scholarly journals Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

2000 ◽  
Vol 348 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Isabelle VAN SEUNINGEN ◽  
Michaël PERRAIS ◽  
Pascal PIGNY ◽  
Nicole PORCHET ◽  
Jean-Pierre AUBERT
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Start Site ◽  
Transcription Start ◽  
Mucin Gene ◽  
Intron 1 ◽  
Flanking Region

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

scholarly journals Functional analysis of the human annexin I and VI gene promoters

1998 ◽  
Vol 332 (3) ◽  
pp. 681-687 ◽  
Author(s):  
Shaun R. DONNELLY ◽  
Stephen E. MOSS
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Luciferase Reporter ◽  
Gene Promoters ◽  
Start Site ◽  
Luciferase Reporter Gene ◽  
Transcription Start ◽  
A431 Cells ◽  
Annexin I ◽  
Annexin Vi

To gain insight into the molecular basis of annexin gene expression we have analysed the annexin I and VI gene promoters. A previously described 881 bp sequence immediately upstream of the annexin I transcription start site and a similar size fragment proximal to the annexin VI transcription start site both drove expression of the luciferase reporter gene in fibroblasts and epithelial cells. Neither promoter displayed any sensitivity to dexamethasone, suggesting that the putative glucocorticoid response element in the annexin I promoter is non-functional. Consistent with this, endogenous annexin I gene expression was unaffected by dexamethasone at the mRNA and protein levels in A431 cells. A series of 5´ deletions of the two promoters were examined to define the minimal active sequences. For annexin I this corresponded to a sequence approx. 150 bp upstream of the transcription start site that included CAAT and TATA boxes. Unexpectedly, the annexin VI promoter, which also contains CAAT and TATA boxes, was fully active in the absence of these elements, a 53 bp sequence between these boxes and the transcription start site having maximal activity. Electrophoretic mobility-shift assays with nuclear extracts from A431 and HeLa cells with probes corresponding to this region revealed an SP1-binding site. These results show that the annexin I and VI genes have individual modes of transcriptional regulation and that if either annexin I or annexin VI has an anti-inflammatory role, then this is in the absence of steroid-induced gene expression.

scholarly journals Expression of the human oestrogen receptor-alpha gene is regulated by promoter F in MG-63 osteoblastic cells

2003 ◽  
Vol 372 (3) ◽  
pp. 831-839 ◽  
Author(s):  
Elisabetta LAMBERTINI ◽  
Letizia PENOLAZZI ◽  
Silvia GIORDANO ◽  
Laura DEL SENNO ◽  
Roberta PIVA
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcription Start Site ◽  
Thyroid Hormone Receptor ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Start Site ◽  
Mcf7 Cells ◽  
Transcription Start ◽  
Specific Element

(O)estrogen receptor-α (ERα), a hormone-dependent transcription factor belonging to the steroid/thyroid-hormone-receptor superfamily, plays an essential role in the development and maintenance of the skeleton. Here we report the analysis of an unexplored sequence inside the bone-specific distal promoter F (PF) with respect to the regulation of ERα gene expression in bone. This sequence, 785 bp in size, is localized upstream of the assigned transcription start site of exon F, at −117140 bp from the originally described transcription start site +1. It contains a TA reach box, a conventional CAAT box and potential regulatory elements for many transcription factors, including Cbfa1 [OSE2 (osteoblast-specific element) core binding factor], GATA-1 [(A/T)GATA(A/G) binding protein], Sox5 [sex-determining region Y (SRY)-type HMG bOX protein, belonging to a subfamily of DNA-binding proteins with an HMG domain], Sry, AP1 (activator protein 1) and CP2 (activator of γ-globin). It is able to strongly activate the luciferase reporter gene in MG-63 osteoblastic-like cells, but not in MCF7 breast-cancer cells. This is in agreement with different transcripts that we found in the two cell types. The footprinting and electrophoretic mobility-shift assays (EMSAs) showed that, inside the region analysed, there were some sequences that specifically reacted to nuclear proteins isolated from MG-63 cells. In particular, we identified two regions, named PFa and PFb, that do not present binding sites for known transcription factors and that are involved in a strong DNA–protein interaction in MG-63, but not in MCF7, cells. The analysis of three transcription factors (GATA-1, Sry and Sox) that might bind the identified footprinted areas suggested a possible indirect role of these proteins in the regulation of ERα gene expression in bone. These data provide evidence for different promoter usage of the ERα gene through the recruitment of tissue-specific transcription activators and co-regulators.

