scholarly journals Functional characterization of transcriptional regulatory elements in the upstream region and intron 1 of the human S6 ribosomal protein gene

1998 ◽  
Vol 336 (2) ◽  
pp. 327-335 ◽  
Author(s):  
Marianne ANTOINE ◽  
Paul KIEFER
Keyword(s):  
Ribosomal Protein ◽  
Specific Binding ◽  
Core Promoter ◽  
Functional Characterization ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Splice Donor Site ◽  
The Core ◽  
Intron 1

Expression of housekeeping genes involves regulation at comparable levels in a wide spectrum of cells. To define the cis-regulatory elements in the human S6 ribosomal protein (rpS6) gene, we made a series of deletions of the upstream non-transcribed region, including or excluding exon 1 or intron 1 sequences. The mutated rpS6 gene regulatory regions were fused to the chloramphenicol acetyltransferase reporter gene and transfected into HeLa and COS-1 cells. The results have identified three parts of the rpS6 gene that are required for efficient and specific transcription. The core promoter includes only a 40 bp region upstream of the transcription start site and initiation region. Both upstream and intronic elements enhance transcription from the core promoter. Furthermore, mutation of the splice donor site of intron 1 almost completely abolished the enhancing activity of the intronic transcriptional modulator. We used gel retardation assays to identify sequence-specific binding sites in the upstream region and in the proximal half of intron 1. Both common and different nuclear factors that bind the rpS6 gene promoter were identified in extracts from HeLa and COS-1 cells, suggesting that different transcription factors may bind specifically to the same binding region and might be interchangeable in their function to ensure high-level expression of housekeeping genes independently of the cell type.

scholarly journals Identification and characterization of cis-regulatory elements for photoreceptor type-specific transcription in zebrafish

2019 ◽  
Author(s):  
Wei Fang ◽  
Yi Wen ◽  
Xiangyun Wei
Keyword(s):  
Core Promoter ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Protein Coding ◽  
Core Promoters ◽  
Protein Coding Genes ◽  
The Core ◽  
Cell Type Specific ◽  
Identification And Characterization

AbstractTissue-specific or cell type-specific transcription of protein-coding genes is controlled by both trans-regulatory elements (TREs) and cis-regulatory elements (CREs). However, it is challenging to identify TREs and CREs, which are unknown for most genes. Here, we describe a protocol for identifying two types of transcription-activating CREs—core promoters and enhancers—of zebrafish photoreceptor type-specific genes. This protocol is composed of three phases: bioinformatic prediction, experimental validation, and characterization of the CREs. To better illustrate the principles and logic of this protocol, we exemplify it with the discovery of the core promoter and enhancer of the mpp5b apical polarity gene (also known as ponli), whose red, green, and blue (RGB) cone-specific transcription requires its enhancer, a member of the rainbow enhancer family. While exemplified with an RGB cone-specific gene, this protocol is general and can be used to identify the core promoters and enhancers of other protein-coding genes.

scholarly journals Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

1998 ◽  
Vol 18 (11) ◽  
pp. 6191-6200 ◽  
Author(s):  
Yukako Yamabe ◽  
Akira Shimamoto ◽  
Makoto Goto ◽  
Jun Yokota ◽  
Minoru Sugawara ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Control Element ◽  
Mrna Levels

ABSTRACT The regulation of Werner’s syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5′ upstream region (2.8 kb) ofWRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides −67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRNpromoter-luciferase reporter (WRN-Luc) plasmids that contained the 5′-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

scholarly journals Core Element Cloning, Cis-Element Mapping and Serum Regulation of the Human EphB4 Promoter: A Novel TATA-Less Inr/MTE/DPE-Like Regulated Gene

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 997 ◽  
Author(s):  
Pierluigi Scalia ◽  
Stephen J. Williams ◽  
Antonio Giordano
Keyword(s):  
Recombinant Dna ◽  
Core Promoter ◽  
Gene Promoter ◽  
Bioinformatic Analysis ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Tyrosine Kinase Receptor ◽  
Recombinant Dna Technology ◽  
Core Element ◽  
Cis Acting

