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M Beato

Publications and source records attributed to M Beato.

At least 19 recordsLinked to original sources
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

Transcriptional control by steroid hormones.

Gene regulation by steroid hormones leads to induction or repression of particular sets of genes. These effects are mediated by intracellular hormone receptors that, in the unliganded state, are maintained in an inactive form by unknown mechanisms possibly involving association with other cellular proteins. Induction of the mouse mammary tumor virus (MMTV) requires binding of the hormone receptor to a complex hormone-responsive element (HRE) located between 75 and 190 bp upstream from the start of transcription. The interaction of several receptor molecules with the four receptor binding sites in the HRE is highly cooperative on circular DNA molecules and each individual site is needed for optimal induction. In chromatin the HRE is precisely organized in phased nucleosomes. Following hormone treatment and receptor binding, changes in chromatin structure are detected that correlate with binding of transcription factors, including nuclear factor I, to the MMTV promoter. However, though nuclear factor I acts as a basal transcription factor on the MMTV promoter it does not cooperate with the hormone receptors in terms of binding to free DNA, and mutation of the nuclear factor I binding site does not eliminate hormonal stimulation. This residual induction is mediated by octamer motifs, upstream of the TATA box, that bind the ubiquitous transcription factor OTF-1. Mutation of these octamer motifs does not influence basal transcription in vitro, but completely abolishes the stimulatory effect of progesterone receptor.

Amino Acid Sequence↗
PubMed1992

Human CC10, the homologue of rabbit uteroglobin: genomic cloning, chromosomal localization and expression in endometrial cell lines.

Human and rat cDNAs to Clara Cell 10 kDa protein (CC10) have been previously isolated. Comparison of the amino acid sequences showed that CC10 is homologous to rabbit uteroglobin. Here we present further evidence that human CC10 is the human counterpart of rabbit uteroglobin. We have isolated the gene and have mapped its genomic localization to chromosome 11q11-qter. Sequence analysis of the 5'-flanking region reveals that the homology between the human and the rabbit gene starts at the first exon/intron boundary and extends up to -1.4 kb. A second region of 0.74 kb from -1.77 to -2.51 kb in the human 5'-flanking gene region is homologous to rabbit sequences that include four progesterone receptor binding sites which have been implicated in progesterone regulation of rabbit uteroglobin gene expression in endometrium. Sequence alignment of this region on the nucleotide level shows that only two weak progesterone receptor binding sites are partially conserved. In addition, close inspection of the human and rabbit promoters reveals that the estrogen responsive element and two recently identified cis elements of the rabbit promoter located between -177 and -258 bp are also absent in the human uteroglobin promoter. Despite these differences in the 5'-flanking regions of the genes, we report that the human uteroglobin mRNA is expressed in a human cell line of endometrial origin indicating that human uteroglobin is expressed in the uterus like its rabbit homologue. Thus, it appears that human uteroglobin is not only a marker for lung Clara cells but also an endometrial differentiation marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1992

Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer.

The progesterone-binding protein uteroglobin has been expressed in Escherichia coli in an unfused, soluble form. Like mature uteroglobin from rabbit endometrium (UG), the E.coli produced uteroglobin (UG1) dimerizes in vitro, forms an antiparallel dimer with Cys3-Cys69' and Cys69-Cys3' disulfide bonds and binds progesterone under reducing conditions. In order to analyze the dimerization and the reduction dependence of progesterone binding in more detail, we separately replaced cysteine 3 and cysteine 69 by serines. Under reducing conditions, both uteroglobin variants (UG1-3Ser and UG1-69Ser) bind progesterone with the same affinity as the wild-type suggesting that both cysteine residues are not directly involved in progesterone binding. In contrast to the wild-type protein, both cysteine variants also bind progesterone with high affinity in the absence of reducing agents. In addition, UG1-3Ser and UG1-69Ser both form covalently linked homodimers. Thus, unnatural Cys69-69' and Cys3-3' disulfide bonds exist in UG1-3Ser and UG1-69Ser, respectively. These data together with computer models based on X-ray diffraction data strongly support the idea that progesterone reaches its binding site located in an internal hydrophobic cavity via a hydrophobic tunnel along helices 1 and 4. Under non-reducing conditions the tunnel is closed by two disulfide bridges (Cys3-Cys69' and Cys69-Cys3') that lie in the most flexible region of the dimer. Reduction or replacement of a cysteine residue enables conformational changes that open the channel allowing progesterone to enter.

Amino Acid Sequence↗
PubMed1992

Elements of the rabbit uteroglobin promoter mediating its transcription in epithelial cells from the endometrium and lung.

