Tissue-specific DNA Methylation Research Articles

Cellular Reprogramming Erases Aberrant DNA Methylation and the Malignant Phenotype in Chronic Myeloid Leukemia

Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the presence of the Philadelphia chromosome deriving from the genetic translocation t(9;22)(q34;q11.2) that encodes for the BCR-ABL fusion gene. BCR-ABL is a constitutively active tyrosine kinase believed to be the primary genetic event driving CML development (Daley and Baltimore, 1988; Huettner et al., 2000). Tyrosine Kinase Inhibitors (TKI) targeting the BCR-ABL kinase have revolutionized CML therapy, however, they fail to fully eradicate the disease due to the presence of a drug-resistant stem cell pool that sustains continued growth of the malignant cells. Indeed, discontinuation of TKI results in relapse and/or disease progression.It has been shown that the emergence of leukemic clones resistant to TKI and responsible for CML evolution is correlated with aberrant DNA methylation (Machova Polakova et al., 2013). DNA methylation is a key epigenetic signature implicated in regulation of gene expression (Robertson, 2001), that occurs predominantly within CpG dinucleotides. CpG-rich regions (namely CpG islands) are frequently located within promoter regions (~70%) of human protein-coding genes (Illingworth et al., 2010). Methylation of CpG-rich promoters negatively correlates with gene expression levels and it is considered an important regulatory mechanism for long-term gene silencing (Herman and Baylin, 2003). Although numerous studies have established a link between aberrant promoter DNA methylation and cancer (Costello et al., 2000; Feinberg et al., 2006), the impact of DNA methylation in CML is still poorly understood (Yamazaki et al., 2012), particularly due to the lack of the specific animal models.In this study we tested the functional relevance of aberrant methylome in CML development and investigated the possibility that BCR-ABL oncogene triggers DNA methylation changes leading to an aggressive leukemic phenotype. To this end, we have combined cellular reprogramming (Amabile and Meissner, 2009; Carette et al., 2010; Kumano et al., 2012; Miyoshi et al., 2010; Takahashi et al., 2007), due to its ability to erase tissue-specific DNA methylation and to re-establish an embryonic stem-like DNA methylation state (Mikkelsen et al., 2008), with a previously developed BCR-ABL inducible murine model (Koschmieder et al., 2005). Using this approach, we demonstrate that the presence of a single genetic aberration is sufficient to trigger DNA methylation changes and leads to an aggressive leukemic phenotype. We show that by resetting the normal DNA methylation profile of primary human CML cells through cellular reprogramming, we are able to re-establish normal myeloid differentiation, despite the persistence of the native genetic lesion. Finally, by combining a comprehensive genome-scale methylation analysis with cellular reprogramming of leukemic cells, we elucidate the sequential events driving leukemogenesis and reveal the reciprocal interplay of genetic and epigenetic mechanisms during malignant transformation.In conclusion, these results dissect the role of DNA methylation alterations in CML development and warrant an application of demethylating agents such as 5-azacytidine as adjuvant treatment in therapeutic approaches to CML. DisclosuresMartinelli:Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau.

In utero bisphenol A concentration, metabolism, and global DNA methylation across matched placenta, kidney, and liver in the human fetus

While urine has been an easily accessible and feasible matrix for human biomonitoring, analytical measurements in internal tissues and organs can provide more accurate exposure assessments to understand disease etiology. This is especially important for the endocrine active compound, bisphenol A (BPA), where studies investigating internal doses at sensitive periods of human development are currently lacking. Herein, BPA concentrations, BPA-specific metabolizing enzyme gene expression, and global DNA methylation were characterized across three matched tissues from elective pregnancy terminations of 2nd trimester human fetuses: the placenta, liver, and kidney (N=12 each; N=36 total). Compared to liver (free: 0.54–50.5ngg−1), BPA concentrations were lower in matched placenta (<0.05–25.4ngg−1) and kidney (0.08–11.1ngg−1) specimens. BPA-specific metabolism gene expression of GUSB, UGT2B15, STS, and SULT1A1 differed across each tissue type; however, conjugation and deconjugation expression patterns were similar across the fetus. Average LINE1 and CCGG global methylation were 58.3% and 59.2% in placenta, 79.5% and 66.4% in fetal liver, and 77.9% and 77.0% in fetal kidney, with significant tissue-specific DNA methylation differences in both LINE1 (p-value<0.001) and CCGG content (p-value<0.001). Total BPA concentrations were positively associated with global methylation for the placenta only using the LINE1 assay (p-value: 0.002), suggesting organ-specific biological effects after fetal exposure. Utilizing sensitive human clinical specimens, results are informative for BPA toxicokinetics and toxicodynamics assessment in the developing human fetus.

