| The remodeling of the chromatin structure mediated by histone posttranslationalmodifications plays a crucial role in the expression regulation of eukaryotic genes. Among thehistone posttranslational modifications, the histone acetylation catalyzed by histoneacetyltransferases (HATs) and histone deacetylases (HDACs) plays a critical role in generegulation and hence has been intensively studied. The reversible acetylation/deacetylationmodification of lysine residues in core histone tails mediated by these two enzymes modulatesthe initiation of transcription, thus leads to the activation/suppression of gene expression.Transcriptional coactivators p300/CBP and P/CAF, which possess histone acetyltransferaseactivities, are involved in transcriptional regulation of many genes by distinct mechanisms. WT1 gene encodes a zinc finger protein, which plays an important role in the regulationof growth and differentiation. WT1 proteins are mainly involved in the development ofkidney and gonad. Moreover, WT1 proteins are associated with the early development ofhematopoietic system. Apart from Wilms' tumor, alterations of WT1 gene have been shown tobe associated with several developmental abnormality syndromes and other tumors such asleukemias, mesotheliomas, gonadoblastoma, etc. Therefore, the elucidation of themechanisms of WT1 transcriptional regulation will be helpful not only in studies ofembryonic development, but also in the establishing theoretical bases for the cure of WT1-associated diseases. The aim of this study was to clarify whether the reversible histoneacetylation/deacetylation modification participates in the regulation of WT1 gene transcriptionand to explore its molecular mechanisms. By applying a series of transient transfection andrelative luciferase activity assays, as well as semiquantitative RT-PCR, we found that histoneacetyltransferases p300/CBP not only promoted the activities of the WT1 promoter andintronic enhancer, but also increased the expression of endogenous WT1 mRNA. E1A proteinrepressed the promotion of WT1 transcription by p300, while the p300/CBP-binding defectiveE1A delta 2-36 did not. This result further confirmed the effects of p300/CBP in promotingthe WT1 gene expression. Our results also indicated that p300/CBP and transcription factorsSp1, c-Myb, and Ets-1 synergistically activated WT1 reporter gene transcription. By using theplasmids with opposite orientations of insertion of the WT1 intronic enhancer, p300/CBP wasshown to be able to activate WT1 reporter gene transcription independent the orientation ofWT1 intronic enhancer. In addition, by using the HAT domain-deleted p300 mutant, weshowed that the histone acetyltransferase activity of p300/CBP was important in regulation ofWT1 gene expression. With the chromatin immunoprecipitation assay (ChIP), we found thatp300/CBP enhanced the acetylation of the histone H3 at the WT1 intronic enhancer.Furthermore, our results showed that another histone acetyltransferase P/CAF could alsoactivate WT1 reporter gene expression and this process required the synergism betweenP/CAF and p300/CBP. The histone acetyltransferase activities of P/CAF and p300/CBP wereboth important in the activation of WT1 reporter gene expression. With regard to histonedeacetylases, we tested the influences of six human histone deacetylases (HDAC1-6) on WT1gene expression. The results showed that HDAC4 and HDAC5 were able to not only repressthe activity of the WT1 promoter/intronic enhancer/luciferase reporter gene, but alsodecreased the expression of endogenous WT1 mRNA. By transient transfection and reportergene assay, we demonstrated that HDAC4 and HDAC5 repressed the promotion of WT1reporter gene transcription by p300. Data presented in this thesis demonstrated, for the first time, that the reversible histoneacetylation/deacetylation modification was involved in the transcriptional regulation of WT1gene. The molecular mechanisms of histone acetylation in WT1 gene regulation were brieflyexploited. This study provides further insight into the mechanisms of transcriptional...
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