Transcription factor Sp1 binds to and activates a human hsp70 gene promoter

1989 ◽  
Vol 9 (9) ◽  
pp. 4099-4104
Author(s):  
W D Morgan
Keyword(s):  
Transcription Factor ◽  
Constitutive Expression ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Transcription Factor Sp1 ◽  
Dnase I Footprinting ◽  
Proximal Site ◽  
Human Hsp70

I investigated the binding of purified transcription factor Sp1 from HeLa cells to the human hsp70 promoter by DNase I footprinting. Three binding sites were detected within the upstream promoter region, including one located 46 base pairs upstream of the transcription start, between the TATA box and the proximal CCAAT box element. In vitro transcription demonstrated that the proximal site is capable of responding to Sp1-dependent stimulation. These results suggest that Sp1 might contribute to constitutive expression in vivo and might also be involved in the various regulatory responses that affect this gene.

scholarly journals Transcription factor Sp1 binds to and activates a human hsp70 gene promoter.

1989 ◽  
Vol 9 (9) ◽  
pp. 4099-4104 ◽  
Author(s):  
W D Morgan
Keyword(s):  
Transcription Factor ◽  
Constitutive Expression ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Transcription Factor Sp1 ◽  
Dnase I Footprinting ◽  
Proximal Site ◽  
Human Hsp70

I investigated the binding of purified transcription factor Sp1 from HeLa cells to the human hsp70 promoter by DNase I footprinting. Three binding sites were detected within the upstream promoter region, including one located 46 base pairs upstream of the transcription start, between the TATA box and the proximal CCAAT box element. In vitro transcription demonstrated that the proximal site is capable of responding to Sp1-dependent stimulation. These results suggest that Sp1 might contribute to constitutive expression in vivo and might also be involved in the various regulatory responses that affect this gene.

scholarly journals Integrin α3β1 Promotes Invasive and Metastatic Properties of Breast Cancer Cells through Induction of the Brn-2 Transcription Factor

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 480
Author(s):  
Rakshitha Pandulal Miskin ◽  
Janine S. A. Warren ◽  
Abibatou Ndoye ◽  
Lei Wu ◽  
John M. Lamar ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Transcription Factor ◽  
Cancer Cells ◽  
Cell Invasion ◽  
Breast Cancer Cells ◽  
Gene Promoter ◽  
Akt Inhibitor ◽  
Integrin Α3β1

In the current study, we demonstrate that integrin α3β1 promotes invasive and metastatic traits of triple-negative breast cancer (TNBC) cells through induction of the transcription factor, Brain-2 (Brn-2). We show that RNAi-mediated suppression of α3β1 in MDA-MB-231 cells caused reduced expression of Brn-2 mRNA and protein and reduced activity of the BRN2 gene promoter. In addition, RNAi-targeting of Brn-2 in MDA-MB-231 cells decreased invasion in vitro and lung colonization in vivo, and exogenous Brn-2 expression partially restored invasion to cells in which α3β1 was suppressed. α3β1 promoted phosphorylation of Akt in MDA-MB-231 cells, and treatment of these cells with a pharmacological Akt inhibitor (MK-2206) reduced both Brn-2 expression and cell invasion, indicating that α3β1-Akt signaling contributes to Brn-2 induction. Analysis of RNAseq data from patients with invasive breast carcinoma revealed that high BRN2 expression correlates with poor survival. Moreover, high BRN2 expression positively correlates with high ITGA3 expression in basal-like breast cancer, which is consistent with our experimental findings that α3β1 induces Brn-2 in TNBC cells. Together, our study demonstrates a pro-invasive/pro-metastatic role for Brn-2 in breast cancer cells and identifies a role for integrin α3β1 in regulating Brn-2 expression, thereby revealing a novel mechanism of integrin-dependent breast cancer cell invasion.

