A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations

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 intraand 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.[Supplemental material is available online at www.genome.org.]The past few years have seen an exciting development in technologies to address long-range chromatin interactions in vivo (3C, 4C, 5C, 3C-chip, ACT, open-ended 3C to name a few) (Dekker et al. 2002;Dostie et al. 2006;Ling et al. 2006;Lomvardas et al. 2006;Simonis et al. 2006;Wurtele and Chartrand 2006;Zhao et al. 2006). These techniques have revolutionized our concepts about key aspects of transcriptional regulation. However, all of these methodologies, to date, investigate physical proximities between chromatin elements without specifically identifying the protein components that may bridge them. Separate assays must then be carried out in order to address the potential role of specific regulatory factor(s) in mediating such long-range chromatin interactions. Moreover, when it comes to knowing whether a specific protein has the property to mediate physical pairing between distant chromatin elements, there have not yet been tools to specifically monitor this question.In the present work, we combine multiple techniques to construct a novel 6C (combined 3C-ChIP-cloning) assay to address the question of long-range chromatin interactions mediated by specific proteins. To demonstrate how this methodology can work, we focus the assay on the protein Enhancer of Zeste (EZH2). This protein is a key member of the Polycomb protein complex (PcG) that regulates long-term gene silencing in multiple organisms. PcG proteins play crucial roles during early embryonic development (Sparmann and van Lohuizen 2006; Ringrose 2007) and are important for the self-renewal and pluripotency of embryonic stem cells (Bernstein et al. 2006;Sparmann and van Lohuizen 2006). Among the PcG proteins, EZH2 is particularly important since it contains the histone methyltransferase (HMTase) activity that catalyzes trimethylation of histone H3 at Lys 27 (H3-K27) (Cao et al. 2002;Cao and Zhang 2004). This mark is required for attracting key components of PcG-mediated gene repression, and this activity is shown to ...

The role of nuclear organization in cancer

“…These genes are silenced in a promoter methylationindependent manner. However, there is evidence that methylation does play a part, as the PcG protein EZH2 directly controls promoter hypermethylation at target genes and PcGs fold hypermethylated DNA into loops, which appear to enforce epigenetic silencing [87][88][89]. PcG proteins thus mediate their repressive effect by inducing classical epigenetic modifications and architectural changes of the chromatin.…”

Dissection of gene regulatory networks in embryonic stem cells by means of high-throughput sequencing

“…Genomic DNA, however, is organized in a three-dimensional manner that -most probably -also influences the gene expression program and cellular fate. Methods have been developed to reveal intra-and interchromosomal interactions, such as the 3C or the 6C technologies (Dekker et al, 2002;Tiwari et al, 2008). 3C generates a library of DNA fragments that reflects literally all interactions and could be utilized in combination with massive parallel sequencing for the analysis of the three-dimensional chromatin structure genome-wide.…”

Spatial Organization of Epigenomes

“…Genetic and biochemical studies show that PRCs control transcription in pluripotent cells by catalytic and non-catalytic mechanisms [65,66]. They are shown to induce chromatin compaction [65][66][67], but can also form long-range contacts within and between chromosomes in vivo, supporting their potential role in regulating gene expression through genome architecture. For example, deletion of a PRC2 subunit that leads to H3K27me2/3 depletion correlates with chromatin decompaction at HOX loci as detected by FISH [66].…”