Contrasting roles for MyoD in organizing myogenic promoter structures during embryonic skeletal muscle development

Abstract

Background Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage-specific genes. The lineage-determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation-specific genes is established remains unclear. Results Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co-activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification prior to myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co-immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP. Conclusion MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo.

MyoD phosphorylation on multiple C terminal sites regulates myogenic conversion activity

“…MyoD temporally orchestrates expression of multiple subsets of genes during the specification and differentiation stages of myogenesis [19]. Additionally, MyoD has been found to bind at sites throughout the genome where it induces histone modifications rather than gene expression [20].…”

“…Thus, for MyoD, DNA binding affinity does not always correlate with transcriptional activation and instead, the timing and location of cofactor association may be the critical determinant for temporal and spatial activity. For example, genome-wide analysis has shown that MyoD transcriptional activity is temporally regulated through a direct repression of promoter-bound MyoD [19]. A comparison of the genome-wide promoter occupancy of both WT and phospho-mutant MyoD may reveal clues as to potential differential phospho-regulation of distinct downstream MyoD targets.…”

Temporal regulation of chromatin during myoblast differentiation

“…Interestingly, while genetic knockout studies confirm a role for Prmt5 in post-natal differentiation, they also show that Prmt5 is not required for embryonic skeletal muscle differentiation (85). However, molecular evidence demonstrates both Prmt5 and dimethylated H3R8 on the myogenin promoter in isolated somites dissected from E8.25-E9.5 embryos (86), suggesting that Prmt5 and H3R8 dimethylation are likely contributing to embryonic gene activation even if not absolutely required.…”

“…This is facilitated, at least in part, by the continued association of MyoD with HDAC proteins at promoters that are not expressed until later in differentiation, even though other differentiation-specific genes are no longer bound by HDACs and co-repressors and are being actively transcribed (86, 91, 92). The functionality of this interaction was revealed by experiments demonstrating that ectopic expression of myogenin and Mef2D prevented HDAC association with promoters expressed at later times and resulted in premature expression of these genes (91).…”

“…Brg1 is essential for driving proliferating myoblasts into differentiation (108) and via ChIP has been implicated as being present at myogenic gene regulatory sequences in developing somites and limb buds (86, 92). Mechanistically, the alteration of chromatin structure by SWI/SNF enzymes at differentiation-specific genes results in stable interaction of MyoD and Mef2 transcription factors, and likely facilitates the binding of additional transcription factors (46, 92).…”