Loss of p300/CBP leads to chromatin decondensation and changes in distribution of histone marks in R-DCKO nuclei.

A. Electron micrographs of nuclei in the ONL of P22 retinas. Compared to Cre neg control littermates, compound heterozygotes (p300 CH and Cbp CH) show slight increases in euchromatin (light areas within nuclei). In R-DCKO nuclei areas of euchromatin are greatly increased, and electron-dense heterochromatin appears reduced. B. Heterochromatin was quantified as a percentage of the total nuclear area in 50 nuclei from 10 micrographs for each genotype. Error bars = 1 SD. Differences from Cre neg values were significant at pC & D. Comparison of immunoreactivity patterns for repressive histone marks H3K9me3 (green in panel C, white in insets) and H3K27me3 (green in panel D, white in insets) in control (left image) and R-DCKO (right image) retinas confirm loss of the characteristic rod chromatin condensation pattern in R-DCKO outer retina cells. Anti-PKC-alpha (red) marks bipolar cells. E & F. Comparison of immunoreactivity patterns for acetylated histone H3 (AcH3, green in panel D) and H4 (AcH4, green in panel E) reveals the redistribution of these activation marks in R-DCKO cells, corresponding to loss of the characteristic peripheral rod euchromatin distribution pattern. DNA is counterstained with Draq-5 (red). Scale bars: cross-sections = 20 µm, insets = 10 µm. G. Western blots of acid-extracted retinal histones from 15-week-old Cre-negative (1) or R-DCKO (2) retinas. CB, Coomassie blue stained gel. AcH3, blot stained for acetylated histone H3; AcH4, blot stained for acetylated histone H4. H. Quantification of band fluorescence intensities for AcH3 levels relative to total H2B levels, and AcH4 levels relative to total H3 levels at P20 did not show significant differences between Cre neg and R-DCKO samples.