DNA Methyltransferase Probing of Chromatin Structure Within Populations and on Single Molecules

Abstract

Non-invasive methods for mapping chromatin structure are necessary for creating an accurate view of genome function and dynamics in vivo. Ectopic induction of cytosine-5 DNA methyltransferases (C5 MTases) in Saccharomyces cerevisiae is a powerful technique for probing chromatin structure with minimal disruption to yeast physiology. Accessibility of MTases to their cognate sites is impaired based on the strength and span of the protein-DNA interaction to be probed. Methylated cytosines that resist chemical deamination are detected positively by the PCR-based technique of bisulfite genomic sequencing. PCR amplicons can be sequenced directly yielding an average m(5)C frequency or accessibility of each target site within the population, a technique termed methyltransferase accessibility protocol (MAP). More recently, the sequencing of cloned molecules in MAP for individual templates (MAPit) enables assignment of the methylation status of each target site along a continuous DNA strand from a single cell. The unique capability to score methylation at multiple sites in single molecules permits detection of inherent structural variability in chromatin. Here, MAPit analysis of the repressed and induced PHO5 promoter of budding yeast, using a C5 MTase with dinucleotide recognition specificity, reveals considerable cell-to-cell heterogeneity in chromatin structure. Substantial variation is observed in the extent to which the MTase gains entry to each of the nucleosomes positioned at PHO5, suggesting differences in their intrinsic thermodynamic stability in vivo. MAPit should be readily adaptable to the analysis of chromatin structure and non-histone protein-DNA interactions in a variety of model systems.

WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix

“…To simultaneously map chromatin structure and m 5 CpG of a larger region of the WIF1 promoter (see Figure 1A), we performed single-molecule MAPit methylation footprinting (41,42) in the C33A, CaSki and SiHa cell lines (Figure 3). In this experiment, nuclei were probed with and without M.CviPI, a DNA methyltransferase that methylates only GC dinucleotides (43).…”

Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma

“…Our laboratory has developed a high-resolution footprinting technique, termed MAPit (methyltransferase accessibility protocol for individual templates). MAPit exploits exogenous addition of DNA methyltransferases (DNMTs), to probe accessibility of DNA in chromatin (Kladde et al 1996;Xu et al 1998b;Kilgore et al 2007;Pardo et al 2009). This technique has been used to simultaneously map DNA methylation and nucleosome positions on single molecules in many gene-specific studies (Kilgore et al 2007;Wolff et al 2010;Delmas et al 2011;Pardo et al 2011a;You et al 2011;Yang et al 2012;Darst et al 2013), and more recently, genome wide (Kelly et al 2012).…”

High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation

“…1C). The MAPit assay is ideally suited to examine the dynamics of protein-DNA interactions during cellular differentiation because individual templates are analyzed, yielding information about the fractional occupancy at specific transcription factor binding sites at specific time points (35). We isolated nuclei from MEL cells at different time points after induction of differentiation with DMSO.…”

“…The MAPit procedure allows detection of protein occupancy within cell nuclei on individual DNA templates and thus yields information with respect to the dynamics of protein-DNA interactions within a cell population (35). The MAPit data demonstrate that a tandem MARE within LCR HS2 is occupied by MARE-binding proteins in a high percentage of cells across the population of undifferentiated and differentiated MEL cells.…”

“…In this study, we analyzed protein-DNA interaction patterns in the murine ␤-globin gene locus during differentiation of murine erythroleukemia (MEL) cells using the methyltransferase accessibility protocol for individual templates (MAPit) (35). The MAPit procedure allows detection of protein occupancy within cell nuclei on individual DNA templates and thus yields information with respect to the dynamics of protein-DNA interactions within a cell population (35).…”