title: Simultaneous single-molecule mapping of protein-DNA interactions and DNA methylation by MAPit.
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.
authors: Department of Biochemistry and Molecular Biology and UF Shands Cancer Center Program in Cancer Genetics, Epigenetics and Tumor Virology, University of Florida College of Medicine, Gainesville, Florida, USA.
authors: Carolina E
authors: CE
authors: Pardo
authors: Russell P
authors: RP
authors: Darst
authors: Nancy H
authors: NH
authors: Nabilsi
authors: Amber L
authors: AL
authors: Delmas
authors: Michael P
authors: MP
authors: Kladde
mesh_terms: D000818:Animals; D001483:Base Sequence; D002843:Chromatin; D018899:CpG Islands; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry; D018983:DNA Footprinting / Q000379:methods*; D019175:DNA Methylation; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000737:chemistry*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D002843:Chromatin; D011506:Proteins; D003596:Cytosine; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases