Global DNA Methylation Analysis Research Articles

Highlights of This Issue

T-cell based immunotherapy seems to be a promising approach as a new treatment for multiple myeloma. Fichtner and colleagues evaluated an impact of immunosuppression and antigen-expression on the development of specific CD8+ T-cell responses against CTA in patients with plasmacell-dyscrasias. The antigen-specific immunoresponses correlated with the expression of the antigen and an early stage of disease. In conclusion, T-cell-based immunotherapy might be performed in an early stage of disease, where the tumor-associated immunosuppression is low and a prophylactic immune response against antigens that were not yet expressed might be possible to establish.Oxidative stress rescue mechanisms have the potential to increase cancer susceptibility, cause chemoresistance, and affect outcome. Despite being a regulator of the main mediator of the redox stress response, NRF2, the Kelch-like ECH-associated protein 1 (KEAP1) has not been extensively studied in breast cancer. Hartikainen and colleagues report associations of KEAP1 gene SNPs with breast cancer risk and survival. In addition to being associated with increased breast cancer risk, the investigated polymorphisms modified the effects of radiotherapy and tamoxifen treatment on patient survival. The knowledge of the involvement of NRF2 pathway in BC lays ground to its translation in clinical use.Natural killer cell lymphomas (NKCLs) are rare but aggressive neoplasms, and little is known concerning the pathogenesis. Küçük and colleagues performed a global analysis of DNA methylation and identified a set of genes recurrently transcriptionally silenced through aberrant promoter hypermethylation. The list is enriched in previously reported tumor-suppressor genes. Many of these genes may contribute to neoplastic transformation by deregulating critical functions such as NK-cell activation/homeostasis and apoptosis. For example, BIM is frequently silenced and its reactivation leads to cell death and increased chemosensitivity in NKCLs. Another interesting finding is the frequent promoter methylation of asparagine synthetase that may predict sensitivity to asparaginase treatment, suggesting the possibility of adjustment in asparaginase containing chemotherapeutic regimens.HGF/MET signaling is frequently activated in human colorectal cancer (CRC) and the mechanism by which the activation of this pathway is not fully elucidated. By using immunohistochemistry, Liu and colleagues found that decreased BATF2 correlates with tumor progression and poor CRC patient prognosis as well as MET expression. In vitro, BATF2 downregulates MET expression through its interaction with c-Jun/AP-1. MET inhibitors in combination with IFN-β produce a synergistic cytotoxicity both in vitro and in vivo. This study suggests that IFNs, as an adjuvant in combination with MET inhibitors, may serve as a novel therapeutic strategy for CRC patients.

Analysis of Global DNA Methylation in Irradiated Fig Selections

Fruits from fig tree (Ficus carica L.) are economically important worldwide, and rank among the top 20 fruits exported by Brazil. However, due to particular features in its floral structure, classical plant breeding procedures that involve hybridization are not possible. Thus, genetic improvement of figs by using mutagens is an important line of research. In this study, five fig selections based on their interesting agronomic features were used. Genetic modifications associated with mutations were not detected by molecular markers. Therefore, it was suggested that certain phenotypes could have resulted from epigenetic modifications. The best characterized epigenetic modification is DNA methylation, which can switch genes on or off by acting as a signal. After epigenetic polymorphisms were identified by methylation sensitive amplified polymorphism (MSAP) molecular markers, we quantified the level of DNA methylation in fig selections irradiated with gamma rays and compared it to levels in the commercial cultivar “Roxo-de-Valinhos.” This was achieved by analyzing the global methylation using an Imprint Methylated DNA Quantification kit. The results showed that there were significant differences in global methylation following different treatments, indicating that irradiation was an abiotic factor that could alter the epigenome of plants. Since the material used as a control was also found to be methylated, demethylation of the polymorphic genomic material might account for the phenotypic variations observed among different treatment groups. These results suggest that irradiation is an external factor that is capable of altering epigenetic patterns.

Structure and functional evaluation of porcine NANOG that is a single-exon gene and has two pseudogenes

