Repli-seq sample preparation using cell sorting with cell-permeant dyes
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE246516 | GEO | 2023/11/02
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 an improved method to measure RT genome-wide that enables high resolution analysis of low input samples.
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Project description:Repli-seq sample preparation using cell sorting with cell-permeant dyes
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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: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:DNA replication occurs in units of chromatin higher order organization (Topologically Associating Domains, or TADs; replication domains, or RDs), which self-organize in 3D into sub-nuclear compartments of early or late replicating chromatin. However, identification of cis-elements regulating replication timing (RT) and sub-nuclear compartments has been a major challenge in the field. Through an extensive series of CRISPR mediated deletion and inversions at a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells (mESCs), we have identified cis-regulatory “early replication control elements” (ERCEs) that mediate early replication and A/B compartmentalization. We show that CTCF/cohesin loops, including the TAD boundaries, were dispensable, and multiple internal DNA segments were necessary for the domain’s early replication in a partially redundant fashion. These segments were also sufficient to maintain early replication in the context of large inversions, irrespective of TAD boundaries. High-resolution capture Hi-C of this region revealed three sites of major contact that also displayed prominent chromatin features. Targeted deletion of all three contact points, which do not include the major mapped replication origins, caused a complete shift to late replication and association with compartment B, equivalent to the switch during lineage commitment. Individual and pair-wise deletions confirmed their partial redundancy and interdependency in giving rise to domain-wise RT patterns, and suggest the importance of long-range interactions. ERCEs are enriched in properties of strong or super-enhancers, and regulate gene expression, although transcription itself is not sufficient in driving early replication. In sum, our results have revealed the first cis-elements regulating the temporal and spatial control of DNA replication that is independent of transcription.
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Project description:DNA replication occurs in units of chromatin higher order organization (Topologically Associating Domains, or TADs; replication domains, or RDs), which self-organize in 3D into sub-nuclear compartments of early or late replicating chromatin. However, identification of cis-elements regulating replication timing (RT) and sub-nuclear compartments has been a major challenge in the field. Through an extensive series of CRISPR mediated deletion and inversions at a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells (mESCs), we have identified cis-regulatory “early replication control elements” (ERCEs) that mediate early replication and A/B compartmentalization. We show that CTCF/cohesin loops, including the TAD boundaries, were dispensable, and multiple internal DNA segments were necessary for the domain’s early replication in a partially redundant fashion. These segments were also sufficient to maintain early replication in the context of large inversions, irrespective of TAD boundaries. High-resolution capture Hi-C of this region revealed three sites of major contact that also displayed prominent chromatin features. Targeted deletion of all three contact points, which do not include the major mapped replication origins, caused a complete shift to late replication and association with compartment B, equivalent to the switch during lineage commitment. Individual and pair-wise deletions confirmed their partial redundancy and interdependency in giving rise to domain-wise RT patterns, and suggest the importance of long-range interactions. ERCEs are enriched in properties of strong or super-enhancers, and regulate gene expression, although transcription itself is not sufficient in driving early replication. In sum, our results have revealed the first cis-elements regulating the temporal and spatial control of DNA replication that is independent of transcription.
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Project description:DNA replication occurs in units of chromatin higher order organization (Topologically Associating Domains, or TADs; replication domains, or RDs), which self-organize in 3D into sub-nuclear compartments of early or late replicating chromatin. However, identification of cis-elements regulating replication timing (RT) and sub-nuclear compartments has been a major challenge in the field. Through an extensive series of CRISPR mediated deletion and inversions at a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells (mESCs), we have identified cis-regulatory “early replication control elements” (ERCEs) that mediate early replication and A/B compartmentalization. We show that CTCF/cohesin loops, including the TAD boundaries, were dispensable, and multiple internal DNA segments were necessary for the domain’s early replication in a partially redundant fashion. These segments were also sufficient to maintain early replication in the context of large inversions, irrespective of TAD boundaries. High-resolution capture Hi-C of this region revealed three sites of major contact that also displayed prominent chromatin features. Targeted deletion of all three contact points, which do not include the major mapped replication origins, caused a complete shift to late replication and association with compartment B, equivalent to the switch during lineage commitment. Individual and pair-wise deletions confirmed their partial redundancy and interdependency in giving rise to domain-wise RT patterns, and suggest the importance of long-range interactions. ERCEs are enriched in properties of strong or super-enhancers, and regulate gene expression, although transcription itself is not sufficient in driving early replication. In sum, our results have revealed the first cis-elements regulating the temporal and spatial control of DNA replication that is independent of transcription.
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Project description:DNA replication occurs in units of chromatin higher order organization (Topologically Associating Domains, or TADs; replication domains, or RDs), which self-organize in 3D into sub-nuclear compartments of early or late replicating chromatin. However, identification of cis-elements regulating replication timing (RT) and sub-nuclear compartments has been a major challenge in the field. Through an extensive series of CRISPR mediated deletion and inversions at a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells (mESCs), we have identified cis-regulatory “early replication control elements” (ERCEs) that mediate early replication and A/B compartmentalization. We show that CTCF/cohesin loops, including the TAD boundaries, were dispensable, and multiple internal DNA segments were necessary for the domain’s early replication in a partially redundant fashion. These segments were also sufficient to maintain early replication in the context of large inversions, irrespective of TAD boundaries. High-resolution capture Hi-C of this region revealed three sites of major contact that also displayed prominent chromatin features. Targeted deletion of all three contact points, which do not include the major mapped replication origins, caused a complete shift to late replication and association with compartment B, equivalent to the switch during lineage commitment. Individual and pair-wise deletions confirmed their partial redundancy and interdependency in giving rise to domain-wise RT patterns, and suggest the importance of long-range interactions. ERCEs are enriched in properties of strong or super-enhancers, and regulate gene expression, although transcription itself is not sufficient in driving early replication. In sum, our results have revealed the first cis-elements regulating the temporal and spatial control of DNA replication that is independent of transcription.
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