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. 2013 Nov 4;4(11):2154–2165. doi: 10.18632/oncotarget.1367

Figure 2. Methylation inhibits HOXA2 promoter activity by impairing binding of the transcription activator, p300.

Figure 2

(A)Three HOXA2 promoter deletion clones and two putative p300 binding sites (p300-1 and p300-2) are indicated. Different HOXA2 promoter deletion clones were transfected into TW02 cells (left panel) or different HOXA2 promoter deletion clones and the pCMV/p300 expression clone were co-transfected into 293T cells (right panel). Relative luciferase activities were detected 48hr post-transfection and all the luciferase reporter activities were normalized with respect to renilla activity. The expression levels of p300 and actin (internal control) were examined by Western blotting. (B) ChIP assays were performed with anti-p300 antibody or isotype IgG, using HK1 nuclear extracts treated with or without 10μM 5'Aza. Q-PCR was used to amplify p300-1 (−171~−55), p300-2 (−435~−254), and a downstream region lacking a p300 binding site (−110~+208). (C) DNA pull-down was used to analyze the binding affinity of exogenous Flag-tagged p300 (p300-3F) to wild-type (WT), methylated (ME), mutated (MT) HOXA2 p300-1 and a three copies of p300 consensus (P) biotinylated probes. Anti-Flag antibody was used to examine the amount of bound p300-3F in the immunoprecipitates and 5% inputs. Methylated cytosine is indicated by “m” above C, and mutated sequences were underlined.