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B Turcotte

Publications and source records attributed to B Turcotte.

15 recordsLinked to original sources
PubMed1992

HAP1 positive control mutants specific for one of two binding sites.

The expression of the yeast CYC1 and CYC7 genes is controlled by the HAP1 activator. A GAL4-like zinc finger (residues 1-148) specifies binding to the dissimilar sites UAS1 (of CYC1) and CYC7, and an acidic domain (residues 1307-1483) is essential for activation of transcription. To analyze how HAP1 binds to UAS1 and CYC7, we performed saturation mutagenesis of the DNA-binding domain and recovered mutants with altered activity. Class 1 mutants had a reduced activity at both UAS1 and CYC7, and class 2 mutants selectively eliminated activity at CYC7. Surprisingly, several mutants of both classes exhibited wild-type DNA binding, indicating that they were specifically defective in activation. These positive control (PC) mutants alter residues that bracket the zinc finger. We explain these mutants in a model involving cofactor proteins that bind UAS1 and CYC7 along with HAP1. The existence of PC mutants that only affect activity at CYC7 raises the possibility that different cofactors may exist for UAS1 and CYC7.

Amino Acid Sequence↗
PubMed1991

Control of transcription of the chicken progesterone receptor gene. In vitro and in vivo studies.

To study the promoter of the chicken progesterone receptor (cPR) gene and the relevance of several progestin-responsive elements therein, chimeric genes were constructed which contained the 5'-flanking region of the cPR gene linked to promoterless globin or chloramphenicol acetyltransferase sequences. Cell-specific initiation of transcription was observed in transiently transfected chicken embryo fibroblasts when using 876 base pairs of the cPR gene upstream region. Transcription from these reporter genes could be induced by progestins in the presence of cPR form A but not of form B. In keeping with these data, three in vitro progesterone receptor (PR)-binding sites were identified in the cPR promoter region by DNase I protection assays. However, in vivo, nuclear run-on transcription demonstrated that neither primary stimulation with progestins, nor treatment of secondarily estrogen-stimulated chicks with progestins, glucocorticoids, or androgens resulted in any significant change of cPR gene transcription in the oviduct, thus suggesting a cell- and/or development-specific role for these progestin-responsive elements. Although estrogen is known to increase PR levels in the chick oviduct, this effect does not involve stimulation of PR gene transcription, as demonstrated here by nuclear run-on experiments, the analysis of DNase I hypersensitive sites, and transient cotransfection studies. Since acute withdrawal from estrogen-stimulation markedly decreased the level of cPR mRNAs in chick oviduct when analyzed by Northern blotting, we conclude that estrogen-dependent stimulation of PR levels in the oviduct is a post-transcriptional process.

Animals↗
PubMed1991

Progestin receptors: isoforms and antihormone action.

We present evidence that the two isoforms of A and B of the chicken (cPR) and human progesterone receptor (hPR) originate from two different mRNA populations. One of these encodes the isoforms A which originate by initiation of translation at an in-frame AUG found 127 (cPR) and 165 (hPR) codons downstream of the AUG which gives rise to the isoforms B. Two estrogen-inducible hPR promoters were identified which are responsible for the generation of these two classes of transcripts. Characterization of the cPR promoter suggested the possible existence of cell-type and isoform-specific auto-regulation of cPR transcription and provided evidence that estrogen-induction of cPR expression occurs at a post-transcriptional level. Finally, we demonstrate promoter-specific transcriptional activation by the hPR isoforms A and B, and we discuss the mechanism of action of the anti-progestin RU486.

Animals↗
PubMed1990

Transient expression of human and chicken progesterone receptors does not support alternative translational initiation from a single mRNA as the mechanism generating two receptor isoforms.

