Dario Strbenac

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.

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.

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.

PD-L1 Negative Status is Associated with Lower Mutation Burden, Differential Expression of Immune-Related Genes, and Worse Survival in Stage III Melanoma

Purpose: Understanding why some melanomas test negative for PD-L1 by IHC may have implications for the application of anti-PD-1 therapies in melanoma management. This study sought to determine somatic mutation and gene expression patterns associated with tumor cell PD-L1 expression, or lack thereof, in stage III metastatic melanoma to better define therapeutically relevant patient subgroups. Experimental Design: IHC for PD-L1 was assessed in 52 American Joint Committee on Cancer stage III melanoma lymph node specimens and compared with specimen-matched comprehensive clinicopathologic, genomic, and transcriptomic data. Results: PD-L1–negative status was associated with lower nonsynonymous mutation (NSM) burden (P = 0.017) and worse melanoma-specific survival [HR = 0.28 (0.12–0.66), P = 0.002] in stage III melanoma. Gene set enrichment analysis identified an immune-related gene expression signature in PD-L1–positive tumors. There was a marked increase in cytotoxic T-cell and macrophage-specific genes in PD-L1–positive melanomas. CD8Ahigh gene expression was associated with better melanoma-specific survival [HR = 0.2 (0.05–0.87), P = 0.017] and restricted to PD-L1–positive stage III specimens. NF1 mutations were restricted to PD-L1–positive tumors (P = 0.041). Conclusions: Tumor negative PD-L1 status in stage III melanoma lymph node metastasis is a marker of worse patient survival and is associated with a poor immune response gene signature. Lower NSM levels were associated with PD-L1–negative status suggesting differences in somatic mutation profiles are a determinant of PD-L1–associated antitumor immunity in stage III melanoma. Clin Cancer Res; 22(15); 3915–23. ©2016 AACR.

Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes

The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma.

Transcriptomic analysis of Nodal- and BMP-associated genes during juvenile development of the sea urchin Heliocidaris erythrogramma

Understanding the unusual radial body plan of echinoderms and its relationship to the bilateral plan of other deuterostomes remains a challenge. The molecular processes of embryonic and early larval development in sea urchins are well characterised, but those giving rise to the adult and its radial body remain poorly studied. We used the developmental transcriptome generated for Heliocidaris erythrogramma, a species that forms the juvenile soon after gastrulation, to investigate changes in gene expression underlying radial body development. As coelomogenesis is key to the development of pentamery and juvenile formation on the left side of the larva, we focussed on genes associated with the nodal and BMP2/4 network that pattern this asymmetry. We identified 46 genes associated with this Nodal and BMP2/4 signalling network, and determined their expression profiles from the gastrula, through to rudiment development, metamorphosis and the fully formed juvenile. Genes associated with Nodal signalling shared similar expression profiles, indicating that they may have a regulatory relationship in patterning morphogenesis of the juvenile sea urchin. Similarly, many genes associated with BMP2/4 signalling had similar expression profiles through juvenile development. Further examination of the roles of Nodal- and BMP2/4-associated genes is required to determine function and whether the gene expression profiles seen in H. erythrogramma are due to ongoing activity of gene networks established during early development, or to redeployment of regulatory cassettes to pattern the adult radial body plan.

Nodal and BMP expression during the transition to pentamery in the sea urchin Heliocidaris erythrogramma : insights into patterning the enigmatic echinoderm body plan

BackgroundThe molecular mechanisms underlying the development of the unusual echinoderm pentameral body plan and their likeness to mechanisms underlying the development of the bilateral plans of other deuterostomes are of interest in tracing body plan evolution. In this first study of the spatial expression of genes associated with Nodal and BMP2/4 signalling during the transition to pentamery in sea urchins, we investigate Heliocidaris erythrogramma, a species that provides access to the developing adult rudiment within days of fertilization.ResultsBMP2/4, and the putative downstream genes, Six1/2, Eya, Tbx2/3 and Msx were expressed in the earliest morphological manifestation of pentamery during development, the five hydrocoele lobes. The formation of the vestibular ectoderm, the specialized region overlying the left coelom that forms adult ectoderm, involved the expression of putative Nodal target genes Chordin, Gsc and BMP2/4 and putative BMP2/4 target genes Dlx, Msx and Tbx. The expression of Nodal, Lefty and Pitx2 in the right ectoderm, and Pitx2 in the right coelom, was as previously observed in other sea urchins.ConclusionThat genes associated with Nodal and BMP2/4 signalling are expressed in the hydrocoele lobes, indicates that they have a role in the developmental transition to pentamery, contributing to our understanding of how the most unusual body plan in the Bilateria may have evolved. We suggest that the Nodal and BMP2/4 signalling cascades might have been duplicated or split during the evolution to pentamery.

ClassifyR: an R package for performance assessment of classification with applications to transcriptomics

UNLABELLED Although a large collection of classification software packages exist in R, a new generic framework for linking custom classification functions with classification performance measures is needed. A generic classification framework has been designed and implemented as an R package in an object oriented style. Its design places emphasis on parallel processing, reproducibility and extensibility. Finally, a comprehensive set of performance measures are available to ease post-processing. Taken together, these important characteristics enable rapid and reproducible benchmarking of alternative classifiers. AVAILABILITY AND IMPLEMENTATION ClassifyR is implemented in R and can be obtained from the Bioconductor project: http://bioconductor.org/packages/release/bioc/html/ClassifyR.html.

Differential distribution improves gene selection stability and has competitive classification performance for patient survival

A consistent difference in average expression level, often referred to as differential expression (DE), has long been used to identify genes useful for classification. However, recent cancer studies have shown that when transcription factors or epigenetic signals become deregulated, a change in expression variability (DV) of target genes is frequently observed. This suggests that assessing the importance of genes by either differential expression or variability alone potentially misses sets of important biomarkers that could lead to improved predictions and treatments. Here, we describe a new approach for assessing the importance of genes based on differential distribution (DD), which combines information from differential expression and differential variability into a unified metric. We show that feature ranking and selection stability based on DD can perform two to three times better than DE or DV alone, and that DD yields equivalent error rates to DE and DV. Finally, assessing genes via differential distribution produces a complementary set of selected genes to DE and DV, potentially opening up new categories of biomarkers.

Quantitative Performance Evaluator for Proteomics (QPEP): Web-based Application for Reproducible Evaluation of Proteomics Preprocessing Methods

Tandem mass spectrometry is one of the most popular techniques for quantitation of proteomes. There exists a large variety of options in each stage of data preprocessing that impact the bias and variance of the summarized protein-level values. Using a newly released data set satisfying a replicated Latin squares design, a diverse set of performance metrics has been developed and implemented in a web-based application, Quantitative Performance Evaluator for Proteomics (QPEP). QPEP has the flexibility to allow users to apply their own method to preprocess this data set and share the results, allowing direct and straightforward comparison of new methodologies. Application of these new metrics to three case studies highlights that (i) the summarization of peptides to proteins is robust to the choice of peptide summary used, (ii) the differences between iTRAQ labels are stronger than the differences between experimental runs, and (iii) the commercial software ProteinPilot performs equivalently well at between-sample normalization to more complicated methods developed by academics. Importantly, finding (ii) underscores the benefits of using the principles of randomization and blocking to avoid the experimental measurements being confounded by technical factors. Data are available via ProteomeXchange with identifier PXD003608.