In vitro Nucleosome Assembly Facilitated by the Histone Chaperone SRCAP.

In eukaryotic cells, nucleosomes are octameric structures composed of DNA and histones, and their tandem arrangement ultimately forms chromosomes. Nucleosome assembly is governed in vivo by a series of molecular mechanisms, particularly histone chaperones such as Swr1, which is indispensable for H2A.Z-nucleosome formation. The assessment of nucleosome assembly using micrococcal nuclease (MNase) digestion has been widely accepted. We describe a simple and reproducible protocol for analyzing the chaperone activity of SRCAP (Snf2-related CREBBP activator protein), the histone H2A.Z chaperone. This approach involves purifying a complex of H2A.Z, H2B, and SRCAP via fast protein liquid chromatography (FPLC) only, followed by its assembly with DNA-(H3.1-H4). The formation of nucleosomes is then determined in vitro using electrophoretic mobility shift assay (EMSA) and MNase digestion. This approach can also be adapted to evaluate whether other histone chaperones or regulatory factors promote nucleosome assembly activity. © 2025 Wiley Periodicals LLC. Basic Protocol: In vitro nucleosome assembly facilitated by the histone chaperone SRCAP.