scholarly journals Genetic analysis of a transcriptional activation pathway by using hepatoma cell variants.

1994 ◽  
Vol 14 (11) ◽  
pp. 7086-7094 ◽  
Author(s):  
G A Bulla ◽  
R E Fournier
Keyword(s):  
Somatic Cell ◽  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Ectopic Expression ◽  
Direct Selection ◽  
Gene Encoding ◽  
Activation Pathway ◽  
Aldolase B ◽  
High Level

A hierarchy of liver-enriched transcription factors plays an important role in activating expression of many hepatic genes. In particular, hepatocyte nuclear factor 4 (HNF-4) is a major activator of the gene encoding HNF-1, and HNF-1 itself activates expression of more than 20 liver genes. To dissect this activation pathway genetically, we prepared somatic cell variants that were deficient in expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene, which requires both HNF-1 and HNF-4 for high-level gene activity. This was accomplished in two steps. First, hepatoma transfectants that stably expressed two selectable markers under alpha 1AT promoter control were prepared; second, variant sublines that could no longer express either transgene were isolated by direct selection. In this report, we demonstrate that the variants contain defects in the HNF-4/HNF-1 activation pathway. These defects functioned in trans, as expression of many liver genes was affected, but the variant phenotypes were recessive to wild type in somatic cell hybrids. Three different variant classes could be discriminated by their phenotypic responses to ectopic expression of either HNF-4 or HNF-1. Two variant clones appeared specifically deficient in HNF-4 expression, as transfection with an HNF-4 expression cassette fully restored their hepatic phenotypes. Another line activated HNF-1 in response to forced HNF-4 expression, but activation of downstream genes failed to occur. One clone was unresponsive to either HNF-1 or HNF-4. Using the variants, we demonstrate further that the chromosomal genes encoding alpha 1AT, aldolase B, and alpha-fibrinogen display strict requirements for HNF-1 activation in vivo, while other liver genes were unaffected by the presence or absence of HNF-1 or HNF-4. We also provide evidence for the existence of an autoregulatory loop in which HNF-1 regulates its own expression through activation of HNF-4.

Genetic analysis of a transcriptional activation pathway by using hepatoma cell variants

1994 ◽  
Vol 14 (11) ◽  
pp. 7086-7094
Author(s):  
G A Bulla ◽  
R E Fournier
Keyword(s):  
Somatic Cell ◽  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Ectopic Expression ◽  
Direct Selection ◽  
Gene Encoding ◽  
Activation Pathway ◽  
Aldolase B ◽  
High Level

A hierarchy of liver-enriched transcription factors plays an important role in activating expression of many hepatic genes. In particular, hepatocyte nuclear factor 4 (HNF-4) is a major activator of the gene encoding HNF-1, and HNF-1 itself activates expression of more than 20 liver genes. To dissect this activation pathway genetically, we prepared somatic cell variants that were deficient in expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene, which requires both HNF-1 and HNF-4 for high-level gene activity. This was accomplished in two steps. First, hepatoma transfectants that stably expressed two selectable markers under alpha 1AT promoter control were prepared; second, variant sublines that could no longer express either transgene were isolated by direct selection. In this report, we demonstrate that the variants contain defects in the HNF-4/HNF-1 activation pathway. These defects functioned in trans, as expression of many liver genes was affected, but the variant phenotypes were recessive to wild type in somatic cell hybrids. Three different variant classes could be discriminated by their phenotypic responses to ectopic expression of either HNF-4 or HNF-1. Two variant clones appeared specifically deficient in HNF-4 expression, as transfection with an HNF-4 expression cassette fully restored their hepatic phenotypes. Another line activated HNF-1 in response to forced HNF-4 expression, but activation of downstream genes failed to occur. One clone was unresponsive to either HNF-1 or HNF-4. Using the variants, we demonstrate further that the chromosomal genes encoding alpha 1AT, aldolase B, and alpha-fibrinogen display strict requirements for HNF-1 activation in vivo, while other liver genes were unaffected by the presence or absence of HNF-1 or HNF-4. We also provide evidence for the existence of an autoregulatory loop in which HNF-1 regulates its own expression through activation of HNF-4.