Nanog plays an important role in maintaining the pluripotency of murine and human embryonic stem cells. However, the molecular features and transcriptional regulation of the NANOG gene have not been well investigated in pig. Here, we report, for the first time, that porcine NANOG is encoded by a single exon gene (SEG) mapped on chromosome 1 and has two daughter genes, one pseudogene NANOGP1 on chromosome 5 and one tandem duplicate on chromosome 1. The duplicated pseudogene NANOGP2 has high sequence similarity to NANOG, but does not encode a functional protein due to deletions and in-frame stop codons. The NANOGP1 contains four exons and three introns, but is short of the homeodomain sequence. Transcriptome analysis confirmed that NANOG mRNA in porcine iPS cells is transcribed from the SEG NANOG, but not from NANOGP1, because the NANOGP1 promoter is highly methylated, as confirmed by global DNA methylation analysis. The NANOG protein encoded by NANOG retains N, H, and C1/W/C2 domains. The H domain is required for nuclear translocation, while the C1/W/C2 domain ensures the NANOG regulatory function. Overexpression of NANOG in porcine embryonic fibroblasts promoted upregulation of its target genes SOX2, KLF4, and c-MYC. In conclusion, the functional porcine NANOG that is different in chromosomal structure from mouse and human genes is a single exon gene and encodes the functional NANOG protein that can be specifically regulated by OCT4/SOX2, and can promote the activation of target pluripotent factors in vivo.

The Oligodendrocyte Lineage Transcription Factor 2 (OLIG2) Is Epigenetically Regulated in AML and Suppresses Leukemia Cell Growth

Introduction: DNA methylation differences between normal and cancer tissue that result in differential expression of genes are a hallmark of acute myeloid leukemia (AML). DNA methylation mediated silencing of specific genes, especially transcription factors, can provide a growth advantage for malignant cells. Global DNA methylation analyses have not only led to a better understanding of AML subgroups and the impact of epigenetic aberrations in leukemogenesis, but also to the identification of new epigenetically regulated genes. We and others have recently identified the oligodendrocyte lineage transcription factor 2 (OLIG2) as differentially methylated in AML cell lines compared with normal bone marrow cells.Aim of the study: With the hypothesis that OLIG2, which is not expressed in normal hematopoiesis, may play a role in cancerogenesis as shown for acute lymphoblastic leukemia (Lin et al., Cancer Res. 2005) and malignant glioma (Mehta et al., Cancer Cell 2011), we sought to further dissect the impact of OLIG2 in AML, implementing functional studies and primary samples.Results: First, in a cohort of 93 AML patients, we could confirm previous results by Kröger et al. (Blood 2008) that OLIG2 is differentially methylated: using pyrosequencing, 37 patients (39.8%) showed methylation levels > 25% (range (r): 26-79%) in the 5 CpG containing amplicon of the OLIG2 promoter region, independent of cytogenetic subgroup. In a small subset of 13 patients where expression-data was available, an inverse correlation between OLIG2 DNA methylation and mRNA expression was significant (r2=0.55, p<0.005). This observation was further supported by a highly significant inverse DNA methylation/mRNA expression correlation in 10 leukemia cell lines (r2=0.74, p< 0.002). Moreover, we could demonstrate that this inverse correlation held also true for OLIG2 protein levels in cell lines with strong expression in THP-1 and NB-4, moderate expression in HL-60 and HEL and no expression in U937, KG-1A, PL-21, Kasumi-1, K-562 and Jurkat.Interestingly, while CD 34+ cells from two healthy donors and 10 out of 12 AML patients where protein was available, showed no protein expression, OLIG2 was expressed in 2 patients, both bearing the translocation t(15;17). This corresponds well to OLIG2 expression of cell line NB-4, which also harbours t(15;17). Treatment of non-expressing cell lines PL-21 and U937 with 200 nM 5-aza-2′-deoxycytidine led to robust re-expression of OLIG2, both on mRNA and protein level, strongly implicating DNA methylation as a silencing mechanism in a subset of AML.To investigate the relationship between OLIG2 expression and AML cell growth we used a siRNA transient knock-down in OLIG2 expressing cell lines THP-1 and NB-4. While OLIG2 protein expression measured via densitometry could be strongly reduced to 38% and 45% from pre-treatment levels in THP-1 and NB-4 cells, respectively, no change on cell viability or cell growth was detected. However, stable over-expression of OLIG2 using the lentiviral-vector pLeGO-iG in Kasumi-1 cells, led to a significant growth-inhibition of 32.2% (r: 27.0-37.3%) after 5 days and a 47.7% (r: 30.7-64.6%) increase of apoptotic cells (Annexin-V-staining) as compared to control-vector transfected cells. This negative effect on cell proliferation supports our presumption that OLIG2 could act as a growth-regulator in a subgroup of AML. This could be caused by a direct interaction between OLIG2 and a cell cycle regulator or a transcription factor complex.Conclusion: We show that OLIG2 (I) is in part epigenetically regulated via DNA methylation in AML, resulting in an inverse correlation between DNA methylation and expression; (II) can be re-expressed upon demethylating treatment in cell lines, therefore making it an attractive biomarker to study in AML patients treated with demethylating agents; (III) shows antiproliferative activity in leukemia cell lines and thus should be further studied as a potential tumor suppressor in AML. DisclosuresLübbert:Cephalon / TEVA: Travel support Other.

