Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels.

BACKGROUND: DNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material. RESULTS: Here, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each amplicon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon. Here, we show that the MBPS assay can amplify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1-5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA. CONCLUSIONS: The MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.

DNA Hypermethylation Encroachment at CpG Island Borders in Cancer Is Predisposed by H3K4 Monomethylation Patterns.

Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial methylation encroachment across the 5' or 3' CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer.

Integrated epigenomic analysis stratifies chromatin remodellers into distinct functional groups.

BACKGROUND: ATP-dependent chromatin remodelling complexes are responsible for establishing and maintaining the positions of nucleosomes. Chromatin remodellers are targeted to chromatin by transcription factors and non-coding RNA to remodel the chromatin into functional states. However, the influence of chromatin remodelling on shaping the functional epigenome is not well understood. Moreover, chromatin remodellers have not been extensively explored as a collective group across two-dimensional and three-dimensional epigenomic layers. RESULTS: Here, we have integrated the genome-wide binding profiles of eight chromatin remodellers together with DNA methylation, nucleosome positioning, histone modification and Hi-C chromosomal contacts to reveal that chromatin remodellers can be stratified into two functional groups. Group 1 (BRG1, SNF2H, CHD3 and CHD4) has a clear preference for binding at 'actively marked' chromatin and Group 2 (BRM, INO80, SNF2L and CHD1) for 'repressively marked' chromatin. We find that histone modifications and chromatin architectural features, but not DNA methylation, stratify the remodellers into these functional groups. CONCLUSIONS: Our findings suggest that chromatin remodelling events are synchronous and that chromatin remodellers themselves should be considered simultaneously and not as individual entities in isolation or necessarily by structural similarity, as they are traditionally classified. Their coordinated function should be considered by preference for chromatin features in order to gain a more accurate and comprehensive picture of chromatin regulation.

Replication timing and epigenome remodelling are associated with the nature of chromosomal rearrangements in cancer.

DNA replication timing is known to facilitate the establishment of the epigenome, however, the intimate connection between replication timing and changes to the genome and epigenome in cancer remain largely uncharacterised. Here, we perform Repli-Seq and integrated epigenome analyses and demonstrate that genomic regions that undergo long-range epigenetic deregulation in prostate cancer also show concordant differences in replication timing. A subset of altered replication timing domains are conserved across cancers from different tissue origins. Notably, late-replicating regions in cancer cells display a loss of DNA methylation, and a switch in heterochromatin features from H3K9me3-marked constitutive to H3K27me3-marked facultative heterochromatin. Finally, analysis of 214 prostate and 35 breast cancer genomes reveal that late-replicating regions are prone to cis and early-replication to trans chromosomal rearrangements. Together, our data suggests that the nature of chromosomal rearrangement in cancer is related to the spatial and temporal positioning and altered epigenetic states of early-replicating compared to late-replicating loci.

Acetylated histone variant H2A.Z is involved in the activation of neo-enhancers in prostate cancer.

Acetylation of the histone variant H2A.Z (H2A.Zac) occurs at active promoters and is associated with oncogene activation in prostate cancer, but its role in enhancer function is still poorly understood. Here we show that H2A.Zac containing nucleosomes are commonly redistributed to neo-enhancers in cancer resulting in a concomitant gain of chromatin accessibility and ectopic gene expression. Notably incorporation of acetylated H2A.Z nucleosomes is a pre-requisite for activation of Androgen receptor (AR) associated enhancers. H2A.Zac nucleosome occupancy is rapidly remodeled to flank the AR sites to initiate the formation of nucleosome-free regions and the production of AR-enhancer RNAs upon androgen treatment. Remarkably higher levels of global H2A.Zac correlate with poorer prognosis. Altogether these data demonstrate the novel contribution of H2A.Zac in activation of newly formed enhancers in prostate cancer.

Methylome sequencing in triple-negative breast cancer reveals distinct methylation clusters with prognostic value.

Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, our findings demonstrate the feasibility of profiling the cancer methylome with limited archival tissue to identify regulatory regions associated with cancer.

Regional activation of the cancer genome by long-range epigenetic remodeling.

