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. 2009 Feb 15;20(4):1120–1131. doi: 10.1091/mbc.E08-07-0759

Figure 4.

Figure 4.

HDAC4 suppresses MSE promoter activity via recruitment to the MSE promoter's MEF2 element. (A) Nuclear-localized HDAC4-3SA suppresses expression from a cotransfected MSE:Luciferase expression vector in C2C12 myotubes. Confluent C2C12 cells were transfected with a CMV:HDAC4-3SA expression vector, MSE:luciferase expression vector, and a CMV:β-gal expression vector that was used for normalization. Cells were differentiated in low serum, and 48 h later they were harvested for luciferase and β-gal assays. Reported MSE promoter activity is luciferase activity normalized to β-gal activity. Error bars are standard deviations, n = 3. (B) The MSE promoter's MEF2 site is required for HDAC4-dependent regulation. C2C12 cells were transfected with wild-type or a MEF2 mutant MSE:luciferase expression vector along with either a control or HDAC4-targeting siRNA and a normalizing CMV:β-gal vector as described above. Samples were analyzed as described in A. (C) Autoradiogram showing that overexpression of MEF2 in HEK293 cells increases binding to the MSE promoter's MEF2 element. HEK293 cells were transfected with increasing amounts of CMV:MEF2 or an empty vector (−) and 48 h later they were harvested and nuclear extracts were prepared. Nuclear extracts with (+) and without (−) overexpressed MEF2 were incubated with a radiolabeled oligonucleotide containing the MSE promoter's MEF2 site in the absence (−) or presence (+) of a 50-fold excess of unlabeled MEF2 oligonucleotide (competitor). Samples were resolved on a native polyacrylamide gel that was then dried and exposed to x-ray film. Shown is a representative autoradiogram. (D) ChIP assay shows that after muscle denervation HDAC4 or overexpressed myc-HDAC4 binds to the MSE promoter's MEF2 site but not the Mgn promoter's MEF2 site. Samples were either 5-d denervated TA muscle or 5-d denervated TA muscle that was electroporated with a myc-HDAC4 expression vector 12 d before denervation. Real-time PCR with primers flanking either the Mgn or MSE promoter's MEF2 site was used to quantify the amount of immunoprecipitated DNA.