In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Summary

This protocol describes a battery of methods that includes analytical size-exclusion chromatography to study histone chaperone oligomerization and stability, pull-down assay to unravel histone chaperone-histone interactions, AUC to analyze the stoichiometry of the protein complexes, and histone chaperoning assay to functionally characterize a putative histone chaperone in vitro.

Abstract

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

Introduction

Nucleosomes composed of DNA and histone proteins form the structural unit of chromatin and regulate several critical cellular events. Nucleosomes are dynamically repositioned and remodeled to make DNA accessible to various processes such as replication, transcription, and translation^1,2. Histones that are highly basic either tend to interact with acidic proteins in the cellular milieu or undergo aggregation, thus leading to various cellular defects^3,4,5. A group of dedicated proteins called histone chaperones aid the ....

Protocol

1. Analytical size-exclusion chromatography to elucidate the oligomeric status and stability of histone chaperones

1. Analysis of the oligomeric status of histone chaperones
   1. Equilibrate a 24 mL analytical size-exclusion chromatography (SEC) column with 1.2 column volume (CV), i.e., 28.8 mL of degassed SEC buffer [20 mM of Tris-HCl (pH 7.5), 300 of mM NaCl, and 1 mM of β-mercaptoethanol (β-ME)] at 4 °C (see Table of Materials).
      NOTE: Column type, buffer composition, and buffer pH may be selected based on the protein of interest. The sample injection volume should not exceed 500 µL for a ....

Discussion

This work demonstrates and validates a comprehensive set of protocols for the biochemical and biophysical characterization of a putative histone chaperone. Herein, recombinantly expressed and purified NAP family proteins, AtNRP1 and AtNRP2, and the N-terminal nucleoplasmin domain of AtFKBP53 were used to demonstrate the protocols. The same set of experiments could very well be used to delineate the functional attributes of previously uncharacterized histone chaperones from any organism.

The fi.......

Materials

Name	Company	Catalog Number	Comments
Acetic acid (glacial)	Sigma	A6283
Acrylamide	MP Biomedicals	814326
Agarose	MP Biomedicals	193983
AKTA Pure 25M FPLC	Cytiva	29018226	Instrument for protein purification
Ammonium persulfate (APS)	Sigma	A3678
An-60Ti rotor	Beckman Coulter	361964	Rotor for analytical ultracentrifugation
Bovine serum albumin (BSA)	Sigma	A7030
Chloroform	Sigma	C2432
Coomassie brilliant blue R 250	Sigma	1.15444
Dialysis tubing (7 kDa cut-off)	Thermo Fisher	68700	For dialysing protein samples
Dithiothreitol (DTT)	MP Biomedicals	100597
DNA Loading Dye	New England Biolabs	B7025S
EDTA disodium salt	MP Biomedicals	194822
Electronic balance	Shimadzu	ATX224R
Ethanol	Sigma	E7023
Ethidium bromide (EtBr)	Sigma	E8751
Gel Doc System	Bio-Rad	12009077	For imaging gels after staining
Horizontal gel electrophoresis apparatus	Bio-Rad	1704405	Instrument for agarose gel electrophoresis
Hydrochloric acid (HCl)	Sigma	320331
Imidazole	MP Biomedicals	102033
Magnesium chloride (MgCl2)	Sigma	M8266
Micropipettes	Eppendorf	Z683779	For pipetting of micro-volumes
Mini-PROTEAN electrophoresis system	Bio-Rad	1658000	Instrument for SDS-PAGE
N,N-methylene-bis-acrylamide	MP Biomedicals	800172
Nano drop	Thermo Fisher	ND-2000	For measurement of protein and DNA concentrations
Ni-NTA agarose	Invitrogen	R901-15	Resin beads for pull-down assay
Optima AUC analytical ultracentrifuge	Beckman Coulter	B86437	Instrument for analytical ultracentrifugation
pH Meter	Mettler Toledo	MT30130863
Phenol	Sigma	P4557
Plasmid isolation kit	Qiagen	27104
Proteinase K	Sigma-Aldrich	1.07393
pUC19	Thermo Fisher	SD0061	Plasmid for supercoiling assay
Refrigerated high-speed centrifuge	Thermo Fisher	75002402
SDS-PAGE protein marker	Bio-Rad	1610317
SEDFIT			Free software program for analytical ultracentrifugation data analysis
SEDNTERP			Free software program to estimate viscosity and density of buffer and partial specific volume of a protein
SigmaPrep Spin Columns	Sigma	SC1000	For pull-down assay
Sodium acetate	Sigma	S2889
Sodium chloride (NaCl)	Merck	S9888
Sodium dodecyl sulfate (SDS)	MP Biomedicals	102918
Superdex 200 Increase 10/300 GL	Cytiva	28990944	Column for analytical size-exclusion chromatography
Superdex 75 Increase 10/300 GL	Cytiva	29148721	Column for analytical size-exclusion chromatography
TEMED	Sigma	1.10732
Topoisomerase I	Inspiralis	WGT102	Enzyme used in plasmid supercoiling assay
Tris base	Merck	T1503
Tween-20	Sigma	P1379
Urea	MP Biomedicals	191450
Water bath	Nüve	NB 5	For heat treatment of protein samples
β-mercaptoethanol (β-ME)	Sigma	M6250