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

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Original research article

A brief version of this protocol appeared in:
eLIFE
Mar 2017

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.

Keywords: Nucleosome assembly, Histone chaperones, Chromatin, Micrococcal nuclease, Quantification

Background

The eukaryotic genome is organized into nucleosomes. Nucleosomes are modular and dynamic structures composed of an octameric core of histone proteins, wrapped by 147 bp of DNA (Luger et al., 1997). Nucleosome assembly begins with the deposition of one (H3-H4)2 tetramer onto DNA to form a tetrasome. Subsequent incorporation of H2A-H2B dimers forms a hexasome, and finally the nucleosome. Histones are highly positively charged small proteins that primarily exist as histone dimers at physiological salt concentrations. Because of their charges, histones require chaperones which shuttle them from the cytoplasm to the nucleus, and then aid their deposition onto, or removal from DNA (Gurard-Levin et al., 2014).

Histone chaperones are grouped in families of structurally unrelated proteins, all characterized by high binding affinity for histones (Laskey et al., 1978). In this way, they shield the histone charges and prevent their non-specific interaction with DNA and other cellular factors (Elsässer and D’Arcy, 2013). How histone chaperones participate in these different roles, and the degree of division of labor among histone chaperones remain largely unknown.

This is due to the lack of mechanistic knowledge of histone chaperone function, in particular as histone deposition factors onto DNA. It is therefore critical to develop assays that can measure histone deposition activity, i.e., nucleosome assembly, to be able to fully understand the functions of this class of proteins and the dynamics of histones in cells.

Because histone chaperones are not enzymes per se, it has been challenging to develop reliable assays to measure their histone deposition activity. Most existing assays have used native gel analysis to assay nucleosome assembly (Muthurajan et al., 2016). The readout in these assays is prone to misinterpretation, as histones and DNA can form a variety of complexes and native gel analysis is not sufficient to accurately distinguish between the different histone-DNA complexes.

We have developed a nucleosome assembly and quantitation (NAQ) assay that measures the amount of nucleosome particles formation in vitro. This assay relies on the activity of Micrococcal Nuclease (MNase), an enzyme that digests DNA that is not bound by histone proteins. The subsequent purification of the DNA fragments provides a footprint of the histone-DNA complexes in solution. The characteristic protection of ~150 bp DNA is a signature of intact nucleosome species and can be measured using a Bioanalyzer apparatus (Muthurajan et al., 2016). Data normalization to a loading control DNA allows us to compare and accurately quantify the amount of nucleosomes formed in different samples. The NAQ assay has been successfully used to measure the activity of the chromatin assembly factor 1 (CAF-1) in vitro (Mattiroli et al., 2017a and 2017b), and has the potential to reveal the differential contribution of histone chaperones to nucleosome assembly in cells. This will pave the way for the complete understanding of their functional roles in nucleosome dynamics.

Copyright Mattiroli et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mattiroli, F., Gu, Y. and Luger, K. (2018). Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay. Bio-protocol 8(3): e2714. DOI: 10.21769/BioProtoc.2714.
  2. Mattiroli, F., Gu, Y., Yadav, T., Balsbaugh, J. L., Harris, M. R., Findlay, E. S., Liu, Y., Radebaugh, C. A., Stargell, L. A., Ahn, N. G., Whitehouse, I. and Luger, K. (2017). 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. Elife 6.
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