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1.  Phase II trial of the histone deacetylase inhibitor romidepsin in patients with recurrent/metastatic head and neck cancer 
Oral oncology  2012;48(12):1281-1288.
Patients with advanced squamous cell carcinoma of the head and neck (SCCHN) have limited treatment options. Inhibition of histone deacetylases (HDACs) represents a novel therapeutic approach warranting additional investigation in solid tumors.
A phase II trial of single agent romidepsin, an HDAC inhibitor, was performed in 14 patients with SCCHN who provided consent for pre- and post-therapy samples of accessible tumor, blood and uninvolved oral mucosa. Romidepsin was administered at 13 mg/m2 as a 4-hour intravenous infusion on days 1, 8 and 15 of 28 day cycles, with response assessment by RECIST every 8 weeks.
Objective responses were not observed, although 2 heavily pretreated patients had brief clinical disease stabilization. Observed toxicities were expected, including frequent severe fatigue. Immunohistochemical analysis of 7 pre- and post-treatment tumor pairs demonstrated induction of p21Waf1/Cip1 characteristic of HDAC inhibition, as well as decreased Ki67 staining. Exploratory microarray analyses of mucosal and tumor samples detected changes in gene expression following romidepsin treatment that were most commonly associated with regulation of transcription, cell cycle control, signal transduction, and electron transport. Treatment with romidepsin did not alter the extent of DNA methylation of candidate gene loci (including CDH1 and hMLH1) in SCCHN tumors.
Single agent romidepsin has limited activity for the treatment of SCCHN but can effectively achieve tumor-associated HDAC inhibition. Although tolerability of romidepsin in this setting may be limiting, further evaluation of other HDAC inhibitors in combination with active therapies may be justified.
PMCID: PMC3465519  PMID: 22748449
romidepsin; head and neck cancer; squamous cell carcinoma; histone deacetylase (HDAC) inhibitors; phase II trial
2.  DNA Methylation is Developmentally Regulated for Genes Essential for Cardiogenesis 
DNA methylation is a major epigenetic mechanism altering gene expression in development and disease. However, its role in the regulation of gene expression during heart development is incompletely understood. The aim of this study is to reveal DNA methylation in mouse embryonic hearts and its role in regulating gene expression during heart development.
Methods and Results
We performed the genome‐wide DNA methylation profiling of mouse embryonic hearts using methyl‐sensitive, tiny fragment enrichment/massively parallel sequencing to determine methylation levels at ACGT sites. The results showed that while global methylation of 1.64 million ACGT sites in developing hearts remains stable between embryonic day (E) 11.5 and E14.5, a small fraction (2901) of them exhibit differential methylation. Gene Ontology analysis revealed that these sites are enriched at genes involved in heart development. Quantitative real‐time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2). Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer. Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co‐expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancer's regulating function.
DNA methylation is developmentally regulated for genes essential to heart development, and abnormal DNA methylation may contribute to congenital heart disease.
PMCID: PMC4309105  PMID: 24947998
DNA methylation; DNA methyltransferase 3b; gene expression; heart development; hyaluronan synthase 2
3.  Mosaic Epigenetic Dysregulation of Ectodermal Cells in Autism Spectrum Disorder 
PLoS Genetics  2014;10(5):e1004402.
DNA mutational events are increasingly being identified in autism spectrum disorder (ASD), but the potential additional role of dysregulation of the epigenome in the pathogenesis of the condition remains unclear. The epigenome is of interest as a possible mediator of environmental effects during development, encoding a cellular memory reflected by altered function of progeny cells. Advanced maternal age (AMA) is associated with an increased risk of having a child with ASD for reasons that are not understood. To explore whether AMA involves covert aneuploidy or epigenetic dysregulation leading to ASD in the offspring, we tested a homogeneous ectodermal cell type from 47 individuals with ASD compared with 48 typically developing (TD) controls born to mothers of ≥35 years, using a quantitative genome-wide DNA methylation assay. We show that DNA methylation patterns are dysregulated in ectodermal cells in these individuals, having accounted for confounding effects due to subject age, sex and ancestral haplotype. We did not find mosaic aneuploidy or copy number variability to occur at differentially-methylated regions in these subjects. Of note, the loci with distinctive DNA methylation were found at genes expressed in the brain and encoding protein products significantly enriched for interactions with those produced by known ASD-causing genes, representing a perturbation by epigenomic dysregulation of the same networks compromised by DNA mutational mechanisms. The results indicate the presence of a mosaic subpopulation of epigenetically-dysregulated, ectodermally-derived cells in subjects with ASD. The epigenetic dysregulation observed in these ASD subjects born to older mothers may be associated with aging parental gametes, environmental influences during embryogenesis or could be the consequence of mutations of the chromatin regulatory genes increasingly implicated in ASD. The results indicate that epigenetic dysregulatory mechanisms may complement and interact with DNA mutations in the pathogenesis of the disorder.
