Histone methyltransferase DOT1L maintains identity and restricts cytotoxic potential of CD8 T cells
Muddassir MalikWillem-Jan de LeeuwMuhammad Assad AslamEliza Mari Kwesi‐MaliepaardTeun van den BrandBram van den BroekMaxime KempersLiesbeth HoekmanNatalie ProostTibor van WelsemElzo de WitJannie BorstHeinz JacobsFred van Leeuwen
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The histone methyltransferase DOT1L is emerging as a central epigenetic regulator in immune cells. Loss of DOT1L during development of CD8 T cells in vivo leads to gain of memory-features but has also been reported to compromise CD8 T cell viability and activity. Here, we determined the cell-intrinsic role of DOT1L in mature mouse CD8 T cells. After conditional deletion of Dot1L in vitro, CD8 T cells retained in vivo proliferative capacity and anti-tumor reactivity. Moreover, Dot1L knock-out CD8 T cells showed increased antigen-specific cytotoxicity towards tumor cells in vitro. Mechanistically, loss of DOT1L resulted in an altered cell-identity program with loss of T-cell and gain of NK-cell features. These transcriptional changes were mediated by loss of DOT1L methyltransferase activity in a dose-dependent manner. Our findings show that ablation of DOT1L activity in mature CD8 T cells is well-tolerated and rewires their cell identity towards the NK-cell lineage, concomitantly enhancing intrinsic cytotoxic capacity.Cancer Epigenetics
Histone Methylation
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Histone H3 lysine 36 (H3K36) methylation was identified as a conserved modification from yeast to human. In yeast, biochemical characterization of the SET2 protein and genome wide mapping of H3K36me2 and K36me3 indicate that H3K36 methylation functions in transcription elongation through Set2/Rpd3S pathway.
A number of H3K36 methyltransferases and demethylases have been identified in different species, which underscores the dynamics of H3K36 methylation. As in yeast, H3K36me3 also peaks at the 3’ end of genes in mammals. The genome wide view of H3K36me1 and H3K36me2 is not clear yet. To date, the functional significance of H3K36 methylation remains largely unknown in mammals.
In this thesis, homologs of SET2 in mammals, including Nsd1, Nsd2, Nsd3, and HypB, were studied. Nsd proteins displayed weak methyltransferase activity towards histone H3 in vitro. Their target specificities needs to be further analyzed. In vitro, HypB showed strong activity for histone H3 lysine 36. In vivo, H3K36 trimethylation levels were significantly reduced in HypB knock-down cells, indicating that HypB is a major H3K36 trimethyltransferase. Distribution of H3K36 methylation (mono-, di-, and tri-) were analyzed by immunofluorescence both in human and mouse cells. All three methylation states of showed euchromatic distribution, whereas H3K36 mono- and dimethylation also showed heterochromatic enrichment in terminally differentiated NIH3T3 cells as well. In embryonic stem cells, H3K36 methylation showed an inverse correlation with the expression level of Oct4, a stem cell marker, suggesting a potential role of H3K36 methylation in ES cell differentiation. After induction of differentiation by removing LIF or adding retinoic acid to the culture medium, stem cell genes failed to be repressed and lineage specific genes failed to be activated to the same degree in HypB knockdown cell as observed in mock treated ES cells. The presence of H3K36me3 along Oct4 locus was mapped by CHIP. H3K36me3 was highly enriched in the coding region, and was low upstream of the transcription start site in undifferentiated ES cells. During differentiation, however, H3K36me3 decreased on the coding region and increased slightly on enhancer region of Oct4 in the course of Oct4 repression after differentiation. In all, we propose that H3K36me3 is catalyzed by HypB and has an inverse correlation with Oct4 expression. HypB facilitates ES cell differentiation. The molecular mechanism by which HypB facilitates differentiation requires further investigation.
Histone Methylation
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Methyltransferases are a superfamily of enzymes that transfer methyl groups to proteins, nucleic acids, and small molecules. Traditionally, these enzymes have been shown to carry out a specific modification (mono-, di-, or trimethylation) on a single, or limited number of, amino acid(s). The largest subgroup of this family, protein methyltransferases, target arginine and lysine side chains of histone molecules to regulate gene expression. Although there is a large number of functional studies that have been performed on individual methyltransferases describing their methylation targets and effects on biological processes, no analyses exist describing the spatial distribution across tissues or their differential expression in the diseased heart. For this review, we performed tissue profiling in protein databases of 199 confirmed or putative methyltransferases to demonstrate the unique tissue-specific expression of these individual proteins. In addition, we examined transcript data sets from human heart failure patients and murine models of heart disease to identify 40 methyltransferases in humans and 15 in mice, which are differentially regulated in the heart, although many have never been functionally interrogated. Lastly, we focused our analysis on the largest subgroup, that of protein methyltransferases, and present a newly emerging phenomenon in which 16 of these enzymes have been shown to play dual roles in regulating transcription by maintaining the ability to both activate and repress transcription through methyltransferase-dependent or -independent mechanisms. Overall, this review highlights a novel paradigm shift in our understanding of the function of histone methyltransferases and correlates their expression in heart disease.
