The cohesin complex is a major factor driving the 3-D organization of mammalian genomes at the scale of tens to kilobases to megabases. Recent single-molecule experiments have shown that it can extrude loops of DNA, which are an organizing principle of genome architecture. Together with CTCF, a DNA-binding protein that stalls cohesin’s translocation and defines loop boundaries, and several regulators of cohesin that promote loading, such as NIPBL, or release from chromatin, such as WAPL, cohesin defines interaction domains in chromosomes that affect patterns of gene expression during development and can lead to developmental diseases or cancer when disrupted. Although loop extrusion on naked DNA has been studied, cohesin in cells must navigate nucleosome-packed chromatin fibers that restrict access to binding sites on DNA, self-organize into compartments of similar epigenetic state that are independent of and compete with loop domains, potentially regulate DNA supercoiling, and are decorated with chromatin-associated RNAs. How cohesin and CTCF interact with chromatin in cells is the next frontier in understanding 3-D genome organization. Progress in this arena will require a multi-scale approach, with methods that probe both nucleosome-scale and megabase-scale features. I propose experiments to probe (1) how loop extrusion by cohesin perturbs the local structure of the chromatin fiber; (2) how the local structure of the chromatin fiber, modulated by depletion of linker histones and destabilization of nucleosomes, regulates cohesin’s ability to load and extrude loops; (3) how changes in the balance of supercoiling due to excess cohesin looping affect local nucleosome-nucleosome interactions; and (4) the chromatin-associated RNA interactome of CTCF at RNA-dependent and RNA-independent loop boundaries. To dissect the specific effects of cohesin, its regulators and the chromatin fiber, we will use a combination of stable protein depletion, acute degradation, and pharmacological inhibition in human and mouse cell lines. We will read out changes in chromatin fiber structure and chromatin-associated RNAs using RICC-seq, a method I recently developed for measuring DNA-DNA contacts at sub-nucleosome resolution in intact cells, and using novel technology development to probe the chromatin-associated RNA interactome of specific proteins. These methods will be combined with more established epigenome and transcription profiling tools and with coarse- grained simulations to develop and test multi-scale models for the interaction of loop extrusion machinery with the chromatin fiber. I anticipate that the results of these experiments will shed new light on how loop extrusion and chromatin’s self-association interact in specific contexts, which models for cohesin’s engagement with DNA are relevant to loop extrusion, how supercoiling is disseminated across chromosomes, and how the local molecular context defines loop boundaries. This knowledge may reveal new strategies for compensating transcriptional dysregulation due to mutations in cohesin or its regulators using targetable factors that regulate the chromatin fiber, cohesin’s native substrate.

Project Narrative Recent advances in DNA sequencing-based methods have begun to reveal the molecular factors that explain how the three-dimensional organization of the human genome is disrupted in both cancer and developmental diseases. In this proposal, we study how the cohesin complex, one such factor mutated in cancer and developmental diseases called cohesinopathies, interacts with DNA as it is packaged within cells – wrapped around histone proteins and associated with RNA molecules. We hope that our results will inform how the cohesin complex interacts with other factors that shape the packaging of DNA into chromatin, revealing new strategies for treating dysregulation of genome organization in cancer and cohesinopathies.