Brown adipose tissue (BAT) represents a promising agent to ameliorate obesity and other metabolic disorders. However, the abundance of BAT decreases with age and BAT paucity is a common feature of obese subjects. As brown adipocytes and myoblasts share a common Myf5 lineage origin, elucidating the molecular mechanisms underlying the fate choices of brown adipocytes versus myoblasts may lead to novel approaches to expand BAT mass. Here we identify MyoD as a key negative regulator of brown adipocyte development. CRISPR/CAS9-mediated deletion of MyoD in C2C12 myoblasts facilitates their adipogenic transdifferentiation. MyoD knockout downregulates miR-133 and upregulates the miR-133 target Igf1r, leading to amplification of PI3K–Akt signaling. Accordingly, inhibition of PI3K or Akt abolishes the adipogenic gene expression of MyoD null myoblasts. Strikingly, loss of MyoD converts satellite cell-derived primary myoblasts to brown adipocytes through upregulation of Prdm16, a target of miR-133 and key determinant of brown adipocyte fate. Conversely, forced expression of MyoD in brown preadipocytes blocks brown adipogenesis and upregulates the expression of myogenic genes. Importantly, miR-133a knockout significantly blunts the inhibitory effect of MyoD on brown adipogenesis. Our results establish MyoD as a negative regulator of brown adipocyte development by upregulating miR-133 to suppress Akt signaling and Prdm16.

Protein methyltransferases mediate posttranslational modifications of both histone and nonhistone proteins. Whereas histone methylation is well-known to regulate gene expression, the biological significance of nonhistone methylation is poorly understood. Methyltransferase-like 21c (Mettl21c) is a newly classified nonhistone lysine methyltransferase whose in vivo function has remained elusive. Using a Mettl21c LacZ knockin mouse model, we show here that Mettl21c expression is absent during myogenesis and restricted to mature type I (slow) myofibers in the muscle. Using co-immunoprecipitation, MS, and methylation assays, we demonstrate that Mettl21c trimethylates heat shock protein 8 (Hspa8) at Lys-561 to enhance its stability. As such, Mettl21c knockout reduced Hspa8 trimethylation and protein levels in slow muscles, and Mettl21c overexpression in myoblasts increased Hspa8 trimethylation and protein levels. We further show that Mettl21c-mediated stabilization of Hspa8 enhances its function in chaperone-mediated autophagy, leading to degradation of client proteins such as the transcription factors myocyte enhancer factor 2A (Mef2A) and Mef2D. In contrast, Mettl21c knockout increased Mef2 protein levels in slow muscles. These results identify Hspa8 as a Mettl21c substrate and reveal that nonhistone methylation has a physiological function in protein stabilization.

Numerous methods have been developed in model systems to deplete or inactivate proteins to elucidate their functional roles. In Caenorhabditis elegans, a common method for protein depletion is RNA interference (RNAi), in which mRNA is targeted for degradation. C. elegans is also a powerful genetic organism, amenable to large‐scale genetic screens and CRISPR‐mediated genome editing. However, these approaches largely lead to constitutive inhibition, which can make it difficult to study proteins essential for development or to dissect dynamic cellular processes. Thus, there have been recent efforts to develop methods to rapidly inactivate or deplete proteins to overcome these barriers. One such method that is proving to be exceptionally powerful is auxin‐inducible degradation. In order to apply this approach in C. elegans, a 44–amino acid degron tag is added to the protein of interest, and the Arabidopsis ubiquitin ligase TIR1 is expressed in target tissues. When the plant hormone auxin is added, it mediates an interaction between TIR1 and the degron‐tagged protein of interest, which triggers ubiquitination of the protein and its rapid degradation via the proteasome. Here, we have outlined multiple methods for inducing auxin‐mediated depletion of target proteins in C. elegans, highlighting the versatility and power of this method. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Long‐term auxin‐mediated depletion on plates Support Protocol: Preparation of NGM and NGM‐auxin plates Basic Protocol 2: Rapid auxin‐mediated depletion via soaking Basic Protocol 3: Acute auxin‐mediated depletion in isolated embryos Basic Protocol 4: Assessing auxin‐mediated depletion