Involvement of the ubiquitous Oct-1 transcription factor in hormonal induction of β-casein gene expression

Transcription of the milk protein β-casein gene is induced by the lactogenic hormones Prl (prolactin) and glucocorticoids. Multiple transcription factors involved in this induction have been identified, including the STAT5 (signal transducer and activator of transcription 5) and the GR (glucocorticoid receptor). Our previous studies have identified a binding site for the ubiquitous Oct-1 (octamer-binding transcription factor 1) protein in the lactogenic hormonal regulatory region of the mouse β-casein promoter. In the present study, we report that Oct-1 is indeed expressed and binds to the β-casein promoter in mammary epithelial cells. Oct-1 activates hormonally induced β-casein promoter activity in a dose-dependent manner. Hormonal induction of promoter activity was decreased not only by mutating the Oct-1-binding site from ATTAGCAT to GCTAGCAT, which abolishes Oct-1 binding (50% decrease, P<0.01), but also by changing the site to the consensus Oct-1-binding motif ATTTGCAT (40% decrease, P<0.01). Reversing the Oct-1-binding site reduced hormonal induction by 70% (P<0.01), showing that orientation of Oct-1 binding is also critical in hormonal action. In transient transfection experiments, Oct-1 collaboratively transactivated the β-casein gene promoter with STAT5 and/or GR in the presence of Prl receptor in cells treated with the lactogenic hormones. The C-terminus of Oct-1 was not essential to its function. The results of the present study provide biochemical evidence that the ubiquitous Oct-1 transcription factor may be involved in hormonally regulated, tissue-specific β-casein gene expression.

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Copper Induces Cytoplasmic Retention of Fission Yeast Transcription Factor Cuf1

Eukaryotic Cell ◽

10.1128/ec.5.2.277-292.2006 ◽

2006 ◽

Vol 5 (2) ◽

pp. 277-292 ◽

Cited By ~ 27

Author(s):

Jude Beaudoin ◽

Simon Labbé

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Nuclear Localization ◽

Copper Transport ◽

Nuclear Localization Sequence ◽

Binding Motif ◽

Physical Interaction ◽

Dependent Manner ◽

C Terminus ◽

N Terminus

ABSTRACT Copper homeostasis within the cell is established and preserved by different mechanisms. Changes in gene expression constitute a way of maintaining this homeostasis. In Schizosaccharomyces pombe, the Cuf1 transcription factor is critical for the activation of copper transport gene expression under conditions of copper starvation. However, in the presence of elevated intracellular levels of copper, the mechanism of Cuf1 inactivation to turn off gene expression remains unclear. In this study, we provide evidence that inactivation of copper transport gene expression by Cuf1 is achieved through a copper-dependent, cytosolic retention of Cuf1. We identify a minimal nuclear localization sequence (NLS) between amino acids 11 to 53 within the Cuf1 N terminus. Deletion of this region and specific mutation of the Lys13, Arg16, Arg19, Lys24, Arg28, Lys45, Arg47, Arg50, and Arg53 residues to alanine within this putative NLS is sufficient to abrogate nuclear targeting of Cuf1. Under conditions of copper starvation, Cuf1 resides in the nucleus. However, in the presence of excess copper as well as silver ions, Cuf1 is sequestered in the cytoplasm, a process which requires the putative copper binding motif, 328Cys-X-Cys-X3-Cys-X-Cys-X2-Cys-X2-His342 (designated C-rich), within the C-terminal region of Cuf1. Deletion of this region and mutation of the Cys residues within the C-rich motif result in constitutive nuclear localization of Cuf1. By coexpressing the Cuf1 N terminus with its C terminus in trans and by using a two-hybrid assay, we show that these domains physically interact with each other in a copper-dependent manner. We propose a model wherein copper induces conformational changes in Cuf1 that promote a physical interaction between the Cuf1 N terminus and the C-rich motif in the C terminus that masks the NLS. Cuf1 is thereby sequestered in the cytosol under conditions of copper excess, thereby extinguishing copper transport gene expression.

