Using eHi-C to map chromatin loops in mouse beta cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA749602 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15242985_1.fastq.gz | Fastqsanger.gz | |||
| SRR15242985_2.fastq.gz | Fastqsanger.gz | |||
| SRR15242986_1.fastq.gz | Fastqsanger.gz | |||
| SRR15242986_2.fastq.gz | Fastqsanger.gz | |||
| SRR15242987_1.fastq.gz | Fastqsanger.gz |
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Project description:R-loops are features of chromatin consisting of a strand of DNA hybridized to RNA, as well as the expelled complementary DNA strand. R-loops are enriched at promoters where they have recently been shown to have important roles in modifying gene expression. However, the location of promoter-associated R-loops and the genomic domains they perturb to modify gene expression remain unclear. To resolve this issue, we developed a bisulfite-based approach, bisDRIP-seq, to map R-loops across the genome at near-nucleotide resolution in MCF-7 cells. We found the location of promoter-associated R-loops is dependent on the presence of introns. In intron-containing genes, R-loops are bounded between the transcription start site and the first exon-intron junction. In intronless genes, the 3' boundary displays gene-specific heterogeneity. Moreover, intronless genes are often associated with promoter-associated R-loop formation. Together, these studies provide a high-resolution map of R-loops and identify gene structure as a critical determinant of R-loop formation.
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Project description:The formation of R-loops is a natural consequence of the transcription process, caused by invasion of the DNA duplex by nascent transcripts. These structures have been considered rare transcriptional by-products with potential harmful effects on genome integrity, due to the fragility of the displaced DNA coding strand. However R-loops may also possess beneficial effects as their widespread formation has been detected over CpG island promoters in human genes. Furthermore we have previously shown that R-loops are particularly enriched over G-rich terminator elements. These facilitate RNA polymerase II (Pol II) pausing prior to efficient termination. Here we reveal an unanticipated link between R-loops and RNA interference (RNAi)-dependent H3K9me2 formation over pause site termination regions of mammalian protein coding genes. We show that R-loops induce antisense transcription over these pause elements which in turn lead to the generation of double-strand RNA (dsRNA) and recruitment of Dicer, Ago1, Ago2, and G9a histone lysine methyltransferase (HKMT). Consequently an H3K9me2 repressive mark is formed and Heterochromatin Protein 1γ (HP1γ) is recruited, that reinforces Pol II pausing prior to efficient transcriptional termination. We predict that R-loops promote a chromatin architecture that defines the termination region for a substantial subset of mammalian genes. PolIIS2ph ChIP-seq and input in untreated condition and treated with BIX and RNaseH1 overexpression in HeLa cells. The 4 samples have been multiplexed, pooled and sequenced on 3 lanes of Illumina HiSeq2000.
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Project description:R-loops are transcription by-products that may constitute a threat to genome integrity. In addition to specific enzymes to remove them, eukaryotes rely on a number of mRNP biogenesis factors such as the THO complex, to prevent co-transcriptional R-loop formation. We show in Saccharomyces cerevisiae that R-loops are tightly and specifically linked with histone H3-Ser10 phosphorylation (H3S10P), a mark of chromatin condensation. Importantly, ChIP-chip analyses reveal a clear H3S10P accumulation at the pericentromeric chromatin during the G1-phase of the cell cycle only in R loop-accumulating yeast strains but not in those non-accumulating R-loops, and a significantly higher accumulation during S-phase. Such a difference can also be detected in a number of genes along the genome. ChIP-chip studies were perfomed with antibodies against Histone H3 and the phosphorylated Histone H3 at Serine10 in the yeast S. cerevisiae.
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