The chromosomal distribution of retrotransposon and retroviral insertions is distinctly non-random, suggesting that retroelements actively select integration sites. One hypothesis to explain target site biases is that retroelement integration complexes are tethered to specific chromatin domains through protein-protein interactions. Support for this model comes from the study of the Saccharomyces Ty5 retrotransposons, which integrate preferentially into domains of specialized chromatin (silent chromatin) near the telomeres and silent mating loci. Host- and element-encoded proteins required for Ty5's target specificity include a component of silent chromatin (Sir4p) and a domain near the C-terminus of Ty5 integrase (the targeting domain, TD). Consistent with the targeting model, these proteins interact both in vitro and in yeast two-hybrid assays. This interaction provides an entry point to further dissect targeting mechanisms: (i) The TD is post-translationally modified, and this modification is required for productive interactions with Sir4p. The biochemical nature of this modification will be determined, and host factors important for TD/Sir4p interactions will be identified and characterized for their role in target specificity, TI) modification and the function of silent chromatin. (ii) Mutations in TD lead to a decrease in transposition, suggesting that transposition is regulated by post-translational modification. This hypothesis will be explored, and other regions of the integrase C-terminus will be characterized for their specific role in targeting and transposition. (iii) Two missense mutations in Sir4p abrogate binding to the TD. These mutations also cause a loss in transcriptional silencing at the HM loci, and their role in the function of silent chromatin will be further characterized. (iv) Ty5 target specificity will be altered by replacing the TD with peptide aptamers that bind with high affinity to other DNA-bound proteins. Targeting determinants of Tyl and Ty5 will be swapped to test whether the target specificity of other retroelements can be altered. Collectively, these experiments will help achieve our long-term goals of understanding the retroelement/host relationship and harnessing retroelement targeting mechanisms to improve methods of genome manipulation and retroviral gene transfer.
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