The TFAP2C transcription factor has been shown to downregulate transcription of the universal cell cycle inhibitor p21 cip (CDKN1A). In examining the mechanism of TFAP2C-mediated repression, we have identified a ternary complex at the proximal promoter containing TFAP2C, the oncoprotein Myc, and the trimethylated lysine 4 of histone H3 (H3K4me3) demethylase, KDM5B. We demonstrated that while TFAP2C and Myc can downregulate the CDKN1A promoter independently, KDM5B acts as a corepressor dependent on the other two proteins. All three factors collaborate for optimal CDKN1A repression, which requires the AP-2 binding site at ؊111/؊103 and KDM5B demethylase activity. Silencing of TFAP2C-KDM5B-Myc led to increased H3K4me3 at the endogenous promoter and full induction of CDKN1A expression. Coimmunoprecipitation assays showed that TFAP2C and Myc associate with distinct domains of KDM5B and the TFAP2C C-terminal 270 amino acids (aa) are required for Myc and KDM5B interaction. Overexpression of all three proteins resulted in forced S-phase entry and attenuation of checkpoint activation, even in the presence of chemotherapy drugs. Since each protein has been linked to poor prognosis in breast cancer, our findings suggest that the TFAP2C-Myc-KDM5B complex promotes cell cycle progression via direct CDKN1A repression, thereby contributing to tumorigenesis and therapy failure.

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IntroductionAP-2α is a transcription factor implicated in the regulation of differentiation and proliferation in certain tissues, including the mammary gland. In breast tumours, continued expression of AP-2α has been correlated with a better prognosis, but this is hard to reconcile with a reported role in the upregulation of the ERBB2 oncogene. The existence of TFAP2A isoforms, deriving from alternative first exons and differing in their N-terminal sequence, has been described in some mammals, but their relative abundance and activity has not been investigated in the human breast.MethodsExpression levels of four TFAP2A isoforms were assayed at the level of RNA and protein (via the generation of isoform-specific antibodies) in a panel of breast tumour cell lines and in tissue from normal breast and primary tumour samples. Expression constructs for each isoform were used in reporter assays with synthetic and natural promoters (cyclin D3 and ERBB2) to compare the activation and repression activity of the isoforms.ResultsWe demonstrate that the two isoforms AP-2α 1b and AP-2α 1c, in addition to the originally cloned, AP-2α 1a, are conserved throughout evolution in vertebrates. Moreover, we show that isoform 1c in particular is expressed at levels at least on a par with the 1a isoform in breast epithelial lines and tissues and may be more highly expressed in tamoxifen resistant tumours. The isoforms share a similar transactivation mechanism involving the recruitment of the adaptors CITED2 or 4 and the transactivators p300 or CBP. However, isoform 1b and 1c are stronger transactivators of the ERBB2 promoter than isoform 1a. In contrast, AP-2α 1a is the only isoform able to act as a repressor, an activity that requires an intact sumoylation motif present within the N-terminus of the protein, and which the other two isoforms lack.ConclusionsOur findings suggest that TFAP2A isoforms may be differentially regulated during breast tumourigenesis and this, coupled with differences in their transcriptional activity, may impact on tumour responses to tamoxifen therapy. These data also have implications for the interpretation of tumour studies that seek to correlate outcomes with TFAP2A expression level.