Two isoforms (A and B) of the human (hPR) and chicken (cPR) progesterone receptors originate from a single PR gene. cPR form A results from initiation of translation at a downstream ATG codon (ATG2) which in the cPR cDNA-deduced open reading frame is found 128 amino acids C-terminal to and in-frame with the first ATG codon (ATG1) that gives rise to form B. Our recent observation of an abundant cPR mRNA which encodes only form A suggested to us that the two isoforms are translated from different transcripts (Jeltsch, J. M., Turcotte, B., Garnier, J. M., Lerouge, T., Krozowski, Z., Gronemeyer, H., and Chambon, P. (1990) J. Biol. Chem. 265, 3961-3974). This view is, however, at variance with data obtained by transient transfection with expression vectors containing most of the cPR cDNA (downstream of nucleotide +53) since both isoforms were generated in transiently transfected COS cells (Conneely, O. M., Kettelberger, D. M., Tsai, J. J., Schrader, W. T., and O'Malley, B. W. (1989) J. Biol. Chem. 264, 14062-14064). To further support our above conclusion, vectors containing either hPR or cPR cDNAs were introduced into HeLa and COS-1 cells. Only hPR form B originated from a vector containing the entire cDNA (containing nucleotides 1 to approximately 4400), and form A was produced only from a vector expressing hPR transcripts (nucleotides 814 to approximately 4400) lacking ATG1. Vectors expressing the 5'-untranslated and coding region of the cPR mRNA (nucleotides 29-2921) generated only traces of form A in the two cell lines. Similar traces of form A were observed in COS-1 cells transfected with a vector lacking the 5'-untranslated region. Collectively, these results do not support the hypothesis that similar amounts of the two PR isoforms are generated by alternative initiation of translation on a single PR transcript. We discuss data indicating that for hPR and cPR, isoforms A and B are in fact translated from different mRNAs.

Animals↗
PubMed1990

Characterization of multiple mRNAs originating from the chicken progesterone receptor gene. Evidence for a specific transcript encoding form A.

The structure of the 42-kilobase (kb) long chicken progesterone receptor (cPR) gene and of all six transcripts that are detectable on Northern blots was determined. The first of 8 exons encodes the N-terminal region A/B which is highly divergent among different species and contains a constitutive transcription activation function. The DNA (DBD)- and hormone-binding domains (HBD) are assembled from 2 and 5 exons, respectively, with the individual "zinc fingers" of the DBD encoded by separate exons. In addition to the previously described 4.5-kb cPR mRNA species, alternative polyadenylation, splicing variation, and "5'-truncation" lead to the generation of 5 further mRNAs. Most importantly, this 5'-truncation produces, by an as yet unidentified mechanism, an abundant transcript which encodes form A but not form B of cPR. Lack of splicing at the exon 2 splice-donor and polyadenylation due to a signal site in the second intron generates a previously undetected 3.4-kb mRNA species. The corresponding cDNA was sequenced in its entirety and shown to encode only region A/B and the N-terminal "finger" of the DBD. Alternative polyadenylation upstream of the signal site for the 4.5-kb mRNA is responsible for the appearance of a 3.3-kb mRNA. The longest cPR mRNA (8.2 kb) originates from a transcription termination point more than 3 kb downstream of the 4.5-kb mRNA 3'-end. Finally, the primary sequence of more than 2 kb upstream sequences of the cPR gene, containing several consensus hexamer progestin/glucocorticoid receptor-binding sites (PRE/GRE and putative Sp1 binding motifs, is discussed.

Amino Acid Sequence↗
PubMed1990

Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B.

The human progesterone receptor (hPR) cDNA, synthesized from T47D breast cancer cells, and the hPR gene 5'-flanking region were cloned and sequenced. Comparison of the cDNA-deduced amino acid sequence with other PR homologues demonstrated the modular structure characteristic of nuclear receptors. As in the case of the chicken homologue, there are two hPR forms, A and B, which originate from translational initiation at AUG2 (codon 165) and AUG1, respectively. Northern blot analysis of T47D mRNA using various cDNA derived probes identified two classes of hPR mRNAs, one of which could code for hPR form B, while the other one lacked the 5' region upstream of AUG1. S1 nuclease mapping and primer extension analyses confirmed that the second class of hPR transcripts are initiated between +737 and +842 and thus encode hPR form A, but not form B. By using the hPR gene 5'-flanking sequences as promoter region in chimeric genes, we show that a functional promoter (located between -711 and +31) directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15. Most importantly, initiation of transcription from chimeric genes demonstrated the existence of a second promoter located between +464 and +1105. Transient co-transfection experiments with vectors expressing the human estrogen receptor showed that both promoters were estrogen inducible, although no classical estrogen responsive element was detected in the corresponding sequences. When transiently expressed, the two hPR forms similarly activated transcription from reporter genes containing a single palindromic progestin responsive element (PRE), while form B was more efficient at activating the PRE of the mouse mammary tumor virus long terminal repeat. Transcription from the ovalbumin promoter, however, was induced by hPR form A, but not by form B.

