XB-ART-50022Development. September 1, 2014; 141 (17): 3378-87.
myomiR-dependent switching of BAF60 variant incorporation into Brg1 chromatin remodeling complexes during embryo myogenesis.
Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF60a or BAF60b expression and are rescued by morpholino knockdown of BAF60a or BAF60b. This suggests that myomiRs contribute to select BAF60c for incorporation into the Brg1 complex by specifically targeting the alternative variants BAF60a and BAF60b during embryo myogenesis, and reveals that interactions between tissue-specific non-coding RNAs and chromatin remodeling factors confer robustness to mesodermal lineage determination.
PubMed ID: 25078649
PMC ID: PMC4199139
Genes referenced: banf1 myog smarca4 smarcd1 smarcd2 smarcd3
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|Fig. 1. Expression of BAF60 variants and Brg1/BAF complex composition during somite development. (A) Immunohistochemistry on somite sections of HH12 chick embryos illustrates the expression of all BAF60 variants (green) in epithelial somites. (B) Immunohistochemistry on somite sections of HH20 embryos shows expression of all BAF60 variants in the myotome. DAPI stain (blue) shows cell nuclei. Scale bars: 50 μm. (C) qPCR of HH12 and HH20 somites shows that relative amounts of BAF60a and BAF60b transcripts are decreasing, whereas transcripts encoding BAF60c are increasing during development. (D) CoIP using anti-Brg1 antibody and protein isolated from HH12 or HH20 somites. The amount of BAF60a and BAF60b protein bound to Brg1 decreases over time, whereas the amount of BAF60c variant associated with Brg1 increases in differentiating somites. Input samples are shown. dm, dermomyotome; my, myotome; nc, notochord; nt, neural tube; so, somite.|
|Fig. 5. Knockdown of BAF60a or BAF60b restores myogenesis after miRNA inhibition. (A) Somites injected with AM133 were electroporated with FITC-labeled control-MO or BAF60a-MO, as indicated. In situ hybridization for myogenin transcripts (purple) and detection of FITC-MO (red) shows that myogenic differentiation is inhibited in the presence of AM133 plus control-MO (top row, arrows and arrowheads); whole-mount and section are shown. Co-electroporation of BAF60a-MO with AM133 rescued the expression of myogenin (bottom row). (B) Somites injected with AM1 plus AM206 were electroporated with FITC-labeled control-MO or BAF60b-MO, as indicated. In situ hybridization for myogenin transcripts (purple) and detection of FITC-MO (red) shows that myogenic differentiation is inhibited in the presence of AM1 and AM206 plus control-MO (top row, arrows and arrowheads). Myogenin expression is rescued when BAF60b-MO is co-electroporated with AM1 and AM206 (bottom row, arrows and arrowheads). Scale bar: 50 µm. (C) Summary of phenotypes observed after AM/MO injections. my, myotome; nt, neural tube; nc, notochord.|
|Fig. 6. Model illustrating the expression and regulation of BAF60 variants in embryonic somites by myomiRs. (A) All BAF60 variants are expressed throughout epithelial somites. The coloring indicates different cell lineages; the ventral half contains chondrogenic progenitors (blue) and the dorsal half contains myogenic progenitors (red). (B) In differentiating somites miR-133, miR-1 and miR-206 are expressed in the myotome (green), which is generated from the edges of the dermomyotome (red), as indicated by arrows. We propose that myomiRs decrease the levels of BAF60a and BAF60b protein available to bind to Brg1, thus allowing an increase in BAF60c to be incorporated into the BAF/Brg1 complex. This switch in complex composition permits activation of myogenic differentiation in embryonic myocytes. The continued presence of high levels of BAF60a and BAF60b variants interferes with myogenic differentiation, presumably by displacing BAF60c from the Brg1 chromatin remodeling complex. ES, epithelial somites; DML, dorsomedial lip; VLL, ventrolateral lip; DM, dermomyotome; MY, myotome; NT, neural tube; NC, notochord; SC, sclerotome.|