratiometric Sensors Based on Plasmon-Exciton Coupling.

We numerically studied the optical properties of spherical nanostructures made of an emitter core coated by a silver shell through the generalized Mie theory. When there is a strong coupling between the localized surface plasmon in the metallic shell and the emitter exciton in the core, the extinction spectra exhibit two peaks. Upon adsorption of analytes on these core-shell nanostructures, the intensities of the two peaks change with opposite trends. This property makes them potential sensitive ratiometric sensors. Molecule adsorption on these nanostructures can be quantified through a very simple optical configuration likely resulting in a much faster acquisition time compared with systems based on the traditional metal nanoparticle surface plasmon resonance (SPR) biosensors.

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.

Comparative Multifractal Analysis of Dynamic Infrared Thermograms and X-Ray Mammograms Enlightens Changes in the Environment of Malignant Tumors.

There is growing evidence that the microenvironment surrounding a tumor plays a special role in cancer development and cancer therapeutic resistance. Tumors arise from the dysregulation and alteration of both the malignant cells and their environment. By providing tumor-repressing signals, the microenvironment can impose and sustain normal tissue architecture. Once tissue homeostasis is lost, the altered microenvironment can create a niche favoring the tumorigenic transformation process. A major challenge in early breast cancer diagnosis is thus to show that these physiological and architectural alterations can be detected with currently used screening techniques. In a recent study, we used a 1D wavelet-based multi-scale method to analyze breast skin temperature temporal fluctuations collected with an IR thermography camera in patients with breast cancer. This study reveals that the multifractal complexity of temperature fluctuations superimposed on cardiogenic and vasomotor perfusion oscillations observed in healthy breasts is lost in malignant tumor foci in cancerous breasts. Here we use a 2D wavelet-based multifractal method to analyze the spatial fluctuations of breast density in the X-ray mammograms of the same panel of patients. As compared to the long-range correlations and anti-correlations in roughness fluctuations, respectively observed in dense and fatty breast areas, some significant change in the nature of breast density fluctuations with some clear loss of correlations is detected in the neighborhood of malignant tumors. This attests to some architectural disorganization that may deeply affect heat transfer and related thermomechanics in breast tissues, corroborating the change to homogeneous monofractal temperature fluctuations recorded in cancerous breasts with the IR camera. These results open new perspectives in computer-aided methods to assist in early breast cancer diagnosis.

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.

Structural organization of human replication timing domains.

Recent analysis of genome-wide epigenetic modification data, mean replication timing (MRT) profiles and chromosome conformation data in mammals have provided increasing evidence that flexibility in replication origin usage is regulated locally by the epigenetic landscape and over larger genomic distances by the 3D chromatin architecture. Here, we review the recent results establishing some link between replication domains and chromatin structural domains in pluripotent and various differentiated cell types in human. We reconcile the originally proposed dichotomic picture of early and late constant timing regions that replicate by multiple rather synchronous origins in separated nuclear compartments of open and closed chromatins, with the U-shaped MRT domains bordered by "master" replication origins specified by a localized (∼200-300 kb) zone of open and transcriptionally active chromatin from which a replication wave likely initiates and propagates toward the domain center via a cascade of origin firing. We discuss the relationships between these MRT domains, topologically associated domains and lamina-associated domains. This review sheds a new light on the epigenetically regulated global chromatin reorganization that underlies the loss of pluripotency and the determination of differentiation properties.

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.

Ubiquitous human 'master' origins of replication are encoded in the DNA sequence via a local enrichment in nucleosome excluding energy barriers.

As the elementary building block of eukaryotic chromatin, the nucleosome is at the heart of the compromise between the necessity of compacting DNA in the cell nucleus and the required accessibility to regulatory proteins. The recent availability of genome-wide experimental maps of nucleosome positions for many different organisms and cell types has provided an unprecedented opportunity to elucidate to what extent the DNA sequence conditions the primary structure of chromatin and in turn participates in the chromatin-mediated regulation of nuclear functions, such as gene expression and DNA replication. In this study, we use in vivo and in vitro genome-wide nucleosome occupancy data together with the set of nucleosome-free regions (NFRs) predicted by a physical model of nucleosome formation based on sequence-dependent bending properties of the DNA double-helix, to investigate the role of intrinsic nucleosome occupancy in the regulation of the replication spatio-temporal programme in human. We focus our analysis on the so-called replication U/N-domains that were shown to cover about half of the human genome in the germline (skew-N domains) as well as in embryonic stem cells, somatic and HeLa cells (mean replication timing U-domains). The 'master' origins of replication (MaOris) that border these megabase-sized U/N-domains were found to be specified by a few hundred kb wide regions that are hyper-sensitive to DNase I cleavage, hypomethylated, and enriched in epigenetic marks involved in transcription regulation, the hallmarks of localized open chromatin structures. Here we show that replication U/N-domain borders that are conserved in all considered cell lines have an environment highly enriched in nucleosome-excluding-energy barriers, suggesting that these ubiquitous MaOris have been selected during evolution. In contrast, MaOris that are cell-type-specific are mainly regulated epigenetically and are no longer favoured by a local abundance of intrinsic NFRs encoded in the DNA sequence. At the smaller few hundred bp scale of gene promoters, CpG-rich promoters of housekeeping genes found nearby ubiquitous MaOris as well as CpG-poor promoters of tissue-specific genes found nearby cell-type-specific MaOris, both correspond to in vivo NFRs that are not coded as nucleosome-excluding-energy barriers. Whereas the former promoters are likely to correspond to high occupancy transcription factor binding regions, the latter are an illustration that gene regulation in human is typically cell-type-specific.

Large replication skew domains delimit GC-poor gene deserts in human.

Besides their large-scale organization in isochores, mammalian genomes display megabase-sized regions, spanning both genes and intergenes, where the strand nucleotide composition asymmetry decreases linearly, possibly due to replication activity. These so-called skew-N domains cover about a third of the human genome and are bordered by two skew upward jumps that were hypothesized to compose a subset of "master" replication origins active in the germline. Skew-N domains were shown to exhibit a particular gene organization. Genes with CpG-rich promoters likely expressed in the germline are over represented near the master replication origins, with large genes being co-oriented with replication fork progression, which suggests some coordination of replication and transcription. In this study, we describe another skew structure that covers ∼13% of the human genome and that is bordered by putative master replication origins similar to the ones flanking skew-N domains. These skew-split-N domains have a shape reminiscent of a N, but split in half, leaving in the center a region of null skew whose length increases with domain size. These central regions (median size ∼860 kb) have a homogeneous composition, i.e. both a null and constant skew and a constant and low GC content. They correspond to heterochromatin gene deserts found in low-GC isochores with an average gene density of 0.81 promoters/Mb as compared to 7.73 promoters/Mb genome wide. The analysis of epigenetic marks and replication timing data confirms that, in these late replicating heterochomatic regions, the initiation of replication is likely to be random. This contrasts with the transcriptionally active euchromatin state found around the bordering well positioned master replication origins. Altogether skew-N domains and skew-split-N domains cover about 50% of the human genome.