Alternatively, acetyl groups may facilitate selleckchem Rapamycin the interactions of chromatin with specific transcription factors containing bromodomains, which recognize and bind acetylated amino acids of other pro teins, including histone tails. The acetylation and deacetylation of histones is controlled by opposing pro tein families called histone acetyltransferases and histone deacetylases. Here we show that several RGC specific genes, which decrease in expression after ONC, exhibit a decrease in promoter histone acetylation. This deacetylation is accompanied by an increase in both HDAC2 and HDAC3 expression and the translocation of HDAC3 to the nuclei of dying RGCs. Additionally, inhibition of HDAC activity is able to prevent the ONC mediated silencing of at least one RGC specific gene and attenuate the level of RGC death.

These results represent one of the first documen tations of epigenetic changes associated with neuronal cell death and may provide insight into some of the earli est changes occurring in dying RGCs. Results Nuclear HDAC activity is increased after ONC Nuclear HDAC activity was measured in retinal nuclear protein extracts isolated at 1, 3, 5, and 7 days following ONC. No significant change was detected in fellow control eyes. Experimental eyes exhib ited an 50% increase in activity at day 5 post ONC. Activity was also significantly higher than con trol eyes at 7 days post ONC, although lower than day 5 experimental eyes. Nuclear HDAC activity in experimental and control eyes was completely inhibited by trichostatin A, indicating the presence of pre dominantly class I and II HDACs in this fraction.

Dose dependent inhibition of nuclear HDAC activity was also performed with different inhibitors to help eval uate which HDACs were active both before and after ONC. TSA mediated inhibition of control and crush nuclear extracts from day 5 retinas showed a dose depen dent decrease in HDAC activity with an IC50 ranging from 15 to 22 nM. A similar loss of activity was observed in extracts treated with the selective class I inhibitor, valproic acid. To further refine which class I HDACs may be contributing to the retinal nuclear activity, we repeated this experiment using apici din, which is selective for HDACs 2 and 3. HDAC activity was also nearly completely inhibited with apicidin, yield ing an IC50 ranging from 1. 88 to 2. 55 nM.

These values agreed with the reported IC50 value for HDAC3 using this inhibitor and suggest that HDACs 2 and 3 contribute the majority of nuclear HDAC activity in the retina. In addition to measuring nuclear HDAC activity, we also characterized which HDACs were expressed in the mouse retina by both mRNA and protein analysis. HDACs 1 5 were selectively examined because they Batimastat have all been reported to be active in nuclei and are known to affect histone acetylation levels.