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Nucleic Acids Res. 2013 Apr 1;41(6):3563-75. doi: 10.1093/nar/gkt067. Epub 2013 Feb 8.

Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub.

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1
Institute of Gene Biology of the Russian Academy of Sciences, 119334 Moscow, Russia, University of Oslo, Center for Medical Studies in Russia, 119334 Moscow, Russia.

Abstract

The current progress in the study of the spatial organization of interphase chromosomes became possible owing to the development of the chromosome conformation capture (3C) protocol. The crucial step of this protocol is the proximity ligation-preferential ligation of DNA fragments assumed to be joined within nuclei by protein bridges and solubilized as a common complex after formaldehyde cross-linking and DNA cleavage. Here, we show that a substantial, and in some cases the major, part of DNA is not solubilized from cross-linked nuclei treated with restriction endonuclease(s) and sodium dodecyl sulphate and that this treatment neither causes lysis of the nucleus nor drastically affects its internal organization. Analysis of the ligation frequencies of the mouse β-globin gene domain DNA fragments demonstrated that the previously reported 3C signals were generated predominantly, if not exclusively, in the insoluble portion of the 3C material. The proximity ligation thus occurs within the cross-linked chromatin cage in non-lysed nuclei. The finding does not compromise the 3C protocol but allows the consideration of an active chromatin hub as a folded chromatin domain or a nuclear compartment rather than a rigid complex of regulatory elements.

PMID:
23396278
PMCID:
PMC3616722
DOI:
10.1093/nar/gkt067
[Indexed for MEDLINE]
Free PMC Article
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