The EphB4 gene encodes for a transmembrane tyrosine kinase receptor involved in embryonic blood vessel differentiation and cancer development. Although EphB4 is known to be regulated at the post-translational level, little is known about its gene regulation. The present study describes the core promoter elements’ identification and cloning, the cis-regulatory elements’ mapping and the serum regulation of the human EphB4 gene promoter region. Using bioinformatic analysis, Sanger sequencing and recombinant DNA technology, we analyzed the EphB4 gene upstream region spanning +40/−1509 from the actual transcription start site (TSS) and proved it to be a TATA-less gene promoter with dispersed regulatory elements characterized by a novel motif-of-ten element (MTE) at positions +18/+28, and a DPE-like motif and a DPE-like-repeated motif (DRM) spanning nt +27/+30 and +32 +35, respectively. We also mapped both proximal (multiple Sp1) and distal (HoxA9) trans-activating/dispersed cis-acting transcription factor (TF)-binding elements on the region we studied and used a transient transfection reporter assay to characterize its regulation by serum and IGF-II using EphB4 promoter deletion constructs with or without the identified new DNA-binding elements. Altogether, these findings shed new light on the human EphB4 promoter structure and regulation, suggesting mechanistic features conserved among Pol-II TATA-less genes phylogenetically shared from Drosophila to Human genomes.

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.

scholarly journals Functional characterization of transcriptional regulatory elements in the upstream region of the yeast GLK1 gene

1999 ◽  
Vol 343 (2) ◽  
pp. 319-325 ◽  
Author(s):  
Pilar HERRERO ◽  
Leticia FLORES ◽  
Tamara de la CERA ◽  
Fernando MORENO
Keyword(s):  
Glucose Repression ◽  
Functional Characterization ◽  
Carbon Sources ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Covalent Modification ◽  
Upstream Region ◽  
Yeast Saccharomyces Cerevisiae ◽  
Constitutive Activation ◽  
Glucose Depletion

The glucokinase gene GLK1 of the yeast Saccharomyces cerevisiae is transcriptionally regulated in response to the carbon source of the growth medium. Northern-blot analysis shows that the GLK1 gene is expressed at a basal level in the presence of glucose, de-repressed more than 6-fold under conditions of sugar limitation and more than 25-fold under conditions of ethanol induction. lacZ fusions of the GLK1 gene promoter were constructed and a deletion analysis was performed in order to identify the cis-acting regulatory elements of the promoter that controls GLK1 gene expression. First, the expression seemed to be mediated mainly by one GCR1 and three stress-responsive element (STRE) activating elements. Secondly, an ethanol repression autoregulation (ERA)/twelve-fold TA repeat (TAB) repressor element was identified within the promoter region of the GLK1 gene. A specific and differential protein binding to the STRE was observed with extracts from de-repressed and repressed cells. No differential binding to the GCR1 or ERA/TAB elements was observed with extracts from de-repressed and repressed cells, but, in both cases, the binding was competed for by an excess of the unlabelled GLK1GCR1 andGLK1ERA sequence. The transcription factors Msn2 and Msn4, which bind to the GLK1 upstream region through the STRE, contribute to inductive activation. The transcription factor Gcr1, which binds through the GCR1 element, contributes to constitutive activation. In order to achieve the severe glucose repression of GLK1, constitutive repressor factors acting through the ERA/TAB element must counteract constitutive activation generated by Gcr1 binding to the GCR1 element. Full expression of the GLK1 gene is produced by inductive activation of three STRE when Msn2 and Msn4 proteins are translocated to the nucleus by covalent modification. The combinatorial effect of the entire region leads to the regulated transcription of GLK1, i.e., silent in media with glucose and other preferred carbon sources, such as fructose or mannose, and increased levels of expression upon glucose depletion.

scholarly journals Genomic environments scale the activities of diverse core promoters

2021 ◽  
Author(s):  
Clarice K.Y. Hong ◽  
Barak A. Cohen
Keyword(s):  
Core Promoter ◽  
Classical Model ◽  
Regulatory Elements ◽  
Sequence Motifs ◽  
Core Promoters ◽  
The Core ◽  
Genomic Location ◽  
Basal Transcriptional Machinery ◽  
Promoter Specificity ◽  
Genomic Locations