Histone Methylation
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INTRODUCTION Histone methyltransferases catalyze the addition of one or more methyl groups to a specific lysine or arginine residue within histones. Currently, there is a great deal of interest in histone methyltransferases, because mutations and misregulation of the genes encoding these proteins have been linked to various cancers and other diseases. Many genes encoding putative histone methyltransferases have been identified in eukaryotes, but the proteins they encode have not been functionally characterized. This protocol describes an in vitro assay for histone methyltransferase activity that uses bacterial cell extracts in which expression of a methyltransferase of interest is induced. In many cases, purification of the enzyme is unnecessary, making this experiment ideal for pilot studies. Bacterial cell extract containing the methyltransferase of interest is incubated with S-adenosyl-L-[ methyl - 3 H]-methionine and various histone substrates, many of which are commercially available. Incorporation of the methyl - 3 H can be measured easily by scintillation counting. The labeled substrate is visualized by SDS-polyacrylamide gel electrophoresis (PAGE) followed by fluorography. This allows the substrate specificity and activity of a histone methyltransferase of interest to be readily characterized.
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Recent studies have implicated that H3K9 lysine methyltransferases (KMTs) and polycomb repressive complex2 (PRC2) act in a cross-talk. However, a connection between GLP, one of the well-known H3K9 KMTs and the EZH2, one of the core members of PRC2, has not been established. Here we not only demonstrated that there was interaction between the GLP and the EZH2 proteins in vivo, but also provided evidence that the activity of EZH2 was effected by down-regulation of GLP. In light of these observations, western blotting, co-immunoprecipitation and qRT-PCR were performed to explore in GLP siRNA KGN cells. The specific interaction between GLP and EZH2 was demonstrated with co-immunoprecipitation. Our results also indicate that the decreased level of GLP participate in the modulation of EZH2 function in vivo.Taken together, our findings identified that an unanticipated interplay between the two histone lysine methyltransferases, which is implicated in regulating of a subset of developmental genes.
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G9a is a histone lysine methyltransferase responsible for the methylation of histone H3 lysine 9. The discovery of A-366 arose from a unique diversity screening hit, which was optimized by incorporation of a propyl-pyrrolidine subunit to occupy the enzyme lysine channel. A-366 is a potent inhibitor of G9a (IC50: 3.3 nM) with greater than 1000-fold selectivity over 21 other methyltransferases.
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Coding control: Protein arginine methyltransferases (PRMTs) and histone lysine methyltransferases (HKMTs) are epigenetic enzymes involved in regulation of gene expression and cellular processes. A new series of histone/protein methyltransferase inhibitors based on the 1,5-diphenyl-1,4-pentadien-3-one scaffold is reported. The described compounds showed various degrees of selectivity against the tested PRMTs (PRMT1 and CARM1) and HKMT (SET7).
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Protein methylation
Histone Methylation
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Abstract Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification deposited by the Set2 methyltransferases. Recent findings show that over-expression or mutation of Set2 enzymes promotes cancer progression, however, mechanisms of H3K36me are poorly understood. Set2 enzymes show spurious activity on histones and histone tails, and it is unknown how they obtain specificity to methylate H3K36 on the nucleosome. In this study, we present 3.8 Å cryo-EM structure of Set2 bound to the mimic of H2B ubiquitinated nucleosome. Our structure shows that Set2 makes extensive interactions with the H3 αN, the H3 tail, the H2A C-terminal tail and stabilizes DNA in the unwrapped conformation, which positions Set2 to specifically methylate H3K36. Moreover, we show that ubiquitin contributes to Set2 positioning on the nucleosome and stimulates the methyltransferase activity. Notably, our structure uncovers interfaces that can be targeted by small molecules for development of future cancer therapies.
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Epigenetic research has recently become one of the hotspots in the field of bioscience and drug design. DNA methylation and histone methylation serve a critical function in influencing gene expression and genome function. The inhibition of DNA and histone methyltransferases (DNMTs and HMTs) is a promising approach for the therapeutic treatment of numerous diseases, including cancer. This work reviews the recent achievements in methyltransferase crystallographic structure resolution and bioactive inhibitor screening. We discuss the features of DNA and HMT structures, as well as the mechanism and structure–function relationship of transferase inhibitors, to elucidate how methyltransferase and inhibitor interactions occur both internally and externally. This study briefly reviews the biological function, as well as the inhibitor discovery and development, of DNA/histone methyltransferases. Keywords: DNA methyltransferase, drug discovery, epigenetic therapy, histone methyltransferase, inhibitor.
Cancer Epigenetics
DNA methyltransferase
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