The rabbit uteroglobin gene is specifically expressed in certain epithelial cells of ontogenetically unrelated origin. In the endometrium, expression is restricted to the glandular and luminal epithelium and is inducible by progesterone and estradiol. In the lung, Clara cells lining the bronchiolar epithelium show constitutive expression of uteroglobin, which is modulated by glucocorticoids. To explore the molecular basis for this cell type specificity, we have transiently transfected the uteroglobin promoter region fused to the chloramphenicol acetyl transferase gene (CAT gene) in the endometrial cell line Ishikawa; in the human lung cell line NCI-H441, which shows morphological Clara cell characteristics; in HeLa cells; and in three fibroblast cell lines. The uteroglobin promoter efficiently drives expression of the CAT gene in Ishikawa and NCI-H441 cells, but not in HeLa and fibroblast cells. To identify the responsible elements we have analyzed progressive promoter 5'-deletion mutants and randomly generated linker scanning mutants spanning the sequence from -258 to -14 of the uteroglobin promoter. Transfection experiments reveal seven mutation-sensitive regions located around -30, -70, -95, -130, -190, -230, and -255. Several mutants display strong cell type-specific phenotypes. Most significantly, the integrity of the region around -190 is essential for full CAT gene expression in Ishikawa cells, but not in NCI-H441 cells.

Animals↗
PubMed1991

Novel upstream elements and the TATA-box region mediate preferential transcription from the uteroglobin promoter in endometrial cells.

To understand the mechanisms responsible for endometrium-specific expression of the uteroglobin gene, we have compared transcription from the uteroglobin promoter in a human endometrial cell line (Ishikawa) and in HeLa cells. In transient transfection experiments and in nuclear extracts, sequences from -395 to +14 of the uteroglobin gene are able to promote transcription of a reporter gene more efficiently in Ishikawa cells than in HeLa cells relative to the RSV or the SV40 early promoter. Analysis of progressive 5'-deletion mutants identifies three promoter regions, -258/-220, -205/-177, and -96/-35, that are important for preferential transcription in endometrial cells. DNase I footprinting experiments with nuclear extracts from Ishikawa and HeLa cells reveal a series of defined protections overlapping these regions. The relative intensity of individual protections differs between the two cell lines. Oligonucleotide competition experiments suggest that similar factor(s) bind(s) to the two relevant upstream regions of the promoter that share no homology to known regulatory elements. A protection over the TATA-box is detected only with extracts from Ishikawa cells. Band shift experiments show that an Ishikawa-specific factor binds to sequences overlapping the TATA-box region that are partially conserved in other endometrium-expressed genes. We propose that novel transcription factors mediate endometrium-specific expression of the uteroglobin gene in conjunction with a tissue-specific factor that binds to the TATA-box region.

Base Sequence↗
PubMed1991

Ubiquitous transcription factor OTF-1 mediates induction of the MMTV promoter through synergistic interaction with hormone receptors.

Steroid hormones induce transcription from the mouse mammary tumor virus (MMTV) promoter by complex mechanisms requiring binding of the hormone receptors to the hormone responsive element (HRE) of the long terminal repeat region. Here we show that the MMTV promoter contains two degenerated octamer motifs immediately upstream of the TATA box that together bind OTF-1 (Oct-1, NFIII) with an affinity similar to the octamer consensus. In transfection experiments, mutation of these octamer motifs interferes with the hormonal response of the MMTV promoter. In vitro, these mutations do not influence basal transcription but completely abolish the stimulatory effect of purified progesterone receptor. Progesterone receptor and glucocorticoid receptor bound to the HRE facilitate binding of OTF-1 to the two octamer motifs. Thus, OTF-1 is a natural mediator of hormonal induction of the MMTV promoter and acts through cooperation with the hormone receptors for binding to DNA.

Base Sequence↗
PubMed1991

Cell-specific, developmentally and hormonally regulated expression of the rabbit uteroglobin transgene and the endogenous mouse uteroglobin gene in transgenic mice.

