Simultaneous Single‐Molecule Mapping of Protein‐DNA Interactions and DNA Methylation by MAPit
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Abstract
Sites of protein binding to DNA are inferred from footprints or spans of protection against a probing reagent. In most protocols, sites of accessibility to a probe are detected by mapping breaks in DNA strands. As discussed in this unit, such methods obscure molecular heterogeneity by averaging cuts at a given site over all DNA strands in a sample population. The DNA methyltransferase accessibility protocol for individual templates (MAPit), an alternative method described in this unit, localizes protein?DNA interactions by probing with cytosine?modifying DNA methyltransferases followed by bisulfite sequencing. Sequencing individual DNA products after amplification of bisulfite?converted sequences permits assignment of the methylation status of every enzyme target site along a single DNA strand. Use of the GC?methylating enzyme M.CviPI allows simultaneous mapping of chromatin accessibility and endogenous CpG methylation. MAPit is therefore the only footprinting method that can detect subpopulations of molecules with distinct patterns of protein binding or chromatin architecture and correlate them directly with the occurrence of endogenous methylation. Additional advantages of MAPit methylation footprinting as well as considerations for experimental design and potential sources of error are discussed. Curr. Protoc. Mol. Biol. 95:21.22.1?21.22.18. © 2011 by John Wiley & Sons, Inc.
Keywords: chromatin; nucleosomes; DNA methylation; DNA methyltransferases; footprinting; single?molecule analysis
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Probing Mammalian Nuclear Chromatin with DNMTs
- Support Protocol 1: Verification of Methylation of DNA by M.CviPI
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
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Basic Protocol 1: Probing Mammalian Nuclear Chromatin with DNMTs
Materials
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Figure 21.22.1 MAPit overview for mapping m5 CG and chromatin accessibility in mammalian nuclei. Nuclei are isolated from cultured cells of interest grown under desired experimental conditions. Isolated nuclei are then probed with M.CviPI, a DNA methyltransferase that methylates cytosines at accessible GC sites, i.e., nucleosome‐free or unbound by non‐histone proteins. After purification of DNA, cytosines will either be unmethylated (unfilled symbols), modified at CG by endogenous DNMTs (m5 CG; black‐filled circles), or modified at GC by M.CviPI probe (G‐m5 C; red‐filled inverted triangles). Purified DNA is then subjected to chemical conversion by bisulfite ion in which unmethylated C in denatured DNA is deaminated to U, whereas methylated C is not. During subsequent PCR of bisulfite‐treated DNA, U is converted through replicative complementarity to T and m5 C to C. Sequencing after cloning individual DNA molecules from the bulk PCR product provides a single‐molecule readout of the methylation status at C in each CG and GC site. This output is representative of the combined endogenous methylation and chromatin accessibility at a single locus within the original nuclei. Sequence alignment and visual representation of methylation status are efficiently obtained computationally, e.g., with MethylViewer (Pardo et al., ). In the shown view in the bottom panel, each horizontal line indicates a single sequenced DNA molecule decorated with symbols representing the methylation status of each CG, GC, and overlapping CGC as defined (key at bottom right). View Image -
Figure 21.22.2 MAPit analysis of the TSS region of human SIM2 in MCF10A cells. Nuclei (106 ) were probed with 10 U of wild‐type M.CviPI for 30 min at 37°C. SIM2 is expressed in MCF‐10A cells. Each horizontal line represents 524 bp of chromatin from a single cell. Circles represent CG sites and triangles represent GC sites. Black filled circles and red filled triangles, represent m5 CG and G‐m5 C, respectively. GCG sites are represented by both gray triangles and circles. GCG site methylation cannot rigorously be discriminated as being placed by endogenous or exogenous DNMT, but this can often be inferred from context (see Anticipated Results for discussion). Blue highlighted areas represent 147 bp of contiguous M.CviPI DNA footprint. Note that about half of the alleles have relatively high levels of endogenous methylation (black filled circles). Based on molecules from cells not treated with M.CviPI, it can be inferred that gray GCG sites in these densely methylated MCF‐10A alleles were likely methylated by endogenous DNMTs. The other half of the molecules is almost free of endogenous methylation but shows an accessible, nucleosome‐length region high in M.CviPI methylation (red triangles) highlighted in red. No other technique can determine this bipartite pattern of chromosome structure. The high accessibility to M.CviPI is probably due to histone depletion near the TSS. In contrast, this putative histone‐free region is flanked by protected spans of median length ∼150 bp. Numbers at the right of each molecule depiction indicate the percentage of C conversion to T in non‐CG and non‐GC sequences. Nucleotides that failed to convert or reverted to a C during PCR amplification are indicated by vertical blue tick marks. View Image
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| Key References | |
| Kladde et al., 1996. See above. | |
| First demonstration of the utility of M.SssI for detection of nucleosome position and transcription factor binding. | |
| Fatemi et al., 2005. See above. | |
| First demonstrations of the use of C‐5 DNMTs in single‐molecule footprinting. | |
| Jessen et al., 2006. See above. | |
| First documented use of MAPit with M.CviPI, yielding simultaneous detection of chromatin accessibility and endogenous m5CG at the single‐molecule level. | |
| Kilgore et al., 2007. See above. | |
| Development of MethylViewer program for rapid analysis of MAPit datasets. | |
| Pardo et al., 2010. See above. | |
| Internet Resources | |
| http://dna.leeds.ac.uk/methylviewer/ | |
| Site for download of MethylViewer program and detailed usage instructions. |
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