Characterization of a proximal element in the rat preadipocyte factor-1 (Pref-1) gene promoter

Preadipocyte factor-1 (Pref-1) was shown to negatively regulate adipocyte d ifferentiation. We recently reported that ZOG, a rat homolog of Pref-1, was specifically expressed in the adrenal zona glomerulosa. Results of the inv estigation of Pref-1 expression in preadipocyte and in undifferentiated adr enal cortex suggested that down-regulation of Pref-1 gene was closely corre lated with the differentiation process. In this study we demonstrate that a n upstream region (from -76 to -47) of the rat Pref-1 gene was essential fo r its expression in adrenocortical carcinoma-derived H295R cells. A nucleot ide sequence found in this region, GCGTGGGCGTGGGCGGGGG (Egr/GC-box), seemed to contain three elements, two early growth response (Egr) elements and on e GC-box, overlapping each other. Mutations of four or five nucleotides in a 7-nucleotides-stretch in the midst of the Egr/GC-box eliminated the bindi ng of Sp1/3, abolished the activation by Egr-factor(s) and diminished the P ref-1 promoter activity. When mutations were introduced into the outside of the middle portion, the binding of Sp1/3 to the Egr/GC-box was abolished s imilarly. However, the decrease in the promoter activity was less than that found with the construct mutated at the middle. These results indicated th at an element present at the 7-nucleotides-stretch in the midst of the Egr/ GC-box might be important for the Pref-1 promoter activity, and this proxim al element was possibly activated by a still-unidentified nuclear factor(s) . This element would function as the promoter of the Pref-1 gene in H295R c ells, but not in HeLa cells.