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.

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.

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.

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].

Targeting Sp1 Transactivation In Waldenstrom's Macroglobulinemia: a Novel Therapeutic Option

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 120-120
Author(s):  
Mariateresa Fulciniti ◽  
Samir B. Amin ◽  
Varuna Mohan ◽  
Guang Yang ◽  
Puru Nanjappa ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Growth ◽  
Board Of Directors ◽  
Research Funding ◽  
Luciferase Reporter ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
Advisory Committees ◽  
Growth And Survival ◽  
Cell Growth And Survival

Abstract Abstract 120 The transcription factor Sp1 transactivates expression of genes containing proximal GC/GT-rich promoter elements controlling cell differentiation, cell cycle and apoptosis affecting growth and survival of tumor cells. Based on previous observation that key multiple myeloma (MM) cell growth and survival genes such as NF-kB p65, IGF-IR, VEGF, and IL-6 are controlled by Sp proteins, we have previously investigated and observed high Sp1 expression and activity in MM cells and confirmed its role in MM by Sp1 knock down using both siRNA and lentiviral shRNA constructs specific for Sp1. We further evaluated the role of Sp-1 in WM and observed high nuclear Sp1 protein expression along with increased Sp1 activity in WM cells compared to normal peripheral blood mononuclear cells (PBMC). Moreover, adhesion of WM cells to bone marrow stromal cells (BMSC) further induces Sp1 activity in WM cells. Based on these data, we have investigated the anti-WM activity of Terameprocol (TMP), a small molecule suitable for clinical application,which specifically competes with Sp1-specific DNA binding domains within gene promoter regions. It disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity without direct effect on its expression. We have confirmed inhibition of both basal and BMSC-induced binding and transcriptional activity of Sp1 in WM cells using an ELISA assay specific for measuring Sp1 binding activity and using Sp1 sensitive luciferase reporter plasmid. TMP treatment did not affect Sp1 protein levels. Importantly, TMP significantly inhibited WM cell growth in a dose-dependent fashion (IC50 between 5–20 μ M at 24 hours) and was able to overcome the protective effects of BMSCs. TMP activates the mitochondrial apoptotic pathway via induction of caspase-3, -9 and -7 and PARP cleavage but without caspase-8 activation. TMP treatment also led to downregulation of expression of survivin, a known anti-apoptotic gene transcriptionally regulated by Sp1. We have also confirmed in vivo activity of TMP in a murine xenograft model of MM. Finally based on the data suggesting that both dexamethasone and revlimid increase Sp1 activity, we have combined TMP with these agents and observed synergistic activity on cell growth and survival. In conclusion, our results demonstrate Sp1 as an important transcription factor in WM and provides preclinical rationale for clinical development of TMP as a specific Sp1 inhibitor alone and in combination with conventional and novel agents in WM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Generation of Subgenomic RNA Directed by a Satellite RNA Associated with Bamboo Mosaic Potexvirus: Analyses of Potexvirus Subgenomic RNA Promoter

2000 ◽  
Vol 74 (22) ◽  
pp. 10341-10348 ◽  
Author(s):  
Yun-Shien Lee ◽  
Yau-Heiu Hsu ◽  
Na-Sheng Lin
Keyword(s):  
Transcription Initiation ◽  
Nonstructural Protein ◽  
Core Promoter ◽  
Potato Virus X ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Compensatory Mutation ◽  
Satellite Rna ◽  
Subgenomic Rna

ABSTRACT Satellite RNA of bamboo mosaic potexvirus (satBaMV), a single-stranded positive-sense RNA encoding a nonstructural protein of 20 kDa (P20), depends on bamboo mosaic potexvirus (BaMV) for replication and encapsidation. A full-length cDNA clone of satBaMV was used to examine the sequences required for the synthesis of potexvirus subgenomic RNAs (sgRNAs). Subgenomic promoter-like sequences (SGPs), 107 nucleotides (nt) upstream from the capsid protein (CP) gene of BaMV-V, were inserted upstream of the start codon of the P20 gene of satBaMV. Insertion of SGPs gave rise to the synthesis of sgRNA of satBaMV in protoplasts ofNicotiana benthamiana and leaves of Chenopodium quinoa when coinoculated with BaMV-V genomic RNA. Moreover, both the satBaMV cassette and its sgRNA were encapsidated. From analysis of the SGPs by deletion mutation, we concluded that an SGP contains one core promoterlike sequence (nt −30 through +16), two upstream enhancers (nt −59 through −31 and −91 through −60), and one downstream enhancer (nt +17 through +52), when the transcription initiation site is taken as +1. Site-directed mutagenesis and compensatory mutation to disrupt and restore potential base pairing in the core promoter-like sequence suggest that the stem-loop structure is important for the function of SGP in vivo. Likewise, the insertion of a putative SGP of the BaMV open reading frame 2 gene or a heterologous SGP of potato virus X resulted in generation of an sgRNA. The satBaMV cassette should be a useful tool to gain insight into sequences required for the synthesis of potexvirus sgRNAs.

scholarly journals Hsa_circRNA_000166 facilitated cell growth and limited apoptosis through targeting miR-326 / LASP1 axis in colorectal cancer

2020 ◽  
Author(s):  
Qin Hao ◽  
Zhongtao Zhang
Keyword(s):  
Colorectal Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Direct Interaction ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Western Blot Assay ◽  
Apoptosis Assay ◽  
Geo Database

