Biology subjects

Rivera-Mulia, J. C.

Publications and source records attributed to Rivera-Mulia, J. C..

12 recordsLinked to original sources

bioRxiv2026

REPLAY: A reproducible and user-friendly application for DNA replication timing analysis from Repli-seq data

BackgroundDNA replication timing (RT) is a fundamental feature of genome organization that is regulated in a cell-type-specific manner and frequently altered in disease. Repli-seq is the standard approach for genome-wide RT profiling; however, its analysis typically requires multiple independent tools and custom scripts, limiting reproducibility, portability, and accessibility, particularly for users without computational expertise. In addition, existing workflows often lack standardization and require substantial user intervention. ResultsWe developed REPLAY, a fully automated, reproducible, and user-friendly application for replication timing analysis. REPLAY is distributed as a standalone executable that enables end-to-end processing from compressed FASTQ files to genome-wide RT profiles without requiring software installation or programming experience. Through an intuitive graphical interface, users can configure analysis parameters, including input and output directories, reference genome, normalization strategy (quantile, median, or interquartile range), and smoothing. The application integrates all processing steps--quality control, trimming, alignment, binning, RT log2 calculation, normalization, smoothing, and visualization-- within a single automated workflow. Application of REPLAY to publicly available datasets demonstrate accurate reconstruction of RT profiles and high reproducibility across samples. ConclusionsREPLAY offers a portable, reproducible, and accessible solution for the analysis of RT data. By eliminating the need for command-line tools and complex installations, it lowers the entry barrier enabling standardized analysis across diverse research settings.

genomics↗

bioRxiv2025

The long-standing relationship between replication timing, gene expression, and chromatin accessibility is maintained in early mouse embryogenesis

Replication timing (RT) is the temporal program of genome duplication that is coordinated with 3D genome organization and gene expression1,2. How and when RT is established during mammalian development are questions that remain unsolved. Four recent studies provided the first insights; however, they reached to contradictory conclusions. Nakatani et al. reported a poorly defined RT in mouse zygotes that is progressively consolidated by the 4-cell stage3; Takahashi et al. and Halliwell, et al. reported a complete RT absence in the zygote with abrupt emergence at later stages4,5; finally, Xu et al.6 reported a RT already established in the zygote. Discrepancies might reflect differences in computational methods and thresholds applied to sparse single-cell RT data. More recently, Shetty et al. concluded that RT is established in the zygote, and that transcriptional activation and chromatin accessibility emerge at late replicating regions7. Their conclusions challenge the long-standing relationship between gene expression and early replication1,2,8-10 and contradict previous studies3-6. This critique demonstrates that methodological flaws and artifacts, stemming from inadequate sample acquisition and data processing led to unreliable RT inference, rendering the conclusions on RT establishment and its inverse correlation with gene expression erroneous. Moreover, re-analysis of their data, with stage-matched controls from previous studies3,5, demonstrates that the canonical relationship between RT, transcriptional activity, and chromatin accessibility are indeed still standing during the early mammalian embryogenesis.

developmental biology↗

bioRxiv2025

PARTAGE: Parallel analysis of replication timing and gene expression

The human genome is partitioned into functional compartments that replicate at specific times during the S-phase. This temporal program, referred to as replication timing (RT), is co-regulated with the 3D genome organization, is cell type-specific, and changes during development in coordination with gene expression. Moreover, RT alterations are linked to abnormal gene expression, genome instability, and structural variation in multiple diseases, including cancer. However, mechanistic links between RT, large-scale 3D genome architecture, and transcriptional regulation remain poorly understood. A major limitation is that current approaches require the separate profiling of RT and transcriptomes from independent batches of samples, obscuring the complex co-regulation between the epigenome and transcriptome. Here, we developed PARTAGE, a multiomics approach that enables joint profiling of copy number variation (CNV), RT, and gene expression from the same sample, providing a more accurate integrative view of the complex relationships between RT and gene regulation.

genomics↗

bioRxiv2025

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

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

genomics↗

bioRxiv2022

Optimized Repli-seq: Improved DNA Replication Timing Analysis by Next-Generation Sequencing

