Sumoylation of the basic helix-loop-helix transcription factor sharp-1 regulates recruitment of the histone methyltransferase G9a and function in myogenesis.

Sumoylation is an important post-translational modification that alters the activity of many transcription factors. However, the mechanisms that link sumoylation to alterations in chromatin structure, which culminate in tissue specific gene expression, are not fully understood. In this study, we demonstrate that SUMO modification of the transcription factor Sharp-1 is required for its full transcriptional repression activity and function as an inhibitor of skeletal muscle differentiation. Sharp-1 is modified by sumoylation at two conserved lysine residues 240 and 255. Mutation of these SUMO acceptor sites in Sharp-1 does not impact its subcellular localization but attenuates its ability to act as a transcriptional repressor and inhibit myogenic differentiation. Consistently, co-expression of the SUMO protease SENP1 with wild type Sharp-1 abrogates Sharp-1-dependent inhibition of myogenesis. Interestingly, sumoylation acts as a signal for recruitment of the co-repressor G9a. Thus, enrichment of G9a, and histone H3 lysine 9 dimethylation (H3K9me2), a signature of G9a activity, is dramatically reduced at muscle promoters in cells expressing sumoylation-defective Sharp-1. Our findings demonstrate how sumoylation of Sharp-1 exerts an impact on chromatin structure and transcriptional repression of muscle gene expression through recruitment of G9a.

Epigenetic regulation of skeletal muscle development and differentiation.

Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. Formation of skeletal muscle involves a series of steps in which cells are committed towards the myogenic lineage, undergo expansion to give rise to myoblasts that differentiate into multinucleated myotubes, and mature to form adult muscle fibers. The commitment, proliferation, and differentiation of progenitor cells involve both genetic and epigenetic changes that culminate in alterations in gene expression. Members of the Myogenic regulatory factor (MRF), as well as the Myocyte Enhancer Factor (MEF2) families control distinct steps of skeletal muscle proliferation and differentiation. In addition, -growing evidence indicates that chromatin modifying enzymes and remodeling complexes epigenetically reprogram muscle promoters at various stages that preclude or promote MRF and MEF2 activites. Among these, histone deacetylases (HDACs), histone acetyltransferases (HATs), histone methyltransferases (HMTs) and SWI/SNF complexes alter chromatin structure through post-translational modifications to impact MRF and MEF2 activities. With such new and emerging knowledge, we are beginning to develop a true molecular understanding of the mechanisms by which skeletal muscle development and differentiation is regulated. Elucidation of the mechanisms by which epigenetic regulators control myogenesis will likely provide a new foundation for the development of novel therapeutic drugs for muscle dystrophies, ageing-related regeneration defects that occur due to altered proliferation and differentiation, and other malignancies.

G9a mediates Sharp-1-dependent inhibition of skeletal muscle differentiation.

Sharp-1, a basic helix-loop-helix transcription factor, is a potent repressor of skeletal muscle differentiation and is dysregulated in muscle pathologies. However, the mechanisms by which it inhibits myogenesis are not fully understood. Here we show that G9a, a lysine methyltransferase, is involved in Sharp-1-mediated inhibition of muscle differentiation. We demonstrate that G9a directly interacts with Sharp-1 and enhances its ability to transcriptionally repress the myogenin promoter. Concomitant with a differentiation block, G9a-dependent histone H3 lysine 9 dimethylation (H3K9me2) and MyoD methylation are apparent upon Sharp-1 overexpression in muscle cells. RNA interference-mediated reduction of G9a or pharmacological inhibition of its activity erases these repressive marks and rescues the differentiation defect imposed by Sharp-1. Our findings provide new insights into Sharp-1-dependent regulation of myogenesis and identify epigenetic mechanisms that could be targeted in myopathies characterized by elevated Sharp-1 levels.

SUMO modification of Stra13 is required for repression of cyclin D1 expression and cellular growth arrest.

Stra13, a basic helix-loop-helix (bHLH) transcription factor is involved in myriad biological functions including cellular growth arrest, differentiation and senescence. However, the mechanisms by which its transcriptional activity and function are regulated remain unclear. In this study, we provide evidence that post-translational modification of Stra13 by Small Ubiquitin-like Modifier (SUMO) dramatically potentiates its ability to transcriptionally repress cyclin D1 and mediate G(1) cell cycle arrest in fibroblast cells. Mutation of SUMO acceptor lysines 159 and 279 located in the C-terminal repression domain has no impact on nuclear localization; however, it abrogates association with the co-repressor histone deacetylase 1 (HDAC1), attenuates repression of cyclin D1, and prevents Stra13-mediated growth suppression. HDAC1, which promotes cellular proliferation and cell cycle progression, antagonizes Stra13 sumoylation-dependent growth arrest. Our results uncover an unidentified regulatory axis between Stra13 and HDAC1 in progression through the G(1)/S phase of the cell cycle, and provide new mechanistic insights into regulation of Stra13-mediated transcriptional repression by sumoylation.

Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation.

Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. The proliferation and differentiation of muscle precursor cells require the concerted activity of myogenic regulatory factors including MyoD. In addition, chromatin modifiers mediate dynamic modifications of histone tails that are vital to reprogramming cells toward terminal differentiation. Here, we provide evidence for a unique dimension to epigenetic regulation of skeletal myogenesis. We demonstrate that the lysine methyltransferase G9a is dynamically expressed in myoblasts and impedes differentiation in a methyltransferase activity-dependent manner. In addition to mediating histone H3 lysine-9 di-methylation (H3K9me2) on MyoD target promoters, endogenous G9a interacts with MyoD in precursor cells and directly methylates it at lysine 104 (K104) to constrain its transcriptional activity. Mutation of K104 renders MyoD refractory to inhibition by G9a and enhances its myogenic activity. Interestingly, MyoD methylation is critical for G9a-mediated inhibition of myogenesis. These findings provide evidence of an unanticipated role for methyltransferases in cellular differentiation states by direct posttranslational modification of a transcription factor.