Indiana University-Purdue University Indianapolis (IUPUI) / The epigenetic state of any cell is, in part, regulated by the interaction of DNA with nuclear histones. Histone tails can be modified in a number of ways that impact on the availability of DNA to interact with transcriptional complexes, including methylation, acetylation, phosphorylation, ubiquituination, and sumoylation. Histones are acetylated by a large family of enzymes, histone acetyl transferases (HATs), and deacetylated by the histone deacetylases (HDACs). Acetylated histones are generally considered markers of genomic regions that are actively being transcribed, whereas deacetylated and methylated histones are generally markers of regions that are inactive. The goal of the present study was to 1) study the epigenetic state with regard to the presence of euchromatin and heterochromatin in the developing chick and murine retina, 2) study and compare the localization patterns of the classical HDACs in the developing chick and murine retina with respect retinal progenitors and early differentiated cell types 3) to test the hypothesis that overall HDAC activity is required for dividing retinal progenitors to leave the cell cycle and differentiate. Our results showed that the classical HDACs were ubiquitously expressed in the developing chick and murine retinas. Species specific differences as well as stage dependent variations were observed in the localization of the HDACs in the cell types that were studied in the chick and murine retina. Our preliminary results also showed that HDAC inhibition may lead to the inability of the cell types to leave the cell cycle and a subsequent increase in the number of progenitor cells present in the developing chick retina.

Indiana University-Purdue University Indianapolis (IUPUI) / Epigenetic alterations have been shown to control cell type specification and differentiation leading to the changes in chromatin structure and organization of many genes. HDACs have been well documented to play an important role in both neurogenesis and gliogenesis in ganglionic eminence and cortex-derived cultures. However, the role of HDACs in glial cell type specification and differentiation in the optic nerve has not been well described. As a first step towards understanding their role in glial cell type specification, we have examined histone acetylation and methylation levels as well as the expression levels and patterns of the classical HDACs in both murine and chick optic nerve. Analysis of mRNA and protein levels in the developing optic nerve indicated that all 11 members of the classical HDAC family were expressed, with a majority declining in expression as development proceeded. Based on the localization pattern in both chick and murine optic nerve glial cells, we were able to group the classical HDACs: predominantly nuclear, nuclear and cytoplasmic, predominantly cytoplasmic. Nuclear expression of HDACs during different stages of development studied in this project in both murine and chick optic nerve glial cells suggests that HDACs play a role in stage-dependent changes in gene expression that accompany differentiation of astrocytes and oligodendrocytes. Examination of localization pattern of the HDACs is the first step towards identifying the specific HDACs involved directly in specification and differentiation of glia in optic nerve.

Indiana University-Purdue University Indianapolis (IUPUI) / Sequestration at the aggresome and degradation through autophagy are two approaches by which a cell can counteract the toxic effect of misfolded proteins. Tuberous sclerosis (TS) and cancer cells can become dependent on autophagy for survival due to the high demand for protein synthesis, thus making protein clearance a potential therapeutic target. Because of its histone deacetylase (HDAC) inhibitory activity, we hypothesized that 4-phenylbutyrate (4-PBA) inhibits HDAC6 and aggresome formation to induce TS cell death. We found that 4-PBA treatment increases cell death and reduces bortezomib-induced aggresome formation. To link these results with HDAC inhibition we used two other HDAC inhibitors, trichostatin A (TSA) and tubastatin, and found that they also reduce bortezomib-induced protein aggregation. Because tubulin is a target of HDAC6, we next measured the effect of the HDAC inhibitors and 4-PBA treatment on tubulin acetylation. As expected, tubastatin increased tubulin acetylation but surprisingly TSA and 4-PBA did not. Because 4-PBA did not significantly inhibit HDAC6, we next hypothesized that 4-PBA was alternatively inducing autophagy and increasing aggresome clearance. Surprisingly, autophagy inhibition did not prevent the 4-PBA-induced reduction in protein aggregation. In conclusion, we found 4-PBA to induce cell death and reduce aggresome levels in TS cells, but we found no link between these phenomena. We next hypothesized that loss of the Ral guanine nucleotide exchange factor Rgl2 induces cell death via autophagy inhibition in pancreatic adenocarcinoma (PDAC) cells. KRas is mutationally activated in over 90% of PDACs and directly activates Rgl2. Rgl2 activates RalB, a known regulator of autophagy, and Rgl2 has been shown to promote PDAC cell survival. We first confirmed that loss of Rgl2 does increase cell death in PDAC cells. Initial experiments using doubly tagged fluorescent p62 and LC3 (autophagy markers) suggested that loss of Rgl2 inhibited autophagosome accumulation, but after developing a more sophisticated quantitation method we found loss of Rgl2 to have no effect. We also measured endogenous LC3 levels, and these experiments confirmed loss of Rgl2 to have no effect on autophagy levels. Therefore, loss of Rgl2 increases cell death in PDAC cells, but does not have a significant effect on autophagy.