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Korenjak, Michael (2006): Purification and Characterization of Retinoblastoma like Factor-containing Protein Complexes from Drosophila melanogaster. Dissertation, LMU München: Faculty of Biology
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Abstract

The Retinoblastoma protein (pRb) was the first tumor suppressor protein to be identified. It is the founding member of the so called pRb or pocket protein family, comprising two additional members (p107 and p130) in mammalian cells, and its best characterized function is the regulation of the E2F family of transcription factors. Today, the pRb-E2F network represents one of the best understood pathways implicated in cell cycle regulation and differentiation. Pocket proteins negatively regulate the transactivation properties of E2F proteins by two mechanisms: First, binding of pocket proteins to E2F masks the E2F transactivation domain and thereby impairs transcriptional activation. Second, pocket proteins interact with several chromatin modifying and chromatin binding proteins and recruit these proteins to E2F target genes, where they help to establish a repressive chromatin conformation. In this work, advantage was taken of the relative simplicity of the Drosophila melanogaster pRb-E2F network to purify and functionally characterize native pRb repressor complexes. Two related multisubunit complexes that only differ in their pocket protein subunit (RBF1 or RBF2) have been purified from Drosophila embryo nuclear extract. These complexes contain several novel pocket protein-associated polypeptides and localize to transcriptionally silent regions on Drosophila polytene chromosomes. Moreover, they specifically associate with deacetylated histone tails, which are a hallmark of transcriptionally silent chromatin. In cycling Drosophila S2 cells, the purified complexes redundantly repress the expression of a certain class of E2F target genes implicated in differentiation and development, whereas they do not control the expression of cell cycle-regulated E2F targets. Interestingly, the isolated complexes seem to be highly conserved between different organisms. Genes encoding the Caenorhabditis elegans homologs of the complex subunits act within the same genetic pathway involved in vulval cell fate determination and they functionally cooperate in different developmental processes. Furthermore, a complex with striking homology to the Drosophila complexes also exists in human cells. In the light of the specific repression of developmentally regulated E2F target genes in cycling Drosophila cells, it is conceivable that the complexes prevent the uncontrolled expression of genes important during differentiation. Since the C. elegans homologs of the complex subunits are also involved in cell fate determination, this might be a highly conserved feature of the isolated complexes.