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Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where “x” is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation. Only very few enzymes are known to catalyze protein histidine methylation. Here, the authors show that METTL9 is responsible for most 1-methylhistidine modifications in mouse and human proteomes, and characterize METTL9′s substrate specificity and potential cellular functions.

Subsets of endogenous retroviruses (ERVs) are derepressed in mouse embryonic stem cells (mESCs) deficient for Setdb1, which catalyzes histone H3 lysine 9 trimethylation (H3K9me3). Most of those ERVs, including IAPs, remain silent if Setdb1 is deleted in differentiated embryonic cells; however they are derepressed when deficient for Dnmt1 , suggesting that Setdb1 is dispensable for ERV silencing in somatic cells. However, H3K9me3 enrichment on ERVs is maintained in differentiated cells and is mostly diminished in mouse embryonic fibroblasts (MEFs) lacking Setdb1. Here we find that distinctive sets of ERVs are reactivated in different types of Setdb1 -deficient somatic cells, including the VL30-class of ERVs in MEFs, whose derepression is dependent on cell-type-specific transcription factors (TFs). These data suggest a more general role for Setdb1 in ERV silencing, which provides an additional layer of epigenetic silencing through the H3K9me3 modification. Previous studies suggest that DNA methylation is the main mechanism to silence endogenous retroviruses (ERVs) in somatic cells. Here the authors provide evidence that distinctive sets of ERVs are silenced by Setdb1 in different types of somatic cells, suggesting a general function in ERV silencing.

Genome stability relies on epigenetic mechanisms that enforce repression of endogenous retroviruses (ERVs). Current evidence suggests that distinct chromatin-based mechanisms repress ERVs in cells of embryonic origin (histone methylation dominant) vs. more differentiated cells (DNA methylation dominant). However, the latter aspect of this model has not been tested. Remarkably, and in contrast to the prevailing model, we find that repressive histone methylation catalyzed by the enzyme SETDB1 is critical for suppression of specific ERV families and exogenous retroviruses in committed B-lineage cells from adult mice. The profile of ERV activation in SETDB1-deficient B cells is distinct from that observed in corresponding embryonic tissues, despite the loss of repressive chromatin modifications at all ERVs. We provide evidence that, on loss of SETDB1, ERVs are activated in a lineage-specific manner depending on the set of transcription factors available to target proviral regulatory elements. These findings have important implications for genome stability in somatic cells, as well as the interface between epigenetic repression and viral latency.

Histone H3 lysine 9 (H3K9) methylation is a crucial epigenetic mark of heterochromatin formation and transcriptional silencing. G9a is a major mammalian H3K9 methyltransferase at euchromatin and is essential for mouse embryogenesis. Here we describe the roles of G9a in germ cell development. Mutant mice in which G9a is specifically inactivated in the germ‐lineage displayed sterility due to a drastic loss of mature gametes. G9a ‐deficient germ cells exhibited perturbation of synchronous synapsis in meiotic prophase. Importantly, mono‐ and di‐methylation of H3K9 (H3K9me1 and 2) in G9a ‐deficient germ cells were significantly reduced and G9a‐regulated genes were overexpressed during meiosis, suggesting that G9a‐mediated epigenetic gene silencing is crucial for proper meiotic prophase progression. Finally, we show that H3K9me1 and 2 are dynamically and sex‐differentially regulated during the meiotic prophase. This genetic and biochemical evidence strongly suggests that a specific set of H3K9 methyltransferase(s) and demethylase(s) coordinately regulate gametogenesis.

Lysine methylation has been extensively studied in histones, where it has been shown to provide specific epigenetic marks for the regulation of gene expression; however, the molecular mechanism and physiological function of lysine methylation in proteins other than histones remains to be fully addressed. To better understand the substrate diversity of lysine methylation, S-adenosylmethionine (SAM) derivatives with alkyne-moieties have been synthesized. A selenium-based SAM analog, propargylic Se-adenosyl-l-selenomethionine (ProSeAM), has a wide spectrum of reactivity against various lysine methyltransferases (KMTs) with sufficient stability to support enzymatic reactions in vitro. By using ProSeAM as a chemical probe for lysine methylation, we identified substrates for two seven-beta-strand KMTs, METTL21A and METTL10, on a proteomic scale in mammalian cells. METTL21A has been characterized as a heat shock protein (HSP)-70 methyltransferase. Mammalian METTL10 remains functionally uncharacterized, although its ortholog in yeast, See1, has been shown to methylate the translation elongation factor eEF1A. By using ProSeAM-mediated alkylation followed by purification and quantitative MS analysis, we confirmed that METTL21A labels HSP70 family proteins. Furthermore, we demonstrated that METTL10 also methylates the eukaryotic elongation factor EF1A1 in mammalian cells. Subsequent biochemical characterization revealed that METTL10 specifically trimethylates EF1A1 at lysine 318 and that siRNA-mediated knockdown of METTL10 decreases EF1A1 methylation levels in vivo. Thus, our study emphasizes the utility of the synthetic cofactor ProSeAM as a chemical probe for the identification of non-histone substrates of KMTs.

