A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations.
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.
Tiwari
Vijay K
VK
Cancer Biology Division, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University Medical Institutions, Baltimore, Maryland 21231, USA.
Cope
Leslie
L
McGarvey
Kelly M
KM
Ohm
Joyce E
JE
Baylin
Stephen B
SB
D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D002877:Chromosomes, Human / Q000737:chemistry / Q000378:metabolism*; D016207:Cytokines; D004268:DNA-Binding Proteins / Q000502:physiology*; D015536:Down-Regulation / Q000235:genetics; D000071221:Enhancer of Zeste Homolog 2 Protein; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D063151:Polycomb Repressive Complex 2; D063146:Polycomb-Group Proteins; D011487:Protein Conformation; D012097:Repressor Proteins / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*
D002843:Chromatin; D016207:Cytokines; D004268:DNA-Binding Proteins; C504459:FILIP1L protein, human; D047908:Intracellular Signaling Peptides and Proteins; D063146:Polycomb-Group Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2