Investigation of the role of GTGT motifs in fly and mouse Polycomb/Trithorax Response Elements and their non- coding RNAs

• Applicant: Professorin Dr. Leonie Ringrose
• Subject Area: General Genetics and Functional Genome Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 404308913

Polycomb group (PcG) and Trithorax group (TrxG) proteins are epigenetic gene regulatory proteins that are essential to life. These proteins work antagonistically on several hundred target genes, and maintain active (TrxG) or silent (PcG) expression states. These proteins bind to their target loci via cis- regulatory DNA elements termed Polycomb/Trithorax Response Elements (PRE/TREs). Aberrant expression of PcG and TrxG proteins or mutation of PRE/TREs can cause developmental defects, metabolic disease and cancer. The PcG and TrxG proteins are highly conserved between flies and vertebrates, but the DNA sequences of PRE/TREs show little apparent similarity between species. Fly PRE/TREs are relatively well- characterised. In contrast, in mammals, the DNA sequence "rules" of these elements remain elusive and controversial. There is currently no known DNA sequence motif that has been shown to have an analogous function in both fly and vertebrate PRE/TREs. Many fly and vertebrate PRE/TREs are transcribed into developmentally regulated non-coding (nc) RNAs, many of which have important roles in modulating PcG and TrxG function, and several of which have been implicated in disease. However their mechanisms of action are poorly understood. Understanding how PcG and TrxG proteins interact with PRE/TREs and their ncRNAs in molecular terms will be profoundly important for our understanding of human health and disease. The aim of this proposal is to determine whether the GTGT motif, which is highly enriched in vertebrate and fly PRE/TREs and their ncRNAs, plays an analogous role in both species, and whether it modulates levels or properties of ncRNAs. I present extensive preliminary data supporting a role for GTGT motifs at PRE/TREs and documenting the establishment of all necessary tools for the project. We will now systematically investigate the role of GTGT motifs in selected fly and mouse PRE/TREs using reporter assays. PRE/TRE sequence variants are integrated at an identical genomic location in each species, enabling quantitative comparisons at different stages of development and differentiation.This is the first study in which vertebrate PRE/TRE reporters integrated at the same genomic location are used to study ncRNA function at PRE/TREs. Furthermore our precise engineering of an endogenous Drosophila locus to evaluate PRE/TRE function is the first of its kind in the fly field. As such both the mouse and fly projects will break substantial new ground. Finally, our assays are not limited to the study of GTGT motifs: the techniques described here can be used to address any PRE/TRE sequence variant in a highly quantitative manner in both mouse and fly. Thus this project has the potential to deliver comprehensive quantitative insights into the relationship between nucleic acid sequence features and function of PRE/TREs in vertebrates and flies.

• DFG Programme: Research Grants