Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

• Published in: Molecular systems biology Vol. 12; no. 12; pp. 891 - n/a
• Main Authors: Brant, Lilija, Georgomanolis, Theodore, Nikolic, Milos, Brackley, Chris A, Kolovos, Petros, Ijcken, Wilfred, Grosveld, Frank G, Marenduzzo, Davide, Papantonis, Argyris
• Format: Journal Article
• Language: English
• Published: England EMBO Press 01.12.2016; John Wiley and Sons Inc; Springer Nature
• Subjects: Animals; Archives & records; Artificial chromosomes; Cell Nucleus - genetics; Chromatin; chromatin looping; chromosome conformation capture; Chromosomes; Chromosomes, Human - chemistry; Chromosomes, Human - genetics; Computer applications; Conformation; cross‐linking; Deoxyribonucleic acid; DNA; DNA damage; Experiments; Genomes; High-Throughput Nucleotide Sequencing - methods; Human Umbilical Vein Endothelial Cells; Humans; Interphase; K562 Cells; Mammals; Mammals - genetics; Methylation; Microscopy; Models, Genetic; nuclear compartments; nuclear organization; Nuclei; Nuclei (cytology); Nucleic Acid Conformation; Oligonucleotide Array Sequence Analysis; Organic chemistry; Physiology; Regulatory sequences; Sequence Analysis, DNA - methods; Stem cells; Structure-function relationships; chromosome conformation capture; cross‐linking; nuclear organization; chromatin looping; nuclear compartments
• ISSN: 1744-4292; 1744-4292
• DOI: 10.15252/msb.20167311

Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure‐to‐function relationship. However, all 3C‐based methods rely on chemical cross‐linking to stabilize spatial interactions. This step remains a “black box” as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed “i3C”, a novel approach for capturing spatial interactions without a need for cross‐linking. We apply i3C to intact nuclei of living cells and exploit native forces that stabilize chromatin folding. Using different cell types and loci, computational modeling, and a methylation‐based orthogonal validation method, “TALE‐iD”, we show that native interactions resemble cross‐linked ones, but display improved signal‐to‐noise ratios and are more focal on regulatory elements and CTCF sites, while strictly abiding to topologically associating domain restrictions. Synopsis i3C captures chromatin folding in intact nuclei without a need for cross‐linking and reveals that native interactions resemble cross‐linked ones, yet they display improved signal‐to‐noise ratios and highlight how cis‐elements and TADs contribute to 3D genomic organization. i3C addresses the issue of potential biases introduced in 3C‐based studies by formaldehyde cross‐linking and harsh treatments. This protocol is robust, sensitive, and significantly faster than the conventional approach, and relies on native forces to preserve spatial interactions in uncross‐linked nuclei. Overall, i3C interaction profiles resemble conventional ones and highlight the contribution of both active and inactive regulatory regions in chromatin looping, as well as that of the restrictions imposed by topologically associating domain boundaries. i3C complements the existing toolkit and can prove especially useful for analyzing dense interaction matrices at high resolution. i3C captures chromatin folding in intact nuclei without a need for cross‐linking and reveals that native interactions resemble cross‐linked ones, yet they display improved signal‐to‐noise ratios and highlight how cis‐elements and TADs contribute to 3D genomic organization.