Biology subjects

Audit, B.

Publications and source records attributed to Audit, B..

12 recordsLinked to original sources

bioRxiv2026

High-resolution single-molecule replication profiling of the human genome

Despite the considerable progress made in recent years thanks to the rise of long-read sequencing, high-resolution, single molecule (SM)-based replication profiling of large genomes has remained out of reach. Here, we present a replication labelling strategy consisting in repeated pulse-labelling of asynchronously growing cells with the thymidine analogue bromodeoxyuridine (BrdU) that greatly enhances the number of detectable replication tracks. We show that the multipulse BrdU labelling protocol, in association with ForkML, a machine-learning method translating BrdU signals in nanopore reads into oriented and positioned replication forks, enables the establishment of a SM replication map of the entire human genome at kilobase resolution.

genomics↗

bioRxiv2026

An extension of Modular Response Analysis for global perturbations and robust connectivity inference of gene regulatory networks.

Understanding how gene regulatory networks respond to global cell perturbations remains a central challenge in systems biology and network inference. Modular Response Analysis (MRA) provides a mathematical framework to infer gene-to-gene directed connectivity graphs from perturbation experiments; however, classical MRA captures direct gene-to-gene influences, and does not explicitly account for global stimuli that simultaneously change the graph. Here, we introduce MRA+, an extension of MRA, that incorporates the effect of global perturbations into gene-to-gene graph inference. MRA+ assumes a sequential experimental design in which targeted gene perturbations are followed by the application of a global stimulus, enabling the separation of connectivity changes from direct gene induction. The method estimates network connectivity under induced conditions and quantifies gene-specific induction strengths, which represent contributions to expression changes arising from mechanisms external to the inferred network. In the case of single-cell expression data, we present a bootstrap strategy to assess the robustness of inferred connectivity coefficients and propose a complementary criterion based on sign stability to interpret weak or non-significant estimates. Together, these developments provide a general framework for robust inference of gene connectivity graphs in the presence of global perturbations, applicable to diverse biological and experimental contexts.

systems biology↗

bioRxiv2026

A Closed-Form Bayesian Framework for DNA Replication Reveals Intrinsic Origin Timing and Activation Delays

I.We present an analytical framework for modeling eukaryotic DNA replication that, given experimental Replication Fork Directionality (RFD) data, enables Bayesian inference of origin number, activation delay [Formula] and intrinsic timing ({lambda}i), (the mean replication time if each origin were isolated). By deriving closed-form expressions for RFD and Mean Replication Timing (MRT) under exponential and a specific Weibull firing-time distributions as functions of [Formula] and ({lambda}i), we eliminate the need for stochastic simulations. These analytical results reveal that RFD, as a ratio of fork directions, is invariant under joint rescaling of intrinsic timing and fork speed; absolute intrinsic timing can nonetheless be inferred when fork speed is independently measured. We demonstrate that under exponential firing distribution for the origin, the observed efficiency (Ei), i.e. the probability for an origin to fire which accounts for nearby origin, is simply MRT(xi)/{lambda}i. The closed-form RFD expressions allow to use a Bayesian method that achieves 0.96-0.99 correlation with yeast RFD profiles and resolves ~780 origins in S. cerevisiae. Our framework identifies about 150 origins with biologically significant delays ([≥] 3 minute), revealing regulated activation kinetics undetectable by existing methods. By quantifying how origin intrinsic timing and delays shape replication timing landscapes, this work confirms yeast as a paradigm organism for studying DNA replication control mechanisms.

biophysics↗

bioRxiv2024

Nanotiming: telomere-to-telomere DNA replication timing profiling by nanopore sequencing

Current temporal studies of DNA replication are either low-resolution or require complex cell synchronisation and/or sorting procedures. Here we introduce Nanotiming, a nanopore sequencing-based method producing high-resolution, telomere-to-telomere replication timing (RT) profiles of eukaryotic genomes by interrogating changes in intracellular dTTP concentration during S phase through competition with its analogue bromodeoxyuridine triphosphate (BrdUTP) for incorporation into replicating DNA. Nanotiming solely demands the labelling of asynchronously growing cells with an innocuous dose of BrdU during one doubling time followed by BrdU quantification along nanopore reads. We demonstrate in yeast S. cerevisiae that Nanotiming precisely reproduces RT profiles generated by reference methods in wild-type and mutant cells inactivated for known RT determinants, for one-tenth of the cost. Nanotiming is simple, accurate, inexpensive, amenable to large-scale analyses, and is capable of unveiling RT at individual telomeres, revealing that Rif1 iconic telomere regulator directly delays the replication only of telomeres with specific subtelomeric elements.

genomics↗

bioRxiv2023

Decoding Nucleosome-Depleted Regions: Insights from Epigenetic Marks, Nucleosome Size, and Thermodynamic Modelling

