Objective In the human genome, many G-rich sequences have the potential to form 4-stranded non-canonical secondary structures known as G-quadruplexes (G4s). G4 structures have been visualized in nuclei by immunofluorescence and mapped genome-wide to regulatory chromatin regions linked to elevated transcription using G4 chromatin immunoprecipitation and high-throughput sequencing (G4 ChIP-seq). G4s are implicated in gene regulation, DNA replication and genome instability, yet little is known about their protein interaction partners or role in regulating genome activity. We therefore propose to develop G4 Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins (G4 RIME) methodologies to elucidate the G4 interactome within a native chromatin context. ChIP-seq of protein candidates coupled with G4 ChIP-seq will verify binding at endogenous G4 structures. Using established biophysical and biochemical techniques, we will investigate binding modes, recruitment and effects on G4 dynamics. Consecutive chromatin immunoprecipitation steps using antibodies targeting characteristic chromatin marks as well as G4 ChIP-seq supported by bioinformatics analysis of genomic databases will identify proteins involved in transcriptional regulation. We will then test the consequence of stabilising G4s with small molecule ligands on the G4 interactome and how this is modulated during genomic stress. Cells genetically deficient in G4-interacting proteins will enable detailed exploration of G4 formation and transcriptional effects. This highly interdisciplinary project will provide a wide range of excellent training opportunities exploiting the Balasubramanian group’s unique position with laboratories at the Cancer Research UK Cambridge Institute (CRUK CI) and the Department of Chemistry. Overall, the studies will generate novel functional insights in the G4 biology that may ultimately lead to the identification of mechanisms and pathways for new anti-cancer agents. Fields of science natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsproteomicsnatural sciencesbiological sciencesgeneticsDNAmedical and health sciencesclinical medicineoncologynatural sciencesbiological sciencesgeneticsgenomesnatural sciencesbiological sciencesgeneticsepigenetics Programme(s) H2020-EU.1.3. - EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions Main Programme H2020-EU.1.3.2. - Nurturing excellence by means of cross-border and cross-sector mobility Topic(s) MSCA-IF-2016 - Individual Fellowships Call for proposal H2020-MSCA-IF-2016 See other projects for this call Funding Scheme MSCA-IF-EF-ST - Standard EF Coordinator THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE Net EU contribution € 183 454,80 Address TRINITY LANE THE OLD SCHOOLS CB2 1TN Cambridge United Kingdom See on map Region East of England East Anglia Cambridgeshire CC Activity type Higher or Secondary Education Establishments Links Contact the organisation Opens in new window Website Opens in new window Participation in EU R&I programmes Opens in new window HORIZON collaboration network Opens in new window Total cost € 183 454,80