Inhibition of Epigenetic Modifiers LSD1 and HDAC1 Blocks Rod Photoreceptor Death in Mouse Models of Retinitis Pigmentosa.

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.

Noggin regulates foregut progenitor cell programming, and misexpression leads to esophageal atresia.

Esophageal atresia (EA/TEF) is a common congenital abnormality present in 1 of 4000 births. Here we show that atretic esophagi lack Noggin (NOG) expression, resulting in immature esophagus that contains respiratory glands. Moreover, when using mouse esophageal organoid units (EOUs) or tracheal organoid units (TOUs) as a model of foregut development and differentiation in vitro, NOG determines whether foregut progenitors differentiate toward esophageal or tracheal epithelium. These results indicate that NOG is a critical regulator of cell fate decisions between esophageal and pulmonary morphogenesis, and its lack of expression results in EA/TEF.

Activin Signals through SMAD2/3 to Increase Photoreceptor Precursor Yield during Embryonic Stem Cell Differentiation.

In vitro differentiation of mouse embryonic stem cells (ESCs) into retinal fates can be used to study the roles of exogenous factors acting through multiple signaling pathways during retina development. Application of activin A during a specific time frame that corresponds to early embryonic retinogenesis caused increased generation of CRX+ photoreceptor precursors and decreased PAX6+ retinal progenitor cells (RPCs). Following activin A treatment, SMAD2/3 was activated in RPCs and bound to promoter regions of key RPC and photoreceptor genes. The effect of activin on CRX expression was repressed by pharmacological inhibition of SMAD2/3 phosphorylation. Activin signaling through SMAD2/3 in RPCs regulates expression of transcription factors involved in cell type determination and promotes photoreceptor lineage specification. Our findings can contribute to the production of photoreceptors for cell replacement therapy.

Identification and prediction of alternative transcription start sites that generate rod photoreceptor-specific transcripts from ubiquitously expressed genes.

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.

Genome-wide mapping of histone H3K9me2 in acute myeloid leukemia reveals large chromosomal domains associated with massive gene silencing and sites of genome instability.

A facultative heterochromatin mark, histone H3 lysine 9 dimethylation (H3K9me2), which is mediated by histone methyltransferases G9a/GLP (EHMT2/1), undergoes dramatic rearrangements during myeloid cell differentiation as observed by chromatin imaging. To determine whether these structural transitions also involve genomic repositioning of H3K9me2, we used ChIP-sequencing to map genome-wide topography of H3K9me2 in normal human granulocytes, normal CD34+ hematopoietic progenitors, primary myeloblasts from acute myeloid leukemia (AML) patients, and a model leukemia cell line K562. We observe that H3K9me2 naturally repositions from the previously designated "repressed" chromatin state in hematopoietic progenitors to predominant association with heterochromatin regions in granulocytes. In contrast, AML cells accumulate H3K9me2 on previously undefined large (> 100 Kb) genomic blocks that are enriched with AML-specific single nucleotide variants, sites of chromosomal translocations, and genes downregulated in AML. Specifically, the AML-specific H3K9me2 blocks are enriched with genes regulated by the proto-oncogene ERG that promotes stem cell characteristics. The AML-enriched H3K9me2 blocks (in contrast to the heterochromatin-associated H3K9me2 blocks enriched in granulocytes) are reduced by pharmacological inhibition of the histone methyltransferase G9a/GLP in K562 cells concomitantly with transcriptional activation of ERG and ETS1 oncogenes. Our data suggest that G9a/GLP mediate formation of transient H3K9me2 blocks that are preserved in AML myeloblasts and may lead to an increased rate of AML-specific mutagenesis and chromosomal translocations.

Histone Deacetylase 1 Is Essential for Rod Photoreceptor Differentiation by Regulating Acetylation at Histone H3 Lysine 9 and Histone H4 Lysine 12 in the Mouse Retina.

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.

LSD1-Mediated Demethylation of H3K4me2 Is Required for the Transition from Late Progenitor to Differentiated Mouse Rod Photoreceptor.

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.

Cell type-specific epigenetic signatures accompany late stages of mouse retina development.

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.

Developmentally regulated linker histone H1c promotes heterochromatin condensation and mediates structural integrity of rod photoreceptors in mouse retina.

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.

Stage and gene specific signatures defined by histones H3K4me2 and H3K27me3 accompany mammalian retina maturation in vivo.

The epigenetic contribution to neurogenesis is largely unknown. There is, however, growing evidence that posttranslational modification of histones is a dynamic process that shows many correlations with gene expression. Here we have followed the genome-wide distribution of two important histone H3 modifications, H3K4me2 and H3K27me3 during late mouse retina development. The retina provides an ideal model for these studies because of its well-characterized structure and development and also the extensive studies of the retinal transcriptome and its development. We found that a group of genes expressed only in mature rod photoreceptors have a unique signature consisting of de-novo accumulation of H3K4me2, both at the transcription start site (TSS) and over the whole gene, that correlates with the increase in transcription, but no accumulation of H3K27me3 at any stage. By in silico analysis of this unique signature we have identified a larger group of genes that may be selectively expressed in mature rod photoreceptors. We also found that the distribution of H3K4me2 and H3K27me3 on the genes widely expressed is not always associated with their transcriptional levels. Different histone signatures for retinal genes with the same gene expression pattern suggest the diversities of epigenetic regulation. Genes without H3K4me2 and H3K27me3 accumulation at any stage represent a large group of transcripts never expressed in retina. The 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. In addition to the developmental patterns seen in wild type retina, the dramatic changes of histone modification in the retinas of mutant animals lacking rod photoreceptors provide a tool to study the epigenetic changes in other cell types and thus describe a broad range of epigenetic events in a solid tissue in vivo.

