title: Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C.
abstract: BACKGROUND
  Several recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication.
  
  
  RESULTS
  We describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines.
  
  
  CONCLUSIONS
  TM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, 98195, WA, USA. ferhatay@uw.edu.
authors.forename: Ferhat
authors.initials: F
authors.lastname: Ay
authors.affiliation: Veterans Affairs Palo Alto Health Care System, Stanford University Medical School, Palo Alto, 94304, CA, USA. thanvu@gmail.com.
authors.forename: Thanh H
authors.initials: TH
authors.lastname: Vu
authors.affiliation: Veterans Affairs Palo Alto Health Care System, Stanford University Medical School, Palo Alto, 94304, CA, USA. mjzeitz@gmail.com.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Zeitz
authors.affiliation: Mines ParisTech, PSL-Research University, CBIO-Centre for Computational Biology, 35 rue St Honoré, Fontainebleau, 77300, France. nelle.varoquaux@ensmp.fr.|Institut Curie, Paris, F-75248, France. nelle.varoquaux@ensmp.fr.|U900, INSERM, ParisF-75248, France. nelle.varoquaux@ensmp.fr.
authors.forename: Nelle
authors.initials: N
authors.lastname: Varoquaux
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, 94305, CA, USA. carette@stanford.edu.
authors.forename: Jan E
authors.initials: JE
authors.lastname: Carette
authors.affiliation: Mines ParisTech, PSL-Research University, CBIO-Centre for Computational Biology, 35 rue St Honoré, Fontainebleau, 77300, France. jean-philippe.vert@mines-paristech.fr.|Institut Curie, Paris, F-75248, France. jean-philippe.vert@mines-paristech.fr.|U900, INSERM, ParisF-75248, France. jean-philippe.vert@mines-paristech.fr.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Vert
authors.affiliation: Veterans Affairs Palo Alto Health Care System, Stanford University Medical School, Palo Alto, 94304, CA, USA. arhoffman@stanford.edu.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Hoffman
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, 98195, WA, USA. william-noble@uw.edu.|Department of Computer Science and Engineering, University of Washington, Seattle, 98195, WA, USA. william-noble@uw.edu.
authors.forename: William S
authors.initials: WS
authors.lastname: Noble
mesh_terms: D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics*; D015894:Genome, Human; D006801:Humans; D007938:Leukemia / Q000235:genetics* / Q000473:pathology; D015183:Restriction Mapping
chemical_list: D002843:Chromatin