Abstract
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.
Highlights
Gene expression in higher eukaryotes is tightly controlled by complex cis-regulatory systems consisting of multiple enhancers modulating the transcription levels of a target gene
The functional expression of all genes is regulated by proteins, namely transcription factors that bind to specific areas of DNA known as regulatory regions
Whereas most DNA in our genome is normally bound by other proteins and packaged into units called nucleosomes, a specific subset of tissue-specific regulatory regions is responsible for tissue-specific gene expression; these active regions are nucleosome-depleted and bound by transcription factors
Summary
Gene expression in higher eukaryotes is tightly controlled by complex cis-regulatory systems consisting of multiple enhancers modulating the transcription levels of a target gene. Nucleosomes flanking active regulatory regions usually carry histone modifications, such as H3K27Ac and H3K4me1 [2], and in human and other vertebrates, active promoters and enhancers may have dynamic hyper- or hypomethylated CpG dinucleotides, denoting inactive and active enhancers respectively, whereby changes in methylation usually accompany changes in activity [3] All of these features, to some extent, correlate significantly with the expression of the target gene(s) that they control [1,4]. It is conceivable that the joint interpretation of cancer genomes and cancer transcriptomes, both of which can be sequenced directly from tumor biopsies down to the single cell level, will require the intermediate layer of the “functional genome” to permit us to understand how the epigenomic changes drive changes in gene expression To profile this chromatin layer, alternative approaches are required to progress towards smaller cell populations
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