Loss of histone H3.3 results in DNA replication defects and altered origin dynamics in C. elegans.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA591001 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Histone H3.3 is a replication-independent variant of histone H3 with important roles in development, differentiation and fertility. Here we show that loss of H3.3 results in replication defects in Caenorhabditis elegans embryos at elevated temperatures. To characterize these defects, we adapt methods to determine replication timing, map replication origins, and examine replication fork progression. Our analysis of the spatiotemporal regulation of DNA replication shows that despite the very rapid embryonic cell cycle, the genome is replicated from early and late firing origins and is partitioned into domains of early and late replication. We find that under temperature stress conditions, additional replication origins become activated. Moreover, loss of H3.3 results in altered replication fork progression around origins, which is particularly evident at stress-activated origins. These replication defects are accompanied by replication checkpoint activation, a delayed cell cycle, and increased lethality in checkpoint-compromised embryos. Our comprehensive analysis of DNA replication in C. elegans reveals the genomic location of replication origins and the dynamics of their firing, and uncovers a role of H3.3 in the regulation of replication origins under stress conditions.
2020-09-28 | GSE140804 | GEO
Project description:Histone H3.3 is a replication-independent variant of histone H3 with important roles in development, differentiation, and fertility. Here, we show that loss of H3.3 results in replication defects in Caenorhabditis elegans embryos at elevated temperatures. To characterize these defects, we adapt methods to determine replication timing, map replication origins, and examine replication fork progression. Our analysis of the spatiotemporal regulation of DNA replication shows that despite the very rapid embryonic cell cycle, the genome is replicated from early and late firing origins and is partitioned into domains of early and late replication. We find that under temperature stress conditions, additional replication origins become activated. Moreover, loss of H3.3 results in altered replication fork progression around origins, which is particularly evident at stress-activated origins. These replication defects are accompanied by replication checkpoint activation, a delayed cell cycle, and increased lethality in checkpoint-compromised embryos. Our comprehensive analysis of DNA replication in C. elegans reveals the genomic location of replication origins and the dynamics of their firing, and uncovers a role of H3.3 in the regulation of replication origins under stress conditions.
| S-EPMC7706726 | biostudies-literature
Project description:Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans, and has been implicated in many important biological processes including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components, or in H3.3 encoding genes, have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3 null mouse models through classical genetic approaches. We found H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres and pericentromeric regions of chromosomes leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development. RNA-seq in embryos at E10.5 comparing 3 samples with the following genotype Trp53-/-; H3f3afl/-; H3f3bfl/-; Sox2-CreTg/0 to three samples with the following genotype Trp53-/-; H3f3afl/+; H3f3bfl/+; Sox2-CreTg/0
2015-06-12 | E-GEOD-69771 | biostudies-arrayexpress
Project description:Histone H3.3 is a highly conserved histone H3 replacement variant in metazoans, and has been implicated in many important biological processes including cell differentiation and reprogramming. Germline and somatic mutations in H3.3 genomic incorporation pathway components, or in H3.3 encoding genes, have been associated with human congenital diseases and cancers, respectively. However, the role of H3.3 in mammalian development remains unclear. To address this question, we generated H3.3 null mouse models through classical genetic approaches. We found H3.3 plays an essential role in mouse development. Complete depletion of H3.3 leads to developmental retardation and early embryonic lethality. At the cellular level, H3.3 loss triggers cell cycle suppression and cell death. Surprisingly, H3.3 depletion does not dramatically disrupt gene regulation in the developing embryo. Instead, H3.3 depletion causes dysfunction of heterochromatin structures at telomeres, centromeres and pericentromeric regions of chromosomes leading to mitotic defects. The resulting karyotypical abnormalities and DNA damage lead to p53 pathway activation. In summary, our results reveal that an important function of H3.3 is to support chromosomal heterochromatic structures, thus maintaining genome integrity during mammalian development.
