Chromatin Immunoprecipitation (ChIP) analysis of the MMP-9 promoter.

<p>(A) HDAC1 recruitment on MMP-9 promoter in response to IFNβ. HT1080 cells were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. ChIP assays, using antibodies specific to p65, histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR performed within the linear range of amplification (determined to be 38 cycles). Input chromatin (1%) was removed from samples prior to immunoprecipitation and subjected to PCR to control for any variation in starting material. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of four independent experiments. (B) HDAC1 recruitment on MMP-9 promoter in response to IFNβ is prevented in vivo by rendering AP-1 site non-functional. HT1080 cells were stably transfected with −660 bp MMP-9 promoter luciferase reporter constructs, either with or without proximal AP-1 site mutated. Resulting stable lines were treated with IFNβ for 12 hrs, PMA for 4 hrs, or both. Chromatin immunoprecipitation (ChIP) assays, using antibodies specific to histone deacetylase (HDAC)-1, or acetylated histone-3 (aH3) were performed, followed by PCR. PCR primers were designed to include the region of interest of the MMP-9 promoter and a section of the luciferase reporter, to select against amplification of endogenous wild type MMP-9 promoter region. Band intensities were normalized to input and the relative band intensities were calculated. The relative band intensities are shown under each band with respect to the strongest band being represented as 1. Data shown is representative of three independent experiments. (C) AP-1 binding to the proximal site is required for HDAC1 induced repression of MMP-9 HT1080 cells were co-transfected in 6-well dishes in triplicates with either −154 MMP-9 promoter deletion construct containing a point mutation in the proximal AP-1 site, which renders the binding site non-functional, or with wild type −154 MMP-9 construct and varying amounts of a HDAC1 expression construct. Cell extracts were assayed for firefly luciferase activities, and normalized by protein concentration to account for any cell concentration differences between samples. P-values were calculated using statistical analysis software. As indicated above the bars, * indicates P-values of significance (50 ng = 0.000698 and 100 ng = 0.000885, significant difference) and # indicates P-values of no significance (50 ng = 0.0763 and 100 ng = 0.882, no significant difference) with significant values being <0.01.