Locus specific analysis of PcG/TrxG proteins using Bio-tagging technology

Abstract

Locus specific analysis of PcG/TrxG using Bio-tagging technology During development many cell types are generated by specific transcription programs that involve activation of gene expression at the level of promoters, enhancers and transcription factors. The identities of these different cell types are characterized by distinct sets of active and inactive genes, and need to be maintained through cell division. To achieve this, the cell type specific expression pattern has to be stably transmitted to the daughter cells. Epigenetic “cellular memory” mechanisms are often involved. In eukaryotes several chromatin-associated protein complexes have been identified, one of these systems comprises the Polycomb group (PcG) and the trithorax group (trxG) complexes. These widely conserved proteins act by locally modifying chromatin to maintain the transcriptional status of their target genes. The PcG and trxG bind as multi-protein complexes to regulatory elements called PREs (Polycomb response elements) in or near their target genes. A PRE can thus be considered an epigenetic switchable element upon which the PcG and trxG act antagonistically to maintain either silencing (PcG) or activation (trxG). Until now only a few distinct PcG complexes have been biochemically identified, but there is ample in vivo evidence that the composition of the PcG is different at different target genes and in different tissues. In addition, the purified complexes composition varies in some extent at different developmental times and depending on the protocol used for the purification. In all cases, biochemical isolations of complexes were performed using whole Drosophila embryos, Drosophila cell lines or mammalian cell lines; thus the purified complexes represent an average of all complexes at all target genes. Altogether, the memory system seems to be much more complicated than previously thought. Additional factors that come in to action at certain points in development might exist, giving a degree of specificity to Pc/TrxG action. So far, little has been done to establish procedures for the differential isolation and characterization of tissue specific, developmental time specific, or locus specific PcG or TrxG complexes. The aim of this thesis work was to design a system to look at the components of chromatin when still bound to the DNA in a locus, time and tissue specific manner. During this work a four-component system was developed based on the utilization of a protein bait and its binding site in the chromatin to co-purify proteins bound to transgenic PRE, giving locus-specificity to the system. To control time or tissue specificity, the protein bait expression was induced by Gal4. One more level of control was introduced by tagging the bait with a Bio-tag recognized and in vivo biotinylated by an enzyme that is also inducible expressed. All system components were introduced in D. melanogaster flies and the generated system was tested proving to be biologically feasible. When tested for protein purification many methodological problems were encountered that affected the efficiency of the system at various levels. Each problem was confronted separately and many experiments were conducted to find a better solution for each experimental step that would lead to a better output. However, protein pull-downs after system optimization resulted in no quantitative enrichment of PRE bound proteins. Nevertheless, thanks to the characterization and system optimization performed in this work, critical steps for functioning of this type of systems were identified. Alternative solutions to some aspects of the system designed will be based in the future on the findings of this work.