Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay

Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a subset of histone chaperones promotes histone deposition in vivo. However, it has been challenging to characterize this activity, due to the lack of quantitative assays. Here we developed a quantitative nucleosome assembly (NAQ) assay to measure the amount of nucleosome formation in vitro. This assay relies on a Micrococcal nuclease (MNase) digestion step that yields DNA fragments protected by the deposited histone proteins. A subsequent run on the Bioanalyzer machine allows the accurate quantification of the fragments (length and amount), relative to a loading control. This allows us to measure nucleosome formation by following the signature DNA length of similar to 150 bp. This assay finally enables the characterization of the nucleosome assembly activity of different histone chaperones, a step forward in the understanding of the functional roles of these proteins in vivo.