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.

The cytokine IL-10 antagonizes pathways that control () infection. Nevertheless, the impact of IL-10 during infection has been difficult to decipher because loss-of-function studies in animal models have yielded only mild phenotypes. We have discovered that the transcription factor basic helix-loop-helix family member e40 (Bhlhe40) is required to repress expression during infection. Loss of Bhlhe40 in mice results in higher expression, higher bacterial burden, and early susceptibility similar to that observed in mice lacking IFN-γ. Deletion of in mice reverses these phenotypes. Bhlhe40 deletion in T cells or CD11c cells is sufficient to cause susceptibility to Bhlhe40 represents the first transcription factor found to be essential during infection to specifically regulate expression, revealing the importance of strict control of IL-10 production by innate and adaptive immune cells during infection. Our findings uncover a previously elusive but significant role for IL-10 in pathogenesis.

Lin et al. show that Bhlhe40 expression identifies encephalitogenic CD4+ T helper cells and define a pertussis toxin–IL-1–Bhlhe40 pathway active in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis.

PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.

The glycine tRNA genes in silkworm Bombyx mon contain two regulatory regions upstream of the transcription start site as identified by direct transcription of 5' deletion mutants, transcription competition, gel mobility shift assays, and footprinting. A positive regulatory region is present in the immediate 5' flanking sequences of the four tRNAGIY clones studied. This region is essential for cell-free transcription in homologous extracts. A negative regulatory region is present farther upstream, and transcription competition experiments indicate its presence in three of the four clones exmned.The posterior silk gland of the silkworm Bombyx mori is a highly specialized organ involved in the synthesis of enormous amounts of the silk protein fibroin. Fibroin is rich in glycine (43%), alanine (28%), serine (12%), and tyrosine (5%) (1). To gear the system for synthesis of large quantities of fibroin, there is a functional adaptation in the silk glands of the B. mori larvae (2). The tRNA population in the posterior silk gland becomes highly enriched for the tRNA species cognate to the predominant amino acids in fibroin (3-5). To understand how the posterior silk gland produces such high levels of defined tRNA species, we have undertaken the study of regulation of expression of glycine tRNA genes. No tRNAGlY species that are exclusive to the silk gland have been identified so far (6-8), unlike the silk gland-specific tRNAAla (9, 10). It was therefore important to compare the relative transcriptional efficiencies of more than one tRNAly gene. In this paper, we report the presence of two regulatory regions for these tRNA GlY genes. An upstream positive regulatory region, within 40 base pairs (bp) of the transcription start site, is required for cell-free transcription of the clones studied. A negative regulatory element is present further upstream in three of the four clones examined. MATERIALS AND METHODSConstruction of Deletion Mutants. The six tRNAGly genes used in this study were a gift from A. Fournier. These clones were isolated from a B. mori genomic library in the phage A vector Charon 4. We have subcloned one of these, pBma2 (8) as an EcoRI-Sau3Al fragment in pUC18, which was designated pR8. The deletion mutants were constructed from this subclone. The 5' deletion constructs include pX3 (-2 to +108), pSX (-40 to +108), and pKX (-150 to +108). The subclone pA3 has all of the upstream sequences of pR8 (-300) and the coding region up to the Sma I site (+53). These constructs are listed in Fig. 1B.Preparation of Nuclear Extracts and in Vitro Transcription. Nuclear extracts from posterior silk glands of the fifth instar larvae were prepared as described for HeLa cell extracts (11) with modifications. The glands were homogenized in a buffer containing 2 M sucrose, 10o (vol/vol) glycerol, 10 mM Hepes (pH 7.9), 15 mM KCI, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride. The homogenate was layered on a 3-ml cushion of the same buffer and centrifuged at 26,000 rpm for 1 hr at 40C in a Beckman SW-4...