Epigenetic gene deregulation in cancer commonly occurs through chromatin repression and promoter hypermethylation of tumor-associated genes. However, the mechanism underpinning epigenetic-based gene activation in carcinogenesis is still poorly understood. Here, we identify a mechanism of domain gene deregulation through coordinated long-range epigenetic activation (LREA) of regions that typically span 1 Mb and harbor key oncogenes, microRNAs, and cancer biomarker genes. Gene promoters within LREA domains are characterized by a gain of active chromatin marks and a loss of repressive marks. Notably, although promoter hypomethylation is uncommon, we show that extensive DNA hypermethylation of CpG islands or "CpG-island borders" is strongly related to cancer-specific gene activation or differential promoter usage. These findings have wide ramifications for cancer diagnosis, progression, and epigenetic-based gene therapies.

Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA.

The complex relationship between DNA methylation, chromatin modification, and underlying DNA sequence is often difficult to unravel with existing technologies. Here, we describe a novel technique based on high-throughput sequencing of bisulfite-treated chromatin immunoprecipitated DNA (BisChIP-seq), which can directly interrogate genetic and epigenetic processes that occur in normal and diseased cells. Unlike most previous reports based on correlative techniques, we found using direct bisulfite sequencing of Polycomb H3K27me3-enriched DNA from normal and prostate cancer cells that DNA methylation and H3K27me3-marked histones are not always mutually exclusive, but can co-occur in a genomic region-dependent manner. Notably, in cancer, the co-dependency of marks is largely redistributed with an increase of the dual repressive marks at CpG islands and transcription start sites of silent genes. In contrast, there is a loss of DNA methylation in intergenic H3K27me3-marked regions. Allele-specific methylation status derived from the BisChIP-seq data clearly showed that both methylated and unmethylated alleles can simultaneously be associated with H3K27me3 histones, highlighting that DNA methylation status in these regions is not dependent on Polycomb chromatin status. BisChIP-seq is a novel approach that can be widely applied to directly interrogate the genomic relationship between allele-specific DNA methylation, histone modification, or other important epigenetic regulators.

Acetylation of H2A.Z is a key epigenetic modification associated with gene deregulation and epigenetic remodeling in cancer.

Histone H2A.Z (H2A.Z) is an evolutionarily conserved H2A variant implicated in the regulation of gene expression; however, its role in transcriptional deregulation in cancer remains poorly understood. Using genome-wide studies, we investigated the role of promoter-associated H2A.Z and acetylated H2A.Z (acH2A.Z) in gene deregulation and its relationship with DNA methylation and H3K27me3 in prostate cancer. Our results reconcile the conflicting reports of positive and negative roles for histone H2A.Z and gene expression states. We find that H2A.Z is enriched in a bimodal distribution at nucleosomes, surrounding the transcription start sites (TSSs) of both active and poised gene promoters. In addition, H2A.Z spreads across the entire promoter of inactive genes in a deacetylated state. In contrast, acH2A.Z is only localized at the TSSs of active genes. Gene deregulation in cancer is also associated with a reorganization of acH2A.Z and H2A.Z nucleosome occupancy across the promoter region and TSS of genes. Notably, in cancer cells we find that a gain of acH2A.Z at the TSS occurs with an overall decrease of H2A.Z levels, in concert with oncogene activation. Furthermore, deacetylation of H2A.Z at TSSs is increased with silencing of tumor suppressor genes. We also demonstrate that acH2A.Z anti-correlates with promoter H3K27me3 and DNA methylation. We show for the first time, that acetylation of H2A.Z is a key modification associated with gene activity in normal cells and epigenetic gene deregulation in tumorigenesis.

Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA methylation analysis reveal CpG sequence coverage bias.

DNA methylation primarily occurs at CpG dinucleotides in mammals and is a common epigenetic mark that plays a critical role in the regulation of gene expression. Profiling DNA methylation patterns across the genome is vital to understand DNA methylation changes that occur during development and in disease phenotype. In this study, we compared two commonly used approaches to enrich for methylated DNA regions of the genome, namely methyl-DNA immunoprecipitation (MeDIP) that is based on enrichment with antibodies specific for 5'-methylcytosine (5MeC), and capture of methylated DNA using a methyl-CpG binding domain-based (MBD) protein to discover differentially methylated regions (DMRs) in cancer. The enriched methylated DNA fractions were interrogated on Affymetrix promoter tiling arrays and differentially methylated regions were identified. A detailed validation study of 42 regions was performed using Sequenom MassCLEAVE technique. This detailed analysis revealed that both enrichment techniques are sensitive for detecting DMRs and preferentially identified different CpG rich regions of the prostate cancer genome, with MeDIP commonly enriching for methylated regions with a low CpG density, while MBD capture favors regions of higher CpG density and identifies the greatest proportion of CpG islands. This is the first detailed validation report comparing different methylated DNA enrichment techniques for identifying regions of differential DNA methylation. Our study highlights the importance of understanding the nuances of the methods used for DNA genome-wide methylation analyses so that accurate interpretation of the biology is not overlooked.

