G1 S-REGULATED E2F-CONTAINING PROTEIN COMPLEXES BIND TO THE MOUSE THYMIDINE KINASE GENE PROMOTER/

By performing DNase I footprint analysis, we had identified three dist inct protein binding sequences (MT1, MT2, and MT3) located on the mous e thymidine kinase (TK) upstream promoter (Dou, Q.-P., Fridovich-Keil, J. L., and Pardee, A. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,11 57-1161). Here we report that MT2 includes an E2F-like binding site (G TTCGCGGGCAAA), as shown by the following evidence. (i) Mt2 bound speci fically to an affinity-purified fusion human E2F protein. (ii) Both MT 2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to simi lar or identical nuclear protein complexes. (iii) Formation of both th ese DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-spe cific complex (E2F.G0/G1) was replaced by an S phase-specific complex( es) (E2F.S), whereas ''free'' E2F increased after the G1/S transition. (iv) Pulse inhibition of protein synthesis with cycloheximide interch anged these complexes with similar kinetics. (v) When MT2-shifted E2F. G0/Gl, E2F.S, and free E2F were eluted and analyzed by Western blot as say using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositi ons of these MT2-bound complexes were also investigated. Studies using specific antibodies revealed that p107, a retinoblastoma-like protein , was present in both E2F.G0/G1 and E2F.S, whereas cyclin E.cyclin A.c dk2 were only present in E2F.S complex(es). These data suggest that re moval of the p107-containing E2F.G0/Gl complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent t ranscription of the mouse TK gene.