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

TY - JOUR

T1 - Measuring Nucleosome Assembly Activity with the Nucleosome Assembly and Quantification (NAQ) Assay

AU - Mattiroli, Francesca

AU - Gu, Yajie

AU - Luger, Karolin

PY - 2018/2/5

Y1 - 2018/2/5

N2 - 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.

AB - 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.

U2 - 10.21769/BioProtoc.2714

DO - 10.21769/BioProtoc.2714

M3 - Article

C2 - 29516027

SN - 2331-8325

VL - 8

JO - Bio-Protocol

JF - Bio-Protocol

IS - 3

ER -