Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

Summary

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

Abstract

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Introduction

Gene expression in mammalian cells is tightly and dynamically regulated, and transcription is one of the key steps. Gene transcription is mainly regulated by transcription factors and histones. A transcription factor is a protein that binds to specific DNA sequences and controls gene transcription. These factors either promote or inhibit the recruitment of RNA polymerase II (PolII), which initiates mRNA synthesis from genomic DNA as a template¹. Histone modifications such as acetylation and methylation of histone tail residues positively and negatively affect gene transcription by changing the chromatin structure². Since....

Protocol

1. Preparation of Reagents

1. Make 18.5% paraformaldehyde (PFA) solution. Add 0.925 g of PFA, 4.8 mL of water (use ultrapurified water throughout the protocol) and 35 µL of 1 M KOH in a 50 mL conical plastic tube. Close the cap tightly and heat the tube in a 400-600 mL glass beaker containing approximately 200 mL of water using a microwave. Remove the tube before the water starts boiling and vortex the tube to dissolve PFA. Allow PFA to cool to room temperature and store on ice.
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Discussion

Although sonication is commonly used to obtain fragmented chromatin, it is time-consuming and cumbersome to identify reproducible conditions. In this protocol, we used MNase digestion because enzyme digestion should be easier to identify reproducible conditions. A brief sonication step after MNase digestion (see step 2.2) was necessary to break the cell membrane and to release the digested chromatin. Therefore, the sonication power in our protocol should be as low as possible. We use the same sonication conditions for al.......

Materials

Name	Company	Catalog Number	Comments
0.5 M EDTA (pH 8.0)	Thermo Scientific	AM9010
2 M KCl	Thermo Scientific	AM9010
2X iQ SYBR Green supermix	Bio-Rad	1706862
5 M NaCl	Thermo Scientific	AM9010
50 bp DNA ladder	New England Biolabs	N3236S
Agarose	Research Product International	A20090
Branched octylphenoxy poly(ethyleneoxy)ethanol	Millipore Sigma	I8896	IGEPAL CA-630
ChIP-grade protein G magnetic beads	Cell signaling technology	9006S
Chromatin Immunoprecipitation (ChIP) Dilution Buffer	Millipore Sigma	20-153	Buffer composition: 0.01% SDS, 1.1% Triton X- 100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl.
Gel Loading Dye Purple (6X)	New England Biolabs	B7024S
Glycine	Bio-Rad	161-0724	Electropheresis grade
Glycogen	Millipore Sigma	G1767	19-22 mg/mL
Halt Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100x)	Thermo Scientific	78445
High Salt Immune Complex Wash Buffer	Millipore Sigma	20-155	Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl.
Histone H3K4me3 antibody (pAb)	Active Motif	39915
LiCl Immune Complex Wash Buffer	Millipore Sigma	20-156	Buffer composition: 0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1.
Low Salt Immune Complex Wash Buffer	Millipore Sigma	20-154	Buffer composition: 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl.
Magna GrIP Rack (8 well)	Millipore Sigma	20-400	Any kind of magnetic separation stands that are compatible with a 1.5 mL tube is fine.
Micrococcal nuclease	New England Biolabs	M0247S	comes with 10 x buffer (500 mM Tris-HCl, 50 mM CaCl2, pH 7.9 @ 25 °C) and 100 x BSA (10 mg/ml)
NaHCO3	JT Baker	3506-01
Normal rabbit IgG	Millipore Sigma	12-370
PIPES	Millipore Sigma	P6757
Proteinase K	Millipore Sigma	3115887001
Real-time PCR system	Bio-Rad	CFX96, C1000
RNA pol II CTD phospho Ser5 antibody	Active Motif	39749
SDS	Boehringer Mannheim	100155	Electropheresis grade
sodium acetate	Millipore Sigma	S5636
Sonicator equipped with a microtip probe	QSONICA	Q700	Any kind of sonicators that are compatible with a 1.5 mL tube is fine.
UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)	Thermo Scientific	15593031	pH 8.05