The mechanism of transcriptional regulation of metastasis-associated protein 1 gene expression during hepatocarcinogenesis

학위논문 (박사)-- 서울대학교 대학원 : 약학과, 2014. 2. 이미옥.Expression of metastasis-associated protein 1 (MTA1) gene correlates with the degree of invasion and metastasis in hepatocellular carcinoma (HCC)however, little is known about the transcriptional regulation of the MTA1 gene. The first part of this study identified the MTA1 gene as a target of p53-induced transrepression. The MTA1 promoter contains three putative p53 response elements (p53REs), which were repressed by the p53-inducing drug 5-fluorouracil (5-FU). Notably, 5-FU treatment decreased MTA1 expression only in p53 wild-type cells. p53 and histone deacetylases 1/2 were recruited to the p53REs, but H3K9Ac was released after 5-FU treatment. Proteomics analysis of the p53 repressor complex, which was pulled down by the MTA1 promoter, revealed that the poly(ADP-ribose) polymerase 1 (PARP-1) was part of the complex. Interestingly, p53 was poly(ADP-ribose)ylated by PARP-1, and the p53-mediated transrepression of the MTA1 gene required poly(ADP-ribose)ylation of p53. In summary, we report a novel function for poly(ADP-ribose)ylation of p53 in the gene-specific regulation of the transcriptional mode of p53 on the promoter of MTA1. Expression of MTA1 is induced by hepatitis B virus X protein (HBx)however, little is known about the transcriptional regulation of MTA1 gene expression. In second part of this study we report that the 5΄-flanking region of the human MTA1 promoter contains two CpG islands. Transient expression of HBx in Chang liver cells increased the methylation of the CpG island1 from 18% to 49% when measured by bisulfite-modified direct sequencing. Chromatin immunoprecipitation showed that HBx recruited DNMT3a and DNMT3b to the CpG island1. In silico analysis of CpG island1 predicted the existence of putative p53-binding sequences. p53 was pulled down by a DNA probe encoding the p53-binding sequences but not by the methylated DNA probe. The mouse MTA1 promoter also contains a CpG island encoding a p53-binding sequence of which p53 binding was decreased in the presence of HBx, and expression of MTA1 and DNMT3 was increased in the liver of HBx-transgenic mice. Comparison of MTA1 and DNMT3a expression in human normal liver and HCC specimens produced a significant correlation coefficient >0.5 (r = 0.5686, P = 0.0001) for DNMT3a, and a marginally significant coefficient (r = 0.3162, P = 0.0103) for DNMT3b. These data show that HBx induces methylation of CpG island in the MTA1 promoter, which interferes with DNA binding of p53 in the specific DNA region. This result may explain the molecular mechanism responsible for the induction of MTA1 gene expression by HBx.Docto