Methylation‐Sensitive Single‐Molecule Analysis of Chromatin Structure
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Abstract
Methylation?sensitive single?molecule analysis of chromatin structure is a high?resolution method for studying nucleosome positioning. As described in this unit, this method allows for the analysis of the chromatin structure of unmethylated CpG islands or in vitro?remodeled nucleosomes by treatment with the CpG?specific DNA methyltransferase SssI (M.SssI), followed by bisulfite sequencing of individual progeny DNA molecules. Unlike nuclease?based approaches, this method allows each molecule to be viewed as an individual entity instead of an average population. Curr. Protoc. Mol. Biol. 89:21.17.1?21.17.16. © 2010 by John Wiley & Sons, Inc.
Keywords: chromatin remodeling; nucleosome positioning; methylation footprint
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Treatment of Nuclei with M.SssI
- Basic Protocol 2: Single Molecule Methylation‐Based Analysis of Nucleosomal DNA Accessibility Alterations Catalyzed by Chromatin‐Remodeling Proteins In Vitro
- Basic Protocol 3: Bisulfite Conversion of Unmethylated Cytosine Residues to Thymidine
- Alternate Protocol 1: Rapid Bisulfite Conversion
- Basic Protocol 4: PCR and Cloning to Obtain Single‐Molecule Resolution of Promoter Architecture
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
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Basic Protocol 1: Treatment of Nuclei with M.SssI
Materials
Basic Protocol 2: Single Molecule Methylation‐Based Analysis of Nucleosomal DNA Accessibility Alterations Catalyzed by Chromatin‐Remodeling Proteins In Vitro
Materials
Basic Protocol 3: Bisulfite Conversion of Unmethylated Cytosine Residues to Thymidine
Materials
Alternate Protocol 1: Rapid Bisulfite Conversion
Materials
Basic Protocol 4: PCR and Cloning to Obtain Single‐Molecule Resolution of Promoter Architecture
Materials
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Figure 21.17.1 Schematic of M.SssI footprinting. First chromatin is treated with M.SssI. This enzyme methylates all CpG sites in purified DNA, but it cannot methylate the same sites when they are assembled into nucleosomes or are associated with tight‐binding factors. Next, the DNA is purified, the sequences are bisulfite‐converted, and individual molecules are cloned. Patches that are inaccessible to M.SssI are revealed. Red circles indicate CpG sites that are methylated and white circles indicate sites that are unmethylated. View Image -
Figure 21.17.2 Schematic of the protocols in this unit. The procedure can start with , which describes nuclei purification and treatment of nuclei with M.SssI, or with , which discusses in vitro remodeling and treatment of the remodeled products with M.SssI. These two protocols are then followed by bisulfite conversion ( or the or commercially available kits) and PCR amplification and cloning of individual molecules (). View Image -
Figure 21.17.3 Images of cells before (A ) and after (B ) lysis of the cell membrane. (A) Microscopic image of cells prior to lysis of the cell membrane show round phase bright cells. (B) Microscopic image of cells after lysis of the cell membrane by incubation with NP‐40. After lysis, no phase bright cell membranes are apparent and nuclei are smaller with irregular outlines. View Image -
Figure 21.17.4 Schematic for the bisulfite conversion of DNA. During bisulfite treatment of DNA all unmethylated cytosines (C) are converted into uracils (U). All methylated cytosines remain unchanged. After the first PCR amplification cycle the Us are complemented with As (adenine) in the antisense strand and the methylated Cs are complemented with Gs (guanine). Then, after subsequent rounds of PCR, the Us in the sense strand become Ts (thymidine) and the methylated Cs in the sense strand remain Cs. Therefore, at the end of the whole process, unmethylated Cs become Ts and methylated Cs remain Cs. View Image -
Figure 21.17.5 Primer design for amplification of bisulfate‐converted DNA. First, convert all Cs that are not part of a CpG site to Ts in the genomic sequence. Then, design a forward primer that is complementary to the antisense strand. This primer should not contain any CpGs and should end in a converted C, if possible. The primer should be 18 to 30 bp and have a melting temperature above 50°C. Do the same for the reverse primer, but have it complement the sense strand. CpG sites are marked in red and primer is marked in blue. View Image -
Figure 21.17.6 Methylation of mononucleosomes with increasing amounts of M.SssI. Open circles represent CpG sites that were inaccessible to M.SssI and closed circles indicate CpG sites that were methylated by M.SssI. If too little M.SssI is used or if incubation times are too short, intermittent methylation patterns will be seen, as well as protection patterns which are >150‐bp per nucleosome (panels A and B ). If the experiment works correctly, then a protection pattern of 150 bp per nucleosome will be observed as patches of open CpG sites (panel C ). View Image
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Literature Cited
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| Internet Resources | |
| http://biq‐analyzer.bioinf.mpi‐sb.mpg.de/ | |
| This program aligns bisulfite‐converted sequences and removes poorly converted and duplicate sequences. |
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