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Regulation of the nitrate reductase operon narKGHJI by the cAMP-dependent regulator GlxR in Corynebacterium glutamicum

Microbiology ◽

10.1099/mic.0.044552-0 ◽

2011 ◽

Vol 157 (1) ◽

pp. 21-28 ◽

Cited By ~ 13

Author(s):

Taku Nishimura ◽

Haruhiko Teramoto ◽

Koichi Toyoda ◽

Masayuki Inui ◽

Hideaki Yukawa

Keyword(s):

Nitrate Reductase ◽

Binding Site ◽

Corynebacterium Glutamicum ◽

Promoter Region ◽

Promoter Activity ◽

Reductase Activity ◽

Receptor Protein ◽

Binding Motif ◽

Dependent Manner ◽

Mobility Shift

The Corynebacterium glutamicum anaerobic nitrate reductase operon narKGHJI is repressed by a transcriptional regulator, ArnR, under aerobic conditions. A consensus binding site of the cAMP receptor protein (CRP)-type regulator, GlxR, was recently found upstream of the ArnR binding site in the narK promoter region. Here we investigated the involvement of GlxR and cAMP in expression of the narKGHJI operon in vivo. Electrophoretic mobility shift assays showed that the putative GlxR binding motif in the narK promoter region is essential for the cAMP-dependent binding of GlxR. Promoter-reporter assays showed that mutation in the GlxR binding site resulted in significant reduction of narK promoter activity. Furthermore, a deletion mutant of the adenylate cyclase gene cyaB, which is involved in cAMP synthesis, exhibited a decrease in both narK promoter activity and nitrate reductase activity. These results demonstrated that C. glutamicum GlxR positively regulates narKGHJI expression in a cAMP-dependent manner.

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Cyclosporin A-Sensitive Transcription Factor Egr-3 Regulates Fas Ligand Expression

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.3744 ◽

1998 ◽

Vol 18 (7) ◽

pp. 3744-3751 ◽

Cited By ~ 118

Author(s):

Paul R. Mittelstadt ◽

Jonathan D. Ashwell

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Dna Binding ◽

Cyclosporin A ◽

Binding Site ◽

Transient Expression ◽

Promoter Activity ◽

Fas Ligand ◽

Regulatory Element ◽

Ligand Expression

ABSTRACT Activation-induced transcriptional upregulation of the ligand for Fas (FasL) and the resulting apoptosis of Fas-bearing cells constitute essential steps in a host of normal and pathological processes. Here we describe an activation-inducible cis-acting regulatory element in the fasL promoter that is required for gene expression. Oligonucleotide competition and antibody supershift analyses identified two activation-induced DNA-binding species: Egr-1 (NGFI-A, krox-24, zif268, TIS-8), a transcription factor that has been implicated in growth, differentiation, and apoptosis; and Egr-3 (PILOT), a transcription factor of no previously known function. Activation-induced expression of Egr-3, like that of FasL, was inhibited by cyclosporin A, whereas expression of Egr-1 was unaffected. Transient expression of Egr-3 alone increased fasL promoter activity in a cyclosporin A-insensitive manner, whereas expression of Egr-1 had little effect. Moreover, endogenous fasL mRNA was induced in nonlymphoid cells by forced expression of Egr-3 in the absence of any other stimulus. These studies identify a critical Egr family-binding site in the fasL promoter and demonstrate that activation-induced Egr-3, but not Egr-1, directly upregulatesfasL transcription in response to activating stimuli.

