E2F-HDAC complexes negatively regulate the tumor suppressor gene ARHI in breast cancer.

ARHI is a maternally imprinted tumor suppressor gene whose expression is markedly downregulated in breast cancer. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased lysine 9 methylation of histone H3. An ARHI promoter segment that spanned bases -420 to +58 (designated the P2 region) exhibits significantly higher promoter activity in normal cells than in cancer cells. To better understand the molecular mechanisms contributing to this differential transcriptional activity, we sought to identify transcription factors that bind to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies can supershift the A2-protein complexes, whereas anti-E2F2 and 6 antibodies cannot, demonstrating that the A2 fragment interacts with specific members of the E2F family proteins. When compared with normal breast epithelial cells, breast cancer cells have significantly elevated expression of E2F1, 4 and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3 cells were cotransfected with an ARHI/luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4. While the retinoblastoma protein, pRB, alone had no effect on ARHI promoter activity, repression by E2F1, but not E2F4, was enhanced by the coexpression of pRB. Taken together, our results suggest that E2F1, 4 and their complexes with HDAC play an important role in downregulating the expression of the tumor suppressor gene ARHI in breast cancer cells.