Epigenetic deregulation across chromosome 2q14.2 differentiates normal from prostate cancer and provides a regional panel of novel DNA methylation cancer biomarkers.

BACKGROUND: Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers. METHODS: We used highly sensitive DNA methylation headloop PCR assays that can detect 10 to 25 pg of methylated DNA with a specificity of at least 1:1,000, and chromatin immunoprecipitation assays to investigate regional epigenetic remodeling across 2q14.2 in prostate cancer, in a cohort of 195 primary prostate tumors and 90 matched normal controls. RESULTS: Prostate cancer cells exhibit concordant deacetylation and methylation of histone H3 Lysine 9 (H3K9Ac and H3K9me2, respectively), and localized DNA hypermethylation of EN1, SCTR, and INHBB and corresponding loss of H3K27me3. EN1 and SCTR were frequently methylated (65% and 53%, respectively), whereas INHBB was less frequently methylated. CONCLUSIONS: Consistent with LRES in colorectal cancer, we found regional epigenetic remodeling across 2q14.2 in prostate cancer. Concordant methylation of EN1 and SCTR was able to differentiate cancer from normal (P < 0.0001) and improved the diagnostic specificity of GSTP1 methylation for prostate cancer detection by 26%. IMPACT: For the first time we show that DNA methylation of EN1 and SCTR promoters provide potential novel biomarkers for prostate cancer detection and in combination with GSTP1 methylation can add increased specificity and sensitivity to improve diagnostic potential.

Evaluation of affinity-based genome-wide DNA methylation data: effects of CpG density, amplification bias, and copy number variation.

DNA methylation is an essential epigenetic modification that plays a key role associated with the regulation of gene expression during differentiation, but in disease states such as cancer, the DNA methylation landscape is often deregulated. There are now numerous technologies available to interrogate the DNA methylation status of CpG sites in a targeted or genome-wide fashion, but each method, due to intrinsic biases, potentially interrogates different fractions of the genome. In this study, we compare the affinity-purification of methylated DNA between two popular genome-wide techniques, methylated DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain-based capture (MBDCap), and show that each technique operates in a different domain of the CpG density landscape. We explored the effect of whole-genome amplification and illustrate that it can reduce sensitivity for detecting DNA methylation in GC-rich regions of the genome. By using MBDCap, we compare and contrast microarray- and sequencing-based readouts and highlight the impact that copy number variation (CNV) can make in differential comparisons of methylomes. These studies reveal that the analysis of DNA methylation data and genome coverage is highly dependent on the method employed, and consideration must be made in light of the GC content, the extent of DNA amplification, and the copy number.

Consolidation of the cancer genome into domains of repressive chromatin by long-range epigenetic silencing (LRES) reduces transcriptional plasticity.

Silencing of individual genes can occur by genetic and epigenetic processes during carcinogenesis, but the underlying mechanisms remain unclear. By creating an integrated prostate cancer epigenome map using tiling arrays, we show that contiguous regions of gene suppression commonly occur through long-range epigenetic silencing (LRES). We identified 47 LRES regions in prostate cancer, typically spanning about 2 Mb and harbouring approximately 12 genes, with a prevalence of tumour suppressor and miRNA genes. Our data reveal that LRES is associated with regional histone deacetylation combined with subdomains of different epigenetic remodelling patterns, which include re-enforcement, gain or exchange of repressive histone, and DNA methylation marks. The transcriptional and epigenetic state of genes in normal prostate epithelial and human embryonic stem cells can play a critical part in defining the mode of cancer-associated epigenetic remodelling. We propose that consolidation or effective reduction of the cancer genome commonly occurs in domains through a combination of LRES and LOH or genomic deletion, resulting in reduced transcriptional plasticity within these regions.

