A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations
- 1 Cancer Biology Division, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University Medical Institutions, Baltimore, Maryland 21231, USA;
- 2 Biometry and Clinical Trials Division, The Johns Hopkins University Medical Institutions, Baltimore, Maryland 21231, USA;
- 3 Program in Cellular and Molecular Medicine, The Johns Hopkins University Medical Institutions, Baltimore, Maryland 21231, USA
Abstract
We describe construction of a novel modification, “6C,” of chromatin looping assays that allows specific proteins that may mediate long-range chromatin interactions to be defined. This approach combines the standard looping approaches previously defined with an immunoprecipitation step to investigate involvement of the specific protein. The efficacy of this approach is demonstrated by using a Polycomb group (PcG) protein, Enhancer of Zeste (EZH2), as an example of how our assay might be used. EZH2, as a protein of the PcG complex, PRC2, has an important role in the propagation of epigenetic memory through deposition of the repressive mark, histone H3, lysine 27, tri-methylation (H3K27me3). Using our new 6C assay, we show how EZH2 is a direct mediator of long-range intra- and interchromosomal interactions that can regulate transcriptional down-regulation of multiple genes by facilitating physical proximities between distant chromatin regions, thus targeting sites within to PcG machinery.
Footnotes
-
↵4 Corresponding author.
↵4 E-mail sbaylin{at}jhmi.edu; fax (410) 614-9884.
-
[Supplemental material is available online at www.genome.org.]
-
Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.073452.107.
-
- Received October 22, 2007.
- Accepted March 19, 2008.
- Copyright © 2008, Cold Spring Harbor Laboratory Press