The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type-specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell analysis of RT has been developed as an alternative, these methods are low throughput and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease.

genomics↗

bioRxiv2019

Replication Timing and Transcription Identifies a Novel Fragility Signature Under Replication Stress

Common fragile sties (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Therefore, the molecular mechanism underlying CFS instability remains unclear. Here, we explored the transcriptional profile and temporal order of DNA replication (replication timing, RT) of cells under replication stress conditions. We show that the RT of only a small portion of the genome is affected by replication stress, and that CFSs are enriched for delayed RT. We identified a signature for chromosomal fragility, comprised of replication stress-induced delay in RT of early/mid S-phase replicating regions within actively transcribed large genes. This fragility signature enabled precise mapping of the core fragility region. Furthermore, the signature enabled the identification of novel fragile sites that were not detected cytogenetically, highlighting the improved sensitivity of our approach for identifying fragile sites. Altogether, this study reveals a link between altered DNA replication and transcription of large genes underlying the mechanism of CFS expression. Thus, investigating the RT and transcriptional changes in cancer may contribute to the understanding of mechanisms promoting genomic instability in cancer.

genetics↗

bioRxiv2019

An integrative ENCODE resource for cancer genomics

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

genomics↗

bioRxiv2019

Variable Retention of Differentiation-specific DNA Replication Timing in Human Pediatric Leukemia

Human B-lineage precursor acute lymphoid leukemias (BCP-ALLs) comprise a group of genetically and clinically distinct disease entities with features of differentiation arrest at known stages of normal B-lineage differentiation. We previously showed BCP-ALL cells display unique and clonally heritable DNA-replication timing (RT) programs; i.e., programs describing the variable order of replication of megabase-scale chromosomal units of DNA in different cell types. To determine the extent to which BCP-ALL RT programs mirror or deviate from specific stages of normal human B-cell differentiation, we transplanted immunodeficient mice with quiescent normal human CD34+ cord blood cells and obtained RT signatures of the regenerating B-lineage populations. We then compared these with RT signatures for leukemic cells from a large cohort of BCP-ALL patients. The results identify BCP-ALL subtype-specific features that resemble specific stages of B-cell differentiation and features that appear associated with relapse. These results suggest the genesis of BCP-ALL involves alterations in RT that reflect clinically relevant leukemia-specific genetic and/or epigenetic changes. SUMMARYGenome-wide DNA replication timing profiles of >100 pediatric leukemic samples and normally differentiating human B-lineage cells isolated from xenografted immunodeficient mice were generated. Comparison of these identified potentially clinically relevant features that both match and deviate from the normal profiles.

cancer biology↗

bioRxiv2018

RT States: systematic annotation of the human genome using cell type-specific replication timing programs

The replication timing (RT) program has been linked to many key biological processes including cell fate commitment, 3D chromatin organization and transcription regulation. Significant technology progress now allows to characterize the RT program in the entire human genome in a high-throughput and high-resolution fashion. These experiments suggest that RT changes dynamically during development in coordination with gene activity. Since RT is such a fundamental biological process, we believe that an effective quantitative profile of the local RT program from a diverse set of cell types in various developmental stages and lineages can provide crucial biological insights for a genomic locus. In the present study, we explored recurrent and spatially coherent combinatorial profiles from 42 RT programs collected from multiple lineages at diverse differentiation states. We found that a Hidden Markov Model with 15 hidden states provide a good model to describe these genome-wide RT profiling data. Each of the hidden state represents a unique combination of RT profiles across different cell types which we refer to as \"RT states\". To understand the biological properties of these RT states, we inspected their relationship with chromatin states, gene expression, functional annotation and 3D chromosomal organization. We found that the newly defined RT states possess interesting genome-wide functional properties that add complementary information to the existing annotation of the human genome.\n\nAUTHOR SUMMARYThe replication timing (RT) program is an important cellular mechanism and has been linked to many key biological processes including cell fate commitment, 3D chromatin organization and transcription regulation. Significant technology progress now allows us to characterize the RT program in the entire human genome. Results from these experiments suggest that RT changes dynamically across different developmental stages. Since RT is such a fundamental biological process, we believe that the local RT program from a diverse set of cell types in various developmental stages can provide crucial biological insights for a genomic locus. In the present study, we explored combinatorial profiles from 42 RT programs collected from multiple lineages at diverse differentiation states. We developed a statistical model consist of 15 \"RT states\" to describe these genome-wide RT profiling data. To understand the biological properties of these RT states, we inspected the relationship between RT states and other types of functional annotations of the genome. We found that the newly defined RT states possess interesting genome-wide functional properties that add complementary information to the existing annotation of the human genome.

