Fig. 3: P300-CtBP/HDAC3 modulates H3K27ac/H4K8ac homeodynamics in the E93 enhancer and promoter.
From: Histone acetylation homeodynamics navigates cell survival and apoptosis
a and a’ Immunofluorescence (IF) staining of Cyt-c and A-cas 3 after P300-i, and P300-i & UAS-E93 in the salivary glands using Flp-out line at 8-12 h APF. The fluorescence intensity statistics of clone cells compared to wt cells in a (a’), mean ± SD; n = 4 independent clone cell and wt cell. one-way ANOVA: different lowercase letters are significantly different (P < 0.05). b, c’ After UAS-P300, UAS-P300 & E93-i, and UAS-P300 & UAS-Diap1 at the EW, DAPI staining in the fat body using ppl-Gal4 (b) and Flp-out (c) line. The size of the cell nucleus statistics of clone cells compared to wt cells in c (c’), mean ± SD; n = 6 independent clone cell and wt cell. one-way ANOVA: different lowercase letters are significantly different (P < 0.05). d and d’ After UAS-P300, UAS-P300 & E93-i, and UAS-P300 & UAS-Diap1 at the EW, IF staining of A-cas 3 and Cyt-c in the salivary glands using Flp-out line (d). The fluorescence intensity statistics of clone cells compared to wt cells (d’), mean ± SD; n = 4 independent clone cell and wt cell. one-way ANOVA: different lowercase letters are significantly different (P < 0.05). e Diagram showing potential modifications of CtBP/HDAC3-P300-modulated histone acetylation targeting on E93. Red font indicates CtBP-regulated histone acetylation modifications screened in Supplementary Fig. 6b. f The Integrative Genomics Viewer (IGV) tracks showing global H3K27ac and H4K8ac at the E93 gene locus at 96 h AEL and 6 h APF, respectively. R1-R5, region 1-region 5 of E93B nucleotide (−9059– + 7875 bp); green fonts and boxes, 5’ UTR of E93A/B; light brown fonts and boxes, exon of E93A/B. g Dual luciferase activity driven by the 20E-induced enhancer and basic promoter of E93B. R1, 20E-induced enhancer; R3, basic promoter. Mean ± SD; n = 3 independent samples. Two-tailed paired t test: ***p < 0.001. ns, not significant. h Enrichment of EcR and Pol Ⅱ on the 20E-induced enhancer (R1) and basic promoter (R5) detected by ChIP (Chromatin Immunoprecipitation)-qPCR, after global P300-i at 6 h APF using tub-Gal4, tub-Gal4>wt was used as control. Primers for ChIP-qPCR were located in peak-R1 and peak-R3. Mean ± SD; n = 3 independent samples. Two-tailed paired t test: *p < 0.05, **p < 0.01. i and j Detection of the GFP signal indicating the activity of 20E-induced E93 enhancer in CtBP-i, HDAC3-i, UAS-P300, and UAS-EcR-B1 (i) and UAS-CtBP, UAS-HDAC3, P300-i, and UAS-EcR-B1DN (j) clone cells of the salivary glands using Flp-out line at the EW and 8-12 h APF, respectively. k Diagram showing that P300-CtBP/HDAC3 modulates H3K27ac/H4K8ac dynamics, which regulate 20E-induced E93 expression. EcRE, ecdysone response element. Source data are provided as a Source Data file. The genotypes are provided in Supplementary Table 3.
