Transcriptional repressors and corepressors play key roles in establishing systems of positional information and in mediating responses to extracellular signals during development. Many of these factors organize large chromosomal domains into transcriptionally silent states. In some cases, these states are self-propagating and survive many mitotic cycles, while in other cases, silencing is short lived and readily reversed. Silent domains are nucleated by cis-regulatory modules in the DNA and may spread from these modules in a manner that is dependent upon repressor or corepressor oligomerization. However, the connections between repressor/corepressor oligomerization, spreading, and silencing are not well understood. The research proposed here will explore the mechanisms of repression by two Drosophila regulatory factors, Groucho (Gro) and Yan, with well-established oligomerization functions.
Specific aim 1 is to determine if Gro spreading is required for repression. Gro is a corepressor with multiple essential roles in embryonic and imaginal development - its human orthologs have equally important roles in human development. It contains a highly conserved oligomerization domain termed the Q domain. Mutations in this domain that prevent oligomerization prevent repression. Furthermore, chromatin immunoprecipitation assays in cultured cells and wing discs demonstrate that Gro repression targets are associated with large Gro-bound domains many kilobases in length. This proposal seeks to establish firm links between Gro oligomerization, spreading, and repression. Gro rescue constructs with specific defects in oligomerization will be developed, and fly lines carrying these mutant alleles will be subjected to phenotypic and chromatin immunoprecipitation analysis to determine the structure and function of Gro-bound domains.
Specific aim 2 is to determine the role of Yan oligomerization in repression and Ras signaling. Yan and it human ortholog Tel are DNA binding transcriptional repressors with essential roles in Ras signaling. Ras activation leads to the phosphorylation of Yan by MAP kinase and the subsequent export of Yan from the nucleus resulting in derepression of Ras pathway target genes. Yan contains a SAM domain, which forms a head to tail protein polymer that is required for repression in S2 cells and also appears to control reception of the Ras signal. New alleles of yan that encode proteins with increased oligomerization affinity will be developed and tested in both cultured cells and the embryo to determine the mechanistic connections between Yan polymerization, repression, and Ras signaling.
The two factors under investigation here, Gro and Yan, each have human orthologs with many connections to human cancer. For example, Tel, the human ortholog of Yan, is a frequent target of chromosomal translocations in leukemia, while up-regulation of mammalian Gro has been linked to lung adenocarcinomas. Therefore, the mechanisms to be uncovered in this research will be relevant to human disease.
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