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Epigenetic modifiers are increasingly being investigated as potential therapeutics to modify and overcome disease phenotypes. Diseases of the nervous system present a particular problem as neurons are postmitotic and demonstrate relatively stable gene expression patterns and chromatin organization. We have explored the ability of epigenetic modifiers to prevent degeneration of rod photoreceptors in a mouse model of retinitis pigmentosa (RP), using rd10 mice of both sexes. The histone modification eraser enzymes lysine demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1) are known to have dramatic effects on the development of rod photoreceptors. In the RP mouse model, inhibitors of these enzymes blocked rod degeneration, preserved vision, and affected the expression of multiple genes including maintenance of rod-specific transcripts and downregulation of those involved in inflammation, gliosis, and cell death. The neuroprotective activity of LSD1 inhibitors includes two pathways. First, through targeting histone modifications, they increase accessibility of chromatin and upregulate neuroprotective genes, such as from the Wnt pathway. We propose that this process is going in rod photoreceptors. Second, through nonhistone targets, they inhibit transcription of inflammatory genes and inflammation. This process is going in microglia, and lack of inflammation keeps rod photoreceptors alive. SIGNIFICANCE STATEMENT Retinal degenerations are a leading cause of vision loss. RP is genetically very heterogeneous, and the multiple pathways leading to cell death are one reason for the slow progress in identifying suitable treatments for patients. Here we demonstrate that inhibition of LSD1and HDAC1 in a mouse model of RP leads to preservation of rod photoreceptors and visual function, retaining of expression of rod-specific genes, and with decreased inflammation, cell death, and Müller cell gliosis. We propose that these epigenetic inhibitors cause more open and accessible chromatin, allowing expression of neuroprotective genes. A second mechanism that allows rod photoreceptor survival is suppression of inflammation by epigenetic inhibitors in microglia. Manipulation of epigenetic modifiers is a new strategy to fight neurodegeneration in RP., (Copyright © 2021 the authors.)

Transcriptome complexity is substantially increased by the use of multiple transcription start sites for a given gene. By utilizing a rod photoreceptor-specific chromatin signature, and the RefSeq database of established transcription start sites, we have identified essentially all known rod photoreceptor genes as well as a group of novel genes that have a high probability of being expressed in rod photoreceptors. Approximately half of these novel rod genes are transcribed into multiple mRNA and/or protein isoforms through alternative transcriptional start sites (ATSS), only one of which has a rod-specific epigenetic signature and gives rise to a rod transcript. This suggests that, during retina development, some genes use ATSS to regulate cell type and temporal specificity, effectively generating a rod transcript from otherwise ubiquitously expressed genes. Biological confirmation of the relationship between epigenetic signatures and gene expression, as well as comparison of our genome-wide chromatin signature maps with available data sets for retina, namely a ChIP-on-Chip study of Polymerase-II (Pol-II) binding sites, ChIP-Seq studies for NRL- and CRX- binding sites and DHS (University of Washington data, available on UCSC mouse Genome Browser as a part of ENCODE project) fully support our hypothesis and together accurately identify and predict an array of new rod transcripts. The same approach was used to identify a number of TSS that are not currently in RefSeq. Biological conformation of the use of some of these TSS suggests that this method will be valuable for exploring the range of transcriptional complexity in many tissues. Comparison of mouse and human genome-wide data indicates that most of these alternate TSS appear to be present in both species, indicating that our approach can be useful for identification of regulatory regions that might play a role in human retinal disease.

Histone acetylation has a regulatory role in gene expression and is necessary for proper tissue development. To investigate the specific roles of histone deacetylases (HDACs) in rod differentiation in neonatal mouse retinas, we used a pharmacological approach that showed that inhibition of class I but not class IIa HDACs caused the same phenotypic changes seen with broad spectrum HDAC inhibitors, most notably a block in the differentiation of rod photoreceptors. Inhibition of HDAC1 resulted in increase of acetylation of lysine 9 of histone 3 (H3K9) and lysine 12 of histone 4 (H4K12) but not lysine 27 of histone 3 (H3K27) and led to maintained expression of progenitor-specific genes such as Vsx2 and Hes1 with concomitant block of expression of rod-specific genes. ChiP experiments confirmed these changes in the promoters of a group of progenitor genes. Based on our results, we suggest that HDAC1-specific inhibition prevents progenitor cells of the retina from exiting the cell cycle and differentiating. HDAC1 may be an essential epigenetic regulator of the transition from progenitor cells to terminally differentiated photoreceptors., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Epigenetic modifiers can work in concert with transcription factors to control the transition of cells from proliferating progenitors into quiescent terminally differentiated cells. This transition involves changes in histone methylation and one of the key regulators of this is the H3K4me2/1 histone demethylase LSD1. Here, we show that the highest expression of LSD1 occurs in postmitotic retinal cells during the peak period of rod photoreceptor differentiation. Pharmacological inhibition of LSD1 in retinal explants cultured from PN1 to PN8 had three major effects. It prevented the normal decrease in expression of genes associated with progenitor function, it blocked rod photoreceptor development, and it increased expression of genes associated with other retinal cell types. The maintained expression of progenitor genes was associated with a maintained level of H3K4me2 over the gene and its promoter. Among the genes whose expression was maintained was Hes1, a repressor known to block rod photoreceptor development. The inhibition of rod photoreceptor gene expression occurred in spite of the normal expression of transcription factors CRX and NRL, and the normal accumulation of H3K4me2 marks over the promoter and gene body. We suggest that LSD1 acts in concert with a series of nuclear receptors to modify chromatin structure and repress progenitor genes as well as to inhibit ectopic patterns of gene expression in the differentiating postmitotic retinal cells.

We have used ChIP-seq to map the distribution of two important histone H3 modifications, H3K4me2 and H3K27me3, over the whole genome at multiple time points during late mouse retina development. We merged these data with our previous retina developmental expression profiles and show that there are several epigenetic signatures specific for different functional groups of genes. The main conclusion from our study is that epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. Rod photoreceptor-specific genes have a striking signature, a de novo accumulation of H3K4me2 and a complete absence of H3K27me3. We were able to use this signature in an unbiased search of the whole genome and identified essentially all the known rod photoreceptor genes as well as a group of novel genes that have a high probability of being rod photoreceptor specific. Comparison of our genome-wide chromatin signature maps with available data sets for Polymerase-II (Pol-II) and CRX binding sites and DNase1 Hypersensitive Sites (DHS) for retina shows great agreement. Because our approach is not dependent on high levels of gene expression, it provides a new way of identifying cell type-specific genes, particularly genes that may be involved in retinal diseases.

Mature rod photoreceptor cells contain very small nuclei with tightly condensed heterochromatin. We observed that during mouse rod maturation, the nucleosomal repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult retina. At the same time, the total level of linker histone H1 increased reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a dramatic increase in H1c. Genetic elimination of the histone H1c gene is functionally compensated by other histone variants. However, retinas in H1c/H1e/H1(0) triple knock-outs have photoreceptors with bigger nuclei, decreased heterochromatin area, and notable morphological changes suggesting that the process of chromatin condensation and rod cell structural integrity are partly impaired. In triple knock-outs, nuclear chromatin exposed several epigenetic histone modification marks masked in the wild type chromatin. Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. During retina development, the H1c gene and its promoter acquired epigenetic patterns typical of rod-specific genes. Our data suggest that histone H1c gene expression is developmentally up-regulated to promote facultative heterochromatin in mature rod photoreceptors.