Cell-growth regulation of the hamster dihydrofolate reductase gene promoter by transcription factor Sp1

Eur J Biochem. 1997 Oct 1;249(1):13-20. doi: 10.1111/j.1432-1033.1997.00013.x.

Abstract

The dihydrofolate reductase (DHFR) gene (dhfr) promoter contains cis-acting elements for the transcription factors Sp1 and E2F. Given the ability of Sp1 to activate the dhfr promoter, we have evaluated the contribution of Sp1 to the cell-growth regulation of the dhfr gene. Using gel-mobility assays performed with DNA probes from the minimal promoter of the hamster dhfr gene and nuclear extracts from cultured hamster cells (CHO K1) we show that the binding of Sp1 to the dhfr promoter is cell-growth-phase regulated. Accordingly, dhfr transcription and mRNA levels in K1 cells increase upon serum stimulation. Cytological detection of Sp1 by immunofluorescence reveals a decrease of this protein in the process leading to the G0 state, and an increase upon serum stimulation of quiescent cells. These results were confirmed by western blot analysis. It is concluded that Sp1 progressively binds to the hamster dhfr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell cycle. The role of Sp1 in the specific control of dhfr during the cell cycle was confirmed in vivo using cell lines derived from dhfr-negative cells transfected with dhfr plasmids carrying either the wild-type or mutated Sp1-binding or E2F-binding sites in the dhfr minimal promoter.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • CHO Cells
  • Carrier Proteins*
  • Cell Cycle / genetics
  • Cell Cycle Proteins*
  • Cell Division / genetics*
  • Cricetinae
  • DNA Primers / genetics
  • DNA-Binding Proteins*
  • E2F Transcription Factors
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic*
  • RNA, Heterogeneous Nuclear / genetics
  • RNA, Heterogeneous Nuclear / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Retinoblastoma-Binding Protein 1
  • Sp1 Transcription Factor / metabolism*
  • Tetrahydrofolate Dehydrogenase / genetics*
  • Transcription Factors / metabolism
  • Transfection

Substances

  • Carrier Proteins
  • Cell Cycle Proteins
  • DNA Primers
  • DNA-Binding Proteins
  • E2F Transcription Factors
  • RNA, Heterogeneous Nuclear
  • RNA, Messenger
  • Retinoblastoma-Binding Protein 1
  • Sp1 Transcription Factor
  • Transcription Factors
  • Tetrahydrofolate Dehydrogenase