scholarly journals p53-Mediated Repression of Alpha-Fetoprotein Gene Expression by Specific DNA Binding

1999 ◽  
Vol 19 (2) ◽  
pp. 1279-1288 ◽  
Author(s):  
Kathleen C. Lee ◽  
Alison J. Crowe ◽  
Michelle Craig Barton
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Western Blot ◽  
P53 Protein ◽  
Transient Transfection ◽  
Alpha Fetoprotein ◽  
Single Mutation ◽  
Hepatic Differentiation ◽  
Aberrant Expression ◽  
Tissue Specific

ABSTRACT Aberrant expression of the alpha-fetoprotein (AFP) gene is characteristic of a majority of hepatocellular carcinoma cases and serves as a diagnostic tumor-specific marker. By dissecting regulatory mechanisms through electromobility gel shift, transient-transfection, Western blot, and in vitro transcription analyses, we find that AFP gene expression is controlled in part by mutually exclusive binding of two trans-acting factors, p53 and hepatic nuclear factor 3 (HNF-3). HNF-3 protein activates while p53 represses AFP transcription through sequence-specific binding within the previously identified AFP developmental repressor domain. A single mutation within the DNA binding domain of p53 protein or a mutation of the p53 DNA binding element within the AFP developmental repressor eliminates p53-repressive effects in both transient-transfection and cell-free expression systems. Coexpression of p300 histone acetyltransferase, which has been shown to acetylate p53 and increase specific DNA binding, amplifies the p53-mediated repression. Western blot analysis of proteins present in developmentally staged, liver nuclear extracts reveal a one-to-one correlation between activation of p53 protein and repression of AFP during hepatic development. Induction of p53 in response to actinomycin D or hypoxic stress decreases AFP expression. Studies in fibroblast cells lacking HNF-3 further support a model for p53-mediated repression that is both passive through displacement of a tissue-specific activating factor and active in the presence of tissue-specific corepressors. This mechanism for p53-mediated repression of AFP gene expression may be active during hepatic differentiation and lost in the process of tumorigenesis.

scholarly journals E2a-Pbx1 induces aberrant expression of tissue-specific and developmentally regulated genes when expressed in NIH 3T3 fibroblasts.

1997 ◽  
Vol 17 (3) ◽  
pp. 1503-1512 ◽  
Author(s):  
X Fu ◽  
M P Kamps
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Ectopic Expression ◽  
Cellular Transformation ◽  
Transactivation Domain ◽  
Developmentally Regulated ◽  
Aberrant Expression ◽  
Tissue Specific ◽  
3T3 Fibroblasts ◽  
Nih 3T3

The E2a-Pbx1 oncoprotein contains the transactivation domain of E2a joined to the DNA-binding homeodomain (HD) of Pbx1. In mice, E2a-Pbx1 transforms T lymphoblasts and fibroblasts and blocks myeloblast differentiation. Pbx1 and E2a-Pbx1 bind DNA as heterodimers with other HD proteins whose expression is tissue specific. While the transactivation domain of E2a is required for all forms of transformation, DNA binding by the Pbx1 HD is essential for blocking myeloblast differentiation but dispensable for fibroblast or T-lymphoblast transformation. These properties suggest (i) that E2a-Pbx1 causes cellular transformation by activating gene transcription, (ii) that transcription of E2a-Pbx1 target genes is normally regulated by ubiquitous Pbx proteins and tissue-specific partners, and (iii) that DNA-binding mutants of E2a-Pbx1 activate a subset of all gene targets. To test these predictions, genes induced in NIH 3T3 fibroblasts by E2a-Pbx1 were identified and examined for tissue- and stage-specific expression and their differential abilities to be upregulated by E2a-Pbx1 in NIH 3T3 fibroblasts and myeloblasts and by a DNA-binding mutant of E2a-Pbx1 in NIH 3T3 cells. Of 12 RNAs induced by E2a-Pbx1, 4 encoded known proteins (a J-C region of the immunoglobulin kappa light chain, natriuretic peptide receptor C, mitochondrial fumarase, and the 3',5'-cyclic nucleotide phosphodiesterase, PDE1A) and 5 encoded new proteins related to angiogenin, ion channels, villin, epidermal growth factor repeat proteins, and the human 2.19 gene product. Expression of many of these genes was tissue specific or developmentally regulated, and most were not expressed in fibroblasts, indicating that E2a-Pbx1 can induce ectopic expression of genes associated with lineage-specific differentiation.

scholarly journals PXR-Mediated Upregulation of CYP3A Expression by Herb Compound Praeruptorin C fromPeucedanum praeruptorumDunn

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Ling Huang ◽  
Qian Wu ◽  
Yu-Hua Li ◽  
Yi-Tao Wang ◽  
Hui-Chang Bi
Keyword(s):  
Gene Expression ◽  
Catalytic Activity ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Pregnane X Receptor ◽  
Transient Transfection ◽  
The Other ◽  
Primary Hepatocytes ◽  
Cyp3a4 Substrate

