Single-molecule Imaging of SWI/SNF Chromatin Remodelers Reveal Multi-modal and Cancer-mutant-specific Landscape of DNA-binding Dynamics

Abstract

Chromatin remodeling, carried out by multi-subunit remodeler complexes, alleviates topological constraints posed by nucleosomes to regulate genome access. Although mutations in the SWI/SNF subfamily of remodelers are implicated in >20% of human cancers, how misregulation of their intranuclear dynamics could underpin cancer remains poorly understood. Combining single-molecule tracking and fluorescence correlation spectroscopy, we probed the live-cell dynamics of three key subunits common to all major human SWI/SNF remodeler complexes (BAF57, BAF155 and BRG1), revealing temporally distinct modes characteristic of free and chromatin-associated diffusion and chromatin-binding. Quantifying residence times of the fully assembled remodeler complex further resolved one transient and two stable binding fractions. Moreover, super-resolved density mapping of single-molecule binding using a newly devised strategy, termed STAR, revealed heterogeneous, nanoscale remodeler binding "hotspots" across the nucleoplasm where multiple binding events preferentially cluster, with particular enrichment of consecutive longer-lived stable binding. Importantly, we showed that the bromodomain plays a key role in modulating the enhancement of remodeler binding dynamics in a DNA-accessibility-dependent manner, but does not facilitate targeting to hyperacetylated chromatin. Finally, we compared the chromatin-binding dynamics of seven common BRG1 mutants implicated in various cancers across tumor types, and uncovered systematic alterations in residence time, binding frequency, fraction of time bound, targeting efficiency and number of binding "hotspots" unique to each point/truncation mutant. Collectively, our findings shed critical insight into the multi-modal landscape regulating the spatio-temporal organizational dynamics of SWI/SNF remodelers to selectively modulate genome accessibility, and could potentially serve as quantitative, mutant-specific signatures for cancers associated with remodeling misregulation. SIGNIFICANCE STATEMENTUsing two complementary approaches, we performed, to our knowledge, the first single-molecule quantification of live-cell dynamics of the fully assembled human SWI/SNF remodeler complex by correlating three key common subunits, and uncovered distinct roles of the bromodomain in modulating chromatin binding/targeting in a DNA-accessibility-dependent manner. Our super-resolved mapping of chromatin-binding also revealed intranuclear "hotspots" where remodelers bind repeatedly in nanometer-scale clusters, as a potential strategy to promote remodeling at these loci. By leveraging previously under-explored parameters, our findings revealed a broader and multi-modal landscape that regulates SWI/SNF-mediated remodeling dynamics in space and time, and established the biophysical basis for aberrant remodeler-chromatin interactions associated with seven mutants implicated in various cancers, which could potentially serve as their unique identifying yardsticks.