Figure 7. Phosphorylation of MyoD by p38-γ directs the assembly of a repressive transcriptional complex at the Myogenin promoter. (A and B) Endogenous H3K9-2me (A) and endogenous KMT1A (B) ChIP assays on a differentiating (24 h) C2C12 myoblast and 10T1/2 fibroblast extracts expressing p38-γ, MKK6EE and p38-γ, or empty vector. MyoDwt and MyoDmut were expressed as indicated for 10T1/2 fibroblasts ChIPs. (C) Nuclear extracts from proliferating 293T cells expressing wild-type or mutant MyoD in the presence or absence of Myc-KMT1A (or Flag-KMT1A; not depicted) were immunoprecipitated for MyoD and immunoblotted for KMT1A. Equal exposure times are shown. Input immunoblots are found in Fig. S3 H. Fold change in the relative association of KMT1A with wild-type versus mutant MyoD is also shown after quantification and normalization to immunoprecipitated MyoD. Error bars represent ±SEM for n = 3. Asterisk denotes significance (P < 0.02). (D) Endogenous KMT1A ChIP assays from proliferating p38-γ+/+ and p38-γ−/− satellite cell–derived myoblasts. PCR was performed on the Igh enhancer as a control. Fold changes for each condition relative to empty vector (C2C12), wild-type MyoD (10T1/2), or p38-γ+/+ (satellite cells) are shown below representative gels. Error bars represent ±SEM for n = 3. H3K9-2me, dimethylated histone H3 Lys9; MyoDwt, wild-type MyoD; MyoDmut, MyoD [S199A/S200A] mutant.
Figure 7.

Phosphorylation of MyoD by p38-γ directs the assembly of a repressive transcriptional complex at the Myogenin promoter. (A and B) Endogenous H3K9-2me (A) and endogenous KMT1A (B) ChIP assays on a differentiating (24 h) C2C12 myoblast and 10T1/2 fibroblast extracts expressing p38-γ, MKK6EE and p38-γ, or empty vector. MyoDwt and MyoDmut were expressed as indicated for 10T1/2 fibroblasts ChIPs. (C) Nuclear extracts from proliferating 293T cells expressing wild-type or mutant MyoD in the presence or absence of Myc-KMT1A (or Flag-KMT1A; not depicted) were immunoprecipitated for MyoD and immunoblotted for KMT1A. Equal exposure times are shown. Input immunoblots are found in Fig. S3 H. Fold change in the relative association of KMT1A with wild-type versus mutant MyoD is also shown after quantification and normalization to immunoprecipitated MyoD. Error bars represent ±SEM for n = 3. Asterisk denotes significance (P < 0.02). (D) Endogenous KMT1A ChIP assays from proliferating p38-γ+/+ and p38-γ−/− satellite cell–derived myoblasts. PCR was performed on the Igh enhancer as a control. Fold changes for each condition relative to empty vector (C2C12), wild-type MyoD (10T1/2), or p38-γ+/+ (satellite cells) are shown below representative gels. Error bars represent ±SEM for n = 3. H3K9-2me, dimethylated histone H3 Lys9; MyoDwt, wild-type MyoD; MyoDmut, MyoD [S199A/S200A] mutant.

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