Macrophage development and activation involve coordinated intron retention in key inflammatory regulators.

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.

Enduring epigenetic landmarks define the cancer microenvironment.

The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.

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.

Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma.

BACKGROUND: Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality. RESULTS: Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37 × 10(6)-86 × 10(6) unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing. CONCLUSIONS: Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer.

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer-promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.

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.

Androgen receptor expression predicts breast cancer survival: the role of genetic and epigenetic events.

BACKGROUND: Breast cancer outcome, including response to therapy, risk of metastasis and survival, is difficult to predict using currently available methods, highlighting the urgent need for more informative biomarkers. Androgen receptor (AR) has been implicated in breast carcinogenesis however its potential to be an informative biomarker has yet to be fully explored. In this study, AR protein levels were determined in a cohort of 73 Grade III invasive breast ductal adenocarcinomas. METHODS: The levels of Androgen receptor protein in a cohort of breast tumour samples was determined by immunohistochemistry and the results were compared with clinical characteristics, including survival. The role of defects in the regulation of Androgen receptor gene expression were examined by mutation and methylation screening of the 5' end of the gene, reporter assays of the 5' and 3' end of the AR gene, and searching for miRNAs that may regulate AR gene expression. RESULTS: AR was expressed in 56% of tumours and expression was significantly inversely associated with 10-year survival (P = 0.004). An investigation into the mechanisms responsible for the loss of AR expression revealed that hypermethylation of the AR promoter is associated with loss of AR expression in breast cancer cells but not in primary breast tumours. In AR negative breast tumours, mutation screening identified the same mutation (T105A) in the 5'UTR of two AR negative breast cancer patients but not reported in the normal human population. Reporter assay analysis of this mutation however found no evidence for a negative impact on AR 5'UTR activity. The role of miR-124 in regulating AR expression was also investigated, however no evidence for this was found. CONCLUSION: This study highlights the potential for AR expression to be an informative biomarker for breast cancer survival and sets the scene for a more comprehensive investigation of the molecular basis of this phenomenon.

DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.

Widespread Aberrant Alternative Splicing despite Molecular Remission in Chronic Myeloid Leukaemia Patients.

Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease.

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.

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.

Methylome and transcriptome maps of human visceral and subcutaneous adipocytes reveal key epigenetic differences at developmental genes.

Adipocytes support key metabolic and endocrine functions of adipose tissue. Lipid is stored in two major classes of depots, namely visceral adipose (VA) and subcutaneous adipose (SA) depots. Increased visceral adiposity is associated with adverse health outcomes, whereas the impact of SA tissue is relatively metabolically benign. The precise molecular features associated with the functional differences between the adipose depots are still not well understood. Here, we characterised transcriptomes and methylomes of isolated adipocytes from matched SA and VA tissues of individuals with normal BMI to identify epigenetic differences and their contribution to cell type and depot-specific function. We found that DNA methylomes were notably distinct between different adipocyte depots and were associated with differential gene expression within pathways fundamental to adipocyte function. Most striking differential methylation was found at transcription factor and developmental genes. Our findings highlight the importance of developmental origins in the function of different fat depots.

Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten.

BACKGROUND: Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing. RESULTS: In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×). CONCLUSIONS: Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.

Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA.

