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

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.

Dynamics of chromatin accessibility and epigenetic state in response to UV damage

“…Furthermore, we also employed an assay for transposase-accessible chromatin (ATAC) on untreated and UV-irradiated cells as an independent measure of chromatin accessibility, revealing that the FAIRE-seq results were largely reproducible ( Fig. S2M) (Buenrostro et al, 2013(Buenrostro et al, , 2015Davie et al, 2015). To further augment these results, nucleosome occupancy was investigated by assessing total H3 enrichments at such sites by ChIP-qPCR.…”

Identification and in silico modeling of enhancers reveals new features of the cardiac differentiation network

“…Of note, an alternative method, ATAC-seq (Buenrostro et al, 2013), which identifies nucleosome-free regions that are accessible to bacterial (Tn5) transposase, has recently been shown to be well suited for limited amounts of chromatin (Davie et al, 2015). It could therefore be used as a complementary approach to H3K27ac ChIP-seq on BiTS chromatin preparations.…”

JAK/STAT signalling mediates cell survival in response to tissue stress

“…JAK/STAT activity is not required for compensatory proliferation JAK/STAT has been reported to play a role in promoting cell proliferation in wild-type (Bach et al, 2003;Tsai and Sun, 2004;Mukherjee et al, 2005), tumorous (Classen et al, 2009;Wu et al, 2010;Davie et al, 2015;Bunker et al, 2015;Amoyel et al, 2014) and surgically injured discs (Katsuyama et al, 2015). To test whether JAK/STAT is generally required to drive compensatory proliferation, we quantified mitotic events after Eiger stimulation in discs with impaired JAK/STAT signalling.…”

“…A proliferative function for JAK/STAT was suggested by early studies of tissue growth during disc development (Bach et al, 2003;Tsai and Sun, 2004;Mukherjee et al, 2005). Subsequently, JAK/STAT has been widely implicated in promoting proliferation of neoplastic cells (Classen et al, 2009;Wu et al, 2010;Davie et al, 2015;Bunker et al, 2015;Amoyel et al, 2014) and aberrant nonautonomous proliferation of wild-type cells in fly models of tumorigenesis (Vaccari and Bilder, 2005;Herz et al, 2006;Moberg et al, 2005). Activation of Upd gene transcription upon tissue damage has been linked to compensatory proliferation in imaginal discs and adult guts (Jiang et al, 2009;Pastor-Pareja et al, 2008;Katsuyama et al, 2015;Wu et al, 2010;Lin et al, 2009;Bunker et al, 2015;Santabárbara-Ruiz et al, 2015).…”

“…In fly tumours, STATresponsive enhancers have been mapped (Davie et al, 2015), but few target genes regulating tissue size have been described (Tsai and Sun, 2004;Betz et al, 2008;Hasan et al, 2015). However, several studies suggest that developmental functions of JAK/STAT are mediated by the transcriptional repressors Chinmo (Flaherty et al, 2010), Zfh1 (Ohayon et al, 2009;Leatherman and Dinardo, 2008) and Zfh2 (Perea et al, 2013;Guarner et al, 2014;Ayala-Camargo et al, 2013).…”