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.
CONCLUSION: 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.