Prediction of disease-related genes based on weighted tissue-specific networks by using DNA methylation.

BackgroundPredicting disease-related genes is one of the most important tasks in bioinformatics and systems biology. With the advances in high-throughput techniques, a large number of protein-protein interactions are available, which make it possible to identify disease-related genes at the network level. However, network-based identification of disease-related genes is still a challenge as the considerable false-positives are still existed in the current available protein interaction networks (PIN).ResultsConsidering the fact that the majority of genetic disorders tend to manifest only in a single or a few tissues, we constructed tissue-specific networks (TSN) by integrating PIN and tissue-specific data. We further weighed the constructed tissue-specific network (WTSN) by using DNA methylation as it plays an irreplaceable role in the development of complex diseases. A PageRank-based method was developed to identify disease-related genes from the constructed networks. To validate the effectiveness of the proposed method, we constructed PIN, weighted PIN (WPIN), TSN, WTSN for colon cancer and leukemia, respectively. The experimental results on colon cancer and leukemia show that the combination of tissue-specific data and DNA methylation can help to identify disease-related genes more accurately. Moreover, the PageRank-based method was effective to predict disease-related genes on the case studies of colon cancer and leukemia.ConclusionsTissue-specific data and DNA methylation are two important factors to the study of human diseases. The same method implemented on the WTSN can achieve better results compared to those being implemented on original PIN, WPIN, or TSN. The PageRank-based method outperforms degree centrality-based method for identifying disease-related genes from WTSN.

Tissue-specific DNA methylation profiles regulate liver-specific expression of the APOA1/C3/A4/A5 cluster and can be manipulated with demethylating agents on intestinal cells

Objective: The tissue-specific expression profiles of genes within the APOA1/C3/A4/A5 cluster play an important role in lipid metabolism regulation. We hypothesize that the tissue-specific expression of the APOA1/C3/A4/A5 gene cluster will show an inverse pattern with DNA methylation, and that repression in non- or low-expressing tissue, such as the intestine, can be reversed using epigenetic drugs. Methods and results: We analyzed DNA samples from different human adult tissues (liver, intestine, leukocytes, brain, kidney, pancreas, muscle and sperm) using the Infinium HumanMethyation450 BeadChip array. DNA methylation profiles in APOA1/C3/A4/A5 gene cluster were confirmed by bisulfite PCR and pyrosequencing. To determine whether the observed tissue-specific methylation was associated with the expression profile we exposed intestinal TC7/Caco-2 cells to the demethylating agent 5-Aza-2′-deoxycytidine and monitored intestinal APOA1/C3/A4/A5 transcript re-expression by RT-qPCR. The promoters of APOA1, APOC3 and APOA5 genes were less methylated in liver compared to other tissues, and APOA4 gene was highly methylated in most tissues and partially methylated in liver and intestine. In TC7/Caco-2 cells, 5-Aza-2′-deoxycytidine treatment induced a decrease between 37 and 24% in the methylation levels of APOA1/C3/A4/A5 genes and a concomitant re-expression mainly in APOA1, APOA4 and APOA5 genes ranging from 22 to 600%. Conclusions: We have determined the methylation patterns of the APOA1/C3/A4/A5 cluster that may be directly involved in the transcriptional regulation of this cluster. DNA demethylation of intestinal cells increases the RNA levels especially of APOA1, APOA4 and APOA5 genes.

Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.

