CLONING AND CHARACTERIZATION OF A NOVEL BINDING-FACTOR (GMEB-2) OF THE GLUCOCORTICOID MODULATORY ELEMENT

The 21-base pair glucocorticoid modulatory element (GME) of the rat ty rosine aminotransferase gene is the only cis-acting element known to m odulate the transcriptional activity of receptors bound to glucocortic oid response elements. Specifically, the GME increases the activity of complexes bound both by physiological concentrations of glucocorticoi ds, due to a left shift in the dose-response curve, and by saturating concentrations of anti-glucocorticoids. For this reason, the nuclear p rotein(s) that has been demonstrated to bind to the GME is of major in terest as a possible transcription factor with hitherto undescribed pr operties. Subsequent studies indicated that not one but two proteins o f 88 and 67 kDa (= GMEB-1 and -2, respectively) formed a heteromeric c omplex with double-stranded GME oligonucleotides in gel shift assays a nd participated in the expression of GME activity (Oshima, H., Szapary , D., and Simons, S. S., Jr. (1995) J. Biol. Chem. 270, 21895-21910). Here, we report the use of polymerase chain reaction of degenerate oli gonucleotides and 5'- and 3'-rapid amplification of cDNA ends to clone two cDNAs of 2.0 and 1.9 kilobase pairs that probably result hom alte rnative splicing. Both cDNAs encoded open reading frames containing al l four previously sequenced peptides. The longer 2.0-kilobase pair cDN A encoded an open reading frame for an acidic, 529-amino acid protein and afforded a major 67-kDa and a minor 58-kDa protein after in vitro transcription/translation. Both proteins were recognized by a mono-epi topic antibody raised against a peptide of GMEB-2. The in vitro transl ated protein bound to GME DNA in gel shift assays. However, the bindin g to GME DNA increased markedly after mixing with authentic GMEB-1 to give a gel-shifted complex that was similar to that derived hom HTC ce ll cytosol. GMEB-2 shares a unique domain (KDWKR) with proteins derive d from diverse organisms as follows: Drosophila (DEAF-I), rat (Suppres sin), and Caenorhabditis elegans (three unknown open reading frames). Collectively, these data suggest that the 67-kDa GMEB-2 not only is an important factor for the modulation of glucocorticoid receptor bound to glucocorticoid response elements but also may belong to a novel fam ily of transcription factors.