Global DNA methylation analysis of human atherosclerotic plaques reveals extensive genomic hypomethylation and reactivation at imprinted locus 14q32 involving induction of a miRNA cluster.

We conducted a genome-wide analysis to identify differentially methylated genes in atherosclerotic lesions. DNA methylation at promoters, exons and introns was identified by massive parallel sequencing. Gene expression was analysed by microarrays, qPCR, immunohistochemistry and western blots. Globally, hypomethylation of chromosomal DNA predominates in atherosclerotic plaques and two-thirds of genes showing over 2.5-fold differential in DNA methylation are up-regulated in comparison to healthy mammary arteries. The imprinted chromatin locus 14q32 was identified for the first time as an extensively hypomethylated area in atherosclerosis with highly induced expression of miR127, -136, -410, -431, -432, -433 and capillary formation-associated gene RTL1. The top 100 list of hypomethylated promoters exhibited over 1000-fold enrichment for miRNAs, many of which mapped to locus 14q32. Unexpectedly, also gene body hypermethylation was found to correlate with stimulated mRNA expression. Significant changes in genomic methylation were identified in atherosclerotic lesions. The most prominent gene cluster activated via hypomethylation was detected at imprinted chromosomal locus 14q32 with several clustered miRNAs that were up-regulated. These results suggest that epigenetic changes are involved in atherogenesis and may offer new potential therapeutic targets for vascular diseases.

High-frequency aberrantly methylated targets in pancreatic adenocarcinoma identified via global DNA methylation analysis using methylCap-seq.

BackgroundExtensive reprogramming and dysregulation of DNA methylation is an important characteristic of pancreatic cancer (PC). Our study aimed to characterize the genomic methylation patterns in various genomic contexts of PC. The methyl capture sequencing (methylCap-seq) method was used to map differently methylated regions (DMRs) in pooled samples from ten PC tissues and ten adjacent non-tumor (PN) tissues. A selection of DMRs was validated in an independent set of PC and PN samples using methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), and methylation sensitive restriction enzyme-based qPCR (MSRE-qPCR). The mRNA and expressed sequence tag (EST) expression of the corresponding genes was investigated using RT-qPCR.ResultsA total of 1,131 PC-specific and 727 PN-specific hypermethylated DMRs were identified in association with CpG islands (CGIs), including gene-associated CGIs and orphan CGIs; 2,955 PC-specific and 2,386 PN-specific hypermethylated DMRs were associated with gene promoters, including promoters containing or lacking CGIs. Moreover, 1,744 PC-specific and 1,488 PN-specific hypermethylated DMRs were found to be associated with CGIs or CGI shores. These results suggested that aberrant hypermethylation in PC typically occurs in regions surrounding the transcription start site (TSS). The BSP, MSP, MSRE-qPCR, and RT-qPCR data indicated that the aberrant DNA methylation in PC tissue and in PC cell lines was associated with gene (or corresponding EST) expression.ConclusionsOur study characterized the genome-wide DNA methylation patterns in PC and identified DMRs that were distributed among various genomic contexts that might influence the expression of corresponding genes or transcripts to promote PC. These DMRs might serve as diagnostic biomarkers or therapeutic targets for PC.

Genome-wide DNA methylation as an epigenetic consequence of Epstein-Barr virus infection of immortalized keratinocytes.

The oral cavity is a persistent reservoir for Epstein-Barr virus (EBV) with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns regulated by epigenetic modifications involving DNA methylation and chromatin structure. Regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may result in long-lasting host epigenetic reprogramming. To identify epigenetic events following EBV infection, a transient infection model was established to map epigenetic changes in telomerase-immortalized oral keratinocytes. EBV-infected oral keratinocytes exhibited a predominantly latent viral gene expression program with some lytic or abortive replication. Calcium and methylcellulose-induced differentiation was delayed in EBV-positive clones and in clones that lost EBV compared to uninfected controls, indicating a functional consequence of EBV epigenetic modifications. Analysis of global cellular DNA methylation identified over 13,000 differentially methylated CpG residues in cells exposed to EBV compared to uninfected controls, with CpG island hypermethylation observed at several cellular genes. Although the vast majority of the DNA methylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquired DNA methylation within the gene body while those with decreased transcript levels acquired DNA methylation near the transcription start site. Treatment with the DNA methyltransferase inhibitor, decitabine, restored expression of some hypermethylated genes in EBV-infected and EBV-negative transiently infected clones. Overall, these observations suggested that EBV infection of keratinocytes leaves a lasting epigenetic imprint that can enhance the tumorigenic phenotype of infected cells. Here, we show that EBV infection of oral keratinocytes led to CpG island hypermethylation as an epigenetic scar of prior EBV infection that was retained after loss of the virus. Such EBV-induced epigenetic modification recapitulated the hypermethylated CpG island methylator phenotype (CIMP) observed in EBV-associated carcinomas. These epigenetic alterations not only impacted gene expression but also resulted in delayed calcium and methylcellulose-induced keratinocyte differentiation. Importantly, these epigenetic changes occurred in cells that were not as genetically unstable as carcinoma cells, indicating that EBV infection induced an epigenetic mutator phenotype. The impact of this work is that we have provided a mechanistic framework for how a tumor virus using the epigenetic machinery can act in a "hit-and-run" fashion, with retention of epigenetic alterations after loss of the virus. Unlike genetic alterations, these virally induced epigenetic changes can be reversed pharmacologically, providing therapeutic interventions to EBV-associated malignancies.

