scholarly journals Characterization and Regulation of the Rat and Human Ghrelin Promoters

Endocrinology ◽  
2005 ◽  
Vol 146 (3) ◽  
pp. 1611-1625 ◽  
Author(s):  
Wei Wei ◽  
Guiyun Wang ◽  
Xiang Qi ◽  
Ella W. Englander ◽  
George H. Greeley
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Core Promoter ◽  
Gh3 Cells ◽  
Ags Cells ◽  
The Core ◽  
Intron 1 ◽  
Proximal Site ◽  
Exon 1

Ghrelin is a recently discovered stomach hormone and endogenous ligand for the GH secretagogue receptor. The aim of these studies is to elucidate molecular mechanisms underlying regulation of the ghrelin gene. Distal and proximal transcription initiation sites are present. A short transcript, a product of the proximal site, showed a more widespread distribution. Two sets of 5′-upstream segments of the rat and human ghrelin genes were cloned and sequenced. Rat promoter segments upstream of the distal site showed highest activity in kidney (COS-7) and stomach (AGS) cells, whereas human promoter segments upstream of the proximal site showed highest activity in AGS and pituitary (GH3) cells in transient transfection assays. For the human, the core promoter spanned −667 to −468 bp, including the noncoding exon 1 and a short 5′ sequence of intron 1. For the rat, the core promoter spanned −581 to −469 bp, and inclusion of exon 1 and a short 5′-sequence of intron 1 reduced activity by 67%. Mutation of initiator-like elements in the rat lowered activity by 20–50%, whereas in the human, all activity was abolished. Overexpression of upstream stimulatory factors increased ghrelin core promoter activity. Fasting increases stomach ghrelin expression, glucagon-a fasting-induced hormone, increased ghrelin expression in vivo in rats, and promoter activity by approximately 25–50%. Together, these findings indicate that structural differences between the rat and human ghrelin core promoters may account in part for the differences in their transcriptional regulation. Nonetheless, upstream stimulatory factor and glucagon exert similar effects on regulation of rat and human ghrelin promoters.

Genomic structure and promoter characterization of the human Sprouty4 gene, a novel regulator of lung morphogenesis

2004 ◽  
Vol 287 (1) ◽  
pp. L52-L59 ◽  
Author(s):  
Wei Ding ◽  
Saverio Bellusci ◽  
Wei Shi ◽  
David Warburton
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Core Promoter ◽  
Genomic Structure ◽  
Inducible Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Significant Similarity ◽  
The Core ◽  
Flanking Region

The expression of Sprouty4 ( Spry4), an intracellular FGF receptor antagonist, shows a temporally and spatially restricted pattern in embryonic lung and is induced by ERK signaling. To clarify the molecular mechanisms regulating Spry4 transcription, the genomic structure of the human Sprouty4 ( hSpry4) gene was first determined by using the GenomeWalker kit. The hSpry4 gene spans > 14 kb and is organized in three exons and two introns. Multiple transcription start sites were subsequently mapped by 5′-rapid amplification of cDNA ends. Analysis of up to 4 kb of sequence in the 5′-flanking region of the gene showed the presence of multiple potential transcription factor binding sites but no TATA or CAAT boxes. Transient transfection using luciferase reporter gene constructs with progressive deletions of the hSpry4 5′-flanking region revealed that the core promoter activity is located within the proximal 0.4-kb region, whereas the minimal ERK-inducible promoter activity is between −69 and −31. Homology analysis further showed that the core promoter region of the hSpry4 gene exhibits significant similarity to the 5′-flanking region of the mouse gene.

scholarly journals Investigation of the Wilson gene ATP7B transcriptional start site and the effect of core promoter alterations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Clemens Höflich ◽  
Angela Brieger ◽  
Stefan Zeuzem ◽  
Guido Plotz
Keyword(s):  
Wilson Disease ◽  
Transcription Initiation ◽  
Transcriptional Activity ◽  
Core Promoter ◽  
Transcriptional Start Site ◽  
Copper Metabolism ◽  
Biologically Relevant ◽  
The Core ◽  
Translational Start

