Role of the composite glucocorticoid response element in proliferin gene expression

Abstract

A binding site for the glucocorticoid receptor in the serum-inducible proliferin gene promoter has been reported to function as a composite glucocorticoid response element when fused to a minimal promoter. We now show that this element can also act as a glucocorticoid-independent negative regulator of transcription, both as an isolated element fused to a minimal promoter and within the context of the proliferin gene promoter. Furthermore, this element is recognized by a factor in mouse fibroblast cell extracts that is distinct from the glucocorticoid receptor and from AP-1, both of which have previously been shown to be able to bind to this site. The ability of this element to repress serum-inducible proliferin promoter activity is dependent on the position of this element with respect to the adjacent serum response region, and on the activity of a positive regulatory element located further upstream in the proliferin promoter.

FIG. 1

Activity of the cGRE in mouse fibroblasts. Duplicate transfections of mouse L fibroblasts (maintained in 15% serum) were carried out with the alcohol dehydrogenase minimal promoter linked to CAT (Adh-CAT), and this same vector in which one copy (plfG-CAT) or three copies (plfG3-CAT) of the PLF cGRE, or one or three copies of the PLF promoter sequence from – 274 to – 252, were inserted. CAT reporter activity, measured as the average conversion of chloramphenicol (C) to acetylated forms (AcC) for the duplicate samples, was: Adh-CAT, 12.3%; plfG-CAT, 10.9%; plfG3-CAT, 2.2%; – 274/– 252-CAT, 69.9%; and (– 272/ – 252) × 3-CAT, 78.2%.

FIG. 2

Activity of PLF promoter mutants. A series of PLF promoter-CAT reporter constructs with progressive 5′ deletions was transfected into mouse L cells. Conversion of chloramphenicol (C) to acetylated forms (AcC) for cells maintained in 0.5% (-) or 20% ( + ) serum, respectively, was: 5′ – 670, 3.2% and 73.1%; 5′ – 268, 3.6% and 59.2%; 5′ – 256, 1.6% and 9.3%; 5′ –2 44, 1.3% and 15.2%; 5′ – 232, 2.3% and 47.2%.

FIG. 3

Activity of the cGRE in the PLF promoter. The wild-type PLF promoter 5′ (– 670) and a linker-substitution mutant – 251 / – 232 in this – 670 promoter background were linked to the CAT gene at position + 61 in the PLF 5′ untranslated region. These DNA constructs were transfected into mouse L cells. After transfection, cultures were maintained for 2 days in 0.5% or 15% serum. Cell extracts were prepared and assayed for CAT enzymatic activity. Conversion of chloramphenicol (C) to acetylated forms (AcC) in 0.5% serum (−) and 15% serum (+), respectively, was 3.3% and 41.5% for wild-type, and 14.0% and 55.3% for the cGRE mutant.

FIG. 4

Protein binding to the cGRE/negative element. Double-stranded oligonucleotides corresponding to the wild type (lane 1) or mutant (lane 2) PLF negative element (from – 254 to – 230) were radiolabeled and incubated with mouse L cell whole-cell extracts. The mutated cGRE was altered at residues – 243 through – 234. Specific binding (arrow) was determined by competition with a 50-fold molar excess of unlabeled double-stranded wild-type (lane 3) or mutant (lane 4) negative element. Additional competition binding reactions included a 50-fold molar excess of the PLF gene AP-1 element (lane 5), the PLF positive element from – 274 to – 253 (lane 6), or five copies of the tyrosine aminotransferase GRE (lane 7). Parallel reactions were also incubated with normal rabbit serum (lane 8), an antiserum against c-fos (lane 9), or a monoclonal antibody against the glucocorticoid receptor (lane 10). Bound and free DNA were separated by polyacrylamide gel electrophoresis.

FIG. 5

Interaction of the cGRE/negative element and the serum response region. The PLF serum response region from – 234 to – 204 alone (SR), the serum response region with the cGRE/negative element (– 256 to – 204, designated cGRE-SR), and the serum response region with the cGRE/negative element separated by an additional 5 bp were fused to the herpes virus thymidine kinase minimal promoter. These DNAs were transfected into mouse L cells, which were then maintained for 2 days in 0.5% or 15% serum. Conversion of chloramphenicol to acetylated forms in 0.5% serum (–) and 15% serum ( + ), respectively, was 4.1% and 91.3% for – 234/– 204; 0.5% and 6.6% for – 256/– 204; and 3.0% and 61.9% for – 256/– 204 ( + 5 bp).

FIG. 6

Schematic diagram of the PLF gene transcriptional regulatory region. Four elements have been defined in the region spanning from – 204 to – 268, including two elements (AP-I and Sph I-like) that constitute the serum response region (6,20), the cGRE/negative element (shown with the boundaries and corresponding sequence sufficient for the glucocorticoid-independent negative regulatory activity determined in this report), and the upstream positive element.