The ultra-sensitive Nodewalk technique identifies virtual and real chromatin networks regulating MYC in 3D
Noriyuki Sumida1*, Emmanouil G Sifakis1, Barbara A Scholz1, Alejandro Fernandez-Woodbridge4, Narsis A Kiani2, David Gomez-Cabrero2, J Peter Svensson3, Jesper Tegner2, Anita Göndör1 and Rolf Ohlsson1*
1Department of Microbiology, Tumor and Cell Biology, Nobels väg 16, Karolinska Institutet, SE-171 77 Stockholm, Sweden; 2Unit of Computational Medicine, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, L8:05, SE-171 76, Stockholm, Sweden; 3Department of Biosciences and Nutrition, Novum, Karolinska Institutet, S-141 83 Huddinge, Sweden; 4Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden
The link between stochastic nuclear processes, such as transcription, and their relationship to dynamic higher order chromatin states is not well understood due to poor sensitivity/resolution of currently available techniques. Here we describe the versatile Nodewalk technique, which includes isothermal, linear amplification and a >400,000-fold single-step enrichment of interacting sequences, to display a >500-fold increase in sensitivity over competing methods. Nodewalk quantitatively identifies a central core of inter-connected enhancer hubs impinging on MYC in colon cancer cells. While this data suggests the existence of complex enhancer hubs to simultaneously drive MYC transcription, analyses of aliquots of 300 cells revealed stochastic variation in the enhancer network and mutually exclusive patterns of enhancer-MYC interactions. Analysis of variations in 3D chromatin structures in small cell populations uncovered, moreover, dynamic changes in the configuration of the boundary region between topological-associated domains containing MYC to demonstrate the plasticity of its transcriptional regulation in 3D.