Increased Transcription of the Neuropeptide Neuromedin U by Ets-2 and C-Myb Not B-Myb in Human Myeloid Leukemia Cells Promotes Their Proliferation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2254-2254
Author(s):  
Julia Gambone ◽  
Stephanie Dusaban ◽  
Susan Shetzline
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Transcription Start Site ◽  
Myeloid Leukemia ◽  
Luciferase Activity ◽  
Hematopoietic Cells ◽  
Luciferase Reporter ◽  
Start Site ◽  
Transcription Start

Abstract Neuromedin U (NmU) is a 25-amino acid neuropeptide that is expressed at high levels in the brain, gut, and bone marrow. In the brain, NmU modulates the central control of feeding and bone mass. In the intestine, NmU regulates the contraction of smooth muscle, local blood flow, and ion transport. In the bone marrow, the physiological function of NmU remains ill-defined. To determine the physiological function of NmU in hematopoietic cells, we utilized K562-MERT cells, which express a tamoxifen-inducible dominant negative Myb (MERT), and cell culture assays. We chose to test the function of NmU in a cell line that expresses high levels of the proto-oncogene c-myb because c-Myb encodes a transcription factor that regulates cell proliferation, survival, and differentiation through the transactivation of its target genes. By inhibiting endogenous Myb activity in K562-MERT cells by tamoxifen in methylcellulose cultures, we observed a 6-fold decrease in cell number compared to untreated K562-MERT cells. Supplementing the tamoxifen treated methylcellulose cultures of K562-MERT cells with NmU resulted in a 3.6-fold increase in K562-MERT cell number compared to K562-MERT cells treated with only tamoxifen. In liquid cultures using primary cells from patients with acute myeloid leukemia, we observed more cells in the cultures with NmU than when NmU was absent from the cultures. Finally, silencing NmU gene expression in K562 cells via RNA interference decreased the proliferation of these cells. Collectively, these data demonstrate that NmU functions to promote the proliferation of hematopoietic cells. The ability of NmU to rescue cell growth in tamoxifen treated K562-MERT cells and Myb’s role in regulating hematopoietic cell proliferation led us to hypothesize that Myb mediates hematopoietic cell proliferation in part by directly regulating NmU gene expression. To test this hypothesis, we examined the DNA sequence upstream of NmU’s predicted transcription start site (as noted in Genbank accession #NM_006681) for potential Myb response elements (MREs). We identified eleven potential MREs within the first 2kb upstream of NmU’s transcription start site. Of these MREs, five were identified as canonical (PyAAC(G/T)G). Our search also identified potential Ets-2 binding motifs within the human NmU promoter region, which were of interest because Myb has been reported to cooperate with Ets-2 in the regulation of c-kit and CD34 gene expression. To determine if any of the potential MREs within the NmU promoter were functional, we first completed in vitro assays using luciferase reporter constructs followed by in vivo assays using chromatin immunoprecipitation (ChIP) assays. The luciferase reporter constructs were generated such that the first 2kb upstream of NmU’s transcription start site was inserted upstream of the luciferase gene in pGL3-basic to yield pGL3-NmU. When pGL3-NmU was co-transfected into 293T cells with a c-Myb expression construct, we observed an average of 15-fold induction of luciferase activity compared to empty vector. There was no change in luciferase activity when 293T cells were co-transfected with the c-Myb isoform B-Myb compared to empty vector, suggesting that c-Myb and not B-Myb interact with the MREs within the NmU promoter to induce its expression. Mutation of either MRE9 or 10, which are distal to NmU’s transcription start site, in pGL3-NmU prevented c-Myb from inducing luciferase gene expression, demonstrating that MRE9 and 10 are functional. When Ets-2 was co-transfected with pGL3-NmU into 293T cells, a 10-fold increase in luciferase activity was observed suggesting that Ets-2 and c-Myb may cooperate to increase the transcription of NmU. To determine the physiologic relevance of our in vitro luciferase studies, we performed ChIP assays. Following immunoprecipitation of the cross-linked chromatin with either anti-c-Myb or anti-Ets-2, a PCR product of the recovered DNA was observed using primers that flanked MRE9, MRE10, and one Ets-2 site, demonstrating that in vivo c-Myb and Ets-2 interact directly with NmU’s promoter. Studies are underway to determine whether c-Myb and Ets-2 cooperate to induce NmU gene expression. Further characterization of the regulation of NmU gene expression in normal and malignant hematopoietic cells may yield new clues to Myb’s role in leukemogenesis and could suggest new therapeutic targets in human myeloid leukemia cells.

