Non-coding RNA of Polycomb/Trithorax Response Elements

The highly conserved Polycomb (PcG) and Trithorax (TrxG) group proteins constitute an epigenetic "cellular memory" system that is essential for maintaining the correct identity of both stem cells and differentiated cells. Despite enormous progress in recent years defining the biochemical properties of the PcG and TrxG proteins, and mapping their target sites in whole genomes, the mechanisms of PcG and TrxG silencing and activation remain obscure. Still less do we understand transitions between these states, and their stable inheritance from one cell generation to the next, a question that lies at the heart of epigenetic research. PcG and TrxG proteins maintain gene activity by binding to Polycomb/Trithorax response elements (PRE/TREs). In flies, several hundred genes are regulated by PRE/TREs, but these elements are best characterised in the Hox genes of the Bithorax complex. During embryogenesis, the Hox PRE/TREs are transcribed in to non coding RNA. Recent studies have examined the role of this non coding transcription in regulation of PRE/TRE elements. Currently, there is strong disagreement as to whether the noncoding transcripts are important for silencing or activation of PRE/TRE associated genes. We have recently published a detailed review of this field, and have shown that, by taking developmental timing into account, some of these apparently conflicting results can be resolved. The project proposed here aims to study noncoding PRE/TRE transcription in the context of developmental timing in Drosophila melaongaster, and consists of two parts, namely 1) Quantitative characterisation of PRE/TRE non- coding RNAs during development. Using quantitative real time PCR, in situ hybridisation and transgenic reporter assays we aim to quantify the effect of PRE/TRE non-coding transcription on mRNA transcription at different developmental stages. 2) Purification of PRE/TRE non-coding RNAs and their binding partners at different developmental stages. We will use "StreptoTag" affinity purification of PRE/TRE non-coding RNAs in vitro and in transgenic flies, to identify binding partners at different developmental stages. The results of this work will have implications not only for understanding the role of non coding transcription in PRE/TRE regulation, but also for placing these events in their correct developmental context.

How do cells remember their identities over cell generations? What happens when they forget? Part of the answer lies in two groups of proteins called Polycomb and Trithorax, which work together to keep genomes under control. These crucial gatekeepers of cell identity must know when to keep things stable, and when to allow change. Derailment of these decisions can lead to cancer. We have discovered a fascinating molecular mechanism, based on RNA molecules, that helps to make these decisions. Switching the direction of transcription of these regulatory RNAs signals a switch for the Polycomb and Trithorax proteins from "stay as you are" to "all change".