5-Aminolaevulinate synthase gene promoter contains two cAMP-response element (CRE)-like sites that confer positive and negative responsiveness to CRE-binding protein (CREB)

2001 ◽  
Vol 353 (2) ◽  
pp. 307-316 ◽  
Author(s):  
Luciana E. GIONO ◽  
Cecilia L. VARONE ◽  
Eduardo T. CÁNEPA
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Phosphorylation Site ◽  
Gene Promoter ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Putative Transcription Factor

The first and rate-controlling step of the haem biosynthetic pathway in mammals and fungi is catalysed by the mitochondrial-matrix enzyme 5-aminolaevulinate synthase (ALAS). The purpose of this work was to explore the molecular mechanisms involved in the cAMP regulation of rat housekeeping ALAS gene expression. Thus we have examined the ALAS promoter for putative transcription-factor-binding sites that may regulate transcription in a cAMP-dependent protein kinase (PKA)-induced context. Applying both transient transfection assays with a chloramphenicol acetyltransferase reporter gene driven by progressive ALAS promoter deletions in HepG2, and electrophoresis mobility-shift assays we have identified two putative cAMP-response elements (CREs) at positions -38 and -142. Functional analysis showed that both CRE-like sites were necessary for complete PKA induction, but only one for basal expression. Co-transfection with a CRE-binding protein (CREB) expression vector increased PKA-mediated induction of ALAS promoter transcriptional activity. However, in the absence of co-transfected PKA, CREB worked as a specific repressor for ALAS promoter activity. A CREB mutant deficient in a PKA phosphorylation site was unable to induce expression of the ALAS gene but could inhibit non-stimulated promoter activity. Furthermore, a DNA-binding mutant of CREB did not interfere with ALAS promoter basal activity. Site-directed-mutagenesis studies showed that only the nearest element to the transcription start site was able to inhibit the activity of the promoter. Therefore, we conclude that CREB, through its binding to CRE-like sites, mediates the effect of cAMP on ALAS gene expression. Moreover, we propose that CREB could also act as a repressor of ALAS transcription, but is able to reverse its role after PKA activation. Dephosphorylated CREB would interfere in a spatial-disposition-dependent manner with the transcriptional machinery driving inhibition of gene expression.

scholarly journals Pituitary homeobox 1 (Pitx1) stimulates rat LHβ gene expression via two functional DNA-regulatory regions

2005 ◽  
Vol 35 (1) ◽  
pp. 145-158 ◽  
Author(s):  
Qiaorong Jiang ◽  
Kyeong-Hoon Jeong ◽  
Cheryl D Horton ◽  
Lisa M Halvorson
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Gene Promoter ◽  
Orphan Nuclear Receptor ◽  
Mobility Shift ◽  
Regulatory Regions ◽  
Steroidogenic Factor 1 ◽  
Reproductive Axis ◽  
Functional Dna ◽  
Mobility Shift Assay

Luteinizing hormone (LH) plays a central role in the reproductive axis, stimulating both gonadal steroid biosynthesis and the development of mature gametes. Over the past decade, significant progress has been made in characterizing the transcription factors and associated DNA-regulatory sites which mediate expression of the LH β-subunit gene (LHβ). One of these factors, pituitary homeobox 1 (Pitx1), has been shown to stimulate LHβ gene promoter activity, both alone and in synergy with the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response gene 1 (Egr-1). Prior reports have attributed the Pitx1 response to a cis-element located at position -101 in the rat LHβ gene promoter. While investigating the role of Pitx1 in regulating rat LHβ gene expression, we observed a small, but significant, residual Pitx1 response despite mutation or deletion of this site. In the studies presented here, we identify the presence of a second functional Pitx1 region spanning positions −73 to −52 in the rat LHβ gene promoter. Based on electrophoretic mobility shift assay, Pitx1 binds to both the initially described 5′Pitx1 site as well as this putative 3′Pitx1 region. In transient transfection analysis, mutation of the LHβ-3′Pitx1 site significantly blunted Pitx1 responsiveness, with elimination of the Pitx1 response in a construct containing mutations in both Pitx1 cis-elements. We also analyzed the importance of each of these Pitx1 sites for providing functional synergy with SF-1 and with Egr-1. We observed a markedly decreased synergistic response with mutation of the 5′Pitx1 site with further loss following mutation of the 3′Pitx1 site. In contrast, functional interaction between Pitx1 and Egr-1 persisted with mutation of both Pitx1 regions. We conclude that Pitx1 stimulates the rat LHβ gene promoter via two Pitx1 DNA-regulatory regions. These results further our understanding of the molecular mechanisms that regulate expression of this critical reproductive gene promoter.

