A NOVEL E2F BINDING-PROTEIN WITH MYC-TYPE HLH MOTIF STIMULATES E2F-DEPENDENT TRANSCRIPTION BY FORMING A HETERODIMER

The human embryonal carcinoma cells NEC14 can be induced to differenti ate morphologically by the addition of 10(-2) M N, N'-hexamethylene-bi s-acetamide and cease to grow in several days. Transcription factors o f the E2F/DP family have been shown to be closely related to the regul ation of cell proliferation. To analyse cellular proteins which intera ct with E2F in NEC14 cells, cDNA clones encoding E2F binding proteins were isolated from a lambda ZAP II NEC14 cell library with the P-32-la beled GST (Glutathione S-transferase)-E2F-l fusion protein as a probe. One of the clones encodes E2FBP1 which has the helix-loop-helix (HLH) motif, but lacks the basic domain and the zipper structure usually fo und at N- and C-terminal sides to the HLH motif, respectively, The arr angement of amino acids in the helix 1 and helix 2 regions is quite si milar to those of Mxi and Mad, but different from those of E2F-1 and D P-1, Western blot analysis of the immunoprecipitates prepared with ant i-E2FBP1 antibody showed that E2FBP1 associates with both E2F-1 and DP -1 in vivo. E2FBP1 alone has no DNA binding activity, but bind to the E2F site through heterodimerization with E2F-1 but not with DP-1, Alth ough E2FBP1 lacks the transactivation domain, it stimulates E2F site-d ependent transcription in cooperation with E2F-1.