ABSTRACT
<p><b>OBJECTIVE</b>To investigate the regulatory mechanisms of acetylated p53 in the expression of microtubule-associated protein-2 (MAP2) in neuronal differentiation of P19 cells induced by all-trans retinoic acid (RA).</p><p><b>METHODS</b>Neuronal differentiation of P19 cells was initiated with 4-day RA treatment. Immunofluorescence, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, and map2 promoter driven luciferase assay were performed to detect the expression and relative promoter activity of MAP2 in those RA-treated cells. Real-time PCR-based chromatin immunoprecipitation assay (ChIP) was carried out to reveal the specific recruitment of acetylated p53 onto its binding sites on map2 promoter.</p><p><b>RESULTS</b>The expression of MAP2 was markedly increased in RA-induced P19 cells. The map2 mRNA increased 34-fold after 4 days of RA treatment and 730-fold 2 days after the treatment, compared with the cells without RA treatment (control). p53 was recruited to the promoter of map2 gene in acetylated form and thereby enhanced its promoter activity. p300/CBP associated factor (PCAF) was found induced in RA-treated cells and enriched in the nucleus, which might contribute to the acetylation of p53 in the regulation of map2 gene.</p><p><b>CONCLUSIONS</b>Acetylated p53 may participate in regulating the expression of map2 in RA-induced differentiation of P19 cells. PCAF is possibly involved in this process by mediating the acetylation of p53.</p>
Subject(s)
Animals , Mice , Base Sequence , Cell Line , DNA Primers , Gene Expression Regulation , Microtubule-Associated Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tretinoin , Pharmacology , Tumor Suppressor Protein p53 , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To verify the binding of p53 to p21WAF1/CIP1 gene promoter and detect its binding to hsp90 beta gene promoter in vivo.</p><p><b>METHODS</b>Chromatin immunoprecipitation and PCR analysis were used to measure specific gene regulation sequence and Western blot analysis to investigate p53 protein.</p><p><b>RESULTS</b>The p53 binding sequences on the promoters of p21WAF1/CIP1 and hsp90 beta gene were found in the p53 antibody immunoprecipitated DNA fragments and p53 was detected in the immunoprecipitated samples.</p><p><b>CONCLUSIONS</b>p53 binds to promoters of p21WAF1/CIP1 and hsp90 beta gene in vivo, and regulates the expression of the two genes.</p>
Subject(s)
Humans , Binding Sites , Chromatin , Genetics , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins , Genetics , Metabolism , Gene Expression Regulation , HSP90 Heat-Shock Proteins , Genetics , Metabolism , Polymerase Chain Reaction , Methods , Precipitin Tests , Methods , Promoter Regions, Genetic , Protein Binding , Transcription, Genetic , Tumor Suppressor Protein p53 , Genetics , Metabolism , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To study the role of a BTB/POZ domain protein in the expression of hsp90alpha gene.</p><p><b>METHODS</b>The eukaryotic expression plasmids of sense- and antisense-GAGA related protein (GRP) or empty vector were transfected into Jurkat cells with pREP4 episomal vector plasmids carrying the hsp90alpha promoter sequence from -1756 to +37 and control plasmids pMCAT. Total RNA was extracted. The relative promoter activity of hsp90alpha-CAT reporter gene was determined by competitive RT-PCR assay.</p><p><b>RESULTS</b>GRP markly increased the relative promoter activity of hsp90alpha-CAT reporter gene during heat shock.</p><p><b>CONCLUSION</b>GRP may promote the expression of hsp90alpha gene by participating in chromatin remolding.</p>
Subject(s)
Animals , Humans , Amino Acid Motifs , Checkpoint Kinase 1 , Cloning, Molecular , DNA , Metabolism , DNA, Complementary , Metabolism , DNA-Binding Proteins , Metabolism , Drosophila , Genetics , Drosophila Proteins , Genetics , Gene Expression Regulation , HSP90 Heat-Shock Proteins , Genetics , Heat-Shock Response , Genetics , Homeodomain Proteins , Chemistry , Genetics , Metabolism , Repressor Proteins , Genetics , Transcription Factors , Genetics , Two-Hybrid System TechniquesABSTRACT
