PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (71)
 

Clipboard (0)
None

Select a Filter Below

Year of Publication
more »
1.  Epigenome data release: a participant-centered approach to privacy protection 
Genome Biology  2015;16(1):142.
Large-scale epigenome mapping by the NIH Roadmap Epigenomics Project, the ENCODE Consortium and the International Human Epigenome Consortium (IHEC) produces genome-wide DNA methylation data at one base-pair resolution. We examine how such data can be made open-access while balancing appropriate interpretation and genomic privacy. We propose guidelines for data release that both reduce ambiguity in the interpretation of open-access data and limit immediate access to genetic variation data that are made available through controlled access.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0723-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-015-0723-0
PMCID: PMC4504083  PMID: 26185018
2.  Brave new epigenomes: the dawn of epigenetic engineering 
Genome Medicine  2015;7(1):59.
Editorial summary
New methods for epigenome editing now make it possible to manipulate the epigenome in living cells with unprecedented specificity and efficiency. These ground-breaking approaches are beginning to yield novel insights into the function of individual chromatin marks in the context of cellular phenotype.
doi:10.1186/s13073-015-0185-8
PMCID: PMC4472160  PMID: 26089986
4.  Assessment of RainDrop BS-seq as a method for large-scale, targeted bisulfite sequencing 
Epigenetics  2014;9(5):678-684.
We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.
doi:10.4161/epi.28041
PMCID: PMC4063826  PMID: 24518816
DNA methylation; EWAS; RainDrop BS-seq; epigenetic biomarker; microdroplet PCR; targeted bisulfite sequencing
5.  Accurate Measurement of 5-Methylcytosine and 5-Hydroxymethylcytosine in Human Cerebellum DNA by Oxidative Bisulfite on an Array (OxBS-Array) 
PLoS ONE  2015;10(2):e0118202.
The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The “methylation” level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3%. We performed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3% and 42%. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.
doi:10.1371/journal.pone.0118202
PMCID: PMC4338296  PMID: 25706862
6.  An Integrative Multi-scale Analysis of the Dynamic DNA Methylation Landscape in Aging 
PLoS Genetics  2015;11(2):e1004996.
Recent studies have demonstrated that the DNA methylome changes with age. This epigenetic drift may have deep implications for cellular differentiation and disease development. However, it remains unclear how much of this drift is functional or caused by underlying changes in cell subtype composition. Moreover, no study has yet comprehensively explored epigenetic drift at different genomic length scales and in relation to regulatory elements.
Here we conduct an in-depth analysis of epigenetic drift in blood tissue. We demonstrate that most of the age-associated drift is independent of the increase in the granulocyte to lymphocyte ratio that accompanies aging and that enrichment of age-hypermethylated CpG islands increases upon adjustment for cellular composition. We further find that drift has only a minimal impact on in-cis gene expression, acting primarily to stabilize pre-existing baseline expression levels. By studying epigenetic drift at different genomic length scales, we demonstrate the existence of mega-base scale age-associated hypomethylated blocks, covering approximately 14% of the human genome, and which exhibit preferential hypomethylation in age-matched cancer tissue. Importantly, we demonstrate the feasibility of integrating Illumina 450k DNA methylation with ENCODE data to identify transcription factors with key roles in cellular development and aging. Specifically, we identify REST and regulatory factors of the histone methyltransferase MLL complex, whose function may be disrupted in aging.
In summary, most of the epigenetic drift seen in blood is independent of changes in blood cell type composition, and exhibits patterns at different genomic length scales reminiscent of those seen in cancer. Integration of Illumina 450k with appropriate ENCODE data may represent a fruitful approach to identify transcription factors with key roles in aging and disease.
