Abstract
The folding mechanisms of the mammalian genome package our genetic material into the nucleus, and in doing so, dictate its appropriate replication and expression. Chromosome conformation capture technology has enabled the dissection of the folding principles of the cellular genome. This has led to a better understanding of the role played by architectural proteins in forming and dissolving 3D-chromatin-structure. These assays are based on the principle of crosslinking distant cellular sites that are proximal to each other in 3D space using formaldehyde followed by digestion of formed hybrid DNA junctions. Invading viruses, such as the lytic parvovirus Minute Virus of Mice (MVM), establish distinct replication centers within the nuclear environment at cellular sites that preferentially undergo DNA damage, but do not integrate into the cellular DNA. We have adapted chromosome conformation capture technology to study the trans-interaction between MVM and the cellular genome, which we have dubbed V3C, which can be extended to a whole-genome analysis we term V3C-seq. This protocol describes the procedure for performing, as well as analyzing V3C-seq assays, and can be adapted for mapping the cellular interaction sites of any non-integrating DNA virus.
Keywords: Chromosome conformation capture, Parvovirus, DNA damage response, Topologically associating domains, Fragile sites, Host-pathogen genome interactions
Background
Chromosome conformation capture technologies have helped us gain significant insights into the folding principles of the mammalian genome, helping identify the short-range promoter-enhancer loops that underlie epigenetic regulation of gene expression, as well as long-range multi-loop domains that form sub-chromosomal chromatin compartments (Dekker et al., 2013; Denker and de Laat, 2016). While these technologies have helped define important rules related to the patterns of cis-folding of the cellular genome, their implementation in studying the trans-interaction between the genome of an invading DNA virus and its target cell remained largely unstudied. In this protocol, we describe the procedure for studying the interactome of the parvovirus Minute Virus of Mice (Figure 1). Owing to its 5 kilobase genome, the application of a modified Circular Chromosome Conformation Capture (4C) assay combined with high-throughput sequencing (Van De Werken et al., 2012) allowed the generation of a high resolution map of the sites on cellular DNA where MVM localizes during infection (Majumder et al., 2018).Figure 1. Schematic of the V3C-seq assay with approximate timeline
Materials and Reagents
Equipment
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Procedure
Data analysis
Recipes
Acknowledgments
Work in the Pintel Laboratory is supported by NIH grants AI046458 and AI116595 to DJP. KM is supported by a Ruth L Kirschstein Postdoctoral Individual National Research Service Award (F32) AI131468. This procedure was modified from published 4C and 3C-seq studies (Dekker et al., 2013; Denker and de Laat, 2016). Detailed procedures are published in Majumder et al. (2018) and sequencing data have been deposited in the GEO database under the accession number GSE112957.
Competing interests
The authors declare no competing financial interest.
References
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