scholarly journals Amino acid limitation regulates gene expression

1999 ◽  
Vol 58 (3) ◽  
pp. 625-632 ◽  
Author(s):  
Alain Bruhat ◽  
Céline Jousse ◽  
Pierre Fafournoux
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Regulation Of Gene Expression ◽  
Essential Amino Acids ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Control Of Gene Expression

In mammals, the plasma concentration of amino acids is affected by nutritional or pathological conditions. For example, an alteration in the amino acid profile has been reported when there is a deficiency of any one or more of the essential amino acids, a dietary imbalance of amino acids, or an insufficient intake of protein. We examined the role of amino acid limitation in regulating mammalian gene expression. Depletion of arginine, cystine and all essential amino acids leads to induction of insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA and protein expression in a dose-dependent manner. Moreover, exposure of HepG2 cells to amino acids at a concentration reproducing the amino acid concentration found in portal blood of rats fed on a low-protein diet leads to a significantly higher (P < 0·0002) expression of IGFBP-1. Using CCAAT/enhancer-binding protein homologous protein (CHOP) induction by leucine deprivation as a model, we have characterized the molecular mechanisms involved in the regulation of gene expression by amino acids. We have shown that leucine limitation leads to induction of CHOP mRNA and protein. Elevated mRNA levels result from both an increase in the rate of CHOP transcription and an increase in mRNA stability. We have characterized two elements of the CHOP gene that are essential to the transcriptional activation produced by an amino acid limitation. These findings demonstrate that an amino acid limitation, as occurs during dietary protein deficiency, can induce gene expression. Thus, amino acids by themselves can play, in concert with hormones, an important role in the control of gene expression.

scholarly journals The RNA-Binding Protein CsrA Controls Virulence in Erwinia amylovora by Regulating RelA, RcsB, and FlhD at the Posttranscriptional Level

2019 ◽  
Vol 32 (10) ◽  
pp. 1448-1459 ◽  
Author(s):  
Jae Hoon Lee ◽  
Veronica Ancona ◽  
Tiyakhon Chatnaparat ◽  
Ho-wen Yang ◽  
Youfu Zhao
Keyword(s):  
Molecular Mechanisms ◽  
Erwinia Amylovora ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Direct Interaction ◽  
Antagonistic Effect ◽  
Site Directed Mutagenesis ◽  
Mobility Shift ◽  
Posttranscriptional Level

CsrA, an RNA-binding protein, binds to target transcripts and alters their translation or stability. In Erwinia amylovora, CsrA positively regulates the expression of type III secretion system (T3SS), exopolysaccharide amylovoran, and motility. In this study, the global effect of CsrA and its noncoding small RNA (ncsRNA) csrB in E. amylovora was determined by RNA-seq, and potential molecular mechanisms of CsrA-dependent virulence regulation were examined. Transcriptomic analyses under the T3SS-inducing condition revealed that mutation in the csrA gene led to differential expression of more than 20% of genes in the genome. Among them, T3SS genes and those required for cell growth and viability were significantly downregulated. On the other hand, the csrB mutant exhibited significant upregulation of most major virulence genes, suggesting an antagonistic effect of csrB on CsrA targets. Direct interaction between CsrA protein and csrB was further confirmed through the RNA electrophoretic mobility shift assay (REMSA). However, no direct interaction between CsrA and hrpL and hrpS transcripts was detected, suggesting that HrpL and HrpS are not targets of CsrA, whereas three CsrA targets (relA, rcsB, and flhD) were identified and confirmed by REMSA, site-directed mutagenesis, and LacZ reporter gene assays. These findings might partially explain how CsrA positively controls E. amylovora virulence by targeting major regulators at the posttranscriptional level.

