Human glucocorticoid receptor 1A promoter and splice variants

Details Human glucocorticoid receptor 1A promoter and splice variants Human glucocorticoid receptor 1A promoter and splice variants

2000-04-19

2003-11-18

Eyler, Yvonne (Department: 1646)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S069100, C435S091200, C435S375000, C435S320100, C536S023100, C536S024310, C536S024330, C530S300000, C530S350000

Reexamination Certificate

active

06649341

ABSTRACT:

This invention pertains to the location of a human glucocorticoid receptor gene promoter region and to three splice variants of the human glucocorticoid receptor gene; and the use of the promoter region and of the splice variants to improve the diagnosis and treatment of leukemia.
The use of naturally occurring substances, such as hormones, to treat cancer has certain advantages. Although side effects can occur, the effects are usually less severe than those caused by cytotoxic chemotherapy. Unfortunately most cancers are not effectively controlled by hormonal therapy, but exceptions include certain hormonally-dependent breast cancers that can be treated with the anti-estrogen tamoxifen, and acute promyelocytic leukemia that is responsive to all-trans retinoic acid. Additionally, some lymphoid malignancies can be effectively treated with glucocorticoid steroid hormones, hormones that control a variety of metabolic and developmental processes. See R. R. Denton et al., “Differential Autoregulation of Glucocorticoid Receptor Expression in Human T- and B-Cell Lines,”
Endocrinology
, vol. 133, pp. 248-256 (1993). Certain types of B- and T-cell acute lymphoblastic leukemia (“ALL”) are particularly sensitive to glucocorticoid hormonal therapy. Glucocorticoids affect lymphoid malignancies due to the induction of programmed cell death, or apoptosis, of immature lymphocytes. See C. W. Distelhorst, “Basic and Clinical Studies of Glucocorticosteroid Receptors in Lymphoid Malignancies,” pp. 494-515 in W. V. Vedeckis (ed.)
Hormones and Cancer
(1996).
The cytolytic effect of glucocorticoids is mediated by the glucocorticoid receptor (GR). Upon entering the cell, glucocorticoids bind to the soluble intracellular receptor protein GR, causing an alteration in GR structure. This alteration in structure converts the unactivated receptor to the activated form that both binds to specific DNA sequences and facilitates transcription of glucocorticoid-responsive genes. The transcribed glucocorticoid-induced mRNA messages are then transported into the cytoplasm and translated into specific proteins. The changes in concentration and types of the intracellular proteins modulate a variety of intracellular processes. GR concentration has been shown to correlate with sensitivity to steroid treatment in vitro. See J. N. Vanderbilt et al., “Intracellular Receptor Concentration Limits Glucocorticoid-Dependent Enhancer Activity,”
Mol. Endocrinol
., vol. 1, pp. 68-74(198
7
). Additionally, an in vivo study of large numbers of patients with ALL found that a low GR level in lymphoblasts isolated at the initial diagnosis was significantly correlated with a poor response to therapy, shorter duration of remission, and a poor overall prognosis. Distelhorst (1996).
Studies have shown that chronic glucocorticoid treatment of cells that do not respond by apoptosis resulted in a decrease in expression (or down-regulation) of the GR gene, as evidenced by decreased levels of GR mRNA and protein. See S. Okret et al., “Down-Regulation of Glucocorticoid Receptor mRNA by Glucocorticoid Hormones and Recognition by the Receptor of a Specific Binding Sequence Within a Receptor cDNA Clone,”
Proc. Natl. Acad. Sci. USA
, vol. 83, pp. 5899-5903 (1986); and Dong et al., “Regulation of Glucocorticoid Receptor Expression: Evidence for Transcriptional and Posttranslational Mechanisms,”
Mol. Endocrinol
., vol. 2, pp. 1256-1264 (1988). By contrast, in cells that undergo apoptosis upon glucocorticoid treatment, such as immature thymocytes, T-lymphocytes, and leukemic T-lymphoblasts, glucocorticoid treatment caused a dramatic increase in the levels of GR mRNA and protein levels, indicating an increase in expression (or up-regulation) of the GR gene. Denton et al. (1993). The molecular mechanisms that control down-regulation and up-regulation of the GR gene are not understood.
