Bivalent domains enforce transcriptional memory of DNA methylated genes in cancer cells

Abstract

Silencing of multiple cancer-related genes is associated with de novo methylation of linked CpG islands. Additionally, bivalent histone modification profiles characterized by the juxtaposition of active and inactive histone marks have been observed in genes that become hypermethylated in cancer. It is unknown how these ambiguous epigenetic states are maintained and how they interrelate with adjacent genomic regions with different epigenetic landscapes. Here, we present the analysis of a set of neighboring genes, including many frequently silenced in colon cancer cells, in a chromosomal region at 5q35.2 spanning 1.25 Mb. Promoter DNA methylation occurs only at genes maintained at a low transcriptional state and is characterized by the presence of bivalent histone marks, namely trimethylation of lysines 4 and 27 in histone 3. Chemically induced hyperacetylation and DNA demethylation lead to up-regulation of silenced genes in this locus yet do not resolve bivalent domains into a domain-wide active chromatin conformation. In contrast, active genes in the region become down-regulated after drug treatment, accompanied by a partial loss of chromatin domain boundaries and spreading of the inactive histone mark trimethylated lysine 27 in histone 3. Our results demonstrate that bivalent domains mark the promoters of genes that will become DNA methylated in adult tumor cells to enforce transcriptional silence. These bivalent domains not only remain upon drug induced gene reactivation, but also spread over adjacent CpG islands. These results may have important implications in understanding and managing epigenetic therapies of cancer.colorectal cancer ͉ DNA methylation ͉ epigenetic memory ͉ transcriptional silencing I t is now clear that epigenetic events, in cooperation with genetic events, are involved in every step of tumorigenesis and play a critical role in the disruption of key cellular pathways deregulated in human cancers (1, 2).De novo methylation of CpG islands is associated with the transcriptional silencing of many cancer-related genes (1, 2). The promoter regions of silenced genes, including those with promoter DNA methylation, contain specific histone modifications, which are a signature of transcriptional inactivation (3). Additionally, the DNA methylation mark itself can be read by specific proteins that can alter chromatin structure (4). Thus, a cross-talk exists between DNA methylation and histone modifications to orchestrate transcriptional silencing.A growing body of evidence suggests that the two main cell memory systems implicated in the maintenance of a stem cell state, Trithorax (Trx) and Polycomb Group (PcG) proteins, may be involved in tumor-associated aberrant gene silencing and promoter DNA methylation (5, 6). In this context, the active mark, methylated lysine 4, together with the silent mark, methylated lysine 27, have been found to coexist over the promoter regions of DNA methylated genes in human cancer cells (7). This epigenetic landscape is similar to the bivalent domains characterized by th...

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.…”

Long-range epigenetic silencing at 2q14.2 affects most human colorectal cancers and may have application as a non-invasive biomarker of disease

“…It is interesting that, hypermethylation of these CpG islands was accompanied by long-range epigenetic silencing (LRES) of a 4-Mb chromosomal region mapping at 2q14.2 affecting all the genes regardless of whether their promoters were associated with CpG islands and if these were methylated or not (Frigola et al, 2006). Beyond this first evidence supporting, the existence of a new mechanism of gene inactivation in cancer, later studies indicate that LRES may also occur in other chromosomal regions and cancers (Stransky et al, 2006;Hesson et al, 2007;Hitchins et al, 2007;Rodriguez et al, 2008). This region encompasses the Engrailed-1 (EN1) gene encoding a homeobox transcription factor.…”

Dynamics of bivalent chromatin domains upon drug induced reactivation and resilencing in cancer cells

“…First group consists of silenced genes exhibiting methylated CpG island-promoter; this group includes EN1, SCTR and INHBB (2q14.2) and HRH2, CPLX2 and SNCB (5q35.2) that express at low levels in normal colon cells. 3,6 A second group consisted of genes with unmethylated CpG island-promoter, and includes DDX18, INSIG2, PTPN4, RALB, TSN (2q14.2), SFXN1 and THOC3 (5q35.2). These genes tend to be downregulated in colorectal tumors (as compared with the normal colon cells) but still retain high expression levels.…”

“…12,13 Different studies have reported that in genes that become silenced in cancer cells, the repressive mark H3K27me3 co-exists with active marks (H3K4me3 and H3K4me2). 6,14,15 These two opposite modifications participate in the mitotic inheritance of lineagespecific gene expression programs and have key developmental functions. Its co-localization is considered characteristic of stem cells and is believed to keep developmental regulator genes poised for induction.…”

“…These data suggest that improvement of epigenetic therapies may be achieved by designing strategies with long lasting effects. [3][4][5][6][7][8][9][10] Histone modifications can lead to either activation or repression depending upon which residues are modified and the type of modification. 11 For instance, in histone 3, dimethylaton and trimethylation of lysine 4 (H3K4me3) are associated with transcriptional activity, while trimethylation of lysine 27 (H3K27me3) is characteristic of silenced promoters.…”