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 m5C 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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Kladde, M. P., Xu, M., and Simpson, R. T. (1996) Direct study of DNA-protein interactions in repressed and active chromatin in living cells. EMBO J. 15, 6290–6300.
Xu, M., Simpson, R. T., and Kladde, M. P. (1998) Gal4p-mediated chromatin remodeling depends on binding site position in nucleosomes but does not require DNA replication. Mol. Cell. Biol. 18, 1201–1212.
Jessen, W. J., Dhasarathy, A., Hoose, S. A., Carvin, C. D., Risinger, A. L., and Kladde, M. P. (2004) Mapping chromatin structure in vivo using DNA methyltransferases. Methods 33, 68–80.
Jessen, W. J., Hoose, S. A., Kilgore, J. A., and Kladde, M. P. (2006) Active PHO5 chromatin encompasses variable number of nucleosomes at individual promoters. Nat. Struct. Mol. Biol. 13, 256–263.
Hoose, S. A. and Kladde, M. P. (2006) DNA methyltransferase probing of DNA-protein interactions, in Methods Mol. Biol. "Gene Mapping, Discovery, and Expression: Methods and Protocols." (Bina, M., ed.), Humana, Totowa, NJ, pp. 225–259.
Kilgore, J. A., Hoose, S. A., Gustafson, T. L., Porter, W., and Kladde, M. P. (2007) Single-molecule and population probing of chromatin structure using DNA methyltransferases. Methods 41, 320–332.
Duan, R., Porter, W., Samudio, I., Vyhlidal, C., Kladde, M., and Safe, S. (1999) Transcriptional activation of c-fos protooncogene by 17-β-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition. Mol. Endocrinol. 13, 1511–1521.
Dong, L., Wang, W., Wang, F., Stoner, M., Reed, J. C., Harigai, M., Samudio, I., Kladde, M. P., Vyhlidal, C., and Safe, S. (1999) Mechanisms of transcriptional activation of bcl-2 gene expression by 17β-estradiol in breast cancer cells. J. Biol. Chem. 274, 32099–32107.
Vyhlidal, C., Samudio, I., Kladde, M. P., and Safe, S. (2000) Transcriptional activation of transforming growth factor α by estradiol: requirement for both a GC-rich site and an estrogen response element half-site. J. Mol. Endocrinol. 24, 329–338.
Samudio, I., Vyhlidal, C., Wang, F., Stoner, M., Chen, I., Kladde, M., Barhoumi, R., Burghardt, R., and Safe, S. (2001). Transcriptional activation of deoxyribonucleic acid polymerase α gene expression in MCF-7 cells by 17β-estradiol. Endocrinol 142, 1000–1008.
Fatemi, M., Pao, M. M., Jeong, S., Gal-Yam, E. N., Egger, G., Weisenberger, D. J., and Jones, P. A. (2005) Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level. Nucleic Acids Res. 33, e176.
Gal-Yam, E. N., Jeong, S., Tanay, A., Egger, G., Lee, A. S., and Jones, P. A. (2006) Constitutive nucleosome depletion and ordered factor assembly at the GRP78 promoter revealed by single molecule footprinting. PLoS Genet. 2, e160.
Renbaum, P., Abrahamove, D., Fainsod, A., Wilson, G., Rottem, S., and Razin, A. (1990) Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA from Spiroplasma sp. strain MQ-1 (M.SssI). Nucleic Acids Res. 18, 1145–1152.
Xu, M., Kladde, M. P., Van Etten, J. L., and Simpson, R. T. (1998) Cloning, characterization and expression of the gene coding for a cytosine-5-DNA methyltransferase recognizing GpC sites. Nucleic Acids Res. 26, 3961–3966.
Chan, S. H., Zhu, Z., Van Etten, J. L., and Xu, S. Y. (2004) Cloning of CviPII nicking and modification system from Chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification. Nucleic Acids Res. 32, 6187–6199.
Frommer, M., MacDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W., Molloy, P. L., and Paul, C. L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89, 1827–1831.
Clark, S. J., Harrison, J., Paul, C. L., and Frommer, M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990–2997.
Polach, K. J. and Widom, J. (1995) Mechanism of protein access to specific DNA sequences in chromatin: a dynamic equilibrium model for gene regulation. J. Mol. Biol. 254, 130–149.
Polach, K. J. and Widom, J. (1996) A model for the cooperative binding of eukaryotic regulatory proteins to nucleosomal target sites. J. Mol. Biol. 258, 800–812.
Li, G. and Widom, J. (2004) Nucleosomes facilitate their own invasion. Nat. Struct. Mol. Biol. 11, 763–769.
Li, G., Levitus, M., Bustamante, C., and Widom, J. (2005) Rapid spontaneous accessibility of nucleosomal DNA. Nat. Struct. Mol. Biol. 12, 763–769.
Kladde, M. P. and Simpson, R. T. (1994) Positioned nucleosomes inhibit Dam methylation in vivo. Proc. Natl. Acad. Sci. USA 91, 1361–1365.
Colella, S., Shen, L., Baggerly, K. A., Issa, J. P., and Krahe, R. (2003) Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. Biotechniques 35, 146–150.
Tost, J., Dunker, J., and Gut, I. G. (2003) Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing. Biotechniques 35, 152–156.
Biggar, S. R. and Crabtree, G. R. (2001) Cell signaling can direct either binary or graded transcriptional responses. EMBO J. 20, 3167–3176.
Warnecke, P. M., Stirzaker, C., Song, J., Grunau, C., Melki, J. R., and Clark, S. J. (2002) Identification and resolution of artifacts in bisulfite sequencing. Methods 27:101–107.
Caserta, M., Zacharias, W., Nwankwo, D., Wilson, G. G., and Wells, R. D. (1987) Cloning, sequencing, in vivo promoter mapping, and expression in Escherichia coli of the gene for the HhaI methyltransferase. J. Biol. Chem. 262, 4770–4777.
Carvin, C. D., Dhasarathy, A., Friesenhahn, L. B., Jessen, W. J., and Kladde, M. P. (2003) Targeted cytosine methylation for in vivo detection of protein-DNA interactions. Proc. Natl. Acad. Sci. USA 100, 7743–7748.
Hayatsu, H. (1976) Bisulfite modification of nucleic acids and their constituents. Prog. Nucleic Acids Res. 16, 75–124.
Kladde, M. P. and Simpson, R. T. (1998) Rapid detection of functional expression of C-5-DNA methyltransferases in yeast. Nucleic Acids Res. 26, 1354–1355.
Dean, F. B., Nelson, J. R., Giesler, T. L., and Lasken, R. S. (2001) Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Research 11, 1095–1099.
Acknowledgments
We are grateful to Randy Morse for the plasmid containing the estrogen-inducible activator, Steve Hanes for the plasmid containing the minimal GAL1 promoter with lexO sites, and the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for high-throughput sequencing. This work was supported by Public Health Service grant CA095525 from the National Cancer Institute to M.P.K. and in part by a Texas Higher Education Coordinating Board ARP award to M.P.K.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Pardo, C., Hoose, S.A., Pondugula, S., Kladde, M.P. (2009). DNA Methyltransferase Probing of Chromatin Structure Within Populations and on Single Molecules. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 523. Humana Press. https://doi.org/10.1007/978-1-59745-190-1_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-190-1_4
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-873-7
Online ISBN: 978-1-59745-190-1
eBook Packages: Springer Protocols