scholarly journals Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

1996 ◽  
Vol 313 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Françoise LEVAVASSEUR ◽  
Jocelyne LIÉTARD ◽  
Kohei OGAWA ◽  
Nathalie THÉRET ◽  
Peter D. BURBELO ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Basement Membranes ◽  
Major Protein ◽  
Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

scholarly journals Interdependent Recruitment of SAGA and Srb Mediator by Transcriptional Activator Gcn4p

2005 ◽  
Vol 25 (9) ◽  
pp. 3461-3474 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Fan Zhang ◽  
Gwo Jiunn Hwang ◽  
Mark J. Swanson ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Wild Type ◽  
General Transcription Factors ◽  
Tata Element ◽  
High Level ◽  
Target Promoters ◽  
Activation Sequence

ABSTRACT Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

scholarly journals Phosphorylated forms of GAL4 are correlated with ability to activate transcription.

1990 ◽  
Vol 10 (9) ◽  
pp. 4623-4629 ◽  
Author(s):  
L M Mylin ◽  
M Johnston ◽  
J E Hopper
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gene Activation ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Transcriptional Activator Protein ◽  
Mel Gene ◽  
High Level

GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L. Mylin, P. Bhat, and J. Hopper, Genes Dev. 3:1157-1165, 1989). Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation. We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II. To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M. Johnston and J. Dover, Proc. Natl. Acad. Sci. USA 84:2401-2405, 1987; Genetics 120;63-74, 1988). The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species. Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II. Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.

Regulation of the chondrocyte phenotype by β-catenin

Development ◽  
2002 ◽  
Vol 129 (23) ◽  
pp. 5541-5550
Author(s):  
Je-Hwang Ryu ◽  
Song-Ja Kim ◽  
Seon-Hee Kim ◽  
Chun-Do Oh ◽  
Sang-Gu Hwang ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Monolayer Culture ◽  
Nuclear Translocation ◽  
Articular Chondrocytes ◽  
Glycogen Synthase ◽  
Ectopic Expression ◽  
Activator Protein 1 ◽  
Chondrocyte Phenotype

β-Catenin regulates important biological processes, including embryonic development and tumorigenesis. We have investigated the role ofβ-catenin in the regulation of the chondrocyte phenotype. Expression ofβ-catenin was high in prechondrogenic mesenchymal cells, but significantly decreased in differentiated chondrocytes both in vivo and in vitro. Accumulation of β-catenin by the inhibition of glycogen synthase kinase-3β with LiCl inhibited chondrogenesis by stabilizing cell-cell adhesion. Conversely, the low level of β-catenin in differentiated articular chondrocytes was increased by post-translational stabilization during phenotypic loss caused by a serial monolayer culture or exposure to retinoic acid or interleukin-1β. Ectopic expression of β-catenin or inhibition of β-catenin degradation with LiCl or proteasome inhibitor caused de-differentiation of chondrocytes. Transcriptional activation ofβ-catenin by its nuclear translocation was sufficient to cause phenotypic loss of differentiated chondrocytes. Expression pattern of Jun, a known target gene of β-catenin, is essentially the same as that of β-catenin both in vivo and in vitro suggesting that Jun and possibly activator protein 1 is involved in the β-catenin regulation of the chondrocyte phenotype.

scholarly journals Long Non-Coding RNA CCAT2 Activates RAB14 and Acts as an Oncogene in Colorectal Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Dalu Wang ◽  
Zhilong Li ◽  
Hongzhuan Yin
Keyword(s):  
Colorectal Cancer ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Noncoding Rna ◽  
Cell Cycle Progression ◽  
Ectopic Expression ◽  
Direct Interaction ◽  
Migration And Invasion

Here, we investigated the clinicopathological and prognostic potential of the long noncoding RNA Colon Cancer-Associated Transcript 2 (CCAT2) in human colorectal cancer (CRC). We used qPCR to quantify CCAT2 levels in 44 pairs of CRC tissues and adjacent nontumor and healthy colon mucosa tissues, and in several CRC cell lines (SW620, SW480, HT-29, LOVO, HCT116 and DLD-1) and normal human colorectal epithelial cells (HFC). We assessed the effects of CCAT2 overexpression or knockdown on the proliferation, migration and invasion by SW620 and LOVO cells using CCK-8, transwell, and wound−healing assays, respectively. We also investigated the potential interaction between CCAT2 and TAF15 through RNA pull down and rescue experiments. Lastly, we evaluated the expression of the cell cycle progression markers and GSK3β signaling pathway proteins using Western blotting. Our results showed that CCAT2 was upregulated in CRC tissues and cell lines as com-pared to controls. Ectopic expression of CCAT2 promoted CRC cell proliferation, migration and invasion, likely through direct interaction with TAF15, transcriptional activation of RAB14, and activation of the AKT/GSK3β signaling pathway. In vivo, CCAT2 promoted CRC cell growth and metastasis in nude mice. Taken together, these results highlight the actions of CCAT2 as a CRC oncogene.

