Investigating The Molecular Mechanisms For Dynamic Regulation Of Epigenetics By Metabolites And Its Role In Tumorigenesis

IDH1and IDH2are the most commonly mutated metabolic genes yet identified in human cancers. Tumor-derived IDH1/2mutations simultaneously cause loss of its normal activity, the production of α-ketoglutarate (α-KG), and gain of a neomorphic activity, the reduction of α-KG to2-hydroxyglutarate (2-HG).2-HG is structurally similar to a-KG and acts as an antagonist of α-KG to competitively inhibit multiple α-KG-dependent dioxygenases, including the JmjC domain-containing histone demethylases (KDMs) and the TET (ten-eleven translocation) family of DNA hydroxylases. Altered epigenetic regulation is currently considered to be a major mechanism whereby IDH mutation and2-HG exert their oncogenic effects.The TET family of a-KG-dependent dioxygenases catalyzes the sequential oxidation of5-methylcytosine (5mC) to5-hydroxymethylcytosine (5hmC),5-formylcytosine (5fC), and5-carboxylcytosine (5caC), leading to eventual DNA demethylation. By performing immunohistochemistry (IHC) and dot-blot hybridization, we found that the levels of5hmC were dramatically reduced in various human cancers, such as breast, liver, lung, pancreatic, and prostate cancers when compared with the matched surrounding normal tissues. Associated with the5hmC decrease was the substantial reduction of the expression of all three TET genes, revealing a possible mechanism for the reduced5hmC in cancer cells. The decrease of5hmC was also observed during tumor development in different genetically engineered mouse models. Together, our results identify5hmC as a biomarker whose decrease is broadly and tightly associated with tumor development.Next, we have manipulated the in vivo level of α-KG by intraperitoneally injecting glucose, glutamate or glutamine into mice. Surprisingly, we found that5hmC was dynamically changed in the liver and other metabolic tissues of mice after injection with glucose, glutamate or glutamine. Notably, there was a significant positive correlation between the levels of α-KG and5hmC in mouse livers after administration. Moreover, we treated mouse embryonic stem cells E14with vitamin C, and observed that vitamin C could rapidly increase the 5hmC levels in E14cells, while a series of dioxygenase inhibitors (such as CoCl2, DFO and DMOG) could effectively inhibit the effect of vitamin C on increasing5hmC. This result suggests that vitamin C may enhance the activity of TETs by maintaining or increasing intracellular levels of divalent iron ion (Fe2+), thereby contributing to the accumulation of5hmC. Together, these data reveal that the activity of TET hydroxylases and thus the5hmC levels can be dynamically regulated by diverse factors under physiological conditions.To sum up, three major implications for my PhD thesis are listed as follows. First, IDH1/2mutations lead to accumulation of2-HG, which inhibits the activities of a-KG-dependent dioxygenases such as histone demethylases and TET hydroxylases, leading to genome-wide histone and DNA methylation alterations. Second, the levels of5hmC are dramatically reduced in multiple human tumors, suggesting that5hmC could be a valuable biomarker for diagnosis of many cancer types. Third,5hmC as a demethylation intermediate can be dynamically regulated by the activity of TET hydroxylases under diverse physiological conditions.