Dg. Phinney et al., QUANTITATIVE-ANALYSIS OF THE CONTRIBUTION MADE BY 5'-FLANKING AND 3'-FLANKING SEQUENCES TO THE TRANSCRIPTIONAL REGULATION OF JUNB BY GROWTH-FACTORS, Oncogene, 9(8), 1994, pp. 2353-2362
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.