methyl-CpG Immunoprecipitation Research Articles

Aberrant DNA methylation patterns in microsatellite stable human colorectal cancers define a new marker panel for the CpG island methylator phenotype.

A distinct group of colorectal carcinomas (CRCs) referred to as the "CpG island methylator phenotype" (CIMP) shows an extremely high incidence of de novo DNA methylation and may share common pathological, clinical or molecular features. However, there is limited consensus about which CpG islands (CGIs) define a CIMP, particularly in microsatellite stable (MSS) carcinomas. To study this phenotype in a systematic manner, we analyzed genome-wide CGI DNA methylation profiles of 19 MSS CRC using methyl-CpG immunoprecipitation (MCIp) and hybridization on 244K CGI oligonucleotide microarrays, determined KRAS and BRAF mutation status and compared disease-related DNA methylation changes to chromosomal instability as detected by microarray-based comparative genomic hybridization. Results were validated using mass spectrometry analysis of bisulfite-converted DNA at a subset of 76 individual CGIs in 120 CRC and 43 matched normal tissue samples. Both genome-wide profiling and CpG methylation fine mapping segregated a group of CRC showing pronounced and frequent de novo DNA methylation of a distinct group of CGIs that only partially overlapped with previously established classifiers. The CIMP group defined in our study revealed significant association with colon localization, either KRAS or BRAF mutation, and mostly minor chromosomal losses but no association with known histopathological features. Our data provide a basis for defining novel marker panels that may enable a more reliable classification of CIMP in all CRCs, independently of the MS status.

Stability of the human sperm DNA methylome to folic acid fortification and short-term supplementation.

Do short-term and long-term exposures to low-dose folic acid supplementation alter DNA methylation in sperm? No alterations in sperm DNA methylation patterns were found following the administration of low-dose folic acid supplements of 400 μg/day for 90 days (short-term exposure) or when pre-fortification of food with folic acid and post-fortification sperm samples (long-term exposure) were compared. Excess dietary folate may be detrimental to health and DNA methylation profiles due to folate's role in one-carbon metabolism and the formation of S-adenosyl methionine, the universal methyl donor. DNA methylation patterns are established in developing male germ cells and have been suggested to be affected by high-dose (5 mg/day) folic acid supplementation. This is a control versus treatment study where genome-wide sperm DNA methylation patterns were examined prior to fortification of food (1996-1997) in men with no history of infertility at baseline and following 90-day exposure to placebo (n = 9) or supplement containing 400 μg folic acid/day (n = 10). Additionally, pre-fortification sperm DNA methylation profiles (n = 19) were compared with those of a group of post-fortification (post-2004) men (n = 8) who had been exposed for several years to dietary folic acid fortification. Blood and seminal plasma folate levels were measured in participants before and following the 90-day treatment with placebo or supplement. Sperm DNA methylation was assessed using the whole-genome and genome-wide techniques, MassArray epityper, restriction landmark genomic scanning, methyl-CpG immunoprecipitation and Illumina HumanMethylation450 Bead Array. Following treatment, supplemented individuals had significantly higher levels of blood and seminal plasma folates compared to placebo. Initial first-generation genome-wide analyses of sperm DNA methylation showed little evidence of changes when comparing pre- and post-treatment samples. With Illumina HumanMethylation450 BeadChip arrays, no significant changes were observed in individual probes following low-level supplementation; when compared with those of the post-fortification cohort, there were also few differences in methylation despite exposure to years of fortified foods. Illumina HumanMethylation450 BeadChip data from this study have been submitted to the NCBI Gene Expression Omnibus under the accession number GSE89781. This study was limited to the number of participants available in each cohort, in particular those who were not exposed to early (pre-1998) fortification of food with folic acid. While genome-wide DNA methylation was assessed with several techniques that targeted genic and CpG-rich regions, intergenic regions were less well interrogated. Overall, our findings provide evidence that short-term exposure to low-dose folic acid supplements of 400 μg/day, over a period of 3 months, a duration of time that might occur during infertility treatments, has no major impact on the sperm DNA methylome. This work was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR: MOP-89944). The authors have no conflicts of interest to declare.

LOC283731 promoter hypermethylation prognosticates survival after radiochemotherapy in IDH1 wild-type glioblastoma patients.

MGMT promoter methylation status is currently the only established molecular prognosticator in IDH wild-type glioblastoma multiforme (GBM). Therefore, we aimed to discover novel therapy-associated epigenetic biomarkers. After enrichment for hypermethylated fractions using methyl-CpG-immunoprecipitation (MCIp), we performed global DNA methylation profiling for 14 long-term (LTS; >36 months) and 15 short-term (STS; 6-10 months) surviving GBM patients. Even after exclusion of the G-CIMP phenotype, we observed marked differences between the LTS and STS methylome. A total of 1,247 probes in 706 genes were hypermethylated in LTS and 463 probes in 305 genes were found to be hypermethylated in STS patients (p values < 0.05, log2 fold change ± 0.5). We identified 13 differentially methylated regions (DMRs) with a minimum of four differentially methylated probes per gene. Indeed, we were able to validate a subset of these DMRs through a second, independent method (MassARRAY) in our LTS/STS training set (ADCY1, GPC3, LOC283731/ISLR2). These DMRs were further assessed for their prognostic capability in an independent validation cohort (n = 62) of non-G-CIMP GBMs from the TCGA. Hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome (p = 0.03). The prognostic performance of LOC283731 promoter hypermethylation was confirmed in a third independent study cohort (n = 89), and was independent of gender, performance (KPS) and MGMT status (p = 0.0485, HR = 0.63). Intriguingly, the prediction was most pronounced in younger GBM patients (<60 years). In conclusion, we provide compelling evidence that promoter methylation status of this novel gene is a prognostic biomarker in IDH1 wild-type/non-G-CIMP GBMs.

