A new human GR gene sequence (hGR 1Ap/e), which is distinct from the previously identified human GR promoter and coding sequences, has been isolated and characterized. The hGR 1Ap/e sequence is approximately 31 kbp upstream of the human GR coding sequence. This sequence (2,056 bp) contains a novel promoter (the hGR 1A promoter; 1,075 bp) and untranslated exon sequence (hGR exon 1A sequence; 981 bp). Alternative splicing produces three different hGR 1A-containing transcripts, 1A1, 1A2, and 1A3. GR transcripts containing exon 1A1, 1A2, 1B, and 1C are expressed at various levels in many cancer cell lines, while the exon 1A3-containing GR transcript is expressed most abundantly in blood cell cancer cell lines. Glucocorticoid hormone treatment causes an up-regulation of exon 1A3-containing GR transcripts in CEM-C7 T-lymphoblast cells and a down-regulation of exon 1A3-containing transcripts in IM-9 B-lymphoma cells. Deoxyribonuclease I footprinting using CEM-C7 cell nuclear extract reveals four footprints in the promoter region and two intraexonic footprints. Much of the basal promoter-activating function is found in the +41/+269 sequence, which contains two deoxyribonuclease I footprints (FP5 and FP6). When this sequence is cloned into the pXP-1 luciferase reporter gene, hormone treatment causes a significant increase in luciferase activity in Jurkat T cells that are cotransfected with a GR expression vector. FP5 is an interferon regulatory factor-binding element, and it contributes significantly to basal transcription rate, but it is not activated by steroid. FP6 resembles a glucocorticoid response element and can bind GRbeta. This novel hGR 1Ap/e sequence may have future applications for the diagnosis, prognosis, and treatment of T-cell leukemia and lymphoma.

Glucocorticoid (GC) hormones are widely used in the treatment of acute lymphoblastic leukemia (ALL). Whereas a high level of GC receptor (GR) protein is associated with the sensitivity of ALL cells to steroid-mediated apoptosis, the auto-up-regulation of human (h)GR mRNA and protein is also found in hormone-sensitive ALL cell lines. We have characterized the hGR gene-proximal promoters for DNA sequences and transcription factors required for hormone responsiveness in T lymphoblasts. Sequences at -4559/-4525 and -2956/-2916, relative to the translation start site, function as strong composite GC response units (GRUs). Both GRUs include adjacent protein recognition sequences for the c-Myb transcription factor and the GR as a DNA cassette. An Ets-binding sequence overlaps the GR-binding site in the -4559/-4525 GRU, whereas an Ets-binding site present in the -2956/-2916 GRU does not overlap the GR/c-Myb-binding cassette. The Ets protein family member, PU.1, blocks hormonal activation of the -4559/-4525 GR/c-Myb-binding cassette but does not interfere with the responsiveness of the -2956/-2916 GRU. Thus, the hGR 1A GRU (described previously), the -4559/-4525 GRU, and the -2956/-2916 GRU have a similar structure and can mediate cell type-specific hormonal auto-up-regulation of hGR promoter activity in steroid-sensitive ALL cells. However, subtle differences in the GRU architecture result in differential sensitivity of the promoters to Ets family members such as PU.1. The architecture of the GRU and the spectrum of specific transcription factors present in different types of ALL might allow the development of a tailored therapy to enhance steroid sensitivity in ALL patients.

No abstract

Glucocorticoids up-regulate the glucocorticoid receptor (GR) in the human T-lymphoblast cell line CEM-C7. One mechanism for the up-regulation of the GR protein is the well-known up-regulation of GR transcripts. We have investigated the effect of other factors on the up-regulation. At least three promoters (1A, 1B, and 1C) exist, which give rise to GR transcripts with different exon 1 sequences. Transcripts with different exon 1 sequences have similar stabilities. Glucocorticoids have little, if any, effect on mRNA stability. In transfection experiments of the GR-deficient mouse fibroblast cell line E8.2, different exon 1 sequences furthermore caused no significant differences in the translational efficiencies of GR transcripts. However, the ratio between the concentrations of the glucocorticoid receptor B (GR-B) isoform and the glucocorticoid receptor A (GR-A) isoform was higher for transcripts containing the exon 1A3 sequence arising from promoter 1A than in transcripts containing exon 1 sequences from promoters 1B and 1C. Because the GR-B isoform is more active in transactivation then GR-A, this would tend to fine-tune glucocorticoid responsiveness of CEM-C7 cells, which express exon 1A3-containing transcripts. We also found that glucocorticoids do not decrease the stability of the GR protein in CEM-C7 cells. In contrast to other cell lines that downregulate GR expression in response to glucocorticoids, CEM-C7 lymphoblasts possess three mechanisms ensuring high glucocorticoid responsiveness: an up-regulation of GR mRNA by glucocorticoids, no destabilization of GR protein by glucocorticoids, and a high activity of promoter 1A with concomitant high expression of the GR-B isoform.

At least three promoters (1A, 1B, and 1C) control the expression of mRNA transcripts for the human glucocorticoid receptor (hGR) protein. An hGR 1A promoter/exon sequence (-218/+269) contains at least 12 deoxyribonuclease (DNase) I footprints that contain bound protein. Whereas four of these footprints (FP6, FP7, FP8, and FP11) contain bound hGR in protein-DNA complexes that are formed, only two (FP7 and FP11) appear to be important for the up-regulation of hGR 1A promoter/exon activity in T-lymphoblasts. Furthermore, the activity of these DNA elements depends upon the promoter context, leading to a redundant and complex regulation of expression of the hGR 1A promoter/exon. FP7 appears to be required for hormonal responsiveness in the absence of upstream sequences (+41/+191), whereas the hormonal responsiveness of FP11 requires a functional, adjacent FP12 DNA sequence. FP12 contains overlapping binding sites for the protooncogene transcription factors c-Myb and c-Ets. It seems likely that binding of either c-Myb or c-Ets to FP12 is necessary for the direct or indirect binding of the hGR to FP11 (a nonconsensus glucocorticoid response element), and the resultant steroid-responsiveness of the hGR 1A promoter/exon sequence. We propose that the identity of the accessory transcription factor bound to FP12 (c-Myb or c-Ets) may determine the nature of regulation (positive or negative) of hGR gene expression by hormone, and that this may be important for hormone-induced apoptosis in T cell acute lymphoblastic leukemia.