Cloning and Characterization of a Novel Binding Factor (GMEB-2) of the Glucocorticoid Modulatory Element

Abstract

The 21-base pair glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene is the only cis-acting element known to modulate the transcriptional activity of receptors bound to glucocorticoid response elements. Specifically, the GME increases the activity of complexes bound both by physiological concentrations of glucocorticoids, due to a left shift in the dose-response curve, and by saturating concentrations of anti-glucocorticoids. For this reason, the nuclear protein(s) that has been demonstrated to bind to the GME is of major interest as a possible transcription factor with hitherto undescribed properties. Subsequent studies indicated that not one but two proteins of 88 and 67 kDa ‫؍(‬ GMEB-1 and -2, respectively) formed a heteromeric complex with double-stranded GME oligonucleotides in gel shift assays and participated in the expression of GME activity (Oshima, H., Szapary, D., and Simons, S. S., Jr. (1995) J. Biol. Chem. 270, 21893-21910). Here, we report the use of polymerase chain reaction of degenerate oligonucleotides and 5-and 3-rapid amplification of cDNA ends to clone two cDNAs of 2.0 and 1.9 kilobase pairs that probably result from alternative splicing. Both cDNAs encoded open reading frames containing all four previously sequenced peptides. The longer 2.0-kilobase pair cDNA 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-epitopic antibody raised against a peptide of GMEB-2. The in vitro translated protein bound to GME DNA in gel shift assays. However, the binding to GME DNA increased markedly after mixing with authentic GMEB-1 to give a gel-shifted complex that was similar to that derived from HTC cell cytosol. GMEB-2 shares a unique domain (KDWKR) with proteins derived from diverse organisms as follows: Drosophila (DEAF-I), rat (Suppressin), 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 family of transcription factors.

Differential modulation of glucocorticoid and progesterone receptor transactivation

“…The GME is part of the upstream enhancer region of the endogenous TAT gene and its effects with GR appear to be mediated by two proteins, GMEB-1 and -2, that bind to the GME as a heterooligomer (Oshima et al, 1995). The presence of the GME in other genes is, unfortunately, difficult to predict due to the frequency with which the four base binding sequence occurs (Oshima et al, 1995;Zeng et al, 1998), usually as a tandem repeat with a flexible spacing of from 1 to as much as 15 bp (Christensen et al, 1999). Therefore, the frequency with which the GME occurs in other regulated genes to afford possible differences between PR-and GR-mediated induction is currently unknown.…”

“…2C, 0.44 ± 0.10 fold left-shift, n=4, P = 0.011), the partial agonist activity of Dex-Mes decreases by 46 ± 3% (n = 4, P=0.0008), while the V max of PR-mediated transactivation is further reduced by 77 ± 5% (n = 4, P = 0.0005). Thus, even though the presence of the GME in the regulated reporter construct does not alter PR induction properties, overexpression of GMEB-2, which binds to the GME sequence (Oshima et al, 1995;Zeng et al, 1998) and to GR (Kaul et al, 2000), exerts effects that are GMEB-2 dose-dependent. In summary, two transactivation properties of PR and GR (i.e., EC 50 and partial agonist activity of antisteroids) respond equally to high GMEB-2 concentrations but in opposite manners to low GMEB-2 while the response of V max with each receptor is the same.…”

“…Two proteins, GMEB-1 and -2, are involved in the expression of GME activity with GR (Oshima et al, 1995;Zeng et al, 1998;Kaul et al, 2000). Overexpression of each GMEB, either alone or in combination, causes a right-shift in the GR dose-response curve of a GMEGREtkLUC reporter in CV-1 cells, presumably due to squelching.…”

“…The factors that we selected are: GME, GMEB2, Ubc9, and STAMP. GME, the first modulatory factor identified , is a cis-acting DNA sequence that was isolated from the rat tyrosine aminotransferase (TAT) gene and acts in concert with two proteins that bind to the GME, i.e., GMEB-1 and -2 (Oshima et al, 1995;Zeng et al, 1998;Chen et al, 2002;Chen et al, 2004). GMEB-1 and -2 are also involved in parvovirus replication (Christensen et al, 1999;Christensen et al, 2001).…”

Liver gene expression in rats in response to the peroxisome proliferator-activated receptor-α agonist ciprofibrate

“…Hnf1 and its transcriptional coactivator, Dcoh, are also involved in the regulation of a number of other genes including some acute-phase inflammatory response genes described above (7,14). Two of the upregulated transcription factors are known to be involved in downregulation of immune and inflammatory responses: nuclease-sensitive element binding protein (Nsep1) represses HLA class II gene expression (65), and glucocorticoid modulatory element binding protein 2 (Gmeb2) modulates transcriptional activity of the glucocorticoid receptor (73). Thus, these transcription factors may mediate immune modulatory effects of ciprofibrate.…”