INTEGRATIVE GENOMIC CHARACTERIZATION OF LOWER GRADE GLIOMAS

BACKGROUND: Lower grade gliomas (LGG) are infiltrative brain tumors that include astrocytomas, oligodendrogliomas and oligoastrocytomas, grades II and III. LGGs almost always progress, typically to glioblastoma (GBM, grade IV), and are uniformly fatal. The Cancer Genome Atlas (TCGA) is conducting a comprehensive molecular analysis of LGG, incorporating genetic and genomic alterations, DNA methylation profiles, and RNA and proteomic signatures. The rich, integrated clinical data of TCGA provides an outstanding platform to uncover biomarkers of therapy response and outcome. METHODS: We analyzed 289 lower grade gliomas for exome sequence to uncover somatic mutations; DNA copy number alterations; RNA sequencing for expression and gene fusion; DNA methlyation; microRNA expression; and protein level and phosphorylation. Supervised and unsupervised clustering was performed to segregate LGGs into robust molecular categories. RESULTS: Among 289 LGGs, exome sequencing identified 19 significantly mutated genes, including IDH1, TP53, ATRX, CIC, FUBP1, NOTCH1, PIK3CA, NF1, PIK3R1, ARID1A, PTEN, SMARCA4 and EGFR. Frequent arm-level deletions were 1p, 19q, 13q, 9p, 10q, 10p, 4q and 4p; arm-level gains included 7q, 7p, 19p and 11q. There were 15 significant genomic amplifications, among which were 7p11.2 (EGFR), 12q14.1 (CDK4), 1q32.1 (MDM4), 8q24.21 (MYC), 12p13.32 (CCND2) and 4q12 (PDGFR). Among 28 deletions were 9p21.3 (CDKN2A, CDKN2B), 19q13.42 (TFPT, ZNF331), 2q37.3, 10q26.2 (FGFR2, DUX4) and 14q24.3 (TSHR, GPHN).Global analysis of DNA methylation identified 5 stable clusters, with one showing substantial hypomethylation. Gene expression profiling identified 4 stable clusters. On integrative analysis, IDH1/2 wt LGGs had features of “pre-GBM”: they formed distinct hypomethylation and gene expression clusters; had molecular alterations typical of GBM, such as EGFR amplifications, PTEN mutations, CDKN2A loss and RTK gene fusions; were mostly grade III astrocytomas; and had short survivals. IDH1/2 mutant LGGs were hypermethylated. One subgroup was enriched for 1p/19q co-deletion, CIC and FUBP mutations, and oligodendrogliomas. Another subset was enriched for TP53 and ATRX mutations and astrocytomas. These groups clustered separately on gene expression analysis. A fourth gene expression cluster had elements of these two IDH1/2 mutant subgroups and the longest survival. A cluster-of-cluster analysis of mRNA, copy number, miRNA and methylation analyses reinforced the clear separation of three molecular classes of LGG based on the status of IDH1/2 and 1p/19q. CONCLUSIONS: These data illustrate a potential stratification of LGGs that emphasizes molecular characteristics. IDH wt LGG have molecular alterations and clinical behavior similar to GBMs, whereas IDH mutant LGGs segregate based on 1p/19q status. SECONDARY CATEGORY: Tumor Biology.

