scholarly journals v-src induction of the TIS10/PGS2 prostaglandin synthase gene is mediated by an ATF/CRE transcription response element.

1994 ◽  
Vol 14 (10) ◽  
pp. 6531-6539 ◽  
Author(s):  
W Xie ◽  
B S Fletcher ◽  
R D Andersen ◽  
H R Herschman
Keyword(s):  
Transcription Factor ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Temperature Sensitive ◽  
3T3 Cells ◽  
Ras Gene ◽  
Prostaglandin Synthase ◽  
E Box ◽  
Src Activation

We recently reported the cloning of a mitogen-inducible prostaglandin synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.

scholarly journals v-src induction of the TIS10/PGS2 prostaglandin synthase gene is mediated by an ATF/CRE transcription response element

1994 ◽  
Vol 14 (10) ◽  
pp. 6531-6539
Author(s):  
W Xie ◽  
B S Fletcher ◽  
R D Andersen ◽  
H R Herschman
Keyword(s):  
Transcription Factor ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Temperature Sensitive ◽  
3T3 Cells ◽  
Ras Gene ◽  
Prostaglandin Synthase ◽  
E Box ◽  
Src Activation

We recently reported the cloning of a mitogen-inducible prostaglandin synthase gene, TIS10/PGS2. In addition to growth factors and tumor promoters, the v-src oncogene induces TIS10/PGS2 expression in 3T3 cells. Deletion analysis, using luciferase reporters, identifies a region between -80 and -40 nucleotides 5' of the TIS10/PGS2 transcription start site that mediates pp60v-src induction in 3T3 cells. This region contains the sequence CGTCACGTG, which includes overlapping ATF/CRE (CGTCA) and E-box (CACGTG) sequences. Gel shift-oligonucleotide competition experiments with nuclear extracts from cells stably transfected with a temperature-sensitive v-src gene demonstrate that the CGTCACGTG sequence can bind proteins at both the ATF/CRE and E-box sequences. Dominant-negative CREB and Myc proteins that bind DNA, but do not transactivate, block v-src induction of a luciferase reporter driven by the first 80 nucleotides of the TIS10/PGS2 promoter. Mutational analysis distinguishes which TIS10/PGS2 cis-acting element mediates pp60v-src induction. E-box mutation has no effect on the fold induction in response to pp60v-src. In contrast, ATF/CRE mutation attenuates the pp60v-src response. Antibody supershift and methylation interference experiments demonstrate that CREB and at least one other ATF transcription factor in these extracts bind to the TIS10/PGS2 ATF/CRE element. Expression of a dominant-negative ras gene also blocks TIS10/PGS2 induction by v-src. Our data suggest that Ras mediates pp60v-src activation of an ATF transcription factor, leading to induced TIS10/PGS2 expression via the ATF/CRE element of the TIS10/PGS2 promoter. This is the first description of v-src activation of gene expression via an ATF/CRE element.

FIGalpha, a germ cell specific transcription factor involved in the coordinate expression of the zona pellucida genes

Development ◽  
1997 ◽  
Vol 124 (24) ◽  
pp. 4939-4947 ◽  
Author(s):  
L. Liang ◽  
S.M. Soyal ◽  
J. Dean
Keyword(s):  
Transcription Factor ◽  
Zona Pellucida ◽  
Reporter Genes ◽  
Single Copy ◽  
Luciferase Reporter ◽  
Embryonic Fibroblasts ◽  
Transcription Start Sites ◽  
Helix Loop Helix ◽  
E Box ◽  
Dna Complex

The mouse zona pellucida is composed of three glycoproteins, ZP1, ZP2 and ZP3, encoded by single-copy genes whose expression is temporally and spatially restricted to oocytes. All three proteins are required for the formation of the extracellular zona matrix and female mice with a single disrupted zona gene lack a zona and are infertile. An E-box (CANNTG), located approximately 200 bp upstream of the transcription start sites of Zp1, Zp2 and Zp3, forms a protein-DNA complex present in oocytes and, to a much lesser extent, in testes. It has been previously shown that the integrity of this E-box in Zp2 and Zp3 promoters is required for expression of luciferase reporter genes microinjected into growing oocytes. The presence of the ubiquitous transcription factor E12 in the complex was used to identify a novel basic helix-loop-helix protein, FIGalpha (Factor In the Germline alpha) whose expression was limited to oocytes within the ovary. The ability of FIGalpha to transactivate reporter genes coupled to each of the three mouse zona promoters in heterologous 10T(1/2) embryonic fibroblasts suggests a role in coordinating the expression of the three zona pellucida genes during oogenesis.

scholarly journals Leukemia-Related Transcription Factor TEL Is Negatively Regulated through Extracellular Signal-Regulated Kinase-Induced Phosphorylation

