Single-molecule and population probing of chromatin structure using DNA methyltransferases

Abstract

Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein-DNA interactions. Here we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite genomic sequencing. The methyltransferase accessibility protocol (MAP) determines the relative frequency at which the enzyme accesses each of its target sites over an entire population of PCR amplified product. While MAP yields much quantitative information about relative accessibility of a region of chromatin, a complementary singlemolecule view of methyltransferase accessibility, termed MAP for individual templates (MAP-IT), is provided by analysis of cloned PCR products. Absolute rather than relative methylation frequencies in a region are obtained by summing the methylation status at each site over a cohort of clones. Moreover, as the integrity of individual molecules is maintained in MAP-IT, unique information about the distribution of multiple footprints along continuous regions is gleaned. In principle, the population MAP and single-molecule MAP-IT strategies can be used to analyze chromatin structure in a variety of model systems. Here we describe the application of MAP in living S. cerevisiae cells and MAP-IT in the analysis of a mammalian tumor suppressor gene in nuclei. This application of MAP-IT provides the first means to simultaneously determine CpG methylation of mammalian genes and their overlying chromatin structure in the same single DNA molecule.

Identification of DNMT1 Selective Antagonists Using a Novel Scintillation Proximity Assay

“…5-AZA (6 M) was used as a positive control. The DNA was deaminated as previously described (28). After bisulfite treatment, the RASSF1A promoter sequence was amplified from the deaminated DNA samples using forward primer 5Ј-GGGTTTTATAGTTTTTGTATTTAGGTTTTT-3Ј and reverse primer 5Ј-CAACTCAATAAACTCAAACTCCCCC-3Ј with M13 sites and a hot start DNA Taq Polymerase.…”

Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules

“…Pioneering work by Michael Kladde and colleagues has demonstrated the ability of methyltransferase-based footprinting to determine nucleosome positioning in yeast and mammalian cells (Xu et al 1998;Jessen et al 2004Jessen et al , 2006Kilgore et al 2007;Pardo et al 2010). Using next generation sequencing, we describe a genome-wide nucleosome footprinting method termed NOMe-seq (nucleosome occupancy and methylome sequencing), which uses a GpC methyltransferase (M.CviPI) (Xu et al 1998) to obtain nucleosome positioning information based on enzyme accessibility to GpC sites, while obtaining endogenous DNA methylation information at the same time from CpG sites.…”

Local Depletion of DNA Methylation Identifies a Repressive p53 Regulatory Region in the NEK2 Promoter

“…To gain insight into the chromatin structure associated with loss of p53 and how it relates to DNA methylation, we conducted MAPit, a single molecule, high resolution assay for chromatin accessibility (36). MAPit exploits the ability of the M.CviPI enzyme (37) to probe chromatin structure by methylating accessible GC sites that are not protected by histones (i.e.…”