Background Heterochromatin is a fundamental component of eukaryotic chromosome architecture, crucial for genome stability and cell type-specific gene regulation. In mammalian nuclei, heterochromatin forms condensed B compartments, distinct from the transcriptionally active euchromatic A compartments. Histone H3 lysine 9 and lysine 27 trimethylation (H3K9me3 and H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Previously, we found that the redistribution of H3K27me3 following the loss of H3K9 methylation contributes to heterochromatin maintenance, while the simultaneous loss of both H3K27me3 and H3K9 methylation induces heterochromatin decondensation in mouse embryonic fibroblasts. However, the spatial positioning of B compartments largely persists, suggesting additional mechanisms are involved. Results In this study, we investigated the role of H2AK119 monoubiquitylation (uH2A), a repressive chromatin mark deposited by Polycomb Repressive Complex 1 (PRC1), in maintaining heterochromatin structure following the loss of H3K9 and H3K27 methylation. We observed that uH2A and H3K27me3 are independently enriched in B compartments after H3K9 methylation loss. Despite the absence of H3K9me3 and H3K27me3, uH2A remained localized and contributed to heterochromatin retention. These results suggest that PRC1-mediated uH2A functions independently and cooperatively with H3K27me3 to maintain heterochromatin organization originally created by H3K9 methylation. Conclusion Our findings highlight a compensatory role for uH2A in preserving heterochromatin structure after the loss of other repressive chromatin modifications. The PRC1–uH2A pathway plays a critical role in maintaining the integrity of B compartments and suggests that heterochromatin architecture is supported by a network of redundant epigenetic mechanisms in mammalian cells.

The binding of PGC7 to maternal chromatin, which is important for methylation maintenance during embryogenesis, is shown to be dependent on a particular histone modification, H3K9me2. Protecting against DNA demethylation During early embryogenesis, the paternal and maternal genomes undergo loss of the DNA modification 5-methylcytosine (5mC), but with different time courses. The maternal factor PGC7 is thought to be involved in protecting the maternal genome from demethylation. Here, the association of PGC7 with maternal chromatin is shown to be dependent on a particular histone modification, dimethylated histone H3 lysine 9 (H3K9me2). PGC7 binding can inhibit the binding of Tet3, an enzyme that converts 5mC into 5-hydroxymethylcytosine (5hmC). The modification of DNA by 5-methylcytosine (5mC) has essential roles in cell differentiation and development through epigenetic gene regulation 1 . 5mC can be converted to another modified base, 5-hydroxymethylcytosine (5hmC), by the tet methylcytosine dioxygenase (Tet) family of enzymes 2 , 3 . Notably, the balance between 5hmC and 5mC in the genome is linked with cell-differentiation processes such as pluripotency and lineage commitment 4 , 5 , 6 , 7 . We have previously reported that the maternal factor PGC7 (also known as Dppa3, Stella) is required for the maintenance of DNA methylation in early embryogenesis, and protects 5mC from conversion to 5hmC in the maternal genome 8 , 9 . Here we show that PGC7 protects 5mC from Tet3-mediated conversion to 5hmC by binding to maternal chromatin containing dimethylated histone H3 lysine 9 (H3K9me2) in mice. In addition, imprinted loci that are marked with H3K9me2 in mature sperm are protected by PGC7 binding in early embryogenesis. This type of regulatory mechanism could be involved in DNA modifications in somatic cells as well as in early embryos.

