Involvement of histone deacetylases in transcriptional repression of Opn and Col11a2 promoters by EWSR1-DDIT3.

<p>(<b>A</b> and <b>B</b>) Derepression of Opn (<b>A</b>) and Col11a2 (<b>B</b>) promoter activity by HDAC inhibitor, trichostatin A (TSA). C3H10T1/2 cells in duplicate plates were cotransfected with promoter reporter plasmids plus EWSR1-DDIT3 expression vectors. Cells in one plate were assayed for luciferase activity 24 h after treatment with TSA and compared with the cells from the other plate that were not treated with TSA. Luciferase activities from TSA-treated cells relative to those from TSA-untreated cells are shown as fold derepression. Experiments in duplicate were repeated at least three times, and the results are shown as averages ± SE. Asterisks (*) indicate statistical significance (<i>p</i><0.05) calculated by unpaired <i>t</i>-test, with <i>p</i> values of 0.0001 for Opn and 0.0277 for Col11a2. (<b>C</b> and <b>D</b>) Transient ChIP assays using an antibody against HDAC1 or normal IgG. C3H10T1/2 cells were transfected with Opn (<b>C</b>) and Col11a2 (<b>D</b>) luciferase reporter plasmids plus EWSR1-DDIT3 expression vectors. Promoter DNA fragments containing the C/EBP site (open box) were immunoprecipitated with an antibody against HDAC1. Relative values reflecting protein–DNA interactions were calculated by adjusting corresponding signal intensities to those of input levels. Experiments in duplicate were repeated at least three times, and the results are shown as averages below each band. Relative values of immunoprecipitated Opn and Col11a2 promoter fragments significantly increased after EWSR1-DDIT3 overexpression from 0.49 to 1.06 and 0.17 to 0.63, respectively. Asterisks (*) indicate statistical significance (<i>p</i><0.05) calculated by unpaired <i>t</i>-test, with <i>p</i> values of 0.0005 for Opn and 0.0021 for Col11a2. As indicated by the arrows, the forward PCR primers are promoter sequence-specific primers and locate upstream to the C/EBP site (open box), while the reverse PCR primer pGL3 is a plasmid-specific primer.