Role of RNAi components in PcG-dependent silencing

In the nuclei of all eukaryotic cells, genomic DNA is highly folded and compacted with histone and non-histone proteins in a dynamic polymer called chromatin. Chromatin-modifying proteins organize chromatin into accessible (euchromatin) and inaccessible (heterochromatin) sub-domains thereby regulating expression of specific genes. Moreover compartmentalization of genes in specific nuclear domains contributes to regulatory changes in gene expression. During development, cell-type-specific transcription programs have to be stably transmitted to maintain cell identity. Silencing of a specific set of genes involves multiple layers of regulation, acting at all levels of chromatin packaging from nucleosomes to folding of chromosomal domains in the nucleus. Polycomb (PcG) and thrithorax Group (trxG) proteins maintain memory of gene expression states by acting at cis-regulatory elements termed PcG response elements (PRE). PRE-mediated silencing is enhanced by trans-interactions among chromosomes, suggesting that PREs may be organized in regulatory nuclear compartments. Moreover, PcG proteins mediate cosuppression, a phenomenon in which silencing among dispersed homologue sequences occur. Silencing is regulated either at the transcriptional level (TGS) via chromatin remodelling or at the post- transcriptional level (PTGS) where it exhibits molecular hallmarks typical of RNA-interference (RNAi). In Drosophila melanogaster, homologues of RNAi-defective mutants affect PTGS as well as PcG-dependent silencing, suggesting a possible link between PcG-action and RNA-mediated gene silencing processes. In this project we will investigate the nature of PRE in Drosophila and the role of RNAi components in the Polycomb pathway of gene silencing and nuclear compartmentalization. One- and two-colour fluorescence in situ hybridisation (FISH) of polytene chromosomes and whole mount embryos will be applied to study nuclear positioning of single or multiple genes. Combining this method to immunostaining of proteins allow to identify co- localization of given proteins with specific DNA sequences (immuno-FISH). RNA FISH will be combined to DNA FISH to analyze co-localization of RNA compounds to specific DNA sequences.