Histone proteins, assembled with DMA to form nucleosomes, are the basic building blocks of chromatin. The N-terminal domains of these proteins, histone tails, are thought to make flexible contacts with the DMA 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, methylation, and phosphorylation. 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. In this grant, we propose to exploit the strengths of Tetrahymena biology to better understand the role of chromatin in programmed DNA rearrangements. Elucidating the complete profile of histone modifications, the enzyme systems that add and remove these modifications, and understanding the physiological substrates of these activities are of paramount importance. Previously we have demonstrated that chromodomain-containing proteins (Pddp's), which are necessary for DNA elimination, read methylated lysine marks, in particular histone H3 lysine 9. However, another key player in the process of DNA elimination has recently been revealed, namely small RNAs, which are a hallmark feature of RNAi-like silencing phenomena. These small RNAs may provide sequence specificity by guiding factors such as Pddlp to heterochromatic regions in the developing macronucleus to facilitate DNA elimination. Our long-term objective is to establish the link between these two entities in the Tetrahymena system, and this will be partially pursued by the purification of chromatin complexes that reside at sequences to be eliminated. The availability of the Tetrahymena genome sequence (which has recently been completed), will greatly assist our investigation for chromatin binding and modifying proteins. In particular, numerous novel chromodomain proteins have been identified thus far, which will be bacterially-expressed and tested for their ability to bind specific histone modifications by anisotropy and thermodynamic studies. We will continue to excavate the genome for additional factors that play a role in this unique silencing process and determine their functions. DNA elimination is the certainly most extreme form of gene silencing, and however unique this system may be, the 'rules' that emerge from these studies may also apply to heterochromatin-induced gene inactivation in other organisms.
Showing the most recent 10 out of 15 publications
