The level of fibronectin (FN) gene transcription in resting rat 3Y1 cells is very high but decreases steeply after growth stimulation by serum or by the induction of EIA expression. To study the mechanism of this ElA-mediated down-regulation, the 5' flanking regions of the FN gene with various deletions and substitutions were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and introduced into resting 3Y1 cells with ElA expression plasmids. The results indicate that the G10 stretch located from nucleotide position -239 to -230 and two GC boxes from position -105 to -95 and position -54 to -44 are the primary ElA-responsive elements for repression of the FN gene. Two GC boxes also contain a GIO stretch that is interrupted by the presence of an internal C residue. These sequences overlap with the Spl motif GGGCGG. The ElA gene of human adenovirus type 2 (Ad2) and adenovirus type 5 generates two major species of mRNAs with sizes of 13 and 12S that encode proteins of 289 and 243 amino acid residues (289R and 243R). These proteins have identical N-and C-terminal sequences but differ by the presence of an internal 46-amino-acid sequence in the 289R protein. The domain for transactivation of a variety of viral and cellular promoters resides in this internal sequence specific to the 289R protein (2), and the domain for repression of the enhancer-dependent promoters (4,18,46) resides in the first exon common to these proteins. The ElA gene has the ability to transform primary rodent cells in collaboration with a second oncogene (38,53). This transforming function may be closely related to its functions for immortalizing primary cells (11, 23) and inducing cellular DNA synthesis in quiescent cells (1,28,36,43 ElA expression (20). This is in contrast to mouse embryo-derived AKR-2B cells (3), NIH 3T3 cells (40), and the human fibrosarcoma cell line HT-1080 (13), in which expression levels of FN, its receptor, and actin increased coordinately shortly after growth stimulation of quiescent cells by serum. FN is a large glycoprotein of the extracellular matrix which plays a central role in cell anchorage through cell-extracellular matrix interaction (24, 39, 51) and connects intracellular actin filaments to the extracellular matrix by binding to its receptor (7,25). The fact that FN expression is required for cell proliferation is somewhat paradoxical. Cell adhesion to the substratum seems to be required in order for quiescent cells to exit from the Go phase, while the loosening of cell adhesion is required for cell cycle progression transiently stimulated by extracellular proteases (8,45) or by growth factors (21,22). The latter process is mediated by the structural modification of focal contacts via intracellular mediators. The level of FN expression is greatly reduced in v-src-transformed cells (15) and in primary rat kidney cells transformed by the EJ Ha-ras and the ElA genes (26).In the present study, the ElA-responsive elements for repression of FN gene transcription in rat 3Y1 cells were invest...