Author Summary
Older mothers have a higher than expected risk of having a child with an autism spectrum disorder (ASD). The reason for this increased risk is unknown. The eggs of older mothers are more prone to abnormalities of chromosome numbers, suggesting this as one possible mechanism of the increased ASD risk. Age is also associated with a loss of control of epigenetic regulatory patterns that govern gene expression, indicating a second potential mechanism. To test both possibilities, we sampled cells from the same developmental origin as the brain, and performed genome-wide tests looking for unusual chromosome numbers and DNA methylation patterns. The studies were performed on individuals with ASD and typically developing controls, all born to mothers at least 35 years of age at the time of birth. We found the cells from individuals with ASD to have changes in DNA methylation at a number of loci, especially near genes encoding proteins known to interact with those already implicated in ASD. We conclude that epigenetic dysregulation occurring in gametes or early embryonic life may be one of the contributors to the development of ASD.
PMCID: PMC4038484  PMID: 24875834
4.  Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of Polycomb target genes 
Genome Biology  2013;14(3):R25.
DNA methylation and the Polycomb repression system are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear.
By genome-wide mapping of the Polycomb Repressive Complex 2-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and Polycomb Repressive Complex 2 from Polycomb target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression.
An intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining Polycomb Repressive Complex 2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease.
PMCID: PMC4053768  PMID: 23531360
DNA methylation; H3K27me3; Polycomb; PRC2; regulation of transcription
5.  HSC commitment–associated epigenetic signature is prognostic in acute myeloid leukemia 
The Journal of Clinical Investigation  2014;124(3):1158-1167.
Acute myeloid leukemia (AML) is characterized by disruption of HSC and progenitor cell differentiation. Frequently, AML is associated with mutations in genes encoding epigenetic modifiers. We hypothesized that analysis of alterations in DNA methylation patterns during healthy HSC commitment and differentiation would yield epigenetic signatures that could be used to identify stage-specific prognostic subgroups of AML. We performed a nano HpaII-tiny-fragment-enrichment-by-ligation-mediated-PCR (nanoHELP) assay to compare genome-wide cytosine methylation profiles between highly purified human long-term HSC, short-term HSC, common myeloid progenitors, and megakaryocyte-erythrocyte progenitors. We observed that the most striking epigenetic changes occurred during the commitment of short-term HSC to common myeloid progenitors and these alterations were predominantly characterized by loss of methylation. We developed a metric of the HSC commitment–associated methylation pattern that proved to be highly prognostic of overall survival in 3 independent large AML patient cohorts, regardless of patient treatment and epigenetic mutations. Application of the epigenetic signature metric for AML prognosis was superior to evaluation of commitment-based gene expression signatures. Together, our data define a stem cell commitment–associated methylome that is independently prognostic of poorer overall survival in AML.
PMCID: PMC3934187  PMID: 24487588
6.  Genetic Recombination Is Targeted towards Gene Promoter Regions in Dogs 
PLoS Genetics  2013;9(12):e1003984.
The identification of the H3K4 trimethylase, PRDM9, as the gene responsible for recombination hotspot localization has provided considerable insight into the mechanisms by which recombination is initiated in mammals. However, uniquely amongst mammals, canids appear to lack a functional version of PRDM9 and may therefore provide a model for understanding recombination that occurs in the absence of PRDM9, and thus how PRDM9 functions to shape the recombination landscape. We have constructed a fine-scale genetic map from patterns of linkage disequilibrium assessed using high-throughput sequence data from 51 free-ranging dogs, Canis lupus familiaris. While broad-scale properties of recombination appear similar to other mammalian species, our fine-scale estimates indicate that canine highly elevated recombination rates are observed in the vicinity of CpG rich regions including gene promoter regions, but show little association with H3K4 trimethylation marks identified in spermatocytes. By comparison to genomic data from the Andean fox, Lycalopex culpaeus, we show that biased gene conversion is a plausible mechanism by which the high CpG content of the dog genome could have occurred.