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The Zinc Finger Transcription Factor Gli2 Mediates Bone Morphogenetic Protein 2 Expression in Osteoblasts in Response to Hedgehog Signaling

Molecular and Cellular Biology ◽

10.1128/mcb.02214-05 ◽

2006 ◽

Vol 26 (16) ◽

pp. 6197-6208 ◽

Cited By ~ 93

Author(s):

Ming Zhao ◽

Mei Qiao ◽

Stephen E. Harris ◽

Di Chen ◽

Babatunde O. Oyajobi ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Bone Morphogenetic Protein ◽

Hedgehog Signaling ◽

Osteoblast Differentiation ◽

Promoter Activity ◽

Bone Morphogenetic Protein 2 ◽

Dependent Manner ◽

Genetic Ablation ◽

Morphogenetic Protein

ABSTRACTBone morphogenetic protein 2 (BMP-2) plays a critical role in osteoblast function. InDrosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression ofdpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. We hypothesize that Gli2 regulates BMP-2 gene transcription and thus osteoblast differentiation. In the present study, we show that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in osteoblast precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal osteoblast differentiation.

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MON-711 Induction of Apolipoprotein A1 Gene Expression by the Rare Sugar Allulose

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.830 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Shrina Parekh

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Promoter Activity ◽

Gene Promoter ◽

Apolipoprotein A1 ◽

Mrna Levels ◽

Dependent Manner ◽

Rare Sugar ◽

Transcription Factor Sp1 ◽

I Gene

Abstract Apolipoprotein A-I (apo A-I) is the primary protein component of high-density lipoprotein (HDL) and has many well documented properties which promote cardiovascular health. However, clinical trials designed to increase HDL levels by preventing its catabolism have failed in their primary endpoints in decreasing the risk of cardiovascular disease. Alternative strategies to increase de-novo apo A-I production may be more attractive. We recently demonstrated that the rare sugar allulose decreases oxidative stress and endoplasmic reticulum stress in both endothelial cells and hepatocytes. During these studies we demonstrated that allulose also induces apo A-I secretion by HepG2 cells. Apo A-I, albumin, and SP1 levels were measured by Western blot. Apo A-I and glyceraldehyde-3-phosphate (GAPDH) mRNA levels were measured by quantitative real-time polymerase chain reaction. The effect of allulose on apo A-I promoter activity was measured using transient transfection assays with several plasmids containing various segments and mutations in the apo A-I gene promoter. Apo A-I protein and mRNA levels in cells treated with allulose increased more than two-fold in a dose-dependent manner. These changes were due to the ability of allulose to induce apo A-I gene promoter activity. Using a series of deletion constructs, an allulose-response element was identified in the apo A-I gene promoter which was previously shown to confer induction of apo A-I gene expression by insulin and epidermal growth factor (EGF), the insulin response core element (IRCE). Mutation of the IRCE decreased the ability of allulose and insulin to induce apo A-I promoter activity. Allulose treatment also increased expression of the transcription factor SP1, which had been shown previously be essential for the effects of insulin and EGF on apo A-I promoter activity. In conclusion, allulose increased apo A-I gene expression in HepG2 hepatocytes. This effect was mediated by the IRCE in the apo A-I gene promoter and the transcription factor SP1. The rare sugar allulose may have novel anti-atherogenic properties, in part, by increasing HDL levels.

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DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells

Genes ◽

10.3390/genes12060853 ◽

2021 ◽

Vol 12 (6) ◽

pp. 853

Author(s):

Siti Aisyah Faten Mohamed Sa’dom ◽

Sweta Raikundalia ◽

Shaharum Shamsuddin ◽

Wei Cun See Too ◽

Ling Ling Few

Keyword(s):

Dna Methylation ◽

Transcription Factor ◽

Binding Site ◽

Promoter Activity ◽

Cytosine Methylation ◽

Cpg Island ◽

Cpg Islands ◽

Site Directed Mutagenesis ◽

Choline Kinase ◽

Mcf 7

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

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Hesx1 homeodomain protein represses transcription as a monomer and antagonises transactivation of specific sites as a homodimer

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0280193 ◽

2002 ◽

Vol 28 (3) ◽

pp. 193-205 ◽

Cited By ~ 10

Author(s):

