scholarly journals Cis-acting elements regulating the placenta-specific promoter of the bovine Cyp19 gene

2000 ◽  
Vol 25 (3) ◽  
pp. 265-273 ◽  
Author(s):  
C Kalbe ◽  
R Furbass ◽  
M Schwerin ◽  
J Vanselow
Keyword(s):  
Reporter Gene ◽  
Point Mutations ◽  
Regulatory Elements ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Cis Acting ◽  
Bovine Placenta ◽  
Cyp19 Gene ◽  
Key Enzyme

Cyp19 encodes aromatase cytochrome P450, the key enzyme of oestrogen biosynthesis. In the bovine placenta, the majority of Cyp19 transcripts include a 5' untranslated region which is encoded by exon 1.1; this suggests that its 5'-flanking region is the predominant placental promoter. The aim of the present investigation was to examine the promoter activity of this region and to map cis-acting regulatory elements in order to improve our understanding of the complex regulation of this gene within the placenta. As an initial approach, human JEG-3 choriocarcinoma cells were transiently transfected with reporter-gene constructs consisting of different 5'-flanking sequences of exon 1.1 fused to the luciferase gene as a reporter. To localise and further characterise functional cis-acting elements, targeted point mutations and electrophoretic mobility-shift experiments were used. The data demonstrate, for the first time, (1) that the bovine exon 1.1 5'-flanking sequence is an active promoter, (2) that 404 bp of this region are sufficient for constitutive reporter-gene expression in JEG-3 cells and (3) that the region includes at least two different enhancer elements; the data also suggest (4) that one of these elements consists of the E-box motif CATGTG and that the second enhancer element includes the half-site hexameric sequence AGGTCA and additional nucleotides flanking this element upstream.

scholarly journals Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499 ◽  
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

scholarly journals Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Lidia A. Daimiel ◽  
María E. Fernández-Suárez ◽  
Sara Rodríguez-Acebes ◽  
Lorena Crespo ◽  
Miguel A. Lasunción ◽  
...  
Keyword(s):  
Cellular Response ◽  
Binding Protein ◽  
Regulatory Element ◽  
Cholesterol Biosynthesis ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Gene Characterization ◽  
Cis Acting

DHCR24 (3β-hydroxysterol Δ24-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at −98/−90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5′-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression.

Identification of a novelcis-acting element for fibroblast-specific transcription of theFSP1 gene

1998 ◽  
Vol 275 (2) ◽  
pp. F306-F314 ◽  
Author(s):  
Hirokazu Okada ◽  
Theodore M. Danoff ◽  
Andreas Fischer ◽  
Jesus M. Lopez-Guisa ◽  
Frank Strutz ◽  
...  
Keyword(s):  
S100 Protein ◽  
Luciferase Reporter ◽  
Flanking Sequence ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Cis Acting ◽  
First Intron ◽  
Nuclear Extracts ◽  
Ef Hands ◽  
Gel Mobility Shift

The FSP1 gene encodes a filament-binding S100 protein with paired EF hands that is specifically expressed in fibroblasts. This led us to look for cis-acting elements in the FSP1 promoter that might engage nuclear transcription factors unique to fibroblasts. The first exon of FSP1 is noncoding, therefore, a series of luciferase reporter minigenes were created containing varying lengths of 5′-flanking sequence, the first intron, and the noncoding region of the second exon. A position and promoter-dependent proximal element between −187 and −88 bp was shown to be active in fibroblasts but not in epithelium. Sequence in the first intron from +777 to +964 had an enhancing effect that was not cell type specific. Hsv TK reporter constructs driven by this promoter/intron cassette in transgenic mice were coexpressed appropriately with FSP1 in tissue fibroblasts. Gel mobility shift competitor assays identified a novel domain, FTS-1 (fibroblast transcription site-1; TTGAT from −177 to −173 bp), that specifically interacts with nuclear extracts from fibroblasts. The necessity of this binding site was confirmed by site-specific mutagenesis. Database searches also turned up putative FTS-1 sites in the early promoter regions of other fibroblast expressed proteins, including the α1 and α2(I), and α1(III) collagens and the αSM-actin gene. We hypothesize that the selective engagement of FTS-1 elements may contribute to the mesenchymal phenotype of fibroblasts and perhaps other dedifferentiated cells.