Author Summary
Recombination in mammalian genomes tends to occur within highly localized regions known as recombination hotspots. These hotspots appear to be a ubiquitous feature of mammalian genomes, but tend to not be shared between closely related species despite high levels of DNA sequence similarity. This disparity has been largely explained by the discovery of PRDM9 as the gene responsible for localizing recombination hotspots via recognition and binding to specific DNA motifs. Variation within PRDM9 can lead to changes to the recognized motif, and hence changes to the location of recombination hotspots thought the genome. Multiple studies have shown that PRDM9 is under strong selective pressure, apparently leading to a rapid turnover of hotspot locations between species. However, uniquely amongst mammals, PRDM9 appears to be dysfunctional in dogs and other canids. In this paper, we investigate how the loss of PRDM9 has affected the fine-scale recombination landscape in dogs and contrast this with patterns seen in other species.
PMCID: PMC3861134  PMID: 24348265
7.  Bidding the CpG island goodbye 
eLife  2013;2:e00593.
Experiments on seven vertebrates suggest that identifying the locations of islands of non-methylated DNA provides more insights into evolutionarily-conserved epigenetic regulatory elements than studies of CpG islands.
PMCID: PMC3582987  PMID: 23467495
CpG islands; DNA methylation; Epigenetics; Chromatin; Evolutionary conservation; Chicken; Human; Mouse
8.  Cytosine methylation changes in enhancer regions of core pro-fibrotic genes characterize kidney fibrosis development 
Genome Biology  2013;14(10):R108.
One in eleven people is affected by chronic kidney disease, a condition characterized by kidney fibrosis and progressive loss of kidney function. Epidemiological studies indicate that adverse intrauterine and postnatal environments have a long-lasting role in chronic kidney disease development. Epigenetic information represents a plausible carrier for mediating this programming effect. Here we demonstrate that genome-wide cytosine methylation patterns of healthy and chronic kidney disease tubule samples obtained from patients show significant differences.
We identify differentially methylated regions and validate these in a large replication dataset. The differentially methylated regions are rarely observed on promoters, but mostly overlap with putative enhancer regions, and they are enriched in consensus binding sequences for important renal transcription factors. This indicates their importance in gene expression regulation. A core set of genes that are known to be related to kidney fibrosis, including genes encoding collagens, show cytosine methylation changes correlating with downstream transcript levels.
Our report raises the possibility that epigenetic dysregulation plays a role in chronic kidney disease development via influencing core pro-fibrotic pathways and can aid the development of novel biomarkers and future therapeutics.
PMCID: PMC4053753  PMID: 24098934
9.  Epigenetic Functions of Smchd1 Repress Gene Clusters on the Inactive X Chromosome and on Autosomes 
Molecular and Cellular Biology  2013;33(16):3150-3165.
The Smchd1 gene encodes a large protein with homology to the SMC family of proteins involved in chromosome condensation and cohesion. Previous studies have found that Smchd1 has an important role in CpG island (CGI) methylation on the inactive X chromosome (Xi) and in stable silencing of some Xi genes. In this study, using genome-wide expression analysis, we showed that Smchd1 is required for the silencing of around 10% of the genes on Xi, apparently independent of CGI hypomethylation, and, moreover, that these genes nonrandomly occur in clusters. Additionally, we found that Smchd1 is required for CpG island methylation and silencing at a cluster of four imprinted genes in the Prader-Willi syndrome (PWS) locus on chromosome 7 and genes from the protocadherin-alpha and -beta clusters. All of the affected autosomal loci display developmentally regulated brain-specific methylation patterns which are lost in Smchd1 homozygous mutants. We discuss the implications of these findings for understanding the function of Smchd1 in epigenetic regulation of gene expression.
PMCID: PMC3753908  PMID: 23754746
10.  Astrogenomics: big data, old problems, old solutions? 
Genome Biology  2013;14(8):129.
PMCID: PMC4053852  PMID: 23953643
11.  Differential epigenome-wide DNA methylation patterns in childhood obesity-associated asthma 
Scientific Reports  2013;3:2164.