<p><b>OBJECTIVE</b>To identify novel binding proteins of hSNF5, a subunit of chromatin remodeling complex in human fetal brain.</p><p><b>METHODS</b>The yeast two-hybrid system was used for this study. Positive cDNA clones were sequenced. Sequence homology and putative functional domains were analyzed and compared with databank.</p><p><b>RESULTS</b>Nine positive clones obtained were analyzed, among which the sequence of one clone was 97% homologous to the 3' mRNA of a hypothetical protein FLJ20643, while other four clones were related to protein coding sequences existed in the GenBank. The rest four clones were not in frame with any known protein coding sequence.</p><p><b>CONCLUSIONS</b>Clones encoding for hSNF5 binding protein exists in cDNA library of human brain. These proteins may recruit chromatin remodeling complex via hSNF5 to modulate the transcription of their target gene and the related cell functions.</p>
Subject(s)
Humans , Brain , Cell Biology , Metabolism , Chromosomal Proteins, Non-Histone , Cloning, Molecular , DNA, Complementary , Genetics , DNA-Binding Proteins , Genetics , Embryo, Mammalian , SMARCB1 Protein , Trans-Activators , Transcription Factors , Genetics , Transcription, Genetic , Two-Hybrid System TechniquesABSTRACT
<p><b>OBJECTIVE</b>A CAT reporter plasmid (pBLCAT3alpha1) driven by the promoter of hsp90alpha was in vitro assembled into chromatin to investigate the transcription activity of the reporter gene upon heat shock.</p><p><b>METHODS</b>A competitive RT-PCR-based technique was used to quantify the promoter activity of hsp90alpha gene on chromatin or naked DNA templates in vitro.</p><p><b>RESULTS</b>The in vitro transcription efficiency was first optimized by using different amounts of whole cell extracts from heat shock-treated HeLa cells. In vitro chromatin assembly was carried out with purified components of chromatin assembly associated factors, core histones and CAT reporter gene driven by the promoter of hsp90alpha gene. Results showed that chromatin formation repressed the in vitro transcription of the gene.</p><p><b>CONCLUSION</b>The heat shock induced transcription of hsp90alpha gene on chromatin template is more efficient than that on naked DNA in vitro.</p>
Subject(s)
Humans , Chromatin Assembly and Disassembly , Genetics , Genes, Reporter , Genetics , HSP90 Heat-Shock Proteins , Genetics , Heat-Shock Response , Genetics , Transcription, GeneticABSTRACT
<p><b>OBJECTIVE</b>To study the effect of Rac-MEKK-JNK (Rac-mitogen activated protein kinase kinase kinase-C-jun N-terminal protein kinase) signal pathway on heat shock-induced hsp90 beta gene expression and the impact of Hsp90 on the regulation of the pathway.</p><p><b>METHODS</b>DN-Rac, DN-MEKK or DN-JNK were cotransfected with hsp90 beta CAT reporter plasmid beta 3.1 into Jurkat or LETPa-2 cells individually, the CAT mRNA expression was then determined quantitatively by competitive RT-PCR based system. Western blot was carried out to detect the expression level and phosphorylation of c-Jun in Jurkat and LETPa-2 cells that were transfected with DN-Rac, DN-MEKK or DN-JNK. By in vitro kinase activity assay and Western blot, the effect of geldnamycin (GA) on heat induced JNK activity were evaluated.</p><p><b>RESULTS</b>In Jurkat cell transfected with DN-Rac, DN-MEKK or DN-JNK, heat shock induced relative CAT mRNA expression level was decreased to (72.8 +/- 5)%, (60 +/- 13.2)% and (47.7 +/- 12.1)% of the control respectively; while in LETPa-2 cell hsp90 beta 3.1 reporter gene expression was accordingly suppressed to (16.17 +/- 5.1)%, (50.2 +/- 8.7)% and (47.5 +/- 10)% of control. C-Jun expression and phosphorylation were inhibited by the transfection of either one of DN-Rac, DN-MEKK or DN-JNK. With GA treatment, heat shock induced JNK activity was repressed, while the expression level of JNK or c-Jun was not obviously changed.</p><p><b>CONCLUSIONS</b>Rac-MEKK-JNK pathway promotes heat shock induced hsp90 beta gene expression and hsp90 may participate in the regulation of heat shock activated Rac-MEKK-JNK signal pathway in both Jurkat and LETPa-2 cells.</p>
