Selective Sp1 Binding Is Critical for Maximal Activity of the Human c-kit Promoter

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4138-4149 ◽  
Author(s):  
Gyeong H. Park ◽  
Howard K. Plummer ◽  
Geoffrey W. Krystal
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Core Promoter ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Kit Gene ◽  
Core Promoter Activity

Abstract The receptor tyrosine kinase c-kit is necessary for normal hematopoiesis, the development of germ cells and melanocytes, and the pathogenesis of certain hematologic and nonhematologic malignancies. To better understand the regulation of the c-kit gene, a detailed analysis of the core promoter was performed. Rapid amplification of cDNA ends (RACE) and RNase protection methods showed two major transcriptional initiation sites. Luciferase reporter assays using 5′ promoter deletion-reporter constructs containing up to 3 kb of 5′ sequence were performed in hematopoietic and small-cell lung cancer cell lines which either did or did not express the endogenous c-kit gene. This analysis showed the region 83 to 124 bp upstream of the 5′ transcription initiation site was crucial for maximal core promoter activity. Sequence analysis showed several potential Sp1 binding sites within this highly GC-rich region. Gel shift and DNase footprinting showed that Sp1 selectively bound to a single site within this region. Supershift studies using an anti-Sp1 antibody confirmed specific Sp1 binding. Site-directed mutagenesis of the −93/−84 Sp1 binding site reduced promoter-reporter activity to basal levels in c-kit–expressing cells. Cotransfection into DrosophilaSL2 cells of a c-kit promoter-reporter construct with an Sp1 expression vector showed an Sp1 dose-dependent enhancement of expression that was markedly attenuated by mutation of the −93/−84 site. These results indicate that despite the fact that the human c-kit promoter contains multiple potential Sp1 sites, Sp1 binding is a selective process that is essential for core promoter activity.

Selective Sp1 Binding Is Critical for Maximal Activity of the Human c-kit Promoter

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4138-4149
Author(s):  
Gyeong H. Park ◽  
Howard K. Plummer ◽  
Geoffrey W. Krystal
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Core Promoter ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Kit Gene ◽  
Core Promoter Activity

The receptor tyrosine kinase c-kit is necessary for normal hematopoiesis, the development of germ cells and melanocytes, and the pathogenesis of certain hematologic and nonhematologic malignancies. To better understand the regulation of the c-kit gene, a detailed analysis of the core promoter was performed. Rapid amplification of cDNA ends (RACE) and RNase protection methods showed two major transcriptional initiation sites. Luciferase reporter assays using 5′ promoter deletion-reporter constructs containing up to 3 kb of 5′ sequence were performed in hematopoietic and small-cell lung cancer cell lines which either did or did not express the endogenous c-kit gene. This analysis showed the region 83 to 124 bp upstream of the 5′ transcription initiation site was crucial for maximal core promoter activity. Sequence analysis showed several potential Sp1 binding sites within this highly GC-rich region. Gel shift and DNase footprinting showed that Sp1 selectively bound to a single site within this region. Supershift studies using an anti-Sp1 antibody confirmed specific Sp1 binding. Site-directed mutagenesis of the −93/−84 Sp1 binding site reduced promoter-reporter activity to basal levels in c-kit–expressing cells. Cotransfection into DrosophilaSL2 cells of a c-kit promoter-reporter construct with an Sp1 expression vector showed an Sp1 dose-dependent enhancement of expression that was markedly attenuated by mutation of the −93/−84 site. These results indicate that despite the fact that the human c-kit promoter contains multiple potential Sp1 sites, Sp1 binding is a selective process that is essential for core promoter activity.

scholarly journals BTG4 is A Novel p53 Target Gene That Inhibits Cell Growth and Induces Apoptosis

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 217 ◽  
Author(s):  
Na Zhang ◽  
Tinghui Jiang ◽  
Yitao Wang ◽  
Lanyue Hu ◽  
Youquan Bu
Keyword(s):  
Transcription Factor ◽  
Cell Growth ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Colorectal Cancers ◽  
P53 Overexpression