While DNA methylation plays a role in T-helper (Th) cell maturation, its potential dysregulation in the non-atopic Th1-polarized systemic inflammation observed in obesity-associated asthma is unknown. We studied DNA methylation epigenome-wide in peripheral blood mononuclear cells (PBMCs) from 8 obese asthmatic pre-adolescent children and compared it to methylation in PBMCs from 8 children with asthma alone, obesity alone and healthy controls. Differentially methylated loci implicated certain biologically relevant molecules and pathways. PBMCs from obese asthmatic children had distinctive DNA methylation patterns, with decreased promoter methylation of CCL5, IL2RA and TBX21, genes encoding proteins linked with Th1 polarization, and increased promoter methylation of FCER2, a low-affinity receptor for IgE, and of TGFB1, inhibitor of Th cell activation. T-cell signaling and macrophage activation were the two primary pathways that were selectively hypomethylated in obese asthmatics. These findings suggest that dysregulated DNA methylation is associated with non-atopic inflammation observed in pediatric obesity-associated asthma.
PMCID: PMC3712321  PMID: 23857381
12.  Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer 
Nucleic Acids Research  2013;41(16):e157.
5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome-wide assays for 5-hmC determination are needed as many of the techniques for 5-methylcytosine (5-mC) determination, including methyl-sensitive restriction digestion and bisulfite sequencing cannot distinguish between 5-mC and 5-hmC. Glycosylation of 5-hmC residues by beta-glucosyl transferase (β-GT) can make CCGG residues insensitive to digestion by MspI. Restriction digestion by HpaII, MspI or MspI after β-GT conversion, followed by adapter ligation, massive parallel sequencing and custom bioinformatic analysis allowed us determine distribution of 5-mC and 5-hmC at single base pair resolution at MspI restriction sites. The resulting HpaII tiny fragment Enrichment by Ligation-mediated PCR with β-GT (HELP-GT) assay identified 5-hmC loci that were validated at global level by liquid chromatography-mass spectrometry (LC-MS) and the locus-specific level by quantitative reverse transcriptase polymerase chain reaction of 5-hmC pull-down DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. The HELP-GT assay allowed global determination of 5-hmC and 5-mC from low amounts of DNA and with the use of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of determination of this modification in conjugation with conventional methylome analysis.
PMCID: PMC3763560  PMID: 23861445
13.  Large, Male Germ Cell-Specific Hypomethylated DNA Domains With Unique Genomic and Epigenomic Features on the Mouse X Chromosome 
To understand the epigenetic regulation required for germ cell-specific gene expression in the mouse, we analysed DNA methylation profiles of developing germ cells using a microarray-based assay adapted for a small number of cells. The analysis revealed differentially methylated sites between cell types tested. Here, we focused on a group of genomic sequences hypomethylated specifically in germline cells as candidate regions involved in the epigenetic regulation of germline gene expression. These hypomethylated sequences tend to be clustered, forming large (10 kb to ∼9 Mb) genomic domains, particularly on the X chromosome of male germ cells. Most of these regions, designated here as large hypomethylated domains (LoDs), correspond to segmentally duplicated regions that contain gene families showing germ cell- or testis-specific expression, including cancer testis antigen genes. We found an inverse correlation between DNA methylation level and expression of genes in these domains. Most LoDs appear to be enriched with H3 lysine 9 dimethylation, usually regarded as a repressive histone modification, although some LoD genes can be expressed in male germ cells. It thus appears that such a unique epigenomic state associated with the LoDs may constitute a basis for the specific expression of genes contained in these genomic domains.
PMCID: PMC3859323  PMID: 23861320
DNA methylation; primordial germ cell; epigenome; reprogramming; cancer testis antigen
14.  Tissue-specific dysregulation of DNA methylation in aging 
Aging cell  2010;9(4):506-518.
The normal aging process is a complex phenomenon associated with physiological alterations in the function of cells and organs over time. Although an attractive candidate for mediating transcriptional dysregulation, the contribution of epigenetic dysregulation to these progressive changes in cellular physiology remains unclear. In this study, we employed the genome-wide HELP assay to define patterns of cytosine methylation throughout the rat genome, and the LUMA assay to measure global levels of DNA methylation in the same samples. We studied both liver and visceral adipose tissue, and demonstrated significant differences in DNA methylation with age at >5% of sites analyzed. Furthermore, we showed that epigenetic dysregulation with age is a highly tissue-dependent phenomenon. The most distinctive loci were located at intergenic sequences and conserved non-coding elements, and not at promoters nor at CG-dinucleotide dense loci. Despite this, we found that there was a subset of genes at which cytosine methylation and gene expression changes were concordant. Finally, we demonstrated that changes in methylation occur consistently near genes that are involved in metabolism and metabolic regulation, implicating their potential role in the pathogenesis of age-related diseases. We conclude that different patterns of epigenetic dysregulation occur in each tissue over time and may cause some of the physiological changes associated with normal aging.
