Integrated DNA Methylation and Chromatin Structural Analysis at Single-Molecule Resolution
Chromatin limits the accessibility of DNA to trans-acting factors in transcription, replication, and repair. Although transcriptional variation between cells in a population may contribute to survival and disease, most assays of chromatin structure recover only population averages. We have developed DNA methyltransferases (MTases) as probing agents of DNA accessibility in chromatin, either expressed in vivo in budding yeast or as recombinant enzymatic probes of nuclei isolated from mammalian cells. In this chapter, we focus on the use of recombinant MTase (M) M.CviPI to probe chromatin accessibility in nuclei isolated from mammalian cell lines and animal tissue. This technique, named methylation accessibility protocol for individual templates (MAPit), reports protein–DNA interactions at single-molecule resolution. The single-molecule readout allows identification of chromatin subpopulations and rare epigenetic variants within a cell population. Furthermore, the use of M.CviPI in mammalian systems gives a comprehensive view of both chromatin structure and endogenous DNA methylation in a single assay.
Key wordsChromatin DNA methylation DNA methyltransferases Footprinting Nucleosomes Single-molecule methods Transcription
We are grateful to the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for high-throughput sequencing. This work was supported by R01CA155390 from the National Cancer Institute to M.P.K. as well as 2BT01 (Team Science Project) and 1BD03 (Postdoctoral Research Fellowship) from the Florida Department of Health Bankhead-Coley Cancer Research Program to M.P.K. and N.H.N., respectively.
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