Author Summary
Two well-known features of aging are the gradual decline of the body’s ability to regenerate tissues, as well as an increased incidence of diseases like cancer and Alzheimers. One of the most recent exciting findings which may underlie the aging process is a gradual modification of DNA, called epigenetic drift, which is effected by the covalent addition and removal of methyl groups, which in turn can deregulate the activity of nearby genes. However, this study presents the most convincing evidence to date that epigenetic drift acts to stabilize the activity levels of nearby genes. This study shows that instead, epigenetic drift may act primarly to disrupt DNA binding patterns of proteins which regulate the activity of many genes, and moreover identifies specific regulatory proteins with key roles in cancer and Alzheimers. The study also performs the most comprehensive analysis of epigenetic drift at different spatial scales, demonstrating that epigenetic drift on the largest length scales is highly reminiscent of those seen in cancer. In summary, this work substantially supports the view that epigenetic drift may contribute to the age-associated increased risk of diseases like cancer and Alzheimers, by disrupting master regulators of genomewide gene activity.
doi:10.1371/journal.pgen.1004996
PMCID: PMC4334892  PMID: 25692570
7.  Comparative methylome analysis identifies new tumour subtypes and biomarkers for transformation of nephrogenic rests into Wilms tumour 
Genome Medicine  2015;7(1):11.
Background
Wilms tumours (WTs) are characterised by several hallmarks that suggest epimutations such as aberrant DNA methylation are involved in tumour progression: loss of imprinting at 11p15, lack of recurrent mutations and formation of nephrogenic rests (NRs), which are lesions of retained undifferentiated embryonic tissue that can give rise to WTs.
Methods
To identify such epimutations, we performed a comprehensive methylome analysis on 20 matched trios of micro-dissected WTs, NRs and surrounding normal kidneys (NKs) using Illumina Infinium HumanMethylation450 Bead Chips and functionally validated findings using RNA sequencing.
Results
Comparison of NRs with NK revealed prominent tissue biomarkers: 629 differentially methylated regions, of which 55% were hypermethylated and enriched for domains that are bivalent in embryonic stem cells and for genes expressed during development (P = 2.49 × 10-5). Comparison of WTs with NRs revealed two WT subgroups; group-2 WTs and NRs were epigenetically indistinguishable whereas group-1 WTs showed an increase in methylation variability, hypomethylation of renal development genes, hypermethylation and relative loss of expression of cell adhesion genes and known and potential new WT tumour suppressor genes (CASP8, H19, MIR195, RB1 and TSPAN32) and was strongly associated with bilateral disease (P = 0.032). Comparison of WTs and NRs to embryonic kidney highlighted the significance of polycomb target methylation in Wilms tumourigenesis.
Conclusions
Methylation levels vary during cancer evolution. We have described biomarkers related to WT evolution from its precursor NRs which may be useful to differentiate between these tissues for patients with bilateral disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s13073-015-0136-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13073-015-0136-4
PMCID: PMC4354990  PMID: 25763109
8.  Analysis pipelines and packages for Infinium HumanMethylation450 BeadChip (450k) data 
The Illumina HumanMethylation450 BeadChip has become a popular platform for interrogating DNA methylation in epigenome-wide association studies (EWAS) and related projects as well as resource efforts such as the International Cancer Genome Consortium (ICGC) and the International Human Epigenome Consortium (IHEC). This has resulted in an exponential increase of 450k data in recent years and triggered the development of numerous integrated analysis pipelines and stand-alone packages. This review will introduce and discuss the currently most popular pipelines and packages and is particularly aimed at new 450k users.