We have generated a transgenic mouse line by introducing the rabbit uteroglobin gene with 4 kb of 5'-flanking DNA and 1 kb of 3'-flanking DNA into the mouse germ line via microinjection into fertilized oocytes. Expression of the rabbit uteroglobin transgene was examined and compared with the endogenous mouse uteroglobin gene. Both genes are expressed in the lung, male genital tract and uterus. In the lung, mRNA expression is enhanced by glucocorticoids and restricted to the Clara cells that line terminal and respiratory bronchioli. During embryonic lung development, transcripts are first detected at day 17. Expression in the uterus is restricted to the glandular epithelium and can be induced by sequential treatment with estrogens and progesterone. In the uterus of these pseudopregnant mice the level of rabbit uteroglobin transcripts is higher than that of the mouse endogenous uteroglobin transcripts. In the male genital tract, expression of both genes is restricted to the epithelial layers of the vesicular gland, vas deferens and epididymis. Our results indicate that the rabbit uteroglobin gene together with 4 kb of 5'-flanking DNA and 1 kb of 3'-flanking DNA contains the information required for cell type-specific, developmentally, and hormonally regulated expression.

Animals↗
PubMed1991

Neither the endogenous nor a functional steroid hormone receptor binding site transactivate the ribosomal RNA gene promoter in vitro.

The mammalian ribosomal RNA gene promoters exhibit a conserved sequence between positions +1 and +16 that shows a high degree of homology to the response element for glucocorticoids and progestins (GRE/PRE). These sequences bind specifically the glucocorticoid receptor and the progesterone receptor (PR) albeit with lower affinity than a canonical GRE/PRE. Because steroid hormones are known to affect expression of the ribosomal genes, we tested the influence of hormone receptors on the activity of the ribosomal RNA gene promoter in a cell-free transcription assay. Preparations of PR that induce transcription from the mouse mammary tumour virus (MMTV) promoter do not stimulate but slightly inhibit transcription from the ribosomal RNA gene promoter. This weak negative effect is not mediated through binding to the hypothetical GRE/PRE as a mutant promoter that does not bind receptor is equally repressed. Introduction of the functional MMTV GRE/PRE upstream of the basal ribosomal RNA gene promoter does not enhance its transcription in the presence of an active PR. Thus, RNA polymerase I transcription cannot be stimulated in vitro by cis elements and regulatory proteins that are active in RNA polymerase II transcription.

Animals↗
PubMed1991

Identification of residues essential for progesterone binding to uteroglobin by site-directed mutagenesis.

In order to identify amino acids directly involved in progesterone binding to rabbit uteroglobin we have mutated Phe 6, Tyr 21 and Thr 60 by site-directed mutagenesis of the uteroglobin cDNA. These residues have been postulated previously to participate in progesterone binding. High-level expression of the mutated uteroglobin cDNAs in Escherichia coli yields recombinant protein mutants that, like natural uteroglobin, form stable dimers, suggesting that the tertiary structure of the protein has not been altered. Substitution of Phe 6 by Ser or Ala does not change the progesterone binding characteristics. In contrast, replacement of Tyr 21 by Phe or Ala, drastically decreases progesterone binding. In addition, replacement of Thr 60 by Ala reduces the affinity for progesterone by a factor of three. These data suggest a direct interaction of progesterone with these two amino acids and support the idea of direct hydrogen bonding of the carbonyl (C3 and C20) of progesterone with the hydroxyl groups of Tyr 21 and Thr 60, respectively.

Animals↗
PubMed1991

Transcriptional control by nuclear receptors.

Gene regulation by steroid hormones is accomplished by a variety of different mechanisms leading to induction or repression of particular genes. These mechanisms are all mediated by a single class of intracellular hormone receptors, which in the unliganded state are maintained in an inactive form by association with other cellular proteins, including hsp90. Induction of the mouse mammary tumor virus (MMTV) requires binding of the hormone receptor to a hormone-responsive element (HRE) that is precisely organized in a phased nucleosome. After receptor binding, changes in chromatin structure are detected that correlate with binding of transcription factors, including nuclear factor I, to the MMTV promoter. However, although nuclear factor I acts as a basal transcription factor on the MMTV promoter it does not cooperate with the hormone receptors in terms of binding to free DNA, and mutation of the nuclear factor I binding site does not eliminate hormonal stimulation. This residual induction is mediated by octamer motifs upstream of the TATA box that bind the ubiquitous transcription factor OTF-1. Mutation of these octamer motifs does not influence basal transcription in vitro, but completely abolishes the stimulatory effect of progesterone receptor. Glucocorticoids also inhibit expression of many genes. The effect on the gene for the alpha-subunit of chorionic gonadotropin is due to DNA binding competition between the receptor and the protein mediating cAMP induction, whereas repression of the collagenase gene involves an interaction of the receptor with components of the AP1 complex, Jun and Fos.

Amino Acid Sequence↗
PubMed1991

Expression of the uteroglobin promoter in epithelial cell lines from endometrium.

