CpNpG Methylation in Mammalian Cells Enclosed are exciting results by Clark et al., regarding sequence specificity of DNA methylation in mammalian cells in Nature Genetics 10: 20-27 (1995). Moshe Szyf Authors: Susan J. Clark 1,2 ,*, Janet Harrison 1 , Marianne Frommer 3 Addresses: 1 Kanematsu Laboratories, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia, Phone: (612) 886-4972 2 *CSIRO Division of Biomolecular Engineering, Sydney Laboratory, PO Box 184, North Ryde, NSW 2113, Australia, Phone: (612) 886-4972 3 School of Biological Sciences, A12, University of Sydney, NSW 2006, Australia, Phone: (612) 692-2298 Corresponding Author: *Susan J. Clark, Fax: (612) 886-4818 E-mail: clark@pelican.dbe.csiro.au Phone: (612) 886-4972 SUMMARY In vertebrate DNA, 3% to 5% of cytosine residues are present as 5-methylcytosine, and it is generally accepted that essentially all of this methylation occurs at cytosines which are contained in the symmetrical dinucleotide CpG. In this report we demonstrate, using bisulphite genomic sequencing, that the methylation machinery of mammalian cells is capable of both maintenance and de novo methylation at CpNpG sites. The existence of inherited CpNpG methylation in mammalian cells has important implications in gene regulation and in the aetiology of disease. INTRODUCTION Methylation of DNA is used by a wide range of prokaryotes and eukaryotes for the marking or tagging of DNA sequences, as part of a range of epigenetic processes, including restriction/modification systems, DNA replication and repair, and regulation of gene expression. Methylation of cytosine at carbon-5 of the pyrimidine ring occurs subsequent to DNA synthesis by the activity of DNA methyltransferase. Information about the distribution of methylated cytosines in CpG dinucleotides has come almost exclusively from Southern hybridisations on genomic DNA digested with restriction enzymes that are sensitive to 5-methylcytosine or, more recently, from genomic sequencing which can determine the level of methylation of individual cytosines in a population of molecules. However, these methods are not sufficiently sensitive to detect low levels of methylation. Bisulphite genomic sequencing is a new and sensitive method which gives a positive signal for methylated cytosines (1). We have used this powerful technique to examine the distribution of methylated cytosines at sites other than CpG dinucleotides and, in particular, to test for the methylation of CpNpG sites in mammalian cells. RESULTS The methylation profile of plasmid DNA (pBluescript SK+), which had been transfected and stably integrated into the genome of two mouse cell lines, F9 embryonal carcinoma cells and NIH3T3 fibroblasts, was determined by bisulphite genomic sequencing. Genomic DNA for analysis was prepared from F9 and 3T3 cells harvested at least 20 generations after transfection. As the introduced plasmid DNA was methylated at EcoRII sites (CmCA/TGG) by the bacterial dcm methylase and contained no other methylation of cytosines, this strategy allowed us to examine the maintenance of methylation at the