Regulation of transcription by steroid hormones.
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Animals↗
Biomedical subjects
M Beato
Publications and source records attributed to M Beato.
Sp1-mediated transcriptional activation is repressed by Sp3.
Sp1, Sp3 (SPR-2) and Sp4 (SPR-1) are human sequence-specific DNA binding proteins with very similar structural features. In this report, we have analyzed Sp3 in direct comparison with Sp1. We have raised antibodies against both Sp1 and Sp3, and show that Sp3 protein, like Sp1, is expressed in various cell lines. Co-transfection experiments in different mammalian cell lines reveal that in contrast to Sp1 and Sp4, Sp3 is not able to activate several Sp1 responsive promoters. In addition, Sp3 also fails to activate reporter constructs in Drosophila SL2 cells lacking endogenous Sp factors. Instead, we find that Sp3 represses Sp1-mediated activation in a linear dose-dependent manner. A mutant of Sp3 lacking the DNA binding domain does not affect activation by Sp1, suggesting that the inhibition is most likely due to the competition with Sp1 for their common binding sites. To determine if any structurally similar domain of Sp3 is able to replace partially homologous domains of Sp1, we have generated chimeric proteins and tested their activation characteristics in gene transfer experiments. It appears that neither the glutamine-rich domains A and B nor the D domain of Sp1 can be replaced by the homologous regions of Sp3. Our results suggest that Sp3 is an inhibitory member of the Sp family.
Animals↗
Two independent pathways for transcription from the MMTV promoter.
The influence of progesterone receptor (PR) and glucocorticoid receptor (GR) on transcription from the mouse mammary tumour virus (MMTV) promoter was analyzed using cell-free transcription of DNA templates with a G-free cassette. Preincubation of the templates with either PR or GR stimulates the rate of transcription initiation 10-50 fold, whereas the recombinant DNA binding domain of GR is inactive. Mutations that inactivate the nuclear factor I (NFI) binding site, or NFI depletion of the nuclear extract, decrease basal transcription without influencing receptor-dependent induction. Recombinant NFI, but not its DNA-binding domain, restores efficient basal transcription of the depleted extract. Recombinant OTF1 or OTF2, but not the POU domain of OTF1, enhance MMTV transcription independently of NF1. In agreement with this finding, NFI and OTF1 do not cooperate, but rather compete for binding to the wild type MMTV promoter, though they have the potential to bind simultaneously to properly oriented sites. Our results imply the existence of two independent pathways for MMTV transcription: one initiated by NFI and the other dependent on octamer transcription factors. Only the second pathway is stimulated by steroid hormone receptors in vitro.
Base Sequence↗
A functional glucocorticoid-responsive unit composed of two overlapping inactive receptor-binding sites: evidence for formation of a receptor tetramer.
An unusual glucocorticoid-responsive element (called GRE A) was found to mediate the induction of the cytosolic aspartate aminotransferase gene by glucocorticoids and was bound by the glucocorticoid receptor in a DNase I footprinting assay. GRE A consists of two overlapping GREs, each comprising a conserved half-site and an imperfect half-site. The complete unit was able to confer glucocorticoid inducibility to a heterologous promoter (delta MTV-CAT). Mutation of any of the half-sites, including the imperfect ones, abolished inducibility by the hormone, demonstrating that each of the isolated GREs was inactive. In electrophoretic mobility shift assays, purified rat liver glucocorticoid receptor (GR) formed a low-mobility complex with GRE A, presumably containing a GR tetramer. When purified bacterially expressed DBD was used, low-mobility complexes as well as dimer and monomer complexes were formed. In inactive mutated oligonucleotides, no GR tetramer formation was detected. Modification of the imperfect half-sites in order to increase their affinity for GR gave a DNA sequence that bound a GR tetramer in a highly cooperative manner. This activated unit consisting of two overlapping consensus GREs mediated glucocorticoid induction with a higher efficiency than consensus GRE.
Aspartate Aminotransferases↗
Two distinct factors bind to the rabbit uteroglobin TATA-box region and are required for efficient transcription.
