Percentages of reads in peaks were found using plotEnrichment in deepTools2117

Percentages of reads in peaks were found using plotEnrichment in deepTools2117. cochlea spiral in vibratome sections of E12-16 temporal bones. Similar to endogenous Sox2 expression, the highest level of Sox2-EGFP immunofluorescence is evident in the prosensory cells of the cochlear duct Rabbit Polyclonal to TSPO as well as the glia of the spiral ganglion in the E12, E14 and E16 cochleae (Fig.?1). In the following study, cochlear ducts were carefully dissected to remove developing spiral ganglion neurons and associated glia. Open in a separate window Figure 1 Sox2-EGFP expression in prosensory cells of the embryonic cochlea. (a) Shows the tissue isolation and FACS pipeline used to generate the cells. Cy3 NHS ester (bCf) Show Sox2-EGFP expression (and (Fig.?2a,g and Supplementary Data?2). Open in a separate window Figure 2 ATAC-seq detects gene regulatory features that include known otic enhancers in Sox2-EGFPloci. Highlighted in are known enhancer regions. Highlighted in are novel open chromatin regions detected only in Sox2-EGFPlocus in E16 Sox2-EGFPis the known 3 enhancer. Highlighted in are 7 open chromatin regions specific to Sox2-EGFPis a region that increased in accessibility in E14.5 vs. E12 Cy3 NHS ester Sox2-EGFPTSS Cy3 NHS ester increased significantly in accessibility in E14.5 when compared with E12 Sox2-EGFPin Fig.?2g). The +268?kb differentially accessible region downstream of is ~300?bp and contains putative Cy3 NHS ester binding sites for members of the Ets, MADS, Zf and Homeobox families as well as 5 consensus binding motifs for the bHLH transcription factor family (and and and corresponded to known dynamics in gene expression68C71 (Fig.?5c). Motif enrichment analysis of peak subsets that increased in accessibility across development showed significant enrichment for motifs of the Six, Rfx, Ctcf and Sox transcription factor families, as well as specific enrichment for some motifs not corresponding to any known regulators of cochlear development: Ebf and Nf1 (Fig.?5e,g, raw data in Supplementary Data?6). Motif enrichment analysis of peak subsets that decreased in accessibility across development also showed significant enrichment for motifs of the Six and Sox families as well as specific enrichment for Tead, Gata, Smad, Gli and Pax transcription factor families (Fig.?5e,g). Open chromatin regions in Sox2-EGFPhigh+ cells of the embryonic cochlear duct map to SNPs in human deafness genes To determine whether open chromatin regions detected in Sox2-EGFPcochlear duct cells mapped to regions of 70% sequence similarity in the human genome (Supplementary Data?8). Over 20,000 SNPs in the DVD overlap the open chromatin regions detected in mouse (Fig.?6a). Most SNPs in the DVD found to overlap with mouse open chromatin regions are intronic and of unknown significance to the pathogenesis of deafness (Fig.?6a). Some SNPs in human deafness genes coincide with transcription factor binding motifs in mouse open chromatin regions. For example, Fig.?6b shows two SNPs in a SIX motif overlapping an open chromatin region detected in Sox2-EGFPindicate two SNPs of unknown significance in intron 1 that potentially affect binding at a SIX motif in a region of high evolutionary conservation (that is orthologous to an ATAC-seq peak (in embryonic mouse Sox2-EGFPinhibition reduces neuromast formation in the zebrafish lateral line78, consistent with a possible role for the Hippo/Yap/Tead pathway in cochlear development. Previous findings in our lab showed that the inhibition of Fgf-signaling in cochlear explant cultures abolishes sensory specification as well as the expression of the downstream targets Etv4 and Etv534, suggesting a role for the Ets family in sensory specification in the cochlea. Mutations in Klf/Sp, Ebf and Nfi family members have been associated with neurodevelopmental defects79C88. Furthermore, defects observed in mutants include hearing loss84,86. Our findings show enrichment of the Ebf1-4 motif in the open chromatin of Sox2-EGFPregulation. Atoh1 is not only necessary for hair cell formation56 but also sufficient to induce hair cell differentiation in a limited population of cochlear cells90. Epigenetic regulation of expression therefore has implications for hair cell regeneration strategies. Previous studies demonstrated a critical 3 enhancer and showed association of this region as well as the promoter and an exonic region with activation-associated histone marks26,91. We find that these regions are accessible in Sox2-EGFPexpression at E14.556 is perhaps primed but unrelated to coincident changes in the accessibility of these regions. Rather, a?+?268?kb distal region containing 5 bHLH binding motifs increased significantly in accessibility in E14.5.