Global DNA methylation and extracellular matrix remodeling in aortic smooth muscle versus endothelial cells in hyperhomocysteinemia (1120.5)

Introduction: Homocysteine (Hcy) is a byproduct of DNA methylation. Increased levels of Hcy (HHcy) have been shown to induce smooth muscle cell (SMC) proliferation and inhibit endothelial cell proliferation. It is well known that HHcy causes increased activation of matrix metalloproteinases, a key factor in vascular remodeling. However, the role of global DNA methylation in regulating aortic matrix remodeling is not known.Hypothesis: In the present study, we hypothesize that global DNA methylation during HHcy regulates the expression of MMPs in mouse aortic SMCs and endothelial cells (MAEC).Methods: Mouse SMC and MAEC were cultured with 80µM Hcy and 100µM Aza deoxycytidine (AzadC), DNA methyltransferase inhibitor.Results: Global DNA methylation analysis by ELISA showed that in HHcy, there was a 2 fold and 1.3 fold increase in %5‐mC in SMC and MAECs respectively. During HHcy, there was an increased expression and activity of MMP9 in SMCs, which depreciated after AzadC treatment. Methylation Specific PCR (MS‐PCR) showed increased methylation status of Hcy metabolism genes, CBS and MTHFR in SMCs and MAECs.Summary: Our results suggest that HHcy causes a greater global hyper‐methylation in aortic SMC than in MAEC. This may suggest that Hcy generated through methylation contributes to vascular remodeling.Grant Funding Source: NIH

S-adenosylmethionine Levels Regulate the Schwann Cell DNA Methylome

Axonal myelination is essential for rapid saltatory impulse conduction in the nervous system, and malformation or destruction of myelin sheaths leads to motor and sensory disabilities. DNA methylation is an essential epigenetic modification during mammalian development, yet its role in myelination remains obscure. Here, using high-resolution methylome maps, we show that DNA methylation could play a key gene regulatory role in peripheral nerve myelination and that S-adenosylmethionine (SAMe), the principal methyl donor in cytosine methylation, regulates the methylome dynamics during this process. Our studies also point to a possible role of SAMe in establishing the aberrant DNA methylation patterns in a mouse model of diabetic neuropathy, implicating SAMe in the pathogenesis of this disease. These critical observations establish a link between SAMe and DNA methylation status in a defined biological system, providing a mechanism that could direct methylation changes during cellular differentiation and in diverse pathological situations.

Simultaneous analysis of global DNA methylation and hydroxymethylation in tissues by liquid chromatography-tandem mass spectrometry

Measuring global DNA methylation and hydroxymethylation is important in researches of effect and mechanism of environmental pollutants exposure. A method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed to simultaneously determine global DNA methylation and hydroxymethylation level in biological tissues. DNA was extracted from tissues and converted into single nucleotide via enzyme digestion. Liquid chromatography coupled to tandem mass spectrometry was used to measure the concentrations of 5-methylcytidine, 5-hydroxymethylcytosine and deoxyguanosine, which were used to calculate global DNA methylation ratios and hydroxymethylation ratios. The results showed that the correlation coefficients were higher than 0.99. The limit of detection ( LOD, S/N = 3) and the limit of quantification (LOQ, S/N = 10) of 5-methylcytidine were 0.015 and 0.045 ng/mL, respectively. They reached 0.001 and 0.003 ng/mL for 5-hydroxymethylcytosine, and were 0.2 and 0.6 ng/mL for deoxyguanosine. The developed method was further successfully applied to investigate global DNA methylation and hydroxymethylation alteration in liver and cerebellum of rats exposed to arsenic via drinking water. This approach could quantitatively detect 5-methylcytidine, 5-hydroxymethylcytosine and deoxyguanosine with high sensitivity, repeatability and stability. Our study provided a means to simultaneously analyze global DNA methylation and hydroxymethylation.

Global DNA methylation and transcriptional analyses of human ESC-derived cardiomyocytes

With defined culture protocol, human embryonic stem cells (hESCs) are able to generate cardiomyocytes in vitro, therefore providing a great model for human heart development, and holding great potential for cardiac disease therapies. In this study, we successfully generated a highly pure population of human cardiomyocytes (hCMs) (>95% cTnT+) from hESC line, which enabled us to identify and characterize an hCM-specific signature, at both the gene expression and DNA methylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation, and stand as an informative and rich resource for investigating cardiac gene functions and disease mechanisms. Moreover, we show that cardiac-structural genes and cardiac-transcription factors have distinct epigenetic mechanisms to regulate their gene expression, providing a better understanding of how the epigenetic machinery coordinates to regulate gene expression in different cell types.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-013-0016-x) contains supplementary material, which is available to authorized users.