The rabbit uteroglobin gene is expressed in a variety of epithelial cell types like the lung Clara cells and the glandular and luminal epithelial cells of the endometrium. Expression in Clara cells is on a high constitutive level, whereas expression in the rabbit endometrium is under tight hormonal control. One important element of the rabbit uteroglobin gene mediating its efficient transcription in two epithelial cell lines from human endometrium (Ishikawa) and lung (NCI-H441) is its noncanonical TATA box (TACA). Here, we show that two factors (TATA core factor [TCF] and TATA palindrome factor [TPF]) different from the TATA-box binding protein bind to the DNA major groove at two adjacent sites within the uteroglobin TATA-box region and that one of them (TCF) is specifically expressed in cell lines derived from uteroglobin-expressing tissues. The binding sites for TCF and TPF, respectively, are both required for efficient transcription in Ishikawa and NCI-H441 cells. Mutation of the TACA box, which we show is a poor TATA box in functional terms, to a canonical TATA motif does not affect TCF and TPF binding. Therefore, we suggest that the function of the unusual cytosine could be to reduce rabbit uteroglobin expression in cells lacking TCF and that the interaction of TATA-box binding protein with the weak TACA site is facilitated in TCF- and TPF-positive cells.
Animals↗
The uteroglobin promoter targets expression of the SV40 T antigen to a variety of secretory epithelial cells in transgenic mice.
Adenocarcinomas derived from the lining epithelia of various organs are the most common malignant tumors in human pathology and about 50% are hormone dependent. The tissue-specific and hormally regulated expression of the rabbit uteroglobin gene is secretory epithelial cells could provide a means of establishing in vivo models for a variety of human tumors originating from such tissues. We have generated trangenic mice inheriting a hybrid gene containing 4.7 kb of the rabbit uteroglobin 5'-flanking sequences fused to the SV40 T antigen encoding region. All transgenic founders examined exhibited bronchio-alveolar adenocarcinomas, probably due to expression of the transgene in Clara cells. Most founders also developed tumors of the submandibular salivary gland, and adenocarcinomas of the stomach. Adenocarcinomas and dysplasias in epithelial cells of the male and female genital tract were found in single founders. Immunohistochemical analysis showed that T antigen expression interfered with stable maintenance of the differentiated phenotype as documented by expression of the endogenous uteroglobin gene. One founder gave rise to a mouse line, UT7.1. Transgenic descendants of UT7.1 developed lung adenocarcinomas and, depending on the genetic background, exhibited tumors of the stomach, the salivary gland and the pancreas. Sporadically male descendants developed prostatic adenocarcinoma whereas females developed dysplasias and adenocarcinomas of the uterus and the oviduct. Thus, the UT7.1 mouse line could be a useful model for several epithelial neoplasias.
Adenocarcinoma↗
Inducible regulatory elements in the human cyclin D1 promoter.
To be able to elucidate the function of cyclin D1 in the control of cell cycle progression and its role as an oncogene in tumorigenesis, it is of paramount importance to understand the mechanisms involved in the regulation of its expression. In the present study, we have cloned the human cyclin D1 gene and analysed the structure and function of 3kb of its 5'-flanking region. Several regulatory regions involved in both basal level and serum-induced expression were identified, two of which turned out to be of particular interest. One of these regions is involved in serum induction and is located 848-944 bp upstream of the initiation site. In agreement with this result, in vivo footprinting revealed a novel, strongly inducible protein binding site around positions -928 to -921. A second constitutively occupied binding site was mapped to a potential CRE at position -52. Cotransfection experiments showed that the cyclin D1 promoter is inducible by c-Jun, and that this induction is mediated predominantly through the protected putative CRE at -52.
Base Sequence↗
Inducible regulatory elements in the human cyclin D1 promoter.
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Base Sequence↗
Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized form MDCK cells and mainly basolaterally from Caco-2 cells.
A complete cDNA encoding rabbit uteroglobin was constructed and expressed in MDCK and Caco-2 cells. The MDCK cells secrete uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco-2 cells secrete uteroglobin mainly to the basolateral side. Both MDCK and Caco-2 cells thus secrete uteroglobin in a non-sorted manner. It has, however, previously been shown that uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563-1573]. This suggests that either the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco-2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type.
