Optimized Repli-seq: An improved DNA Replication Timing Analysis by Next Generation Sequencing
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE196749 | GEO | 2022/12/16
REPOSITORIES: GEO
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Project description: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 developed a sample preparation method to measure RT genome-wide that does not require fixation.
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Project description:Replication timing (RT) control is linked to histone modifications (HMs) and genome architecture. However, HM landscape across cell cycle and its relationship with RT and chromatin modifiers have not been comprehensively assessed. Here we perform detailed replication profiling together with cell cycle profiling of multiple HMs, ATAC-seq, and gene expression. We classify 97% of the genome into four gross chromatin states, including a previously uncharacterized intergenic H3K36me2 state. RT and associated replication features (e.g. initiation zones) quantitatively associate with these chromatin states, HM dynamics, and spatial chromatin structure. Furthermore, overexpression of the histone H3 lysine 9/36 tri-demethylase KDM4A impacts RT across 12% of the genome. RT regulation is strongly linked to KDM4A-associated HMs and enhancer-like elements. Lastly, replication within the intergenic H3K36me2 neighborhoods is sensitive to KDM4A overexpression and is controlled as wide megabase-scale units. In conclusion, these studies establish a critical role for chromatin regulation in directing RT genome-wide.
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Project description: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.
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Project description:Duplication of the genome in mammalian cells occurs in a defined temporal order referred as its replication-timing program (RT). RT is regulated in units of 400-800 Kb referred as replication domains (RDs) and changes dynamically during development. Changes in RT are generally coordinated with transcriptional competence and changes in sub-nuclear position. We generated genome-wide RT profiles for 29 distinct human cell types including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm and neural crest (NC) development. We identified clusters of RDs that replicate at unique times in each stage (RT signatures). Surprisingly, transcriptome data revealed that, despite an overall correlation between early replication and transcriptional activity, most genes that switched RT during differentiation can be expressed when late replicating. Intriguingly, this class of genes was nonetheless induced to high expression levels prior to a late to early RT switch and down-regulated after the switch back to late replication. These results clarify the complex relationship between transcription and RT and identify classes of genes that behave as potential drivers of the RT switch vs. those that may depend upon an RT switch for transcriptional induction. Genome-wide replication timing profiles were constructed from 60 human samples covering 29 distinct cell types including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm and neural crest (NC) development.
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Project description: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 and sub-nuclear 3D architecture 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 of varied genetic subtype and outcome. 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 biologically significant and clinically relevant leukemia-specific epigenetic changes that have potential as a novel genre of prognostic biomarkers.
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Project description: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 and sub-nuclear 3D architecture 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 of varied genetic subtype and outcome. 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 biologically significant and clinically relevant leukemia-specific epigenetic changes that have potential as a novel genre of prognostic biomarkers.
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Project description:The role of DNA sequence in determining replication timing (RT) and chromatin higher order organization remains elusive. To address this question, we have developed an extra-chromosomal replication system consisting of ~200kb human bacteria artificial chromosomes (BACs) modified with Epstein-Barr virus (EBV) replication origin elements (E-BACs). E-BACs were stably maintained as autonomous mini-chromosomes in both HeLa and human induced pluripotent stem cells (hiPSCs) and established their RT de novo. We applied repli-seq to evaluate E-BACs' replication timing.
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Project description:The replication timing program, or the order in which DNA is duplicated during S-phase, is associated with various features of chromosome structure and function, including gene expression, histone modifications, and 3-D compartmentalization of chromatin. 3 cell types, with a total of 6 individual replicates
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Project description:Duplication of the genome in mammalian cells occurs in a defined temporal order referred as its replication-timing program (RT). RT is regulated in units of 400-800 Kb referred as replication domains (RDs) and changes dynamically during development. Changes in RT are generally coordinated with transcriptional competence and changes in sub-nuclear position. We generated genome-wide RT profiles for 29 distinct human cell types including embryonic stem cell (hESC)-derived, primary cells and established cell lines representing intermediate stages of endoderm, mesoderm, ectoderm and neural crest (NC) development. We identified clusters of RDs that replicate at unique times in each stage (RT signatures). Surprisingly, transcriptome data revealed that, despite an overall correlation between early replication and transcriptional activity, most genes that switched RT during differentiation can be expressed when late replicating. Intriguingly, this class of genes was nonetheless induced to high expression levels prior to a late to early RT switch and down-regulated after the switch back to late replication. These results clarify the complex relationship between transcription and RT and identify classes of genes that behave as potential drivers of the RT switch vs. those that may depend upon an RT switch for transcriptional induction.
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