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Integrating RNA-seq and assay for transposase-accessible chromatin by sequencing (ATAC-seq) predicts functionally-relevant chromatin regions

ABSTRACT: Gene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for epigenomic investigations. Integrating epigenomic and transcriptomic data has the potential to reveal the chromatin-mediated mechanisms regulating transcription. However, integrating these two data types remains challenging. We used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We show that insulin causes widespread changes in chromatin accessibility and gene expression. Then, we attempted to integrate ATAC- and RNA-seq data to predict functionally-relevant chromatin regions that control the transcriptional response to insulin. We show that using differential chromatin accessibility can predict functionally-relevant genome regions, but that stratifying differentially-accessible chromatin regions by annotated feature type provides a better prediction of whether a chromatin region regulates gene expression. In particular, our data demonstrate a strong correlation between chromatin regions annotated to distal promoters (1-2 kb from the transcription start site). To test this prediction, we cloned candidate distal promoter regions upstream of luciferase and validated the functional relevance of these chromatin regions. Our data show that distal promoter regions selected by correlations with RNA-seq are more likely to control gene expression. Thus, correlating ATAC- and RNA-seq data can home in on functionally-relevant chromatin regions

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE197224 | GEO | 2022/02/23

REPOSITORIES: GEO

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Project description:Genomic enhancers regulate spatio-temporal gene expression by recruiting specific combinations of transcription factors (TFs). When TFs are bound to active regulatory regions, they displace canonical nucleosomes, making these regions biochemically detectable as nucleosome-depleted regions or accessible/open chromatin. Here we ask whether open chromatin profiling can be used to identify the entire repertoire of active promoters and enhancers underlying tissue-specific gene expression during normal development and oncogenesis in vivo. To this end, we first compare two different approaches to detect open chromatin in vivo using the Drosophila eye primordium as a model system: FAIRE-seq, based on physical separation of open versus closed chromatin; and ATAC-seq, based on preferential integration of a transposon into open chromatin. We find that both methods reproducibly capture the tissue-specific chromatin activity of regulatory regions, including promoters, enhancers, and insulators. Using both techniques, we screened for regulatory regions that become ectopically active during Ras-dependent oncogenesis, and identified 3778 regions that become (over-)activated during tumor development. Next, we applied motif discovery to search for candidate transcription factors that could bind these regions and identified AP-1 and Stat92E as key regulators. We validated the importance of Stat92E in the development of the tumors by introducing a loss of function Stat92E mutant, which was sufficient to rescue the tumor phenotype. Additionally we tested if the predicted Stat92E responsive regulatory regions are genuine, using ectopic induction of JAK/STAT signaling in developing eye discs, and observed that similar chromatin changes indeed occurred. Finally, we determine that these are functionally significant regulatory changes, as nearby target genes are up- or down-regulated. In conclusion, we show that FAIRE-seq and ATAC-seq based open chromatin profiling, combined with motif discovery, is a straightforward approach to identify functional genomic regulatory regions, master regulators, and gene regulatory networks controlling complex in vivo processes. FAIRE-Seq in Drosophila wild type eye-antennal imaginal discs (2 wt strains); ATAC-Seq in Drosophila wild type eye-antennal imaginal discs (3 wt strains) ; FAIRE-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila Ras/Scrib induced eye disc tumors (1 early and 1 late); ATAC-Seq in Drosophila eye discs with Unpaired over-expression (2 biological replicates); CTCF ChIP-seq in Drosophila eye discs; ChIP-seq input in Drosophila eye discs

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Integrating RNA-seq and assay for transposase-accessible chromatin by sequencing (ATAC-seq) predicts functionally-relevant chromatin regions

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