Animals↗
Chromatin structure modulates transcription factor binding to the mouse mammary tumor virus (MMTV) promoter.
The MMTV promoter contains a complex hormone responsive region (HRR) upstream of a binding site for the transcription factor nuclear factor I (NFI). Hormonal induction of MMTV expression requires the integrity of both the HRR and the NFI binding site. However, in vitro NFI acts as a basal transcription factor on the MMTV promoter that does not cooperate but rather competes with the hormone receptors in terms of binding to MMTV-DNA. Fragments that contain the HRR and the NFI binding site have been reconstituted into mononucleosomes. Steroid hormone receptors bind efficiently to these nucleosomes, NFI does not. Therefore it has been postulated that the chromatin structure may be responsible for the inability of NFI to bind to the chromosomally organized inactive MMTV promoter. In vivo DNaseI and methidium-propyl-EDTA-Fe(II) (MPE) digestion pattern indicate the presence of a nucleosome covering the HRR and the NFI binding site. Genomic footprinting shows that in vivo the rotational setting of the MMTV promoter DNA in this nucleosome is identical to that previously reported for reconstituted nucleosomes in which the major grooves of the NFI half palindromes are facing towards the histone octamer and appear not to be accessible to NFI. These results indicate that MMTV promoter sequences are determining nucleosome positioning in vivo and supports the concept that rotational positioning of DNA in this nucleosome constitutively represses the MMTV promoter.
Base Sequence↗
Expression and functional analysis of steroid receptor fragments secreted from Staphylococcus aureus.
Fragments of the DNA-binding domain of the rat glucocorticoid receptor (rGR) and the human estrogen receptor (hER) were expressed in Staphylococcus aureus as a chimeric fusion to protein A by using a modified expression vector with an artificial factor X-cleavage site. The secreted product was isolated by hydrophobic chromatography on Phenyl-Sepharose and purified on DNA-cellulose or by anion-exchange chromatography. After cleavage of the protein A moiety, the purified rGR DNA-binding domain from amino acids 406 to 523 (rGR406-523), binds specifically to a glucocorticoid responsive element as a homodimer but cannot form heterodimers with the DNA-binding domain of the hER. Amino acids 510 to 523 following the zinc finger region, as well as free sulfhydryl-groups are necessary for DNA-binding, which is more efficient when the tripeptide Gly-Gly-Cys is added to the carboxy terminal end. Despite its specific interaction with DNA, rGR406-523 does not activate transcription from the MMTV promoter in a cell-free system that efficiently responds to addition of native GR, suggesting that regions essential for transcriptional activation in vitro are located outside of the DNA-binding domain.
Amino Acid Sequence↗
Steroid hormone receptors: interaction with deoxyribonucleic acid and transcription factors.
Gene regulation by steroid hormones is mediated by binding of the hormone ligand to the corresponding receptor that triggers a complex set of interactions of the hormone receptors with each other, with DNA in chromatin, and with a variety of other proteins. In this review we attempt to summarize what is known about these interactions using as the main example the regulation of mouse mammary tumor virus transcription by glucocorticoids and progestins. We describe in some detail the interaction of monomers and homodimers of the steroid receptors with their recognition sequences, and the molecular mechanism used to discriminate between the responsive elements for glucocorticoids/progestins and estrogens. We then review the interactions between homologous and heterologous hormone receptors on complex hormone regulatory regions, before devoting some attention to the synergistic and inhibitory interactions of hormone receptors with other transcription factors. Finally we briefly summarize some of the possible mechanisms that modulate the molecular interactions of hormone receptors. In addition to ligand binding, these include receptor phosphorylation, changes in DNA topology, and the organization of DNA in nucleosomes. From this overview we draw the tentative conclusion that the specificity of the hormonal response in different cells results from a combination of developmental restrictions both in the accessibility of genomic sequence and in the repertoire of regulatory proteins present in each particular cell. In addition, the array of regulatory sequences in DNA and chromatin determines the precise nature of macromolecular interactions of the receptors that are modulated by their degree of phosphorylation.
