Histone proteins, assembled with DNA to form nucleosomes, are the basic building blocks of chromatin. N-terminal domains of these proteins, histone tails, are thought to make flexible contacts with the DNA that allow for dynamic changes in the accessibility of the underlying genome. These tails are also subjected to a diverse array of post-translational modifications, such as acetylation, and an increasing body of evidence suggests that covalent histone modifications play a fundamental role in modulating chromatin structure with far-reaching implications for human biology and disease. Elucidating the enzyme systems that add and subtract these modifications, and understanding the physiological substrates of these activities are of paramount importance. The finding that bromodomains 'read' acetyl-lysines in histone tails provides an exciting precedence that other chromatin 'velcro' modules may exist that bind to histone tails bearing unique patterns of post-translational marks. We will implement high resolution NMR spectroscopy in conjunction with thermodynamic studies to characterize the basis for specificity of chromodomain binding to modified histone tail(s). In this grant, we propose to exploit the strengths of Tetrahymana biology to better understand the role of chromatin in programmed DNA rearrangementa The existence of multiple chromodomains in the proteins involved in this process (Pddps) suggests that these domains may be involved in generating a unique chromatin structure of germ-line DNA that is to be eliminated. Covalent histone modifications, such as histone methylation, may also be involved, and we hypothesize that chromodomains have evolved to read this mark, perhaps in concert with other histone modifications. Our plan to generate site-directed, methylation-specific histone antibodies is likely to yield invaluable reagents for these and other studies. The fundamental nature of chromatin, and histone modifications in particular, promises to provide links to a large number of DNA-templated processes. Therefore, knowledge learned from this study of the role of histone modifications in programmed DNA elimination in ciliates may be extended to other systems such as RAG-mediated VID/J shuffling and transposition in vertebrates. Whether any of the 'rules' that emerge from these studies will also apply to heterochromatin-induced gene inactivation in other organisms is not known, but remains an exciting possibility.
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