Transient Activation Of Inflammatory Molecules By Epigenetic Modifiers Likely Plays a Role In Expansion Of Transplantable Human Cord Blood Stem/Progenitor Cells

Recent studies indicate that inflammatory molecules, including interferon, possess stimulatory effects which trigger excessive proliferation leading to hematopoietic stem cell (HSC) exhaustion and bone marrow (BM) failure. We hypothesized that transient activation of inflammatory molecules by chromatin modifying agents (CMA) stimulates proliferation of transplantable HSC in culture. We previously demonstrated that sequential in vitro addition of a hypomethylating drug, 5-aza-2'-deoxycytidine (5azaD) followed by addition of a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) during human umbilical cord blood CD34+ cell culture results in expansion of serially transplantable long term HSC (Araki et al. Blood 2007, Exp Hematol 2009). However treatment with valproic acid (VPA), a HDAC inhibitor, only expands short term HSC, which are not serially transplantable (Araki et al. Abst. 827, Blood 2010). This failure of VPA to expand long-term repopulating HSC is not however due to poor BM homing (homing efficiency VPA-expanded cells 1.68 ± 0.35%, n = 10 NOD/SCID mice, 5azaD/TSA-expanded cells 0.39 ± 0.056%, n = 7, p = 0.008). Molecular studies indicate that expression of genes implicated in HSC self-renewal (HoxB4, Bmi1, Ezh2, and GATA2) are equally increased in 5azaD/TSA or VPA expanded CD34+ cells (n = 3, control vs. 5azaD/TSA or control vs. VPA p < 0.05). Similarly, increased HoxB4, Ezh2 and PU.1 protein levels are observed in VPA or 5azaD/TSA expanded cells compared to controls. Our global microarray data (Affymetrix chip HG U133 plus 2.0, n = 3) reveals a set of 113 differentially expressed genes preferentially detectable in 5azaD/TSA expanded cells associated with transplantable HSC expansion in culture and 278 HSC maintenance genes differentially expressed in CD34+ cells expanded in VPA or 5azaD/TSA, but not in control. Differential expression of genes representative of HSC expansion or maintenance gene sets were validated by real time qPCR. Intriguingly, functional pathway analyses of the 113 HSC expansion genes indicate the importance of inflammation signaling pathways (genes such as S100A8, Alox5). Furthermore, global genomic DNA methylation analysis of uncultured vs expanded, enriched CD34+ cells by LINE-1 methylation assay indicates transient hypomethylation of 5azaD/TSA-expanded cells at day 3 (55.4 ± 2.2 vs pre–treatment levels 77.3 ± 1.9) which later returned towards baseline (69.7 ± 2.1), corresponding with recovery of DNMT1 expression. Notably, control cultures or CD34+ cells expanded in VPA do not display changes in LINE-1 methylation (78.5 ± 2.2 and 77.2 ± 2 respectively, n = 2, mean % ± SE of 4 CpG sites). Interestingly, ELISA assays of conditioned medium from 5azaD/TSA-expanded cells reveals that levels of the inflammatory mediator leukotriene B4 (LTB4; synthesized from arachidonic acid by the Alox5 gene products, 5-lipooxygenase and LTA4 hydrolase) are 426.87 ± 101.7 pg/ml, significantly greater than that of control cultures (109.5 ± 11.6 pg/ml; n = 3, p = 0.03). VPA-treated cultures had slightly higher levels than controls (346.8 ± 138.8 pg/ml; p = 0.16). Similarly 5azaD/TSA IL-1β levels were significantly greater than controls (10.1 ± 0.8 pg/ml vs 6.0 ± 0.6 pg/ml, n= 2, triplicate wells, p = 0.05). IL-1β levels from VPA cultures were 10.1 ± 4.5 pg/ml (p = n.s.). Notably, expansion of CD34+CD90+ cells in the presence of 5azaD/TSA is reduced by 47.6% by addition of dexamethasone, an anti-inflammatory agent, without affecting total cell numbers. Furthermore, demethylation of CpG sites of the promoters of inflammatory/stress response genes correspond with higher expression of these genes in CMA-expanded cultures (e.g., S100A8 and Cyp11A1; pre treatment: 76.42% ± 4.91% and 80.73 % ± 4.16%, post 5azaD/TSA treatment 54.5% ± 3.84% and 54.29% ± 3.72% respectively; n = 2, mean ± SE of 3 to 11 CpG sites methylation). S100A8 is a known agonist of toll-like receptor 4, which plays a role in the maintenance and proliferation of endothelial progenitor cells, in keeping with the influence of systemic infection on HSC cycling and of interferon on HSC proliferation. Our studies, for the first time, identify inflammatory molecules regulated by epigenetic mechanisms that are potentially important in governing HSC fate choices. Further studies should determine the mechanisms by which CMAs influence HSC expansion or maintenance through inflammatory and other stress response pathway genes. Disclosures:No relevant conflicts of interest to declare.