2015-06-12 | GSE69771 | GEO
Project description:We developed a new sequencing assay to track the de novo deposition of the histone H3 variants H3.1 and H3.3 during S phase. We use cells stably expressing H3.1-SNAP or H3.3-SNAP, and synchronize them in G1/S by double-thymidine block. The SNAP-tag enables to discriminate newly synthesized histones from preexisting ones, via a quench-chase-capture strategy. We applied this strategy to isolate new H3.1 and H3.3 after releasing cells into S phase, and probed their distribution by MNase digestion and sequencing. We could thus characterize H3.1 and H3.3 dynamics from early to mid S phase at genome-wide resolution. We further applied our method to investigate the consequences of perturbations upon deletion of the H3.3 chaperone HIRA. We used HIRA knockout and control cells, and compared H3.1 and H3.3 distribution to early replication patterns by EdU labeling and sequencing of nascent DNA.
2022-03-31 | E-MTAB-10619 | biostudies-arrayexpress
Project description:Histone chaperones and chromatin remodelers control nucleosome dynamics, essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display an exclusive preference for the H3.3-depositing HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefer the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation, and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated H3.3-H4 deposition via the HIRA pathway for proper epigenetic regulation of the genome.
2022-10-31 | PXD035578 | Pride
Project description:Modifications of histone H3 lysine 27 (H3K27) are strongly correlated with gene activation and gene repression. The histones bearing these modifications are encoded by both the replication-dependent H3 genes, and the replication-independent H3.3 genes. To examine the role that K27 modification of the H3.3 genes plays during development, we use CRISPR/Cas9 to mutate the endogenous H3.3 genes to a non-modifiable residue and performed RNAseq. We find that H3.3K27 is required for Polycomb target gene silencing, but this requirement is at a later stage of development than the requirement for replication-dependent H3K27. Notably, we find no evidence of transcriptional defects in H3.3K27 mutants, despite the strong correlation between H3.3K27 acetylation and active transcription.
2019-01-07 | GSE117703 | GEO
Project description:Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins. In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unkown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that thsese deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism. In this study, we aimed to establish an independent system to investigate how an origin-deficient chromosome is replicated. To this end, we systematically deleted seven well-characterized origins on the left arm of S. cerevisiae chromosome VI and analyzed (1) Orc2 localization during G2/M arrest and (2) BrdU incorporation during synchronous release from G1 arrest into S-phase, and compared the results to wild-type cell signals. For Orc2 ChIP-Chip experiments, Orc-bound DNA was isolated from Orc2-2Xlinker-3XFlag epitope-tagged cells arrested in G2/M using antibodies against Flag. For BrdU ChIP-Chip experiments, actively replicating DNA was isolated from cells harboring a single integrated BrdU incorporation vector released synchronously into 200mM HU using antibodies against BrdU. Immunoprecipitated and input (Orc2) or G1 (BrdU) DNA was then amplified and competitively hybridized to high-resolution strand-specific microarrays covering chromosomes III, VI, and XII.
2014-09-26 | E-GEOD-61743 | biostudies-arrayexpress
Project description:Gene bodies of vertebrates and flowering plants are occupied by histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. The profiles of enrichments in DNA methylation and H3.3 over gene bodies are correlated and both depend similarly on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation. We engineered H3.3 knockdown in Arabidopsis and observed transcription reduction that predominantly affected genes responsive to environmental cues. When H3.3 levels were reduced, gene bodies showed a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome wide distributions of several histone H3 marks, H2A.Z, linker histone H1 and nucleosome densities. We observed that in absence of H3.3, H1 distribution increased in gene bodies. This depends on levels of gene transcription. We propose that H3.3 prevents recruitment of H1, which in turn promotes chromatin folding and antagonizes access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in relation with transcriptional activity.
2017-04-24 | GSE96834 | GEO
Project description:Gene bodies of vertebrates and flowering plants are occupied by histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. The profiles of enrichments in DNA methylation and H3.3 over gene bodies are correlated and both depend similarly on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation. We engineered H3.3 knockdown in Arabidopsis and observed transcription reduction that predominantly affected genes responsive to environmental cues. When H3.3 levels were reduced, gene bodies showed a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome wide distributions of several histone H3 marks, H2A.Z, linker histone H1 and nucleosome densities. We observed that in absence of H3.3, H1 distribution increased in gene bodies. This depends on levels of gene transcription.We propose that H3.3 prevents recruitment of H1, which in turn promotes chromatin folding and antagonizes access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in relation with transcriptional activity.
2017-04-24 | GSE95175 | GEO