Effects of HMGCR deficiency on skeletal muscle development.

Pathogenic variants in HMGCR were recently linked to a limb-girdle muscular dystrophy (LGMD) phenotype. The protein product HMG CoA reductase (HMGCR) catalyzes a key component of the cholesterol synthesis pathway. The two other muscle diseases associated with HMGCR, statin-associated myopathy (SAM) and autoimmune anti-HMGCR myopathy, are not inherited in a Mendelian pattern. The mechanism linking pathogenic variants in HMGCR with skeletal muscle dysfunction is unclear. We knocked down Hmgcr in mouse skeletal myoblasts, knocked down hmgcr in Drosophila, and expressed three pathogenic HMGCR variants (c.1327C>T, p.Arg443Trp; c.1522_1524delTCT, p.Ser508del; and c.1621G>A, p.Ala541Thr) in Hmgcr knockdown mouse myoblasts. Hmgcr deficiency was associated with decreased proliferation, increased apoptosis, and impaired myotube fusion. Transcriptome sequencing of Hmgcr knockdown versus control myoblasts revealed differential expression involving mitochondrial function, with corresponding differences in cellular oxygen consumption rates. Both ubiquitous and muscle-specific knockdown of hmgcr in Drosophila led to lethality. Overexpression of reference HMGCR cDNA rescued myotube fusion in knockdown cells, whereas overexpression of the pathogenic variants of HMGCR cDNA did not. These results suggest that the three HMGCR-related muscle diseases share disease mechanisms related to skeletal muscle development.

MED12 Regulates Human Adipose-Derived Stem Cell Adipogenesis and Mediator Kinase Subunit Expression in Murine Adipose Depots.

The mediator kinase module plays a critical role in the regulation of transcription during metabolic processes. Here we demonstrate that in human adipose-derived stem cells (hASCs), kinase module subunits have distinct mRNA and protein expression profiles during different stages of adipogenesis. In addition, siRNA-mediated loss of MED12 results in decreased adipogenesis as evident through decreased lipid accumulation and decreased expression of PPARγ, a master regulator of adipogenesis. Moreover, the decrease in adipogenesis and reduced PPARγ expression are observed only during the early stages of MED12 knockdown. At later stages, knockdown of MED12 did not have any significant effects on adipogenesis or PPARγ expression. We also observed that MED12 was present in a protein complex with PPARγ and C/EBPα during all stages of adipogenesis in hASCs. In 3T3-L1 preadipocytes and adipocytes, MED12 is present in protein complexes with PPARγ1, C/EBPα, and STAT5A. CDK8, another member of the kinase module, was only found to interact with C/EBPα. We found that the expression of all kinase module subunits decreased in inguinal, gonadal, and retroperitoneal white adipose tissue (WAT) depots in the fed state after an overnight fast, whereas the expression of kinase module subunits remained consistent in mesenteric WAT (mWAT) and brown adipose tissue. These data demonstrate that the kinase module undergoes physiologic regulation during fasting and feeding in specific mouse adipose tissue depots, and that MED12 likely plays a specific role in initiating and maintaining adipogenesis.

Mediator's Kinase Module: A Modular Regulator of Cell Fate.

Selective gene expression is crucial in maintaining the self-renewing and multipotent properties of stem cells. Mediator is a large, evolutionarily conserved, multi-subunit protein complex that modulates gene expression by relaying signals from cell type-specific transcription factors to RNA polymerase II. In humans, this complex consists of 30 subunits arranged in four modules. One critical module of the Mediator complex is the kinase module consisting of four subunits: MED12, MED13, CDK8, and CCNC. The kinase module exists in variable association with the 26-subunit Mediator core and affects transcription through phosphorylation of transcription factors and by controlling Mediator structure and function. Many studies have shown the kinase module to be a key player in the maintenance of stem cells that is distinct from a general role in transcription. Genetic studies have revealed that dysregulation of this kinase subunit contributes to the development of many human diseases. In this review, we discuss the importance of the Mediator kinase module by examining how this module functions with the more recently identified transcriptional super-enhancers, how changes in the kinase module and its activity can lead to the development of human disease, and the role of this unique module in directing and maintaining cell state. As we look to use stem cells to understand human development and treat human disease through both cell-based therapies and tissue engineering, we need to remain aware of the on-going research and address critical gaps in knowledge related to the molecular mechanisms that control cell fate.