scholarly journals Mechanism of ubiquitous expression of mouse uncoupling protein 2 mRNA: control by cis-acting DNA element in 5′-flanking region

1999 ◽  
Vol 340 (2) ◽  
pp. 397-404 ◽  
Author(s):  
Hideki YOSHITOMI ◽  
Kazuto YAMAZAKI ◽  
Isao TANAKA
Keyword(s):  
Promoter Region ◽  
Uncoupling Protein ◽  
Initiation Site ◽  
Computer Assisted ◽  
Placental Alkaline Phosphatase ◽  
Transient Expression Assay ◽  
Transcription Activity ◽  
Cis Acting ◽  
Ucp2 Gene ◽  
Flanking Region

Uncoupling protein (UCP) 2 is a member of the uncoupling-protein family, and it appears to function as an uncoupler of oxidative phosphorylation. To identify cis-acting regulatory elements controlling this gene's expression, we cloned an approx. 6.2-kb region upstream from the translation-initiation site of the mouse UCP2 gene and analysed its transcription activity using chimaeric mouse UCP2 promoter-placental-alkaline-phosphatase (PLAP) reporter-gene constructs. Sequence analysis showed that the 5ʹ-flanking region of the mouse UCP2 gene was not similar to those of mouse UCP1 or UCP3. For the mouse UCP2, the region near the transcription-initiation site lacked the typical TATA box, but was GC-rich, resulting in presence of several potential specificity protein 1 (Sp-1), activator protein (AP)-1 and AP-2 binding sites. The putative regulatory motifs for muscle-regulatory protein (MyoD), brown-fat regulatory element, CCAAT box, cAMP-response element and Y box were also found in the mouse UCP2 promoter region by computer-assisted analysis. From the results of Northern-blot analysis and transient expression assay, we found that the mouse UCP2 gene responded to the cAMP-dependent protein kinase α-catalytic subunit signal activation at the transcription level. Additionally, deletion analysis of the UCP2 promoter-PLAP constructs indicated that the minimal region exhibiting the promoter activity was located between nt -33 and +100, and that a strong enhancer was present within 601 bp of the 5ʹ-promoter region. In particular, the region from nt -233 to -34 significantly induced PLAP activity in the cell lines derived from various tissues and in the primary culture cells of rat brown adipose tissue, suggesting that this region is most important for the ubiquitous expression of mouse UCP2 mRNA. Furthermore, it was shown that two silencer elements were involved in the mouse UCP2 gene; one was located between nt -2746 and -602, and the other was identified in intron 1. These regions deprived the enhancer of the ability to induce PLAP activity. This study shows a fundamental role for positive and negative cis-acting DNA elements in regulating the basal and cAMP-induced transcription activity of the mouse UCP2 gene.

scholarly journals The decapentaplegic core promoter region plays an integral role in the spatial control of transcription.

1995 ◽  
Vol 15 (7) ◽  
pp. 3960-3968 ◽  
Author(s):  
D H Schwyter ◽  
J D Huang ◽  
T Dubnicoff ◽  
A J Courey
Keyword(s):  
Expression Pattern ◽  
Phase Ii ◽  
Promoter Region ◽  
Core Promoter ◽  
Critical Role ◽  
Regulatory Elements ◽  
Drosophila Embryo ◽  
Phase Iii ◽  
Core Promoter Region ◽  
Flanking Region

The Drosophila melanogaster decapentaplegic (dpp) gene encodes a transforming growth factor beta-related cell signaling molecule that plays a critical role in dorsal/ventral pattern formation. The dpp expression pattern in the Drosophila embryo is dynamic, consisting of three phases. Phase I, in which dpp is expressed in a broad dorsal domain, depends on elements in the dpp second intron that interact with the Dorsal transcription factor to repress transcription ventrally. In contrast, phases II and III, in which dpp is expressed first in broad longitudinal stripes (phase II) and subsequently in narrow longitudinal stripes (phase III), depend on multiple independent elements in the dpp 5'-flanking region. Several aspects of the normal dpp expression pattern appear to depend on the unique properties of the dpp core promoter. For example, this core promoter (extending from -22 to +6) is able to direct a phase II expression pattern in the absence of additional upstream or downstream regulatory elements. In addition, a ventral-specific enhancer in the dpp 5'-flanking region that binds the Dorsal factor activates the heterologous hsp70 core promoter but not the dpp core promoter. Thus, the dpp core promoter region may contribute to spatially regulated transcription both by interacting directly with spatially restricted activators and by modifying the activity of proteins bound to enhancer elements.

Activation of an enhancerless gene by chromosomal integration

1986 ◽  
Vol 6 (12) ◽  
pp. 4179-4184
Author(s):  
H Hamada
Keyword(s):  
Promoter Region ◽  
Genomic Dna ◽  
Simian Virus 40 ◽  
Initiation Site ◽  
Simian Virus ◽  
Chromosomal Integration ◽  
Early Promoter ◽  
Sv40 Enhancer ◽  
Active Expression ◽  
Cat Gene

Expression of enhancerless (E-) and enhancer-containing (E+) genes that are chromosomally integrated was examined. An E- plasmid (pE-cat) containing a chloramphenicol acetyltransferase (cat) gene linked to the simian virus 40 (SV40) early promoter or its E+ counterpart plasmid (pE+-cat) containing the SV40 enhancer was cotransfected into thymidine kinase (TK)-deficient L cells with a cloned tk gene. A number of TK+ transformants were isolated, and expression of the cointegrated cat gene in these cell lines was quantitatively determined by the assay of CAT activity. The results indicated unexpectedly that the E- cat gene was as actively expressed as the E+ cat gene. Analysis of CAT mRNA by primer extension indicated that the E- cat gene, as well as the E+ cat gene, was transcribed from the "native" initiation site contained in the SV40 early promoter region. The active expression of the E- cat gene was maintained in secondary TK+ transformants that arose by transfection with genomic DNA from the primary transformant. These results suggest that expression of the integrated E- cat gene is activated by endogenous enhancer elements.