scholarly journals Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Daniel P. Kiesenhofer ◽  
Robert L. Mach ◽  
Astrid R. Mach-Aigner
Keyword(s):  
Trichoderma Reesei ◽  
Transcription Start Site ◽  
Binding Sites ◽  
Inverted Repeat ◽  
Promoter Activity ◽  
Promoter Strength ◽  
Start Site ◽  
Transcription Start ◽  
Content Type ◽  
Sheer Number

ABSTRACTTrichoderma reeseican produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of thecbh1promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in thexyn1promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One suchciselement, an inverted repeat, plays a crucial role in the inducibility of thexyn1promoter. We investigated the impact of the properties of suchciselements by shuffling them by insertion, exchange, deletion, and rearrangement ofciselements in both thecbh1andxyn1promoter. A promoter-reporter assay using theAspergillus nigergoxAgene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase incbh1promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances ofciselements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances ofciselements have large impacts on the strength of thecbh1promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically importantcbh1promoter can be improved byciselement rearrangement.IMPORTANCEIn the present study, we demonstrate that the arrangement ofciselements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances ofciselements to the transcription start site. Furthermore, we found that the configuration ofciselements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement ofciselements is an important factor that should not be overlooked when enhancement of gene expression is desired.

scholarly journals Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 5993-6004 ◽  
Author(s):  
Anne M. L. Barnard ◽  
Jeffrey Green ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Binding Site ◽  
Transcription Regulation ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Start Site ◽  
Transcription Start ◽  
Substitution Mutant

ABSTRACT FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

scholarly journals Identification and characterization of the MUC2 (human intestinal mucin) gene 5′-flanking region: promoter activity in cultured cells

1997 ◽  
Vol 325 (1) ◽  
pp. 259-267 ◽  
Author(s):  
James R. GUM ◽  
James W. HICKS ◽  
Young S. KIM
Keyword(s):  
Cell Lines ◽  
Promoter Activity ◽  
Cultured Cells ◽  
Cell Types ◽  
Luciferase Reporter ◽  
Cell Type Specificity ◽  
Mobility Shift ◽  
Initiation Point ◽  
Mucin Gene ◽  
Flanking Region

The initiation point for MUC2 gene transcription is located within a 7000-base GC-rich region of the mucin gene cluster found on chromosome 11p15.5. The promoter activity of the 5′-flanking region of the MUC2 gene was examined following its cloning into the luciferase-producing pGL2-Basic reporter vector. A short segment comprising bases -91 to -73 relative to the start of transcription was found to be important for basal promoter activity in all cell lines tested. Electrophoretic mobility shift assays demonstrated nuclear protein binding to this region, which contains the consensus CACCC motif (5′-GCCACACCC). This element has been shown to be functionally important in several promoters that are active in diverse cell types. Competition experiments using an Sp1 oligonucleotide and antibody supershift experiments indicated that both Sp1 and other Sp1 family members bind to this element. Inclusion of the region between bases -228 and -171 in pGL2-Basic constructs increased normalized luciferase reporter activity by almost 3-fold in C1a cells, which produce relatively high levels of MUC2 mRNA. Significantly lower levels of normalized luciferase activity resulted when the same construct was transfected into cultured cell lines that express low or undetectable levels of MUC2, suggesting a possible role for this region in conferring cell-type specificity of expression. We also demonstrate, using actinomycin D, that the MUC2 mRNA is long-lived, at least in cultured cells. Moreover, no evidence was found that the MUC2 mRNA turned over more rapidly in LS174T cells, which produce relatively low levels of MUC2 mRNA, as compared with C1a cells, which produce high levels of mRNA. Thus a long mRNA half-life appears to be an important mechanism involved in achieving elevated levels of MUC2 mRNA.

