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 ( approximately 30 kb) of and extending approximately 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 approximately 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.