Abstract
Many DNA hypermethylated and epigenetically silenced genes in adult cancers are Polycomb group (PcG) marked in embryonic stem (ES) cells. We show that a large region upstream (∼30 kb) of and extending ∼60 kb around one such gene, GATA-4, is organized—in Tera-2 undifferentiated embryonic carcinoma (EC) cells—in a topologically complex multi-loop conformation that is formed by multiple internal long-range contact regions near areas enriched for EZH2, other PcG proteins, and the signature PcG histone mark, H3K27me3. Small interfering RNA (siRNA)–mediated depletion of EZH2 in undifferentiated Tera-2 cells leads to a significant reduction in the frequency of long-range associations at the GATA-4 locus, seemingly dependent on affecting the H3K27me3 enrichments around those chromatin regions, accompanied by a modest increase in GATA-4 transcription. The chromatin loops completely dissolve, accompanied by loss of PcG proteins and H3K27me3 marks, when Tera-2 cells receive differentiation signals which induce a ∼60-fold increase in GATA-4 expression. In colon cancer cells, however, the frequency of the long-range interactions are increased in a setting where GATA-4 has no basal transcription and the loops encompass multiple, abnormally DNA hypermethylated CpG islands, and the methyl-cytosine binding protein MBD2 is localized to these CpG islands, including ones near the gene promoter. Removing DNA methylation through genetic disruption of DNA methyltransferases (DKO cells) leads to loss of MBD2 occupancy and to a decrease in the frequency of long-range contacts, such that these now more resemble those in undifferentiated Tera-2 cells. Our findings reveal unexpected similarities in higher order chromatin conformation between stem/precursor cells and adult cancers. We also provide novel insight that PcG-occupied and H3K27me3-enriched regions can form chromatin loops and physically interact in cis around a single gene in mammalian cells. The loops associate with a poised, low transcription state in EC cells and, with the addition of DNA methylation, completely repressed transcription in adult cancer cells.
Highlights
Several mammalian Polycomb group (PcG) proteins play crucial roles during early embryonic development, and the PRC2 components EZH2 and EED were shown to be important for the self-renewal and pluripotency of embryonic stem (ES) cells [1,2,3]
The small arrows next to each EcoRI site represent the location and the direction of primers used for the chromosome conformation capture (3C) assay in Figures 1 and 4. (B) The relative crosslinking frequency of different regions interacting with the À18.3 fragment across the GATA-4 locus in undifferentiated Tera-2 cells and is compared to embryonic carcinoma (EC) cells differentiated by ATRA treatment
We demonstrate that distant chromatin elements at the GATA-4 locus interact to form what might be envisioned as a ‘‘pre-repressive chromatin hub’’ in undifferentiated Tera-2 cells where the gene is poised, with presence of RNA Pol II at the start site, in a low to medium expression state (Figure 6)
Summary
Several mammalian Polycomb group (PcG) proteins play crucial roles during early embryonic development, and the PRC2 components EZH2 and EED were shown to be important for the self-renewal and pluripotency of embryonic stem (ES) cells [1,2,3]. These PcG target genes mainly encode transcriptional regulators that have a low basal transcription in pluripotent cells, which is often augmented during cell commitment to differentiation lineages [3,4,9,10,11] The promoters of such genes can bear a combination of active, as well as the PcG inactive, histone modifications in ES cells that led to the proposal that these genes are held in a ‘‘transcription-ready’’ state and are available for induced expression upon receipt of specific developmental cues [11]. Global repression of key developmental regulatory genes by PcG proteins is, crucial for maintaining the pluripotency of ES cells when activation of the involved genes would otherwise lead to onset of differentiation
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