Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Summary

Cleavage under targets and tagmentation (CUT&Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&Tag strategy for the profiling of histone modifications in plants.

Abstract

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Introduction

Transcription factor binding DNA sites and open chromatin associated with histone modification marks serve critical functional roles in regulating gene expression and are the major focuses of epigenetic research¹. Conventionally, chromatin immunoprecipitation assay (ChIP) coupled with deep sequencing (ChIP-seq) have been used for the genome-wide identification of specific chromatin histone modification or DNA targets with specific proteins, and is widely adopted in the field of epigenetics². Cleavage under targets and tagmentation (CUT&Tag) technology was originally developed by the Henikoff Lab to capture the protein-af....

Protocol

1. Prepare transposase and stock solutions (Day 1)

NOTE: In this part, the oligonucleotide adapters are complexed with Tn5 transposase to make active transposase.

1. Dilute primers (primer A, primer B, and primer C) to 100 µM concentration using the annealing buffer (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA) (refer to Table 1 for sequence information of primers and Table 2 for the recipes for working solutions). Store at -20 °C until use.
2. Set up the following two reactions in PCR tubes: Reaction 1 (adaptor AB), 10 µL of 100 µM primer A, 10 µL of 1....

Results

Figure 1 depicts the CUT&Tag workflow. Figure 2 shows the DAPI staining of the intact nuclei. The goal of the "nuclei isolation" step was to obtain the intact nuclei at a sufficient amount for the subsequent CUT&Tag reaction. Figure 3 shows the agarose gel electrophoresis of PCR products. The IgG negative control is required in parallel when setting up the experiment. Compared with the IgG control group, the bulk of the DNA .......

Discussion

Here, we have described CUT&Tag, a technology for generating DNA libraries at high resolution and exceptionally low background using a small number of cells with a simplified procedure compared to chromatin immunoprecipitation (ChIP). Our success with H3K4me3 profiling in cotton leaves suggests that CUT&Tag, which was first designed for animal cells, can also be used for plant cells. Both the Tris buffer system commonly used for ChIP assay⁸ and the HEPES buffer system used for animal CUT&#.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibody
Anti-H3K4me3	Millipore	07-473
Normal rabbit IgG	Millipore	12-370
Chemicals
Bovine Serum Albumin (BSA)			Make 10 mg/ml BSA stock solution. Store at -20°C
digitonin (~50% (TLC)	Sigma-Aldrich	D141	Make 5% digitonin stock solution (200 mg digitonin [~50% purity] to 2 mL DMSO). Note: Sterilize using a 0.22- micron filter. Store at -20°C
dimethyl sulfoxide (DMSO)
chloroform
ethylenediaminetetraacetic acid (EDTA)			Make 0.5 M EDTA (pH = 8.5) stock solution. Note: Making 100 mL of 0.5-M EDTA (pH = 8.5) requires approximately 2 g of sodium hydroxide (NaOH) pellets to adjust the pH
ethanol
GlycoBlue Coprecipitant (15 mg/mL)	Invitrogen	AM9516
magnesium chloride (MgCl2)			Make 1 M MgCl2 stock solution
protease inhibitor cocktail	Calbiochem	539133-1SET
potassium chloride (KCl)			Make 1 M KCl stock solution
phenol:chloroform:isoamyl alcohol (25:24:1,v:v:v)
sodium chloride (NaCl)			Make 5 M NaCl stock solution
spermidine			Make 2 M spermidine stock solution, store at -20°C.
sodium dodecyl sulfate (SDS)			Make 10% SDS stock solution. Note: Do not autoclave; sterilize using a 0.22-micron filter
Tris base			Make 1 M Tris (pH = 8.0) stock solution
Triton X-100			Make 20% Triton X-100 stock solution
Enzyme
Hyperactive pG-Tn5/pA-Tn5 transposase for CUT&Tag	Vazyme	S602/S603	Check the antibody affinity of the protein A or protein G that is fused with the Tn5. Generally speaking, proteins A and G have broad antibody affinity. However, protein A has a relatively higher affinity to rabbit antibodies and protein G has a relatively higher affinity to mouse antibodies. Select the appropriate transposase products that match your antibody.
TruePrep Amplify Enzyme	Vazyme	TD601
Equipment
Centrifuge	Eppendorf	5424R
PCR machine	Applied Biosystems	ABI9700
Orbital shaker	MIULAB	HS-25
NanoDrop One  spectrophotometer	Thermo Scientific	ND-ONE-W