Rearrangement of upstream sequences of the hTERT gene during cellular immortalization.

Telomerase expression, resulting from transcriptional activation of the hTERT gene, allows cells to acquire indefinite proliferative potential during cellular immortalization and tumorigenesis. However, mechanisms of hTERT gene activation in many immortal cell lines and cancer cells are poorly understood. Here, we report our studies on hTERT activation using genetically related pairs of telomerase-negative (Tel(-)) and -positive (Tel(+)) fibroblast lines. First, whereas transiently transfected plasmid reporters did not recapitulate the endogenous hTERT promoter, the promoter in chromosomally integrated bacterial artificial chromosome (BAC) reporters was activated in a subset of Tel(+) cells, indicating that activation of the hTERT promoter required native chromatin context and/or distal regulatory elements. Second, the hTERT gene, located near the telomere of chromosome 5p, was translocated in all three Tel(+) cell lines but not in their parental precrisis cells and Tel(-) immortal siblings. The breakage points were mapped to regions upstream of the hTERT promoter, indicating that the hTERT gene was the target of these chromosomal rearrangements. In two Tel(+) cell lines, translocation of the endogenous hTERT gene appeared to be the major mechanism of its activation as the activity of hTERT promoter in many chromosomally integrated BAC reporters, with intact upstream and downstream neighboring loci, remained relatively low. Therefore, our results suggest that rearrangement of upstream sequences is an important new mechanism of hTERT promoter activation during cellular immortalization. The chromosomal rearrangements likely occurred during cellular crisis and facilitated by telomere dysfunction. Such translocations allowed the hTERT promoter to escape from the native condensed chromatin environment.

Chromatin condensation in terminally differentiating mouse erythroblasts does not involve special architectural proteins but depends on histone deacetylation.

Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation and chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late-stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells.

X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation.

Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop-sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop-sheet linkages.

Epigenetic heterochromatin markers distinguish terminally differentiated leukocytes from incompletely differentiated leukemia cells in human blood.

OBJECTIVE: During terminal cell differentiation, nuclear chromatin becomes condensed and the repertoire of epigentic heterochromatin proteins responsible for chromatin condensation is dramatically changed. In order to identify the chromatin regulatory factors associated with incomplete cell differentiation and impaired chromatin condensation in hematological malignancies, we examined expression levels of major heterochromatin proteins in normal blood cells and cells derived from a number of chronic and acute myeloid leukemia patients exhibiting different degrees of differentiation. METHODS: We used immunoblotting and immunofluorescence to examine the levels and localization of epigenetic heterochromatin factors in isolated cell nuclei and fractionated peripheral blood cells. RESULTS: While the major epigenetic heterochromatin factor, histone H3 methylated at lysine 9, is present in all cell types, its main counterparts, nonhistone proteins, heterochromatin proteins 1 (HP1) alpha, beta, and gamma, are dramatically reduced in peripheral blood leukocytes of normal donors and chronic myeloid leukemia patients, but are substantially increased in the blood of accelerated phase and blast crisis patients. In the terminally differentiated cells, nuclear chromatin accumulates a nucleocytoplasmic serpin, monocyte and neutrophil elastase inhibitor (MNEI). HP1 and MNEI levels inversely correlate in a number of normal and leukemia myeloid cells and show strikingly opposite coordinated changes during differentiation of U937 cell line induced by retinoic acid. CONCLUSIONS: Our results suggest that repression of HP1 and accumulation of MNEI are linked to terminal cell differentiation and that their levels may be monitored in blood cell populations to detect transitions in cell differentiation associated with leukemia progression and treatment.

The end adjusts the means: heterochromatin remodelling during terminal cell differentiation.

All cells that constitute mature tissues in an eukaryotic organism undergo a multistep process of cell differentiation. At the terminal stage of this process, cells either cease to proliferate forever or rest for a very long period of time. During terminal differentiation, most of the genes that are required for cell 'housekeeping' functions, such as proto-oncogenes and other cell-cycle and cell proliferation genes, become stably repressed. At the same time, nuclear chromatin undergoes dramatic morphological and structural changes at the higher-order levels of chromatin organization. These changes involve both constitutively inactive chromosomal regions (constitutive heterochromatin) and the formerly active genes that become silenced and structurally modified to form facultative heterochromatin. Here we approach terminal cell differentiation as a unique system that allows us to combine biochemical, ultrastructural and molecular genetic techniques to study the relationship between the hierarchy of chromatin higher-order structures in the nucleus and its function(s) in dynamic packing of genetic material in a form that remains amenable to regulation of gene activity and other DNA-dependent cellular processes.

Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation.

MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein) is a developmentally regulated chromosomal serpin that condenses chromatin in terminally differentiated avian blood cells. We show that MENT is an effective inhibitor of the papain-like cysteine proteinases cathepsins L and V. In addition, ectopic expression of MENT in mammalian cells is apparently sufficient to inhibit a nuclear papain-like cysteine proteinase and prevent degradation of the retinoblastoma protein, a major regulator of cell proliferation. MENT also accumulates in the nucleus, causes a strong block in proliferation, and promotes condensation of chromatin. Variants of MENT with mutations or deletions within the M-loop, which contains a nuclear localization signal and an AT-hook motif, reveal that this region mediates nuclear transport and morphological changes associated with chromatin condensation. Non-inhibitory mutants of MENT were constructed to determine whether its inhibitory activity has a role in blocking proliferation. These mutations changed the mode of association with chromatin and relieved the block in proliferation, without preventing transport to the nucleus. We conclude that the repressive effect of MENT on chromatin is mediated by its direct interaction with a nuclear protein that has a papain-like cysteine proteinase active site.