FIGURE 5.

HDAC10 binds to the promoter regions of MMP2 and -9 and deacetylates histones H3 and H4 in these regions. A–J, results of ChIP assay. A and B, HDAC10 binds to MMP2 and -9 promoter regions. Top, diagrams of MMP2 and -9 promoter regions. The transcriptional factor binding sites and the primers designed are shown. Bottom, the amount of DNA precipitated by either control IgG or anti-HDAC10 antibody was expressed as a percentage of the total input genomic DNA. C and D, HDAC10 decreases acetylation of histones H3 and H4 in MMP2 and -9 promoter regions. Acetylated histone H3 and H4 antibodies were used to precipitate DNA in ChIP assay. Primers associated with the transcription factor binding sites in MMP2 and -9 promoter regions were used for quantitative real time PCR. The data are from three independent experiments. E and F, reduced acetylation of H3 and H4 blocks the association of RNA polymerase II to the transcription factors binding sites. DNA of HeLa cells in the control group and HDAC10 overexpression group was precipitated by anti-RNA polymerase II (pol II) antibody in a ChIP assay. G and H, HeLa cells overexpressing full-length HDAC10, DAC domain, and LRD were collected and analyzed using a ChIP assay. DNA was precipitated by acetylated histone H3 and H4 antibodies. Primers recognizing MMP2 (−1298 to −1073) (G) and MMP9 (−670 to −486) (H) were used to analyze the amount of DNA precipitated. Columns, results of triplicates; error bars, S.D. *, p < 0.05; **, p < 0.01 versus the control group. I and J, co-immunoprecipitation (IP) assay. Cell lysates of the control and HDAC10 overexpression groups were incubated with anti-FLAG-agarose beads. The immunoprecipitates were analyzed by Western blotting with antibodies recognizing FLAG, AP1, or p65. CREB, cAMP-response element-binding protein. IB, immunoblot.