AbstractPathogenic genetic variants in the ATP7B gene cause Wilson disease, a recessive disorder of copper metabolism showing a significant variability in clinical phenotype. Promoter mutations have been rarely reported, and controversial data exist on the site of transcription initiation (the core promoter). We quantitatively investigated transcription initiation and found it to be located in immediate proximity of the translational start. The effects human single-nucleotide alterations of conserved bases in the core promoter on transcriptional activity were moderate, explaining why clearly pathogenic mutations within the core promoter have not been reported. Furthermore, the core promoter contains two frequent polymorphisms (rs148013251 and rs2277448) that could contribute to phenotypical variability in Wilson disease patients with incompletely inactivating mutations. However, neither polymorphism significantly modulated ATP7B expression in vitro, nor were copper household parameters in healthy probands affected. In summary, the investigations allowed to determine the biologically relevant site of ATP7B transcription initiation and demonstrated that genetic variations in this site, although being the focus of transcriptional activity, do not contribute significantly to Wilson disease pathogenesis.

scholarly journals Binding of Phosphorylated Sp1 Protein to Tandem Sp1 Binding Sites Regulates α2 Integrin Gene Core Promoter Activity

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 678-689 ◽  
Author(s):  
Mary M. Zutter ◽  
Ellen E. Ryan ◽  
Audrey D. Painter
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Binding Sites ◽  
Promoter Activity ◽  
Core Promoter ◽  
K562 Cells ◽  
The Core ◽  
Nuclear Extracts ◽  
Integrin Gene ◽  
Α2β1 Integrin

Abstract The α2β1 integrin, a collagen/laminin receptor, is expressed by a variety of cell types, including epithelial cells, mesenchymal cells, and hematopoietic cells. To understand the molecular mechanisms that regulate expression of the α2β1 integrin in cells with megakaryocytic differentiation, we characterized the 5′ flanking region of the α2 integrin gene and identified three distinct regulatory regions, including a core promoter, a silencer, and megakaryocyte enhancers in the distal 5′ flank (Zutter et al, Blood 96:3006, 1995 and Zutter et al, J Biol Chem 269:463, 1994). We now focus on the core promoter of the α2 integrin gene located between bp −30 and −92 that is required for transcriptional activity of the α2 integrin gene. Sequence analysis identified two Sp1 consensus sites and a potential AP2 site. Gel retardation assays showed that nuclear proteins from uninduced K562 cells and K562 cells induced to become megakaryocytic bound specifically to the core promoter region (bp −30 to bp −92) producing two DNA-protein complexes. In addition, nuclear extracts from cells induced along the megakaryocyte lineage produced a selective increase in the slower migrating complex. Site-directed mutagenesis of the 5′, the 3′, or both Sp1 binding sites suggested that both Sp1 binding sites are required for full promoter activity and for DNA-protein complex formation. DNA footprinting also showed specific protection of the 5′ Sp1 site by nuclear extracts from uninduced K562 cells and protection of both the 5′ and the 3′ Sp1 sites by nuclear extracts from induced K562 cells. Sp1 protein-DNA complex formation was dependent on Sp1 phosphorylation. The faster migrating DNA-protein complex was enhanced by dephosphorylation; the slower migrating DNA-protein complex was diminished or lost.

Binding of Phosphorylated Sp1 Protein to Tandem Sp1 Binding Sites Regulates α2 Integrin Gene Core Promoter Activity

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 678-689 ◽  
Author(s):  
Mary M. Zutter ◽  
Ellen E. Ryan ◽  
Audrey D. Painter
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Binding Sites ◽  
Promoter Activity ◽  
Core Promoter ◽  
K562 Cells ◽  
The Core ◽  
Nuclear Extracts ◽  
Integrin Gene ◽  
Α2β1 Integrin

