Research On The Interactions Between Histone And DNA Methylation In Acute Leukemia And The Regulation Of Wnt Signal Pathway

Acute leukemia is a multiple factor relative disease. Some researches found that the acute leukemia occurrence, development, treatment effect and prognosis are relevant with epigenetics . Epigenetics is refered to the heritable gene expression variations without the DNA sequence changes. It is mainly through several mechanisms such as DNA methylation, histone posttranslational modifications, RNA silence and chromatin remodeling to moderate gene expression, which is also one of the important mechanism of tumour exist. Current researches mainly point on anti-oncogene DNA methylation, histone acetylation and histone methylation separately. It's not clear to the interactions between these different modifications and the mechanisms of gene expression regulating, which in same time, are the recent focus of epigenetic research. Wnt signal pathway (wingless pathway) is a conservative signal pathway, involved in regulating cell growth, migration, differentiation and apoptosis. It is one of the most important signal pathway in tumor occurrence. The abnormal activation of Wnt pathway is closely related to the tumor formation.This project is planned to target the antagonist genes in Wnt pathway. By interfering up and down the balance between DNA methylation and histone methylation patterns, the results would be discussed on the interaction of Jurkat cell DNA and histone H3K9 methylation and on the regulation mechanism of target gene expression, including the research on the Jurkat cell lines biologic activities and its influence to Wnt signal pathway's conduction, revealing the acute leukemia epigenetical pathogenesis, finding some new clues for the early leukemia diagnosis and gene targeting therapy.The project is combined of four parts:1. Adopting the Western Blot method, the total methylation levels of histone H3K9 and H3K4 were detected on acute leukemia patients and seven series of malignant leukemia cell lines, analyzing their relationships among the occurrence, development, treatment effect and the prognosis of acute leukemia.2. The acute lymphocytic leukemia Jurkat lines was set as the research object in which the histone H3K9me3 methylation level was high. ChIP-on-chip(chromatin immunopricipitation on chip) technic was used to screen the Wnt antagonism genes like WNT5A, DKK3 and SFRP2 as target genes. Then these target gene expressions were analyzed by qPCR method and their promoter DNA methylation levels by MSP method. The correlation between histone and DNA methylation and their relationships with Wnt were compared.3.Constructing interference plasmid for histone methyl transferase SUV39h1 on H3K9me3 site, silencing the SUV39h1 gene to reverse H3K9 histone methylation, at the same time, 5 - Aza– CdR as the classic DNA methylation drugs was used to wipe off the target gene promoter DNA methylation. Under the bi-regulating of DNA and histone methylation, methods including Western Blot, MSP and ChIP-qPCR were employed to detect the level changes of corresponding DNA/histone methyl transferase, DNA/histone methylation and target gene expression. The interaction between DNA and histone methylation and their regulation mechanism on target gene expression were then explored.4. As the non-drug group be normal control, the 5-Aza-CdR treatment group and SUV39h1 gene knockout group were cross compared in levels as its growth curves, proliferation activities, clone formation abilities, cell cycles and apoptosis. The express alteration of key effective proteinβ-catenin was detected by Western Blot. The influences of DNA/histone methylation modification on Jurkat cell's biologic activity and Wnt signal pathway were discussed.Conclusions :1. The histone H3K9me3 high methylation and H3K4 low methylation are widely exist in acute leukemia and correlated to leukemia's type, treatment effect and prognosis.2. Jurkat cell's histone H3K9me3 methylation is correlated to promoter DNA hyper-methylation levels of Wnt signal pathway's antagonism genes (WNT5A, DKK3 and SFRP2).3. The drug 5-Aza-CdR can reverse DNA methylation, down-regulate histone H3K9 methylation level, decrease histone methyl transferase SUV39h1 and G9a expression and activate the target gene expression.It also can inhibit Jurkat cell proliferation, block the cell cycle process and induce apoptosis in vitro. These effects may be due to inhibiting the Wnt/β-catenin signal pathway.4. SUV39h1 gene silence can reverse histone H3K9 methylation state, decrease the expression of DNA methyltransferase as DNMT1, DNMT3A and DNMT3B,increase the target gene expression, but there is no significant change of DNA methylation. It can inhibit Jurkat cell proliferation, block the cell cycle process and induce apoptosis in vitro. These effects may be due to inhibiting the Wnt/β-catenin signal pathway.5.The imbalance between DNA and histone H3K9 methylation can independently or collaboratively activate Wnt pathways antagonism genes expression, restrain Wnt signals. H3K9 histone methylation is a more earlier actived event than DNA methylation.