Two transcriptional activators, CCAAT-box-binding transcription factor and heat shock transcription factor, interact with a human hsp70 gene promoter

1987 ◽  
Vol 7 (3) ◽  
pp. 1129-1138
Author(s):  
W D Morgan ◽  
G T Williams ◽  
R I Morimoto ◽  
J Greene ◽  
R E Kingston ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Gene Promoter ◽  
Heat Shock Transcription Factor ◽  
Transcriptional Activators ◽  
Hsp70 Gene ◽  
Nuclear Extracts ◽  
Ccaat Box ◽  
Human Hsp70

We characterized the activity of a human hsp70 gene promoter by in vitro transcription. Analysis of 5' deletion and substitution mutants in HeLa nuclear extracts showed that the basal activity of the promoter depends primarily on a CCAAT-box sequence located at -65. A protein factor, CCAAT-box-binding transcription factor (CTF), was isolated from HeLa nuclear extracts and shown to be responsible for stimulation of transcription in a reconstituted in vitro system. DNase I footprinting revealed that CTF interacts with two CCAAT-box elements located at -65 and -147 of the human hsp70 promoter. An additional binding activity, heat shock transcription factor (HSTF), which interacted with the heat shock element, was also identified in HeLa extract fractions. This demonstrates that the promoter of this human hsp70 gene interacts with at least two positive transcriptional activators, CTF, which is required for CCAAT-box-dependent transcription as in other promoters such as those of globin and herpes simplex virus thymidine kinase genes, and HSTF, which is involved in heat inducibility.

scholarly journals Transcriptional initiation is controlled by upstream GC-box interactions in a TATAA-less promoter.

1990 ◽  
Vol 10 (12) ◽  
pp. 6632-6641 ◽  
Author(s):  
M C Blake ◽  
R C Jambou ◽  
A G Swick ◽  
J W Kahn ◽  
J C Azizkhan
Keyword(s):  
Dihydrofolate Reductase ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Start Site ◽  
Transcriptional Initiation ◽  
Reductase Gene ◽  
Gc Box

Numerous genes contain TATAA-less promoters, and the control of transcriptional initiation in this important promoter class is not understood. We have determined that protein-DNA interactions at three of the four proximal GC box sequence elements in one such promoter, that of the hamster dihydrofolate reductase gene, control initiation and relative use of the major and minor start sites. Our results indicate that although the GC boxes are apparently equivalent with respect to factor binding, they are not equivalent with respect to function. At least two properly positioned GC boxes were required for initiation of transcription. Abolishment of DNA-protein interaction by site-specific mutation of the most proximal GC box (box I) resulted in a fivefold decrease in transcription from the major initiation site and a threefold increase in heterogeneous transcripts initiating from the vicinity of the minor start site in vitro and in vivo. Mutations that separately abolished interactions at GC boxes II and III while leaving GC box I intact affected the relative utilization of both the major and minor initiation sites as well as transcriptional efficiency of the promoter template in in vitro transcription and transient expression assays. Interaction at GC box IV when the three proximal boxes were in a wild-type configuration had no effect on transcription of the dihydrofolate reductase gene promoter. Thus, GC box interactions not only are required for efficient transcription but also regulate start site utilization in this TATAA-less promoter.

scholarly journals Analysis of BvgA Activation of the Pertactin Gene Promoter in Bordetella pertussis

1999 ◽  
Vol 181 (17) ◽  
pp. 5234-5241 ◽  
Author(s):  
Susan M. Kinnear ◽  
Philip E. Boucher ◽  
Scott Stibitz ◽  
Nicholas H. Carbonetti
Keyword(s):  
Virulence Factors ◽  
Bordetella Pertussis ◽  
Whooping Cough ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Start Site ◽  
Reading Frame ◽  
Dnase I Footprinting ◽  
Rapid Amplification