The α2β1 integrin, a collagen/laminin receptor, is expressed by a variety of cell types, including epithelial cells, mesenchymal cells, and hematopoietic cells. To understand the molecular mechanisms that regulate expression of the α2β1 integrin in cells with megakaryocytic differentiation, we characterized the 5′ flanking region of the α2 integrin gene and identified three distinct regulatory regions, including a core promoter, a silencer, and megakaryocyte enhancers in the distal 5′ flank (Zutter et al, Blood 96:3006, 1995 and Zutter et al, J Biol Chem 269:463, 1994). We now focus on the core promoter of the α2 integrin gene located between bp −30 and −92 that is required for transcriptional activity of the α2 integrin gene. Sequence analysis identified two Sp1 consensus sites and a potential AP2 site. Gel retardation assays showed that nuclear proteins from uninduced K562 cells and K562 cells induced to become megakaryocytic bound specifically to the core promoter region (bp −30 to bp −92) producing two DNA-protein complexes. In addition, nuclear extracts from cells induced along the megakaryocyte lineage produced a selective increase in the slower migrating complex. Site-directed mutagenesis of the 5′, the 3′, or both Sp1 binding sites suggested that both Sp1 binding sites are required for full promoter activity and for DNA-protein complex formation. DNA footprinting also showed specific protection of the 5′ Sp1 site by nuclear extracts from uninduced K562 cells and protection of both the 5′ and the 3′ Sp1 sites by nuclear extracts from induced K562 cells. Sp1 protein-DNA complex formation was dependent on Sp1 phosphorylation. The faster migrating DNA-protein complex was enhanced by dephosphorylation; the slower migrating DNA-protein complex was diminished or lost.

Selective Sp1 Binding Is Critical for Maximal Activity of the Human c-kit Promoter

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4138-4149
Author(s):  
Gyeong H. Park ◽  
Howard K. Plummer ◽  
Geoffrey W. Krystal
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Core Promoter ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Kit Gene ◽  
Core Promoter Activity

The receptor tyrosine kinase c-kit is necessary for normal hematopoiesis, the development of germ cells and melanocytes, and the pathogenesis of certain hematologic and nonhematologic malignancies. To better understand the regulation of the c-kit gene, a detailed analysis of the core promoter was performed. Rapid amplification of cDNA ends (RACE) and RNase protection methods showed two major transcriptional initiation sites. Luciferase reporter assays using 5′ promoter deletion-reporter constructs containing up to 3 kb of 5′ sequence were performed in hematopoietic and small-cell lung cancer cell lines which either did or did not express the endogenous c-kit gene. This analysis showed the region 83 to 124 bp upstream of the 5′ transcription initiation site was crucial for maximal core promoter activity. Sequence analysis showed several potential Sp1 binding sites within this highly GC-rich region. Gel shift and DNase footprinting showed that Sp1 selectively bound to a single site within this region. Supershift studies using an anti-Sp1 antibody confirmed specific Sp1 binding. Site-directed mutagenesis of the −93/−84 Sp1 binding site reduced promoter-reporter activity to basal levels in c-kit–expressing cells. Cotransfection into DrosophilaSL2 cells of a c-kit promoter-reporter construct with an Sp1 expression vector showed an Sp1 dose-dependent enhancement of expression that was markedly attenuated by mutation of the −93/−84 site. These results indicate that despite the fact that the human c-kit promoter contains multiple potential Sp1 sites, Sp1 binding is a selective process that is essential for core promoter activity.

scholarly journals The Core Promoter Is a Regulatory Hub for Developmental Gene Expression

2021 ◽  
Vol 9 ◽  
Author(s):  
Anna Sloutskin ◽  
Hila Shir-Shapira ◽  
Richard N. Freiman ◽  
Tamar Juven-Gershon
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Core Promoter ◽  
Developmental Gene ◽  
Core Promoter Region ◽  
Developmental Gene Expression ◽  
The Core ◽  
Regulatory Module