Amino Acid Sequence↗
PubMed1990

Repression of the alpha-fetoprotein gene promoter by progesterone and chimeric receptors in the presence of hormones and antihormones.

Using transient transfection assays, we showed that repression of the alpha-fetoprotein promoter by intact and deletion mutants of the progesterone receptor and by chimeric progesterone/glucocorticoid-estrogen receptors in the presence of their cognate hormones was closely correlated with their ability to bind to a progesterone/glucocorticoid-responsive element. This negative regulation was also observed in the presence of antihormones, providing evidence that receptor-antihormone complexes can bind to their responsive elements in vivo.

Animals↗
PubMed1989

Steroid hormone receptors compete for factors that mediate their enhancer function.

Stimulation of transcription of reporter genes by the progesterone receptor (PR) was inhibited in transfected HeLa cells by co-expressing the estrogen receptor (ER) in an ER-dose- and estrogen-dependent manner. Both the N-terminal A/B region and the hormone binding domain of ER were involved in this inhibition, which was antagonized by antiestrogens and did not appear to involve direct interaction between ER and either reporter gene or PR. ER expression also inhibited activation by the glucocorticoid receptor (GR), and both PR and GR expression inhibited activation by ER, albeit to a lower extent. Similar transcriptional interference was observed between the endogenous PR and ER present in T47D and MCF-7 breast cancer cells transfected with an ER reporter gene. Moreover, transcription of the resident estrogen-induced pS2 gene was partially inhibited by exposing MCF-7 cells to progestins or glucocorticoids. We propose that these observations reflect competition for a functionally limiting transcription factor(s).

Cell Line↗
PubMed1988

Differential regulation of normal and tumor alpha 1-fetoprotein genes in fetal hepatocyte x hepatoma hybrids.

Fetal rat hepatocytes and mouse hepatoma cells actively expressing alpha 1-fetoprotein (AFP) and albumin genes were fused with the use of Sendai virus, and the expression of normal (rat) and tumor (mouse) AFP and albumin genes was analyzed in hybrid clones. The tumor AFP gene and both albumin genes were active in 103 hybrids. Expression of the normal fetal rat AFP gene, however, was maintained in only 3 hybrids, and it was frequently lost or decreased selectively upon subcloning. Furthermore, the normal AFP gene, when expressed, was more reactive than the tumor AFP gene to repression by a glucocorticosteroid hormone. These results suggest constitutive differences in the manner an oncofetal gene is activated and regulated in normal and neoplastic states. AFP gene expression in normal hepatocytes appears to be subordinated to a differentiation program degenerated and bypassed in hepatoma cells.

Albumins↗
PubMed1988

The N-terminal region of the chicken progesterone receptor specifies target gene activation.

Steroid hormone receptors belong to a family of nuclear receptors that trigger transcriptional activation of target genes by specific binding to DNA recognition sequences, usually located in the 5'-flanking region of the target gene. Nuclear receptors appear to be segmented proteins and extensive structure-function analyses have attempted to elucidate the functional significance of individual segments. Two of these regions have been defined as the domains responsible for recognition of responsive elements of target genes (region C) and hormone binding (region E) (refs 2-7). But the functional significance of the N-terminal region (A/B), which diverges extensively even for a given receptor between different species, has remained obscure. We have previously cloned, expressed and analysed the chicken progesterone receptor (cPR) (ref. 8). This receptor and its human homologue from T47D breast cancer cells are unique among the steroid hormone receptors in that two forms, A and B, are present in equal amounts in cytosolic extracts, the latter having the higher molecular weight. For the chicken progesterone receptor, we have presented evidence suggesting that the cPR form A corresponds to an N-terminally truncated form of B (ref. 8). Here we report on the functional difference between the forms A and B in the transcriptional activation of two target genes.

Animals↗
PubMed1987

The chicken progesterone receptor: sequence, expression and functional analysis.