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, regulates expression of its virulence factors via a two-component signal transduction system encoded by the bvgregulatory locus. It has been shown by activation kinetics that several of the virulence factors are differentially regulated. fhais transcribed at 10 min following an inducing signal, whileptx is not transcribed until 2 to 4 h after the inducing signal. We present data indicating that prn is transcribed at 1 h, an intermediate time compared to those offha and ptx. We have identifiedcis-acting sequences necessary for expression ofprn in B. pertussis by usingprn-lac fusions containing alterations in the sequence upstream of the prn open reading frame. In vitro transcription and DNase I footprinting analyses provided evidence to support our hypothesis that BvgA binds to this sequence upstream ofprn to activate transcription from the promoter. Our genetic data indicate that the region critical for prnactivation extends upstream to position −84. However, these data do not support the location of the prn transcription start site as previously published. We used a number of methods, includingprn-lac fusions, reverse transcriptase PCR, and 5′ rapid amplification of cDNA ends, to localize and identify thebvg-dependent 5′ end of the prn transcript to the cytosine at −125 with respect to the published start site.

scholarly journals Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation

2017 ◽  
Author(s):  
Yoo Jin Joo ◽  
Scott B. Ficarro ◽  
Luis M. Soares ◽  
Yujin Chun ◽  
Jarrod A. Marto ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Transcription Reinitiation ◽  
Promoter Dna ◽  
Basal Transcription Factors ◽  
Necessary And Sufficient

AbstractTFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-Binding Protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-Associated Factor (TAF) subunits recognize downstream promoter elements, act as co-activators, and interact with nucleosomes. Here we show that transcription induces stable TAF binding to downstream promoter DNA, independent of upstream contacts, TBP, or other basal transcription factors. This transcription-dependent TAF complex promotes subsequent activator-independent transcription, and promoter response to TAF mutations in vivo correlates with the level of downstream, rather than overall, Taf1 crosslinking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.

scholarly journals In vitro transcription from the human A gamma-globin gene promoter

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1344-1350 ◽  
Author(s):  
M Castle ◽  
D O'Neill ◽  
A Bank
Keyword(s):  
Gene Transcription ◽  
Globin Gene ◽  
Gene Promoter ◽  
K562 Cells ◽  
In Vitro Transcription ◽  
Systematic Analysis ◽  
Gamma Globin ◽  
Octamer Motif

Abstract We report enhanced transcription from the human A gamma-globin gene promoter in nuclear extracts (NE) of erythroleukemia (K562) cells compared with that in HeLa NE. We do not observe differences in transcription levels in the two extracts with nonglobin promoter templates. Our findings, indicating preferential recognition of the globin gene promoter by nuclear factors in K562 cells, are consistent with results of studies previously reported by ourselves and others. A novel finding described here is that the addition of a double-stranded octamer motif oligonucleotide to K562 NE increases the level of transcription from the A gamma-globin gene promoter, suggesting a potential role for an octamer motif-binding factor in the repression of A gamma-globin gene transcription. A cosmid construct containing extensive human gamma- and beta-globin gene promoter and structural sequences as well as upstream control sequences also exhibits higher levels of globin gene transcription in K562 NE than in HeLa NE. Our demonstration of the feasibility of efficient, globin promoter-specific in vitro transcription of this complex template offers a novel approach for the systematic analysis of the effects of putative regulatory factors on globin gene expression in vitro in the context of a genetic environment approximating that found in vivo.

Synergistic activation of a human promoter in vivo by transcription factor Sp1

1991 ◽  
Vol 11 (4) ◽  
pp. 1935-1943
Author(s):  
G M Anderson ◽  
S O Freytag
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Single Copy ◽  
Relative Contribution ◽  
Human Promoter ◽  
Transcription Factor Sp1 ◽  
Synergistic Activation ◽  
Cellular Context