A classical model of gene regulation is that enhancers provide specificity whereas core promoters provide a modular site for the assembly of the basal transcriptional machinery. However, examples of core promoter specificity have led to an alternate hypothesis in which specificity is achieved by core promoters with different sequence motifs that respond differently to genomic environments containing different enhancers and chromatin landscapes. To distinguish between these models, we measured the activities of hundreds of diverse core promoters in four different genomic locations and, in a complementary experiment, six different core promoters at thousands of locations across the genome. Although genomic locations had large effects on expression, the intrinsic activities of different classes of promoters were preserved across genomic locations, suggesting that core promoters are modular regulatory elements whose activities are independently scaled up or down by different genomic locations. This scaling of promoter activities is nonlinear and depends on the genomic location and the strength of the core promoter. Our results support the classical model of regulation in which diverse core promoter motifs set the intrinsic strengths of core promoters, which are then amplified or dampened by the activities of their genomic environments.

scholarly journals Intron-driven gene expression in the absence of a core promoter in Arabidopsis thaliana

2016 ◽  
Author(s):  
Jenna E Gallegos ◽  
Alan B Rose
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Regulatory Elements ◽  
Intron Position ◽  
Mrna Accumulation ◽  
Practical Applications ◽  
Promoter Sequences ◽  
The Core

AbstractIn diverse eukaryotes, certain introns increase mRNA accumulation through the poorly understood mechanism of intron-mediated enhancement (IME). A distinguishing feature of IME is that these introns have no effect from upstream or more than 1 Kb downstream of the transcription start site (TSS). To more precisely define the intron position requirements for IME in Arabidopsis, we tested the effect of the UBQ10 intron on gene expression from 6 different positions surrounding the TSS of a TRP1:GUS fusion. The intron strongly increased expression from all transcribed positions, but had no effect when 204 nt or more upstream of the 5’-most TSS. When the intron was located in the 5’ UTR, the TSS unexpectedly changed, resulting in longer transcripts. Remarkably, deleting 303 nt of the core promoter, including all known TSS’s and all but 18 nt of the 5’ UTR, had virtually no effect on the level of gene expression as long as a stimulating intron was included in the gene. When the core promoter was deleted, transcription initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. Together, these results suggest that certain introns play unexpectedly large roles in directing transcription initiation and represent a previously unrecognized type of downstream regulatory elements for genes transcribed by RNA polymerase II. This study also demonstrates considerable flexibility in the sequences surrounding the TSS, indicating that the TSS is not determined by promoter sequences alone. These findings are relevant in practical applications where introns are used to increase gene expression and contribute to our general understanding of gene structure and regulation in eukaryotes.

scholarly journals Evolution of Regulated Transcription

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1675 ◽  
Author(s):  
Oleg V. Bylino ◽  
Airat N. Ibragimov ◽  
Yulii V. Shidlovskii
Keyword(s):  
Gene Expression ◽  
Core Promoter ◽  
Regulatory Element ◽  
Regulatory System ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Transcription Control ◽  
The Core ◽  
Gradual Evolution ◽  
Upstream Promoter Region

The genomes of all organisms abound with various cis-regulatory elements, which control gene activity. Transcriptional enhancers are a key group of such elements in eukaryotes and are DNA regions that form physical contacts with gene promoters and precisely orchestrate gene expression programs. Here, we follow gradual evolution of this regulatory system and discuss its features in different organisms. In eubacteria, an enhancer-like element is often a single regulatory element, is usually proximal to the core promoter, and is occupied by one or a few activators. Activation of gene expression in archaea is accompanied by the recruitment of an activator to several enhancer-like sites in the upstream promoter region. In eukaryotes, activation of expression is accompanied by the recruitment of activators to multiple enhancers, which may be distant from the core promoter, and the activators act through coactivators. The role of the general DNA architecture in transcription control increases in evolution. As a whole, it can be seen that enhancers of multicellular eukaryotes evolved from the corresponding prototypic enhancer-like regulatory elements with the gradually increasing genome size of organisms.

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