MP29-13 IMPROVING RENAL NEEDLE-BIOPSY ACCURRACY WITH RCC-SPECIFIC DNA METHYLATION MARKERS

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2014MP29-13 IMPROVING RENAL NEEDLE-BIOPSY ACCURRACY WITH RCC-SPECIFIC DNA METHYLATION MARKERS Mehrdad Alemozaffar, Clayton Coolings, Sameer Chopra, Sumeet Syan, Brian Hu, Inderbir Gill, and Gangning Liang Mehrdad AlemozaffarMehrdad Alemozaffar More articles by this author , Clayton CoolingsClayton Coolings More articles by this author , Sameer ChopraSameer Chopra More articles by this author , Sumeet SyanSumeet Syan More articles by this author , Brian HuBrian Hu More articles by this author , Inderbir GillInderbir Gill More articles by this author , and Gangning LiangGangning Liang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.755AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The detection of small renal masses (SRMs) <4cm in size have risen given the widespread use of abdominal imaging. 20-30% of SRMs are benign and workup and management remains controversial with advocates for biopsy, surveillance, or surgical treatment. Thus, accurate characterization of SRMs is needed to determine treatment. Needle biopsy of SRMs have high false negative rates on standard microscopic examination (10–20%) since small amounts of tissue are sometimes obtained. We aimed to identify epigenetic markers to improve the diagnostic value of renal needle biopsies. METHODS Through an IRB approved protocol, needle biopsies were obtained ex-vivo following partial nephrectomy for 100 patients with SRMs. Each lesion underwent 3 pairs of biopsies: 2 from tumor regions and 1 from adjacent normal tissue; one was examined and other was used for DNA extraction. The methylation levels of these samples at select CpG dinucleotides throughout the genome were detected using the Infinium 450K array technology. To elucidate tissue-specific DNA methylation markers, these biological values were then compared to Infinium 450K data derived from 300 categorized renal samples from The Cancer Genome Atlas (TCGA). RESULTS Sixty SRMs have been examined to date. We isolated 9,432 candidate markers of differential DNA methylation with average beta value difference between two sample groups is >0.4 (Figure 1). Current efforts have been made to create an algorithm that allows us to predict the type of tissue based on methylation markers. To date, the algorithm has shown to successfully predict normal and cancerous tissue at a sensitivity rate of 91% and 90%, respectively. CONCLUSIONS Preliminary results suggest significant differences in DNA methylation levels exist at specific sites in the human genome among normal and aberrant kidney tissues. It's likely these biomarkers can be used to distinguish tissue types. We are using statistical approaches to identify a panel of DNA methylation markers to improve predicting phenotype and develop an economically efficient method for distinguishing between benign and cancer tissues. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e309-e310 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Mehrdad Alemozaffar More articles by this author Clayton Coolings More articles by this author Sameer Chopra More articles by this author Sumeet Syan More articles by this author Brian Hu More articles by this author Inderbir Gill More articles by this author Gangning Liang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Tissue specific DNA methylation in normal human breast epithelium and in breast cancer.

Cancer is a heterogeneous and tissue-specific disease. Thus, the tissue of origin reflects on the natural history of the disease and dictates the therapeutic approach. It is suggested that tissue differentiation, mediated mostly by epigenetic modifications, could guide tissue-specific susceptibility and protective mechanisms against cancer. Here we studied breast specific methylation in purified normal epithelium and its reflection in breast cancers. We established genome wide methylation profiles of various normal epithelial tissues and identified 110 genes that were differentially methylated in normal breast epithelium. A number of these genes also showed methylation alterations in breast cancers. We elaborated on one of them, TRIM29 (ATDC), and showed that its promoter was hypo-methylated in normal breast epithelium and heavily methylated in other normal epithelial tissues. Moreover, in breast carcinomas methylation increased and expression decreased whereas the reverse was noted for multiple other carcinomas. Interestingly, TRIM29 regulation in breast tumors clustered according to the PAM50 classification. Thus, it was repressed in the estrogen receptor positive tumors, particularly in the more proliferative luminal B subtype. This goes in line with previous reports indicating tumor suppressive activity of TRIM29 in estrogen receptor positive luminal breast cells in contrast to oncogenic function in pancreatic and lung cancers. Overall, these findings emphasize the linkage between breast specific epigenetic regulation and tissue specificity of cancer.

Genome-Wide Analysis of DNA Methylation in Five Tissues of Zhikong Scallop, Chlamys farreri

DNA methylation plays a vital role in tissue development and differentiation in eukaryotes. Epigenetic studies have been seldom conducted in the extremely diverse and evolutionarily highly successful bilaterian lineage Mollusca. In the present study, we conducted the genome-wide profiling of DNA methylation for five tissues of a bivalve mollusc, Chlamys farreri using the methylation-sensitive amplification polymorphism (MSAP) technique. The methylation levels were quite similar among tissues, ranging from 20.9% to 21.7%. CG methylation was the dominant type (14.9%–16.5%) in the C. farreri genome, but CHG methylation also accounted for a substantial fraction of total methylation (5.1%–6.3%). Relatively high methylation diversity was observed within tissues. Methylation differentiation between tissues was evaluated and 460 tissue-specific epiloci were identified. Kidney differs from the other tissues in DNA methylation profiles. Our study presents the first look at the tissue-specific DNA methylation patterns in a bivalve mollusc and represents an initial step towards understanding of epigenetic regulatory mechanism underlying tissue development and differentiation in bivalves.

