FIGURE 8.

PLZF-RARα represses transcription of CDKN1A epigenetically by histone deacetylation and DNA methylation. A, structure of the human CDKN1A gene promoter. The arrows indicate the locations of the qChIP-PCR primer binding sites. B, qChIP assays showing PLZF-RARα binding at the endogenous CDKN1A proximal promoter using an antibody against RARα. Cells were transfected with the PLZF-RARα expression vector and immunoprecipitated (IP) with an anti-RARα antibody. IgG, control ChIP antibody. C and D, qChIP-PCR assays showing histone modifications at the endogenous CDKN1A proximal promoter. Cells were transfected with the PLZF-RARα expression vector and lysates were immunoprecipitated with the indicated antibodies (IgG, Ac-H3, Ac-H4, H3K4-Me3, or H3K9-Me3). E, Me-DIP (methylated DNA ChIP) assays showing increased DNA methylation at the endogenous CDKN1A proximal promoter following ectopic PLZF-RARα expression. HEK293 cells were transfected with a PLZF-RARα expression vector, lysates were immunoprecipitated (IP) with the antibody recognizing methylated DNA, and precipitated DNA was amplified using the primers indicated in A. F, bisulfite sequencing analysis of the methylated CDKN1A promoter. The proximal promoter sequences of CDKN1A, with potentially methylated CpG sites are shown in gray ovals, and the Sp1 binding GC-boxes shown above. Open ovals, unmethylated CpG; filled ovals, methylated CpG. G, co-immunoprecipitation of PLZF-RARα, MBD3, NuRD (MTA2), DNMT1, DNMT3b, and HP1. Cell lysates from either HEK293 cells transfected with a pcDNA3 vector or a PLZF-RARα expression vector were immunoprecipitated (IP) using anti-PLZF, anti-RARα or anti-IgG antibodies and analyzed by Western blot (WB) with the indicated antibodies. H, qChIP assays showing the proximal promoter binding of PLZF-RARα, MBD3, the NuRD-HDAC3 complex, DNMT1/3b and HP1 proteins in HEK293 cells transfected with the PLZF-RARα expression vector. *, p < 0.05; N.S., not significant; t test.