HDAC4 negatively regulates miR-9-2 promoter by binding Mef2C.

<p>(A) Infection of multipotential L2.3 cells with lentiviral-encoded shRNA specific for HDAC4 leads to increased miR-9 levels. Cultures were infected with lentiviruses and sampled 2 or 3 days later. MiR-9 was assessed using a TaqMan miRNA assay (Life Technologies). Relative Quantity (RQ) was calculated relative to RNU43 endogenous control. *p<0.05 by Student's t-test, compared with 3 days of no virus. (B) Co-immunoprecipitation of Mef2C and HDAC4. A GFP-HDAC4 fusion cDNA was expressed in N2a cells with or without a Mef2C expression clone and immunoprecipitated with antibodies against GFP or MEF2C as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094348#pone.0094348-Li3" target="_blank">[37]</a>. (C) Chromatin immunoprecipitation with anti-HDAC4 enriches Mef2 binding sites upstream of mouse miR-9-2. HDAC4 immunoprecipitated chromatin from homogenized mouse brain was PCR amplified for three predicted Mef2 binding sites (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094348#pone.0094348.s003" target="_blank">Fig. S3</a>). Primer sequences are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094348#pone.0094348.s005" target="_blank">Table S1</a>. Results are expressed as fold increase relative to IgG-immunoprecipitated chromatin (*p<0.05 Student's t-test, compared with IgG).