This group of genes represented a subset of genes that exhibit a reported decrease in expression after ONC. The second group of genes, Bim and cJun, were examined because they undergo an increase in expression phosphatase inhibitor following ONC. ChIP assays, with antibodies against acetylated H4, were performed on retinas from control and crush eyes followed by qPCR to quantify the genomic DNA that was collected. The quan tification of promoter DNA associated with acetylated histone H4 is shown in Figure 9 and is expressed as a ratio of experimental control retinas. All ratios have been nor malized to day 0 controls. Surprisingly, promoter regions for both down regulated and up regulated genes showed a significant decrease in histone acetylation 1 day post ONC, with the exception of the promoter for cJun.

Three days post ONC, however, the acetylation pat tern of promoter histones was changed and only down regulated genes exhibited a decrease in promoter acetyla tion, while promoter H4 acetylation for cJun had remained at day 0 levels and levels for Bim had signif icantly increased 2 fold. A similar pattern of promoter acetylation observed 3 days after ONC was also evident on day 5 post ONC, except that Bim H4 pro moter acetylation had increased further to 3 fold the level detected in day 0 retinas. Inhibition of HDAC activity blocks ONC induced silencing of the Fem1cR3 reporter gene Although promoter histone deacetylation is associated with silenced genes in RGCs, these experiments do not conclusively demonstrate that this epigenetic change is the controlling mechanism for transcriptional downregu lation.

To address this, we examined if inhibitors of HDAC activity could block the ONC mediated downreg ulation of RGC specific gene expression. For these exper iments, we used Fem1cRosa3 mice, which contain the BGeo promoter trap reporter in the first intron of the Fem1c gene. Previously, we showed that mice express BGEO in an RGC specific manner. Additionally, we have observed a 75% decrease in BGEO total protein and enzyme activity, and a 50% decrease in Fem1c tran script levels, by 5 days post ONC. Thus, the R3 reporter allows for the rapid detection and quanti fication of changes in RGC gene expression. HDAC activity in the retina was inhibited by pretreat ment of mice with TSA given as a single intraperitoneal injection, 24 hours before ONC.

Western blot analy sis of AcH4 levels in extracts of total retinal protein confirmed inhibition of HDAC activity, which was exemplified by hyperacetylation of H4. The effects of TSA were detected as quickly as 2 hours after injection and persisted as long as 7 days post injection. R3 gene expression was assessed by BGEO solution assays 5 days post ONC. ONC resulted in a 55 75% decrease in BGEO enzyme activity by 5 days after GSK-3 surgery. TSA treated mice, however, exhibited significantly more activity at this time point than mice receiving no injection or DMSO injections.