Fundamental diversity of human CpG islands at multiple biological levels

CpG islands (CGIs) are commonly used as genomic markers to study the patterns and regulatory consequences of DNA methylation. Interestingly, recent studies reveal a substantial diversity among CGIs: long and short CGIs, for example, exhibit contrasting patterns of gene expression complexity and nucleosome occupancy. Evolutionary origins of CGIs are also highly heterogeneous. In order to systematically evaluate potential diversities among CGIs and ultimately to illuminate the link between diversity of CGIs and their epigenetic variation, we analyzed the nucleotide-resolution DNA methylation maps (methylomes) of multiple cellular origins. We discover novel ‘clusters’ of CGIs according to their patterns of DNA methylation; the stably hypomethylated CGI cluster (cluster I), sperm-hypomethylated CGI cluster (cluster II), and variably methylated CGI cluster (cluster III). These epigenomic CGI clusters are strikingly distinct at multiple biological features including genomic, evolutionary, and functional characteristics. At the genomic level, the stably hypomethylated CGI cluster tends to be longer and harbors many more CpG dinucleotides than those in other clusters. They are also frequently associated with promoters, while CGI clusters II and III mostly reside in intragenic or intergenic regions and exhibit highly tissue-specific DNA methylation. Functional ontology terms and transcriptional profiles co-vary with CGI clusters, indicating that the regulatory functions of CGIs are tightly linked to their heterogeneity. Finally, CGIs associated with distinctive biological processes, such as diseases, aging, and imprinting, occur disproportionately across CGI clusters. These new findings provide an effective means to combine existing knowledge on CGIs into a genomic context while bringing new insights that elucidate the significance of DNA methylation across different biological conditions and demography.

Tissue-specific Leptin promoter DNA methylation is associated with maternal and infant perinatal factors

Leptin a regulator of body weight is involved in reproductive and developmental functions. Leptin promoter DNA methylation (LEP) regulates gene expression in a tissue-specific manner and has been linked to adverse pregnancy outcomes. In non-pathologic human pregnancies, we assessed LEP methylation, genotyped the single nucleotide polymorphism (SNP) rs2167270 in placental (n=81), maternal and cord blood samples (n=60), and examined the association between methylation, genotype, and perinatal factors. Maternal blood LEP methylation was lower in pre-pregnancy obese women (P=0.01). Cord blood LEP methylation was higher in small for gestational age (SGA) (P=4.6×10−3) and A/A genotype (P=1.6×10−4), lower (−1.47, P=0.03) in infants born to pre-pregnancy obese mothers and correlated (P=0.01) with maternal blood LEP. Gender was associated with placental LEP methylation (P=0.05). These results suggest that LEP epigenetic control may be influenced by perinatal factors including: maternal obesity, infant growth, genotype and gender in a tissue-specific manner and may have multigenerational implications.

Variation in DNA Methylation Patterns is More Common among Maize Inbreds than among Tissues

Chromatin modifications, such as DNA methylation, can provide heritable, epigenetic regulation of gene expression in the absence of genetic changes. A role for DNA methylation in meiotically stable marking of repetitive elements and other sequences has been demonstrated in plants. Methylation of DNA is also proposed to play a role in development through providing a mitotic memory of gene expression states established during cellular differentiation. We sought to clarify the relative levels of DNA methylation variation among different genotypes and tissues in maize (Zea mays L.). We have assessed genomewide DNA methylation patterns in leaf, immature tassel, embryo, and endosperm tissues of two inbred maize lines: B73 and Mo17. There are hundreds of regions of differential methylation present between the two genotypes. In general, the same regions exhibit differential methylation between B73 and Mo17 in each of the tissues that were surveyed. In contrast, there are few examples of tissue‐specific DNA methylation variation. Only a subset of regions with tissue‐specific variation in DNA methylation show similar patterns in both genotypes of maize and even fewer are associated with altered gene expression levels among the tissues. Our data indicates a limited impact of DNA methylation on developmental gene regulation within maize.

