Mechanism, regulation and functions of genome folding through loop extrusion by cohesin-nipbl
Since its discovery 150 years ago, DNA has been intensely studied. As a result, it is understood in some detail how genomic DNA is replicated, repaired and segregated, and entire genomes can be sequenced. However, how DNA is folded in chromosomes has remained a mystery. It has been proposed that this is achieved by a process called loop extrusion. This hypothesis proposes that SMC complexes, multi-subunit ATPases present in all kingdoms of life, reel genomic DNA into loops, thus bringing distant loci into proximity. This process is thought to have important structural and regulatory functions, such as the separation of bacterial genomes and the folding of eukaryotic DNA into loops and topologically associating domains (TADs), facilitating sister chromatid resolution, gene regulation and immunoglobulin gene recombination. We discovered recently that in vitro single molecules of the SMC complex cohesin bound to NIPBL (cohesin-NIPBL) indeed rapidly extrude DNA into loops at up to 2.1 kb/s. These results provided the first direct evidence for the hypothesis that cohesin generates chromatin loops and TADs by extrusion. However, the most fundamental questions relating to this process remain unanswered: What is the mechanism of loop extrusion? How is this process controlled? How does cohesin extrude chromatin fibers in cells, and how does this contribute to the processes mentioned above and to other functions? We are proposing to use our single-molecule loop extrusion assay and other techniques to address these questions. Answering these will be essential for understanding genome architecture and regulation, will provide insights into mechanisms of gene regulation and recombination, will reveal how SMC complexes function as molecular ‘motors’, and may provide insight into the etiology of human disorders caused by malfunctioning of cohesin and NIPBL, such as several widespread cancers, rare congenital ‘cohesinopathies’, and trisomies and spontaneous human abortions.