PMCID: PMC2935175  PMID: 20497131
aging; epigenetics; DNA methylation; tissue-specific; liver; visceral adiposity
15.  Convergent and divergent evolution of genomic imprinting in the marsupial Monodelphis domestica 
BMC Genomics  2012;13:394.
Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin specific monoallelic gene expression. It is postulated to have evolved in placental mammals to modulate intrauterine resource allocation to the offspring. In this study, we determined the imprint status of metatherian orthologues of eutherian imprinted genes.
L3MBTL and HTR2A were shown to be imprinted in Monodelphis domestica (the gray short-tailed opossum). MEST expressed a monoallelic and a biallelic transcript, as in eutherians. In contrast, IMPACT, COPG2, and PLAGL1 were not imprinted in the opossum. Differentially methylated regions (DMRs) involved in regulating imprinting in eutherians were not found at any of the new imprinted loci in the opossum. Interestingly, a novel DMR was identified in intron 11 of the imprinted IGF2R gene, but this was not conserved in eutherians. The promoter regions of the imprinted genes in the opossum were enriched for the activating histone modification H3 Lysine 4 dimethylation.
The phenomenon of genomic imprinting is conserved in Therians, but the marked difference in the number and location of imprinted genes and DMRs between metatherians and eutherians indicates that imprinting is not fully conserved between the two Therian infra-classes. The identification of a novel DMR at a non-conserved location as well as the first demonstration of histone modifications at imprinted loci in the opossum suggest that genomic imprinting may have evolved in a common ancestor of these two Therian infra-classes with subsequent divergence of regulatory mechanisms in the two lineages.
PMCID: PMC3507640  PMID: 22899817
Genomic imprinting; Marsupials; Eutherians
16.  Epigenetic Silencing of the Circadian Clock Gene CRY1 is Associated with an Indolent Clinical Course in Chronic Lymphocytic Leukemia 
PLoS ONE  2012;7(3):e34347.
Disruption of circadian rhythm is believed to play a critical role in cancer development. Cryptochrome 1 (CRY1) is a core component of the mammalian circadian clock and we have previously shown its deregulated expression in a subgroup of patients with chronic lymphocytic leukemia (CLL). Using real-time RT-PCR in a cohort of 76 CLL patients and 35 normal blood donors we now demonstrate that differential CRY1 mRNA expression in high-risk (HR) CD38+/immunoglobulin variable heavy chain gene (IgVH) unmutated patients as compared to low-risk (LR) CD38−/IgVH mutated patients can be attributed to down-modulation of CRY1 in LR CLL cases. Analysis of the DNA methylation profile of the CRY1 promoter in a subgroup of 57 patients revealed that CRY1 expression in LR CLL cells is silenced by aberrant promoter CpG island hypermethylation. The methylation pattern of the CRY1 promoter proved to have high prognostic impact in CLL where aberrant promoter methylation predicted a favourable outcome. CRY1 mRNA transcript levels did not change over time in the majority of patients where sequential samples were available for analysis. We also compared the CRY1 expression in CLL with other lymphoid malignancies and observed epigenetic silencing of CRY1 in a patient with B cell acute lymphoblastic leukemia (B-ALL).
PMCID: PMC3314606  PMID: 22470559
17.  DNA methylation profiling using HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) 
Methods (San Diego, Calif.)  2010;52(3):218-222.
The HELP assay is a technique that allows genome-wide analysis of cytosine methylation. Here we describe the assay, its relative strengths and weaknesses, and the transition of the assay from a microarray to massively-parallel sequencing-based foundation.