Elucidating the global and local rules that govern genome-wide nucleosome organisation and chromatin architecture remains a critical challenge. Thermodynamic modelling based on DNA elastic properties predicts the presence of sequence-encoded nucleosome-inhibiting energy barriers (NIEBs) along vertebrate genomes. They delineate in vivo nucleosome-depleted regions (NDRs) flanked by 2-3 well positioned nucleosomes. Here, we compared mouse NIEBs to NDRs observed at CTCF binding sites and active TSSs to reveal specific chromatin organizations. We uncover in MNase-seq chromatin profiles the presence of particles of subnucleosomal length specifically positioned at the border of NIEBs with an enrichment of H3.3 and its modification H3.3 S31Ph, whereas the positioning of nucleosomes bearing H3K27ac appears insensitive to NIEBs. Surprisingly, post-translational modifications affect the size distribution of nucleosomes as seen by MNase digestion and so likely their breathing capability. We implemented an extension of our thermodynamic model allowing for variable particle size and suggest that subnucleomes at NIEB borders would result from the recruitment of chromatin remodellers at NIEBs. Our findings provide new insights into the mechanisms by which the DNA sequence and epigenetic marks shape the nucleosome positioning and breathing.

genomics↗

bioRxiv2021

Inferring the DNA replication origin density landscape in human cells from replication timing and fork directionality data

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. Author SummaryDNA replication is a vital process that produces two identical replicas of DNA from one DNA molecule, ensuring the faithful transmission of genetic information from mother to daughter cells. The synthesis of new DNA strands initiates at multiple sites, termed replication origins, propagates bidirectionally, and terminates by merging of converging strands. Replication initiation continues in unreplicated DNA but is blocked in replicated DNA. Experiments have only given partial information about origin usage. In this work we reveal the exact propensity of any site to initiate replication along human chromosomes. First, we simulate the DNA replication process using approximate origin information, predict the direction and time of replication at each point of the genome, and train a neural network to precisely recover from the predictions the starting origin information. Second, we apply this network to real replication time and direction data, extracting the replication initiation propensity landscape that exactly predicts them. We compare this landscape to independent origin usage data, benchmarking them, and to landscapes of protein factors that mark potential origins. We find that the local abundance of such factors is insufficient to predict replication initiation and we infer to which extent other chromosomal cues locally influence potential origin usage.

biophysics↗

bioRxiv2020

Human ORC/MCM density is low in active genes and correlates with replication time but does not solely define replication 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. ORC and MCM are strongly 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 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 that specify when and where replication initiates in human cells.

cell biology↗

bioRxiv2020

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. Author summaryDNA replication is one of the fundamental cell functions. The genome of eukaryotic organisms is duplicated from multiple positions named replication origins. Single molecule experiments allow to visualise the dynamics of spatio-temporal patterns created during replication process. The dynamic of replication process is regulated by checkpoints. However, the influence and the role of checkpoint regulation in the dynamics of spatio-temporal patterns of DNA replication is not understood. In this work we build a minimal, process-based and data rooted numerical model that allows to decipher the impact of checkpoint regulation on the dynamics of spatio-temporal pattern of DNA replication.

cell biology↗

bioRxiv2020

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 4,964 and 4,485 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.

genomics↗

bioRxiv2019

Active transcription regulates ORC/MCM distribution whereas replication timing correlates with ORC density in human cells

Eukaryotic 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 replication initiation events obtained by Okazaki fragment sequencing. We demonstrate that MCM is displaced from early replicating, actively transcribed gene bodies, while ORC is mainly enriched at active TSS. Late replicating, H4K20me3 containing initiation zones display enhanced ORC and MCM levels. Furthermore, we find early RTDs being primarily enriched in ORC, compared to MCM, indicating that ORC levels are involved in organizing the temporal order of DNA replication. The organizational connection between active transcription and replication competence directly links changes in the transcriptional program to flexible replication patterns, which ensures the cells flexibility to respond to environmental cues.

cell biology↗

bioRxiv2018

Mapping DNA replication with nanopore sequencing

We have harnessed nanopore sequencing to study DNA replication genome-wide at the single-molecule level. Using in vitro prepared DNA substrates, we characterized the effect of bromodeoxyuridine (BrdU) substitution for thymidine on the MinION nanopore electrical signal. Using a neural-network basecaller trained on yeast DNA containing either BrdU or thymidine, we identified BrdU-labelled tracts in yeast cells synchronously entering S phase in the presence of hydroxyurea and BrdU. As expected, the BrdU-labelled tracts coincided with previously identified early-firing, but not late-firing, replication origins. These results open the way to high-throughput, high-resolution, single-molecule analysis of DNA replication in many experimental systems.

genomics↗

bioRxiv2018

Universal temporal rate of DNA replication origin firing: A balance between origin activation and passivation

The time-dependent rate I(t) 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 I(t) 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 I(t) 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.

biophysics↗