Antagonistic effects of phorbol esters on insulin regulation of insulin-like growth factor-binding protein-1 (IGFBP-1) but not glucose-6-phosphatase gene expression

2001 ◽  
Vol 359 (3) ◽  
pp. 611-619 ◽  
Author(s):  
Satish PATEL ◽  
Pamela A. LOCHHEAD ◽  
Graham RENA ◽  
Calum SUTHERLAND
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Map Kinases ◽  
Phorbol Esters ◽  
Binding Protein ◽  
Gene Promoter ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Factor Binding

Glucose-6-phosphatase (G6Pase) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes contain a homologous promoter sequence that is required for gene repression by insulin. Interestingly, this element interacts with members of the forkhead family of transcription factors [e.g. HNF3 (hepatic nuclear factor 3), FKHR (forkhead in rhabdomyosarcoma)] in vitro, while insulin promotes the phosphorylation and inactivation of FKHR in a phosphatidylinositol 3-kinase- and protein kinase B (PKB)-dependent manner. This mechanism has been proposed to underlie insulin action on G6Pase and IGFBP-1 gene transcription. However, we find that treatment of cells with phorbol esters mimics the effect of insulin on G6Pase, but not IGFBP-1, gene expression. Indeed, phorbol ester treatment actually blocks the ability of insulin to repress IGFBP-1 gene expression. In addition, the action of phorbol esters is significantly reduced by inhibition of the p42/p44 mitogen-activated protein (MAP) kinase pathway. However insulin-induced phosphorylation of PKB or FKHR is not affected by the presence of phorbol esters. Therefore we suggest that activation of p42/p44 MAP kinases will reduce the sensitivity of the IGFBP-1 gene promoter, but not the G6Pase gene promoter, to insulin. Importantly, the activation of PKB and phosphorylation of FKHR is not, in itself, sufficient to reduce IGFBP-1 gene expression in the presence of phorbol esters.

scholarly journals Sequential Action of Ets-1 and Sp1 in the Activation of the Human β-1,4-Galactosyltransferase V Gene Involved in Abnormal Glycosylation Characteristic of Cancer Cells

2007 ◽  
Vol 282 (38) ◽  
pp. 27702-27712 ◽  
Author(s):  
Takeshi Sato ◽  
Kiyoshi Furukawa
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Cancer Cells ◽  
Transcriptional Activation ◽  
Promoter Activity ◽  
A549 Cells ◽  
Gene Promoter ◽  
Dominant Negative ◽  
Mobility Shift ◽  
V Gene

Malignant transformation is associated with increased gene expression of β-1,4-galactosyltransferase (β-1,4-GalT) V, which contributes to the biosynthesis of highly branched N-linked oligosaccharides characteristic of cancer cells. Our previous study showed that expression of the human β-1,4-GalT V gene is regulated by Sp1 (Sato, T., and Furukawa, K. (2004) J. Biol. Chem. 279, 39574–39583), and a subsequent study showed that the gene expression is also activated by Ets-1, a product of the oncogene (Sato, T., and Furukawa, K. (2005) Glycoconj. J. 22, 365). Herein we report the mechanism of β-1,4-GalT V gene activation by these transcription factors. The gene expression and promoter activity of β-1,4-GalT V increased when the ets-1 cDNA was transfected into A549 cells, which contain a small amount of Ets-1, but decreased dramatically when the dominant-negative ets-1 cDNA was transfected into HepG2 cells, which contain a large amount of Ets-1. Luciferase assays using deletion constructs of the β-1,4-GalT V gene promoter showed that promoter region –116 to +22 is critical for the transcriptional activation of the gene by Ets-1. Despite the presence of one Ets-1-binding site, which overlapped the Sp1-binding site, electrophoretic mobility shift assays showed that the region bound preferentially to Sp1 rather than to Ets-1. To solve this problem, we examined the transcriptional regulation of the human Sp1 gene by Ets-1 and found that the gene expression and promoter activity of Sp1 are regulated by Ets-1 in cancer cells. Functional analyses of two Ets-1-binding sites in the Sp1 gene promoter showed that only Ets-1-binding site –413 to –404 is involved in the activation of the gene by Ets-1. These results indicate that Ets-1 enhances expression of the β-1,4-GalT V gene through activation of the Sp1 gene in cancer cells.