We recently reported that Praeruptorin C effectively transactivated the mRNA, protein expression, and catalytic activity of CYP3A4 via the CAR-mediated pathway, but whether and how PC could affect the expression and catalytic activity of CYP3A4 via PXR pathway remains unknown. Therefore, in this study, the effect of PC on theCYP3Agene expression was investigated in mice primary hepatocytes after knockdown of PXR by transient transfection of PXR siRNA, and the gene expression, protein expression, and catalytic activity of CYP3A4 in the LS174T cells with PXR overexpression were determined by real-time PCR, western blot analysis, and LC-MS/MS-based CYP3A4 substrate assay, respectively. We found that the level ofCYP3a11gene expression in mouse primary hepatocytes was significantly increased by praeruptorin C, but such an induction was suppressed after knockdown of pregnane X receptor by its siRNA. In PXR-overexpressed LS174T cells, PC significantly enhanced CYP3A4 mRNA, protein expression, and functional activity through PXR-mediated pathway; conversely, no such increase was found in the untransfected cells. These findings suggest that PC can significantly upregulate CYP3A level via the PXR-mediated pathway, and this should be taken into consideration to predict any potential herb-drug interactions between PC, Qianhu, and the other coadministered drugs.

scholarly journals cis and trans elements differ among mouse strains with high and low extrahepatic complement factor B gene expression.

1992 ◽  
Vol 175 (2) ◽  
pp. 471-479 ◽  
Author(s):  
G Garnier ◽  
B Ault ◽  
M Kramer ◽  
H R Colten
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Alternative Pathway ◽  
Constitutive Expression ◽  
Mouse Strains ◽  
Complement Factor ◽  
Class Iii ◽  
Factor B ◽  
Tissue Specific ◽  
Mhc Class Iii

Factor B (Bf), an enzyme of the alternative pathway of complement activation, is one of four major histocompatibility complex (MHC) class III genes. To ascertain the genetic mechanism for tissue-specific constitutive and regulated expression of Bf, we sequenced the regulatory regions 5' of the gene from mice of different H-2 MHC haplotypes and assessed trans-acting factors, specific DNA binding nucleoproteins, in liver and kidney. Striking tissue-specific differences in constitutive expression of Bf were demonstrated in mice of H-2f or H-2z haplotypes when compared with H-2d or H-2u (kidney and intestinal Bf in H-2d or H-2u much greater than H-2f or H-2z). These differences correlated with a point nucleotide substitution 3 bp downstream of the upstream Bf initiation site that affects interaction with a DNA binding protein. This and additional cis differences localize the sequence substitutions responsible for previously identified restriction fragment length polymorphisms among inbred mouse strains and also reveal two previously unrecognized polymorphisms generated by SmaI and HinfI digestion. Evidence for differences in trans was found in a comparison of DNA binding nucleoproteins from kidney, but not liver, of B10.PL when compared with B10.M. These data, together with the high degree of sequence homology between human and mouse Bf 5' flanking regions, should prompt a search for polymorphic restriction sites and cis binding elements in the Bf promoter that could serve as markers of human MHC-associated renal pathology and variants in local MHC class III gene expression.

scholarly journals Tissue-specific gene expression in mouse hepatocytes cultured in growth-restricting medium.

1988 ◽  
Vol 8 (8) ◽  
pp. 3338-3344 ◽  
Author(s):  
T Spiegelberg ◽  
J O Bishop
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Mrna Level ◽  
Alpha Fetoprotein ◽  
Apolipoprotein A1 ◽  
Specific Gene ◽  
Mrna Levels ◽  
Hormonal Stimulation ◽  
Tissue Specific

Culture conditions which maintain hepatocytes in their in vivo state are not known. This hampers the study of liver gene expression and of direct responses of liver genes to hormonal stimulation. We argued that hepatocytes that were unable to divide might retain in vivo characteristics. We therefore plated mouse (BALB/c) hepatocytes on plastic dishes in medium lacking arginine and measured the levels and transcription rates of six tissue-specific mRNAs over a period of days. Alpha-fetoprotein mRNA began to accumulate at about 48 h of culture, and transcription could sometimes be detected after 72 h. The levels and transcription rates of four mRNAs (albumin, alpha-1-antitrypsin, apolipoprotein A1, and major urinary protein [MUP]) fell sharply. The rate of transcription of transferrin mRNA fell less rapidly, and its level remained high, partly due to its longer half-life. The overall pattern of gene expression in the plated cells did not exactly parallel that of either fetal or regenerating liver. The hepatocytes remained responsive to hormonal stimulation. Insulin and dexamethasone each tended to counteract changes in mRNA levels, for example, preventing the accumulation of alpha-fetoprotein mRNA. The effects of insulin were primarily due to changes in transcription rates. Bovine growth hormone and thyroxine elevated the levels of most of the mRNAs. Many of the effects of these hormones, when added singly, could not be ascribed to changes in transcription. The level of MUP mRNA was strongly affected by added hormones. The mRNA level at 5 days was increased by added insulin, dexamethasone, growth hormone, and thyroxine. In the presence of these three hormones, the decay in the transcription rate of the MUP genes was reduced about 10-fold. We conclude that hepatocytes plated under these nongrowing conditions can provide insights into the hormonal responsiveness of tissue-specific genes.

