Jovana Maksimovic

SWAN: Subset-quantile Within Array Normalization for Illumina Infinium HumanMethylation450 BeadChips

DNA methylation is the most widely studied epigenetic mark and is known to be essential to normal development and frequently disrupted in disease. The Illumina HumanMethylation450 BeadChip assays the methylation status of CpGs at 485,577 sites across the genome. Here we present Subset-quantile Within Array Normalization (SWAN), a new method that substantially improves the results from this platform by reducing technical variation within and between arrays. SWAN is available in the minfi Bioconductor package.

Genome-wide DNA methylation analysis identifies hypomethylated genes regulated by FOXP3 in human regulatory T cells

Regulatory T cells (Treg) prevent the emergence of autoimmune disease. Prototypic natural Treg (nTreg) can be reliably identified by demethylation at the Forkhead-box P3 (FOXP3) locus. To explore the methylation landscape of nTreg, we analyzed genome-wide methylation in human naive nTreg (rTreg) and conventional naive CD4(+) T cells (Naive). We detected 2315 differentially methylated cytosine-guanosine dinucleotides (CpGs) between these 2 cell types, many of which clustered into 127 regions of differential methylation (RDMs). Activation changed the methylation status of 466 CpGs and 18 RDMs in Naive but did not alter DNA methylation in rTreg. Gene-set testing of the 127 RDMs showed that promoter methylation and gene expression were reciprocally related. RDMs were enriched for putative FOXP3-binding motifs. Moreover, CpGs within known FOXP3-binding regions in the genome were hypomethylated. In support of the view that methylation limits access of FOXP3 to its DNA targets, we showed that increased expression of the immune suppressive receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), which delineated Treg from activated effector T cells, was associated with hypomethylation and FOXP3 binding at the TIGIT locus. Differential methylation analysis provides insight into previously undefined human Treg signature genes and their mode of regulation.

Bacteriophages in gut samples from pediatric Crohn's disease patients: metagenomic analysis using 454 pyrosequencing.

Background: The role of bacteriophage in Crohn’s disease (CD) is unknown. This study investigated the abundance of phages in ileal and colonic samples from pediatric CD patients and controls. Methods: Ileal and colonic biopsies from 6 CD patients, gut wash samples from 3 CD patients, and ileal biopsies from 6 noninflammatory bowel disease patients (controls) were analyzed for the presence of bacteriophage using 454 high-throughput pyrosequencing. A sequence-independent single-primer amplification method was used to amplify viral sequences. Results: A total of 186,143 high quality reads were obtained from the 4 sample populations. Contigs and sequence clusters (generated from unassembled singletons) were aligned with sequences from the National Center for Biotechnology Information viral reference database and analyzed by MEGAN. The largest number of viral hits was obtained from the CD gut wash samples (n = 691), followed by CD ileal samples (n = 52), control ileum samples (n = 20), and CD colonic samples (n = 1). The most abundant virus sequences identified belonged to the Caudovirales phage. Conclusions: Our study characterized a diverse phage community in the gut of CD patients. In this study, we have identified differences in phage composition between CD patients and control individuals. The large abundance of phages in CD ileum tissue and CD gut wash sample suggests a role of phage in CD development. The role of phage dysbiosis in CD is currently unknown but opens up a new area of research.

missMethyl: an R package for analyzing data from Illumina’s HumanMethylation450 platform

UNLABELLED DNA methylation is one of the most commonly studied epigenetic modifications due to its role in both disease and development. The Illumina HumanMethylation450 BeadChip is a cost-effective way to profile >450 000 CpGs across the human genome, making it a popular platform for profiling DNA methylation. Here we introduce missMethyl, an R package with a suite of tools for performing normalization, removal of unwanted variation in differential methylation analysis, differential variability testing and gene set analysis for the 450K array. AVAILABILITY AND IMPLEMENTATION missMethyl is an R package available from the Bioconductor project at www.bioconductor.org. CONTACT alicia.oshlack@mcri.edu.au SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Condensin I associates with structural and gene regulatory regions in vertebrate chromosomes

The condensin complex is essential for correct packaging and segregation of chromosomes during mitosis and meiosis in all eukaryotes. To date, the genome-wide location and the nature of condensin-binding sites have remained elusive in vertebrates. Here we report the genome-wide map of condensin I in chicken DT40 cells. Unexpectedly, we find that condensin I binds predominantly to promoter sequences in mitotic cells. We also find a striking enrichment at both centromeres and telomeres, highlighting the importance of the complex in chromosome segregation. Taken together, the results show that condensin I is largely absent from heterochromatic regions. This map of the condensin I binding sites on the chicken genome reveals that patterns of condensin distribution on chromosomes are conserved from prokaryotes, through yeasts to vertebrates. Thus in three kingdoms of life, condensin is enriched on promoters of actively transcribed genes and at loci important for chromosome segregation.

