Fibronectin (FN) is an extracellular matrix glycoprotein that serves as a substrate for cell attachment and migration during embryogenesis, organogenesis, metastasis, wound healing, and cellular differentiation. Cells adhere to FN through an integral membrane protein, the fibronectin receptor (FNR) which is composed of two subunits, as an alpha subunit, specific for the FNR, and a beta subunit, shared by a family of extracellular matrix receptors. Regulation of both FN and FNR expression may play a key role in cell placement and migration during development and tissue repair. We have previously demonstrated that expression of both FN and the FNR is stimulated by transforming growth factor-beta (TGF-beta) which also is important for tissue differentiation and wound healing. TGF-beta stimulates expression of the FNR by increasing gene transcription. We propose to isolate and characterize the FNR gene alpha promoter and examine how TGF-beta increases transcription of this promoter. We have isolated a genomic clone and a cDNA encoding the 5' end of the FNR alpha gene, which will be used to identify the transcription start site and promoter of the FNR alpha gene. The promoter will be sequenced and fused to a reporter gene for analysis in transfection experiments. The region(s) in the FNR alpha promoter which mediates the response to TGF-beta will be identified by creating deletions and mutations and analyzing their effect on TGF-beta inducibility in transfection assays. Presumably TGF-beta affects either the synthesis or activity of a nuclear transcription factor(s) that binds to the TGF-beta responsive DNA element(s) in the promoter. The interaction of nuclear factors with TGF-beta responsive elements will be analyzed using DNase I protection and gel retardation assays. TGF-beta treatment may alter the affinity of factors for the TGF-beta responsive element or affect the interaction of factors to interact with other proteins (i.e. RNA polymerase). This will be analyzed using gel retardation assays which provide information on the abundance, affinity, and size of the protein-DNA complex.
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