Amino Acid Sequence↗
Glucocorticoid receptor binding site in the mouse alpha-amylase 2 gene mediates response to the hormone.
Amylase gene expression has been shown to be positively regulated by glucocorticoids. Previous reports have suggested that this effect is indirect. We have addressed this question in a mouse exocrine pancreas cell line, 266-6, in which basal level of expression of amylase mRNA is low but inducible by glucocorticoids. In these cells the effect of glucocorticoids is not inhibited by cycloheximide at early time points. Reporter plasmids containing 224 base pairs of mouse amylase 5'-flanking DNA are positively regulated by glucocorticoids in gene transfer experiments. Glucocorticoid receptor purified from rat liver binds to the amylase promoter from position -56 to -33 and at the start of transcription. Site-directed mutation at the upstream position (-47 to -42) eliminates response to glucocorticoids in transient gene transfer experiments. Thus, glucocorticoid regulation of the mouse amylase gene is a direct effect and is mediated via a receptor binding site in the promoter region of the gene. Inhibition of the hormone response by cycloheximide at later time points after induction suggests the additional requirement for a short-lived factor. The DNA binding domain of the glucocorticoid receptor binds to a single site in the amylase promoter as a monomer, suggesting that both receptor binding sites as well as an additional short-lived factor are required to obtain induction.
Animals↗
Expression of epithelial phenotype is enhanced by v-Ha-ras in rat endometrial cells immortalized by SV40 T antigen.
To study the interplay of steroid hormones and oncogenes in the control of endometrial cell proliferation and differentiation we have generated cell lines derived from rat endometrium by expressing the immortalizing oncogenes adeno E1A or SV40 large T antigen. These lines are positive for mesenchymal markers and contain very few characteristic epithelial proteins. Cell lines expressing a temperature-sensitive mutant of SV40 T antigen exhibit a temperature-dependent morphology and growth behavior, but do not manifest an epithelial phenotype at the non-permissive temperature. Cell lines additionally infected with retroviral vectors carrying the v-Ha-ras oncogene (p21rasArg-12) no longer express collagen type III and recover part of their epithelial potential by expressing cytokeratins and/or cadherin E. Some of these cells also express characteristic decidual marker proteins such as desmin, whereas others express glandular epithelial markers such as uteroglobin. Uteroglobin mRNA levels in these cells are increased by glucocorticoids. The parental temperature-sensitive cells do not contain progesterone receptor but become positive for progesterone receptor at the permissive temperature after infection with the v-Ha-ras-expressing retrovirus. Our results indicate that there is a fluent transition and overlapping between mesenchymal, glandular epithelial and decidual phenotypes of endometrial cells, suggesting that these three cell types are derived from the same stem/precursor cells. The v-Ha-ras oncogene product appears to act on the differentiation pathway at an early step prior to the distinction between decidual and glandular epithelial lineage.
Animals↗
Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes.
Previous analyses of the uteroglobin gene promoter revealed a GT1 box which is also found in the SV40 enhancer. The GT1 element in the context of the uteroglobin promoter is active in Ishikawa cells, a human endometrial cell line, but not in HeLa cells. Here we report the cloning by recognition site screening of two factors (SPR-1 and SPR-2) which bind to this GT1 motif. SPR-1 and SPR-2 are homologues of the transcription factor Sp1. All three proteins are closely related members of a gene family encoding proteins with very similar structural features. Like Sp1, SPR-1 and SPR-2 contain glutamine and serine/threonine rich amino acid stretches. Most significantly, the DNA binding domains of all three proteins are highly conserved and they recognize GT as well as GC boxes identically. SPR-2 mRNA is expressed ubiquitously, whereas SPR-1 transcripts are abundant in the brain but barely detectable in other organs. The possible function of these factors for the activity of the uteroglobin promoter is discussed.
Amino Acid Sequence↗
Artificial steroid hormone response element generated by dam-methylation.
