Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Identification of novel genome organising factors using proximity labeling (#335)

Habib Francis 1 2 3 , Mehdi Sharifi Tabar 1 2 3 , Cynthia Metierre 1 2 , John Rasko 2 3 4 , Chuck Bailey 1 2 3
  1. Cancer and Gene Regulation Laboratory Centenary Institute, The University of Sydney, Camperdown, NSW, 2050, Australia
  2. Gene and Stem Cell Therapy Program Centenary Institute, The University of Sydney, Camperdown, NSW, 2050, Australia
  3. Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, 2006, Australia
  4. Cell and Molecular Therapies, Royal Prince Alfred Hospital, Camperdown, NSW, 2050, Australia

The three-dimensional (3D) configuration of the genome is complex and dynamic.  CCCTC-binding factor (CTCF) plays a central role in this process and mediates its function through interaction with numerous other chromatin binding proteins. Using traditional approaches such as Y2H and AP-MS, a few direct protein interaction partners of CTCF have been identified and their contribution to the 3D structure of the genome has been defined. However, due to several inherent limitations, these techniques cannot identify all protein interactions especially those which occur in a spatial context or temporal manner. Hence, we implemented proximity-dependent biotin identification (BioID), an alternative approach to circumvent these limitations. We have mapped 213 highly confident protein interactions for CTCF, of which 78 were significantly enriched with the N-terminus and 135 with the C-terminus of CTCF. Using biochemical approaches, we have further confirmed the direct interaction of several of these novel interaction partners. Now we are set to investigate the role of these direct interactors in the 3D genome organisation using genomic and proteomics approaches. This study gives fundamental understanding of chromatin organisation within cells and spatiotemporal insights into chromatin dynamics.