doi:10.1016/j.ymeth.2014.08.011
PMCID: PMC4304832  PMID: 25233806
HumanMethylation450; Epigenetics; Analysis pipelines; EWAS; DNA methylation; 450k BeadChip
9.  Probe Lasso: A novel method to rope in differentially methylated regions with 450K DNA methylation data 
The speed and resolution at which we can scour the genome for DNA methylation changes has improved immeasurably in the last 10 years and the advent of the Illumina 450K BeadChip has made epigenome-wide association studies (EWAS) a reality. The resulting datasets are conveniently formatted to allow easy alignment of significant hits to genes and genetic features, however; methods that parse significant hits into discreet differentially methylated regions (DMRs) remain a challenge to implement. In this paper we present details of a novel DMR caller, the Probe Lasso: a flexible window based approach that gathers neighbouring significant-signals to define clear DMR boundaries for subsequent in-depth analysis. The method is implemented in the R package ChAMP (Morris et al., 2014) and returns sets of DMRs according to user-tuned levels of probe filtering (e.g., inclusion of sex chromosomes, polymorphisms) and probe-lasso size distribution. Using a sub-sample of colon cancer- and healthy colon-samples from TCGA we show that Probe Lasso shifts DMR calling away from just probe-dense regions, and calls a range of DMR sizes ranging from tens-of-bases to tens-of-kilobases in scale. Moreover, using TCGA data we show that Probe Lasso leverages more information from the array and highlights a potential role of hypomethylated transcription factor binding motifs not discoverable using a basic, fixed-window approach.
doi:10.1016/j.ymeth.2014.10.036
PMCID: PMC4304833  PMID: 25461817
Differentially methylated regions; DNA methylation; Epigenetics; EWAS; Illumina 450K BeadChip
10.  oxBS-450K: A method for analysing hydroxymethylation using 450K BeadChips 
Highlights
•A method is presented for 5hmC detection and analysis using Infinium 450K BeadChips.•The oxBS-450K method can discriminate between 5mC and 5hmC in human gDNA•5hmC levels were quantified genome-wide in 3 distinct biological samples.•The reported 5hmC signal was validated using mass spectrometry and pyrosequencing.•The effects of differing amounts of input DNA on final 5hmC call rate are discussed.
DNA methylation analysis has become an integral part of biomedical research. For high-throughput applications such as epigenome-wide association studies, the Infinium HumanMethylation450 (450K) BeadChip is currently the platform of choice. However, BeadChip processing relies on traditional bisulfite (BS) based protocols which cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we report the adaptation of the recently developed oxidative bisulfite (oxBS) chemistry to specifically detect both 5mC and 5hmC in a single workflow using 450K BeadChips, termed oxBS-450K. Supported by validation using mass spectrometry and pyrosequencing, we demonstrate reproducible (R2 > 0.99) detection of 5hmC in human brain tissue using the optimised oxBS-450K protocol described here.
doi:10.1016/j.ymeth.2014.08.009
PMCID: PMC4304834  PMID: 25175075
Hydroxymethylation; DNA methylation; 450K BeadChip; Oxidation; Bisulfite conversion; Epigenetics
11.  Advances in epigenome-wide association studies for common diseases 
Trends in Molecular Medicine  2014;20(10):541-543.
Epigenome-wide association studies (EWASs) provide a systematic approach to uncovering epigenetic variants underlying common diseases. Discoveries have shed light on novel molecular mechanisms of disease and enabled the application of epigenetic variants as biomarkers. Here, we highlight the recent advances in this emerging line of research and discuss key challenges for current and future studies.
doi:10.1016/j.molmed.2014.07.002
PMCID: PMC4186777  PMID: 25092140
GWAS; EWAS; DNA methylation; disease mechanism; biomarker
12.  Methylome analysis identifies a Wilms tumor epigenetic biomarker detectable in blood 
Genome Biology  2014;15(8):434.
Background
Wilms tumor is the most common pediatric renal malignancy and there is a clinical need for a molecular biomarker to assess treatment response and predict relapse. The known mutated genes in this tumor type show low mutation frequencies, whereas aberrant methylation at 11p15 is by far the most common aberration. We therefore analyzed the epigenome, rather than the genome, to identify ubiquitous tumor-specific biomarkers.
