scholarly journals Functional Analysis of a Novel cis-Acting Regulatory Region within the Human Ankyrin Gene (ANK-1) Promoter

2010 ◽  
Vol 30 (14) ◽  
pp. 3493-3502 ◽  
Author(s):  
Karina Laflamme ◽  
Ashley N. Owen ◽  
Emily E. Devlin ◽  
Mary Q. Yang ◽  
Clara Wong ◽  
...  
Keyword(s):  
Hereditary Spherocytosis ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Regulatory Sequence ◽  
Cis Acting ◽  
Promoter Sequences ◽  
Promoter Function

ABSTRACT The characterization of atypical mutations in loci associated with diseases is a powerful tool to discover novel regulatory elements. We previously identified a dinucleotide deletion in the human ankyrin-1 gene (ANK-1) promoter that underlies ankyrin-deficient hereditary spherocytosis. The presence of the deletion was associated with a decrease in promoter function both in vitro and in vivo establishing it as a causative hereditary spherocytosis mutation. The dinucleotide deletion is located in the 5′ untranslated region of the ANK-1 gene and disrupts the binding of TATA binding protein and TFIID, components of the preinitiation complex. We hypothesized that the nucleotides surrounding the mutation define an uncharacterized regulatory sequence. To test this hypothesis, we generated a library of more than 16,000 ANK-1 promoters with degenerate sequence around the mutation and cloned the functional promoter sequences after cell-free transcription. We identified the wild type and three additional sequences, from which we derived a consensus. The sequences were shown to be functional in cell-free transcription, transient-transfection, and transgenic mouse assays. One sequence increased ANK-1 promoter function 5-fold, while randomly chosen sequences decreased ANK-1 promoter function. Our results demonstrate a novel functional motif in the ANK-1 promoter.

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

2004 ◽  
Vol 287 (4) ◽  
pp. G822-G829 ◽  
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.

Identification of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence that governs Wnt-1 expression in the murine neural plate

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2735-2746 ◽  
Author(s):  
D.H. Rowitch ◽  
Y. Echelard ◽  
P.S. Danielian ◽  
K. Gellner ◽  
S. Brenner ◽  
...  
Keyword(s):  
Base Pair ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Neural Plate ◽  
Homologous Region ◽  
Regulatory Sequence ◽  
Embryonic Mouse ◽  
Cis Acting ◽  
Evolutionarily Conserved

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(−/−) embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.

cis- and trans-acting regulatory elements of the yeast URA3 promoter

1990 ◽  
Vol 10 (10) ◽  
pp. 5257-5270
Author(s):  
A Roy ◽  
F Exinger ◽  
R Losson
Keyword(s):  
Specific Binding ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Cis Acting ◽  
Cell Extracts ◽  
Dnase I Footprinting ◽  
Pair Sequence ◽  
Dyad Symmetry

Expression of the yeast pyrimidine biosynthetic gene, URA3, is induced three- to fivefold in response to uracil starvation, and this regulation is mediated by the transcriptional activator PPR1 (pyrimidine pathway regulator 1). In this study, we have analyzed the regulatory elements of the URA3 promoter by DNase I footprinting, using partially purified yeast cell extracts, by deletion mutagenesis, and by 5'-end mapping of RNA transcripts. Two DNA-binding activities have been detected, and at least four distinct cis-acting regions have been identified. A region rich in poly(dA-dT) serves as an upstream promoter element necessary for the basal level of URA3 expression. A 16-base-pair sequence with dyad symmetry acts acts as a uracil-controlled upstream activating site (UASURA) and shows a specific binding only with cell extracts from strains overproducing PPR1. This in vitro binding does not require dihydroorotic acid, the physiological inducer of URA3. The TATA region appears to be composed of two functionally distinct (constitutive and regulatory) elements. Two G + A-rich regions surrounding this TATA box bind an unidentified factor called GA-binding factor. The 5' copy, GA1, is involved in PPR1 induction and overlaps the constitutive TATA region. The 3' region, GA2, is necessary for maximal expression. Neither of these GA sequences acts as a UAS in a CYC1-lacZ context. The promoters of the unlinked but coordinately regulated URA1 and URA4 genes contain highly conserved copies of the UASURA sequence, which prompted us to investigate the effects of many point mutations within this UASURA sequence on PPR1-dependent binding. In this way, we have identified the most important residues of this binding site and found that a nonsymmetrical change of these bases is sufficient to prevent the specific binding and to suppress the UASURA activity in vivo. In addition, we showed that UASURA contains a constitutive activating element which can stimulate transcription from a heterologous promoter independently of dihydroorotic acid and PPR1.

scholarly journals Class II box consensus sequences in the HLA-DR alpha gene: transcriptional function and interaction with nuclear proteins.

