The long-term objective is to determine the mechanism by which the packaging of a gene in a heterochromatic form leads to stable inactivation. This type of """"""""off"""""""" regulation is clearly of functional importance in many epigenetic systems, and appears to extend to the regulation of the homeotic loci, whose on/off pattern is critical for normal development in all higher organisms. Many of the chromosomal proteins required for stable silencing are high conserved, and their misfunction can contribute to a disease state. Drosophila melanogaster will be used because of the ease of monitoring and modifying position effect variegation (PEV). A P-element carrying a visible marker (hsp70-white) and a well-characterized test gene (a marked form of hsp26) exhibits PEV when resident in the pericentric heterochromatin, the telomeres, and some sites within the banded region of chromosome 4. The silencing in pericentric heterochromatin and chromosome 4 is sensitive to mutations in HP1 (heterochromatin protein 1), a protein localized to these regions. These variegating transgenes show alterations in chromatin structure. We will carry out a detailed structural analysis in two lines recovered in a screen at 28 degreesC, HS-2 and HS-5, that show complete silencing of the transgenes (which are in pericentric heterochromatin) at 25 degreesC. We will map the nucleosome array and look at limits of accessibility, rates of nuclease digestion, and DNA modification to determine whether or not alterations have occurred in the primary chromatin fiber, or in higher order packaging, or in both, distinguishing among such models for achieving silencing. A second recently identified heterochromatin-specific protein, HP2, will be analyzed by molecular and genetic techniques. Formaldehyde crosslinking of chromatin followed by immunoprecipitation will be used to analyze the pattern of histone acetylation and map the association of HP1 and HP2 with the silent transgenes. The effect of mutations in one chromosomal protein on the association of other will be determined cytologically and biochemically to establish the hierarchy of interactions, and the impact on chromatin structure. Mapping sites of HP1 interaction may indicate DNA sequences that nucleate heterochromatin formation; these will be tested in P-element constructs. A map of transgene function will be generated for chromosome 4, which appears to be made up of interspersed euchromatic and heterochromatic domains. Correlating this map with a sequence map with a sequence map should provide insights into chromosome organization, helping to define the differences between heterochromatin and euchromatin and potentially leading to identification of boundaries between these domains.
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