Characterization of the Calbindin-D28k Gene Proximal Promoter Activity and Regulatory Elements under Charcoal Stripped or Serum-Free Stress Conditions

During embryonic development, the aristaless-type homeodomain protein Alx3 is expressed in the forehead mesenchyme and contributes to the regulation of craniofacial development. In the adult, Alx3 is expressed in pancreatic islets where it participates in the control of glucose homoeostasis. In the present study, we investigated the transcriptional regulation of Alx3 gene expression in these two cell types. We found that the Alx3 promoter contains two E-box regulatory elements, named EB1 and EB2, that provide binding sites for the basic helix–loop–helix transcription factors Twist1, E47, USF (upstream stimulatory factor) 1 and USF2. In primary mouse embryonic mesenchymal cells isolated from the forehead, EB2 is bound by Twist1, whereas EB1 is bound by USF1 and USF2. Integrity of both EB1 and EB2 is required for Twist1-mediated transactivation of the Alx3 promoter, even though Twist1 does not bind to EB1, indicating that binding of USF1 and USF2 to this element is required for Twist1-dependent Alx3 promoter activity. In contrast, in pancreatic islet insulin-producing cells, the integrity of EB2 is not required for proximal promoter activity. The results of the present study indicate that USF1 and USF2 are important regulatory factors for Alx3 gene expression in different cell types, whereas Twist1 contributes to transcriptional transactivation in mesenchymal, but not in pancreatic, cells.

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Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.206-216.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 206-216

Author(s):

Y T Chung ◽

E B Keller

Keyword(s):

Drosophila Melanogaster ◽

Transient Expression ◽

Promoter Activity ◽

Regulatory Element ◽

Regulatory Elements ◽

Proximal Promoter ◽

Base Pairs ◽

Chloramphenicol Acetyltransferase Gene ◽

Constitutive Synthesis

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays.

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Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site

Biochemical Journal ◽

10.1042/bj20070364 ◽

2007 ◽

Vol 407 (1) ◽

pp. 101-111 ◽

Cited By ~ 9

Author(s):

Ian Pearse ◽

Ying X. Zhu ◽

Eleanor J. Murray ◽

Pradeep K. Dudeja ◽

Krishnamurthy Ramaswamy ◽

...

Keyword(s):

Transcription Initiation ◽

Promoter Region ◽

Promoter Activity ◽

Critical Role ◽

Constitutive Expression ◽

Regulatory Elements ◽

Initiation Site ◽

Transcription Initiation Site ◽

Proximal Promoter ◽

Gc Box

We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the −40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, −10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5′ flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at −25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthermore, we identified two positive regulatory elements in the DNA region corresponding to the 5′-UTR (5′-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5′-UTR may co-operate with the 5′-flanking region to achieve the optimal promoter activity.

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Characterization of the human biliverdin reductase gene structure and regulatory elements: promoter activity is enhanced by hypoxia and suppressed by TNF‐α‐activated NF‐κB

The FASEB Journal ◽

10.1096/fj.09-144592 ◽

2010 ◽

Vol 24 (9) ◽

pp. 3239-3254 ◽

Cited By ~ 12

Author(s):

Peter E. M. Gibbs ◽

Tihomir Miralem ◽

Mahin D. Maines

Keyword(s):

Gene Structure ◽

Promoter Activity ◽

Regulatory Elements ◽

Biliverdin Reductase ◽

Tnf Α ◽

Reductase Gene

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Identification and characterization of the minimal 5′-regulatory region of the human riboflavin transporter-3 (SLC52A3) in intestinal epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00342.2014 ◽

2015 ◽

Vol 308 (2) ◽

pp. C189-C196 ◽

Cited By ~ 12

Author(s):

Abhisek Ghosal ◽

Subrata Sabui ◽

Hamid M. Said

Keyword(s):

Epithelial Cells ◽

Transgenic Mice ◽

Promoter Activity ◽

Intestinal Epithelial Cells ◽

Regulatory Region ◽

Regulatory Elements ◽

Intestinal Epithelial ◽

Identification And Characterization

The human riboflavin (RF) transporter-3 (product of the SLC52A3 gene) plays an important role in intestinal RF absorption. Our aims in this study were to identify the minimal 5′-regulatory region of the SLC52A3 gene and the regulatory element(s) involved in its activity in intestinal epithelial cells, as well as to confirm promoter activity and establish physiological relevance in vivo in transgenic mice. With the use of transiently transfected human intestinal epithelial HuTu 80 cells and 5′-deletion analysis, the minimal SLC52A3 promoter was found to be encoded between −199 and +8 bp (using the start of the transcription start site as position 1). Although several putative cis-regulatory elements were predicted in this region, only the stimulating protein-1 (Sp1) binding site (at position −74/−71 bp) was found to play a role in promoter activity, as indicated by mutational analysis. Binding of Sp1 to the minimal SLC52A3 promoter was demonstrated by means of EMSA and supershift assays and by chromatin immunoprecipitation analysis. Studies with Drosophila SL2 cells (which lack Sp activity) confirmed the importance of Sp1 in driving the activity of the SLC52A3 minimal promoter; they further showed that Sp3 can also do the activation. Finally, with the use of luciferase gene fusions, the activity of the cloned SLC52A3 promoter was confirmed in vivo in transgenic mice. These studies report, for the first time, on the identification and characterization of the SLC52A3 promoter and also demonstrate the importance of Sp1 in regulating its activity in intestinal epithelial cells.

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Genome-wide Functional Characterization of Escherichia coli Promoters and Regulatory Elements Responsible for their Function

10.1101/2020.01.04.894907 ◽

2020 ◽

Cited By ~ 8

Author(s):

Guillaume Urtecho ◽

Kimberly D. Insigne ◽

Arielle D. Tripp ◽

Marcia Brinck ◽

Nathan B. Lubock ◽

...