To understand the molecular mechanism of endometrial differentiation we have initiated an analysis of the uteroglobin promoter. Uteroglobin is normally expressed in endometrial tissues under the control of ovarian hormones. In gene transfer experiments with the Ishikawa cell line, derived from a human endometrial adenocarcinoma, we have identified several regions in the promoter of the uteroglobin gene that are responsible for its endometrium-specific expression. To evaluate the generality of these findings, we have begun cloning the promoter regions of potential endometrial markers, including the rat, mouse, and human uteroglobin gene. In the rat, expression of the uteroglobin-like gene, CC10, is dominant in the lung but is also observed in the endometrium of progesterone treated animals. A comparison of the 5'-flanking sequence of the rat and rabbit uteroglobin gene resulted in the detection of similarities and differences that could explain their differential expression in vivo. To substantiate these findings we have established several cell lines from rat endometrium using murine retroviral vectors containing a positive selection marker and various viral oncogenes, such as SV40 large T antigen, adenovirus E1A, and Ha-ras. Cell lines immortalized by SV40 T-antigen were subsequently transformed with the Ha-ras oncogene. Several cell lines exhibit properties of epithelial endometrial cells. Two cell lines generated with a temperature sensitive mutant of the SV40 large T-antigen grow as transformed cells at the permissive temperature, but differentiate upon shifting to the non-permissive temperature. These rat endometrial cell lines should be useful for the analysis of endometrium-specific gene expression and as model systems for endometrial carcinoma.

Animals↗
PubMed1991

Functional interaction of hybrid response elements with wild-type and mutant steroid hormone receptors.

Steroid hormone receptors can be divided into two subfamilies according to the structure of their DNA binding domains and the nucleotide sequences which they recognize. The glucocorticoid receptor and the progesterone receptor (PR) recognize an imperfect palindrome (glucocorticoid responsive element/progesterone responsive element [GRE/PRE]) with the conserved half-sequence TGTYCY, whereas the estrogen receptor (ER) recognizes a palindrome (estrogen responsive element) with the half-sequence TGACC. A series of symmetric and asymmetric variants of these hormone responsive elements (HREs) have been tested for receptor binding and for the ability to mediate induction in vivo. High-resolution analysis demonstrates that the overall number and distribution of contacts with the N-7 position of guanines and with the phosphate backbone of various HREs are quite similar for PR and ER. However, PR and glucocorticoid receptor, but not ER, are able to contact the 5'-methyl group of thymines found in position 3 of HREs, as shown by potassium permanganate interference. The ER mutant HE84, which contains a single amino acid exchange, Glu-203 to Gly, in the knuckle of ER, creates a promiscuous ER that is able to bind to GRE/PREs by contacting this thymine. Elements with the sequence GGTCAcagTGTYCT that represent hybrids between an estrogen response element and a GRE/PRE respond to estrogens, glucocorticoids, and progestins in vivo and bind all three wild-type receptors in vitro. These hybrid HREs could serve to confer promiscuous gene regulation.

Animals↗
PubMed1991

Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene.

Expression of the vitellogenin genes in avian and amphibian liver is regulated by estrogens. The DNA elements mediating estrogen induction of the various vitellogenin genes of chicken and Xenopus encompass one or more copies of a 13-mer palindromic sequence called the estrogen-responsive element (ERE). Here we show that upon incubation with the purified estrogen receptor (ER) from calf uterus the Xenopus vitellogenin A2 gene yields a DNase-I footprint over the ERE between -331 and -319. This element does not mediate the response to glucocorticoids or progestins in T47D cells. The three guanine residues in each half of the palindrome are protected against methylation by dimethylsulfate after incubation with ER, but not with glucocorticoid (GR) or progesterone (PR) receptors. In contrast, the chicken vitellogenin II gene exhibits multihormonal regulation by estrogens, progestins, and glucocorticoids in T47D and MCF7 cells. Regulation is mediated by the DNA region between -721 and -591 that contains four binding sites for hormone receptors, as demonstrated by DNase-I footprints and methylation protection experiments. The two distal and most proximal binding sites are recognized by ER, GR, and PR, whereas the central binding site is only bound by ER and GR. At suboptimal concentrations, estrogens and progestins or glucocorticoids act synergistically. In experiments using a DNA fragment containing an ERE adjacent to a glucocorticoid-responsive element/progesterone-responsive element, ER and PR bind synergistically to their corresponding sites, perhaps explaining the functional synergism of both hormones. Thus, two very different regulatory elements are used to mediate estrogen induction of related genes in chickens and amphibians.

Animals↗