Discovery of Transcription Factors and Regulatory Regions Driving In Vivo Tumor Development by ATAC-seq and FAIRE-seq Open Chromatin Profiling

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.
Author Summary 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 (histones) 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. We use two techniques to identify these open chromatin regions, in a normal tissue and a RasV12 induced cancer tissue. We discovered a remarkable change in the accessible regulatory landscape between these two tissues, with several thousand regions becoming more accessible in the cancer tissue. We identified two transcription factors known to be involved in cancer (AP-1 and Stat92E) controlling these newly accessible regulatory regions. Finally, we introduced a mutation resulting in Stat92E becoming non-functional in the cancer tissue, which decreased the severity of the tumor. Our study shows that open chromatin profiling can be used to identify complex in vivo processes, and we shed new light on Ras dependent cancer development.