Transcriptional regulation of human abraxas brother protein 1 expression by yin yang 1

2020 ◽  
Author(s):  
Pan Song ◽  
Jian Hong ◽  
Yuan Wang ◽  
Xuelian Yao ◽  
Yiqun Zhan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Start Codon ◽  
Mobility Shift ◽  
Cis Acting ◽  
Yin Yang 1 ◽  
Activating Transcription Factor 4 ◽  
Yin Yang

Abraxas brother protein 1 (ABRO1) is a subunit of the deubiquitinating enzyme BRCC36-containing isopeptidase complex and plays important roles in cellular responses to stress by interacting with its binding partners, such as ubiquitin-specific peptidase 7, p53, activating transcription factor 4, THAP-domain containing 5, and serine hydroxymethyltransferase. However, the transcriptional regulation of ABRO1 remains unexplored. In this study, we identified and characterized the core regulatory elements of the human ABRO1 gene and mapped them to the ABRO1 promoter region. Additionally, 5′ rapid amplification of cDNA ends revealed that the transcriptional start site (TSS) was located −13 bp upstream from the start codon. Reporter gene, chromatin immunoprecipitation, and electrophoretic mobility shift assays demonstrated that ABRO1 transcription was regulated through cis-acting elements located in the region −89 to −59 bp upstream of the ABRO1 TSS and that these elements were targeted by yin yang 1 transcription factor (YY1). Moreover, YY1 overexpression increased human ABRO1 mRNA and protein expression, and small-interfering RNA-mediated downregulation of YY1 attenuated ABRO1 expression. These results suggested that YY1 positively regulated human ABRO1 expression by binding to cis-acting elements located in the ABRO1 TSS.

Functional analysis of the promoter of a sea urchin metallothionein gene

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1142-1150 ◽  
Author(s):  
Peter Cserjesi ◽  
Peter Fairley ◽  
Bruce P. Brandhorst
Keyword(s):  
Sea Urchin ◽  
Regulatory Element ◽  
Gene Promoter ◽  
Flanking Sequence ◽  
Core Element ◽  
Sequence Element ◽  
Enhancer Element ◽  
Cis Acting ◽  
Conserved Sequence ◽  
Gc Box

The 5′-flanking region of the metallothionein (MT) gene LpMT1 of the sea urchin Lytechinus pictus includes three copies of a conserved sequence that includes the metal-responsive element (MRE) consensus core sequence required for heavy metal induction of other MT genes, a GC box, a G box of a putative basal level enhancer element which includes another MRE core element, and a poly(C) tract. A fragment of LpMT1 DNA from nucleotides + 31 to −309 fused to a chloramphenicol acetyltransferase reporter gene was inducible with cadmium after injecton into L. pictus embryos. This induced activity was greatly reduced in a deletion mutant which retained only 195 base pairs of 5′-flanking sequence, including the proximal pair of MREs and the G box, but excluding the poly(C) tract, GC box, and distal MRE. A potent human hMT-IIA gene promoter is marginally functional in L. pictus embryos. In contrast, the LpMT1 promoter is active in HeLa cells and in embryos of the sea urchin Strongylocentrotus purpuratus. The hMT-IIA gene may lack a cis-acting sequence element required for expression of MT genes in L. pictus embryos. The LpMT1 promoter is a powerful, inducible, promiscuous promoter useful for driving the expression of heterologous genes in sea urchin embryos.Key words: metallothionein, sea urchin, transcriptional regulation, induction by heavy metals, metal regulatory element.