SUV39H is the major histone H3 lysine 9 (H3K9)-specific methyltransferase that targets pericentric regions and is crucial for assembling silent heterochromatin. SUV39H recognizes trimethylated H3K9 (H3K9me3) via its chromodomain (CD), and enriched H3K9me3 allows SUV39H to target specific chromosomal regions. However, the detailed targeting mechanisms, especially for naïve chromatin without preexisting H3K9me3, are poorly understood. Here we show that Suv39h1’s CD (Suv39h1-CD) binds nucleic acids, and this binding is important for its function in heterochromatin assembly. Suv39h1-CD had higher binding affinity for RNA than DNA, and its ability to bind nucleic acids was independent of its H3K9me3 recognition. Suv39h1 bound major satellite RNAs in vivo, and knockdown of major satellite RNAs lowered Suv39h1 retention on pericentromere. Suv39h1 mutational studies indicated that both the nucleic acid–binding and H3K9me–binding activities of Suv39h1-CD were crucial for its pericentric heterochromatin assembly. These results suggest that chromatin-bound RNAs contribute to creating SUV39H’s target specificity. Plants, animals and fungi store much of their DNA tightly packed with proteins in a form named heterochromatin. This arrangement helps to inactivate genes that are not needed in specific cells or at specific times, and provides a way to protect the genetic material from damage. Heterochromatin tends to form when an enzyme called a lysine methyltransferase chemically modifies some of the proteins associated with the DNA, which are known as histones. This enzyme modifies only some of the histones to get the process started, while a second protein then binds to the modified histones and causes more of the DNA to become packaged up as heterochromatin. In 2012, researchers reported that the version of the lysine methyltransferase enzyme from yeast binds to RNA molecules via a portion known as its chromodomain. Moreover, the enzyme needed to bind to RNA to help heterochromatin to form. A similar mechanism also occurs in fruit flies, another organism that is commonly studied in the laboratory. However, it was not clear if it happened in mammals like mice and humans. Now, Shirai, Kawaguchi et al. – who include many of the researchers involved in the 2012 study – report that the corresponding enzyme from mice can also bind to RNA molecules via its chromodomain. Further experiments showed that this activity was closely linked with the enzyme’s ability to target the correct histones and efficiently form heterochromatin. The first experiments were conducted using purified enzymes in the laboratory, while follow-up experiments looked at the enzyme’s activity within mouse cells. Other studies have previously reported that mutant mice lacking the lysine methyltransferase enzyme have defective heterochromatin, tend to die young and have genetic instabilities that are associated with an increased risk of tumors and male infertility. The new findings of Shirai, Kawaguchi et al. reveal that the mechanism behind the establishment of heterochromatin has mostly likely been conserved over a billion years of evolution, which is when yeast and mammals last shared a common ancestor. By revealing more about how mammalian cells can protect their DNA, these new findings could also mark an important step toward understanding and preventing birth defects that are caused when an embryo’s genetic material becomes damaged.

Andrew Feinberg and colleagues identify large chromatin domains marked by histone H3 lysine 9 dimethylation in human and mouse cells. These blocks show tissue specificity, and their appearance is correlated with differentiation status. Higher eukaryotes must adapt a totipotent genome to specialized cell types with stable but limited functions. One potential mechanism for lineage restriction is changes in chromatin, and differentiation-related chromatin changes have been observed for individual genes 1 , 2 . We have taken a genome-wide view of histone H3 lysine 9 dimethylation (H3K9Me2) and find that differentiated tissues show surprisingly large K9-modified regions (up to 4.9 Mb). These regions are highly conserved between human and mouse and are differentiation specific, covering only ∼4% of the genome in undifferentiated mouse embryonic stem (ES) cells, compared to 31% in differentiated ES cells, ∼46% in liver and ∼10% in brain. These modifications require histone methyltransferase G9a and are inversely related to expression of genes within the regions. We term these regions large organized chromatin K9 modifications (LOCKs). LOCKs are substantially lost in cancer cell lines, and they may provide a cell type–heritable mechanism for phenotypic plasticity in development and disease.

In vivo reprogramming provokes a wide range of cell fate conversion. Here, we discover that in vivo induction of higher levels of OSKM in mouse somatic cells leads to increased expression of primordial germ cell (PGC)-related genes and provokes genome-wide erasure of genomic imprinting, which takes place exclusively in PGCs. Moreover, the in vivo OSKM reprogramming results in development of cancer that resembles human germ cell tumors. Like a subgroup of germ cell tumors, propagated tumor cells can differentiate into trophoblasts. Moreover, these tumor cells give rise to induced pluripotent stem cells (iPSCs) with expanded differentiation potential into trophoblasts. Remarkably, the tumor-derived iPSCs are able to contribute to non-neoplastic somatic cells in adult mice. Mechanistically, DMRT1, which is expressed in PGCs, drives the reprogramming and propagation of the tumor cells in vivo. Furthermore, the DMRT1-related epigenetic landscape is associated with trophoblast competence of the reprogrammed cells and provides a therapeutic target for germ cell tumors. These results reveal an unappreciated route for somatic cell reprogramming and underscore the impact of reprogramming in development of germ cell tumors. The mechanisms by which in vivo expression of the Yamanaka transcription factors (OSKM) renders somatic cells permissive for differentiation remain unclear. Here, the authors show that in vivo reprogramming using OSKM generates germ cell tumors and drives acquisition of totipotency-like features in somatic cells through DMRT1.