E2F4-RB AND E2F4-P107 COMPLEXES SUPPRESS GENE-EXPRESSION BY TRANSFORMING-GROWTH-FACTOR-BETA THROUGH E2F BINDING-SITES

Transforming growth factor beta (TGF-beta) causes growth arrest in mos t cell types, TGF-beta induces hypophosphorylation of retinoblastoma s usceptibility gene 1 product (RE), which sequesters E2F factors needed for progression into S phase of the cell cycle, thereby leading to ce ll cycle arrest at G(1), It is possible, however, that the E2F-RB comp lex induced by TGF-beta may bind to E2F sites and suppress expression of specific genes whose promoters contain E2F binding sites, We show h ere that TGF-beta treatment of HaCaT cells induced the formation of E2 F4-RB and E2F4-p107 complexes, which are capable of binding to E2F sit es, Disruption of their binding to DNA with mutation in the E2F sites did not change the expression from promoters of E2F1, B-myb, or HsORC1 genes in cycling HaCaT cells. However, the same mutation stimulated 5 - to 6-fold higher expression from all three promoters in cells treate d with TGF-beta, These results suggest that E2F binding sites play an essential role in the transcription repression of these genes under TG F-beta treatment, Consistent with their repression of TGF-beta-induced gene expression, introduction of E2F sites into the promoter of cycli n-dependent kinase inhibitor p15(INK4B) gene effectively inhibited its induction by TGF-beta, Experiments utilizing Gal4-RB and Gal4-p107 ch imeric constructs demonstrated that either RE or p107 could directly r epress TGF-beta induction of p15(INK4B) gene when tethered to p15(INK4 B) promoter through Gal4 DNA binding sites. Therefore, E2F functions t o bring RE and p107 to E2F sites and represses gene expression by TGF- beta, These results define a specific function for E2F4-RB and E2F4-p1 07 complexes in gene repression under TGF-beta treatment, which may co nstitute an integral part of the TGF-beta-induced growth arrest progra m.