Fig. 5.

Epigenomic analyses indicate that β-catenin promotes MyoD association with chromatin. (A) H3Kac and H3K4me3 ChIP-seq was performed using chromatin from wild-type myoblasts treated with control or Wnt3a-containing media for 24 h. Homer analysis was used to identify the top 10 motifs that are enriched in promoter regions that show increased levels of H3Kac and H3K4me3 (Wnt/L cell≥threefold) after Wnt3a induction (represented as target sequences with motif/total sequences with motif). (B) MyoD ChIP-seq analyses of wild-type and β-catenin-null primary myoblasts treated with Wnt3a medium for 24 h. Gene ontology (GO) analysis was performed on the set of genes showing increased MyoD binding after Wnt3a treatment (Wnt/L cell≥twofold) in wild-type (left) and β-catenin CRISPR-null (right) myoblasts. Wild-type myoblasts show significant enrichment of muscle-associated GO categories; β-catenin-null myoblasts do not show enrichment in any muscle-specific categories. (C) The top 15 motifs (Homer analysis) seen in promoter regions showing increased MyoD binding (Wnt/L cell≥twofold) after Wnt3a treatment of wild-type myoblasts (target sequences with motif/total sequences with motifs). Statistical analysis: multiple t-tests with P<0.05 considered significant. (D,E) Wnt3a increases binding of β-catenin and MyoD to the proximal promoters of the myomaker and myogenin genes, as shown by ChIP-qPCR analysis. Chromatin from wild-type primary myoblasts treated with control or Wnt3a-containing medium for 48 h was used for ChIP with β-catenin (D) or MyoD (E) antibodies. qPCR was used to assess enrichment of the myomaker promoter in both β-catenin- and MyoD-ChIP samples; the myogenin promoter was used as a control for MyoD-ChIP and the Axin2 enhancer was used as a control for β-catenin-ChIP. Data were first normalized to a control non-target locus (rDNA1) and then to the mock ChIP with preimmune IgG, set to a value of 1. n=3. Data are mean±s.e.m. *P<0.05, **P<0.001.