Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes.

Histone H3 Lys 9 (H3-K9) methylation is a crucial epigenetic mark for transcriptional silencing. G9a is the major mammalian H3-K9 methyltransferase that targets euchromatic regions and is essential for murine embryogenesis. There is a single G9a-related methyltransferase in mammals, called GLP/Eu-HMTase1. Here we show that GLP is also important for H3-K9 methylation of mouse euchromatin. GLP-deficiency led to embryonic lethality, a severe reduction of H3-K9 mono-and dimethylation, the induction of Mage-a gene expression, and HP1 relocalization in embryonic stem cells, all of which were phenotypes of G9a-deficiency. Furthermore, we show that G9a and GLP formed a stoichiometric heteromeric complex in a wide variety of cell types. Biochemical analyses revealed that formation of the G9a/GLP complex was dependent on their enzymatic SET domains. Taken together, our new findings revealed that G9a and GLP cooperatively exert H3-K9 methyltransferase function in vivo, likely through the formation of higher-order heteromeric complexes.[Keywords: Histone; methylation; G9a; GLP] Supplemental material is available at http://www.genesdev.org.

The pluripotency determining gene, Oct-3/4 (also called Pou5f1) undergoes post implantation silencing in a process mediated by the histone methyltransferase (HMT) G9a. Microarray analysis now shows that this enzyme may operate as a master regulator that inactivates multiple early embryonic genes by bringing about methylated-histone H3K9 heterochromatinization and de novo DNA methylation. Genetic studies in differentiating ES cells demonstrate that a point mutation in the G9a SET domain prevents heterochromatinization, but still allows de novo methylation, while biochemical and functional studies indicate that G9a itself is capable of bringing about de novo methylation through its ankyrin (ANK) domain, by recruiting Dnmt3a/3b independently of its HMT activity. These modifications appear to be programmed for carrying out two separate biological functions, with histone methylation blocking target-gene reactivation in the absence of transcriptional repressors, while DNA methylation prevents reprogramming to the undifferentiated state.

In serum, mouse embryonic stem cells (mESCs) fluctuate between a naive inner cell mass (ICM)-like state and a primed epiblast-like state, but when cultured with inhibitors of the mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 pathways (2i), they are harnessed exclusively in a distinct naive pluropotent state, the ground state, that more faithfully recapitulates the ICM. Understanding the mechanism underlying this naive pluripotent state will be critical for realizing the full potential of ESCs. We show here that PRDM14, a PR-domain-containing transcriptional regulator, ensures naive pluripotency through a dual mechanism: antagonizing activation of the fibroblast growth factor receptor (FGFR) signaling by the core pluripotency transcriptional circuitry, and repressing expression of de novo DNA methyltransferases that modify the epigenome to a primed epiblast-like state. PRDM14 exerts these effects by recruiting polycomb repressive complex 2 (PRC2) specifically to key targets and repressing their expression.

Although it is known that OCT4-NANOG are required for maintenance of pluripotent cells in vitro, the upstream signals that regulate this circuit during early development in vivo have not been identified. Here we demonstrate, for the first time, signal transducers and activators of transcription 3 (STAT3)-dependent regulation of the OCT4-NANOG circuitry necessary to maintain the pluripotent inner cell mass (ICM), the source of in vitro-derived embryonic stem cells (ESCs). We show that STAT3 is highly expressed in mouse oocytes and becomes phosphorylated and translocates to the nucleus in the four-cell and later stage embryos. Using leukemia inhibitory factor (Lif )-null embryos, we found that STAT3 phosphorylation is dependent on LIF in four-cell stage embryos. In blastocysts, interleukin 6 (IL-6) acts in an autocrine fashion to ensure STAT3 phosphorylation, mediated by janus kinase 1 (JAK1), a LIF-and IL-6-dependent kinase. Using genetically engineered mouse strains to eliminate Stat3 in oocytes and embryos, we firmly establish that STAT3 is essential for maintenance of ICM lineages but not for ICM and trophectoderm formation. Indeed, STAT3 directly binds to the Oct4 and Nanog distal enhancers, modulating their expression to maintain pluripotency of mouse embryonic and induced pluripotent stem cells. These results provide a novel genetic model of cell fate determination operating through STAT3 in the preimplantation embryo and pluripotent stem cells in vivo.