A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples

BackgroundGrowing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (≥1 μg) DNA samples, limiting the analysis of small tissue samples. Here we present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA.ResultsWe have developed a methodology utilizing MBD2b/MBD3L1 enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized tiling arrays, which we termed MeKL-chip. Kinase modification in combination with the addition of PEG has increased ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng of DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2,498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing.ConclusionsMeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs.

Tissue-specific DNA methylation profiles in newborns

BackgroundEpidemiological studies demonstrate that foetal growth restriction and low birth weight affect long-term health. Derangements in tissue-specific epigenetic programming of foetal and placental tissues are a suggested underlying mechanism of which DNA methylation is best understood. DNA methylation has been mostly investigated in DNA from white blood cells. To improve baseline understanding of tissue-specific DNA methylation, we examined variation in DNA methylation profiles of the imprinted foetal growth genes IGF2 and H19 in three different tissues from the same newborn obtained at the same time.FindingsWe obtained DNA from umbilical cord blood mononuclear cells (MNC; CD34+ and CD34–, n = 6), foetal side of the placenta (n = 5) and umbilical cord Wharton jelly (n = 5). DNA methylation of the IGF2 differentially methylated region (DMR) and H19 DMR was measured using quantitative mass spectrometry. Analysis of variance testing showed no statistical difference between total mean methylation of CD34+ and CD34– MNC. Further comparisons were made with the pooled total MNC fraction. Mean IGF2 DMR methylation of Wharton jelly was 1.3 times higher (P = 0.001) than mean methylation of the pooled MNC. Placental mean methylation was 0.8 times lower (P <0.001) and Wharton jelly 0.9 times lower (P <0.001) than the pooled MNC of H19 DMR.ConclusionThe total MNC fraction is a rather homogeneous cell population for methylation studies of imprinted genes in umbilical cord blood white blood cells, but may not always reflect the methylation levels of IGF2 and H19 in other organs.

Abstract 1111: Whole genome bisulfite sequencing reveals tissue-specific DNA methylation in a normal mouse.

Abstract Epigenetic alteration is a hallmark of cancer. In particular, compared to normal cells, the DNA methylomes of cancer cells often exhibit two contrasting phenomena: 1) hypomethylation of large regions of the genome, notably at regions of late replicating DNA; and 2) hypermethylation of focal sites, frequently at CpG-island containing promoters. While this epigenetic variation between cancer cells and normal counterparts has been well documented, the variation between different normal cell types is still unknown. In an effort to better understand the epigenetic variation of a normal individual, we profiled DNA methylation using whole genome bisulfite sequencing in 16 tissues isolated from an individual mouse. We observed a unique distribution of CpG methylation for each tissue, which cluster based on cell lineage and type. Most somatic tissues exhibit a small but consistent decrease in methylation at late-replicating regions of the genome, suggesting the beginnings of global hypomethylation. Our global analysis identified only one-eighth of the genome as tissue-specific differentially methylated regions. Remarkably, the vast majority of these regions exhibit hallmarks of cis-regulatory activity, including: small size (&amp;lt;500 bp), DNase I hypersensitivity, enhancer chromatin modifications (H3K4me1/H3K27ac), and evolutionary conservation. Our results reveal that most somatic cells are primed for global hypomethylation in regions of the genome that are typically hypomethylated in cancer cells, supporting previous observations that hypomethylation may be an indicator of cell age. In addition, our results in normal tissue suggest that hypomethylation in cancer cells could alter transcriptional regulation through disruption of cis-regulatory activity. Citation Format: Gary C. Hon, Yin Shen, David F. McCleary, Lee Edsall, Samantha Kuan, Bing Ren. Whole genome bisulfite sequencing reveals tissue-specific DNA methylation in a normal mouse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1111. doi:10.1158/1538-7445.AM2013-1111

ERG Induces Epigenetic Activation of Tudor Domain-Containing Protein 1 (TDRD1) in ERG Rearrangement-Positive Prostate Cancer

