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.

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Positive and Negative Regulation of the Rat Vasopressin Gene Promoter*

Endocrinology ◽

10.1210/endo.138.12.5639 ◽

1997 ◽

Vol 138 (12) ◽

pp. 5266-5274 ◽

Cited By ~ 56

Author(s):

Yasumasa Iwasaki ◽

Yutaka Oiso ◽

Hidehiko Saito ◽

Joseph A. Majzoub

Keyword(s):

Gene Transcription ◽

Consensus Sequence ◽

Negative Regulation ◽

Luciferase Assay ◽

Luciferase Reporter ◽

Dominant Negative Mutant ◽

Deletion Mutants ◽

Glucocorticoid Responsive Element ◽

Responsive Element ◽

Vasopressin Gene

Abstract To study the transcriptional regulation of the vasopressin gene in vitro, 3 kb of the 5′ regulatory region of the rat vasopressin gene was isolated and subcloned, along with a series of various deletion mutants, into vectors containing the luciferase reporter gene. After transfecting these genes transiently into the human choriocarcinoma cell line JEG-3 along with a glucocorticoid receptor (GR) expression vector, transcriptional activity was quantitated using the luciferase assay. Forskolin, 8-bromo-cAMP, and protein kinase A catalytic subunit expression all markedly increased transcription from the 3-kb promoter. Analyses with deletion mutants of the promoter showed that two cAMP-responsive element (CRE)-like sequences (−227 to −220 bp and −123 to −116 bp) contribute to this positive regulation. Expression of KCREB, a dominant negative mutant of the cAMP-responsive element binding protein (CREB), suggested the involvement of CREB. Transfection of the activator protein 2 (AP2) DNA consensus sequence partially blocked transcription. Dexamethasone suppressed forskolin-stimulated expression. The negative effect of glucocorticoid was GR dependent and may be mediated by a mechanism not involving GR binding to DNA because it was independent of the putative glucocorticoid-responsive element previously reported in the vasopressin promoter (−622 to −608 bp) and was preserved in the shorter promoter constructs in which no glucocorticoid-responsive element-like sequence was found. Our data suggest that several trans-acting factors including CREB, AP2, and GR are likely to be involved in vasopressin gene transcription and that the positive and negative regulation of vasopressin gene transcription is complex.

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Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function

Molecular and Cellular Biology ◽

10.1128/mcb.3.7.1295-1309.1983 ◽

1983 ◽

Vol 3 (7) ◽

pp. 1295-1309

Author(s):

R W Scott ◽

S M Tilghman

Keyword(s):

Transient Expression ◽

Hela Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Alpha Fetoprotein ◽

Tata Box ◽

Deletion Mutants ◽

Promoter Function ◽

Flanking Region

The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a heterologous system precludes its identification.

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gadd153/Chop10, a potential target gene of the transcriptional repressor ATF3.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6700 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6700-6707 ◽

Cited By ~ 118

Author(s):

C D Wolfgang ◽

B P Chen ◽

J L Martindale ◽

N J Holbrook ◽

T Hai

Keyword(s):

Binding Sites ◽

Target Gene ◽

Mrna Level ◽

Transcriptional Repressor ◽

Transient Transfection ◽

Negative Regulation ◽

Present Evidence ◽

Ccl4 Treatment

Recently, we demonstrated that the function of ATF3, a stress-inducible transcriptional repressor, is negatively regulated by a bZip protein, gadd153/Chop10. In this report, we present evidence that ATF3 can repress the expression of its own inhibitor, gadd153/Chop10. First, ATF3 represses a chloramphenicol acetyltransferase reporter gene driven by the gadd153/Chop10 promoter when assayed by a transfection assay in vivo and a transcription assay in vitro. Second, the gadd153/Chop10 promoter contains two functionally important binding sites for ATF3: an AP-1 site and a C/EBP-ATF composite site, a previously unidentified binding site for ATF3. The absence of either site reduces the ability of ATF3 to repress the promoter. Third, overexpression of ATF3 by transient transfection results in a reduction of the endogenous gadd153/Chop10 mRNA level. Fourth, as described previously, ATF3 is induced in the liver upon CCl4 treatment. Intriguingly, we show in this report that gadd153/Chop10 mRNA is not present in areas where ATF3 is induced. Taken together, these results strongly suggest that ATF3 represses the expression of gadd153/Chop10. The mutual negative regulation between ATF3 and gadd153/Chop10 is discussed.

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Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function.

