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

Abstract

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 ~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.

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

“…Next, we tested whether APLF AD facilitates nucleosome formation. Using the nucleosome assembly and quantitation (NAQ) assay ( 29 ), we monitored nucleosome formation upon incubation of the octamer-mix with 207–base pair (bp) DNA fragments in the presence of APLF AD , followed by digestion with micrococcal nuclease (MNase). Addition of APLF AD to the histones caused increased protection of DNA fragments of 125 to 160 bp in a dose-dependent manner, consistent with nucleosome formation ( Fig.…”

DNA-mediated association of two histone-bound complexes of yeast Chromatin Assembly Factor-1 (CAF-1) drives tetrasome assembly in the wake of DNA replication

“…The procedure for measuring nucleosome assembly activity in vitro with the nucleosome assembly and quantification (NAQ) assay is described in more detail at Bio-protocol ( Mattiroli et al, 2018b ). The assembly assay was carried out as described above containing 200 nM of 207 bp DNA, 200 nM (H3-H4) 2 , 400 nM H2A-H2B and titration of CAF-1 (100-200-400-800 nM).…”

Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

“…Next, we tested whether APLF AD facilitates nucleosome formation. Using the nucleosome assembly and quantitation (NAQ) assay( 29 ), we monitored nucleosome formation upon incubation of the octamer-mix with 207 bp DNA fragments in the presence of APLF AD , followed by digestion with micrococcal nuclease (MNase). Addition of APLF AD to the histones caused increased protection of DNA fragments of 125-160 bp in a dose-dependent manner, consistent with nucleosome formation (Fig.…”

“…The gel was stained with SYBR™ Gold Nucleic Acid Gel Stain (Invitrogen) before visualization using a Molecular Imager Gel Doc XR System (Bio-Rad). Quantification of protected DNA fragments was performed using Bioanalyzer High sensitivity DNA chips, as previously described( 29 ). The significance of the increase of bands in the nucleosomal size range was tested using one-tailed Students’ t -test in MATLAB 2016 (The MathWorks, Inc.).…”

“…Nucleosome assembly reactions were carried out as in the chaperone assay described above, with the following modifications: the reactions were run in 25 mM Tris pH 7, 300 mM NaCl (reaction buffer) using a 165 bp DNA fragment, all concentrated protein stocks were diluted in reaction buffer before use, concentration of octamer-mix was used at 3 µM, and the incubation was performed using untagged wild-type APLF AD in a total reaction volume of 12 µL. After incubation of the octamer-mix with or without APLF AD and DNA in reaction buffer, 4 µL of reaction mixture was transferred to fresh Quantification of protected DNA fragments was performed using Bioanalyzer High sensitivity DNA chips, as previously described (29). The significance of the increase of bands in the nucleosomal size range was tested using one-tailed Students' t-test in MATLAB 2016 (The MathWorks, Inc.).…”