Fig. 1.
ATOH1 binds to pre-established and de novo distal regulatory elements in differentiating hair cells. (A) Schematic of early mouse organ of Corti development. Photomicrograph of E13.5 cochlear whole mount expressing Cdkn1b-GFP (p27Kip1-GFP) within the prosensory domain. Hair cell differentiation as seen in an E17.5 cross-section of organ of Corti expressing ATOH1-GFP, stained with antibody against the hair cell marker MYO7A, and counter stained by DAPI. The cartoon depicts Atoh1-dependent, Notch-mediated lateral inhibition. Atoh1 is up-regulated in the undifferentiated progenitors (E13.5), which then differentiate into a mosaic of hair cells and supporting cells. (Scale bar, 50 μm.) (B) Venn diagram illustrating the quantitative comparison of chromatin accessibility at the 64,803 total distal elements present in sensory progenitors and nascent hair cells (FDR < 0.1). A total of 18,681 distal elements are significantly more accessible in the nascent hair cells (hair cell–enriched distal elements) relative to 21,662 enriched sites in sensory progenitors and 24,460 sites remaining unchanged between the two populations. (C) Hair cell–enriched distal elements (18,681), shown as heatmaps, are subclustered into “pre-established” and “de novo” sites (see Results) (μATACseq, IDR < 0.1). Heatmaps also indicate the presence of H3K4me1 and ATOH1 binding at the 18,681 hair cell–enriched distal elements. The dashed line separates the ATOH1 targets from non-ATOH1 targets in each group (IDR < 0.1). (D) Motif enrichment analysis of the 2,904 pre-established ATOH1 targets (Left). GO results associated with the pre-established ATOH1 targets suggest that enhancers for Notch signaling components are poised to be activated in the sensory progenitors (Middle). Hes6 locus serves as an example of the pre-established ATOH1 targets, which are already open, and H3K4me1-labeled, in the sensory progenitors (Right, gray bar). (E) Motif enrichment analysis of the 3,264 de novo ATOH1 targets (Left) indicating that POU-domain transcription factors are more enriched in the de novo ATOH1 targets compared to the pre-established ATOH1 targets. GO results indicate that ATOH1 targets within the de novo group are associated with hair cell differentiation and mechanosensation (Middle). The Gfi1 locus is representative of distal elements within the group of de novo ATOH1 targets, which are not open or labeled by active histone modifications in the sensory progenitor cells but become open and active (H3K27ac) following hair cell differentiation (Right, gray bar).