Molecular and Cellular Biology ◽

10.1128/mcb.3.7.1295 ◽

1983 ◽

Vol 3 (7) ◽

pp. 1295-1309 ◽

Cited By ~ 25

Author(s):

R W Scott ◽

S M Tilghman

Keyword(s):

Transient Expression ◽

Hela Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Alpha Fetoprotein ◽

Tata Box ◽

Deletion Mutants ◽

Promoter Function ◽

Flanking Region

The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a heterologous system precludes its identification.

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On the interaction of cytosolic complexes of the glucocorticoid receptor and synthetic glucocorticoid responsive element

Problems of Endocrinology ◽

10.14341/probl199844341-44 ◽

1998 ◽

Vol 44 (3) ◽

pp. 41-44

Author(s):

E. I. Solenov

Keyword(s):

Glucocorticoid Receptor ◽

Conformational Changes ◽

Gene Promoter ◽

The Other ◽

Synthetic Oligonucleotide ◽

Sense Strand ◽

Antisense Sequence ◽

Glucocorticoid Responsive Element ◽

Responsive Element ◽

Synthetic Glucocorticoid

The constant of affinity between glucocorticoid receptor (GR) complex isolated together with triamcinolone and a synthetic oligonucleotide glucocorticoid-responsive element (GRE) corresponding to the GRE-1 sequence of the rat thyrosine aminotransferase gene promoter is assessed. Singlestranded oligonucleotides with the straight sequence (Kd 0.2 ± 0.006 pM) possess the highest affinity for GR. The affinity of single-stranded oligonucleotides with antisense sequence is far lower (Kd 1.5 ± 0.2 pM), but is superior to that of double-stranded oligo GRE (Kd 12.0+ 1.1 pM). The results suggest that the complexes are formed mainly with the sense strand of DNA; on the other hand, it is probable that the reaction of glucocorticoid receptor with GRE promotes conformational changes of DNA in this gene region.

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In vivo search for butyrate responsive sequences using transgenic mice carrying A gamma gene promoter mutants

Blood ◽

10.1182/blood.v88.3.1079.1079 ◽

1996 ◽

Vol 88 (3) ◽

pp. 1079-1083 ◽

Cited By ~ 28

Author(s):

BS Pace ◽

Q Li ◽

G Stamatoyannopoulos

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Messenger Rna ◽

Globin Gene ◽

Gene Promoter ◽

Gamma Globin ◽

Amino Butyric Acid ◽

Transgenic Lines ◽

Responsive Element

Abstract We describe an in vivo approach, in transgenic mice, aimed to identify promoter elements responsible for the induction of gamma globin expression by butyrate. Transgenic lines carrying human A gamma gene promoter truncations at position -141, -201, -382, and -730 A gamma were treated with alpha amino butyric acid (alpha ABA), and effects on gamma globin expression were analyzed at the messenger RNA level. No induction of gamma gene expression was observed in animals carrying promoters truncated at positions -141, -201, or -382 A gamma, suggesting either that butyrate response elements (BRE) are not located in the proximal gamma gene promoter or, if they were, they require the cooperation of upstream sequences for gamma gene induction. Two animals from one line carrying the -730 A gamma truncation responded to alpha ABA treatment with significant increases in gamma gene expression, indicating that a BRE is located between position -382 and -730 region of the A gamma gene promoter. Because the maximum induction by alpha ABA is observed in transgenic mice carrying a A gamma gene promoter extending to nucleotide -1350, it is likely that another butyrate responsive element is located between -730 and -1350 of the A gamma gene promoter. These results indicate that the transgenic mouse model can be used for identification of DNA regions that contain cis elements involved in gamma globin gene inducibility.

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Molecular analysis of the distal enhancer of the mouse alpha-fetoprotein gene.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3848 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3848-3856 ◽

Cited By ~ 26

Author(s):

J H Millonig ◽

J A Emerson ◽

J M Levorse ◽

S M Tilghman

Keyword(s):

Transgenic Mice ◽

Protein Binding ◽

Binding Site ◽

Fetal Liver ◽

Specific Protein ◽

Transient Transfection ◽

Alpha Fetoprotein ◽

Distal Enhancer ◽

Enhancer Function

The mouse alpha-fetoprotein (AFP) gene is transcribed at high levels in the visceral endoderm of the yolk sac and fetal liver and at much lower rates in the endoderm of the fetal gut. Expression of the gene in vivo requires the presence of at least one of three enhancers which lie in its 5' flanking region. In this report, we establish that the most distal AFP enhancer directed consistent expression of a linked AFP minigene in all three endodermal tissues in transgenic mice. The enhancer is composed of three domains, each of which is essential for full enhancer function by transient transfection assays. DNase I footprinting identified three regions of the enhancer which are protected by human hepatoma nuclear extracts, one of which corresponded to a consensus site for HNF-3 binding. Site-directed mutations in this site caused a 10-fold reduction in enhancer function by transient transfection. In transgenic mice, however, the mutation resulted in sporadic expression of the transgene, dependent on the site of integration. A similar acquisition of position-dependent sporadic expression of the transgene was observed with a mutation in a second protein binding site, despite the fact that this mutation had very little effect on enhancer function as assessed by transient transfection. These studies underscore the value of examining the functions of specific protein binding sites in vivo.