Aberrant de novo methylation of the p16INK4A CpG island is initiated post gene silencing in association with chromatin remodelling and mimics nucleosome positioning.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly enriched in CpG island associated promoter regions. DNA hypermethylation and histone repression correlate with gene silencing, however, the dynamics of this process are still largely unclear. The tumour suppressor gene p16(INK4A) is inactivated in association with CpG island methylation during neoplastic progression in a variety of cancers, including breast cancer. Here, we investigated the temporal progression of DNA methylation and histone remodelling in the p16(INK4A) CpG island in primary human mammary epithelial cell (HMEC) strains during selection, as a model for early breast cancer. Silencing of p16(INK4A) has been previously shown to be necessary before HMECs can escape from selection. Here, we demonstrate that gene silencing occurs prior to de novo methylation and histone remodelling. An increase in DNA methylation was associated with a rapid loss of both histone H3K27 trimethylation and H3K9 acetylation and a gradual gain of H3K9 dimethylation. Interestingly, we found that regional-specific 'seeding' methylation occurs early after post-selection and that the de novo methylation pattern observed in HMECs correlates with the apparent footprint of nucleosomes across the p16(INK4A) CpG island. Our results demonstrate for the first time that p16(INK4A) gene silencing is a precursor to epigenetic suppression and that subsequent de novo methylation initially occurs in nucleosome-free regions across the p16(INK4A) CpG island and this is associated with a dynamic change in histone modifications.

Transcriptional gene silencing promotes DNA hypermethylation through a sequential change in chromatin modifications in cancer cells.

It is well established that DNA hypermethylation of tumor suppressor and tumor-related genes can occur in cancer cells and that each cancer subtype has specific gene sets that are commonly susceptible to methylation and silencing. Glutathione S-transferase (GSTP1) is one example of a gene that is hypermethylated and inactivated in the majority of prostate cancers. We previously reported that hypermethylation of the GSTP1 CpG island promoter in prostate cancer cells is initiated by a combination of transcriptional gene silencing (by removal of the Sp1 sites) and seeds of methylation that, instead of being constantly removed because of demethylation associated with transcription, acts as a catalyst for the spread of methylation across the CpG island. In this study, we now demonstrate that the seeds of DNA methylation also play an important role in initiating chromatin modification. Our results address a number of central questions about the temporal relationship between gene expression, DNA hypermethylation, and chromatin modification in cancer cells. We find that for the GSTP1 gene, (a). histone acetylation is independent of gene expression, (b). histone deacetylation is triggered by seeds of DNA methylation, (c). the spread of DNA hypermethylation across the island is linked to MBD2 and not MeCP2 binding, and (d). histone methylation occurs after histone deacetylation and is associated with extensive DNA methylation of the CpG island. These findings have important implications for understanding the biochemical events underlying the mechanisms responsible for abnormal hypermethylation of CpG island-associated genes in cancer cells.

Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells.

Understanding what triggers hypermethylation of tumour suppressor genes in cancer cells is critical if we are to discern the role of methylation in the oncogenic process. CpG sites in CpG island promoters, that span most tumour suppressor genes, remain unmethylated in the normal cell, despite the fact that CpG sites are the prime target for de novo methylation by the DNA methyltransferases. The CpG island-associated with the GSTP1 gene is an intriguing example of a CpG rich region which is susceptible to hypermethylation in the majority of prostate tumours and yet is unmethylated in the normal prostate cell. In this study we evaluate a number of factors purported to be involved in hypermethylation to test their role in triggering hypermethylation of GSTP1 in prostate cancer DU145 and LNCaP cells. We find that hypermethylation is not associated with (1) elevated expression of the DNA methyltranferases, or (2) removal of Sp1 transcription factor binding sites in the CpG island or (3) removal of CpG island boundary elements or (4) prior gene silencing. Instead our results support a model that requires a combination of prior gene silencing and random "seeds" of methylation to trigger hypermethylation of the GSTP1 gene in the prostate cancer cell. We propose that the GSTP1 gene is initially silenced in the prostate cancer and random sites of methylation accumulate that result in subsequent hypermethylation and chromatin remodelling.