Results
Methylome analysis of matched normal kidney and Wilms tumor identifies 309 preliminary methylation variable positions which we translate into three differentially methylated regions (DMRs) for use as tumor-specific biomarkers. Using two novel algorithms we show that these three DMRs are not confounded by cell type composition. We further show that these DMRs are not methylated in embryonic blastema but are intermediately methylated in Wilms tumor precursor lesions. We validate the biomarker DMRs using two independent sample sets of normal kidney and Wilms tumor and seven Wilms tumor histological subtypes, achieving 100% and 98% correct classification, respectively. As proof-of-principle for clinical utility, we successfully use biomarker DMR-2 in a pilot analysis of cell-free circulating DNA to monitor tumor response during treatment in ten patients.
Conclusions
These findings define the most common methylated regions in Wilms tumor known to date which are not associated with their embryonic origin or precursor stage. We show that this tumor-specific methylated DNA is released into the blood circulation where it can be detected non-invasively showing potential for clinical utility.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0434-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0434-y
PMCID: PMC4310621  PMID: 25134821
13.  The Dynamics of DNA Methylation Covariation Patterns in Carcinogenesis 
PLoS Computational Biology  2014;10(7):e1003709.
Recently it has been observed that cancer tissue is characterised by an increased variability in DNA methylation patterns. However, how the correlative patterns in genome-wide DNA methylation change during the carcinogenic progress has not yet been explored. Here we study genome-wide inter-CpG correlations in DNA methylation, in addition to single site variability, during cervical carcinogenesis. We demonstrate how the study of changes in DNA methylation covariation patterns across normal, intra-epithelial neoplasia and invasive cancer allows the identification of CpG sites that indicate the risk of neoplastic transformation in stages prior to neoplasia. Importantly, we show that the covariation in DNA methylation at these risk CpG loci is maximal immediately prior to the onset of cancer, supporting the view that high epigenetic diversity in normal cells increases the risk of cancer. Consistent with this, we observe that invasive cancers exhibit increased covariation in DNA methylation at the risk CpG sites relative to normal tissue, but lower levels relative to pre-cancerous lesions. We further show that the identified risk CpG sites undergo preferential DNA methylation changes in relation to human papilloma virus infection and age. Results are validated in independent data including prospectively collected samples prior to neoplastic transformation. Our data are consistent with a phase transition model of carcinogenesis, in which epigenetic diversity is maximal prior to the onset of cancer. The model and algorithm proposed here may allow, in future, network biomarkers predicting the risk of neoplastic transformation to be identified.
Author Summary
DNA methylation is a covalent modification of DNA which can regulate how active genes are. DNA methylation is altered at many genomic loci in cancer cells, leading to widespread functional disruption. Importantly, DNA methylation alterations across the genome are seen even in early carcinogenesis. Although the pattern of DNA methylation change during carcinogenesis has been studied at individual genomic loci, no study has yet analysed how these patterns change at a systems-level, specifically how do DNA methylation patterns at pairs of genomic sites change during disease progression. Doing so can shed light on how the epigenetic diversity of cell populations changes during the carcinogenic process. This study performs a systems-level analysis of the dynamic changes in DNA methylation correlation pattern during cervical carcinogenesis, demonstrating that epigenetic diversity is maximal just prior to the onset of cancer. Importantly, this supports the view that the risk of cancer development is closely related to an increase in epigenetic diversity in apparently healthy cells. In addition, the study provides a computational algorithm which successfully identifies the altered genomic sites confering the risk of cervical cancer.
doi:10.1371/journal.pcbi.1003709
PMCID: PMC4091688  PMID: 25010556
14.  Sequence of a Complete Chicken BG Haplotype Shows Dynamic Expansion and Contraction of Two Gene Lineages with Particular Expression Patterns 
PLoS Genetics  2014;10(6):e1004417.
Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5′ untranslated regions (5′UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.