scholarly journals Cis- and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression

1993 ◽  
Vol 13 (6) ◽  
pp. 3202-3212
Author(s):  
L Guerrini ◽  
S S Gong ◽  
K Mangasarian ◽  
C Basilico
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Hela Cells ◽  
Promoter Activity ◽  
Regulatory Element ◽  
Amino Acid Starvation ◽  
Asparagine Synthetase ◽  
Start Site ◽  
Transcription Start ◽  
Nuclear Extracts

We have previously shown that asparagine synthetase (AS) mRNA expression can be dramatically up-regulated by asparagine deprivation in ts11 cells, mutants of BHK hamster cells which encode a temperature-sensitive AS. The expression of AS mRNA was also induced upon starvation for one of several essential amino acids in HeLa cells. We also showed that regulation of AS mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. Here we report the analysis of the elements in the human AS promoter region important for its basal activity and activation by amino acid starvation. Our results indicate that a DNA fragment spanning from nucleotides -164 to +44 of the AS promoter is sufficient for uninduced and induced gene expression. Mutations in a region located 15 to 30 bp downstream from the major transcription start site that shows good homology to a sequence in the first exon of c-fos implicated as a negative regulatory element resulted in a significant increase in basal gene expression but did not affect regulation. Interestingly, this region binds single-stranded-DNA-binding proteins that are specific for the AS coding strand. Mutations in either one of two putative binding sites for transcription factor Sp1, in a region of approximately 60 bp where many minor RNA start sites are located, or at the major transcription start site decreased promoter activity, but significant induction by amino acid starvation was still observed. Strikingly, mutations centered around nucleotide -68 not only decreased the basal promoter activity but also abolished amino acid regulation. This DNA region contains the sequence 5'-CATGATG-3', which we call the amino acid response element (AARE), that can bind a factor(s) present in HeLa cells nuclear extracts that is not capable of binding to an AS promoter with mutations or deletions of the AARE. This finding is in line with the hypothesis that transcriptional activation of AS gene expression is mediated through the binding of a positive regulatory element. We did not detect changes in the level of binding of this factor to the AARE by using nuclear extracts from HeLa cells grown under starved conditions, suggesting that activation of this factor(s) results from posttranslational modification or complexing with other proteins that do not affect its DNA-binding properties.

scholarly journals Alpha-thalassemia resulting from deletion of regulatory sequences far upstream of the alpha-globin structural genes

Blood ◽  
1991 ◽  
Vol 78 (6) ◽  
pp. 1589-1595
Author(s):  
L Romao ◽  
L Osorio-Almeida ◽  
DR Higgs ◽  
J Lavinha ◽  
SA Liebhaber
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Globin Gene ◽  
Regulatory Sequences ◽  
Start Site ◽  
Structural Genes ◽  
Transcription Start ◽  
Alpha Thalassemia ◽  
Alpha Globin ◽  
Globin Gene Expression

We describe an alpha-thalassemia determinant in which alpha-globin expression is silenced by a deletion located 27 kb 5′ to the transcription start site of the alpha 2-globin gene. This alpha- thalassemic determinant, (alpha alpha)MM, is a member of a newly described group of thalassemic mutations resulting from deletion of locus-controlling sequences critical to globin gene expression.

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