Tissue-specific gene expression in mouse hepatocytes cultured in growth-restricting medium

1988 ◽  
Vol 8 (8) ◽  
pp. 3338-3344
Author(s):  
T Spiegelberg ◽  
J O Bishop
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Mrna Level ◽  
Alpha Fetoprotein ◽  
Apolipoprotein A1 ◽  
Specific Gene ◽  
Mrna Levels ◽  
Hormonal Stimulation ◽  
Tissue Specific

Culture conditions which maintain hepatocytes in their in vivo state are not known. This hampers the study of liver gene expression and of direct responses of liver genes to hormonal stimulation. We argued that hepatocytes that were unable to divide might retain in vivo characteristics. We therefore plated mouse (BALB/c) hepatocytes on plastic dishes in medium lacking arginine and measured the levels and transcription rates of six tissue-specific mRNAs over a period of days. Alpha-fetoprotein mRNA began to accumulate at about 48 h of culture, and transcription could sometimes be detected after 72 h. The levels and transcription rates of four mRNAs (albumin, alpha-1-antitrypsin, apolipoprotein A1, and major urinary protein [MUP]) fell sharply. The rate of transcription of transferrin mRNA fell less rapidly, and its level remained high, partly due to its longer half-life. The overall pattern of gene expression in the plated cells did not exactly parallel that of either fetal or regenerating liver. The hepatocytes remained responsive to hormonal stimulation. Insulin and dexamethasone each tended to counteract changes in mRNA levels, for example, preventing the accumulation of alpha-fetoprotein mRNA. The effects of insulin were primarily due to changes in transcription rates. Bovine growth hormone and thyroxine elevated the levels of most of the mRNAs. Many of the effects of these hormones, when added singly, could not be ascribed to changes in transcription. The level of MUP mRNA was strongly affected by added hormones. The mRNA level at 5 days was increased by added insulin, dexamethasone, growth hormone, and thyroxine. In the presence of these three hormones, the decay in the transcription rate of the MUP genes was reduced about 10-fold. We conclude that hepatocytes plated under these nongrowing conditions can provide insights into the hormonal responsiveness of tissue-specific genes.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

scholarly journals Aberrant expression of USF2 in refractory rheumatoid arthritis and its regulation of proinflammatory cytokines in Th17 cells

2020 ◽  
Vol 117 (48) ◽  
pp. 30639-30648
Author(s):  
Dan Hu ◽  
Emily C. Tjon ◽  
Karin M. Andersson ◽  
Gabriela M. Molica ◽  
Minh C. Pham ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
T Cells ◽  
Transcription Factor ◽  
Proinflammatory Cytokines ◽  
Th17 Cells ◽  
Gene Set Enrichment Analysis ◽  
Aberrant Expression ◽  
Signature Genes ◽  
The Impact

IL-17–producing Th17 cells are implicated in the pathogenesis of rheumatoid arthritis (RA) and TNF-α, a proinflammatory cytokine in the rheumatoid joint, facilitates Th17 differentiation. Anti-TNF therapy ameliorates disease in many patients with rheumatoid arthritis (RA). However, a significant proportion of patients do not respond to this therapy. The impact of anti-TNF therapy on Th17 responses in RA is not well understood. We conducted high-throughput gene expression analysis of Th17-enriched CCR6+CXCR3−CD45RA−CD4+T (CCR6+T) cells isolated from anti-TNF–treated RA patients classified as responders or nonresponders to therapy. CCR6+T cells from responders and nonresponders had distinct gene expression profiles. Proinflammatory signaling was elevated in the CCR6+T cells of nonresponders, and pathogenic Th17 signature genes were up-regulated in these cells. Gene set enrichment analysis on these signature genes identified transcription factor USF2 as their upstream regulator, which was also increased in nonresponders. Importantly, short hairpin RNA targetingUSF2in pathogenic Th17 cells led to reduced expression of proinflammatory cytokines IL-17A, IFN-γ, IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as transcription factor T-bet. Together, our results revealed inadequate suppression of Th17 responses by anti-TNF in nonresponders, and direct targeting of the USF2-signaling pathway may be a potential therapeutic approach in the anti-TNF refractory RA.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

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