The development of multicellular organisms and the uniqueness of each cell are achieved by distinct transcriptional programs. Multiple processes that regulate gene expression converge at the core promoter region, an 80 bp region that directs accurate transcription initiation by RNA polymerase II (Pol II). In recent years, it has become apparent that the core promoter region is not a passive DNA component, but rather an active regulatory module of transcriptional programs. Distinct core promoter compositions were demonstrated to result in different transcriptional outputs. In this mini-review, we focus on the role of the core promoter, particularly its downstream region, as the regulatory hub for developmental genes. The downstream core promoter element (DPE) was implicated in the control of evolutionarily conserved developmental gene regulatory networks (GRNs) governing body plan in both the anterior-posterior and dorsal-ventral axes. Notably, the composition of the basal transcription machinery is not universal, but rather promoter-dependent, highlighting the importance of specialized transcription complexes and their core promoter target sequences as key hubs that drive embryonic development, differentiation and morphogenesis across metazoan species. The extent of transcriptional activation by a specific enhancer is dependent on its compatibility with the relevant core promoter. The core promoter content also regulates transcription burst size. Overall, while for many years it was thought that the specificity of gene expression is primarily determined by enhancers, it is now clear that the core promoter region comprises an important regulatory module in the intricate networks of developmental gene expression.

Core promoter-selective RNA polymerase II transcription

2006 ◽  
Vol 73 ◽  
pp. 225-236 ◽  
Author(s):  
Petra Gross ◽  
Thomas Oelgeschläger
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Promoter Sequence ◽  
Tata Box ◽  
Promoter Elements ◽  
The Core ◽  
Core Promoter Sequence ◽  
Rnap Ii

The initiation of mRNA synthesis in eukaryotic cells is a complex and highly regulated process that requires the assembly of general transcription factors and RNAP II (RNA polymerase II; also abbreviated as Pol II) into a pre-initiation complex at the core promoter. The core promoter is defined as the minimal DNA region that is sufficient to direct low levels of activator-independent (basal) transcription by RNAP II in vitro. The core promoter typically extends approx. 40 bp up- and down-stream of the start site of transcription and can contain several distinct core promoter sequence elements. Core promoters in higher eukaryotes are highly diverse in structure, and each core promoter sequence element is only found in a subset of genes. So far, only TATA box and INR (initiator) element have been shown to be capable of directing accurate RNAP II transcription initiation independent of other core promoter elements. Computational analysis of metazoan genomes suggests that the prevalence of the TATA box has been overestimated in the past and that the majority of human genes are TATA-less. While TATA-mediated transcription initiation has been studied in great detail and is very well understood, very little is known about the factors and mechanisms involved in the function of the INR and other core promoter elements. Here we summarize our current understanding of the factors and mechanisms involved in core promoter-selective transcription and discuss possible pathways through which diversity in core promoter architecture might contribute to combinatorial gene regulation in metazoan cells.

Characterization of factors that direct transcription of rat ribosomal DNA

1990 ◽  
Vol 10 (6) ◽  
pp. 3105-3116
Author(s):  
S D Smith ◽  
E Oriahi ◽  
D Lowe ◽  
H F Yang-Yen ◽  
D O'Mahony ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Rna Polymerase I ◽  
Promoter Element ◽  
Novikoff Hepatoma ◽  
Direct Transcription ◽  
Core Promoter Element ◽  
The Core ◽  
Polymerase I

The protein components that direct and activate accurate transcription by rat RNA polymerase I were studied in extracts of Novikoff hepatoma ascites cells. A minimum of at least two components, besides RNA polymerase I, that are necessary for efficient utilization of templates were identified. The first factor, rat SL-1, is required for species-specific recognition of the rat RNA polymerase I promoter and may be sufficient to direct transcription by pure RNA polymerase I. Rat SL-1 directed the transcription of templates deleted to -31, the 5' boundary of the core promoter element (+1 being the transcription initiation site). The second factor, rUBF, increased the efficiency of template utilization. Transcription of deletion mutants indicated that the 5' boundary of the domain required for rUBF lay between -137 and -127. Experiments using block substitution mutants confirmed and extended these observations. Transcription experiments using those mutants demonstrated that two regions within the upstream promoter element were required for optimal levels of transcription in vitro. The first region was centered on nucleotides -129 and -124. The 5' boundary of the second domain mapped to between nucleotides -106 and -101. DNase footprint experiments using highly purified rUBF indicated that rUBF bound between -130 and -50. However, mutation of nucleotides -129 and -124 did not affect the rUBF footprint. These results indicate that basal levels of transcription by RNA polymerase I may require only SL-1 and the core promoter element. However, higher transcription levels are mediated by additional interactions of rUBF, and possibly SL-1, bound to distal promoter elements.

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.

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