This study is one of the first to interrogate and integrate multi-study clinical prostate cancer expression data with complex multi-layered epigenome data. We have built a comprehensive prostate cancer epigenome map to answer important questions on the prevalence and mode of action of LRES in prostate cancer. Our data reveal that LRES is a common event in prostate cancer and affects a significant proportion of the cancer genome.
One of the main features common to LRES genomic locations is the overlap with regions of genomic deletion or LOH, reported in prostate and other cancers37–39
. It is widely accepted that genetic and/or epigenetic processes can silence single genes involved in tumourigenesis. We now propose that LOH and LRES, acting either independently or simultaneously on different alleles, can also result in regional gene suppression in cancer. The common overlap of LOH/genomic deletion and LRES regions may reflect the presence of genes that play a role in cancer, with loss of expression providing a growth advantage. Indeed, over a third of LRES regions harbour known tumour-suppressor, tumour-related13,39–46
and miRNA genes47–51
. A mechanistic relationship connecting genomic deletion and/or LOH to LRES is not clear. There may be underlying chromosomal features that predispose these genomic regions to either regional epigenetic silencing or deletion, or epigenetic silencing may itself pre-dispose a region to subsequent deletion. It has been suggested that double strand breaks and DNA repair may lead to epigenetic remodelling and histone modification52,53
or conversely that compromised chromatin is less efficient for DNA double-strand break repair and prone to chromosomal aberration54
In addition to genomic features, we investigated epigenomic features associated with LRES regions in prostate cancer cells. The overriding feature was an overall depletion of H3K9 acetylation that occurred, not only in neighbouring genes that were active in normal prostate, but also in genes that were silent in normal prostate or hES cells. In addition to global deacetylation, we were surprised to find distinct combinations of epigenetic silencing marks, spanning multiple genes in domains within each of the LRES regions. Three main types of epigenetically distinct cancer-associated domains were found (summarised in ). First, “Re-enforcement” of repressive marks to a more definitively repressed state; re-enforcement occurs in regions that are predominately suppressed in normal prostate and hES cells, and are marked by even lower levels of H3K9ac, an enrichment of H3K9me2 and higher H3K27me3 levels, and in some cases localised DNA hypermethylation. Second, “Gain” of multiple repressive marks in regions that were clearly active and associated with H3K9 hyperacetylation in normal prostate and hES cells; these repressive marks include a complete lack of H3K9ac and presence of H3K27me3 and also can include elevated H3K9me2 and DNA hypermethylation. Third, “Exchange” of repressive marks is seen in genes that are inactive or lowly expressed in normal prostate and hES cells; “exchange” commonly involves a relative lack of the H3K27me3 mark and higher DNA methylation especially in genes normally bearing bivalent marks in hES cells, or a combination of active (H3K9ac) and repressive (H3K27me3) marks in PrEC cells. In some cases however, especially in non CpG island-associated genes, a lack of DNA methylation and elevated H3K27me3 and H3K9me2 marks is observed. The transcriptional state of the gene in the normal cell commonly predicts the mode of epigenetic remodelling observed in the cancer cell. We propose that all three major remodelling patterns that occur within LRES regions contribute to a consolidation or reduction of the accessible genome potentially available for any normal transcriptional response in the cancer cell. Across the whole genome we found that for all epigenetic marks adjacent pairs were more likely to differ concordantly in cancer. This supports the concept that many local chromosomal regions are under coordinated epigenetic control and that the stringent criteria we have applied to identify LRES regions has selected a subset of a more general phenomenon.
Consolidation of the cancer epigenome into domains of repressive chromatin by LRES
Single genes have been reported recently to undergo different modes of epigenetic reprogramming, most notably “epigenetic switching” which occurs in developmental genes that are silent and associated with H3K27me3 in normal cells, but in cancer, these genes are susceptible to DNA methylation and lose the polycomb mark55
. However, epigenetic switching has not been reported to occur in clusters. Clustering of chromatin marks does occur to some extent in normal cells. For example the organisation of the genome into euchromatin and heterochromatin is well established and G and R banding is thought to be associated with enrichment or depletion of repressive histone marks56
. More recently, two studies described a single epigenetic mark that formed domains in the normal (mouse) genome; H3K9me2 domains (LOCKs) were found to be acquired during normal cell differentiation and were associated with gene silencing over large regions57
; and domains of H3K27me3 (BLOCs) appeared to span silent genes in normal fibroblasts58
. Chromatin of undifferentiated hES cells is less condensed and has higher plasticity compared to that of the differentiated cell59–62
We propose that, LRES in cancer results in a yet further consolidation of the genome to a more definitive epigenetic repressive state across large domains, affecting a large variety of epigenetic marks resulting in reduced transcriptional plasticity. In fact, some LRES regions appear to expand into neighbouring genes in metastatic disease, suggesting a role for LRES in tumour progression, that is potentially seeded by epigenetic silencing of a critical gene or genes involved in cancer initiation. Our study has important implications in development of epigenetic-based cancer treatment strategies that may be required to re-activate genes in chromosomal domains that are overlayed with multiple repressive epigenetic marks.