scholarly journals Delineation of Upstream Signaling Events in the Salmonella Pathogenicity Island 2 Transcriptional Activation Pathway

2004 ◽  
Vol 186 (14) ◽  
pp. 4694-4704 ◽  
Author(s):  
Charles C. Kim ◽  
Stanley Falkow
Keyword(s):  
Transcriptional Activation ◽  
Minimal Medium ◽  
Pathogenicity Island ◽  
Secretion System ◽  
Host Cells ◽  
Bacterial Survival ◽  
Activation Pathway ◽  
Intracellular Environment

ABSTRACT Survival and replication in the intracellular environment are critical components of the ability of Salmonella enterica serovar Typhimurium to establish systemic infection in the murine host. Intracellular survival is mediated by a number of genetic loci, including Salmonella pathogenicity island 2 (SPI2). SPI2 is a 40-kb locus encoding a type III secretion system that secretes effector molecules, which permits bacterial survival and replication in the intracellular environment of host cells. A two-component regulatory system, ssrAB, is also encoded in SPI2 and controls expression of the secretion system and effectors. While the environmental signals to which SPI2 responds in vivo are not known, activation of expression is dependent on OmpR and can be stimulated in vitro by chelation of cations or by a shift from rich to acidic minimal medium. In this work, we demonstrated that SPI2 activation is associated with OmpR in the phosphorylated form (OmpR-P). Mutations in envZ and ackA-pta, which disrupted two distinct sources of OmpR phosphorylation, indicated that SPI2 activation by chelators or a shift from rich to acidic minimal medium is largely dependent on functional EnvZ. In contrast, the PhoPQ pathway is not required for SPI2 activation in the presence of OmpR-P. As in the case of in vitro stimulation, SPI2 expression in macrophages correlates with the presence of OmpR-P. Additionally, EnvZ, but not acetyl phosphate, is required for maximal expression of SPI2 in the intracellular environment, suggesting that the in vitro SPI2 activation pathway is the same as that used in vivo.

Butyrate induces selective transcriptional activation of a hypomethylated embryonic globin gene in adult erythroid cells

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1536-1542
Author(s):  
LJ Burns ◽  
JG Glauber ◽  
GD Ginder
Keyword(s):  
Sodium Butyrate ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Gene Activation ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Hemoglobin Switching ◽  
High Level

An animal model of hemoglobin switching has been developed in which anemic adult chickens are treated with 5-azacytidine and sodium butyrate or alpha-aminobutyric acid, thereby resulting in activation of the embryonic rho-globin gene in adult erythroid cells. In vitro nuclear runoff transcription assays using erythroid nuclei from treated birds show that the mechanism of activation of the rho-globin gene is transcriptional whereas no transcriptional activation of the embryonic epsilon-globin gene occurs. The action of 5-azacytidine appears to be as an inhibitor of DNA methylation because other S-phase active cytotoxic drugs, when substituted for 5-azacytidine, do not cause demethylation of the embryonic globin genes, nor do they allow transcriptional activation to occur. Embryonic rho-globin gene activation in this model is not due to selection of primitive erythroid cells since a subpopulation of primitive erythroid cells is not evident either morphologically or when cells are probed for embryonic and adult globin RNA by in situ hybridization. These studies show that demethylation by 5-azacytidine is a prerequisite but not sufficient cis- regulatory event for a high level of transcriptional activation of the embryonic rho-globin gene in adult erythroid cells in vivo. The possible basis for the selective transcriptional activation by sodium butyrate in this system is discussed.

scholarly journals Role for Hsp90-Associated Cochaperone p23 in Estrogen Receptor Signal Transduction

