scholarly journals The functional domains of the murine Thy-1 gene promoter.

1991 ◽  
Vol 11 (4) ◽  
pp. 2216-2228 ◽  
Author(s):  
E Spanopoulou ◽  
V Giguere ◽  
F Grosveld
Keyword(s):  
Gene Promoter ◽  
Initiation Site ◽  
Functional Domains ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Multiple Binding Sites ◽  
Nuclear Factors ◽  
Gel Mobility Shift ◽  
Ccaat Box ◽  
Enhancer Sequences

The Thy-1 gene promoter resembles a "housekeeping" promoter in that it is located within a methylation-free island, lacks a canonical TATA box, and displays heterogeneity in the 5'-end termini of the mRNA. Using transgenic mice, we show that this promoter does not confer any tissue specificity and is active only in a position-dependent manner. It can only be activated in a tissue-specific manner by elements that lie downstream of the initiation site. We have analyzed the functional domains of the minimal Thy-1 promoter and show that the dominant promoter elements consist of multiple binding sites for the transcription factor Sp1, an inverted CCAAT box, and sequences proximal to the transcription start site. DNase I and gel mobility shift assays show the binding of a number of nuclear factors to these elements, including Sp1 and CP1. Our results show that the structure of this promoter only permits productive interactions of the two transcription factors Sp1 and CP1 with the basal transcription machinery in the presence of enhancer sequences.

scholarly journals The functional domains of the murine Thy-1 gene promoter

1991 ◽  
Vol 11 (4) ◽  
pp. 2216-2228
Author(s):  
E Spanopoulou ◽  
V Giguere ◽  
F Grosveld
Keyword(s):  
Gene Promoter ◽  
Initiation Site ◽  
Functional Domains ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Multiple Binding Sites ◽  
Nuclear Factors ◽  
Gel Mobility Shift ◽  
Ccaat Box ◽  
Enhancer Sequences

The Thy-1 gene promoter resembles a "housekeeping" promoter in that it is located within a methylation-free island, lacks a canonical TATA box, and displays heterogeneity in the 5'-end termini of the mRNA. Using transgenic mice, we show that this promoter does not confer any tissue specificity and is active only in a position-dependent manner. It can only be activated in a tissue-specific manner by elements that lie downstream of the initiation site. We have analyzed the functional domains of the minimal Thy-1 promoter and show that the dominant promoter elements consist of multiple binding sites for the transcription factor Sp1, an inverted CCAAT box, and sequences proximal to the transcription start site. DNase I and gel mobility shift assays show the binding of a number of nuclear factors to these elements, including Sp1 and CP1. Our results show that the structure of this promoter only permits productive interactions of the two transcription factors Sp1 and CP1 with the basal transcription machinery in the presence of enhancer sequences.

scholarly journals Regulation of the Rat Thyrotropin Receptor Gene by the Methylation-Sensitive Transcription Factor GA-Binding Protein

1998 ◽  
Vol 12 (8) ◽  
pp. 1241-1249 ◽  
Author(s):  
Norihiko Yokomori ◽  
Masato Tawata ◽  
Tukasa Saito ◽  
Hiroki Shimura ◽  
Toshimasa Onaya
Keyword(s):  
Transcription Factor ◽  
Binding Protein ◽  
Gene Promoter ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Cpg Sites ◽  
Nuclear Extracts ◽  
Tshr Gene ◽  
Gel Mobility Shift ◽  
Ga Binding Protein

Abstract The GA-binding protein (GABP), a transcription factor with a widespread tissue distribution, consists of two subunits,α and β1, and acts as a potent positive regulator of various genes. The effect of GABP on transcription of the TSH receptor (TSHR) gene in rat FRTL-5 thyroid cells has now been investigated. Both deoxyribonuclease I footprint analysis and gel mobility-shift assays indicated that bacterially expressed glutathione S-transferase fusion proteins of GABP subunits bind to a region spanning nucleotides (nt) −116 to −80 of the TSHR gene. In gel mobility-shift assays, nuclear extracts of FRTL-5 cells and FRT cells yielded several specific bands with a probe comprising nt −116 to− 80. Supershift assays with antibodies to GABPα and to GABPβ1 showed that GABP was a component of the probe complexes formed by the nuclear extracts. Immunoblot analysis confirmed the presence of both GABP subunits in the nuclear extracts. A reporter gene construct containing the TSHR gene promoter was activated, in a dose-dependent manner, in FRTL-5 cells by cotransfection with constructs encoding both GABPα and GABPβ1. Both GABP binding to and activation of the TSHR gene promoter were prevented by methylation of CpG sites at nt −93 and− 85. These CpG sites were highly methylated (>82%) in FRT cells and completely demethylated in FRTL-5 cells, consistent with expression of the TSHR gene in the latter, but not the former. These results suggest that GABP regulates transcription of the TSHR gene in a methylation-dependent manner and that methylation of specific CpG sites and the methylation sensitivity of GABP contribute to the failure of FRT cells to express the endogenous TSHR gene.

