Cloning and Characterization of a GC-Box Binding Protein, G10BP-1, Responsible for Repression of the Rat Fibronectin Gene

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, ␣5␤1, 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 G 1 . Expression of FN mRNA is inversely correlated with G10BP-1 expression, and the level decreased steeply during G 1 -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.Fibronectin (FN) is a large glycoprotein of the extracellular matrix that binds to its cell surface receptor, ␣5␤1, a member of the integrin superfamily, as a dimer (2,15,39). FN consists of multiple rodlike domains, and each domain binds to a component of the extracellular matrix such as collagen and heparin and to its receptor (16). The cytoplasmic domain of the receptor binds to bundles of actin filaments known as stress fibers indirectly through binding to the attachment proteins (14). The binding of FN to its receptor at so-called focal contact sites therefore connects the cytoskeleton with the extracellular matrix (3, 17, 21, 39), governing cell shape, adhesion, and movement. FN is also involved in the regulation of cell proliferation and differentiation through organization of the extracellular matrix and cytoskeleton (7, 15).The expression level of FN is closely linked to the growth potential of cells. The level increases when cells cease growing, and senescent cells inevitably express FN at high levels (11,28,33). On the contrary, FN expression is greatly inhibited upon neoplastic transformation (10,20,35), and most tumor cells express ...

Transcriptional repression of the human fibronectin gene in laryngeal squamous cell carcinoma cells

“…This high molecular weight glycoprotein contains 1±3% of total cellular protein (Yamada and Olden 1978), the function of which is primarily adhesive, contributing to both cellcell and cell-substrate interaction (Olden and Yamada 1977;Mautner et al 1977). A major source of interest in FN is the ®nding that its expression level is reduced in most of the tumor cells, facilitating their anchorageindependent growth by still unclari®ed mechanisms (Suzuki et al 1995;Gu and Oliver 1995;Oda et al 1998;Taylor et al 1998;Suzuki et al 1998). Although suppressed expression of cell surface FN is often reported after malignant transformation, no systematic examination of the sequence preceding the initiation codon has speci®cally been performed in tumors of the upper aerodigestive tract so far.…”

Leukemia/Lymphoma-related Factor, a POZ Domain-containing Transcriptional Repressor, Interacts with Histone Deacetylase-1 and Inhibits Cartilage Oligomeric Matrix Protein Gene Expression and Chondrogenesis

“…Yeast one-hybrid screening has proven to be an effective tool to identify DNA-binding proteins (40,41), including the rat fibronectin gene (42) and Fgf3 promoter (43). To identify proteins that interact with the NRE in the promoter region of the COMP gene, we screened a yeast expression cDNA library using a tandem repeat of four NREs as bait.…”

Human Papillomavirus Type 16 E7 Oncoprotein Represses Transcription of Human Fibronectin

“…The adenovirus E1A protein, which shares amino acid sequence similarity with portions of E7, represses rat FN transcription. This repression is mediated by G10BP, a negative regulator of Sp1, and is abrogated by disruption of the pRB-binding domain in E1A (25)(26)(27)38). Although the human and rat FN promoters seem to be activated by Sp1, the two promoters differ in terms of the arrangement and C content of their G-rich sequences.…”