Uwe Strähle

Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene

Two glucocorticoid response elements (GREs) located 2.5 kb upstream of the transcription initiation site of the tyrosine aminotransferase gene were identified by gene transfer experiments and shown to bind to purified glucocorticoid receptor. Although the proximal GRE has no inherent capacity by itself to stimulate transcription, when present in conjunction with the distal GRE, this element synergistically enhances glucocorticoid induction of gene expression. Cooperativity of the two GREs is maintained when they are transposed upstream of a heterologous promoter. An oligonucleotide of 22 bp representing the distal GRE is sufficient to confer glucocorticoid inducibility. As evidenced by the mapping of DNAase I hypersensitive sites, local alterations in the structure of chromatin at the GREs take place as a consequence of hormonal treatment.

Synergistic action of the glucocorticoid receptor with transcription factors.

Steroid responsive elements (SRE) have been mapped at variable positions relative to the transcription start site and are often adjacent to binding sites of transcription regulatory proteins. In order to define the role of these transcriptional control sequences in the induction process, we inserted the previously defined 15‐bp glucocorticoid response element (GRE) or 15‐bp estrogen response element (ERE) immediately upstream of the TATA box of the thymidine kinase promoter, deleting all distal promoter elements. Both ERE and GRE confer inducibility by the respective hormone to the truncated promoter. These data suggest that the steroid receptor protein, possibly in conjunction with the TATA box binding protein, is able to form an active transcription complex. In contrast, the GRE when inserted 351 bp upstream of the start site of transcription of the tyrosine aminotransferase gene (TAT) is not capable of mediating hormone inducibility. Inducibility can be attained at this position by either two GREs or a single GRE in combination with a CCAAT motif. A cluster of point mutations in the CCAAT box abolishes hormone inducibility, strongly suggesting a synergistic action between the glucocorticoid receptor and the factor recognizing the CCAAT motif. The CCAAT box can be replaced by a CACCC box, an NF I and an SP1 binding site, thus demonstrating that synergistic action is not restricted to the CCAAT box binding protein. These combinations of a GRE with different transcription factor binding sites show a pronounced cell‐type‐dependent glucocorticoid induction of expression.

Glucocorticoid- and progesterone-specific effects are determined by differential expression of the respective hormone receptors

ALTHOUGH glucocorticoids and progestins control vastly different physiological processes, the receptors mediating the effects of these hormones interact with the same DNA sequences1–4. Transfer experiments involving synthetic genes5 and in vitro binding studies6 have shown that progesterone and glucocorticoid receptors both recognize the same 15-base pair DNA element (TGTACAGGATGTTCT), raising the question of how the two steroids affect gene expression selectively. We considered the possibility that their selectivity arises from either the differential expression of the receptors in target cells or the differential dependence of receptor function on additional transcription factors. To test these alternatives we introduced a progesterone-receptor expression pi asm id into the rat hepatoma cell line Fto2B-3 which contains glucocorticoid receptor but is devoid of progesterone receptor. We report that expression of the progesterone receptor in Fto2B-3 cells renders endogenous glucocorticoid-regulated genes inducible by progestins. Our data show that the responsive-ness of a cell to external stimuli can be reprogrammed by the expression of a single transcription factor and that differential expression of hormone receptors is at least one mechanism by which steroid-specific gene activation is achieved.

Glucocorticoid receptor binds cooperatively to adjacent recognition sites.

In order to define the mechanism of synergistic induction mediated by multiple glucocorticoid response elements (GRE), the affinity of the glucocorticoid receptor to a single or duplicated GRE was analyzed by gel retardation, nitrocellulose filter binding and by footprinting experiments. Direct measurement of the relative affinity and indirect determination by competition showed greater than 10‐fold higher affinity of the glucocorticoid receptor to a duplicated GRE when compared to a single element. Maximal stability of the GRE‐receptor complex was obtained using two closely spaced GREs positioned on the same side of the DNA helix. Increasing the distance or changing the helical position of the GREs considerably increased the off rate of the receptor. DNase I footprinting shows in addition to the protection of the GRE region, an altered pattern in the nonprotected intervening DNA indicating structural alteration of the DNA helix by the receptor bound to adjacent GREs.

Expression of the mouse glucocorticoid receptor and its role during development

Genes encoding enzymes involved in gluconeogenesis are activated in liver shortly after birth by the synergistic effect of glucagon and glucocorticoids. This induction is achieved by the synergistic action of hormone responsive and liver-specific enhancer elements. In the case of glucocorticoids, this enhancer is composed of a glucocorticoid-response element (GRE) and a number of cell-specific hepatocyte nuclear factor 3 (HNF-3) binding sites. The GRE binds the ligand-activated glucocorticoid receptor (GR) which is ubiquitously expressed and the HNF-3 element binds a cell-specific protein factor. To further understand the role of cell-specific glucocorticoid signalling in the perinatal period and earlier during development we have studied the expression of the mouse GR gene. The gene has been cloned and fully characterized. Expression of the gene is controlled by at least three promoters, one of which is only active in T-lymphocytes. Expression of GR mRNA has been detected back to day 9.5 of mouse development. The role of GR during mouse development has been further analysed by disruption of the GR gene in vivo by homologous recombination in mouse embryonic stem cells.

Steroid Response Elements: Composite Structure and Definition of a Minimal Element

One of the striking features of hormonal regulation is the specifity of the response. One element contributing to this specificity is the ligand itself. Since the sequence of the receptor proteins is known by cloning of their cDNAs it is clear that each steroid hormone binds to a different polypeptide (Evans, 1988). Comparison of different parts of the receptor have shown that the most conserved part of the protein is the region interacting with the DNA (the hormone response element, HRE) whereas the part forming the hormone binding domain is less well conserved but still shows considerable homology.

Steroid Response Elements. Definition of a Minimal Promoter and Interaction with Other Activating Sequences

Control of gene expression by steroid hormones is exerted mainly by modulation of the transcriptional activity of the regulated genes. The response to the various steroid hormones is highly specific. It is mediated by binding of the hormone to its specific receptor which, after binding, attains the capacity to recognize a specific DNA region on regulated genes. This receptor-DNA interaction eventually leads in a still-unknown way to an increase of the rate of transcription of the target gene (Yamamoto, 1985).