Numerous studies have been conducted on the active methylation of the lysines in histone tails as early components of a chromatin remodeling process that leads to alterations in gene expression. The specific methylation of histone H3 lysine 9 (H3K9) in the promoter region of genes is one of the most important chromatin marks and is commonly associated with nucleosomal DNA methylation and gene silencing. Recent studies have shown that demethylation of the histone H3K9 chromatin mark is mediated by iron (Fe)-, oxygen-, ascorbic acid-, and oxoglutarate-dependent oxidative demethylation, which is catalyzed by a family of dioxygenases (JMJD2A-D and JHDM2A) that contain the Jumonji C (JmjC) protein domain. The affinity constant of nickel ions binding to this JmjC facial triad is predicted to be at least 3 orders of magnitude greater than that of Fe. Our goals and hypothesis of this grant are that nickel ions, as well as other hypoxia signaling insults (hypoxia and deferoxamine), inactivate the JmjC-containing dioxygenases that catalyze the demethylation of H3K9, which leads to chromatin remodeling, DNA methylation, silencing of tumor suppressors genes, and, ultimately, carcinogenesis. We will study the alterations of the mono, di- and tri-methylation patterns of H3K9 following a prolonged exposure to nickel, hypoxia and deferoxamine by Western blot. ChIP- on-chip assays will be employed to determine which gene promoters are associated with alterations of H3K9 methylations following nickel exposure. To link the observed changes in the distribution of histone H3K9 methylations to the inactivation of specific H3K9 demethylases, the ChIP-on-chip assays will also be performed in stable transfectants with RNAi-directed knockdown of JHDM2A or JMJD2A. We will study whether the increase of H3K9 methylation at the gene promoters identified will lead to the establishment of DNA methylation, using methylation-specific PCR. We will also study the general effects of altering the expression of histone H3K9 demethylases alone, by overexpression or RNAi knockdown, or in combination with nickel exposure, on cell transformation and proliferation in human BEAS-2B cells. It will also be of interest to study the roles of nickel-induced gene silencing in the transformation of Beas-2B cells by nickel. We will purify the JMJD2A and JHDM2A enzymes that are expressed in insect cells. Using these purified enzymes, we will determine the effects of nickel and other transition metal ions on the enzymatic activity of these recombinant proteins in vitro. We will also examine the ability of isotopic 63Ni ions to become associated with recombinant JMJD2A and JHDM2A in cells transiently transfected with JMJD2A or JHDM2A expression vectors, and correlate nickel ion incorporation with the loss of enzyme activity as measured by an in vitro demethylation assay. We will determine the number of nickel binding sites and metal dissociation constants for iron and nickel in JMJD2A and JHDM2A. XAS will also be utilized to determine the metal binding sites (iron and nickel ions) in these histone demethylases.
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