The complete mRNA sequence of the chicken progesterone receptor (cPR) has been determined. Expression of the cloned cDNA both in vivo and in vitro produces a protein that has the same apparent mol. wt on SDS--polyacrylamide gels as the 'natural' cPR form B (109 kd) as determined by immunoblotting and photoaffinity labelling. When expressed in HeLa or in Cos-1 cells the 'cloned' cPR displays hormone binding characteristics indistinguishable from the 'natural' receptor and, in the presence of progestins, exhibits 'tight nuclear binding'. A protein corresponding in size to the cPR form A (79 kd) could be detected by expressing in vivo and in vitro an N-terminally truncated cPR starting at methionine 128. A protein of the same apparent mol. wt results from internal initiation during in vitro translation. In contrast, such a protein was barely detectable after in vivo expression of the cPR cDNA in Cos-1 cells. These results suggest that form A is generated by an oviduct cell specific process involving either internal initiation of translation and/or proteolysis in the vicinity of methionine-128. The cPR contains two highly conserved regions C and E, a characteristic of the steroid/thyroid hormone receptor supergene family. By expression of a series of cPR deletion mutants, region E could be defined as the hormone binding domain whereas region C is indispensable for the tight nuclear association of the progestin-receptor complex. In the presence of progestins, the cloned cPR efficiently trans-activates transcription from the long terminal repeat region (LTR) of the mouse mammary tumor virus (MMTV). Deletion of the entire N-terminal region A/B or of the hormone binding domain E results in a 100-fold reduction of transcriptional activation. No stimulation of transcription can be detected when the C-terminal deletion extends into region C, indicating that this region is involved in the recognition of the hormone responsive element (HRE) of the MMTV LTR.

Amino Acid Sequence↗
PubMed1986

DNase I hypersensitivity and methylation of the 5'-flanking region of the alpha 1-fetoprotein gene during developmental and glucocorticoid-induced repression of its activity in rat liver.

Three major regions of DNase I hypersensitivity (DH) were found in alpha 1-fetoprotein (AFP) chromatin of rat liver. DH site I is located at the transcription initiation site and associated with ongoing AFP transcription. DH site II is located 2.5 kb upstream from the cap site: it is developmental stage-dependent but dissociable from ongoing AFP transcription. DH site III, 3.7 kb upstream from the cap site, behaves as hepatocyte-constitutive. DH sites are present in similar regions of liver albumin chromatin. Dexamethasone-induced AFP gene repression is accompanied by the selective loss of AFP DH site I, a likely result of glucocorticoid receptors binding to a DNA recognition sequence located 5'-adjacent to DH site I. Sl nuclease-hypersensitive sites were found on naked superhelical AFP and albumin DNA, but do not appear to contribute DH sites in liver chromatin. The extent of hypomethylation of HpaII sites at the 5'-end of the AFP gene correlates positively with the level of potential and actual expression of the gene. We conclude that developmental and hormonal regulation of the AFP gene is confined within congruent to 4 kb of 5'-flanking DNA, and we discuss possible hierarchical interactions among DH sites, in relation to DNA methylation and replication.

Aging↗
PubMed1985

Rat alpha 1-fetoprotein messenger RNA: 5'-end sequence and glucocorticoid-suppressed liver transcription in an improved nuclear run-off assay.

Cloned cDNA fragments spanning nearly the entire coding regions of rat AFP and albumin genes were used in liver nuclear run-off assays. Under standard assay conditions, transcription signals detected with 5' probes were systematically stronger than with 3' probes. Heparin eliminated this phenomenon, which suggests that nuclear run-off assays are subject to in vitro reinitiation occurring preferentially in promoter gene regions. Transcription in the presence of heparin indicates that very few polymerases are engaged on the AFP gene in adult rat liver. Dexamethasone treatment of developing rat liver results in the loss of transcribing polymerases from all regions of the AFP gene. Albumin gene transcription is unaffected. Inhibition of liver protein synthesis with cycloheximide does not modify the AFP gene suppressive action of dexamethasone. Glucocorticoid hormone receptors may thus directly interact with the AFP locus, blocking polymerase initiation. We also report the sequence analysis of rat AFP mRNA, which reveals the existence of two potential initiation codons on this molecule.

Albumins↗