G1/S-regulated E2F-containing protein complexes bind to the mouse thymidine kinase gene promoter

J Biol Chem. 1994 Jan 14;269(2):1306-13.

Abstract

By performing DNase I footprint analysis, we had identified three distinct protein binding sequences (MT1, MT2, and MT3) located on the mouse 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, 1157-1161). Here we report that MT2 includes an E2F-like binding site (GTTCGCGGGCAAA), as shown by the following evidence. (i) MT2 bound specifically to an affinity-purified fusion human E2F protein. (ii) Both MT2 and an authentic E2F site (TTTCGCGCGCTTT) bound specifically to similar or identical nuclear protein complexes. (iii) Formation of both these DNA-protein complexes were cell cycle-dependent: a G0/G1 phase-specific 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 interchanged these complexes with similar kinetics. (v) When MT2-shifted E2F.G0/G1, E2F.S, and free E2F were eluted and analyzed by Western blot assay using a specific antiserum to human E2F-1, two forms of murine E2F (62 and 66 kDa) were observed from all three complexes. The compositions 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.cdk2 were only present in E2F.S complex(es). These data suggest that removal of the p107-containing E2F.G0/G1 complex, a candidate repressor, from the MT2 site in late G1 may be essential for S phase-dependent transcription of the mouse TK gene.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Binding, Competitive
  • Carrier Proteins*
  • Cell Cycle Proteins*
  • Cell Cycle*
  • Consensus Sequence
  • Cyclins / metabolism
  • DNA-Binding Proteins / metabolism*
  • E2F Transcription Factors
  • E2F1 Transcription Factor
  • Gene Expression Regulation, Enzymologic*
  • Macromolecular Substances
  • Mice
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism
  • Promoter Regions, Genetic*
  • Protein Binding
  • RNA, Messenger / genetics
  • Recombinant Fusion Proteins
  • Retinoblastoma-Binding Protein 1
  • Thymidine Kinase / genetics*
  • Transcription Factor DP1
  • Transcription Factors / metabolism*

Substances

  • Arid4a protein, mouse
  • Carrier Proteins
  • Cell Cycle Proteins
  • Cyclins
  • DNA-Binding Proteins
  • E2F Transcription Factors
  • E2F1 Transcription Factor
  • E2f1 protein, mouse
  • Macromolecular Substances
  • Nuclear Proteins
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Retinoblastoma-Binding Protein 1
  • Transcription Factor DP1
  • Transcription Factors
  • Thymidine Kinase