bioinformatics↗

bioRxiv2018

Identification of cis elements for spatio-temporal control of DNA replication

The temporal order of DNA replication (replication timing, RT) is highly coupled with genome architecture, but cis-elements regulating spatio-temporal control of replication have remained elusive. We performed an extensive series of CRISPR mediated deletions and inversions and high-resolution capture Hi-C of a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells. Whereas CTCF mediated loops and chromatin domain boundaries were dispensable, deletion of three intra-domain prominent CTCF-independent 3D contact sites caused a domain-wide delay in RT, shift in sub-nuclear chromatin compartment and loss of transcriptional activity, These \"early replication control elements\" (ERCEs) display prominent chromatin features resembling enhancers/promoters and individual and pair-wise deletions of the ERCEs confirmed their partial redundancy and interdependency in controlling domain-wide RT and transcription. Our results demonstrate that discrete cis-regulatory elements mediate domain-wide RT, chromatin compartmentalization, and transcription, representing a major advance in dissecting the relationship between genome structure and function.\n\nHighlightsO_LIcis-elements (ERCEs) regulate large scale chromosome structure and function\nC_LIO_LIMultiple ERCEs cooperatively control domain-wide replication\nC_LIO_LIERCEs harbor prominent active chromatin features and form CTCF-independent loops\nC_LIO_LIERCEs enable genetic dissection of large-scale chromosome structure-function.\nC_LI

molecular biology↗

bioRxiv2017

Allele-specific control of replication timing and genome organization during development

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus X castaneus mouse crosses and exploited the high single nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (nuclear RNA-seq) and chromatin accessibility (ATAC-seq). We also present HARP: a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of 6 different hybrid mESC clones with different genomes (C57BL/6, 129/sv and CAST/Ei), parental configurations and gender revealed significant RT asynchrony between alleles across ~12 % of the autosomal genome linked to sub-species genomes but not to parental origin, growth conditions or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not SNP density, gene expression, imprinting or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types including extraembryonic endoderm stem (XEN) cells, 4 male and female primary mouse embryonic fibroblasts (MEFs) and neural precursors (NPCs) differentiated in vitro from mESCs with opposite parental configurations. Surprisingly, we found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs was largely lost in all differentiated cell types, coordinated with a more uniform Hi-C compartment arrangement, suggesting that genome organization of homologues converges to similar folding patterns during cell fate commitment.

cell biology↗

bioRxiv2017

Replication Timing Networks: a novel class of gene regulatory networks

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program and is regulated during development, coordinated with 3D genome organization and transcriptional activity. However, transcription and RT are not sufficiently coordinated to predict each other, suggesting an indirect relationship. Here, we exploit genome-wide RT profiles from 15 human cell types and intermediate differentiation stages derived from human embryonic stem cells to construct different types of RT regulatory networks. First, we constructed networks based on the coordinated RT changes during cell fate commitment to create highly complex RT networks composed of thousands of interactions that form specific functional sub-network communities. We also constructed directional regulatory networks based on the order of RT changes within cell lineages and identified master regulators of differentiation pathways. Finally, we explored relationships between RT networks and transcriptional regulatory networks (TRNs), by combining them into more complex circuitries of composite and bipartite networks, revealing novel trans interactions between transcription factors and downstream RT changes that were validated with ChIP-seq data. Our findings suggest a regulatory link between the establishment of cell type specific TRNs and RT control during lineage specification.

systems biology↗