Our long-term goal is to understand the mechanism used to package large portions of the eukaryotic genome into a heterochromatic form, analyzing the proteins involved, the marking of domains, and the means of blocking the spread of heterochromatin into euchromadc regions. Malfunctions in the heterochromatin system can lead to misregulation of gene expression, as welt as chromosome instability, and can be a contributing factor in many health problems, including cancer and genetically-based developmental disabilities. All of the work will be carried out in Drosophila, a model system that allows genetic, cytological, and biochemical approaches; in particular, position effect variegation (PEV) is being used as an indicator of heterochromatin packaging.
First specific aim : HP2, a newly identified heterochromatin protein, interacts with HPI by a number of criteria and is co-distributed with HPI, being associated with the pericentric heterochromatin and small fourth chromosome. We will examine the impact of mutations in HP2 on heterochromatin formation and gene silencing, looking at the structure of polytene and metaphase chromosomes and assessing changes in PEV both alone and in conjunction with mutations in known heterochromatin proteins HP1, SU(VAR)3-9 and SU(VAR)3-7. A tagged version of HP2 will be used to recover heterochromatin protein complexes and search for additional protein components. These studies will contribute to both a functional and a structural model of heterochromatin assembly. Second specific aim: The P[hsp26-pt, hsp70-w] transposable element is sensitive to chromatin packaging, giving a full red eye when inserted into a euchromadc domain, and a variegating phenotype when inserted into a heterochromafic domain. Several lines of evidence, including use of this P element, have demonstrated that the small fourth chromosome is composed of interspersed heterochromatic and euchromatic domains. Loss of DNA adjacent to a P element in a euchromatic domain, showing a red eye, can cause a switch in phenotype to a variegating eye, implying the loss of a barrier to the spread of adjacent heterochromatin. The chromatin structure in the putative barrier region wilt be mapped by additional genetic analysis, by mapping of hypersensitive sites, and by chromatin immunoprecipitation to identify the heterochromatin / euchromatin boundary; selected DNA fragments will be tested for barrier function. Genomic approaches will be used to analyze the DNA sequence patterns in such regions. This work should provide insights into chromatin organization, helping to define the differences between heterochromatin and euchromatin, and leading to better understanding of the nature of the barriers that lie between these domains. ? ?
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