Using the interaction of steroid hormone receptors with their palindromic response elements as an example, we show here that cloning in dam+ bacterial strains can lead to artifactual results due to methylation of adenine residues at the N-6 position. Substitution of the T by an A in the third position of the half palindromes of the hormone responsive element TGTTCT(1) yields a functional element only when amplification is made in dam+ bacteria. Mutant palindromes methylated at the N-6 position of this adenine exhibit the same affinity for progesterone and glucocorticoid receptors as the consensus response element, whereas their unmethylated counterpart binds with negligible affinity. These observations underline the significance of hydrophobic interactions between receptors and the major groove of the DNA for discrimination among various responsive elements, and point to the importance of using dam- bacterial strains for the correct identification of the nucleotide sequence of cis-acting elements.
Base Sequence↗
Interplay of steroid hormone receptors and transcription factors on the mouse mammary tumor virus promoter.
The mouse mammary tumor virus (MMTV) promoter, that responds to glucocorticoids and progestins, contains a complex hormone response element (HRE) in the long terminal repeat (LTR) region covered by a phased nucleosome. Hormone treatment leads to alterations in chromatin structure that make the HRE region more accessible to digestion by DNase I and permit binding of transcription factors, including nuclear factor I (NFI), immediately downstream of the HRE. NFI acts as a basal transcription factor on the MMTV promoter in vitro but competes with the hormone receptors in terms of binding to free DNA. In uninduced chromatin, the precise positioning of the DNA double helix on the surface of the histone octamer precludes binding of NFI to its cognate sequence while still allowing recognition of the HRE by the hormone receptors. We postulate that receptor binding to the nucleosomally organized MMTV promoter disrupts the chromatin structure enabling NFI binding and subsequent formation of a stable transcription complex. Whether the receptor remains bound to DNA during induction or is displaced by NFI is not conclusively known, but our evidence supports a "hit and run" mechanism. NFI is not the only factor involved in hormonally induced transcription of the MMTV promoter. Two degenerated octamer motifs located immediately upstream of the TATA box are recognized by the ubiquitous transcription factor OTF-1 (Oct-1, NFIII), and are also important. In vitro, mutations in these motifs do not influence basal transcription, but completely abolish the stimulatory effect of purified progesterone receptor. Progesterone receptor bound to the HRE facilitates binding of OTF-1 to the two octamer motifs. Thus, OTF-1 is a natural mediator of progesterone induction of the MMTV promoter and acts through cooperation with the hormone receptor for binding to DNA.
Animals↗
Regulation of androgen receptor mRNA and protein level by steroid hormones in human mammary cancer cells.
The regulation of the human androgen receptor (AR) by steroid hormones in human mammary cancer cells was investigated using immunocytochemical and ligand binding assays for its protein and Northern blot analyses for the corresponding mRNA. MFM-223 cells contain high levels of ARs and are growth-inhibited by dihydrotestosterone (DHT). The AR protein is down-regulated to 57% of the control by 10 nM DHT after 24 h, and the corresponding mRNA is also reduced. The nonsteroidal antiandrogen hydroxyflutamide had no effect on the AR level, whereas after incubation with 1 microM cyproterone acetate a slight down-regulation was observed. The AR level was restored completely after release from a 7 day treatment with DHT. However, only 60% of the control level was restored, if the cells wer grown in the presence of DHT for 6 weeks. In androgen-pretreated cells the proliferation rate remained decreased even after the withdrawal of DHT. Concomitantly the distinct growth inhibition was lost. Transfection experiments demonstrated a reduced activity of the residual androgen receptor in these pretreated cells. In addition to the AR, EFM-19 cells also contain significant amounts of estrogen and progesterone receptors. EFM-19 cells are not growth inhibited by physiological concentrations of DHT. Autoregulation of AR was also found in this cell line. Additionally, reduced levels of AR protein and mRNA were found in EFM-19 cells after treatment with the synthetic progestin R5020. The maximum effect of R5020 was observed at the high concentration of 1 microM. Estrogen treatment with 10 nM 17 beta-estradiol for 3 days reduced the AR level only by 25%.
Blotting, Northern↗