Many eucaryotic promoters contain multiple binding sites for sequence-specific DNA-binding proteins. In some cases, these proteins have been shown to interact synergistically to activate transcription. In this study, we address the possibility that the transcription factor Sp1 can synergistically activate a native human promoter in a cellular context that closely resembles that of a single-copy gene. Using DNase I footprinting with affinity-purified Sp1, we show that the human argininosuccinate synthetase (AS) promoter contains three sites that bind Sp1 with different affinities. These binding sites were mutated to abolish Sp1 binding, individually and in all possible combinations, to generate a series of AS promoter-chloramphenicol acetyltransferase (CAT) expression constructs. Mutations designed to increase Sp1 binding were also introduced at each site. The in vivo transcriptional activity of these mutant AS promoter-CAT constructs was then measured in stably transfected human RPMI 2650 cell lines. Our results show that each of the three Sp1-binding sites contributes to full activation of the human AS promoter and that the relative contribution of each site correlates well with its in vitro affinity for Sp1. More importantly, we find that the three Sp1-binding sites when present in the same promoter activate transcription to a level that is 8 times greater than would be expected given their individual activities in the absence of the other two sites. Thus, we provide direct evidence that Sp1-binding sites in their native context in a human promoter can interact synergistically in vivo to activate transcription. The ability to activate transcription synergistically may be the reason that many cellular promoters have multiple Sp1-binding sites arranged in tandem and in close proximity.

scholarly journals NFATC3 promotes IRF7 transcriptional activity in plasmacy­­toid dendritic cells

2016 ◽  
Vol 213 (11) ◽  
pp. 2383-2398 ◽  
Author(s):  
Musheng Bao ◽  
York Wang ◽  
Ying Liu ◽  
Peiqing Shi ◽  
Hongbo Lu ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Transcription Factor ◽  
Nuclear Translocation ◽  
Constitutive Expression ◽  
Toll Like Receptor ◽  
Cpg Dna ◽  
Specialized Function

Plasmacytoid dendritic cells (pDCs) rapidly produce large amounts of type 1 interferon (IFN) after Toll-like receptor 7 and 9 engagements. This specialized function of type 1 IFN production is directly linked to the constitutive expression of IRF7, the master transcription factor for type 1 IFN production. However, the IRF7 regulatory network in pDCs remains largely unknown. In this study, we identify that the transcription factor NFATC3 specifically binds to IRF7 and enhances IRF7-mediated IFN production. Furthermore, knockout of NFATC3 greatly reduced the CpG DNA–induced nuclear translocation of IRF7, which resulted in impaired type 1 IFN production in vitro and in vivo. In addition, we found that NFATC3 and IRF7 both bound to type 1 IFN promoters and that the NFAT binding site in IFN promoters was required for IRF7-mediated IFN expression. Collectively, our study shows that the transcription factor NFATC3 binds to IRF7 and functions synergistically to enhance IRF7-mediated IFN expression in pDCs.

scholarly journals In vitro analysis of the pea chloroplast 16S rRNA gene promoter.

1989 ◽  
Vol 9 (12) ◽  
pp. 5650-5659 ◽  
Author(s):  
E Sun ◽  
B W Wu ◽  
K K Tewari
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Topoisomerase I ◽  
Transcriptional Start Site ◽  
Gene Promoter ◽  
In Vitro Transcription ◽  
Rrna Gene ◽  
Start Site

A cloned pea chloroplast 16S rRNA gene promoter has been characterized in detail by use of a homologous in vitro transcription system that contains a highly purified chloroplast RNA polymerase. The in vivo and in vitro 16S rRNA transcriptional start site has been identified to be a T on the plus strand, 158 bases upstream of the mature 5' end of the gene. BAL 31 deletions of the 16S rRNA leader region demonstrated that the bases between -66 to +30 relative to the transcriptional start site (+1) are necessary for specific 16S transcription. Disruption of canonical TTGACA or TATAAT elements within this region caused complete transcriptional inactivation and prevented protein binding. The topological requirement for 16S transcription was examined by using a construct that synthesized a transcript from the 16S promoter and released it from a pea plastid putative terminator sequence. This minigene was relaxed in vitro with a topoisomerase I from pea chloroplast. It was shown that the 16S promoter was most active when the minigene plasmid was supercoiled.

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