Regulatory elements of the EKLF gene that direct erythroid cell-specific expression during mammalian development

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4078-4083 ◽  
Author(s):  
Li Xue ◽  
Xiaoyong Chen ◽  
Yanjie Chang ◽  
James J. Bieker
Keyword(s):  
Transcription Initiation ◽  
Fetal Liver ◽  
Critical Role ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Proximal Promoter ◽  
Erythroid Cell ◽  
Mammalian Development ◽  
Specific Expression ◽  
Position Effects

Abstract Erythroid Krüppel-like factor (EKLF) plays an essential role in enabling β-globin expression during erythroid ontogeny. It is first expressed in the extraembryonic mesoderm of the yolk sac within the morphologically unique cells that give rise to the blood islands, and then later within the hepatic primordia. The BMP4/Smad pathway plays a critical role in the induction of EKLF, and transient transfection analyses demonstrate that sequences located within less than 1 kb of its transcription initiation site are sufficient for high-level erythroid-specific transcription. We have used transgenic analyses to verify that 950 bp located adjacent to the EKLF start site of transcription is sufficient to generate lacZ expression within the blood islands as well as the fetal liver during embryonic development. Of particular importance are 3 regions, 2 of which overlap endogenous erythroid-specific DNase hypersensitive sites, and 1 of which includes the proximal promoter region. The onset of transgene expression mimics that of endogenous EKLF as it begins by day 7.5 (d7.5) to d8.0. In addition, it exhibits a strict hematopoietic specificity, localized only to these cells and not to the adjacent vasculature at all stages examined. Finally, expression is heterocellular, implying that although these elements are sufficient for tissue-specific expression, they do not shield against the position effects of adjacent chromatin. These analyses demonstrate that a surprisingly small DNA segment contains all the information needed to target a linked gene to the hematopoietic compartment at both early and later stages of development, and may be a useful cassette for this purpose.

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

2007 ◽  
Vol 407 (1) ◽  
pp. 101-111 ◽  
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.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

scholarly journals Specific expression of the human CD4 gene in mature CD4+ CD8- and immature CD4+ CD8+ T cells and in macrophages of transgenic mice

1994 ◽  
Vol 14 (2) ◽  
pp. 1084-1094
Author(s):  
Z Hanna ◽  
C Simard ◽  
A Laperrière ◽  
P Jolicoeur
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Transcription Initiation ◽  
Critical Role ◽  
Regulatory Elements ◽  
T Cell Subsets ◽  
Double Positive ◽  
Specific Expression

The CD4 protein plays a critical role in the development and function of the immune system. To gain more insight into the mechanism of expression of the human CD4 gene, we cloned 42.2 kbp of genomic sequences comprising the CD4 gene and its surrounding sequences. Studies with transgenic mice revealed that a 12.6-kbp fragment of the human CD4 gene (comprising 2.6 kbp of 5' sequences upstream of the transcription initiation site, the first two exons and introns, and part of exon 3) contains the sequences required to support the appropriate expression in murine mature CD4+ CD8- T cells and macrophages but not in immature double-positive CD4+ CD8+ T cells. Expression in CD4+ CD8+ T cells was found to require additional regulatory elements present in a T-cell enhancer fragment recently identified for the murine CD4 gene (S. Sawada and D. R. Littman, Mol. Cell. Biol. 11:5506-5515, 1991). These results suggest that expression of CD4 in mature and immature T-cell subsets may be controlled by distinct and independent regulatory elements. Alternatively, specific regulatory elements may control the expression of CD4 at different levels in mature and immature T-cell subsets. Our data also indicate that mouse macrophages contain the regulatory factors necessary to transcribe the human CD4 gene.

scholarly journals Genomic organization and transcriptional analysis of the human l-glutaminase gene

2003 ◽  
Vol 370 (3) ◽  
pp. 771-784 ◽  
Author(s):  
Cristina PÉREZ-GÓMEZ ◽  
José M. MATÉS ◽  
Pedro M. GÓMEZ-FABRE ◽  
Antonio del CASTILLO-OLIVARES ◽  
Francisco J. ALONSO ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Genomic Organization ◽  
Core Promoter ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Genome Project ◽  
Transcriptional Analysis ◽  
Cdna Clones ◽  
Mobility Shift ◽  
Specific Expression