scholarly journals GATA augments GNRH-mediated increases in Adcyap1 gene expression in pituitary gonadotrope cells

2013 ◽  
Vol 51 (3) ◽  
pp. 313-324 ◽  
Author(s):  
Robin L Thomas ◽  
Natalie M Crawford ◽  
Constance M Grafer ◽  
Weiming Zheng ◽  
Lisa M Halvorson
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Proximal Promoter ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Mobility Shift ◽  
Subunit Expression ◽  
Mobility Shift Assay

Pituitary adenylate cyclase-activating polypeptide 1 (PACAP or ADCYAP1) regulates gonadotropin biosynthesis and secretion, both alone and in conjunction with GNRH. Initially identified as a hypothalamic-releasing factor, ADCYAP1 subsequently has been identified in pituitary gonadotropes, suggesting it may act as an autocrine–paracrine factor in this tissue. GNRH has been shown to increase pituitaryAdcyap1gene expression through the interaction of CREB and jun/fos with CRE/AP1cis-elements in the proximal promoter. In these studies, we were interested in identifying additional transcription factors and cognatecis-elements which regulateAdcyap1gene promoter activity and chose to focus on the GATA family of transcription factors known to be critical for both pituitary cell differentiation and gonadotropin subunit expression. By transient transfection and electrophoretic mobility shift assay analysis, we demonstrate that GATA2 and GATA4 stimulateAdcyap1promoter activity via a GATAcis-element located at position −191 in the ratAdcyap1gene promoter. Furthermore, we show that addition of GATA2 or GATA4 significantly augments GNRH-mediated stimulation ofAdcyap1gene promoter activity in the gonadotrope LβT2 cell line. Conversely, blunting GATA expression with specific siRNA inhibits the ability of GNRH to stimulate ADCYAP1 mRNA levels in these cells. These data demonstrate a complex interaction between GNRH and GATA on ADCYAP1 expression, providing important new insights into the regulation of gonadotrope function.

scholarly journals PKC-dependent stimulation of the human MCT1 promoter involves transcription factor AP2

2009 ◽  
Vol 296 (2) ◽  
pp. G275-G283 ◽  
Author(s):  
Seema Saksena ◽  
Alka Dwivedi ◽  
Ravinder K. Gill ◽  
Amika Singla ◽  
Waddah A. Alrefai ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Core Promoter ◽  
Human Colon ◽  
Short Chain Fatty Acids ◽  
Site Directed Mutagenesis ◽  
Monocarboxylate Transporter ◽  
Dependent Manner ◽  
Stimulation Of

Monocarboxylate transporter (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFA) such as butyrate in the human colon. Previous studies from our laboratory have demonstrated that phorbol ester, PMA (1 μM, 24 h), upregulates butyrate transport and MCT1 protein expression in human intestinal Caco-2 cells. However, the molecular mechanisms involved in the transcriptional regulation of MCT1 gene expression by PMA in the intestine are not known. In the present study, we showed that PMA (0.1 μM, 24 h) increased the MCT1 promoter activity (−871/+91) by approximately fourfold. A corresponding increase in MCT1 mRNA abundance in response to PMA was also observed. PMA-induced stimulation of MCT1 promoter activity was observed as early as 1 h and persisted until 24 h, suggesting that the effects of PMA are attributable to initial PKC activation. Kinase inhibitor and phosphorylation studies indicated that these effects may be mediated through activation of the atypical PKC-ζ isoform. 5′-deletion studies demonstrated that the MCT1 core promoter region (−229/+91) is the PMA-responsive region. Site-directed mutagenesis studies showed the predominant involvement of potential activator protein 2 (AP2) binding site in the activation of MCT1 promoter activity by PMA. In addition, overexpression of AP2 in Caco-2 cells significantly increased MCT1 promoter activity in a dose-dependent manner. These findings showing the regulation of MCT1 promoter by PKC and AP2 are of significant importance for an understanding of the molecular regulation of SCFA absorption in the human intestine.

scholarly journals Regulation of the Rat Thyrotropin Receptor Gene by the Methylation-Sensitive Transcription Factor GA-Binding Protein

