GSE60623
Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C
Background: DNA in the nucleus of a living cell carries out its functions in the context  of a complex, three-dimensional chromatin architecture. Several recently  developed methods, each an extension of the chromatin conformation capture (3C)  assay, have enabled the genome-wide profiling of chromatin contacts between  pairs of loci in yeast, fruit fly, human and mouse. 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.  Here we describe a novel assay to map genome-wide chromatin contacts, tethered  multiple 3C (TM3C), that involves a simple protocol of restriction enzyme  digestion and religation of fragments upon agarose gel beads followed by deep  DNA paired-end sequencing. In addition to identifying contacts between pairs  of loci, TM3C enables identification of contacts among more than two loci  simultaneously.  Results: 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, as well as a low noise-to-signal ratio between inter- and  intrachromosomal contacts. We also confirm that TM3C captures several known  cell type-specific contacts, ploidy shifts and translocations, such as Ph+  formation in KBM7. Furthermore, we develop a two-phase mapping strategy that  separately maps chimeric subsequences within a single read, allowing us to  identify contacts involving three or four loci simultaneously, potentially  corresponding to combinatorial regulation events.  This mapping strategy also  greatly increases the number of distinct binary contacts identified and,  therefore, the coverage obtained for a fixed number of mapped reads. We  confirm a subset of the triplet contacts involving the IGF2-H19  imprinting control region (ICR) using PCR analysis for KBM7 cells.  Assaying the genome architecture of a near-haploid cell line allows us  to create 3D models of a human cell line without averaging  signal from two homologous copies of a chromosome.  Our 3D models of KBM7 show 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  complex chromatin loops that may play a role in regulating reciprocal  expression of the IGF2 and H19 genes. Overall design: Analysis of the spatial organization of two human cell lines (KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line) using tethered multiple 3C (TM3C), a novel and simple protocol for ascertaining genome architecture which can be used to identify simultaneous contacts among three or four loci in addition to binary contacts that can be identified using traditional chromosome conformation capture coupled with next generation sequencing (Hi-C).