Mechanisms of Gene Silencing

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DESCRIPTION (Verbatim from the applicant's abstract): The long-term goal of this research is to understand the mechanisms involved in gene regulation. Proper control of gene expression is essential during the life span of an organism; misregulation of expression can lead to developmental abnormalities and disease. Heterochromatin protein one, HP1, is an evolutionary conserved chromatin protein that is a component of silent chromatin. This proposal will focus on the molecular mechanisms of HP1 -mediated silencing at euchromatic locations. Recent data suggest that HP1 is essential for silencing genes within euchromatic regions of chromosomes. In mice, HP1 has been shown to interact with transcriptional corepressors that associate with the promoter regions of many genes. In humans, a reduction in HP1 correlates with metastasis of breast cancer cells, possibly due to the loss of silencing of genes required for the metastatic phenotype. Drosophila offers the cytology and genetics not available in mammalian systems to determine the role of HP1 in the regulation of euchromatic genes. Experiments proposed will test the following hypotheses: 1) HP1 nucleates the formation of heterochromatic domains that spread along the chromosome, silencing neighboring genes. To dissect the mechanisms of heterochromatin formation and spreading, HP1 will be tethered to 256 tandem sites within euchromatin. Genetic, cytological, and biochemical experiments will he used to determine the properties of the silent chromatin formed, the extent of spreading, and the identity of additional factors recruited to the region. Results will be compared with those known for centric heterochromatin. 2) HP1 acts as a short-range transcriptional silencer. HP1 will be tethered to 8 tandem sites strategically located within promoter regions of well-characterized genes. The balance between transcriptional activation and silencing will be explored by expression and chromatin structural analyses. The results will determine whether mechanisms of short-range silencing are similar to those responsible for heterochromatin mediated silencing. 3) HP1 is essential for silencing euchromatic genes. Euchromatic genes regulated by HP1 have not been identified in any organism to date. A combination of cytology, chromatin immunoprecipitation experiments, and genetics will be used to identify euchromatic genes regulated by HP1. An analysis of these genes will allow for comparisons with genes artificially silenced by HP1, and genes silenced by HP1 in heterochromatin.