QUANTITATIVE-ANALYSIS OF THE CONTRIBUTION MADE BY 5'-FLANKING AND 3'-FLANKING SEQUENCES TO THE TRANSCRIPTIONAL REGULATION OF JUNB BY GROWTH-FACTORS

We have identified four DNAase I-hypersensitive regions (DHRs) at the junB locus, DHR1 is located between sequences - 100 and + 250, DHR2 is centered at - 1000, DHR3 at - 1650, and DHR4 at + 2040 relative to th e junB transcriptional start site. Sequence analysis of these DHRs rev ealed two serum response elements at - 1452 and + 2091, two cyclic AMP response elements at + 2071 and + 2116, and a 12-O-tetradecanoylphorb ol-13-acetate (TPA) response element at - 949. To study the contributi on made by these cis-elements to junB transcriptional regulation, we s tably transfected a recombinant mouse junB gene (JBSV4) containing the intact junB coding sequences, 6.3 kb of 5'-flanking DNA, and 2.0 kb o f 3'-flanking DNA into Rat1A cells. The pattern of DHRs identified at the mouse junB locus was re-established at the JBSV4 locus. By directl y comparing JBSV4 and rat junB mRNA levels, we found that these genes were induced to equivalent levels by serum, TPA, cyclic AMP, platelet- derived growth factor, epidermal growth factor, and basic fibroblastic growth factor. These results established that JBSV4 resides in a phys ical environment within chromatin that closely mimics that of the junB locus, and contains the necessary sequence information to recapitulat e the transcriptional regulation of junB. By analysing a series of rec ombinant mouse junB genes containing deletion mutations in 5'-flanking and 3'-flanking sequences, we provide a quantitative assessment of th e contribution these sequences make to junB induction by different reg ulatory agents.