BackgroundOverexpression of ERG transcription factor due to genomic ERG-rearrangements defines a separate molecular subtype of prostate tumors. One of the consequences of ERG accumulation is modulation of the cell’s gene expression profile. Tudor domain-containing protein 1 gene (TDRD1) was reported to be differentially expressed between TMPRSS2:ERG-negative and TMPRSS2:ERG-positive prostate cancer. The aim of our study was to provide a mechanistic explanation for the transcriptional activation of TDRD1 in ERG rearrangement-positive prostate tumors.Methodology/Principal FindingsGene expression measurements by real-time quantitative PCR revealed a remarkable co-expression of TDRD1 and ERG (r2 = 0.77) but not ETV1 (r2<0.01) in human prostate cancer in vivo. DNA methylation analysis by MeDIP-Seq and bisulfite sequencing showed that TDRD1 expression is inversely correlated with DNA methylation at the TDRD1 promoter in vitro and in vivo (ρ = −0.57). Accordingly, demethylation of the TDRD1 promoter in TMPRSS2:ERG-negative prostate cancer cells by DNA methyltransferase inhibitors resulted in TDRD1 induction. By manipulation of ERG dosage through gene silencing and forced expression we show that ERG governs loss of DNA methylation at the TDRD1 promoter-associated CpG island, leading to TDRD1 overexpression.Conclusions/SignificanceWe demonstrate that ERG is capable of disrupting a tissue-specific DNA methylation pattern at the TDRD1 promoter. As a result, TDRD1 becomes transcriptionally activated in TMPRSS2:ERG-positive prostate cancer. Given the prevalence of ERG fusions, TDRD1 overexpression is a common alteration in human prostate cancer which may be exploited for diagnostic or therapeutic procedures.

The human placenta methylome

Tissue-specific DNA methylation is found at promoters, enhancers, and CpG islands but also over larger genomic regions. In most human tissues, the vast majority of the genome is highly methylated (>70%). Recently, sequencing of bisulfite-treated DNA (MethylC-seq) has revealed large partially methylated domains (PMDs) in some human cell lines. PMDs cover up to 40% of the genome and are associated with gene repression and inactive chromatin marks. However, to date, only cultured cells and cancers have shown evidence for PMDs. Here, we performed MethylC-seq in full-term human placenta and demonstrate it is the first known normal tissue showing clear evidence of PMDs. We found that PMDs cover 37% of the placental genome, are stable throughout gestation and between individuals, and can be observed with lower sensitivity in Illumina 450K Infinium data. RNA-seq analysis confirmed that genes in PMDs are repressed in placenta. Using a hidden Markov model to map placental PMDs genome-wide and compare them to PMDs in other cell lines, we found that genes within placental PMDs have tissue-specific functions. For regulatory regions, methylation levels in promoter CpG islands are actually higher for genes within placental PMDs, despite the lower overall methylation of surrounding regions. Similar to PMDs, polycomb-regulated regions are hypomethylated but smaller and distinct from PMDs, with some being hypermethylated in placenta compared with other tissues. These results suggest that PMDs are a developmentally dynamic feature of the methylome that are relevant for understanding both normal development and cancer and may be of use as epigenetic biomarkers.

Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscape

BackgroundDNA methylation is an epigenetic modification that changes with age in human tissues, although the mechanisms and specificity of this process are still poorly understood. We compared CpG methylation changes with age across 283 human blood, brain, kidney, and skeletal muscle samples using methylation arrays to identify tissue-specific age effects.ResultsWe found age-associated CpGs (ageCGs) that are both tissue-specific and common across tissues. Tissue-specific ageCGs are frequently located outside CpG islands with decreased methylation, and common ageCGs show the opposite trend. AgeCGs are significantly associated with poorly expressed genes, but those with decreasing methylation are linked with higher tissue-specific expression levels compared with increasing methylation. Therefore, tissue-specific gene expression may protect against common age-dependent methylation. Distinguished from other tissues, skeletal muscle ageCGs are more associated with expression, enriched near genes related to myofiber contraction, and closer to muscle-specific CTCF binding sites. Kidney-specific ageCGs are more increasingly methylated compared to other tissues as measured by affiliation with kidney-specific expressed genes. Underlying chromatin features also mark common and tissue-specific age effects reflective of poised and active chromatin states, respectively. In contrast with decreasingly methylated ageCGs, increasingly methylated ageCGs are also generally further from CTCF binding sites and enriched within lamina associated domains.ConclusionsOur data identified common and tissue-specific DNA methylation changes with age that are reflective of CpG landscape and suggests both common and unique alterations within human tissues. Our findings also indicate that a simple epigenetic drift model is insufficient to explain all age-related changes in DNA methylation.

