Skeletal muscles constitute the most abundant organ in an adult human, about 40% of its total body mass. Most skeletal muscles are composed of a mixture of myofibers with distinct contractile, metabolic, resistance to fatigue properties, as well as differential vulnerability in pathophysiological situations. These different myofibers can be classified as slow or fast subtypes that selectively express genes responsible for their specific properties. The most widely used classification of myofibers types is based on their Myosin heavy chain (MYH) expression profile. MYH, one of the most abundant proteins present in adult myofibers, is a major determinant of myofiber speed of contraction. Each of the mammalian MYH isoform is coded by a specific gene and adult slow-type myofibers express Myh7 (also known as MyHCI, beta or slow), adult fast-type myofibers express Myh2 (MyHCIIA), Myh1 (MyHCIIX), Myh4 (MyHCIIB) or Myh13 (MyHCeo). During embryonic development Myh7 and two specific fast Myh (fMyh) genes, Myh3 (MyHCemb) and Myh8 (MyHCperi) are expressed. The fMyh genes (Myh3, Myh2, Myh1, Myh4, Myh8, and Myh13) are organized as a cluster within a 350kb region on mouse chromosome 11.
The mechanisms controlling the robust coordinated expression of fMyh genes in the hundred nuclei of a myofiber are not understood. The clustered organization and strict temporal regulation of the fMyh locus shows similarities with that of the human beta-globin locus. Special regulatory elements called super enhancers (SE) have been shown to control high expression levels for cell lineage identity genes. These SE are composed of multiple enhancer elements spanning 10 to 50 kb of DNA and allowing efficient expression of associated genes.
To characterize the cis-regulatory elements required for the complex regulation of the specific fMyh genes the authors performed snATAC-seq and 4C-seq experiments with adult skeletal muscles and identified a 42kb opened chromatin region interacting in an exclusive manner with the activated fMyh promoter at the locus through 3D chromatin looping as revealed by 4C-seq experiments.
A mouse rainbow transgenic line including this SE recapitulates the spatio-temporal expression of endogenous Myh2, Myh1 and Myh4 genes. The authors further show by CRISPR/Cas9 editing that in situ deletion of this 42kb SE region prevents expression of fetal Myh8 and adult fMyh genes at the locus leading to fetal myofibers devoid of sarcomeres, unable to contract and precluding breathing at birth.
The authors tested the hypothesis of promoter competition for the shared SE and show that absence of Myh1 and Myh4 leads to increased expression of Myh2, Myh8 or Myh13 in specific subregions of limb muscles, suggesting that competition between the promoters leads to the activation of a single gene at the locus. Combined with their past work (Dos Santos et al, 2020) these results suggest that each nucleus bi-allelically expresses only one gene at the locus, the other genes being silent. These experiments further suggest that the super enhancer at the locus forms multimolecular assemblies by liquid-liquid phase separation, allowing aggregation of the transcriptional machinery at a single promoter and that the fMyh super enhancer is responsible for the non-stochastic robust coordinated fMyh gene expression in the hundred of body myonuclei present in adult myofibers.
Legend: The fMyh Super Enhancer is composed of seven enhancer elements (1-7) recruiting TF and cofactors allowing the nuclear formation of a phase separation condensate in myonuclei and allowing robust fMyh expression, adapted from (Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934-947). Left, during fetal development the SE contacts Myh8 allowing its robust expression. Right, during post-natal development the transcription factors and cofactors expressed in distinct fast subtypes myonuclei favorize contacts between the SE and a single promoter at the locus. In those myonuclei robust bi-allelic expression of adult Myh1, Myh2 or Myh4 is achieved, while transcription of the other genes is silenced.
Matthieu Dos Santos, Stéphanie Backer, Frédéric Auradé, Matthew Man-Kin Wong, Maud Wurmser, Rémi Pierre, Francina Langa, Marcio Do Cruzeiro, Alain Schmitt, Jean-Paul Concordet, Athanassia Sotiropoulos, F.Jeffrey Dilworth, Daan Noordermeer, Frédéric Relaix, Iori Sakakibara, Pascal Maire. Nature Communications. Online 24 Feb. 2022. DOI: 10.1038/s41467-022-28666-1
This work was financed by ANR and AFM grants.