Single-molecule analysis of chromatin: changing the view of genomes one molecule at a time.
Wrapping DNA into chromatin provides a wealth of regulatory mechanisms that ensure normal growth and development in eukaryotes. Our understanding of chromatin structure, including nucleosomes and non-histone protein-DNA interactions, has benefited immensely from nuclease and chemical digestion techniques. DNA-bound proteins, such as histones or site-specific factors, protect DNA against nuclease cleavage and generate large nucleosomal or small regulatory factor footprints. Chromatin subject to distinct modes of regulation often coincides with sites of nuclease hypersensitivity or nucleosome positioning. An inherent limitation of cleavage-based analyses has been the inability to reliably analyze regions of interest when levels of digestion depart from single-hit kinetics. Moreover, cleavage-based techniques provide views that are averaged over all the molecules in a sample population. Therefore, in cases of occupancy of multiple regulatory elements by factors, one cannot define whether the factors are bound to the same or different molecules in the population. The recent development of DNA methyltransferase-based, single-molecule MAP-IT technology overcomes limitations of ensemble approaches and has opened numerous new avenues in chromatin research. Here, we review the strengths, limitations, applications and future prospects of MAP-IT ranging from structural issues to mechanistic questions in eukaryotic chromatin regulation.
Pondugula
Santhi
S
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610-3633, USA.
Kladde
Michael P
MP
D002843:Chromatin / Q000737:chemistry*; D015254:DNA Modification Methylases; D016678:Genome; D015336:Molecular Probe Techniques
D002843:Chromatin; D015254:DNA Modification Methylases