Abstract Background: Circular RNAs(circRNAs) belong to non-coding RNAs and widely expressed in a variety of cell species, including cancers. However, the function and mechanism of circRNAs in colorectal cancer (CRC) has not been well investigated. Methods: Microarray data of CRC from Gene Expression Omnibus (GEO) database was used to obtain DEGs. QRT-PCR and western blot assay were performed to determine the mRNA and protein levels of multiple genes, respectively. Cell growth and apoptosis assay were conducted to measure CRC cell proliferation and apoptosis, respectively. Luciferase assay was utilized to confirm the direct interaction between hsa_circRNA_000166 and miR-326. Results: We downloaded and analyzed the circRNA expression profile of CRC from the GEO database and identified 181 differentially expressed circRNAs between 10 pairs of CRC and adjacent normal tissues. Interestingly, we observed that the expression of hsa_circRNA_000166 was the top increased among these circRNAs. Then, we confirmed an upregulation of hsa_circRNA_000166 in CRC tissues and cell lines and observed that higher expression of hsa_circRNA_000166 was associated with poor 5-year survival rate of patients with CRC. Cell growth and apoptosis assay revealed that hsa_circRNA_000166 regulated the cell growth and apoptosis in CRC cell lines. Furthermore, we identified that hsa_circRNA_000166 targeted miR-326/LASP1 pathway using bioinformatic analysis and luciferase reporter assay. Finally, overexpression of miR-326 could sufficiently rescued the aberrant cell growth and apoptosis in CRC cell lines. Conclusion: Taken together, our results indicated that downregulation of hsa_circRNA_000166 inhibited the cell growth and facilitated apoptosis during CRC development by sponging miR-326 / LASP1 pathway.

scholarly journals Architecture of TAF11/TAF13/TBP complex suggests novel regulatory state in General Transcription Factor TFIID function

2017 ◽  
Author(s):  
Kapil Gupta ◽  
Aleksandra A. Watson ◽  
Tiago Baptista ◽  
Elisabeth Scheer ◽  
Anna L. Chambers ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Supporting Cell ◽  
Tata Box ◽  
Integrative Approach ◽  
Regulatory State ◽  
Histone Fold ◽  
General Transcription Factor

AbstractGeneral transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-binding domain (CTID) in TAF13 which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

scholarly journals Mutagenesis Analysis of ABCG2 Gene Promoter of Zebrafish (Danio Rerio)

2020 ◽  
Vol 3 (2) ◽  
pp. a53-59
Author(s):  
NABILA ZURAIN BINTI MD YUSNI ◽  
LEONARD WHYE KIT LIM ◽  
HUNG HUI CHUNG
Keyword(s):  
Breast Cancer ◽  
Transcription Factor ◽  
Danio Rerio ◽  
Chemotherapeutic Agent ◽  
Chemotherapeutic Agents ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Directed Mutagenesis ◽  
Cancer Resistance ◽  
Abcg2 Gene

Breast cancer is the commonest cancer among women worldwide and the probability of a woman dying from breast cancer is high (about 1 in 38 of total human population (2.6%)).The main factor for mortality is due to the resistance of this particular disease to chemotherapeutic agents. One of the most well-known proteins to be found to correlate significantly with breast cancer resistance to chemotherapeutic agent is the ATP-binding cassette super-family G member 2 (ABCG2). Knowledge on ABCG2 gene regulation is still lacking in terms of how the increased cytotoxic levels are closely related to induce a hype in gene transcript levels and ultimately cause of the reduction in chemotherapeutic agents. The approach taken in this study is through mutational analysis of selected transcription factor governing the expression of ABCG2. In order to achieve this, a previously cloned ABCG2 promoter which has been isolated (around 1500 bp in size) from Danio rerio and inserted into pGL3.0 plasmid, was subjected to site-directed mutagenesis. Selected transcription factor which is AP-1 was successfully mutated by deletion of 5'- TGACGCG -3' sequence at position 1113 bp from TSS+1 where it would bind in order to define their role in ABCG2 physiological function. Sequencing result after site-directed mutagenesis shows high similarities about 98% with ABCG2 gene of Danio rerio. Upon validation, it was found that the intended AP-1 binding site has been mutated. In future work, the mutated clone here will be subjected to transfection analysis where dual-luciferase assay will be conducted to verify the loss of activity from the ABCG2 promoter upon mutation of the targeted AP-1 site. Hence, the mutagenesis analysis of ABCG2 promoter are able to provide information on the involvement of AP-1 transcription factor in multidrug resistance mechanism of breast cancer and thus will be a potential target for chemotherapeutic agent.

scholarly journals ACOX1, regulated by C/EBPα and miR-25-3p, promotes bovine preadipocyte adipogenesis

2021 ◽  
Author(s):  
Feng Zhang ◽  
Qi Xiong ◽  
Hu Tao ◽  
Yang Liu ◽  
Nian Zhang ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Mobility Shift ◽  
Promoter Deletion Analysis ◽  
Factor C ◽  
Peroxisomal Fatty Acid

Acyl-Coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme in peroxisomal fatty acid β-oxidation of fatty acids. Previous studies have reported that ACOX1 was correlated with the meat quality of livestock, while the role of ACOX1 in intramuscular adipogenesis of beef cattle and its transcriptional and post-transcriptional regulatory mechanisms remain unclear. In the present study, gain-of-function and loss-of-function assays demonstrated that ACOX1 positively regulated the adipogenesis of bovine intramuscular preadipocytes. The C/EBPα-binding sites in the bovine ACOX1 promoter region at -1142 to -1129 bp, -831 to -826 bp, and -303 to -298 bp were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) further showed that these three regions are C/EBPα-binding sites, both in vitro and in vivo, indicating that C/EBPα directly interacts with the bovine ACOX1 promoter and inhibits its transcription. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the ACOX1 3’untranslated region (3’UTR). Taken together, our findings suggest that ACOX1, regulated by transcription factor C/EBPα and miR-25-3p, promotes adipogenesis of bovine intramuscular preadipocytes via regulating peroxisomal fatty acid β-oxidation.

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