Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Summary

Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Abstract

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer¹. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells^2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/α-ketoglutarate-dependent dioxygenases^{2, 3}. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development^{2, 5-10}. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC¹¹. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically¹².

Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by β-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.

Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Protocol

1. Genomic DNA Fragmentation

Fragment genomic DNA using sonication to a desired size range suited for the genome-wide sequencing platform. (We usually sonicate to ~300 bp.) Verify the size distribution of the fragmented genomic DNA on 1% agarose gel (Figure 1).

2. DNA Preparation

Determine the starting DNA amounts based on the abundance of 5-hmC in genomic DNA. Since 5-hmC levels vary significantly in different tissue types, starting DNA amounts depend on the 5-hmC levels of the samples. Please refer to Table 1 for examples.

3. β-....

Discussion

5-hydroxymethylcytosine (5-hmC) is a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. The method presented here is for determining the genome-wide distribution of 5-hmC. We use T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment, and sequencing of 5-hmC-containing DNA fragments in ma.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
5M Sodium chloride (NaCl)	Promega	V4221
0.5M pH8.0 Ethylenediaminetetraacetic acid (EDTA)	Promega	V4231
1M Trizma base (Tris) pH7.5	Invitrogen	15567-027)
HEPES 1M, pH7.4	Invitrogen	15630
Magnesium chloride (MgCl2) 1M	Ambion	AM9530G
Dimethyl sulfoxide (DMSO)	Sigma	D8418
Tween 20	Fisher BioReagents	BP337-100
DBCO-S-S-PEG3-Biotin conjugate	Click Chemistry Tools	A112P3
1,4-Dithiothreitol, ultrapure (DTT) Superpure	Invitrogen	15508-013
QIAquick Nucleotide Removal Kit	Qiagen	28304
Micro Bio-Spin 6 Column	Bio-Rad	732-6222
Dynabeads MyOne	Invitrogen	650-01
Streptavidin C1
Qiagen MinElute PCR Purification Kit	Qiagen	28004
UltraPure Agarose	Invitrogen	16500500
UDP-6-N3-glucose	Active Motif	55013
Enzyme
β-glucosyltransferase (β-GT)	New England Biolab	M0357
Equipment
Sonication device	Covaris
Desktop centrifuge
Water bath	Fisher Scientific
Gel running apparatus	Bio-Rad
NanoDrop1000	Thermo Scientific
Labquake Tube Shaker	Barnstead
Labquake Tube Shaker	Thermolyne
Magnetic Separation Stand	Promega	Z5342
Qubit 2.0 Fluorometer	Invitrogen
Reagent setup 10 X β-GT Reaction Buffer (500 mM HEPES pH 7.9, 250 mM MgCl2) 2 X Binding and washing (B&W) buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween 20).