Makoto Tachibana

Partitioning and Plasticity of Repressive Histone Methylation States in Mammalian Chromatin

Methylation of position-specific lysine residues in histone N termini is a central modification for regulating epigenetic transitions in chromatin. Each methylatable lysine residue can exist in a mono-, di-, or trimethylated state, thereby extending the indexing potential of this particular modification. Here, we examine all possible methylation states for histone H3 lysine 9 (H3-K9) and lysine 27 (H3-K27) in mammalian chromatin. Using highly specific antibodies together with quantitative mass spectrometry, we demonstrate that pericentric heterochromatin is selectively enriched for H3-K27 monomethylation and H3-K9 trimethylation. This heterochromatic methylation profile is dependent on the Suv39h histone methyltransferases (HMTases) but independent of the euchromatic G9a HMTase. In Suv39h double null cells, pericentric heterochromatin is converted to alternative methylation imprints and accumulates H3-K27 trimethylation and H3-K9 monomethylation. Our data underscore the selective presence of distinct histone lysine methylation states in partitioning chromosomal subdomains but also reveal a surprising plasticity in propagating methylation patterns in eukaryotic chromatin.

SET Domain-containing Protein, G9a, Is a Novel Lysine-preferring Mammalian Histone Methyltransferase with Hyperactivity and Specific Selectivity to Lysines 9 and 27 of Histone H3

The covalent modification of histone tails has regulatory roles in various nuclear processes, such as control of transcription and mitotic chromosome condensation. Among the different groups of enzymes known to catalyze the covalent modification, the most recent additions are the histone methyltransferases (HMTases), whose functions are now being characterized. Here we show that a SET domain-containing protein, G9a, is a novel mammalian lysine-preferring HMTase. Like Suv39 h1, the first identified lysine-preferring mammalian HMTase, G9a transfers methyl groups to the lysine residues of histone H3, but with a 10–20-fold higher activity. It was reported that lysines 4, 9, and 27 in H3 are methylated in mammalian cells. G9a was able to add methyl groups to lysine 27 as well as 9 in H3, compared with Suv39 h1, which was only able to methylate lysine 9. Our data clearly demonstrated that G9a has an enzymatic nature distinct from Suv39 h1 and its homologue h2. Finally, fluorescent protein-labeled G9a was shown to be localized in the nucleus but not in the repressive chromatin domains of centromeric loci, in which Suv39 h1 family proteins were localized. This finding indicates that G9a may contribute to the organization of the higher order chromatin structure of non-centromeric loci.

Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET.

Endogenous retroviruses (ERVs), retrovirus-like elements with long terminal repeats, are widely dispersed in the euchromatic compartment in mammalian cells, comprising ∼10% of the mouse genome. These parasitic elements are responsible for >10% of spontaneous mutations. Whereas DNA methylation has an important role in proviral silencing in somatic and germ-lineage cells, an additional DNA-methylation-independent pathway also functions in embryonal carcinoma and embryonic stem (ES) cells to inhibit transcription of the exogenous gammaretrovirus murine leukaemia virus (MLV). Notably, a recent genome-wide study revealed that ERVs are also marked by histone H3 lysine 9 trimethylation (H3K9me3) and H4K20me3 in ES cells but not in mouse embryonic fibroblasts. However, the role that these marks have in proviral silencing remains unexplored. Here we show that the H3K9 methyltransferase ESET (also called SETDB1 or KMT1E) and the Krüppel-associated box (KRAB)-associated protein 1 (KAP1, also called TRIM28) are required for H3K9me3 and silencing of endogenous and introduced retroviruses specifically in mouse ES cells. Furthermore, whereas ESET enzymatic activity is crucial for HP1 binding and efficient proviral silencing, the H4K20 methyltransferases Suv420h1 and Suv420h2 are dispensable for silencing. Notably, in DNA methyltransferase triple knockout (Dnmt1-/-Dnmt3a-/-Dnmt3b-/-) mouse ES cells, ESET and KAP1 binding and ESET-mediated H3K9me3 are maintained and ERVs are minimally derepressed. We propose that a DNA-methylation-independent pathway involving KAP1 and ESET/ESET-mediated H3K9me3 is required for proviral silencing during the period early in embryogenesis when DNA methylation is dynamically reprogrammed.

Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice

We previously reported that primordial germ cells (PGCs) in mice erase genome-wide DNA methylation and histone H3 lysine9 dimethylation (H3K9me2), and instead acquire high levels of tri-methylation of H3K27 (H3K27me3) during their migration, a process that might be crucial for the re-establishment of potential totipotency in the germline. We here explored a cellular dynamics associated with this epigenetic reprogramming. We found that PGCs undergo erasure of H3K9me2 and upregulation of H3K27me3 in a progressive, cell-by-cell manner, presumably depending on their developmental maturation. Before or concomitant with the onset of H3K9 demethylation, PGCs entered the G2 arrest of the cell cycle, which apparently persisted until they acquired high H3K27me3 levels. Interestingly, PGCs exhibited repression of RNA polymerase II-dependent transcription, which began after the onset of H3K9me2 reduction in the G2 phase and tapered off after the acquisition of high-level H3K27me3. The epigenetic reprogramming and transcriptional quiescence were independent from the function of Nanos3. We found that before H3K9 demethylation, PGCs exclusively repress an essential histone methyltransferase, GLP, without specifically upregulating histone demethylases. We suggest the possibility that active repression of an essential enzyme and subsequent unique cellular dynamics ensures successful implementation of genome-wide epigenetic reprogramming in migrating PGCs.

PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos

The modification of DNA by 5-methylcytosine (5mC) has essential roles in cell differentiation and development through epigenetic gene regulation. 5mC can be converted to another modified base, 5-hydroxymethylcytosine (5hmC), by the tet methylcytosine dioxygenase (Tet) family of enzymes. Notably, the balance between 5hmC and 5mC in the genome is linked with cell-differentiation processes such as pluripotency and lineage commitment. 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. 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.

H3K9 methyltransferase G9a and the related molecule GLP.

The discovery of Suv39h1, the first SET domain-containing histone lysine methyltransferase (HKMT), was reported in 2000. Since then, research on histone methylation has progressed rapidly. Among the identified HKMTs in mammals, G9a and GLP are the primary enzymes for mono- and dimethylation at Lys 9 of histone H3 (H3K9me1 and H3K9me2), and exist predominantly as a G9a-GLP heteromeric complex that appears to be a functional H3K9 methyltransferase in vivo. Recently, many important studies have reported that G9a and GLP play critical roles in various biological processes. The physiological relevance of G9a/GLP-mediated epigenetic gene regulation is discussed.

G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription.

Methylation of DNA and lysine 9 of histone H3 (H3K9) are well‐conserved epigenetic marks for transcriptional silencing. Although H3K9 methylation directs DNA methylation in filamentous fungi and plants, this pathway has not been corroborated in mammals. G9a and GLP/Eu‐HMTase1 are two‐related mammalian lysine methyltransferases and a G9a/GLP heteromeric complex regulates H3K9 methylation of euchromatin. To elucidate the function of G9a/GLP‐mediated H3K9 methylation in the regulation of DNA methylation and transcriptional silencing, we characterized ES cells expressing catalytically inactive mutants of G9a and/or GLP. Interestingly, in ES cells expressing a G9a‐mutant/GLP complex that does not rescue global H3K9 methylation, G9a/GLP‐target genes remain silent. The CpG sites of the promoter regions of these genes were hypermethylated in such mutant ES cells, but hypomethylated in G9a‐ or GLP‐KO ES cells. Treatment with a DNA methyltransferase inhibitor reactivates these G9a/GLP‐target genes in ES cells expressing catalytically inactive G9a/GLP proteins, but not the wild‐type proteins. This is the first clear evidence that G9a/GLP suppresses transcription by independently inducing both H3K9 and DNA methylation.

Methyl-CpG Binding Domain 1 (MBD1) Interacts with the Suv39h1-HP1 Heterochromatic Complex for DNA Methylation-based Transcriptional Repression

Cytosine methylation and posttranslational modifications of the amino termini of the core histones in the nucleosome provide epigenetic codes for genome regulation. In the nucleus, not only is the DNA methylated, but the methylated DNA is also interpreted by methyl-CpG binding domain (MBD) proteins. MBD1 possesses an MBD involved in mediating DNA methylation-dependent transcriptional repression. The MBD of MBD1 binds a symmetrically methylated CpG sequence, but the precise roles of this domain have not been investigated. In addition, little is understood about the state of histone modifications within MBD1-containing heterochromatin on methylated gene promoters. Here we show that histone H3 methylase Suv39h1 and the methyl lysine-binding protein HP1 directly interact with MBD of MBD1 in vitro and in cells. Suv39h1 was found to enhance MBD1-mediated transcriptional repression via MBD but not via the C-terminal transcriptional repression domain of MBD1. Furthermore, MBD1 links to histone deacetylases through Suv39h1, resulting in methylation and deacetylation of histones for gene inactivation. These data indicate that MBD1 may tether the Suv39h1-HP1 complex to methylated DNA regions, suggesting the presence of a pathway from DNA methylation to the modifications of histones for epigenetic gene regulation.

Cloning of mice to six generations

Mice have been cloned by nuclear transfer into enucleated oocytes, and here we describe the reiterative cloning of mice to four and six generations in two independent lines. Successive generations showed no signs of prematureageing, as judged by gross behaviouralparameters, and there was no evidence of shortening of telomeres at the ends of chromosomes, normally an indicator of cellular senescence — in fact, these appeared to increase slightly in length. This increase is surprising, given that the number of mitotic divisions greatly exceeds that of sexually produced animals and that any deleterious effects of cloning might be expected to be amplified in sequentially cloned mice. Our results offer a new approach to the study of organismal ageing.

Functional dynamics of H3K9 methylation during meiotic prophase progression

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.