PMCID: PMC2954265  PMID: 20434563
Cytosine methylation; CpG island; epigenetic
18.  Allele-specific transcriptional elongation regulates monoallelic expression of the IGF2BP1 gene 
Random monoallelic expression contributes to phenotypic variation of cells and organisms. However, the epigenetic mechanisms by which individual alleles are randomly selected for expression are not known. Taking cues from chromatin signatures at imprinted gene loci such as the insulin-like growth factor 2 gene 2 (IGF2), we evaluated the contribution of CTCF, a zinc finger protein required for parent-of-origin-specific expression of the IGF2 gene, as well as a role for allele-specific association with DNA methylation, histone modification and RNA polymerase II.
Using array-based chromatin immunoprecipitation, we identified 293 genomic loci that are associated with both CTCF and histone H3 trimethylated at lysine 9 (H3K9me3). A comparison of their genomic positions with those of previously published monoallelically expressed genes revealed no significant overlap between allele-specifically expressed genes and colocalized CTCF/H3K9me3. To analyze the contributions of CTCF and H3K9me3 to gene regulation in more detail, we focused on the monoallelically expressed IGF2BP1 gene. In vitro binding assays using the CTCF target motif at the IGF2BP1 gene, as well as allele-specific analysis of cytosine methylation and CTCF binding, revealed that CTCF does not regulate mono- or biallelic IGF2BP1 expression. Surprisingly, we found that RNA polymerase II is detected on both the maternal and paternal alleles in B lymphoblasts that express IGF2BP1 primarily from one allele. Thus, allele-specific control of RNA polymerase II elongation regulates the allelic bias of IGF2BP1 gene expression.
Colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. Moreover, association of individual alleles with both active (H3K4me3) and silent (H3K27me3) chromatin modifications (allelic bivalent chromatin) or with RNA polymerase II also fails to identify monoallelically expressed gene loci. The selection of individual alleles for expression occurs in part during transcription elongation.
PMCID: PMC3174113  PMID: 21812971
20.  Experimental Approaches to the Study of Epigenomic Dysregulation in Ageing 
Experimental gerontology  2010;45(4):255-268.
In this review, we describe how normal ageing may involve the acquisition of epigenetic errors over time, akin to the accumulation of genetic mutations with ageing. We describe how such experiments are currently performed, their limitations technically and analytically and their application to ageing research.
PMCID: PMC2838972  PMID: 20060885
21.  Widespread Hypomethylation Occurs Early and Synergizes with Gene Amplification during Esophageal Carcinogenesis 
PLoS Genetics  2011;7(3):e1001356.
Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia, and adenocarcinoma, the combinatorial effect of these changes is unknown. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We find that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation synergizes with gene amplification and leads to significant upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation is observed at a restricted number of loci and, in combination with hemi-allelic deletions, leads to downregulatation of selected transcripts during multistep progression. We also observe that epigenetic regulation during epithelial carcinogenesis is not restricted to traditionally defined “CpG islands,” but may also occur through a mechanism of differential methylation outside of these regions. Finally, validation of novel upregulated targets (CXCL1 and 3, GATA6, and DMBT1) in a larger independent panel of samples confirms the utility of integrative analysis in cancer biomarker discovery.
Author Summary
The incidence of esophageal adenocarcinoma (EA) is increasing at an alarming pace in the United States. Distinct pathological stages of Barrett's metaplasia and low- and high-grade dysplasia can be seen preceding malignant transformation. These precursor lesions provide a unique in vivo model for deepening our understanding the early steps in human neoplasia. By integrating genome-wide DNA methylation, copy number, and transcriptomic datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we are uniquely able to define the molecular events associated progression of Barrett esophagus. We show that the predominant change during this process is loss of DNA methylation. We show that this global hypomethylation occurs very early during the process and is seen even in preinvasive lesions. This loss of DNA methylation drives carcinogenesis by cooperating with gene amplifications in upregulating proteins during this process. Finally we uncovered proteins that upregulated by loss of methylation or gene amplification (CXCL1 and 3, GATA6, and DMBT1) and show their relevance by validating their levels in larger independent panel of samples, thus confirming the utility of integrative analysis in cancer biomarker discovery.
PMCID: PMC3069107  PMID: 21483804
22.  Distinct factors control histone variant H3.3 localization at specific genomic regions 
Cell  2010;140(5):678-691.
The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem (ES) cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence, and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres, and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.
PMCID: PMC2885838  PMID: 20211137
23.  Opposing Roles of Dnmt1 in Early- and Late-Stage Murine Prostate Cancer▿  
Molecular and Cellular Biology  2010;30(17):4159-4174.
