The aim of this research is to understand how stable states of gene expression are maintained during development. Molecular, genetic and biochemical methods will be used to study proteins that control transcription of homeotic genes in Drosophila melanogaster. Homeotic genes determine the identities of segments in the fly. Selective expression of homeotic genes is needed throughout fly development. Segmentation gene products control the initial patterns of homeotic gene expression in 2- hour-old embryos, but they decay by about 4 hours. The Polycomb group (PcG) proteins are transcriptional repressors that then maintain homeotic expression patterns during the rest of development. Current models suggest that PcG proteins recognize the initial off states of homeotic genes established in early embryos and they maintain repression through stable association and action of protein complexes in the local chromatin. This research will investigate molecular roles of PcG protein complexes and their individual components. Two major types of PcG complexes have been identified; one contains the PcG proteins extra sex combs (ESC) and Enhancer of zeste [E(Z)] and the other contains Polycomb (PC), polyhomeotic (PH), Posterior sex combs (PSC) and Sex comb on midleg (SCM). The experiments described in this proposal will seek to define molecular activities and components of the ESC-E(Z) complex and the molecular function of SCM with respect to the other major PcG complex.
These aims will be approached using a combination of protein purification and characterization, site-directed and random mutagenesis, expression and manipulation of transgenes, protein interaction tests, and immunostaining of chromosomes. Additional studies will investigate in vivo interactions between the two types of PcG complexes. Every PcG repressor so far cloned from Drosophila has homologs in mammals. These are functional homologs since knockout mutations in these genes produce homeotic defects in mouse embryos that resemble the developmental defects in the corresponding fly mutants. Thus, an understanding of PcG mechanisms of repression in flies should provide clues about similar developmental controls in higher organisms. PcG repressors have also been implicated in lymphomagenesis in mice, in abnormal cell proliferation in tissue culture cells, and in normal processes of hematopoiesis. Knowledge about the fly PcG components and mechanisms may contribute to a better understanding of normal blood cell development and processes that underlie certain human cancers.
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