Cloning and characterization of a GC-box binding protein, G10BP-1, responsible for repression of the rat fibronectin gene.
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.
Oda
E
E
Department of Biological Science and Technology, Science University of Tokyo, Noda-shi, Chiba 278, Japan.
Shirasuna
K
K
Suzuki
M
M
Nakano
K
K
Nakajima
T
T
Oda
K
K
D017500:Adenovirus E1A Proteins / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002460:Cell Line; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005353:Fibronectins / Q000235:genetics*; D016193:G1 Phase; D005786:Gene Expression Regulation; D005838:Genotype; D006147:Guanine; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D016196:S Phase; D012441:Saccharomyces cerevisiae
D017500:Adenovirus E1A Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005353:Fibronectins; C113768:G10BP-1 protein, rat; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D006147:Guanine