Subject(s)
Humans , Benzoquinones , Cell Line, Tumor , Genes, Reporter , HSP90 Heat-Shock Proteins , Genetics , Hot Temperature , JNK Mitogen-Activated Protein Kinases , Lactams, Macrocyclic , Leukemia, T-Cell , Pathology , Mitogen-Activated Protein Kinase Kinases , Physiology , Mitogen-Activated Protein Kinases , Physiology , Protein Kinase C , Physiology , Quinones , Pharmacology , Signal Transduction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To explore GAGA-like element binding protein in human cells.</p><p><b>METHODS</b>Yeast one-hybrid system was used to screen the GAGA-like element binding proteins in HTLV-1 transformed Jurkat cell cDNA fusion library. Total RNA extracted from Jurkat cells was first labeled by reverse transcription, and was taken as cDNA probe to hybridize with the candidate positive clones.</p><p><b>RESULTS</b>9 positive clones were obtained, and 6 out of the 9 clones were positively hybridized with the cDNA probe.</p><p><b>CONCLUSIONS</b>6 candidate clones encoding for GAGA-like element binding proteins were obtained from Jurkat cells for further investigation.</p>
Subject(s)
Humans , Carrier Proteins , Genetics , Cloning, Molecular , DNA, Complementary , Genetics , DNA-Binding Proteins , Drosophila Proteins , HSP90 Heat-Shock Proteins , Genetics , Homeodomain Proteins , Genetics , Human T-lymphotropic virus 1 , Genetics , Jurkat Cells , Leukemia, T-Cell , Metabolism , Pathology , Transcription Factors , Genetics , Two-Hybrid System TechniquesABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of p53 binding site (+31/+60) of hsp90 beta gene on its transcriptional regulation.</p><p><b>METHODS</b>The binding site was first inserted into pBS-SK. After the plasmid annealing and elongation with mutagenic and selective primers, nuclease digestion and bacteria transformation was performed twice to select the positive mutated plasmid. Electrophoretic mobility shift assays (EMSA) was employed to detect the binding of hsp90 beta gene fragment containing mutated p53 binding site and Jurkat cell nuclear extract transfected by p53 expression vector.</p><p><b>RESULTS</b>The sequence analysis profile confirmed a successful mutation of two bases on the core sequence of the second half binding site. EMSA results showed the specific DNA-protein complex band disappeared after the mutation.</p><p><b>CONCLUSIONS</b>The core sequence of p53 binding site plays a key role in the trans binding of p53 to hsp90 beta gene.</p>
Subject(s)
Humans , Binding Sites , HSP90 Heat-Shock Proteins , Genetics , Leukemia, T-Cell , Pathology , Mutagenesis, Site-Directed , Mutation , Transcription, Genetic , Transcriptional Activation , Tumor Suppressor Protein p53 , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To establish a RT-PCR system for detecting mRNA expression level of 4 hsp genes in human cells.</p><p><b>METHODS</b>RT-PCR system was established with gene cloning, gene recombination, in vitro transcription and RT-PCR techniques; The detection of expression level of 4 hsp genes in SW13 cell was carried out with this system.</p><p><b>RESULTS</b>In SW13 cell, hsp70 and hsp90 alpha were typical heat shock induced genes, while hsp60 and hsp90 beta were efficiently expressed and further induced by heat-shock to various extent.</p><p><b>CONCLUSIONS</b>In our hands novel RT-PCR system can be used to detect mRNA expression level of 4 human hsp genes.</p>