In mammals, glutaminase (GA) is expressed in most tissues, but the regulation of organ-specific expression is largely unknown. Therefore, as an essential step towards studying the regulation of GA expression, the human liver-type GA (hLGA) gene has been characterized. LGA genomic sequences were isolated using the genome walking technique. Analysis and comparison of these sequences with two LGA cDNA clones and the Human Genome Project database, allowed the determination of the genomic organization of the LGA gene. The gene has 18 exons and is approx. 18kb long. All exon/intron junction sequences conform to the GT/AG rule. Progressive deletion analysis of LGA promoter—luciferase constructs indicated that the core promoter is located between nt −141 and +410, with several potential regulatory elements: CAAT, GC, TATA-like, Ras-responsive element binding protein and specificity protein 1 (Sp1) sites. The minimal promoter was mapped within +107 and +410, where only an Sp1 binding site is present. Mutation experiments suggested that two CAAT recognition elements near the transcription-initiation site (-138 and −87), play a crucial role for optimal promoter activity. Electrophoretic mobility-shift assays confirmed the importance of CAAT- and TATA-like boxes to enhance basal transcription, and demonstrated that HNF-1 motif is a significant distal element for transcriptional regulation of the hLGA gene.

scholarly journals Specific expression of the human CD4 gene in mature CD4+ CD8- and immature CD4+ CD8+ T cells and in macrophages of transgenic mice.

1994 ◽  
Vol 14 (2) ◽  
pp. 1084-1094 ◽  
Author(s):  
Z Hanna ◽  
C Simard ◽  
A Laperrière ◽  
P Jolicoeur
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Cd8 T Cells ◽  
Transcription Initiation ◽  
Critical Role ◽  
Regulatory Elements ◽  
T Cell Subsets ◽  
Double Positive ◽  
Specific Expression

The CD4 protein plays a critical role in the development and function of the immune system. To gain more insight into the mechanism of expression of the human CD4 gene, we cloned 42.2 kbp of genomic sequences comprising the CD4 gene and its surrounding sequences. Studies with transgenic mice revealed that a 12.6-kbp fragment of the human CD4 gene (comprising 2.6 kbp of 5' sequences upstream of the transcription initiation site, the first two exons and introns, and part of exon 3) contains the sequences required to support the appropriate expression in murine mature CD4+ CD8- T cells and macrophages but not in immature double-positive CD4+ CD8+ T cells. Expression in CD4+ CD8+ T cells was found to require additional regulatory elements present in a T-cell enhancer fragment recently identified for the murine CD4 gene (S. Sawada and D. R. Littman, Mol. Cell. Biol. 11:5506-5515, 1991). These results suggest that expression of CD4 in mature and immature T-cell subsets may be controlled by distinct and independent regulatory elements. Alternatively, specific regulatory elements may control the expression of CD4 at different levels in mature and immature T-cell subsets. Our data also indicate that mouse macrophages contain the regulatory factors necessary to transcribe the human CD4 gene.

scholarly journals Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene.

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

scholarly journals Structure, Promoter Analysis, and Chromosomal Localization of the Murine H+/K+-ATPase α2 Subunit Gene

2001 ◽  
Vol 12 (12) ◽  
pp. 2554-2564
Author(s):  
Wenzheng Zhang ◽  
Teresa Kuncewicz ◽  
Sandra C. Higham ◽  
Bruce C. Kone
Keyword(s):  
Transcription Initiation ◽  
Collecting Duct ◽  
Critical Role ◽  
Distal Colon ◽  
Initiation Site ◽  
Complete Sequence ◽  
Thick Ascending Limb ◽  
Specific Expression ◽  
Duct Cells ◽  
Collecting Duct Cells

ABSTRACT. The H+/K+-ATPase α2 subunit (HKα2) of distal colon and renal collecting ducts plays a critical role in potassium and acid-base homeostasis. The isolation and complete sequence of the murine HKα2 gene are reported. The HKα2 gene contains 23 exons and spans 23.5 kb of genomic DNA. The exon/intron organization is comparable to that of the human ATP1AL1 gene. Primer extension and 5′-rapid amplification of cDNA ends of distal colon RNA were used to map the transcription initiation site. Fluorescence in situ hybridization analysis localized the HKα2 gene to murine chromosome 14C3. Sequence analysis of 7.2 kb of the 5′-flanking region revealed numerous consensus sites for transcription factors, including two potential glucocorticoid response elements. Transient transfection of promoter-luciferase constructs demonstrated strong basal HKα2 promoter activity in renal collecting duct cells but not in fibroblasts or in a medullary thick ascending limb of Henle’s loop cell line. Deletion analysis revealed that the proximal 0.2 kb of the promoter was sufficient to confer activity in collecting duct cells. These data should prove important in elucidation of the mechanisms controlling the differential, tissue-specific expression of the HKα2 gene.