Global analysis of DNA methylation changes during progression of oral cancer

Earlier studies involving a priori gene selection have identified promoter regions deregulated by DNA methylation changes in oral squamous cell cancers (OSCCs) and precancers. Interrogation of global DNA methylation patterns for such specimens has not been reported, though such analyses are needed to uncover novel molecular factors driving disease. We evaluated global DNA methylation patterns for 30 biopsies obtained from 10 patients undergoing surgical removal of an OSCC or carcinoma in situ (CIS). From a disease field in each patient, we collected (i) dysplastic, (ii) CIS or OSCC, and (iii) adjacent normal biopsies. DNA isolated from each biopsy was profiled for methylation status using the Illumina HumanMethylation27K platform. Our data demonstrate that aberrant methylation of promoter CpG islands exists across oral precancer and OSCC genomes. Non-hierarchical clustering of all methylation data revealed distinct methylation patterns between the normal and the CIS/OSCC tissues (with results for dysplastic biopsies split between groups). Multiple genes exhibiting recurrent aberrant DNA methylation were found for both dysplastic and CIS/OSCC groups, and included enrichment for genes found in the WNT and MAPK signaling pathways. In identifying aberrant DNA methylation at the earliest stages of oral precancer and finding recurring epigenetic disruption of specific genes/pathways across our analyzed cohort, we see evidence that CpG methylation changes may play a role in oral cancer progression and that global DNA methylation analyses may have significant utility in wider studies that seek to derive biomarkers or potentially druggable targets to improve oral cancer outcomes.

Persistent gene expression changes in NAc, mPFC, and OFC associated with previous nicotine or amphetamine exposure

Highly addictive drugs like nicotine and amphetamine not only change an individual's behaviour in the short and long-term, they also induce persistent changes in neuronal excitability and morphology. Although research has started to examine the epigenetic changes that occur immediately after drug exposure, there has been little investigation into the persistent modifications to the epigenome that likely moderate the stable maintenance of the neurological changes. Male Long-Evans rats were administered amphetamine, nicotine, or saline for 14 consecutive days, given a 14 day withdrawal period, and then sacrificed. DNA from the mPFC, OFC, and nucleus accumbens (NAc) was used for global DNA methylation analysis and RNA from the same brain regions was used for gene expression analysis. Following the two-week withdrawal period, exposure to amphetamine or nicotine was associated with a decrease in global DNA methylation in each brain region examined. Previous exposure to nicotine was associated with changes in expression of 16 genes (NAc:6, mPFC:5, OFC:5) whereas exposure to amphetamine was associated with changes in expression of 25 genes (NAc:13, OFC:8, mPFC:4). The persistent epigenetic changes associated with exposure to amphetamine and nicotine were region and drug dependent, and differ from the latent epigenetic changes that occur immediately after drug exposure. The changes in DNA methylation are consistent with the gene expression results and provide further support to the notion that DNA methylation is the key regulatory mechanism for experience dependent changes.

Global DNA methylation and transcriptional analyses of human ESC-derived cardiomyocytes.

With defined culture protocol, human embryonic stem cells (hESCs) are able to generate cardiomyocytes in vitro, therefore providing a great model for human heart development, and holding great potential for cardiac disease therapies. In this study, we successfully generated a highly pure population of human cardiomyocytes (hCMs) (>95% cTnT+) from hESC line, which enabled us to identify and characterize an hCM-specific signature, at both the gene expression and DNA methylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation, and stand as an informative and rich resource for investigating cardiac gene functions and disease mechanisms. Moreover, we show that cardiac-structural genes and cardiac-transcription factors have distinct epigenetic mechanisms to regulate their gene expression, providing a better understanding of how the epigenetic machinery coordinates to regulate gene expression in different cell types.

Abstract B32: Dynamic whole-genome methylomes reveal hidden diversity of CpG islands at multiple biological levels

Abstract CpG islands mark epigenetic regulatory hotspots of mammalian genomes. Numerous studies have demonstrated essential roles of CpG islands in regulation of gene expression, developmental processes, genomic imprinting, aging, and cancer. These regulatory effects rest critically on correct (or the lack of) DNA methylation of CpG islands, and aberrant DNA methylation of CpG islands are hallmarks of cancer. However, how DNA methylation of CpG islands occurs across different cell types, and how variation of CpG islands methylation relates to their functions, remain poorly understood. Here we present a global analysis of DNA methylation of CpG islands in the human genome. We examined variation of CpG island methylation across five complete methylomes of distinctive origins (whole blood, prefrontal cortex, sperm, embryonic stem cells and fibroblast). Our goal was to identify distinctive characteristics of CpG islands with respect to their epigenetic variation in normal tissues. Our analysis reveals that, contrary to the prevailing notion, CpG islands mark the most highly variably methylated regions in the human genome. Many CpG islands exhibit highly methylome-specific patterns of DNA methylation. Remarkably, DNA methylation patterns of CpG islands reflect their distinctive nature at many biological levels, including genomic characteristics such as lengths and nucleotide composition, functional differences as evidenced by distinctive gene ontology enrichments, and evolutionary features. Moreover, CpG islands implicated in cancers and those associated with aging show intriguing differences in their genomic, epigenomic and functional features. These findings indicate distinctive nature of CpG islands at genomic, epigenomic and functional levels. These new knowledge provides novel insights into deciphering the regulatory mechanisms of CpG islands in human health and diseases and may be used to improve empirical studies of DNA methylation variation across different biological conditions, and furthermore ultimately to aid development of better diagnostic markers. Note: This abstract was not presented at the conference. Citation Format: Jia Zeng, Hema Nagrajan, Soojin V. Yi. Dynamic whole-genome methylomes reveal hidden diversity of CpG islands at multiple biological levels. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B32.