Author Summary
Many immune genes are multigene families, presumably in response to pathogen variation. Some multigene families undergo expansion and contraction, leading to copy number variation (CNV), presumably due to more intense selection. Recently, the butyrophilin family in humans and other mammals has come under scrutiny, due to genetic associations with autoimmune diseases as well as roles in immune co-regulation and antigen presentation. Butyrophilin genes exhibit allelic polymorphism, but gene number appears stable within a species. We found that the BG homologues in chickens are very different, with great changes between haplotypes. We characterised one haplotype in detail, showing that there are two single BG genes, one on chromosome 2 and the other in the major histocompatibility complex (BF-BL region) on chromosome 16, and a family of BG genes in a tandem array in the BG region nearby. These genes have specific expression in cells and tissues, but overall are expressed in either haemopoietic cells or tissues. The two singletons have relatively stable evolutionary histories, but the BG region undergoes dynamic expansion and contraction, with the production of hybrid genes. Thus, chicken BG genes appear to evolve much more quickly than their closest homologs in mammals, presumably due to increased pressure from pathogens.
doi:10.1371/journal.pgen.1004417
PMCID: PMC4046983  PMID: 24901252
15.  C2c: turning cancer into chronic disease 
Genome Medicine  2014;6(5):38.
doi:10.1186/gm555
PMCID: PMC4062053  PMID: 24944585
16.  Genome-wide age-related changes in DNA methylation and gene expression in human PBMCs 
Age  2014;36(3):9648.
Aging is a progressive process that results in the accumulation of intra- and extracellular alterations that in turn contribute to a reduction in health. Age-related changes in DNA methylation have been reported before and may be responsible for aging-induced changes in gene expression, although a causal relationship has yet to be shown. Using genome-wide assays, we analyzed age-induced changes in DNA methylation and their effect on gene expression with and without transient induction with the synthetic transcription modulating agent WY14,643. To demonstrate feasibility of the approach, we isolated peripheral blood mononucleated cells (PBMCs) from five young and five old healthy male volunteers and cultured them with or without WY14,643. Infinium 450K BeadChip and Affymetrix Human Gene 1.1 ST expression array analysis revealed significant differential methylation of at least 5 % (ΔYO > 5 %) at 10,625 CpG sites between young and old subjects, but only a subset of the associated genes were also differentially expressed. Age-related differential methylation of previously reported epigenetic biomarkers of aging including ELOVL2, FHL2, PENK, and KLF14 was confirmed in our study, but these genes did not display an age-related change in gene expression in PBMCs. Bioinformatic analysis revealed that differentially methylated genes that lack an age-related expression change predominantly represent genes involved in carcinogenesis and developmental processes, and expression of most of these genes were silenced in PBMCs. No changes in DNA methylation were found in genes displaying transiently induced changes in gene expression. In conclusion, aging-induced differential methylation often targets developmental genes and occurs mostly without change in gene expression.
Electronic supplementary material
The online version of this article (doi:10.1007/s11357-014-9648-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s11357-014-9648-x
PMCID: PMC4082572  PMID: 24789080
Molecular aging; Epigenetics; DNA methylation; Gene expression; PBMCs; Epigenetic biomarkers of aging
17.  Human-specific epigenetic variation in the immunological Leukotriene B4 Receptor (LTB4R/BLT1) implicated in common inflammatory diseases 
Genome Medicine  2014;6(3):19.
Background
Common human diseases are caused by the complex interplay of genetic susceptibility as well as environmental factors. Due to the environment’s influence on the epigenome, and therefore genome function, as well as conversely the genome’s facilitative effect on the epigenome, analysis of this level of regulation may increase our knowledge of disease pathogenesis.
Methods
In order to identify human-specific epigenetic influences, we have performed a novel genome-wide DNA methylation analysis comparing human, chimpanzee and rhesus macaque.