J Quirk ◽

P Brown

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Site ◽

Anterior Pituitary ◽

Homeodomain Protein ◽

Dependent Manner ◽

Gene Promoters ◽

Differentiation Markers ◽

Proximal Half ◽

Dna Binding Site

The homeobox repressor Hesx1, expressed throughout Rathke's pouch and required for normal pituitary development, has been implicated in anterior pituitary pathogenesis in man. Prolonged expression of Hesx1 delays the appearance of anterior pituitary terminal differentiation markers in mice, particularly the gonadotroph hormones. We tested if Hesx1 could modulate gonadotrophin gene expression directly, and found that Hesx1 repressed both common alpha subunit (alpha GSU) and luteinising hormone beta-subunit (LH beta) gene promoters. Repression mapped to the Pitx1 homeodomain protein transactivation site in the proximal alpha GSU promoter, but did not map to the equivalent site on LH beta. Hesx1 repression of the alpha GSU Pitx1 site was overridden by co-transfection of Pitx1. In contrast, Hesx1 antagonised Pitx1 transactivation of LH beta in a dose-dependent manner. This was due to monomeric binding of Hesx1 on alpha GSU and homodimerisation on LH beta. The homodimerisation site comprises the Pitx1 DNA binding site and a proximal binding site, and mutation of either inhibited homodimer formation. Conversion of the LH beta Pitx1 DNA binding site to an alpha GSU-type did not promote homodimer formation, arguing that Hesx1 has pronounced site selectivity. Furthermore, mutation of the proximal half of the homodimerisation site blocked Hesx1 antagonisation of Pitx1 transactivation. We conclude that Hesx1 monomers repress gene expression, and homodimers block specific transactivation sites.

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Fibroblast Growth Factors Regulate Prolactin Transcription via an Atypical Rac-Dependent Signaling Pathway

Molecular Endocrinology ◽

10.1210/me.2003-0167 ◽

2003 ◽

Vol 17 (10) ◽

pp. 1921-1930 ◽

Cited By ~ 12

Author(s):

Twila A. Jackson ◽

David M. Koterwas ◽

Melissa A. Morgan ◽

Andrew P. Bradford

Keyword(s):

Gene Expression ◽

Growth Factors ◽

Signaling Pathway ◽

Promoter Activity ◽

Fibroblast Growth Factors ◽

Dominant Negative ◽

Pituitary Cells ◽

Dependent Manner ◽

Fibroblast Growth ◽

Prl Gene

Abstract Fibroblast growth factors (FGFs) play a critical role in pituitary development and in pituitary tumor formation and progression. We have previously characterized FGF signal transduction and regulation of the tissue-specific rat prolactin (rPRL) promoter in GH4 pituitary cells. FGF induction of rPRL transcription is independent of Ras, but mediated by a protein kinase C-δ (PKCδ)-dependent activation of MAPK (ERK). Here we demonstrate a functional role for the Rho family monomeric G protein, Rac1, in FGF regulation of PRL gene expression via an atypical signaling pathway. Expression of dominant negative Rac, but not RhoA or Cdc42, selectively inhibited FGF-induced rPRL promoter activity. Moreover, expression of dominant negative Rac also attenuated FGF-2 and FGF-4 stimulation of MAPK (ERK). However, in contrast to other Rac-dependent signaling pathways, FGF activation of rPRL promoter activity was independent of the c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase/Akt cascades. FGFs failed to activate JNK1 or JNK2, and expression of dominant negative JNK or Akt constructs did not block FGF-induced PRL transcription. Consistent with the role of PKCδ in FGF regulation of PRL gene expression, activation of the rPRL promoter was blocked by an inhibitor of phospholipase Cγ (PLCγ) activity. FGF treatment also induced rapid tyrosine phosphorylation of PLCγ in a Rac-dependent manner. These results suggest that FGF-2 and FGF-4 activate PRL gene expression via a novel Rac1, PLCγ, PKCδ, and ERK cascade, independent of phosphoinositol-3-kinase and JNK.