1999 ◽  
Vol 19 (5) ◽  
pp. 3748-3759 ◽  
Author(s):  
Roland Knoblauch ◽  
Michael J. Garabedian
Keyword(s):  
Signal Transduction ◽  
Estrogen Receptor ◽  
Transcriptional Activation ◽  
Fluorescent Protein ◽  
Binding Capacity ◽  
Ectopic Expression ◽  
Yeast Cells ◽  
Protein Fusion ◽  
Er Expression

ABSTRACT The mechanism of signal transduction by the estrogen receptor (ER) is complex and not fully understood. In addition to the ER, a number of accessory proteins are apparently required to efficiently transduce the steroid hormone signal. In the absence of estradiol, the ER, like other steroid receptors, is complexed with Hsp90 and other molecular chaperone components, including an immunophilin, and p23. This Hsp90-based chaperone complex is thought to repress the ER’s transcriptional regulatory activities while maintaining the receptor in a conformation that is competent for high-affinity steroid binding. However, a role for p23 in ER signal transduction has not been demonstrated. Using a mutant ER (G400V) with decreased hormone binding capacity as a substrate in a dosage suppression screen in yeast cells (Saccharomyces cerevisiae), we identified the yeast homologue of the human p23 protein (yhp23) as a positive regulator of ER function. Overexpression of yhp23 in yeast cells increases ER transcriptional activation by increasing estradiol binding in vivo. Importantly, the magnitude of the effect of yhp23 on ER transcriptional activation is inversely proportional to the concentration of both ER and estradiol in the cell. Under conditions of high ER expression, ER transcriptional activity is largely independent of yhp23, whereas at low levels of ER expression, ER transcriptional activation is primarily dependent on yhp23. The same relationship holds for estradiol levels. We further demonstrate that yhp23 colocalizes with the ER in vivo. Using a yhp23-green fluorescent protein fusion protein, we observed a redistribution of yhp23 from the cytoplasm to the nucleus upon coexpression with ER. This nuclear localization of yhp23 was reversed by the addition of estradiol, a finding consistent with yhp23’s proposed role as part of the aporeceptor complex. Expression of human p23 in yeast partially complements the loss of yhp23 function with respect to ER signaling. Finally, ectopic expression of human p23 in MCF-7 breast cancer cells increases both hormone-dependent and hormone-independent transcriptional activation by the ER. Together, these results strongly suggest that p23 plays an important role in ER signal transduction.

scholarly journals Identification of seven hydrophobic clusters in GCN4 making redundant contributions to transcriptional activation.

1996 ◽  
Vol 16 (10) ◽  
pp. 5557-5571 ◽  
Author(s):  
B M Jackson ◽  
C M Drysdale ◽  
K Natarajan ◽  
A G Hinnebusch
Keyword(s):  
Amino Acids ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Substantial Reduction ◽  
Activation Function ◽  
Activation Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hydrophobic Residues ◽  
High Level

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. The N-terminal 100 amino acids of GCN4 contains a potent activation function that confers high-level transcription in the absence of the centrally located acidic activation domain (CAAD) delineated in previous studies. To identify specific amino acids important for activation by the N-terminal domain, we mutagenized a GCN4 allele lacking the CAAD and screened alleles in vivo for reduced expression of the HIS3 gene. We found four pairs of closely spaced phenylalanines and a leucine residue distributed throughout the N-terminal 100 residues of GCN4 that are required for high-level activation in the absence of the CAAD. Trp, Leu, and Tyr were highly functional substitutions for the Phe residue at position 45. Combined with our previous findings, these results indicate that GCN4 contains seven clusters of aromatic or bulky hydrophobic residues which make important contributions to transcriptional activation at HIS3. None of the seven hydrophobic clusters is essential for activation by full-length GCN4, and the critical residues in two or three clusters must be mutated simultaneously to observe a substantial reduction in GCN4 function. Numerous combinations of four or five intact clusters conferred high-level transcription of HIS3. We propose that many of the hydrophobic clusters in GCN4 act independently of one another to provide redundant means of stimulating transcription and that the functional contributions of these different segments are cumulative at the HIS3 promoter. On the basis of the primacy of bulky hydrophobic residues throughout the activation domain, we suggest that GCN4 contains multiple sites that mediate hydrophobic contacts with one or more components of the transcription initiation machinery.

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