Results
We have identified that the immunological Leukotriene B4 receptor (LTB4R, BLT1 receptor) is the most epigenetically divergent human gene in peripheral blood in comparison with other primates. This difference is due to the co-ordinated active state of human-specific hypomethylation in the promoter and human-specific increased gene body methylation. This gene is significant in innate immunity and the LTB4/LTB4R pathway is involved in the pathogenesis of the spectrum of human inflammatory diseases. This finding was confirmed by additional neutrophil-only DNA methylome and lymphoblastoid H3K4me3 chromatin comparative data. Additionally we show through functional analysis that this receptor has increased expression and a higher response to the LTB4 ligand in human versus rhesus macaque peripheral blood mononuclear cells. Genome-wide we also find human species-specific differentially methylated regions (human s-DMRs) are more prevalent in CpG island shores than within the islands themselves, and within the latter are associated with the CTCF motif.
Conclusions
This result further emphasises the exclusive nature of the human immunological system, its divergent adaptation even from very closely related primates, and the power of comparative epigenomics to identify and understand human uniqueness.
doi:10.1186/gm536
PMCID: PMC4062055  PMID: 24598577
18.  Using high-density DNA methylation arrays to profile copy number alterations 
Genome Biology  2014;15(2):R30.
The integration of genomic and epigenomic data is an increasingly popular approach for studying the complex mechanisms driving cancer development. We have developed a method for evaluating both methylation and copy number from high-density DNA methylation arrays. Comparing copy number data from Infinium HumanMethylation450 BeadChips and SNP arrays, we demonstrate that Infinium arrays detect copy number alterations with the sensitivity of SNP platforms. These results show that high-density methylation arrays provide a robust and economic platform for detecting copy number and methylation changes in a single experiment. Our method is available in the ChAMP Bioconductor package: http://www.bioconductor.org/packages/2.13/bioc/html/ChAMP.html.
doi:10.1186/gb-2014-15-2-r30
PMCID: PMC4054098  PMID: 24490765
19.  DNA methylation analysis of murine hematopoietic side population cells during aging 
Epigenetics  2013;8(10):1114-1122.
Stem cells have been found in most tissues/organs. These somatic stem cells produce replacements for lost and damaged cells, and it is not completely understood how this regenerative capacity becomes diminished during aging. To study the possible involvement of epigenetic changes in somatic stem cell aging, we used murine hematopoiesis as a model system. Hematopoietic stem cells (HSCs) were enriched for via Hoechst exclusion activity (SP-HSC) from young, medium-aged and old mice and subjected to comprehensive, global methylome (MeDIP-seq) analysis. With age, we observed a global loss of DNA methylation of approximately 5%, but an increase in methylation at some CpG islands. Just over 100 significant (FDR < 0.2) aging-specific differentially methylated regions (aDMRs) were identified, which are surprisingly few considering the profound age-based changes that occur in HSC biology. Interestingly, the polycomb repressive complex -2 (PCRC2) target genes Kiss1r, Nav2 and Hsf4 were hypermethylated with age. The promoter for the Sdpr gene was determined to be progressively hypomethylated with age. This occurred concurrently with an increase in gene expression with age. To explore this relationship further, we cultured isolated SP-HSC in the presence of 5-aza-deoxycytdine and demonstrated a negative correlation between Sdpr promoter methylation and gene expression. We report that DNA methylation patterns are well preserved during hematopoietic stem cell aging, confirm that PCRC2 targets are increasingly methylated with age, and suggest that SDPR expression changes with age in HSCs may be regulated via age-based alterations in DNA methylation.
doi:10.4161/epi.26017
PMCID: PMC3891692  PMID: 23949429
hematopoietic stem cells; aging; epigenetics; methylomics; methylome; Nano-MeDIP-seq; DNA methylation; Sdpr polycomb repressive complex -2 (PCRC2); Nav2; Kiss1r; Hsf4
20.  Age-associated epigenetic drift: implications, and a case of epigenetic thrift? 
Human Molecular Genetics  2013;22(R1):R7-R15.