The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites

1999 ◽  
Vol 112 (21) ◽  
pp. 3691-3702 ◽  
Author(s):  
W.L. Severt ◽  
T.U. Biber ◽  
X. Wu ◽  
N.B. Hecht ◽  
R.J. DeLorenzo ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Antisense Oligonucleotides ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Neuronal Remodeling ◽  
Kinesin Heavy Chain ◽  
Gel Mobility Shift

Ribonucleoprotein particles (RNPs) are thought to be key players in somato-dendritic sorting of mRNAs in CNS neurons and are implicated in activity-directed neuronal remodeling. Here, we use reporter constructs and gel mobility shift assays to show that the testis brain RNA-binding protein (TB-RBP) associates with mRNPs in a sequence (Y element) dependent manner. Using antisense oligonucleotides (anti-ODN), we demonstrate that blocking the TB-RBP Y element binding site disrupts and mis-localizes mRNPs containing (alpha)-calmodulin dependent kinase II (alpha)-CAMKII) and ligatin mRNAs. In addition, we show that suppression of kinesin heavy chain motor protein alters only the localization of (alpha)-CAMKII mRNA. Thus, differential sorting of mRNAs involves multiple mRNPs and selective motor proteins permitting localized mRNAs to utilize common mechanisms for shared steps.

scholarly journals Transition State Regulator AbrB Inhibits Transcription of Bacillus amyloliquefaciens FZB45 Phytase through Binding at Two Distinct Sites Located within the Extended phyC Promoter Region

2008 ◽  
Vol 190 (19) ◽  
pp. 6467-6474 ◽  
Author(s):  
Oliwia Makarewicz ◽  
Svetlana Neubauer ◽  
Corinna Preusse ◽  
Rainer Borriss
Keyword(s):  
Bacillus Amyloliquefaciens ◽  
Promoter Region ◽  
Response Regulator ◽  
Dependent Manner ◽  
Coding Region ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Regulator Protein ◽  
Transition State Regulator ◽  
Gel Mobility Shift

ABSTRACT We have previously identified the phyC gene of Bacillus amyloliquefaciens FZB45, encoding extracellular phytase, as a member of the PhoP regulon, which is expressed only during phosphate starvation. Its σA-dependent promoter is positively and negatively regulated by the phosphorylated PhoP response regulator in a phosphate-dependent manner (O. Makarewicz, S. Dubrac, T. Msadek, and R. Borriss, J. Bacteriol. 188:6953-6965, 2006). Here, we provide experimental evidence that the transcription of phyC underlies a second control mechanism exerted by the global transient-phase regulator protein, AbrB, which hinders its expression during exponential growth. Gel mobility shift and DNase I footprinting experiments demonstrated that AbrB binds to two different regions in the phyC promoter region that are separated by about 200 bp. One binding site is near the divergently orientated yodU gene, and the second site is located downstream of the phyC promoter and extends into the coding region of the phyC gene. Cooperative binding to the two distant binding regions is necessary for the AbrB-directed repression of phyC transcription. AbrB does not affect the transcription of the neighboring yodU gene.

scholarly journals Downregulation of PU.1 Leads to Decreased Expression of Dectin-1 in Alveolar Macrophages during Pneumocystis Pneumonia

2010 ◽  
Vol 78 (3) ◽  
pp. 1058-1065 ◽  
Author(s):  
Chen Zhang ◽  
Shao-Hung Wang ◽  
Chung-Ping Liao ◽  
Shoujin Shao ◽  
Mark E. Lasbury ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Mrna Level ◽  
Gene Promoter ◽  
Pneumocystis Pneumonia ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Mobility Shift Assay

ABSTRACT Dectin-1 is an important macrophage phagocytic receptor recognizing fungal β-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

A cloned human CCAAT-box-binding factor stimulates transcription from the human hsp70 promoter

1990 ◽  
Vol 10 (12) ◽  
pp. 6709-6717
Author(s):  
L S Lum ◽  
L A Sultzman ◽  
R J Kaufman ◽  
D I Linzer ◽  
B J Wu
Keyword(s):  
Initiation Site ◽  
Dependent Manner ◽  
Hsp70 Gene ◽  
Hsp70 Promoter ◽  
Transcriptional Initiation ◽  
Enhancer Binding Protein ◽  
Binding Factor ◽  
Ccaat Box ◽  
Transcriptional Initiation Site ◽  
Human Hsp70

