Histone methyltransferase MLL4 controls myofiber identity and muscle performance through MEF2 interaction
Lin Liu, … , Kai Ge, Zhenji Gan
Lin Liu, … , Kai Ge, Zhenji Gan
Published June 16, 2020
Citation Information: J Clin Invest. 2020;130(9):4710-4725. https://doi.org/10.1172/JCI136155.
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Research Article Metabolism Muscle biology

Histone methyltransferase MLL4 controls myofiber identity and muscle performance through MEF2 interaction

Abstract

Skeletal muscle depends on the precise orchestration of contractile and metabolic gene expression programs to direct fiber-type specification and to ensure muscle performance. Exactly how such fiber type–specific patterns of gene expression are established and maintained remains unclear, however. Here, we demonstrate that histone monomethyl transferase MLL4 (KMT2D), an enhancer regulator enriched in slow myofibers, plays a critical role in controlling muscle fiber identity as well as muscle performance. Skeletal muscle–specific ablation of MLL4 in mice resulted in downregulation of the slow oxidative myofiber gene program, decreased numbers of type I myofibers, and diminished mitochondrial respiration, which caused reductions in muscle fatty acid utilization and endurance capacity during exercise. Genome-wide ChIP-Seq and mRNA-Seq analyses revealed that MLL4 directly binds to enhancers and functions as a coactivator of the myocyte enhancer factor 2 (MEF2) to activate transcription of slow oxidative myofiber genes. Importantly, we also found that the MLL4 regulatory circuit is associated with muscle fiber–type remodeling in humans. Thus, our results uncover a pivotal role for MLL4 in specifying structural and metabolic identities of myofibers that govern muscle performance. These findings provide therapeutic opportunities for enhancing muscle fitness to combat a variety of metabolic and muscular diseases.

Authors

Lin Liu, Chenyun Ding, Tingting Fu, Zhenhua Feng, Ji-Eun Lee, Liwei Xiao, Zhisheng Xu, Yujing Yin, Qiqi Guo, Zongchao Sun, Wanping Sun, Yan Mao, Likun Yang, Zheng Zhou, Danxia Zhou, Leilei Xu, Zezhang Zhu, Yong Qiu, Kai Ge, Zhenji Gan

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Figure 5

MLL4 cooperates with MEF2 to activate slow myofiber gene transcription.

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MLL4 cooperates with MEF2 to activate slow myofiber gene transcription. ...
(A and B) Co-IP experiments were performed by cotransfecting HA-MEF2A and Flag-MLL4 in HEK293 cells, as indicated at the top. Antibodies against the HA or Flag epitope were used for co-IP. Extracts (Input) from the HEK293 cells and proteins from the IP were analyzed by immunoblotting. Representative results for co-IP are shown. n = 3 independent experiments. (C) Co-IP results with extracts prepared from C2C12 myotubes using anti-MLL4 antibody or control IgG. Representative results are shown. n = 3 independent experiments. (D) MLL4 and MEF2D ChIP-Seq tracks from myocytes at the Myh7 locus. Two putative conserved MEF2-binding sites within the cis-proximal enhancer region of the Myh7 gene are shown. (E) MLL4 and MEF2 synergistically activate Myh7 gene promoter. The rat Myh7.Luc.3.5k promoter reporter was used in cotransfection studies in HEK293 cells in the presence of expression vectors indicated. Values represent mean (± SEM) firefly/renilla luciferase activity shown as arbitrary units (AU) normalized (=1.0) to vector control. n = 4 independent experiments. (F) Results of transient transfection performed with rMyh7.Luc.3.5k and truncation mutant of rMyh7.Luc.408 in HEK293 cells in the presence of expression vectors indicated. n = 4–5 independent experiments. (G) Left: MLL4 and MEF2D ChIP-Seq tracks from myocyte at the Myh7b locus. Right: putative conserved MEF2-binding site within the mouse Myh7b promoter regions. (H) Top: site-directed mutagenesis was used to abolish the MEF2 response element. Bottom: mMyh7b.Luc.1k (WT) or MEF2mut.mMyh7b.Luc.1k promoter reporters were used in cotransfection studies in HEK293 cells in the presence of expression vectors indicated. n = 5 independent experiments. Values are represented as mean ± SEM. *P < 0.05 vs. corresponding controls; #P < 0.05 compared with MEF2A alone. P values were determined using 1-way ANOVA coupled to a Fisher’s LSD post hoc test.