1998 ◽  
Vol 12 (8) ◽  
pp. 1241-1249 ◽  
Author(s):  
Norihiko Yokomori ◽  
Masato Tawata ◽  
Tukasa Saito ◽  
Hiroki Shimura ◽  
Toshimasa Onaya
Keyword(s):  
Transcription Factor ◽  
Binding Protein ◽  
Gene Promoter ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Cpg Sites ◽  
Nuclear Extracts ◽  
Tshr Gene ◽  
Gel Mobility Shift ◽  
Ga Binding Protein

Abstract The GA-binding protein (GABP), a transcription factor with a widespread tissue distribution, consists of two subunits,α and β1, and acts as a potent positive regulator of various genes. The effect of GABP on transcription of the TSH receptor (TSHR) gene in rat FRTL-5 thyroid cells has now been investigated. Both deoxyribonuclease I footprint analysis and gel mobility-shift assays indicated that bacterially expressed glutathione S-transferase fusion proteins of GABP subunits bind to a region spanning nucleotides (nt) −116 to −80 of the TSHR gene. In gel mobility-shift assays, nuclear extracts of FRTL-5 cells and FRT cells yielded several specific bands with a probe comprising nt −116 to− 80. Supershift assays with antibodies to GABPα and to GABPβ1 showed that GABP was a component of the probe complexes formed by the nuclear extracts. Immunoblot analysis confirmed the presence of both GABP subunits in the nuclear extracts. A reporter gene construct containing the TSHR gene promoter was activated, in a dose-dependent manner, in FRTL-5 cells by cotransfection with constructs encoding both GABPα and GABPβ1. Both GABP binding to and activation of the TSHR gene promoter were prevented by methylation of CpG sites at nt −93 and− 85. These CpG sites were highly methylated (&gt;82%) in FRT cells and completely demethylated in FRTL-5 cells, consistent with expression of the TSHR gene in the latter, but not the former. These results suggest that GABP regulates transcription of the TSHR gene in a methylation-dependent manner and that methylation of specific CpG sites and the methylation sensitivity of GABP contribute to the failure of FRT cells to express the endogenous TSHR gene.

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

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.

scholarly journals DNA Binding-Independent Induction of IκBα Gene Transcription by PPARα

2002 ◽  
Vol 16 (5) ◽  
pp. 1029-1039 ◽  
Author(s):  
Philippe Delerive ◽  
Karolien De Bosscher ◽  
Wim Vanden Berghe ◽  
Jean-Charles Fruchart ◽  
Guy Haegeman ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Transcriptional Repression ◽  
Energy Homeostasis ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Response Element

Abstract PPARs are ligand-activated transcription factors that regulate energy homeostasis. In addition, PPARs furthermore control the inflammatory response by antagonizing the nuclear factor-κB (NF-κB) signaling pathway. We recently demonstrated that PPARα activators increase IκBα mRNA and protein levels in human aortic smooth muscle cells. Here, we studied the molecular mechanisms by which PPARα controls IκBα expression. Using transient transfection assays, it is demonstrated that PPARα potentiates p65-stimulated IκBα transcription in a ligand-dependent manner. Site-directed mutagenesis experiments revealed that PPARα activation of IκBα transcription requires the NF-κB and Sp1 sites within IκBα promoter. Chromatin immunoprecipitation assays demonstrate that PPARα activation enhances the occupancy of the NF-κB response element in IκBα promoter in vivo. Overexpression of the oncoprotein E1A failed to inhibit PPARα-mediated IκBα promoter induction, suggesting that cAMP response element binding protein-binding protein/p300 is not involved in this mechanism. By contrast, a dominant-negative form of VDR-interacting protein 205 (DRIP205) comprising its two LXXLL motifs completely abolished PPARα ligand-mediated activation. Furthermore, cotransfection of increasing amounts of DRIP205 relieved this inhibition, suggesting that PPARα requires DRIP205 to regulate IκBα promoter activity. By contrast, DRIP205 is not involved in PPARα-mediated NF-κB transcriptional repression. Taken together, these data provide a molecular basis for PPARα-mediated induction of IκBα and demonstrate, for the first time, that PPARα may positively regulate gene transcription in the absence of functional PPAR response elements.

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