scholarly journals Essential Thioredoxin-Dependent Peroxiredoxin System from Helicobacter pylori: Genetic and Kinetic Characterization

2001 ◽  
Vol 183 (6) ◽  
pp. 1961-1973 ◽  
Author(s):  
Laura M. S. Baker ◽  
Ausra Raudonikiene ◽  
Paul S. Hoffman ◽  
Leslie B. Poole
Keyword(s):  
Helicobacter Pylori ◽  
Transcription Initiation ◽  
Reductase Activity ◽  
Critical Role ◽  
Oxygen Toxicity ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Oxygen Sensitive ◽  
H Pylori ◽  
Cellular Peroxide

ABSTRACT Helicobacter pylori, an oxygen-sensitive microaerophile, contains an alkyl hydroperoxide reductase homologue (AhpC, HP1563) that is more closely related to 2-Cys peroxiredoxins of higher organisms than to most other eubacterial AhpC proteins. Allelic replacement mutagenesis revealed ahpC to be essential, suggesting a critical role for AhpC in defending H. pyloriagainst oxygen toxicity. Characterization of the ahpCpromoter region divulged two putative regulatory elements and identified the transcription initiation site, which was mapped to 96 and 94 bp upstream of the initiation codon. No homologue ofahpF, which encodes the dedicated AhpC reductase in most eubacteria, was found in the H. pylori genome. Instead, homologues of Escherichia coli thioredoxin (Trx) reductase (TrxR, HP0825) and Trx (Trx1, HP0824) formed a reductase system forH. pylori AhpC. A second Trx homologue (Trx2, HP1458) was identified but was incapable of AhpC reduction, although Trx2 exhibited disulfide reductase activity with other substrates [insulin and 5,5′-dithiobis(2-nitrobenzoic acid)]. AhpC interactions with each substrate, Trx1 and hydroperoxide, were bimolecular and nonsaturable (infinite V max and Km values) but rapid enough (at 1 × 105 to 2 × 105 M−1 s−1) to suggest an important role for AhpC in cellular peroxide metabolism. AhpC also exhibited a wide specificity for hydroperoxide substrates, which, taken together with the above results, suggests a minimal binding site for hydroperoxides composed of little more than the cysteinyl (Cys49) active site. H. pylori AhpC was not reduced bySalmonella typhimurium AhpF and was slightly more active with E. coli TrxR and Trx1 than was S. typhimurium AhpC, demonstrating the specialized catalytic properties of this peroxiredoxin.

Stat6 Inhibits Human Interleukin-4 Promoter Activity in T Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4529-4538 ◽  
Author(s):  
Steve N. Georas ◽  
John E. Cumberland ◽  
Thomas F. Burke ◽  
Rongbing Chen ◽  
Ulrike Schindler ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Regulatory Elements ◽  
Jurkat Cells ◽  
Proximal Promoter ◽  
Interleukin 4 ◽  
Th2 Cells ◽  
Th Cells

Abstract The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4–secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4–activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.

Aqp1 expression in erythroleukemia cells: genetic regulation of glucocorticoid and chemical induction

1997 ◽  
Vol 273 (5) ◽  
pp. C1562-C1570 ◽  
Author(s):  
Chulso Moon ◽  
Landon S. King ◽  
Peter Agre
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Initiation ◽  
Protein Biosynthesis ◽  
Genetic Regulation ◽  
Water Channel ◽  
Erythroid Differentiation ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Chemical Induction ◽  
Site Mutation

The aquaporin-1 (AQP1) water channel protein is expressed in multiple mammalian tissues by several different developmental programs; however, the genetic regulation is undefined. The proximal promoter of mouse Aqp1 contains multiple putative cis-acting regulatory elements, and mouse erythroleukemia (MEL) cells are a well-characterized model for erythroid differentiation. Corticosteroid or dimethyl sulfoxide (DMSO) exposure induces AQP1 protein expression in MEL cells, and transcriptional regulation was investigated by transient transfections with Aqp1 promoter-reporter constructs. Dexamethasone induction is abrogated by deletion of two glucocorticoid response elements −0.5 kilobases (kb) from the transcription initiation site. Mutation of the GATA element at −0.62 kb has no effect, whereas mutation of the CACCC site at −37 bp significantly reduces DMSO-induced promoter activity. Hydroxyurea induces expression of AQP1 protein without acting through the proximal promoter. The MEL cell line is a reproducible erythroid model system for studying transcriptional regulation of the Aqp1 gene while determining the consequences on AQP1 protein biosynthesis.

Sign in / Sign up

Export Citation Format

Share Document