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, alpha 5 beta 1, a member of the integrin superfamily. The expression level of FN is reduced in most t umor cells, facilitating their anchorage-independent growth by still u nclarified mechanisms. The cDNA clone encoding G-rich sequence binding protein G10BP-1, which is responsible for repression of the rat FN ge ne, was isolated by using a yeast one hybrid screen with the G10 stret ch inserted upstream of the HIS3 and lacZ gene minimal promoters. G10B P-1 comprises 385 amino acids and contains two basic regions and a put ative zipper structure. It has the same specificity of binding to thre e G-rich sequences in the FN promoter and the same size as the G10BP p reviously identified in adenovirus E1A- and E1B-transformed rat cells. Expression of G10BP-1 is cell cycle regulated; the level was almost u ndetectable in quiescent rat 3Y1 cells but increased steeply after gro wth stimulation by serum, reaching a maximum in late G(1). Expression of FN mRNA is inversely correlated with G10BP-1 expression, and the le vel decreased steeply during G(1)-to-S progression. This down regulati on was strictly dependent on the downstream GC box (GCd), and base sub stitutions within GCd abolished the sensitivity of the promoter to G10 BP-1. In contrast, the level of Spl, which competes with G10BP for bin ding to the G-rich sequences, was constant throughout the cell cycle, suggesting that the concentration of G10BP-1 relative to that of Sp1 d etermines the expression level of the FN gene. Preparation of glutathi one 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 it s basic-zipper structure.