1989 ◽  
Vol 9 (1) ◽  
pp. 50-56 ◽  
Author(s):  
P A Sherman ◽  
P V Basta ◽  
T L Moore ◽  
A M Brown ◽  
J P Ting
Keyword(s):  
Protein Binding ◽  
Nuclear Protein ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Class Ii ◽  
Promoter Function ◽  
Hla Dr ◽  
Alpha Gene

The promoter regions of class II major histocompatibility complex genes contain two highly conserved sequences, the X and Y boxes, which may be involved in the control of class II gene expression. In this study, we correlate in vivo functional assays for cis-acting regulatory elements in the HLA-DR alpha gene with in vitro binding assays for trans-acting regulatory proteins. Mutagenesis and transient transfection analyses indicated that both the X and Y boxes were important for HLA-DR alpha promoter function in a B lymphoblastoid cell line. Although specific nuclear protein interactions with the X consensus sequence were not apparent, the Y box, which contained an inverted CCAAT sequence, did bind specifically to at least one nuclear protein. This Y box-binding protein was present in nuclear extracts of all cell types examined, including human B and T cells and HeLa cells. The molecular mass of the protein, as determined by photoactivated protein-DNA cross-linking, was approximately 40 to 50 kilodaltons. Mutagenesis of the Y box that decreased protein binding also decreased promoter activity, implying that protein binding to this DNA sequence is important for DR alpha promoter function.

scholarly journals Class II box consensus sequences in the HLA-DR alpha gene: transcriptional function and interaction with nuclear proteins

1989 ◽  
Vol 9 (1) ◽  
pp. 50-56
Author(s):  
P A Sherman ◽  
P V Basta ◽  
T L Moore ◽  
A M Brown ◽  
J P Ting
Keyword(s):  
Protein Binding ◽  
Nuclear Protein ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Class Ii ◽  
Promoter Function ◽  
Hla Dr ◽  
Alpha Gene

The promoter regions of class II major histocompatibility complex genes contain two highly conserved sequences, the X and Y boxes, which may be involved in the control of class II gene expression. In this study, we correlate in vivo functional assays for cis-acting regulatory elements in the HLA-DR alpha gene with in vitro binding assays for trans-acting regulatory proteins. Mutagenesis and transient transfection analyses indicated that both the X and Y boxes were important for HLA-DR alpha promoter function in a B lymphoblastoid cell line. Although specific nuclear protein interactions with the X consensus sequence were not apparent, the Y box, which contained an inverted CCAAT sequence, did bind specifically to at least one nuclear protein. This Y box-binding protein was present in nuclear extracts of all cell types examined, including human B and T cells and HeLa cells. The molecular mass of the protein, as determined by photoactivated protein-DNA cross-linking, was approximately 40 to 50 kilodaltons. Mutagenesis of the Y box that decreased protein binding also decreased promoter activity, implying that protein binding to this DNA sequence is important for DR alpha promoter function.

scholarly journals Novel Binding Sites for Regulatory Factors in the Human Papillomavirus Type 18 Enhancer and Promoter Identified by In Vivo Footprinting

1998 ◽  
Vol 72 (1) ◽  
pp. 708-716 ◽  
Author(s):  
Paula H. Bednarek ◽  
Betty J. Lee ◽  
Sanjay Gandhi ◽  
Edward Lee ◽  
Benette Phillips
Keyword(s):  
Transcriptional Control ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Human Papillomaviruses ◽  
Regulatory Factors ◽  
Dnase I Footprinting ◽  
E6 And E7 ◽  
In Vivo Footprinting

ABSTRACT The E6 and E7 genes of human papillomaviruses (HPVs) associated with anogenital cancers are largely responsible for the oncogenic activity of these viruses, and regulation of these genes has been intensively studied. Transcription of the E6 and E7 genes is controlled by the viral upstream regulatory region (URR). We have used in vivo footprinting to examine the occupancy by regulatory factors of the HPV type 18 (HPV18) URR enhancer and promoter in the cervical carcinoma cell lines HeLa and C4-II. While corroborating occupancy in vivo of all of the elements previously implicated in the transcriptional control of the HPV18 E6 and E7 genes by in vitro DNase I footprinting, gel retardation assays, and transfection studies, we also detect occupancy in vivo of several enhancer and promoter sequences which have not been previously identified as HPV18 URR regulatory elements. Our data suggest that the HPV18 enhancer and promoter are more densely occupied by DNA-binding proteins than previously thought and raise the possibility that additional, possibly novel factors contribute to transcription of the HPV18 early genes.

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

2015 ◽  
Vol 308 (2) ◽  
pp. C189-C196 ◽  
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.