Ethanol‐Induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain

Abnormal DNA methylation has been observed in promoter regions of a number of genes in human alcoholics. It is unclear whether DNA methylation changes in alcoholics result directly from alcohol consumption or predated the occurrence of alcohol abuse or dependence and whether altered DNA methylation influences gene expression. We investigated ethanol (EtOH)-induced DNA methylation changes in mouse serotonin receptor 3a gene (Htr3a). A 5-day drinking-in-the-dark paradigm was applied to 28 male outbred CD-1 mice (15 EtOH-drinking and 13 water-drinking). The Sequenom MassARRAY approach was used to quantify methylation levels of 8 CpGs around Htr3a transcription start site in trunk blood and 9 brain regions (dorsomedial prefrontal cortex [DMPFC], ventromedial prefrontal cortex, ventral tegmental area, dorsolateral striatum, dorsomedial striatum [DMSTR], ventral striatum, amygdala, hippocampus [HIPPO], and cerebellum). DNA methylation differences between the 2 groups of mice (EtOH- and water-drinking) were analyzed using multivariate analysis of covariance with consideration of EtOH consumption amount. Expression levels of Htr3a in the DMSTR were measured by real-time PCR in 14 EtOH-drinking and 14 water-drinking male CD-1 mice. EtOH drinking increased methylation levels of specific Htr3a promoter CpGs in mouse blood (CpG-27: p = 0.028; CpG+54: p = 0.044) and HIPPO (CpG+151: p = 0.012) but reduced methylation levels of specific Htr3a promoter CpGs in mouse DMSTR (CpG-96: p = 0.020; CpG-27: p = 0.035) and DMPFC (CpG+138: p = 0.011; CpG+151: p = 0.040). Nevertheless, methylation levels of Htr3a promoter CpGs in 6 other brain regions were not significantly altered by EtOH consumption. Additionally, the expression level of Htr3a in the DMSTR was 1.43-fold higher in alcohol-drinking mice than in water-drinking mice (p = 0.044). Our findings indicate that alcohol consumption may induce tissue-specific DNA methylation changes and further suggest that Htr3a promoter methylation levels may be reversely correlated with Htr3a expression levels in specific brain regions such as DMSTR.

Different measures of “genome-wide” DNA methylation exhibit unique properties in placental and somatic tissues

DNA methylation of CpGs located in two types of repetitive elements—LINE1 (L1) and Alu—is used to assess “global” changes in DNA methylation in studies of human disease and environmental exposure. L1 and Alu contribute close to 30% of all base pairs in the human genome and transposition of repetitive elements is repressed through DNA methylation. Few studies have investigated whether repetitive element DNA methylation is associated with DNA methylation at other genomic regions, or the biological and technical factors that influence potential associations. Here, we assess L1 and Alu DNA methylation by Pyrosequencing of consensus sequences and using subsets of probes included in the Illumina Infinium HumanMethylation27 BeadChip array. We show that evolutionary age and assay method affect the assessment of repetitive element DNA methylation. Additionally, we compare Pyrosequencing results for repetitive elements to average DNA methylation of CpG islands, as assessed by array probes classified into strong, weak and non-islands. We demonstrate that each of these dispersed sequences exhibits different patterns of tissue-specific DNA methylation. Correlation of DNA methylation suggests an association between L1 and weak CpG island DNA methylation in some of the tissues examined. We caution, however, that L1, Alu and CpG island DNA methylation are distinct measures of dispersed DNA methylation and one should not be used in lieu of another. Analysis of DNA methylation data is complex and assays may be influenced by environment and pathology in different or complementary ways.

Recurrent Variations in DNA Methylation in Human Pluripotent Stem Cells and Their Differentiated Derivatives

Human pluripotent stem cells (hPSCs) are potential sources of cells for modeling disease and development, drug discovery, and regenerative medicine. However, it is important to identify factors that may impact the utility of hPSCs for these applications. In an unbiased analysis of 205 hPSC and 130 somatic samples, we identified hPSC-specific epigenetic and transcriptional aberrations in genes subject to X chromosome inactivation (XCI) and genomic imprinting, which were not corrected during directed differentiation. We also found that specific tissue types were distinguished by unique patterns of DNA hypomethylation, which were recapitulated by DNA demethylation during in vitro directed differentiation. Our results suggest that verification of baseline epigenetic status is critical for hPSC-based disease models in which the observed phenotype depends on proper XCI or imprinting and that tissue-specific DNA methylation patterns can be accurately modeled during directed differentiation of hPSCs, even in the presence of variations in XCI or imprinting.