Abstract 3807: Integrative genomic profiling of pilocytic astrocytomas reveals location specific differential methylation and expression of brain developmental genes.

Abstract Pilocytic astrocytomas (PA) are the most common brain tumor in pediatric patients and cause significant morbidity, particularly related to chronic neurological deficiencies. They are characterized by activating alterations in the mitogen activated protein kinase (MAPK) pathway, but little else is known about their development. To map the methylation profile of these tumors, we have performed a global DNA methylation analysis on 62 PAs and 7 normal cerebellum samples using Illumina 450K microarrays. Based on these data we have found two subgroups of PA that separate according to tumor location (infratentorial versus supratentorial), and have identified key neural developmental genes that are differentially methylated between the two groups, including NR2E1 and EN2. Integration with gene expression microarray data revealed significant expression differences that were typically associated with a strong positive correlation between methylation and expression. Differential methylation was most commonly identified within the gene body and/or 10 kb upstream/downstream of the gene body, and was, in part, accounted for by differences in the level of 5-hydroxymethylcytosine. We also identified a large number of differentially methylated genes between cerebellar PAs and normal cerebellum, which were again enriched for developmental genes. In addition, we found a significant association between differential methylation and SUZ12 binding sites, suggesting potential disruption of the polycomb repressor complex 2 (PRC2) in PA development. Taken together these data suggest the methylation profile of PA may reflect the cell of origin from which the tumors are derived, and highlights the potential disruption of key developmental regulators during tumorigenesis. These findings have implications for future clinical trials, as they suggest drug sensitivity and response may differ according to tumor location. Citation Format: Sally R. Lambert, Hendrik Witt, Volker Hovestadt, Manuela Zucknick, Marcel Kool, Danita Pearson, Andrey Korshnov, Stefan Pfister, V. Peter Collins, David T. W. Jones. Integrative genomic profiling of pilocytic astrocytomas reveals location specific differential methylation and expression of brain developmental genes. [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 3807. doi:10.1158/1538-7445.AM2013-3807

Analysis of age-related global DNA methylation in chicken.

DNA methylation is an epigenetic modification that plays an important role in the normal development and function of organisms. The level of DNA methylation is species-, tissue-, and organelle-specific, and the methylation pattern is determined during embryogenesis. DNA methylation has also been correlated with age. The aim of this study was to determine the global DNA methylation levels and their correlation with age in the chicken, using a Polish autosexing chicken breed, Polbar. A quantitative technique based on an immunoenzymatic assay was used for global DNA methylation analysis. The results show increased global DNA methylation levels with older Polbar embryos. Global DNA methylation levels decrease with the age of hens in the postembryonic stage. This study expands the current knowledge of the Polbar epigenome and the general knowledge of the function of epigenetic mechanisms in birds.

Protective effects of propolis on female rats’ histopathological, biochemical and genotoxic changes during LPS induced endotoxemia

In recent years, propolis has been the object of extensive research for its antibacterial, antioxidant, anti-inflammatory, and antitumoral activities. This study aims to determine the hepatoprotective efficiency of propolis on experimental endotoxemia in rats.In the current study, fifty adult Sprague Dawley rats (weighing 200–300g) were randomly divided into five groups of ten rats each. Normal saline solution was administered to the rats in the control group, while in the second group LPS (30mg/kg), in the third group propolis (250mg/kg), in the fourth group first propolis and then LPS (30mg/kg), and in the fifth group, first LPS (30mg/kg) and then propolis were given. Six hours after the application, biochemical (MDA levels) and histopathological changes as well as global DNA methylation analysis in the liver tissue samples were determined, while in the blood tissue samples Genomic Template Stability (GTS, %) was evaluated using RAPD-PCR profiles.The results demonstrated that the administration of propolis could have a protective effect against changes of both genomic stability values and methylation profiles, and it minimized the increase in MDA and tissue damage caused by LPS.In conclusion, the application of propolis prior to LPS-induced endotoxemia has shown to reduce hepatic damage.