Keyword(s):

Promoter Activity ◽

Functional Characterization ◽

Regulatory Elements ◽

Machine Learning Algorithms ◽

Regulatory Sequences ◽

Primary Sequence ◽

Entire Genome ◽

E Coli ◽

Full Characterization

SummaryDespite decades of intense genetic, biochemical, and evolutionary characterizations of bacterial promoters, we still lack the basic ability to identify or predict transcriptional activities of promoters using primary sequence. Even in simple, well-characterized organisms such as E. coli there is little agreement on the number, location, and strength of promoters. Here, we use a genomically-encoded massively parallel reporter assay to perform the first full characterization of autonomous promoter activity across the E. coli genome. We measure promoter activity of >300,000 sequences spanning the entire genome and precisely map 2,228 promoters active in rich media. We show that antisense promoters have a profound effect on global transcription and how codon usage has adapted to encode intragenic promoters. Furthermore, we perform a scanning mutagenesis of 2,057 promoters to uncover regulatory sequences responsible for regulating promoter activity. Finally, we show that despite these large datasets and modern machine learning algorithms, the task of predicting promoter activity from primary sequence sequence is still challenging.

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Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00234.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. G822-G829 ◽

Cited By ~ 35

Author(s):

Svetlana M. Nabokina ◽

Hamid M. Said

Keyword(s):

Promoter Region ◽

Promoter Activity ◽

Critical Role ◽

Regulatory Region ◽

Regulatory Elements ◽

Minimal Promoter ◽

Minimal Promoter Region

Transcriptional regulation of expression of the human thiamin transporter-2 (the product of the SLC19A3 gene) is unknown. In this study, we cloned the 5′-regulatory region of the human SLC19A3 gene (2,016 bp), identified the minimal promoter region required for basal activity, demonstrated a critical role for specific cis-regulatory elements in determining the promoter activity, and confirmed activity and physiological relevance of the cloned SLC19A3 promoter in vivo. With the use of transiently transfected human intestinal epithelial Caco-2 cells and 5′-deletion analysis, the minimal promoter region required for basal activity of the SLC19A3 promoter was found to be encoded in a sequence between −77 and +59 by using the start of transcription initiation as position 1. This minimal region was found to contain a number of putative cis-regulatory elements, with a critical role for a stimulating protein-1 (SP1)/GC-box binding site (at position −48/−45 bp) established by means of mutational analysis. With the use of EMSA and supershift assays, the binding of SP1 and SP3 to the minimal promoter region was also demonstrated. In transiently transfected Drosophila SL2 cells, both SP1 and SP3 transactivated the SLC19A3 minimal promoter in a dose-dependent manner and in combination demonstrated an additive stimulatory effect. Functionality of the full-length SLC19A3 promoter was confirmed in vivo in transgenic mice expressing the promoter-luciferase reporter gene. These studies report the first characterization of the SLC19A3 promoter in vitro and in vivo and demonstrate the importance of an SP1 cis-regulatory element in regulating promoter activity of this important human gene.

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Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.206 ◽

1990 ◽

Vol 10 (1) ◽

pp. 206-216 ◽

Cited By ~ 29

Author(s):

Y T Chung ◽

E B Keller

Keyword(s):

Drosophila Melanogaster ◽

Transient Expression ◽

Promoter Activity ◽

Regulatory Element ◽

Regulatory Elements ◽

Proximal Promoter ◽

Base Pairs ◽

Chloramphenicol Acetyltransferase Gene ◽

Constitutive Synthesis

The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays.

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Identification of regulatory elements in the Cyp19 proximal promoter in rat luteal cells

Journal of Molecular Endocrinology ◽

10.1677/jme-07-0026 ◽

2007 ◽

Vol 39 (4) ◽

pp. 211-221 ◽

Cited By ~ 20

Author(s):

Carlos Stocco ◽

Jakub Kwintkiewicz ◽

Zailong Cai

Keyword(s):

Binding Site ◽

Promoter Activity ◽

Regulatory Elements ◽

Binding Activity ◽

Proximal Promoter ◽

Promoter Regions ◽

Pregnant Rats ◽

Response Element ◽

Luteal Cells ◽

Immature Rats

AbstractThe cytochrome P450 aromatase (Cyp19) gene encodes an enzyme of crucial importance in the synthesis of estradiol. Estradiol is luteotropic in the rat. In this species, luteal Cyp19 expression increases progressively during pregnancy and falls before parturition. The mechanisms that control these changes are unknown. Using gel shift assays, we sought to identify the promoter regions that control Cyp19 expression in the rat corpus luteum (CL). The Cyp19 promoter contains a cAMP response element-like sequence (CLS), two nuclear receptor elements half sites (NREs), a GATA binding site, a Yin Yang-1 (YY1) response element, and an activation protein 3 (AP3) binding site. Nuclear extracts were obtained from CL of rats on days 4, 15, and 23 of pregnancy and from the ovaries of immature rats treated with vehicle or a hormone that induces Cyp19 expression in the follicles. CLS was active in immature ovaries but inactive in the CL of pregnant rats, whereas binding to NREs and GATA was observed in both tissues. YY1 was inactive in all samples tested. In the CL, AP3 binding was higher on day 15 of pregnancy when compared with day 4 and day 23 but it was absent in ovaries of immature rats, whereas luteinization increased AP3 binding activity. Mutation of the AP3 site blunted the stimulation of Cyp19 promoter activity in granulosa cells. Our results indicate that CLS is active only in follicles; whereas in the CL, binding to the GATA, NRE, and AP3 sites associates with changes in Cyp19 expression, suggesting that they control Cyp19 promoter activity in luteal cells.

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