BTG4 is the last cloned and poorly studied member of BTG/Tob family. Studies have suggested that BTG4 is critical for the degradation of maternal mRNAs in mice during the process of maternal-to-zygotic transition, and downregulated in cancers, such as gastric cancer. However, the regulatory mechanism of BTG4 and its function in cancers remain elusive. In this study, we have for the first time identified the promoter region of the human BTG4 gene. Serial luciferase reporter assay demonstrated that the core promoter of BTG4 is mainly located within the 388 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the BTG4 promoter contains binding sites for canonical transcription factors, such as Sp1, whereas its first intron contains two overlapped consensus p53 binding sites. However, overexpression of Sp1 has negligible effects on BTG4 promoter activity, and site-directed mutagenesis assay further suggested that Sp1 is not a critical transcription factor for the transcriptional regulation of BTG4. Of note, luciferase assay revealed that one of the intronic p53 binding sites is highly responsive to p53. Both exogenous p53 overexpression and adriamycin-mediated endogenous p53 activation result in the transcriptional upregulation of BTG4. In addition, BTG4 is downregulated in lung and colorectal cancers, and overexpression of BTG4 inhibits cell growth and induces apoptosis in cancer cells. Taken together, our results strongly suggest that BTG4 is a novel p53-regulated gene and probably functions as a tumor suppressor in lung and colorectal cancers.

scholarly journals Identification of a Previously Unrecognized Promoter That Drives Expression of the UXP Transcription Unit in the Human Adenovirus Type 5 Genome

2010 ◽  
Vol 84 (21) ◽  
pp. 11470-11478 ◽  
Author(s):  
Baoling Ying ◽  
Ann E. Tollefson ◽  
William S. M. Wold
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Mrna Level ◽  
A549 Cells ◽  
Initiation Site ◽  
Transcription Unit ◽  
Transcription Initiation Site ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Ccaat Box

ABSTRACT We previously identified an adenovirus (Ad) protein named U exon protein (UXP) encoded by a leftward-strand (l-strand) transcription unit. Here we identify and characterize the UXP promoter. Primer extension and RNase protection assays mapped the transcription initiation site at 32 nucleotides upstream of the UXP gene initiation codon. A series of viral mutants with mutations at two putative inverted CCAAT (I-CCAAT) boxes and two E2F sites were generated. With mutants lacking the proximal I-CCAAT box, the UXP mRNA level decreased significantly to 30% of the Ad type 5 (Ad5) mRNA level as measured by quantitative reverse transcription-PCR. Decreased UXP was also observed by immunoblotting and immunofluorescence. UXP mRNA and protein levels were similar to those of Ad5 for mutants lacking the distal I-CCAAT box or both putative E2F sites. Ad DNA levels were similar in mutant- and wild-type Ad5-infected cells during the late stage of infection, strongly suggesting that the decreased UXP mRNA and protein from mutants lacking the proximal I-CCAAT box was due to decreased promoter activity. Electrophoretic mobility shift assays (EMSA) indicated that a cellular factor binds specifically to the proximal I-CCAAT box of the UXP promoter. An in vitro luciferase reporter assay demonstrated that basal promoter activity lies between bp −158 and +30 of the transcription initiation site. No E1A-mediated promoter transactivation was observed in 293 cells compared with A549 cells. Thus, we propose that there is a previously unidentified Ad5 promoter that drives expression of the UXP transcription unit. This promoter is embedded within the gene for fiber, and it contains a proximal I-CCAAT box critical for UXP mRNA transcription.

scholarly journals DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 853
Author(s):  
Siti Aisyah Faten Mohamed Sa’dom ◽  
Sweta Raikundalia ◽  
Shaharum Shamsuddin ◽  
Wei Cun See Too ◽  
Ling Ling Few
Keyword(s):  
Dna Methylation ◽  
Transcription Factor ◽  
Binding Site ◽  
Promoter Activity ◽  
Cytosine Methylation ◽  
Cpg Island ◽  
Cpg Islands ◽  
Site Directed Mutagenesis ◽  
Choline Kinase ◽  
Mcf 7

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

scholarly journals Stimulated initiation of mitogen-activated protein kinase phosphatase-1 (MKP-1) gene transcription involves the synergistic action of multiple cis-acting elements in the proximal promoter

2004 ◽  
Vol 378 (2) ◽  
pp. 473-484 ◽  
Author(s):  
Stephan RYSER ◽  
Abbas MASSIHA ◽  
Isabelle PIUZ ◽  
Werner SCHLEGEL
Keyword(s):  
Gene Transcription ◽  
Transcription Initiation ◽  
Site Directed Mutagenesis ◽  
Mitogen Activated Protein Kinase ◽  
Proximal Promoter ◽  
In Vivo Studies ◽  
Luciferase Reporter ◽  
Basal Transcription ◽  
Band Shift ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases (MAPKs) are inactivated by a dual specificity phosphatase, MAPK phosphatase-1 (MKP-1). MKP-1 is transcribed as an immediate early response gene (IEG) following various stimuli. In the pituitary cell line GH4C1, MKP-1 gene transcription is strongly induced by thyrotropin-releasing hormone (TRH) as well as by epidermal growth factor (EGF) as a consequence of activated MAPK/extracellular-signal-regulated kinase (ERK) signalling. Intriguingly, reporter gene analysis with the MKP-1 promoter showed strong basal transcription, but only limited induction by TRH and EGF. Site-directed mutagenesis of the reporter construct combined with band-shift and in vivo studies revealed that part of the constitutive activity of the MKP-1 promoter resides in two GC boxes bound by Sp1 and Sp3 transcription factors in the minimal promoter. Basal transcription of transiently transfected luciferase reporter can be initiated by either of the two GC boxes or also by either of the two cAMP/Ca2+ responsive elements or by the E-box present in the proximal promoter. On the other hand, when analysed by stable transfection, the five responsive elements are acting in synergy to transactivate the MKP-1 proximal promoter. We show in this study that the MKP-1 promoter can function as a constitutive promoter or as a rapid and transient sensor for the activation state of MAPKs/ERKs. This dual mode of transcription initiation may have different consequences for the control of a block to elongation situated in the first exon of the MKP-1 gene, as described previously [Ryser, Tortola, van Haasteren, Muda, Li and Schlegel (2001) J. Biol. Chem. 276, 33319–33327].

Characterization of a chicken retinoid X receptor-γ gene promoter and identification of sequences that direct expression in retinal cells

2000 ◽  
Vol 347 (2) ◽  
pp. 485-490
Author(s):  
Clara AMEIXA ◽  
Paul M. BRICKELL
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Neural Retina ◽  
Specific Protein ◽  
Retinoid X Receptor ◽  
Transcription Initiation Site ◽  
Luciferase Reporter ◽  
Enhancer Element ◽  
Extracellular Signals

Development of the cellular complexity of the vertebrate neural retina relies on an intricate interplay between extracellular signals and intracellular factors. In particular, transcription factors play a key role in determining the competence of cells to respond to extracellular signals. We have previously shown that, in the developing chick neural retina, expression of the retinoid X receptor-γ (RXR-γ2) nuclear receptor gene is restricted to photoreceptors. To characterize the mechanisms that regulate expression of this gene in the neural retina, we isolated a chicken RXR-γ genomic clone containing the RXR-γ2 promoter and mapped the transcription initiation site by means of ribonuclease protection. We analysed promoter activity by transient transfection of luciferase reporter gene constructs into cultured cells isolated from embryonic-chick neural retina or facial mesenchyme, which does not normally express detectable RXR-γ2 transcripts. The DNA fragment lying between nucleotides -657 and +37 with respect to the transcription initiation site had basal promoter activity in both cell types. The fragment lying between nucleotides -1198 and -991 directed 10-20-fold higher levels of luciferase activity in neural retina cells, but only basal levels in facial mesenchyme cells. This 208 bp fragment also enhanced the activity of the simian-virus-40 promoter, when placed upstream in either orientation. Electrophoretic-mobility-shift assays using this 208 bp fragment demonstrated the formation of four neural retina-specific protein-DNA complexes. These results indicate that regulation of RXR-γ2 transcription in the developing chick neural retina involves the binding of one or more neural retina-specific protein factors to an enhancer element located approx. 1 kbp upstream of the transcription initiation site.

scholarly journals LUC7L3/CROP inhibits replication of hepatitis B virus via suppressing enhancer II/basal core promoter activity

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yuan Li ◽  
Masahiko Ito ◽  
Suofeng Sun ◽  
Takeshi Chida ◽  
Kenji Nakashima ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Promoter Activity ◽  
Core Promoter ◽  
Basal Core Promoter ◽  
B Virus ◽  
Enhancer Ii ◽  
Core Promoter Activity

scholarly journals The NRF-1/α-PAL transcription factor regulates human E2F6 promoter activity

2004 ◽  
Vol 383 (3) ◽  
pp. 529-536 ◽  
Author(s):  
Zoulika KHERROUCHE ◽  
Yvan DE LAUNOIT ◽  
Didier MONTE
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Core Promoter ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1 ◽  
Dnase I Footprinting ◽  
Dominant Negative Form

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoreticmobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/α-PAL (nuclear respiratory factor-1/α-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/α-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/α-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/α-PAL is implicated in the regulation of basal E2F6 gene expression.

scholarly journals Sp1-Mediated Transcription of the Werner Helicase Gene Is Modulated by Rb and p53

1998 ◽  
Vol 18 (11) ◽  
pp. 6191-6200 ◽  
Author(s):  
Yukako Yamabe ◽  
Akira Shimamoto ◽  
Makoto Goto ◽  
Jun Yokota ◽  
Minoru Sugawara ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Control Element ◽  
Mrna Levels

ABSTRACT The regulation of Werner’s syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5′ upstream region (2.8 kb) ofWRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides −67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRNpromoter-luciferase reporter (WRN-Luc) plasmids that contained the 5′-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2.5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

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