Oxidative stress is a common effect of a number of environmental stressors. Hypoxia can also produce O2*- (superoxide) when the mitochondrial electron transport has been disrupted by lack of oxygen. Recent work related to 2-oxoglutarate (2-OG) dependent dioxygenases makes it clear that there are additional effects of hypoxia and oxidative stress beyond those mediated through HIFs. The 2-oxoglutarate dependent dioxygenases, which regulate epigenetic parameters, include oxidative histone demethylases and TET proteins, the 5-methylcytosine (5mC) hydroxylases. These enzymes require O2 to function and are also affected by oxidative stress. We believe that the inability to hydroxylate 5mC in DNA and demethylate a number of histone lysines due to the inactivation of 2-oxoglutarate dependent dioxygenases, is likely to have major effects on a cell's epigenetic program resulting in inherited aberrations in gene expression. We hypothesize that oxidative stress (and hypoxia) induced by a variety of environmental insults is upstream of persistent and inherited epigenetic changes that can lead to diseases such as cancer, via their ability to transiently inhibit the activity of the 2-oxoglutarate dependent histone demethylases and TET proteins. We will investigate the effect of various chemical agents that produce oxidative stress or hypoxia signaling on the epigenetic program by identifying which of the above dioxygenases is inactivated by oxidative stress and hypoxia. We hypothesize that measuring the mRNA and protein levels of the oxidative demethylases, which are upregulated when their enzymatic activities are inhibited, will identify the demethylases that are affected by oxidative stress or hypoxia. In addition, global levels of histone modifications and DNA methylation and hydroxymethylation will be assessed to confirm the identity of oxidative demethylases that are inhibited. Specificity to oxidative stress will be examined using antioxidants such as reduced ascorbate or vitamin E. We will map on a genome-wide scale epigenetic modifications that are affected by the inhibition of oxidative demethylases using ChIP-Seq. We will also study which histone modification marks, 5mC, 5-hydroxymethylcytosine (5hmC) and gene expression changes persist following removal of the oxidative stress in relevant gene promoters and also selected modifications will be mapped on a genome wide scale using ChIP-Seq. To investigate the effects of persistent chromatin alterations due to the inactivation of oxidative demethylases chromatin accessibility will be assessed on a genome-wide scale using FAIRE-Seq in the absence and presence of oxidative stress/hypoxia with a focus on understanding the persistence of these sites after oxidative stress is terminated for a number of cell generations. Decreases in binding of critical transcription factors due to reduced chromatin accessibility will be predicted by computational approaches and confirmed by genome-wide mapping of candidate factors in the absence and presence of oxidative stress/hypoxia, with a focus on understanding which changes persist after the stress has ceased.
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