BACKGROUND: The discovery that 5-methylcytosine (5mC) can be oxidized to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation (TET) proteins has prompted wide interest in the potential role of 5hmC in reshaping the mammalian DNA methylation landscape. The gold-standard bisulphite conversion technologies to study DNA methylation do not distinguish between 5mC and 5hmC. However, new approaches to mapping 5hmC genome-wide have advanced rapidly, although it is unclear how the different methods compare in accurately calling 5hmC. In this study, we provide a comparative analysis on brain DNA using three 5hmC genome-wide approaches, namely whole-genome bisulphite/oxidative bisulphite sequencing (WG Bis/OxBis-seq), Infinium HumanMethylation450 BeadChip arrays coupled with oxidative bisulphite (HM450K Bis/OxBis) and antibody-based immunoprecipitation and sequencing of hydroxymethylated DNA (hMeDIP-seq). We also perform loci-specific TET-assisted bisulphite sequencing (TAB-seq) for validation of candidate regions. RESULTS: We show that whole-genome single-base resolution approaches are advantaged in providing precise 5hmC values but require high sequencing depth to accurately measure 5hmC, as this modification is commonly in low abundance in mammalian cells. HM450K arrays coupled with oxidative bisulphite provide a cost-effective representation of 5hmC distribution, at CpG sites with 5hmC levels >~10%. However, 5hmC analysis is restricted to the genomic location of the probes, which is an important consideration as 5hmC modification is commonly enriched at enhancer elements. Finally, we show that the widely used hMeDIP-seq method provides an efficient genome-wide profile of 5hmC and shows high correlation with WG Bis/OxBis-seq 5hmC distribution in brain DNA. However, in cell line DNA with low levels of 5hmC, hMeDIP-seq-enriched regions are not detected by WG Bis/OxBis or HM450K, either suggesting misinterpretation of 5hmC calls by hMeDIP or lack of sensitivity of the latter methods. CONCLUSIONS: We highlight both the advantages and caveats of three commonly used genome-wide 5hmC profiling technologies and show that interpretation of 5hmC data can be significantly influenced by the sensitivity of methods used, especially as the levels of 5hmC are low and vary in different cell types and different genomic locations.

Transient and Permanent Reconfiguration of Chromatin and Transcription Factor Occupancy Drive Reprogramming.

Somatic cell reprogramming into induced pluripotent stem cells (iPSCs) induces changes in genome architecture reflective of the embryonic stem cell (ESC) state. However, only a small minority of cells typically transition to pluripotency, which has limited our understanding of the process. Here, we characterize the DNA regulatory landscape during reprogramming by time-course profiling of isolated sub-populations of intermediates poised to become iPSCs. Widespread reconfiguration of chromatin states and transcription factor (TF) occupancy occurs early during reprogramming, and cells that fail to reprogram partially retain their original chromatin states. A second wave of reconfiguration occurs just prior to pluripotency acquisition, where a majority of early changes revert to the somatic cell state and many of the changes that define the pluripotent state become established. Our comprehensive characterization of reprogramming-associated molecular changes broadens our understanding of this process and sheds light on how TFs access and change the chromatin during cell-fate transitions.

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.

Multiplex bisulfite PCR resequencing of clinical FFPE DNA.

BACKGROUND: The clinical utility of DNA methylation as a predictive or prognostic biomarker requires scalable resequencing protocols for bisulfite-converted DNA. Key features of any validation method should be adaptability for low- or high-throughput needs and high reproducibility, and should only require minimal amounts of precious clinical sample as input material. Critically, this method should also deliver robust results when working with bisulfite-converted DNA extracted from formalin-fixed, paraffin-embedded (FFPE) blocks. RESULTS: We report here for the first time on comparison studies between the Fluidigm Access Array system and multiplex assays for multiplex bisulfite PCR resequencing. The requirement of the Fluidigm Access Array system for high template amounts and its sensitivity to variations in template quality rendered it unsuitable for bisulfite PCR applications utilizing FFPE DNA. In response to this limitation, we established a multiplex bisulfite PCR assay capable of delivering robust methylation data using minimal amounts of FFPE clinical DNA. To evaluate the parameters and reproducibility of this assay, 57 amplicons were used to prepare sequencing libraries in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%). Analysis of this data demonstrated that this multiplex assay had high reproducibility (mean standard deviation of 1.4% for methylation values), was low cost, required low sample input (50 ng of DNA or less), and could be scaled for both low- and high-throughput needs. Notably, ExoSAP-IT (exonuclease I) treatment to remove residual primers in bisulfite resequencing libraries appeared to degrade the library and generate a high-molecular weight smear which may impact on the degree of methylation assessed. CONCLUSIONS: Multiplex bisulfite PCR assays represent a convenient and scalable method for validation and screening of methylated DNA regions from archival FFPE DNA. Moreover, the library construction process detailed here can be rapidly optimized and implemented with a minimal amount of work, can be performed using the standard equipment found in any molecular biology laboratory, and can be easily adapted for use on both genomic DNA and bisulfite DNA applications. However, in preparing bisulfite libraries for sequencing, the use of ExoSAP-IT is not recommended due to potential off-target nuclease effects which may impact downstream methylation analysis.

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.

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.