Protocol

In Vivo DNase I, MNase, and Restriction Enzyme Footprinting via Ligation-Mediated Polymerase Chain Reaction (LM-PCR)

Adapted from Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 2nd edition, by Michael F. Carey, Craig L. Peterson, and Stephen T. Smale. CSHL Press, Cold Spring Harbor, NY, USA, 2009.

INTRODUCTION

Genomic footprinting and related methods can reveal important protein-DNA interactions not detected using other methods. This protocol presents procedures for DNase I genomic footprinting, micrococcal nuclease (MNase) mapping of nucleosome positioning, and monitoring nucleosome remodeling via restriction enzyme accessibility. These procedures rely on a technique known as ligation-mediated polymerase chain reaction (LM-PCR), which can be used in concert with a variety of agents that cleave or modify genomic DNA within an intact cell or nucleus, with each agent providing unique insight into the regulation of a gene. LM-PCR is a powerful technique for detecting DNA strand breaks within a complex sample because of its extreme sensitivity and specificity. This general method has proven to be invaluable for several purposes, in particular, in analysis of the properties of an endogenous DNA locus. Coupling LM-PCR to DNase I digestion results in assays that are analogous to in vitro DNase I footprinting or methylation protection. Coupling to MNase digestion allows for the high-resolution analysis of nucleosome positioning. Coupling to restriction enzyme digestion in isolated nuclei provides evidence of nucleosome remodeling. For each of these methods, nuclei must first be isolated by lysis of the cells with a non-ionic detergent, followed by centrifugation. The isolated nuclei are then treated with the appropriate nuclease, followed by purification of the genomic DNA. LM-PCR is then performed on the cleaved genomic DNA.