Studies on the structure of the human GR gene have shown that the mature GR mRNA is coded by nine separate exons in the genomic DNA. See I. J. Encio and S. D. Detera-Wadleigh, “The Genomic Structure of the Human Glucocorticoid Receptor,”
J. Biol. Chem
., Vol. 266, pp. 7182-7188 (1991). Exon 1 is an untranslated exon; i.e., it is transcribed into mRNA but does not code for amino acids in the GR protein. Exon 2 contains the ATG methionine initiator for protein translation. Thus any exon 1 sequences that are spliced onto exon 2 will have no effect on the amino acid sequence of the GR, because the protein coding sequence begins in exon 2, and also because there is an in-frame stop codon located three codons upstream of the initiator ATG in exon 2. Exon 9 codes a long 3′ untranslated region in the GR mRNA that contains two potential polyadenylation addition sites. The two major, mature GR mRNA species are about 7 kilobases and 5 kilobases long, depending upon which polyadenylation site is used. Additionally, an alternative splicing event in exon 9 gives rise to two GR protein forms, GR alpha and GR beta. GR alpha is the major, functional species found in all cell types. The function, if any, of GR beta is not clear.
The GR promoter region, which controls GR gene expression and mRNA synthesis, has been investigated in both the human and mouse. The human GR promoter was found to be ~2.7 kilobase pairs (kbp). See J. Zong et al. “The Promoter and First, Untranslated Exon of the Human Glucocorticoid Receptor Gene are GC Rich but Lack Consensus Glucocorticoid Receptor Element Sites,”
Mol. Cell. Biol
., vol. 10, pp. 5580-5585 (1990) and Y. Nobukuni et al., “Characterization of the Human Glucocorticoid Promoter,”
Biochemistry
, vol. 34, pp. 8207-8214 (1995). This GR promoter is GC-rich and lacks a TATA box and CAAT box, common characteristics of “housekeeping” genes that are constitutively expressed in most cell types.
The GR protein is found in virtually all cells in the human body. Regulatory elements that control GR gene expression have been characterized in the 2.7 kbp human GR promoter region,just upstream from the untranslated exon 1. See Nobukuni et al. (1995); M. B. Breslin and W. V. Vedeckis, “The Glucocorticoid Receptor and c-jun Promoters Contain AP-1 Sites that Bind Different AP-1 Transcription Factors,”
Endocrine
, vol. 5, pp. 15-22 (1996), P. Wei and W. V. Vedeckis, “Regulation of the Glucocorticoid Receptor Gene by the AP-1 Transcription Factor,”
Endocrine
, vol. 7, pp.303-310(1997), and M. B. Breslin and W. V. Vedeckis, “The Human Glucocorticoid Receptor Promoter Upstream Sequences Contain Binding Sites for the Ubiquitous Transcription Factor, Yin Yang 1
,” J. Steroid Biochem. Molec. Biol
., vol. 67, pp. 369-381 (1998). Eleven regions in the first 800 bp of the promoter have been identified that bind protein. Of these eleven, three regions bind the transcription factor Sp1, two regions bind Sp1 and another protein, and one region binds predominantly the transcription factor AP-2, as well as some Sp1. The binding of Sp1 to GC-rich regions is typical of housekeeping, constitutive promoters. See Nobukuni et al. (1995). A single untranslated exon in the 2.7 kbp human GR promoter gene fragment has also been identified. See Zong et al., (1990) and I. J. Encio and Detera-Wadleigh (1991). The start sites for transcription that were identified in the human GR promoter region were somewhat variable, which is another characteristic of a promoter for a housekeeping gene.
The mouse GR promoter and gene structure have also been characterized. See Strähle et al., “At Least Three Promoters Direct Expression of the Mouse Glucocorticoid Receptor Gene,”
Proc. Natl. Acad. Sci. USA
., vol. 89, pp. 6731-6735 (1992). Similar to human, the mouse GR transcript derives from 9 exons, and the ATG methionine initiator is in exon 2. However, two exon 1 sequences that derive from the mouse promoter analogous to the human 2.7 kpb sequence have been identified. The one nearest to exon 2 has been designated exon 1C, and is homologous to the human GR exon 1 sequence. See Nobukuni et al., 1995. Further upstream is untranslated exon 1B, found about 1 kbp upstream from the exon 1C seque

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