A system for CBAF reconstitution reveals roles for BAF47 domains and BCL7 in nucleosome ejection

Abstract

Canonical BAF (CBAF) is an essential 12-protein chromatin-remodeling complex that slides and/or ejects nucleosomes using the alternative catalytic ATP-dependent DNA translocases BRG1 or BRM. Currently, the regulation of BRG1/BRM activity and nucleosome ejection remain incompletely understood. To address this, we developed a system for full CBAF reconstitution and purification, and created a novel nucleosome ejection assay. ARID1A and DPF2 were dispensable for assembly and chromatin remodeling activity, contrasting with prior work. The actin-related protein BAF53A and {beta}-actin components interacted and enhanced DNA translocation, and were required for BCL7A incorporation, which potentiated ejection. BAF47 also regulated ejection, utilizing two stimulatory domains and an autoinhibitory domain. Finally, we provide evidence for direct nucleosome ejection at low nucleosome density on closed circular arrays. Taken together, we provide powerful new tools for CBAF mechanistic investigation and reveal new roles for several CBAF components.