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. 2009 Jul 6;29(18):4982–4993. doi: 10.1128/MCB.00285-09

FIG. 6.

FIG. 6.

Collaboration between PADI4 and HDAC1 in generating a repressive pS2 chromatin environment. (A) Specific-RNAi-encoding vectors decrease HDAC1 expression substantially and PADI4 expression moderately. MDA::ERα cells were stably infected with the pRS/control vector (RNAi), the pRS/PADI4 vector (RNAi PADI4), the pRS/HDAC1 vector (RNAi HDAC1), or both pRS/PADI4 and pRS/HDAC1 (RNAi PADI4 + RNAi HDAC1). After selection the cells were harvested, and quantitative RT-PCR was performed on their mRNA, with primers corresponding to HDAC1 (left panel) or PADI4 (right panel). All transcript levels were normalized with respect to SDHA and then divided by the normalized level recorded for control cells. Values are means of three independent experiments. (B) Chromatin was prepared from MDA::ERα cells expressing a control RNAi, PADI4 RNAi, HDAC1 RNAi, or both PADI4 RNAi and HDAC1 RNAi (RNAi PADI4 + RNAi HDAC1) and treated with 10−8 M E2. Cross-linked chromatin samples were immunoprecipitated with antibodies to HDAC1 (lanes 1 to 4), PADI4 (lanes 5 to 8), or CitH3 [H3R(17+2+8) citrullinated] (lanes 9 to 12). The amount of immunoprecipitated pS2 promoter was quantified by real-time PCR on the basis of the threshold cycle value (CT). Calculation details were done as previously described (20) (see also Materials and Methods). The data are presented as the means ± the standard errors of four independent experiments, and asterisks indicate statistical significance as determined by Dunnett and Mann-Whitney comparisons (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) Chromatin samples were subjected, as described in panel B, to immunoprecipitation with antibodies raised against AcH3 (lanes 1 to 4), dimeH3R17 (lanes 5 to 8), or dimeH4R3 (lanes 9 to 12). The immunoprecipitated pS2 promoter was quantified by real-time PCR on the basis of the CT for each PCR (see reference 20 and Materials and Methods for calculation details). The relative enrichment values shown are means of four independent experiments. Asterisks indicate the statistical significance as determined by Dunnett and Mann-Whitney comparisons (*, P < 0.05; **, P < 0.01; ***, P < 0.001).