Cooperativity of sequence elements mediates tissue specificity of the rat insulin II gene

1990 ◽  
Vol 10 (4) ◽  
pp. 1784-1788
Author(s):  
Y P Hwung ◽  
Y Z Gu ◽  
M J Tsai
Keyword(s):  
Beta Cell ◽  
Tissue Specificity ◽  
Promoter Element ◽  
Heterologous Promoter ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Insulin Promoter ◽  
Sequence Elements ◽  
Flanking Region

The 5'-flanking region of the rat insulin II gene (-448 to +50) is sufficient for tissue-specific expression. To further determine the tissue-specific cis-acting element(s), important sequences defined by linker-scanning mutagenesis were placed upstream of a heterologous promoter and transfected into insulin-producing and -nonproducing cells. Rat insulin promoter element 3 (RIPE3), which spans from -125 to -86, was shown to confer beta-cell-specific expression in either orientation. However, two subregions of RIPE3, RIPE3a and RIPE3b (defined by linker-scanning mutations), displayed only marginal activities. These results suggest that the two subregions cooperate to confer tissue specificity, presumably via their cognate binding factors.

Tissue-specific transcription of the mouse alpha-fetoprotein gene promoter is dependent on HNF-1

1989 ◽  
Vol 9 (10) ◽  
pp. 4204-4212
Author(s):  
M H Feuerman ◽  
R Godbout ◽  
R S Ingram ◽  
S M Tilghman
Keyword(s):  
Specific Binding ◽  
Gene Promoter ◽  
Binding Activity ◽  
Alpha Fetoprotein ◽  
Transient Expression Assay ◽  
Band Shift ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Dnase I Footprinting

Previous work identified four upstream cis-acting elements required for tissue-specific expression of the alpha-fetoprotein (AFP) gene: three distal enhancers and a promoter. To further define the role of the promoter in regulating AFP gene expression, segments of the region were tested for the ability to direct transcription of a reporter gene in transient expression assay. Experiments showed that the region within 250 base pairs of the start of transcription was sufficient to confer liver-specific transcription. DNase I footprinting and band shift assays indicated that the region between -130 and -100 was recognized by two factors, one of which was highly sequence specific and found only in hepatoma cells. Competition assays suggested that the liver-specific binding activity was HNF-1, previously identified by its binding to other liver-specific promoters. Mutation of the HNF-1 recognition site at -120 resulted in a significant reduction in transcription in transfection assays, suggesting a biological role for HNF-1 in the regulation of AFP expression.

Identification of a regulatory region that mediates glucose-dependent induction of the Saccharomyces cerevisiae enolase gene ENO2

1986 ◽  
Vol 6 (7) ◽  
pp. 2287-2297
Author(s):  
R Cohen ◽  
J P Holland ◽  
T Yokoi ◽  
M J Holland
Keyword(s):  
Gene Expression ◽  
Regulatory Region ◽  
Carbon Sources ◽  
Initiation Site ◽  
Deletion Mapping ◽  
Regulatory Regions ◽  
Coding Sequences ◽  
Flanking Region ◽  
Fused Gene ◽  
In Cells

There are two yeast enolase genes, designated ENO1 and ENO2, which are expressed differentially in vegetative cells grown on glucose and in cells grown on gluconeogenic carbon sources. ENO2 is induced more than 20-fold in cells grown on glucose, whereas ENO1 expression is similar in cells grown on glucose and in cells grown on gluconeogenic carbon sources. Sequences within the 5' flanking region of ENO2 which are required for glucose-dependent induction were identified by deletion mapping analysis. These studies were carried out by using a fused gene containing the ENO2 5' flanking sequences and the ENO1 coding sequences. This fused gene undergoes glucose-dependent induction and is expressed at the same level as the resident ENO2 gene in cells grown on glucose or gluconeogenic carbon sources. Expression of fused genes containing deletion mutations within the ENO2 5' flanking region was monitored after integration at the ENO1 locus of a strain carrying a deletion of the resident ENO1 coding sequences. This analysis showed that there are two upstream activation sites located immediately upstream and downstream from a position 461 base pairs upstream from the transcriptional initiation site. Either one of these upstream activation sites is sufficient for glucose-dependent induction and normal gene expression in the presence of gluconeogenic carbon sources. Deletion of both regulatory regions results in a complete loss of gene expression. The regulatory regions function normally in both orientations relative to the coding sequences. Mutant fused genes containing small deletions within the regulatory regions were constructed; these genes were expressed normally in gluconeogenic carbon sources but were not induced in the presence of glucose. Based on this analysis, ENO2 contains a cis-acting regulatory region which is required for gene expression and mediates glucose-dependent induction of gene expression.

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