It is now well established that the genomic landscape of DNA methylation (DNAm) gets altered as a function of age, a process we here call ‘epigenetic drift’. The biological, functional, clinical and evolutionary significance of this epigenetic drift, however, remains unclear. We here provide a brief review of epigenetic drift, focusing on the potential implications for ageing, stem cell biology and disease risk prediction. It has been demonstrated that epigenetic drift affects most of the genome, suggesting a global deregulation of DNAm patterns with age. A component of this drift is tissue-specific, allowing remarkably accurate age-predictive models to be constructed. Another component is tissue-independent, targeting stem cell differentiation pathways and affecting stem cells, which may explain the observed decline of stem cell function with age. Age-associated increases in DNAm target developmental genes, overlapping those associated with environmental disease risk factors and with disease itself, notably cancer. In particular, cancers and precursor cancer lesions exhibit aggravated age DNAm signatures. Epigenetic drift is also influenced by genetic factors. Thus, drift emerges as a promising biomarker for premature or biological ageing, and could potentially be used in geriatrics for disease risk prediction. Finally, we propose, in the context of human evolution, that epigenetic drift may represent a case of epigenetic thrift, or bet-hedging. In summary, this review demonstrates the growing importance of the ‘ageing epigenome’, with potentially far-reaching implications for understanding the effect of age on stem cell function and differentiation, as well as for disease prevention.
doi:10.1093/hmg/ddt375
PMCID: PMC3782071  PMID: 23918660
21.  Integrated virus-host methylome analysis in head and neck squamous cell carcinoma 
Epigenetics  2013;8(9):953-961.
One in six cancers worldwide is caused by infection and human papillomavirus (HPV) is one of the main culprits. To better understand the dynamics of HPV integration and its effect on both the viral and host methylomes, we conducted whole-genome DNA methylation analysis using MeDIP-seq of HPV+ and HPV- head and neck squamous cell carcinoma (HNSCC). We determined the viral subtype to be HPV-16 in all cases and show that HPV-16 integrates into the host genome at multiple random sites and that this process predominantly involves the transcriptional repressor gene (E2) in the viral genome. Comparative analysis identified 453 (FDR ≤ 0.01) differentially methylated regions (DMRs) in the HPV+ host methylome. Bioinformatics characterization of these DMRs confirmed the previously reported cadherin genes to be affected but also revealed new targets for HPV-mediated methylation changes at regions not covered by array-based platforms, including the recently identified super-enhancers.
doi:10.4161/epi.25614
PMCID: PMC3883772  PMID: 23867721
human papillomavirus (HPV); head and neck squamous cell carcinoma (HNSCC); DNA methylation; methylome; epigenome
22.  The good, the bad and the ugly: Epigenetic mechanisms in glioblastoma 
Molecular Aspects of Medicine  2013;34(4):849-862.
Cell type-specific patterns of gene expression reflect epigenetic changes imposed through a particular developmental lineage as well as those triggered by environmental cues within adult tissues. There is great interest in elucidating the molecular basis and functional importance of epigenetic mechanisms in both normal physiology and disease – particularly in cancer, where abnormal ‘-omic’ states are often observed. In this article we review recent progress in studies of epigenetic mechanisms in the most common primary adult brain cancer, glioblastoma multiforme. Three distinct areas are discussed. First, the evidence in support of ongoing ‘normal’ epigenetic processes associated with differentiation – as predicted by ‘cancer stem cell’ models of the disease. Second, identification of site-specific and global epigenetic abnormalities. Third, genetic disruptions directly within the core epigenetic machinery, exemplified by the recently identified mutations within isocitrate dehydrogenase genes IDH1/2 and variant histone genes H3.3/H3F3A. These constitute the ‘good, the bad and the ugly’ of epigenetic mechanisms in cancer.
doi:10.1016/j.mam.2012.06.007
PMCID: PMC3714597  PMID: 22771539
Epigenetics; Glioma; DNA methylation; Central nervous system (CNS); Differentiation; Cancer stem cells
23.  Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors 
Genome Medicine  2013;5(5):49.