The basal promoter of the human hsp70 gene is predominantly controlled by a CCAAT element at position -70 relative to the transcriptional initiation site. We report the isolation of a novel cDNA clone encoding a 114-kDa polypeptide that binds to the CCAAT element of the hsp70 promoter. Expression of this CCAAT-binding factor (CBF) cDNA activated transcription from cotransfected hsp70 promoter-reporter gene constructs in a CCAAT-dependent manner. CCAAT-binding factor shows no homology to the previously identified human CCAAT transcription factor or rat CCAAT/enhancer-binding protein.

scholarly journals Glucocorticoid repression of human with-no-lysine (K) kinase-4 gene expression is mediated by the negative response elements in the promoter

2007 ◽  
Vol 40 (1) ◽  
pp. 3-12 ◽  
Author(s):  
Chunyi Li ◽  
Yan Li ◽  
Yinghui Li ◽  
Hong Liu ◽  
Zhijun Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Level ◽  
Gene Promoter ◽  
Initiation Site ◽  
Binding Activity ◽  
Hek293 Cells ◽  
Mobility Shift ◽  
Transcriptional Initiation ◽  
Response Elements ◽  
Real Time Quantitative Pcr

With-no-lysine (K) kinase-4 (WNK4) is a serine/threonine kinase that plays an essential role in the regulation of fluid and electrolyte homeostasis. The effects of glucocorticoids, key physiological regulators, on the WNK4 gene expression are still unknown. Here, we used dexamethasone (Dex) to treat the human embryo kidney 293 (HEK293) cells and found a decrease of human WNK4 (hWNK4) mRNA level by northern blot and real-time quantitative PCR. After an hWNK4 transcriptional initiation site was located by 5′ rapid amplification of cDNA end assay, a series of 5′-deleted hWNK4 promoter–luciferase constructs were generated by PCR. Transfection of these constructs in COS-7 and HEK293 cells revealed that Dex inhibited the hWNK4 transcriptional activity in glucocorticoid receptor (GR)-dependent pattern. Two negative glucocorticoid response elements (nGREs) were identified at −285 and −337 of the hWNK4 gene promoter and the GR binding activity to them was increased by Dex as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation. In summary, these data demonstrated that hWNK4 was a new glucocorticoid-regulated gene whose expression was inhibited through the interaction of GR with nGREs in the promoter region.

scholarly journals The Zinc Finger-Containing Transcription Factor Gata-4 Is Expressed in the Developing Endocrine Pancreas and Activates Glucagon Gene Expression

2005 ◽  
Vol 19 (3) ◽  
pp. 759-770 ◽  
Author(s):  
Beate Ritz-Laser ◽  
Aline Mamin ◽  
Thierry Brun ◽  
Isabelle Avril ◽  
Valérie M. Schwitzgebel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Exocrine Pancreas ◽  
Gene Promoter ◽  
Control Element ◽  
Mobility Shift ◽  
Insulinoma Cell ◽  
Nuclear Extracts ◽  
Endoderm Development ◽  
Gel Mobility Shift

Abstract Gene inactivation studies have shown that members of the Gata family of transcription factors are critical for endoderm development throughout evolution. We show here that Gata-4 and/or Gata-6 are not only expressed in the adult exocrine pancreas but also in glucagonoma and insulinoma cell lines, whereas Gata-5 is restricted to the exocrine pancreas. During pancreas development, Gata-4 is expressed already at embryonic d 10.5 and colocalizes with early glucagon+ cells at embryonic d 12.5. Gata-4 was able to transactivate the glucagon gene both in heterologous BHK-21 (nonislet Syrian baby hamster kidney) and in glucagon-producing InR1G9 cells. Using gel-mobility shift assays, we identified a complex formed with nuclear extracts from InR1G9 cells on the G5 control element (−140 to −169) of the glucagon gene promoter as Gata-4. Mutation of the GATA binding site on G5 abrogated the transcriptional activation mediated by Gata-4 and reduced basal glucagon gene promoter activity in glucagon-producing cells by 55%. Furthermore, Gata-4 acted more than additively with Forkhead box A (hepatic nuclear factor-3) to trans-activate the glucagon gene promoter. We conclude that, besides its role in endoderm differentiation, Gata-4 might be implicated in the regulation of glucagon gene expression in the fetal pancreas and that Gata activity itself may be modulated by interactions with different cofactors.

Identification of protein-binding sites in the hepatitis B virus enhancer and core promoter domains

1988 ◽  
Vol 8 (12) ◽  
pp. 5159-5165
Author(s):  
S Karpen ◽  
R Banerjee ◽  
A Zelent ◽  
P Price ◽  
G Acs
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Binding Sites ◽  
Core Promoter ◽  
Nuclear Extract ◽  
Mobility Shift ◽  
B Virus ◽  
Gel Mobility Shift ◽  
Enhancer Sequences ◽  
Cat Gene

We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17- and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.

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