This project will characterize the cancer cell lines and tissues common to the Center using newly developed single-molecule tools, together with new methods for chromatin fractionation based on physical properties of mononucleosomes and arrays, to probe chromatin and epigenetic changes in cancer. Recent advances in the understanding of chromatin dynamics in model systems leads us to propose a novel mechanism for chromatin changes in human cancer. It is widely accepted that silencing of tumor suppressor genes is a key step in cancer initiation, and maintenance of tumor suppressor gene silencing often underlies cancer progression. Among the epigenetic mechanisms that are responsible for maintaining this silencing, promoter DNA methylation has strong experimental support. However, the popular hypothesis that DNA methylation silences genes by binding methylcytosine DNA binding proteins and consequent recruitment of his tone modifiers remains to be demonstrated, despite the fact that it has been the dogma for over a decade. We propose that gene silencing instead occurs because DNA methylation and other epigenetic modifications interfere with incorporation or properties ofthe universal his tone variant, H2A.Z. Our work and that of others suggests that H2A.Z destabilizes nucleosomes at promoters and thus favors promoter activation, so that by preventing H2A.Z incorporation or destabilization activity, unscheduled DNA methylation of tumor suppressor gene promoters prevents gene activation. We will test this hypothesis by investigating the genome-wide changes in H2A.Z and assay the physical properties and post-translational modifications of H2A.Z-containing nucleosomes from cancer cells provided by the Materials Core Facility. Our project will apply atomic force microscopy (AFM) and recognition imaging technologies that we have recently used to characterized single native chromatin particles containing the CenHS his tone variant in an ongoing ASU-Hutch collaboration. By following changes in DNA methylation, H2A.Z, and selected post-translational modifications in esophageal and colon cancer cells and tissue samples using both genome-wide and single-molecule methods, we will test our hypothesis, probe epigenetic changes, and correlate these changes with the physical properties measured by the two other projects in the Center.
Whereas genetic changes are essentially irreversible, epigenetic changes, including changes in DNA methylation, his tone modification and variants, are potentially reversible. Therefore, understanding the epigenetic mechanisms responsible for silencing of tumor suppressor genes promises to revolutionize diagnosis, treatment and prevention.
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