In vivo expression and molecular characterization of the porcine slow-myosin heavy chain

1993 ◽  
Vol 106 (1) ◽  
pp. 331-341 ◽  
Author(s):  
K.C. Chang ◽  
K. Fernandes ◽  
G. Goldspink
Keyword(s):  
Myosin Heavy Chain ◽  
Molecular Characterization ◽  
Heavy Chain ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Slow Myosin ◽  
Slow Myosin Heavy Chain ◽  
In Vivo Expression

We report on the molecular characterization of the porcine slow-myosin heavy chain (HC) beta gene and the isolation of its 5′ end cDNA. In vivo expression study, by in situ hybridization and histochemistry, revealed a highly regular rosette pattern of fiber arrangement, with a slow fiber occupying the central core, in all the skeletal muscles examined. This feature can be advantageous in the distinction of primary and secondary fibers in myogenic lineage studies. In the neonatal heart, beta isoform expression is diffuse, with higher expression occurring in the ventricle than in the atrium. Transient transfection assays showed the porcine promoter functions in a muscle- and differentiation stage-specific manner. In the 5′ regulatory region are several putative positive and negative regulatory elements, including a positive and a negative element in close proximity to each other in intron 1.

scholarly journals cis- and trans-acting regulatory elements of the yeast URA3 promoter.

1990 ◽  
Vol 10 (10) ◽  
pp. 5257-5270 ◽  
Author(s):  
A Roy ◽  
F Exinger ◽  
R Losson
Keyword(s):  
Specific Binding ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Cis Acting ◽  
Cell Extracts ◽  
Dnase I Footprinting ◽  
Pair Sequence ◽  
Dyad Symmetry

Expression of the yeast pyrimidine biosynthetic gene, URA3, is induced three- to fivefold in response to uracil starvation, and this regulation is mediated by the transcriptional activator PPR1 (pyrimidine pathway regulator 1). In this study, we have analyzed the regulatory elements of the URA3 promoter by DNase I footprinting, using partially purified yeast cell extracts, by deletion mutagenesis, and by 5'-end mapping of RNA transcripts. Two DNA-binding activities have been detected, and at least four distinct cis-acting regions have been identified. A region rich in poly(dA-dT) serves as an upstream promoter element necessary for the basal level of URA3 expression. A 16-base-pair sequence with dyad symmetry acts acts as a uracil-controlled upstream activating site (UASURA) and shows a specific binding only with cell extracts from strains overproducing PPR1. This in vitro binding does not require dihydroorotic acid, the physiological inducer of URA3. The TATA region appears to be composed of two functionally distinct (constitutive and regulatory) elements. Two G + A-rich regions surrounding this TATA box bind an unidentified factor called GA-binding factor. The 5' copy, GA1, is involved in PPR1 induction and overlaps the constitutive TATA region. The 3' region, GA2, is necessary for maximal expression. Neither of these GA sequences acts as a UAS in a CYC1-lacZ context. The promoters of the unlinked but coordinately regulated URA1 and URA4 genes contain highly conserved copies of the UASURA sequence, which prompted us to investigate the effects of many point mutations within this UASURA sequence on PPR1-dependent binding. In this way, we have identified the most important residues of this binding site and found that a nonsymmetrical change of these bases is sufficient to prevent the specific binding and to suppress the UASURA activity in vivo. In addition, we showed that UASURA contains a constitutive activating element which can stimulate transcription from a heterologous promoter independently of dihydroorotic acid and PPR1.

scholarly journals First insights into the molecular basis association between promoter polymorphisms of the IL1B gene and Helicobacter pylori infection in the Sudanese population: computational approach

2020 ◽  
Author(s):  
Abeer Babiker Idris ◽  
Einas Babiker Idris ◽  
Amany Eltayib Ataelmanan ◽  
Ali Elbagir Ali Mohamed ◽  
Bashir M. Osman Arbab ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Silico ◽  
Expression Patterns ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Computer Algorithms ◽  
Promoter Polymorphisms ◽  
H Pylori

Abstract Background: Helicobacter pylori (H. pylori) infects nearly half of the world’s population with a variation in incidence among different geographic regions. Genetic variants in the promoter regions of the IL1B gene can affect cytokine expression and creates a condition of hypoacidity which favors the survival and colonization of H. pylori. Therefore, the aim of this study was to characterize the polymorphic sites in the 5’- region [-687_+297] of IL1B in H. pylori infection using in silico tools.Results: A total of five nucleotide variations were detected in the 5’-regulatory region [-687_+297] of IL1B which led to the addition or alteration of transcription factor binding sites (TFBSs) or composite regulatory elements (CEs). Genotyping of IL1B-31 C>T revealed a significant association between -31T and susceptibility to H. pylori infection in the studied population (P=0.0363). Comparative analysis showed conservation rates of IL1B upstream [−368_+10] region above 70% in chimpanzee, rhesus monkey, a domesticated dog, cow and rat.Conclusions: In H. pylori-infected patients, three detected SNPs (-338, -155 and -31) located in the IL1B promoter were predicted to alter TFBSs and CE, which might affect the gene expression. These in silico predictions provide insight for further experimental in vitro and in vivo studies of the regulation of IL1B expression and its relationship to H. pylori infection. However, the recognition of regulatory motifs by computer algorithms is fundamental for understanding gene expression patterns.

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