Human erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting regulatory elements

1990 ◽  
Vol 10 (3) ◽  
pp. 930-938
Author(s):  
G L Semenza ◽  
R C Dureza ◽  
M D Traystman ◽  
J D Gearhart ◽  
S E Antonarakis
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Cobalt Chloride ◽  
Fetal Liver ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Flanking Sequence ◽  
Rnase Protection ◽  
Cis Acting

Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. The single-copy EPO gene is normally expressed in liver and kidney, and increased transcription is induced by anemia or cobalt chloride administration. To identify cis-acting DNA sequences responsible for regulated expression, transgenic mice were generated by microinjection of a 4-kilobase-pair (kb) (tgEPO4) or 10-kb (tgEPO10) cloned DNA fragment containing the human EPO gene, 0.7 kb of 3'-flanking sequence, and either 0.4 or 6 kb of 5'-flanking sequence, respectively. tgEPO4 mice expressed the transgene in liver, where expression was inducible by anemia or cobalt chloride, kidney, where expression was not inducible, and other tissues that do not normally express EPO. Human EPO RNA in tgEPO10 mice was detected only in liver of anemic or cobalt-treated mice. Both tgEPO4 and tgEPO10 mice were polycythemic, demonstrating that the human EPO RNA transcribed in liver is functional. These results suggest that (i) a liver inducibility element maps within 4 kb encompassing the gene, 0.4 kb of 5'-flanking sequence, and 0.7 kb of 3'-flanking sequence; (ii) a negative regulatory element is located between 0.4 and 6 kb 5' to the gene; and (iii) sequences required for inducible kidney expression are located greater than 6 kb 5' or 0.7 kb 3' to the gene. RNase protection analysis revealed that human EPO RNA in anemic transgenic mouse liver and hypoxic human hepatoma cells is initiated from several sites, only a subset of which is utilized in nonanemic transgenic liver and human fetal liver.

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.

Constitutive and interleukin-1 (IL-1)-inducible factors interact with the IL-1-responsive element in the IL-6 gene

1990 ◽  
Vol 10 (6) ◽  
pp. 2757-2764
Author(s):  
H Isshiki ◽  
S Akira ◽  
O Tanabe ◽  
T Nakajima ◽  
T Shimamoto ◽  
...  
Keyword(s):  
Phorbol Esters ◽  
Interleukin 1 ◽  
Regulatory Elements ◽  
Enhancer Element ◽  
Cis Acting ◽  
Nuclear Factors ◽  
Two Factors ◽  
Gene Expression Studies ◽  
Responsive Element ◽  
Dyad Symmetry

The interleukin-6 (IL-6) promoter is rapidly and transiently activated with other cytokines, including IL-1, tumor necrosis factor, and platelet-derived growth factor, as well as phorbol esters and agents that increase intracellular cyclic AMP. In this study, we have investigated cis-acting regulatory elements and trans-acting factors responsible for IL-1-induced IL-6 gene expression. Studies on the 5' deletion mutants of the human IL-6 gene suggested that the IL-1-responsive element was mapped within the IL-6 promoter region (-180 to -123) which was homologous to the c-fos serum-responsive enhancer element. Gel retardation assay identified two types of nuclear factors that bound to this region, one constitutive and the other inducible. These two factors recognized a 14-base-pair (bp) palindromic sequence, ACATTGCACAATCT. Furthermore, three copies of this 14-bp palindrome conferred IL-1 responsiveness to the basal enhancerless IL-6 promoter, indicating that a 14-bp-dyad symmetry sequence was an IL-1-responsive element in the IL-6 gene.

Alternative Super-Enhancer States Determine MYC Sensitivity to Notch and Brd4 Inhibitors in T Lymphoblastic Leukemia/Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 863-863
Author(s):  
Hongfang Wang ◽  
Yumi Yashiro-Ohtani ◽  
Chongzhi Zang ◽  
Yinling Joey Wong ◽  
Will Bailis ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Reporter Gene ◽  
Lymphoblastic Leukemia ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Enhancer Element ◽  
Enhancer Region

