Retinal progenitor cells are multipotent throughout development, producing cell types in a conserved order. Early progenitor cells give rise to early born cell types: the ganglion cells, horizontal cells, cones and amacrine cells. Late progenitor cells produce late born cell types: the bipolar cells and Müller glial cells. Rod photoreceptors, the major cell type in the murine retina, are generated both before and after birth, with peak production taking place around birth [43
]. This process is likely controlled by a network of transcription factors that must be expressed at a particular time and in specific cells. For instance, rod photoreceptor development requires activation of Otx2, followed by the expression of Nrl and Crx. Loss of Otx2 or Nrl leads to loss of rod photoreceptor cells, while loss of Crx leads to a failure in differentiation of rod and cone photoreceptor cells [27
]. Neurod1 also promotes rod photoreceptor development, as overexpression of Neurod1 favors the production of rods at the expense of bipolar cells and Müller glia, and Neurod1 is required for the survival of rod photoreceptor cells [35
]. TSA treatment led to a reduction in the expression of all of these key regulators of rod development, and of rod development itself. Downregulation of these regulators took place quickly, within 3 hours, before cell death was observed, 20 hours after TSA treatment. As there was not a general downregulation of gene expression observed on the microarrays, and the effect on the pro rod genes was so rapid, it is unlikely that the effect of TSA on the expression of these key regulators resulted from the cell death effect exerted by TSA. In addition, TSA led to an inhibition of Müller glia development. This may hold generally true in the central nervous system, as inhibition of HDAC activity in multipotent hippocampal neural progenitor cells inhibits glial cell formation [44
]. TSA treatment also led to a reduction in total cell counts due to reduced cell proliferation and increased cell apoptosis. However, the observed approximately 30% reduction in cell numbers does not explain a 3 fold increase in bipolar cells when HDAC activity was inhibited by TSA in the retinal explant cultures. Taken together, these results suggest that loss of HDAC activity can drive the bipolar cell fate at the expense of other fates. It is interesting to consider this effect in light of the fact that all proliferation was inhibited by TSA. Birthdating experiments in the mouse (34) have shown that the vast majority of bipolar cells are generated postnatally from mitotic progenitor cells. The observation of additional bipolar cells following TSA treatment suggests that the bipolar cells in the TSA-treated cultures resulted from fate effects on cells that were postmitotic when the cultures were initiated, and were most likely those cells normally fated to be rods.
Although the function of the transcription factors controlling rod development has been revealed, little is known regarding the regulation of their expression. The results of this study demonstrate that HDACs play an important role in regulating their expression at the initial stages of rod development. Clearly, HDAC activity is required for the expression of a panel of pro-rod genes. The maintenance of expression of Nrl and Crx in the mature retina also requires HDACs, indicating that the function of HDACs in controlling retinal gene expression extends beyond the early developmental stage.
HDACs are generally described as transcriptional repressors. Class I HDACs are ubiquitously expressed in the nucleus. Class II HDACs are capable of shuttling in and out of the nucleus in response to signals. The association of Class II HDACs with the transcription factor MEF2 repressed cardiomyocyte differentiation through the silencing of MEF2-mediated gene transcription. Phosphorylation of Class II HDACs (HDAC 4, 5, 7) caused them to move from the nucleus to the cytoplasm, thus releasing transcription repression, and allowing myocytes to differentiate. However, recent studies have implicated HDACs in the process of transcription activation, providing evidence that HDACs can also act as transcription activators, inducing interferon-stimulated gene expression and the subsequent antiviral response [21
]. These new findings strongly suggest that the relative acetylation level of HDAC target proteins, regulated by the opposing acitivity of HDACs and HATs, determines the transcription states of specific genes, either activating or repressing transcription. Our results support a role of HDACs as transcription activators, as they may directly regulate the promoter activity of Nrl and Crx. However, from the reporter analysis of Nrl and Crx promoters, we can not completely exclude the possibility that TSA acted indirectly on the Nrl and Crx promoters via activation of transcription repressors that in turn act on the Nrl and Crx promoters. In the latter case, a transcription repressor protein would need to be induced, whose expression is normally repressed by HDACs. Given the fast action of TSA to nearly abolish the expression of Nrl and Crx within 3 hours, even in the presence of a protein synthesis inhibitor cycloheximide, it is more likely that the HDACs are transcription activators of these key retinal development regulators.
It is known that HDACs have many non-histone target proteins. Some are nuclear transcription factors, such as GATA4, NFAT, RB, and p53; others are cytoplasmic targets, such as tubulin and hsp90, which were identified as substrates for HDAC6. HDAC6 deacetylates α-tubulin, with a resulting increase in cell motility [11
]. Recently, it was found that HDAC6 mediated acetylation controls the chaperone function of heat shock protein 90 [12
]. The biological function of other Class II HDACs, when they are in the cytoplasm, remains unknown. In our study, TSA treatment resulted in an increased acetylation of histones and other cellular proteins, including tubulin and two other species that are unknown. Interestingly, although TSA-induced histone acetylation was barely appreciable after 3 hours, a significantly enhanced acetylation of non-histone targets was obvious in 3 hours, and the acetylation levels of these non-histone targets was maintained at later time points. The fast acetylation of non-histone targets is consistent with the kinetics of the mRNA downregulation by TSA. These results suggest that modification of non-histone targets by HDACs may mediate an important pathway in the regulation of retinal gene expression. Using a high-affinity antibody against acetyl-lysine groups will facilitate the purification and identification of these non-histone targets of HDACs by mass spectrometry.
Given the multiplicity of family members from the Class I and Class II HDACs, it is desirable to identify individual HDACs responsible for their role in gene expression regulation. With the development of inhibitors specific to individual HDAC family members [45
], it should be possible to address this question in the retina, as well as in other tissues. In addition, analysis of HDAC knockout mice will aid in the identification of the functions of individual HDACs.