Cloning and characterization of a GC-box binding protein, G10BP-1, responsible for repression of the rat fibronectin gene

Mol Cell Biol. 1998 Aug;18(8):4772-82. doi: 10.1128/MCB.18.8.4772.

Abstract

Fibronectin (FN) is an extracellular matrix protein that connects the extracellular matrix to intracellular cortical actin filaments through binding to its cell surface receptor, alpha5beta1, a member of the integrin superfamily. The expression level of FN is reduced in most tumor cells, facilitating their anchorage-independent growth by still unclarified mechanisms. The cDNA clone encoding G-rich sequence binding protein G10BP-1, which is responsible for repression of the rat FN gene, was isolated by using a yeast one-hybrid screen with the G10 stretch inserted upstream of the HIS3 and lacZ gene minimal promoters. G10BP-1 comprises 385 amino acids and contains two basic regions and a putative zipper structure. It has the same specificity of binding to three G-rich sequences in the FN promoter and the same size as the G10BP previously identified in adenovirus E1A- and E1B-transformed rat cells. Expression of G10BP-1 is cell cycle regulated; the level was almost undetectable in quiescent rat 3Y1 cells but increased steeply after growth stimulation by serum, reaching a maximum in late G1. Expression of FN mRNA is inversely correlated with G10BP-1 expression, and the level decreased steeply during G1-to-S progression. This down regulation was strictly dependent on the downstream GC box (GCd), and base substitutions within GCd abolished the sensitivity of the promoter to G10BP-1. In contrast, the level of Sp1, which competes with G10BP for binding to the G-rich sequences, was constant throughout the cell cycle, suggesting that the concentration of G10BP-1 relative to that of Sp1 determines the expression level of the FN gene. Preparation of glutathione S-transferase pulldowns of native proteins from the cell extracts containing exogenously or endogenously expressed G10BP-1, followed by Western blot analysis, showed that G10BP-1 forms homodimers through its basic-zipper structure.

MeSH terms

  • Adenovirus E1A Proteins / genetics
  • Adenovirus E1A Proteins / metabolism
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Cycle Proteins*
  • Cell Line
  • Cell Line, Transformed
  • Cloning, Molecular
  • DNA, Complementary
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Dimerization
  • Fibronectins / genetics*
  • G1 Phase
  • Gene Expression Regulation
  • Genotype
  • Guanine
  • Molecular Sequence Data
  • Promoter Regions, Genetic
  • Rats
  • Rats, Inbred F344
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • S Phase
  • Saccharomyces cerevisiae

Substances

  • Adenovirus E1A Proteins
  • Cell Cycle Proteins
  • DNA, Complementary
  • DNA-Binding Proteins
  • Fibronectins
  • G10BP-1 protein, rat
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Guanine