Fibronectin (FN) is an extracellular matrix glycoprotein which acts as a substrate for cell adhesion and migration during development. FN attaches to cells through a membrane protein, the FN receptor. Antibodies to FN and synthetic peptides which inhibit FN-receptor interaction inhibit gastrulation, block neural crest cell migration, arrest cardiac development and block the fusion of myoblasts to form myotubes. FN and its receptor also appear to be important for lung development. FN expression is regulated by a number of different hormones and growth factors. One of these inducers, cAMP, is important for tissue differentiation and also stimulates synthesis of other extracellular matrix components such as collagen (IV) and laminin. cAMP may then stimulate matrix synthesis during differentiation thereby controlling cell attachment and migration. cAMP simulates FN synthesis by increasing transcription of the gene. The FN gene promoter contains three different cAMP regulatory elements (CREs) which act as binding sites for nuclear transcription factors. These elements differ in affinity for nuclear proteins and they differ functionally in transfection assays. Each CRE exhibited similar cAMP inducibility but only the high affinity CRE stimulated basal expression. The ability to stimulate basal expression was dependent on close proximity of the CRE to a promoter while cAMP inducibility was relatively distance independent. The CREs form multiple nuclear protein-DNA complexes in gel retardation assays. The number and relationship of nuclear proteins in these complexes is not known. We propose to identify proteins in these complexes by photoaffinity crosslinking them to CREs and resolving them by SDS polyacrylamide gel electrophoresis. The relationship between proteins will be examined by peptide mapping. CRE (-260 bp) appears to interact with a protein distinct from the two previously identified CRE binding proteins, CREB and CRE-BP1. CRE (-260) is identical in sequence to the PEA-2 site in the polyoma virus enhancer which appears to be important for control of viral gene expression and DNA replication. Therefore, the previously uncharacterized nuclear protein that interacts with PEA-2 and CRE (-260) could have several functions which may be regulated by cAMP. The CRE (-260) binding protein will be isolated by affinity chromatography and characterized. CRE binding proteins may interact with other nuclear proteins to stimulate transcription, i.e., binding of proteins to a CRE can influence protein binding to the TATA box. We will examine the interaction of FN gene CREs with other FN promoter elements in transfection assays and the interaction between CRE binding proteins and proteins that bind other elements by gel retardation assays. It is then necessary to identify other FN gene promoter elements that are required for basal expression. Such elements will be identified in transfection assays using plasmids with 5'- serial deletions in the FN gene promoter.
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