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

The polycomb repressive complex 2 governs life and death of peripheral T cells

Differentiation of naïve CD4(+) T cells into effector (Th1, Th2, and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by the Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). Here we show that silencing of the Ifng, Gata3, and Il10 loci in naïve CD4(+) T cells is dependent on Ezh2. Naïve CD4(+) T cells lacking Ezh2 were epigenetically primed for overproduction of IFN-γ in Th2 and iTreg and IL-10 in Th2 cells. In addition, deficiency of Ezh2 accelerated effector Th cell death via death receptor-mediated extrinsic and intrinsic apoptotic pathways, confirmed in vivo for Ezh2-null IFN-γ-producing CD4(+) and CD8(+) T cells responding to Listeria monocytogenes infection. These findings demonstrate the key role of PRC2/Ezh2 in differentiation and survival of peripheral T cells and reveal potential immunotherapeutic targets.

Removing unwanted variation in a differential methylation analysis of Illumina HumanMethylation450 array data

Due to their relatively low-cost per sample and broad, gene-centric coverage of CpGs across the human genome, Illuminas 450k arrays are widely used in large scale differential methylation studies. However, by their very nature, large studies are particularly susceptible to the effects of unwanted variation. The effects of unwanted variation have been extensively documented in gene expression array studies and numerous methods have been developed to mitigate these effects. However, there has been much less research focused on the appropriate methodology to use for accounting for unwanted variation in methylation array studies. Here we present a novel 2-stage approach using RUV-inverse in a differential methylation analysis of 450k data and show that it outperforms existing methods.

Genome-scale profiling reveals a subset of genes regulated by DNA methylation that program somatic T-cell phenotypes in humans

The aim of this study was to investigate the dynamics and relationship between DNA methylation and gene expression during early T-cell development. Mononuclear cells were collected at birth and at 12 months from 60 infants and were either activated with anti-CD3 for 24 h or cultured in media alone, and the CD4+ T-cell subset purified. DNA and RNA were co-harvested and DNA methylation was measured in 450 000 CpG sites in parallel with expression measurements taken from 25 000 genes. In unstimulated cells, we found that a subset of 1188 differentially methylated loci were associated with a change in expression in 599 genes (adjusted P value<0.01, β-fold >0.1). These genes were enriched in reprogramming regions of the genome known to control pluripotency. In contrast, over 630 genes were induced following low-level T-cell activation, but this was not associated with any significant change in DNA methylation. We conclude that DNA methylation is dynamic during early T-cell development, and has a role in the consolidation of T-cell-specific gene expression. During the early phase of clonal expansion, DNA methylation is stable and therefore appears to be of limited importance in short-term T-cell responsiveness.

Stability of gene expression and epigenetic profiles highlights the utility of patient-derived paediatric acute lymphoblastic leukaemia xenografts for investigating molecular mechanisms of drug resistance

BackgroundPatient-derived tumour xenografts are an attractive model for preclinical testing of anti-cancer drugs. Insights into tumour biology and biomarkers predictive of responses to chemotherapeutic drugs can also be gained from investigating xenograft models. As a first step towards examining the equivalence of epigenetic profiles between xenografts and primary tumours in paediatric leukaemia, we performed genome-scale DNA methylation and gene expression profiling on a panel of 10 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) tumours that were stratified by prednisolone response.ResultsWe found high correlations in DNA methylation and gene expression profiles between matching primary and xenograft tumour samples with Pearson’s correlation coefficients ranging between 0.85 and 0.98. In order to demonstrate the potential utility of epigenetic analyses in BCP-ALL xenografts, we identified DNA methylation biomarkers that correlated with prednisolone responsiveness of the original tumour samples. Differential methylation of CAPS2, ARHGAP21, ARX and HOXB6 were confirmed by locus specific analysis. We identified 20 genes showing an inverse relationship between DNA methylation and gene expression in association with prednisolone response. Pathway analysis of these genes implicated apoptosis, cell signalling and cell structure networks in prednisolone responsiveness.ConclusionsThe findings of this study confirm the stability of epigenetic and gene expression profiles of paediatric BCP-ALL propagated in mouse xenograft models. Further, our preliminary investigation of prednisolone sensitivity highlights the utility of mouse xenograft models for preclinical development of novel drug regimens with parallel investigation of underlying gene expression and epigenetic responses associated with novel drug responses.