Study Of The Regulation Of Human Ino80Chromatin Remodeling Complex On Cell Cycle&P21^Waf1/Cip1 And The Expression Of Human MOF In Colorectal Carcinoma

Being one of the genetic branches, Epigenetics has been developing rapidly inthe past several decades. As the research getting wider, more and more statisticsreveal that nearly all of human diseases have a relevance to the modificationabnormality in epigenetics. Therefore，it has great significance to study and clarify therole of epigenetic regulation enzymes in gene transcription and their mechanism incancer and other diseases. My research is focus on the study of hIno80and hMOF.The main outlines in this paper are as followed.Ino80chromatin remodeling complex, a member of Ino80subfamily of theSWI/SNF chromatin remodeling superfamily, is highly conserved from Sacchamycescerevisiae to human species. Although it has known that hIno80complex functions inDNA damage repair and therefore leads to cell cycle delay or arrest, the precisemechanism remains unclear yet. In the first place, we got the gene expression profileof hIno80from specific siRNA knockdown cells using microarray analysis. Geneexpression profile revealed that many cell cycle related genes were transcriptionallyregulated by hIno80complex, including p21. With the method of qRT-PCR, westernblot and immunofluorescence staining, we confirmed that p21was negativelyregulated by hIno80for the first time. Thus, we infer that the hIno80regulation of p21may impact the cell cycle directly. Trough Flow Cytometry (FCM), we confirmed theinference that deletion of hIno80indeed delayed G2/M to G1progression. Secondly,Chromatin immunoprecipitation (ChIP) assay further demonstrated that hIno80complex was restricted at-2.3kb and-1.03kb upstream of the p21transcriptional startsite which was considered as p53biding sites. For further study whether thisregulation is in a p53-dependent manner, p53wild type (p53wt) and p53-null HCT116cells were selected as experimental model. We found an obvious increase of p21expression level in hIno80knockdown p53wt cells, but not in p53-null cells.Meanwhile, over-express p53in p53-null cells also resulted in increase of p21expression level. Combining the ChIP assay, we confirmed that hIno80regulates p21 gene transcription in a p53-dependent manner. Finally, to study the effect ofprolonged progression G2/M phase on chromosome stability. Using siRNA or shRNAknockdown assay, we found that hIno80depletion led to abnormal chromosomestability, including polyploid cells and large nuclei. Interestingly, high-levelexpression of p21was observed in most of morphology changed cells, suggesting thatnegative regulation of p21by hIno80complex might be implicated in cell cycle phaseG2/M arrest and abnormal chromosome stability. Taken together, our findings willprovide a theoretical basis to further elucidate the cellular mechanism of hIno80complex.Human MOF (males absent on the first), a member of MYST1（Moz-Ybf2/Sas3-Sas2-Tip60） histone acetyltransferase family, is believed to berequired for histone H4K16acetylation. Here, we first report the involvement ofhMOF expression in clinically diagnosed primary colorectal carcinoma (CRC). Theconsequences illustrated a significant reduction (>2-fold decrease) of hMOF geneexpression in CRC,57%(25/44) tissues of the patients. What`s more, the low-levelexpression of hMOF correlates with lymph node metastasis and clinical stages ofcolorectal cancer. In conclusion, our results suggest that down-regulation of hMOFmay be common in cancer tissue and it may represent a novel biomarker for tumordiagnosis.