Background
Human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) is an emerging disease, representing a distinct clinical and epidemiological entity. Understanding the genetic basis of this specific subtype of cancer could allow therapeutic targeting of affected pathways for a stratified medicine approach.
Methods
Twenty HPV+ and 20 HPV- laser-capture microdissected oropharyngeal carcinomas were used for paired-end sequencing of hybrid-captured DNA, targeting 3,230 exons in 182 genes often mutated in cancer. Copy number alteration (CNA) profiling, Sequenom MassArray sequencing and immunohistochemistry were used to further validate findings.
Results
HPV+ and HPV- oropharyngeal carcinomas cluster into two distinct subgroups. TP53 mutations are detected in 100% of HPV negative cases and abrogation of the G1/S checkpoint by CDKN2A/B deletion and/or CCND1 amplification occurs in the majority of HPV- tumors.
Conclusion
These findings strongly support a causal role for HPV, acting via p53 and RB pathway inhibition, in the pathogenesis of a subset of oropharyngeal cancers and suggest that studies of CDK inhibitors in HPV- disease may be warranted. Mutation and copy number alteration of PI3 kinase (PI3K) pathway components appears particularly prevalent in HPV+ tumors and assessment of these alterations may aid in the interpretation of current clinical trials of PI3K, AKT, and mTOR inhibitors in HNSCC.
doi:10.1186/gm453
PMCID: PMC4064312  PMID: 23718828
24.  Sequencing and comparative analysis of the gorilla MHC genomic sequence 
Major histocompatibility complex (MHC) genes play a critical role in vertebrate immune response and because the MHC is linked to a significant number of auto-immune and other diseases it is of great medical interest. Here we describe the clone-based sequencing and subsequent annotation of the MHC region of the gorilla genome. Because the MHC is subject to extensive variation, both structural and sequence-wise, it is not readily amenable to study in whole genome shotgun sequence such as the recently published gorilla genome. The variation of the MHC also makes it of evolutionary interest and therefore we analyse the sequence in the context of human and chimpanzee. In our comparisons with human and re-annotated chimpanzee MHC sequence we find that gorilla has a trimodular RCCX cluster, versus the reference human bimodular cluster, and additional copies of Class I (pseudo)genes between Gogo-K and Gogo-A (the orthologues of HLA-K and -A). We also find that Gogo-H (and Patr-H) is coding versus the HLA-H pseudogene and, conversely, there is a Gogo-DQB2 pseudogene versus the HLA-DQB2 coding gene. Our analysis, which is freely available through the VEGA genome browser, provides the research community with a comprehensive dataset for comparative and evolutionary research of the MHC.
doi:10.1093/database/bat011
PMCID: PMC3626023  PMID: 23589541
25.  An integrative network algorithm identifies age-associated differential methylation interactome hotspots targeting stem-cell differentiation pathways 
Scientific Reports  2013;3:1630.
Epigenetic changes have been associated with ageing and cancer. Identifying and interpreting epigenetic changes associated with such phenotypes may benefit from integration with protein interactome models. We here develop and validate a novel integrative epigenome-interactome approach to identify differential methylation interactome hotspots associated with a phenotype of interest. We apply the algorithm to cancer and ageing, demonstrating the existence of hotspots associated with these phenotypes. Importantly, we discover tissue independent age-associated hotspots targeting stem-cell differentiation pathways, which we validate in independent DNA methylation data sets, encompassing over 1000 samples from different tissue types. We further show that these pathways would not have been discovered had we used a non-network based approach and that the use of the protein interaction network improves the overall robustness of the inference procedure. The proposed algorithm will be useful to any study seeking to identify interactome hotspots associated with common phenotypes.
doi:10.1038/srep01630
PMCID: PMC3620664  PMID: 23568264

Results 1-25 (71)