A fast Myosin super enhancer dictates muscle fiber phenotype through competitive interactions with Myosin genes

Matthieu Dos Santos,Francina Langa,Daan Noordermeer,Iori Sakakibara,Alain Schmitt,Frédéric Relaix,Frédéric Auradé,Rémi Pierre,Pascal Maire,Marcio Do Cruzeiro,Athanassia Sotiropoulos,Jean-Paul Concordet,F Jeffrey Dilworth,Matthew Man-Kin Wong,Maud Wurmser,Stéphanie Backer

doi:10.1038/s41467-022-28666-1

Abstract

The contractile properties of adult myofibers are shaped by their Myosin heavy chain isoform content. Here, we identify by snATAC-seq a 42 kb super-enhancer at the locus regrouping the fast Myosin genes. By 4C-seq we show that active fast Myosin promoters interact with this super-enhancer by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the super-enhancer recapitulates the endogenous spatio-temporal expression of adult fast Myosin genes. In situ deletion of the super-enhancer by CRISPR/Cas9 editing demonstrates its major role in the control of associated fast Myosin genes, and deletion of two fast Myosin genes at the locus reveals an active competition of the promoters for the shared super-enhancer. Last, by disrupting the organization of fast Myosin, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

Highlights

The contractile properties of adult myofibers are shaped by their Myosin heavy chain isoform content
We further show by CRISPR/Cas[9] editing that in situ deletion of this 42 kb super enhancers (SE) region prevents expression of fetal Myh[8] and adult fast Myh (fMyh) genes at the locus leading to fetal myofibers devoid of sarcomeres, unable to contract and precluding breathing at birth
To identify the regulatory elements controlling the expression of fMyh genes, we performed snATAC-seq experiments with nuclei isolated from adult fast quadriceps and slow soleus[10]

Summary

Introduction

The contractile properties of adult myofibers are shaped by their Myosin heavy chain isoform content. 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[1] These different myofibers can be classified as slow or fast subtypes that selectively express genes responsible for their specific properties[2,3,4]. As identity genes expressed at high levels in specific fast myofiber subtypes, fMyh genes are good candidates to be controlled by a SE in the skeletal muscle lineage. To characterize the cis-regulatory elements required for the complex regulation of the specific fMyh genes we performed snATAC-seq and 4C-seq experiments with adult skeletal muscles and identified a 42-kb 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. Analysis of the phenotype of all forelimbs and hindlimbs muscles in genetic perturbations within the fMyh locus reveals different categories of muscle susceptibility reminiscent of the selective muscle vulnerability observed in different neuromuscular diseases

Methods

Results

Conclusion