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SAT-450 Essential Role of GATA2 in the Negative Regulation of the Prepro-Thyrotropin-Releasing Hormone Gene by Liganded T3 in the Rat Paraventricular Nucleus

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.762 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Go Kuroda ◽

Shigekazu Sasaki ◽

Akio Matsushita ◽

Kenji Ohba ◽

Yuki Sakai ◽

...

Keyword(s):

Transcription Factor ◽

Paraventricular Nucleus ◽

Negative Regulation ◽

Thyrotropin Releasing Hormone ◽

Mobility Shift ◽

Releasing Hormone ◽

Melanocortin 4 Receptor ◽

Mobility Shift Assay ◽

Responsive Element

Abstract T3 inhibits thyrotropin-releasing hormone (TRH) synthesis in hypothalamic paraventricular nucleus (PVN). Although T3 receptor (TR) β2 is known to mediate the negative regulation of prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene indicate the tethering mechanism, where T3-bound TRβ2 interferes with the function of the transcription factor GATA2, which is essential for TSHβ expression. Interestingly, the transcription factor Sim1, a determinant of PVN differentiation in hypothalamus, is reported to induce the expressions of TRβ2 and GATA2. Indeed, our immunohistochemistry revealed the expression of GATA2 in the TRH neuron of the rat PVN. According to the experimental report with transgenic mice, the DNA sequence from nt. -547 to nt. +84 is sufficient for the expression of the prepro-TRH gene in PVN. Using the CAT reporter gene harboring this region, we found that this promoter is activated by GATA2 approximately 6-fold in CV1 cells. The deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRβ2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. T3-dependent repression was maintained after the mutation of the putative negative T3 responsive element (site4). Although the melanocortin 4 receptor signaling is known to stimulate the prepro-TRH promoter via protein kinase A pathway in the PVN, inhibition by T3 was dominant over the 8-bromo-cAMP-induced activation. We observed the in vivo recognition of GATA-RE by GATA2 using chromatin immunoprecipitation assay with CA77 cells, which express endogenous TRH. The electrophoretic mobility shift assay also demonstrated that GATA2 bound to oligonucleotide containing the GATA-RE. These results suggest that, as in the case of the TSHβ gene, GATA2 transactivates the prepro-TRH gene and that T3-bound TRβ2 interferes with its function, resulting in the negative regulation of this gene.

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In vivo search for butyrate responsive sequences using transgenic mice carrying A gamma gene promoter mutants

Blood ◽

10.1182/blood.v88.3.1079.bloodjournal8831079 ◽

1996 ◽

Vol 88 (3) ◽

pp. 1079-1083 ◽

Cited By ~ 1

Author(s):

BS Pace ◽

Q Li ◽

G Stamatoyannopoulos

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Messenger Rna ◽

Globin Gene ◽

Gene Promoter ◽

Gamma Globin ◽

Amino Butyric Acid ◽

Transgenic Lines ◽

Responsive Element

We describe an in vivo approach, in transgenic mice, aimed to identify promoter elements responsible for the induction of gamma globin expression by butyrate. Transgenic lines carrying human A gamma gene promoter truncations at position -141, -201, -382, and -730 A gamma were treated with alpha amino butyric acid (alpha ABA), and effects on gamma globin expression were analyzed at the messenger RNA level. No induction of gamma gene expression was observed in animals carrying promoters truncated at positions -141, -201, or -382 A gamma, suggesting either that butyrate response elements (BRE) are not located in the proximal gamma gene promoter or, if they were, they require the cooperation of upstream sequences for gamma gene induction. Two animals from one line carrying the -730 A gamma truncation responded to alpha ABA treatment with significant increases in gamma gene expression, indicating that a BRE is located between position -382 and -730 region of the A gamma gene promoter. Because the maximum induction by alpha ABA is observed in transgenic mice carrying a A gamma gene promoter extending to nucleotide -1350, it is likely that another butyrate responsive element is located between -730 and -1350 of the A gamma gene promoter. These results indicate that the transgenic mouse model can be used for identification of DNA regions that contain cis elements involved in gamma globin gene inducibility.

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