2004 ◽  
Vol 24 (8) ◽  
pp. 3227-3237 ◽  
Author(s):  
Kazuhiro Maki ◽  
Honoka Arai ◽  
Kazuo Waga ◽  
Ko Sasaki ◽  
Fumihiko Nakamura ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Effect ◽  
Phosphorylation Sites ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT TEL is an ETS family transcription factor that possesses multiple putative mitogen-activated protein kinase phosphorylation sites. We here describe the functional regulation of TEL via ERK pathways. Overexpressed TEL becomes phosphorylated in vivo by activated ERK. TEL is also directly phosphorylated in vitro by ERK. The inducible phosphorylation sites are Ser213 and Ser257. TEL binds to a common docking domain in ERK. In vivo ERK-dependent phosphorylation reduces trans-repressional and DNA-binding abilities of TEL for ETS-binding sites. A mutant carrying substituted glutamates on both Ser213 and Ser257 functionally mimics hyperphosphorylated TEL and also shows a dominant-negative effect on TEL-induced transcriptional suppression. Losing DNA-binding affinity through phosphorylation but heterodimerizing with unmodified TEL could be an underlying mechanism. Moreover, the glutamate mutant dominantly interferes with TEL-induced erythroid differentiation in MEL cells and growth suppression in NIH 3T3 cells. Finally, endogenous TEL is dephosphorylated in parallel with ERK inactivation in differentiating MEL cells and is phosphorylated through ERK activation in Ras-transformed NIH 3T3 cells. These data indicate that TEL is a constituent downstream of ERK in signal transduction systems and is physiologically regulated by ERK in molecular and biological features.

scholarly journals The zinc finger transcription factor ZFHX1A is linked to cell proliferation by Rb–E2F1

2007 ◽  
Vol 408 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Yongqing Liu ◽  
Mary E. Costantino ◽  
Diego Montoya-Durango ◽  
Yujiro Higashi ◽  
Douglas S. Darling ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Dominant Negative ◽  
Proliferating Cells ◽  
Zinc Finger Transcription Factor ◽  
Dominant Negative Form ◽  
Repressor Complex ◽  
E Box ◽  
Cell Cycle Pathway ◽  
Negative Form

ZFHX1A is expressed in proliferating cells in the developing embryo, and in the present study we provide evidence that its expression is confined to proliferating cells through dependence on the Rb (retinoblastoma protein) family/E2F cell cycle pathway. Mutation of the Rb or E2F1 genes lead to induction of ZFHX1A mRNA, implying that the Rb–E2F1 repressor complex is important for repression of ZFHX1A. This repression is associated with recruitment of an E2F–Rb–histone deacetylase repressor complex to the promoter. A dominant-negative form of E2F1 inhibited ZFHX1A expression in p16INK4a(−) cells where Rb is constitutively hyperphosphorylated and inactive, suggesting that E2F can contribute to ZFHX1A transactivation in the absence of functional Rb. ZFHX1A is an E-box-binding transcription factor whose binding sites overlap with those bound by Snail1 and 2, and ZFHX1B/SIP1 (leading to at least partially overlapping function; for example, each of the proteins can repress E-cadherin expression). We found that expression of Snail1 and ZFHX1B/SIP1 is also regulated by E2Fs, but in contrast with ZFHX1A this regulation is Rb-family-independent. Snail2 expression was unaffected by either E2F or the Rb family. We propose that the differential effects of the Rb family/E2F pathway on expression of these E-box-binding proteins are important in maintaining their distinct patterns (and thus distinct functions) during embryogenesis.

scholarly journals Constitutive Activation of the Transcription Factor NF-κB Results in Impaired Borna Disease Virus Replication

2005 ◽  
Vol 79 (10) ◽  
pp. 6043-6051 ◽  
Author(s):  
Soizic Bourteele ◽  
Katja Oesterle ◽  
Stephan Pleschka ◽  
Gunhild Unterstab ◽  
Christina Ehrhardt ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Disease Virus ◽  
Rna Virus ◽  
Dominant Negative ◽  
Interferon Response ◽  
Luciferase Reporter ◽  
Target Cells ◽  
Borna Disease Virus ◽  
Iκb Kinase ◽  
Borna Disease

ABSTRACT The inducible transcription factor NF-κB is commonly activated upon RNA virus infection and is a key player in the induction and regulation of the innate immune response. Borna disease virus (BDV) is a neurotropic negative-strand RNA virus, which replicates in the nucleus of the infected cell and causes a persistent infection that can lead to severe neurological disorders. To investigate the activation and function of NF-κB in BDV-infected cells, we stably transfected the highly susceptible neuronal guinea pig cell line CRL with a constitutively active (IKK EE) or dominant-negative (IKK KD) regulator of the IKK/NF-κB signaling pathway. While BDV titers were not affected in cells with impaired NF-κB signaling, the expression of an activated mutant of IκB kinase (IKK) resulted in a strong reduction in the intracellular viral titer in CRL cells. Electrophoretic mobility shift assays and luciferase reporter gene assays revealed that neither NF-κB nor interferon regulatory factors (IRFs) were activated upon acute BDV infection of wild-type or vector-transfected CRL cells. However, when IKK EE-transfected cells were used as target cells for BDV infection, DNA binding to an IRF3/7-responsive DNA element was detectable. Since IRF3/7 is a key player in the antiviral interferon response, our data indicate that enhanced NF-κB activity in the presence of BDV leads to the induction of antiviral pathways resulting in reduced virus titers. Consistent with this observation, the anti-BDV activity of NF-κB preferentially spread to areas of the brains of infected rats where activated NF-κB was not detectable.

scholarly journals Mutational analysis of the Prt1 protein subunit of yeast translation initiation factor 3.

