Genome-wide mapping implicates R-loops in lineage-specific processes and DNA break hotspot formation in neural stem/progenitor cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA802813 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17862117.fastq.gz | Fastqsanger.gz | |||
| SRR17862118.fastq.gz | Fastqsanger.gz | |||
| SRR17862119.fastq.gz | Fastqsanger.gz | |||
| SRR17862120.fastq.gz | Fastqsanger.gz | |||
| SRR17862121.fastq.gz | Fastqsanger.gz |
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Project description:We elucidated the genome-wide landscape of RNA:DNA hybrid structures called "R-loops" in primary neural stem/progenitor cells to assess their contribution to recurrent classes of DNA break "hotspots".
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Project description:The microRNA biogenesis enzyme Drosha was found to be important for DNA repair and this function appears to be distinct to its role in miRNA-mediated repression. Novel small RNAs were reported previously to be produced from the sequences around a DNA break. Utilising an endonuclease system (AsiSI) we were unable to detect such small RNA around 100 cuts within the endogenous genome. Sequencing of R-loops (DNA:RNA hybrids) was performed and an increase in R-loop formation was observed around many DNA break sites. Loss of Drosha appears to perturb this damage dependent formation of R-loops. RNase H1 over-expression appears to reduce repair at these break sites. Drosha appears to be important for facilitating R-loop formation at DNA break sites to aid in the repair process.
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Project description:Recurrent somatic hotspot mutations of DICER1 appear to be clustered around each of four critical metal binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3’ end of the 5p-miRNA strand of a pre-mRNA hairpin. To investigate the effects of these cancer-associated “hotspot” mutations we engineered mouse Dicer1-deficient ES cells to express wild-type and an allelic series of the mutant human DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal binding site mutations were compared to each other and wild-type human DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation carrying cells were distinct from both wild-type and Dicer1-deficient cells. Further, miRNA profiles showed mutant DICER1 results in a dramatic loss in processing of mature 5p-miRNA strands but were still able to create 3p-strand miRNAs. Messenger-RNA profile changes were consistent with the loss of 5p-strand miRNAs and showed enriched expression for predicted targets of the lost 5p derived miRNAs. We therefore conclude that cancer-associated somatic hotspot mutations of DICER1, affecting any one of four metal binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA-processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p-strand miRNA. We further propose that this resulting 3p-strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations. A total of 28 Affymetrix Mouse Gene ST arrays were done for mRNA expression profiling of various DICER1 mutants (n=14), wildtype controls (n=6), vector only (n=3) and parental cell lines (n=5).
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Project description:Homologous recombination events during meiosis cluster at specific loci called hotspots. Hotspot activation requires accessible chromatin, DNA sequence motifs DNA sequence-specific binding proteins, but how these proteins create a suitable environment for Rec12 recruitment is unknown. Here, we purified the ade6-M26 meiotic recombination hotspot from Schizosaccharomyces pombe and identified novel regulators of hotspot activation by mass spectrometry. Small, circular minichromosomes containing the ade6-M26 hotspot or control allele were transformed into strains permitting synchronous meiotic induction and purified from these cultures at sequential time points. Enrichment of known M26 binding factors Atf1 and Pcr1 in the data set validated the approach. Filtering for chromatin-related proteins enriched in the hotspot revealed candidate hotspot regulators, which were validated using genetic studies. This study highlights the power of unbiased proteomic screens to reveal novel insights into fundamental biological processes, and highlights a critical role in H2AZ turnover in mediating access of transcription factors to their underlying DNA-sequence motifs.
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Project description:Ewing’s sarcoma family of tumors (ESFT) is an aggressive pediatric bone and soft tissue cancer. It is the prototypical example of mesenchymal tumors driven by a fusion oncogene involving the ewing sarcoma break point region 1 (EWSR1) gene, most frequently– EWS-FLI1. We have discovered that loss of EWSR1 leads to accumulation of R-loops, replication stress and impaired homologous recombination, recapitulating breast cancer 1, early onset (BRCA1) deficiency. EWS-FLI1 acts dominant negatively in ESFT to impart the same phenotypes. Further we demonstrate that in ESFT, BRCA1 predominantly associates with the elongating transcription machinery and is unavailable for DNA strand break repair. Gene expression profiling identified upregulated compensatory mechanisms in ESFT cells to process increased R-loops (RNASEH2 and FEN1) and replication stress (Fanconi Anemia). Taken together, our data identifies BRCA1 sequestration due to transcription stress as the mechanistic basis for ESFT chemosensitivity and suggests potential targets for the much lacking second-line therapy.
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Project description:Ewing’s sarcoma family of tumors (ESFT) is an aggressive pediatric bone and soft tissue cancer. It is the prototypical example of mesenchymal tumors driven by a fusion oncogene involving the ewing sarcoma break point region 1 (EWSR1) gene, most frequently– EWS-FLI1. We have discovered that loss of EWSR1 leads to accumulation of R-loops, replication stress and impaired homologous recombination, recapitulating breast cancer 1, early onset (BRCA1) deficiency. EWS-FLI1 acts dominant negatively in ESFT to impart the same phenotypes. Further we demonstrate that in ESFT, BRCA1 predominantly associates with the elongating transcription machinery and is unavailable for DNA strand break repair. Gene expression profiling identified upregulated compensatory mechanisms in ESFT cells to process increased R-loops (RNASEH2 and FEN1) and replication stress (Fanconi Anemia). Taken together, our data identifies BRCA1 sequestration due to transcription stress as the mechanistic basis for ESFT chemosensitivity and suggests potential targets for the much lacking second-line therapy.
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