Previous studies have shown that tumor progression in the transgenic adenocarcinoma of mouse prostate (TRAMP) model is characterized by global DNA hypomethylation initiated during early-stage disease and locus-specific DNA hypermethylation occurring predominantly in late-stage disease. Here, we utilized Dnmt1 hypomorphic alleles to examine the role of Dnmt1 in normal prostate development and in prostate cancer in TRAMP. Prostate tissue morphology and differentiation status was normal in Dnmt1 hypomorphic mice, despite global DNA hypomethylation. TRAMP; Dnmt1 hypomorphic mice also displayed global DNA hypomethylation, but were characterized by altered tumor phenotype. Specifically, TRAMP; Dnmt1 hypomorphic mice exhibited slightly increased tumor incidence and significantly increased pathological progression at early ages and, conversely, displayed slightly decreased tumor incidence and significantly decreased pathological progression at advanced ages. Remarkably, hypomorphic Dnmt1 expression abrogated local and distant site macrometastases. Thus, Dnmt1 has tumor suppressor activity in early-stage prostate cancer, and oncogenic activity in late stage prostate cancer and metastasis. Consistent with the biological phenotype, epigenomic studies revealed that TRAMP; Dnmt1 hypomorphic mice show dramatically reduced CpG island and promoter DNA hypermethylation in late-stage primary tumors compared to control mice. Taken together, the data reveal a crucial role for Dnmt1 in prostate cancer and suggest that Dnmt1-targeted interventions may have utility specifically for advanced and/or metastatic prostate cancer.
PMCID: PMC2937561  PMID: 20584988
24.  DNA Methylation Signatures Identify Biologically Distinct Subtypes in Acute Myeloid Leukemia 
Cancer cell  2010;17(1):13-27.
We hypothesized that DNA methylation distributes into specific patterns in cancer cells, which reflect critical biological differences. We therefore examined the methylation profiles of 344 patients with acute myeloid leukemia (AML). Clustering of these patients by methylation data segregated patients into 16 groups. Five of these groups defined new AML subtypes that shared no other known feature. In addition, DNA methylation profiles segregated patients with CEBPA aberrations from other subtypes of leukemia, defined four epigenetically distinct forms of AML with NPM1 mutations, and showed that established AML1-ETO, CBFb-MYH11, and PML-RARA leukemia entities are associated with specific methylation profiles. We report a 15 gene methylation classifier predictive of overall survival in an independent patient cohort (p < 0.001, adjusted for known covariates).
PMCID: PMC3008568  PMID: 20060365
25.  Lack of Evidence for Green Tea Polyphenols as DNA Methylation Inhibitors in Murine Prostate 
Green tea polyphenols (GTPs) have been reported to inhibit DNA methylation in cultured cells. Here we tested whether oral consumption of GTPs affects normal or cancer specific DNA methylation in vivo, using mice. Wildtype (WT) and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice were administered 0.3% GTPs in drinking water beginning at 4 weeks of age. To monitor DNA methylation, we measured 5-methyl-deoxycytidine (5mdC) levels, methylation of the B1 repetitive element, and methylation of the Mage-a8 gene. Each of these parameters were unchanged in prostate, gut, and liver from WT mice at both 12 and 24 weeks of age, with the single exception of a decrease of 5mdC in the liver at 12 weeks. In GTP-treated TRAMP mice, 5mdC levels and the methylation status of four loci hypermethylated during tumor progression were unaltered in TRAMP prostates at 12 or 24 weeks. Quite surprisingly, GTP treatment did not inhibit tumor progression in TRAMP mice, although known pharmacodynamic markers of GTPs were altered in both WT and TRAMP prostates. We also administered 0.1%, 0.3%, or 0.6% GTPs to TRAMP mice for 12 weeks and measured 5mdC levels and methylation of B1 and Mage-a8 in prostate, gut, and liver tissues. No dose-dependent alterations in DNA methylation status were observed. Genome-wide DNA methylation profiling using the HELP assay also revealed no significant hypomethylating effect of GTP. These data indicate that oral administration of GTPs does not affect normal or cancer-specific DNA methylation in the murine prostate.
PMCID: PMC2789312  PMID: 19934341
green tea polyphenols; TRAMP; prostate cancer; DNA methylation; HELP assay

Results 1-25 (38)