1995 ◽  
Vol 15 (8) ◽  
pp. 4525-4535 ◽  
Author(s):  
D R Evans ◽  
C Rasmussen ◽  
P J Hanic-Joyce ◽  
G C Johnston ◽  
R A Singer ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Gene Transcript ◽  
Missense Mutations ◽  
Wild Type ◽  
Protein Subunit

The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5' end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.

Kv1.5 potassium channel gene regulation by Sp1 transcription factor and oxidative stress

2007 ◽  
Vol 293 (5) ◽  
pp. H2719-H2725 ◽  
Author(s):  
Samuel J. Fountain ◽  
Alex Cheong ◽  
Jing Li ◽  
Naciye Y. Dondas ◽  
Fanning Zeng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Smooth Muscle ◽  
Potassium Channel ◽  
Promoter Activity ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Therapeutic Drug ◽  
Luciferase Reporter Gene ◽  
Sp1 Transcription Factor

KV1.5, a voltage-gated potassium channel, has functional importance in regulating blood vessel tone and cardiac action potentials and is a target for numerous therapeutic drug development programs. Despite the importance of KV1.5, there is little knowledge of the mechanisms controlling expression of its underlying gene, Kcna5. We identified a 5′ flanking region of the murine Kcna5 gene that drives expression of a luciferase reporter gene in primary smooth muscle cells and a smooth muscle cell line. The promoter contained CACCC nucleotide motifs, which we have shown to bind the Sp1 transcription factor in the aorta under physiological conditions in vivo. Inhibition of Sp1- Kcna5 promoter interactions using mithramycin A, a dominant-negative Sp1 mutant, or disruption of the CACCC boxes by mutagenesis inhibited promoter activity. Conversely, expression of exogenous Sp1 augmented promoter activity. Sp1 has known sensitivity to oxidative stress and, consistent with this property, Kcna5 promoter activity was suppressed by hydrogen peroxide-induced oxidative stress. Our results show that Kcna5 promoter activity in vascular smooth muscle is critically dependent on Sp1 regulation via CACCC box motifs and identify mechanisms that potentially influence the expression of KV1.5 channel expression in physiological or pathological conditions.

Angiotensin II-responsive element is the insulin-responsive element in the adipocyte fatty acid synthase gene: role of adipocyte determination and differentiation factor 1/sterol-regulatory-element-binding protein 1c

2001 ◽  
Vol 357 (3) ◽  
pp. 899-904 ◽  
Author(s):  
Suyeon KIM ◽  
Isabelle DUGAIL ◽  
Melissa STANDRIDGE ◽  
Kate CLAYCOMBE ◽  
Joseph CHUN ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fatty Acid ◽  
Angiotensin Ii ◽  
Gene Transcription ◽  
Fatty Acid Synthase ◽  
Luciferase Reporter ◽  
Ang Ii ◽  
Differentiation Factor ◽  
E Box ◽  
Responsive Element

We have previously shown that angiotensin II (Ang II) increases the expression of the gene encoding adipocyte fatty acid synthase (FAS). Here we investigate the mechanism responsible for increased FAS gene transcription by Ang II. We demonstrate that Ang II increased luciferase activity by 3-fold in 3T3-L1 adipocytes transfected with fusion constructs linking the FAS promoter to the luciferase reporter gene. Interestingly, we mapped the Ang II regulatory sequences to the insulin-responsive region (E box) in the proximal FAS promoter. The E box alone was able to mediate Ang II responsiveness when linked to a heterologous promoter. However, this response was lost when mutations that abolished the binding of the E box to its transcription factors were introduced. Using adenoviral overexpression of a dominant-negative form of adipocyte determination and differentiation factor 1 (ADD1), a transcription factor that binds to the insulin-responsive E box, we demonstrated that ADD1 was required for Ang II regulation of the FAS gene in 3T3-L1 adipocytes. Furthermore, ADD1 expression was also up-regulated by Ang II. With the use of transfections as well as glucose transport assays, we further demonstrated that Ang II stimulation of the FAS gene was dependent on glucose. In conclusion, this is the first report that Ang II regulates adipocyte FAS gene transcription via insulin response sequences in a glucose-dependent manner and that this regulation is mediated at least in part via the ADD1 transcription factor.

Mutation P291fsinsC in the transcription factor hepatocyte nuclear factor-1alpha is dominant negative

Diabetes ◽  
1998 ◽  
Vol 47 (8) ◽  
pp. 1231-1235 ◽  
Author(s):  
K. Yamagata ◽  
Q. Yang ◽  
K. Yamamoto ◽  
H. Iwahashi ◽  
J. Miyagawa ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dominant Negative ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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