Tissue-specific splicing of a ubiquitously expressed transcription factor is essential for muscle differentiation

Abstract

Alternate splicing contributes extensively to cellular complexity by generating protein isoforms with divergent functions. However, the role of alternate isoforms in development remains poorly understood. Mef2 transcription factors are essential transducers of cell signaling that modulate differentiation of many cell types. Among Mef2 family members, Mef2D is unique, as it undergoes tissue-specific splicing to generate a muscle-specific isoform. Since the ubiquitously expressed (Mef2Da1) and muscle-specific (Mef2Da2) isoforms of Mef2D are both expressed in muscle, we examined the relative contribution of each Mef2D isoform to differentiation. Using both in vitro and in vivo models, we demonstrate that Mef2D isoforms act antagonistically to modulate differentiation. While chromatin immunoprecipitation (ChIP) sequencing analysis shows that the Mef2D isoforms bind an overlapping set of genes, only Mef2Da2 activates late muscle transcription. Mechanistically, the differential ability of Mef2D isoforms to activate transcription depends on their susceptibility to phosphorylation by protein kinase A (PKA). Phosphorylation of Mef2Da1 by PKA provokes its association with corepressors. Conversely, exon switching allows Mef2Da2 to escape this inhibitory phosphorylation, permitting recruitment of Ash2L for transactivation of muscle genes. Thus, our results reveal a novel mechanism in which a tissue-specific alternate splicing event has evolved that permits a ubiquitously expressed transcription factor to escape inhibitory signaling for temporal regulation of gene expression.

MEF2 Transcription Factors Regulate Distinct Gene Programs in Mammalian Skeletal Muscle Differentiation

“…3B). Curiously, contrary to a recent report (8), acute overexpression of MEF2D did not appear to enhance myotube formation in C2C12 cells, even though this adenovirus encodes the musclespecific ␣2 isoform described in that study.…”

“…These 16 genes were distributed among the MEF2 knockdown gene sets. A more recent study (8) looked at the binding enrichment of two MEF2D splice variants in C2C12 cells using ChIP sequencing. These assays yielded 160 genes with significant MEF2D binding on differentiation day 5, of which 126 were found in our MEF2 knockdown gene sets.…”

“…The lack of conservation between the four individual MEF2 isoforms in their carboxyl-terminal transactivation domain and the dramatically different phenotypes of the various MEF2-deficient vertebrate models suggests that individual MEF2 isoforms regulate distinct gene programs in muscle. MEF2 transcriptional function is further complicated by complex tissue-specific and temporal alternative splicing of mRNA transcripts produced from each of the four Mef2 genes (3,7,8).…”

The MEF2–HDAC axis controls proliferation of mammary epithelial cells and acini formation in vitro

“…We have initially demonstrated that the expression of MEF2-target genes is augmented during acini formation. This behavior resembles that under other differentiation contexts, as in skeletal muscle, where the contribution of the axis has been previously studied Sebastian et al, 2013). Activation of MEF2 transcription is parallel to a dramatic downregulation of HDAC7 protein.…”

Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes

“…For example, Mef2d exon α2 is specifically included in heart and muscle and is known to be regulated by the RBFOX family (Singh et al 2014). Inclusion of the α2 exon alters the phosphorylation and interaction partners of MEF2D to activate transcription of key myogenic genes during myogenesis (Sebastian et al 2013). Importantly, previous studies have demonstrated that inclusion of Mef2d exon α2 is dysregulated in heart failure where RBFOX family members are down-regulated (Singh et al 2014;Gao et al 2016).…”