The long-range goals of this grant proposal are to study the involvement of the hypoxia signaling pathway upon chromatin remodeling, gene silencing, and cell transformation. Hypoxia or agents that mimic hypoxia cause a global loss of histone acetylation and increase the methylation of H3 lysine 9 which is the chromatin mark of gene silencing for the individual nucleosomes. Methylation of H3 lysine 9 leads to DNA methylation and inherited gene silencing. We will study the ability of hypoxia and agents that activate hypoxia signaling (NiCI2, CoCI2, deferoxamine, and DMOG) to induce cell transformation in wild-type or in HIF-1alpha knockout mouse embryo fibroblast (MEF). We will transfect a normal HIF-1alpha construct into MEF cells with a knockout of this gene to study whether we can restore their ability to be transformed by hypoxia and agents that mimic hypoxia. We will also stably transfect mutated HIF-1alpha constructs that will constitutively express HIF in its stabile and or stable/active form into MEF HIF+/- cells and study the effect of stabilized and/or activated HIF- 1alpha on anchorage-independent growth and tumor formation in nude mice. We will investigate global changes in H3K9 acetylation, H3K9 methylation, G9a activity (enzyme that dimethylates H3K9) and DNA methylation in MEF with intact or knockout of HIF-1alpha following exposure to hypoxia or agents that mimic hypoxia. We will also utilize the expression levels of DHFR and DNA mismatch repair gene Mlh1 which are down-regulated by hypoxia signaling to study gene specific effects of chromatin remodeling induced by hypoxia signaling using CHIP assays targeted to their promoters with antibodies specific to H3K9 dimethylation and/or H3K9 acetylation. We have obtained mouse embryonic stem cell wild-type (WT), G9a knockout (G9a-/-) and G9a-/- stably transfected with wild-type G9a (G9a-/- + G9a WT), and we will utilize these cells to address the role of G9a in mediating histone methylation H3K9 and the effects of hypoxia signaling on DNA methylation and DHFR/MIh expression. We will study the reversibility and stability of MEF cells that have already been transformed via the hypoxia pathway to chromatin remodeling agents and R59949 (HIF inhibitor). These studies will allow us to understand the importance of hypoxia signaling and the HIF-1alpha transcription factor in promoting neoplastic cell transformation.
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