Histone lysine methylation, as one of the most important factors in transcriptional regulation, is associated with a various physiological conditions. Using a bioinformatics search, we identified and subsequently cloned mouse SET domain containing 3 (SETD3) with SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) and Rubis-subs-bind domains. SETD3 is a novel histone H3K4 and H3K36 methyltransferase with transcriptional activation activity. SETD3 is expressed abundantly in muscular tissues and, when overexpressed, activates transcription of muscle-related genes, myogenin, muscle creatine kinase (MCK), and myogenic factor 6 (Myf6), thereby inducing muscle cell differentiation. Conversely, knockdown of SETD3 by shRNA significantly retards muscle cell differentiation. In this study, SETD3 was recruited to the myogenin gene promoter along with MyoD where it activated transcription. Together, these data indicate that SETD3 is a H3K4/K36 methyltransferase and plays an important role in the transcriptional regulation of muscle cell differentiation.The conformational structure or molecular charge of the histone core complex can be modified via methylation of the lysine/arginine residue in the histone tail, which affects gene expression and heterochromatin formation (1). Arginine methylation is mediated by PRDM family proteins, which are characterized by the presence of a PR (PRD1-BF1 and RIZ homology) domain at their N terminus, whereas lysine is methylated by histone methyltransferase (HMTase), 4 which commonly harbors the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain (2). By forming complexes with a broad variety of transcription factors, HMTases perform an important role in the regulation of gene expression, stem cell renewal, reproductive organ maturation, and tumorigenesis in mammals (2-5).Additionally, HMTases have been confirmed as crucial to myofibril organization (6), intestinal and pancreatic differentiation (7), and neurogenesis (8) in zebrafish.Muscle differentiation requires sequences of harmonized steps after the commitment of mesodermal progenitor cells to the muscular lineage (9). Under the regulation of diverse modifiers, myoblasts fuse with other neighboring myoblasts to generate multinucleated myotubes (10). During differentiation, the cell cycle is withdrawn and muscle-specific transcription factors activated. Mesodermal precursor cells with muscular lineages are differentiated into skeletal muscle or smooth muscle via the interplay of muscle-specific factors, including MyoD, myogenin, myogenic factor 5 (Myf5), muscle regulatory factor 4 (MRF4), and myocyte enhancer factor-2 (MEF2) (10).Histone modification enzymes have been implicated in muscle cell differentiation through the regulation of musclespecific gene expression (11,12). Chromatin modification enzymes such as histone acetyltransferases, deacetylases (HDACs), and chromatin remodeling factors have recently been reported to regulate MyoD activity during muscle differentiation. For example, the histone acetyltransferases p300 and p300/C...

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Histone H3K9 methyltransferase (HMTase) G9a-mediated transcriptional repression is a major epigenetic silencing mechanism. UHRF1 (ubiquitin-like with PHD and ring finger domains 1) binds to hemimethylated DNA and plays an essential role in the maintenance of DNA methylation. Here, we provide evidence that UHRF1 is transcriptionally downregulated by H3K9 HMTase G9a. We found that increased expression of G9a along with transcription factor YY1 specifically represses UHRF1 transcription during TPA-mediated leukemia cell differentiation. Using ChIP analysis, we found that UHRF1 was among the transcriptionally silenced genes during leukemia cell differentiation. Using a DNA methylation profiling array, we discovered that the UHRF1 promoter was hypomethylated in samples from leukemia patients, further supporting its overexpression and oncogenic activity. Finally, we showed that G9a regulates UHRF1-mediated H3K23 ubiquitination and proper DNA replication maintenance. Therefore, we propose that H3K9 HMTase G9a is a specific epigenetic regulator of UHRF1.

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Relaxin (Rln), a polypeptide hormone of the insulin superfamily, is an ovarian peptide hormone that is involved in a diverse range of physiological and pathological reactions. In this study, we investigated the effect of Rln on bone morphogenetic protein 2 (BMP-2)-induced osteoblast differentiation and bone formation. Expression of Rln receptors was examined in the primary mouse bone marrow stem cells (BMSCs) and mouse embryonic fibroblast cell line C3H/10T1/2 cells by RT-PCR and Western blot during BMP-2-induced osteoblast differentiation. The effect of Rln on osteoblast differentiation and mineralization was evaluated by measuring the alkaline phosphatase activity, osteocalcin production, and Alizarin red S staining. For the in vivo evaluation, BMP-2 and/or Rln were administered with type I collagen into the back of mice, and after 3 weeks, bone formation was analyzed by micro-computed tomography (mCT). Western blot was performed to determine the effect of Rln on osteoblast differentiation-related signaling pathway. Expression of Rxfp 1 in BMSCs and C3H/10T1/2 cells was significantly increased by BMP-2. In vitro, Rln augmented BMP-2-induced alkaline phosphatase expression, osteocalcin production, and matrix mineralization in BMSCs and C3H/10T1/2 cells. In addition, in vivo administration of Rln enhanced BMP-2-induced bone formation in a dose-dependent manner. Interestingly, Rln synergistically increased and sustained BMP-2-induced Smad, p38, and transforming growth factor-b activated kinase (TAK) 1 phosphorylation. BMP-2-induced Runx 2 expression and activity were also significantly augmented by Rln. These results show that Rln enhanced synergistically BMP-2-induced osteoblast differentiation and bone formation through its receptor, Rxfp 1, by augmenting and sustaining BMP-2-induced Smad and p38 phosphorylation, which upregulate Runx 2 expression and activity. These results suggest that Rln might be useful for therapeutic application in destructive bone diseases.

Histone methylation at specific lysine residues is a crucial regulatory process in transcriptional regulation. Using chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis, we found that the H3K9-me2 target gene JAK2 was an important factor during differentiation of the HL-60 promyelocytic leukemia cell line by all- trans -retinoic acid (ATRA) treatment. Here, we report that the H3K9 methyltransferase G9a negatively regulated JAK2 transcription in histone methyltransferase activity and in a YY1-dependent manner during ATRA-mediated leukemia cell differentiation. We found that G9a knockdown repressed ATRA-mediated HL-60 cell differentiation. We demonstrated that G9a interacts with YY1 and is recruited to the JAK2 promoter along with corepressors, including histone deacetylase, that induced H3K9-me2. Repression of JAK2 transcription by G9a decreased H3Y41 phosphorylation and promoted inhibition of the recently identified JAK2-H3Y41P-HP1α pathway-mediated leukemogenesis.

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Edited by Ned ManteiKeywords: SET/TAF-Ib INHAT H3S28 Phosphorylation H3K27me Peptide array a b s t r a c t Significant progress has been made in understanding the relationship between histone modifications and 'reader' molecules and their effects on transcriptional regulation. A previously identified INHAT complex subunit, SET/TAF-Ib, binds to histones and inhibits histone acetylation. To investigate the binding specificities of SET/TAF-Ib to various histone modifications, we employed modified histone tail peptide array analyses. SET/TAF-Ib strongly recognized PRC2-mediated H3K27me1/2/3; however, the bindings were completely disrupted by H3S28 phosphorylation. We have demonstrated that SET/TAF-Ib is sequentially recruited to the target gene promoter ATF3 after the PRC2 complex via H3K27me recognition and may offer additive effects in the repression of the target gene.