Abstract Gain-of-function NOTCH1 mutations are oncogenic drivers in a high fraction of T-cell lymphoblastic leukemia/lymphoma (T-LL). These mutations variously cause increased production or stabilization of the free intracellular domain of NOTCH1, which regulates gene expression by forming a transcription complex with the DNA-binding factor RBPJ and coactivators of the MAML family. Using expression profiling and ChIP-seq, we have shown that NOTCH1/RBPJ complexes activate most target genes by binding to super-enhancers, large regulatory elements that switch on transcription through long-range interactions with gene promoters. MYC is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates MYC is unknown. To understand which regulatory element(s) regulate MYC expression, we used chromatin conformation capture (3C) assays to test the interaction between putative enhancer(s) and the MYC promoter in T-LL cell lines, and reporter gene assays to confirm enhancer function of candidate sites. We identified a distal site located >1 Mb 3’ of human and murine MYC termed the Notch-dependent MYC enhancer (NDME) that binds Notch transcription complexes and physically interacts with the MYC proximal promoter. An ~1 kb DNA fragment containing this site activates a luciferase reporter gene in a Notch-dependent fashion in T-LL cells but not in heterologous cell types. The Notch binding site lies within a large enhancer region (>600 kb in breadth) containing multiple discrete H3K27ac peaks. Remarkably, acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer region and the MYC promoter that correlate with NOTCH1/RBPJ complex binding and MYC expression. T-LL cells selected for resistance to gamma-secretase inhibitors (GSIs) exhibit epigenetic silencing of the NDME and loss of NDME looping interactions with the MYC promoter, yet maintain MYC expression. 3C analysis of GSI resistant cells shows preferential interaction between the MYC promoter and a more 3’ enhancer element recently described as a BRD4-dependent regulator of MYC expression in acute myeloid leukemia cells. In line with this observation, BRD4 antagonists are potent inhibitors of MYC expression in GSI resistant T-LL cells but not GSI-sensitive cells. We also studied a case of Notch-mutated early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL). ChIP-Seq analysis of the leukemic blasts revealed an “AML-like” MYC enhancer chromatin state, and as predicted from our analysis of cell lines, the blasts rapidly down-regulated MYC in response to BRD4 inhibitor but not in response to GSI. These findings suggest that specific MYC chromatin states predict responsiveness to Notch and BRD4 inhibitors, and provide a rationale for use of Notch and BRD4 inhibitor combinations in Notch-mutated leukemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals Intronic and flanking sequences are required to silence enhancement of an embryonic beta-type globin gene.

1996 ◽  
Vol 16 (1) ◽  
pp. 236-246 ◽  
Author(s):  
N J Wandersee ◽  
R C Ferris ◽  
G D Ginder
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Regulatory Elements ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Eukaryotic Gene ◽  
Flanking Sequences ◽  
Silencer Elements

In the course of studying regulatory elements that affect avian embryonic rho-globin gene expression, the multipotential hematopoietic cell line K562 was transiently transfected with various rho-globin gene constructs containing or lacking an avian erythroid enhancer element. Enhanced levels of rho gene expression were seen from those constructs containing an enhancer element and minimal 5' or 3' flanking rho sequences but were not seen from enhancer-containing constructs that included extensive 5' and 3' flanking sequences. Deletion analysis localized 5' and 3' "enhancer-silencing elements" to -2140 to -2000 and +1865 to +2180 relative to the mRNA cap site. A third element required for enhancer silencing was identified within the second intron of the rho gene. The treatment of K562 cells with hemin, which induces erythroid differentiation, partially alleviated the enhancer-silencing effect. The silencer elements were able to block enhancement from a murine erythroid enhancer, but not from a nonerythroid enhancer. Electrophoretic mobility shift assays demonstrated that the transcription factor YY1 is able to bind both the 5' and 3' enhancer silencer elements; a point mutation of the single overlapping YY1/NF-Y binding site in the 3' element completely abolished the enhancer-silencing effect. These results demonstrate a complex enhancer silencer that requires 5' flanking, intronic, and 3' flanking sequences for a single regulatory effect on a eukaryotic gene.

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