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Synergistic activation of ADH2 expression is sensitive to upstream activation sequence 2 (UAS2) orientation, copy number and UAS1-UAS2 helical phasing.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3442 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3442-3449 ◽

Cited By ~ 16

Author(s):

M S Donoviel ◽

N Kacherovsky ◽

E T Young

Keyword(s):

Gene Expression ◽

Binding Site ◽

Reporter Gene ◽

Copy Number ◽

Glucose Repression ◽

Regulatory Protein ◽

Regulatory Region ◽

Upstream Regulatory Region ◽

Specific Sequence

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression. Two cis-acting upstream activation sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site for the transcriptional regulator Adr1p. UAS2 has been shown to be important for ADH2 expression and confers glucose-regulated, ADR1-independent activity to a heterologous reporter gene. An analysis of point mutations within UAS2, in the context of the entire ADH2 upstream regulatory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein. In the context of the ADH2 upstream regulatory region, including UAS1, working in concert with the ADH2 basal promoter elements, UAS2-dependent gene activation was dependent on orientation, copy number, and helix phase. Multimerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent expression of a reporter gene containing the ADH2 basal promoter and coding sequence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1. Inhibition of UAS2-dependent gene expression by Adr1p was also observed with a UAS2-dependent ADH2 reporter gene. This inhibition increased with ADR1 copy number and required the DNA-binding activity of Adr1p. Specific but low-affinity binding of Adr1p to UAS2 in vitro was demonstrated, suggesting that the inhibition of UAS2-dependent gene expression observed in vivo could be a direct effect due to Adr1p binding to UAS2.

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New Insights into the Pleiotropic Drug Resistance Network from Genome-Wide Characterization of the YRR1 Transcription Factor Regulation System

Molecular and Cellular Biology ◽

10.1128/mcb.22.8.2642-2649.2002 ◽

2002 ◽

Vol 22 (8) ◽

pp. 2642-2649 ◽

Cited By ~ 68

Author(s):

Stéphane Le Crom ◽

Frédéric Devaux ◽

Philippe Marc ◽

Xiaoting Zhang ◽

W. Scott Moye-Rowley ◽

...

Keyword(s):

Drug Resistance ◽

Transcription Factor ◽

Binding Site ◽

Time Course ◽

Target Genes ◽

Regulation System ◽

Dependent Manner ◽

Pleiotropic Drug Resistance ◽

Genome Wide

ABSTRACT Yrr1p is a recently described Zn2Cys6 transcription factor involved in the pleiotropic drug resistance (PDR) phenomenon. It is controlled in a Pdr1p-dependent manner and is autoregulated. We describe here a new genome-wide approach to characterization of the set of genes directly regulated by Yrr1p. We found that the time-course production of an artificial chimera protein containing the DNA-binding domain of Yrr1p activated the 15 genes that are also up-regulated by a gain-of-function mutant of Yrr1p. Gel mobility shift assays showed that the promoters of the genes AZR1, FLR1, SNG1, YLL056C, YLR346C, and YPL088W interacted with Yrr1p. The putative consensus Yrr1p binding site deduced from these experiments, (T/A)CCG(C/T)(G/T)(G/T)(A/T)(A/T), is strikingly similar to the PDR element binding site sequence recognized by Pdr1p and Pdr3p. The minor differences between these sequences are consistent with Yrr1p and Pdr1p and Pdr3p having different sets of target genes. According to these data, some target genes are directly regulated by Pdr1p and Pdr3p or by Yrr1p, whereas some genes are indirectly regulated by the activation of Yrr1p. Some genes, such as YOR1, SNQ2, and FLR1, are clearly directly controlled by both classes of transcription factor, suggesting an important role for the corresponding membrane proteins.

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