Fig. 2.

Atoh1 expression and hair cell differentiation is dependent on Atoh1-associated histone acetylation. (A) Schematic shows the locations across the Atoh1 locus (sites 1, 2 and 3; triangles) analyzed by µChIP qPCR for the presence of H3K9ac in FACS-purified E14.5 progenitors (PG) and E17.5 hair cells (HC), as well as control mouse embryonic stem cells, (mESC), cerebellar granule cell precursors (GCPs), and astrocytes. H3K9ac increases upon hair cell differentiation across the Atoh1 locus (sites 1, 2 and 3). Results are mean±s.e.m., *P<0.05 (n=3). (B-D″) HATi blocks Atoh1 upregulation and hair cell differentiation in embryonic organ of Corti explant cultures. Representative images of whole-mount cochlear epithelia from Atoh1-GFP transgenic organ of Corti. Time-mated E13.0 cochlear ducts (B) were cultured in the absence (C,D) or presence (C′,D′) of HAT inhibitor (curcumin) for 24 or 48 h as indicated. Alternatively, organs were cultured in HATi for 24 h, after which the inhibitor was washed out, and organs analyzed after another 24 h (D″). Scale bars: 100 µm. Arrows indicate the approximate origin and direction of progressive Atoh1-GFP expression. (E) Quantification of hair cell number as shown in B-D″. At time point 0 h there were no Atoh1-GFP+ cells. Control organs (black bars) have on average 105±42 Atoh1-GFP+ hair cells by 24 h and 1063±195 cells by 48 h. The presence of HAT inhibitor decreases the number of Atoh1-GFP+ cells (gray bars) to 32±30 by 24 h, and to 113±114 by 48 h of treatment relative to control. Washing out the inhibitor at 24 h restores the number of hair cells to 235±52 (yellow bars) by 48 h. Results are mean±s.e.m., *P<0.05 (n=3). (F) Atoh1 mRNA levels at the 0 h, 24 h, and 48 h time points, and following inhibitor washout, in control and HATi cultures. HATi decreases the relative Atoh1 mRNA levels 95% (from 40.0±3.4 to 12.7±10.0) at 24 h of treatment, and 66% (from 82.0±15.4 to 27.6±16.4) at 48 h compared with control organs. Washout of inhibitor at 24 h, increased Atoh1 mRNA levels 1.9-fold at 48 h compared with time-matched control (from 27.6±16.4 to 52.7±9.6). Atoh1 gene expression is normalized to Gapdh as internal reference. Results are mean±s.e.m., *P<0.05 (n=3). (G) H3K9ac-µChIP qPCR assessment of the Atoh1 transcription start site (TSS) at 0 h, 24 h or 48 h in HATi-treated organotypic cultures. HAT inhibition in nascent hair cells reduced the H3K9ac levels at the Atoh1 transcription start site 37% (from 5.9±1.4% to 3.7±1.1% of input) at 24 h, and 19.8-fold (from 20.9±6.2% to 1.0±0.1%) by 48 h. Washing out the inhibitor increases the H3K9ac level 15.2-fold (from 1.0±0.1% to 16.0±1.0%), to a level almost as high as that of the control organs at 48 h. Results are mean±s.e.m., *P<0.05 (n=3).