NFATc1 Is Transcriptionally Activated in Chronic Lymphocytic Leukemia (CLL) By Promotor DNA-Hypomethylation Which Correlates with in-Vitro Vulnerability to Calcineurin Inhibitors

Chronic lymphocytic leukemia (CLL), the most frequent adult leukemia in Western countries, is characterized by progressive accumulation of mature, monoclonal B lymphocytes in blood, bone marrow, and lymphoid tissues. In the pathogenesis and treatment of CLL, B cell receptor (BCR) signaling plays a crucial role, and aberrations in downstream pathways that become activated in CLL need to be better defined. One downstream target of BCR signaling is NFATc1, a transcription factor with a high oncogenic and transforming potential. Employing a genome-wide comparative DNA methylation analysis the NFATc1 5’ region was identified to be DNA hypomethylated in CLL patient samples. The pilot series comprised CD19+ B-cells from 13 CLL patients without previous treatment (9 with mutated IGHV using a 98% similarity cut-off) and CD19+ B-cells from 6 age-matched healthy donors. DNA methylation was relatively quantified using methyl-CpG immunoprecipitation (MCIp) on custom oligonucleotide promoter arrays covering -8kbp to +2.5kbp of all human transcription start sites. In total 1944 genes showed significant DNA hypomethylation in CLL cells compared to non-malignant B-cells (p<0.05), with NFATc1 ranking at position 36. DNA hypomethylation of the NFATc1 promoter was validated in peripheral blood CD19+ B-cells from 76 CLL patients without previous treatment compared to 14 additional age-matched healthy donors using mass spectrometry based quantitative high resolution measurement of DNA methylation (MASSARRAY). These findings suggest activation of the NFATc1 gene in CLL cells. In fact, NFATc1 mRNA was significantly upregulated in CLL samples compared to non-malignant B-cells, and expression correlated highly significantly with DNA methylation (r=-0.66, p<0.001). Inhibition of NFATc1 in primary CLL cells from patients (n=5) using calcineurin inhibitors like CsA and FK-506 induced significant apoptosis in primary CLL cells in-vitro even when protected by co-culture with bone-marrow stromal cells, suggesting a dependence of CLL cells on the activity of NFATc1. For a mechanistic insight into NFATc1 signal transduction in primary CLL cells and to allow modeling of the signaling network, a panel of phospho-specific antibodies is currently used in flow cytometry. This allows to monitor multiple components of signaling pathways in parallel with high time resolution to understand the impact of NFATc1 upregulation on the cellular signaling networks. The approach will shed further light on how CLL cell survival is regulated how this pathway can be targeted in CLL. DisclosuresNo relevant conflicts of interest to declare.

ABCB1 Expression in Acute Myeloid Leukemia (AML): A Possible Predictive Value for Treatment Resistance?

Introduction: Over the last years, there has been a tremendous increase in understanding acute myeloid leukemia (AML) biology and a great effort has been taken in order to improve AML chemotherapy strategies. However, the growing knowledge of leukemia associated molecular mechanisms just started to translate into improved outcome. With regard to conventional chemotherapy multidrug resistance (MDR) is a persisting problem and the impact of ABCB1 (MDR1) expression is still controversially discussed.Methods: In this study we evaluated the ABCB1 expression using qRT-PCR and gene expression profiling (Affymetrix U133plus2.0 arrays) in 250 diagnostic AML samples derived from patients enrolled on a prospective treatment trial of the German-Austrian AML Study Group (AMLSG 07-04 trial; NCT00151242), in which patients were treated with an intensive anthracycline/cytarabine-based induction therapy. Findings were also evaluated in 154 TCGA AML cases receiving a 7+3 induction treatment (data available at http://cancergenome.nih.gov/) and put into perspective with previous reports. Furthermore, we investigated ABCB1 expression associated gene signatures and examined epigenetic regulation mechanisms by COBRA and methyl-CpG immunoprecipitation sequencing (MCIp-seq) in selected cases.Results: Our global analysis showed that patients who obtained a complete response (CR) following double induction therapy had lower ABCB1 mRNA levels compared to patients with refractory disease (RD) (p=0.07). Regarding cytogenetic AML subtypes, ABCB1 mRNA levels varied among the different cytogenetic groups with the complex karyotype group showing the highest ABCB1 and the inv(16) group the lowest ABCB1 expression levels. A comparison of CR versus RD cases within the cytogenetically determined prognostic groups showed that in the intermediate [CN-AML, t(11q23), and other intermediate risk cytogenetic aberrations (othersinter)] and poor risk groups (complex karyotype and othershigh), RD patients presented with significantly higher ABCB1 mRNA levels (p=0.02). Similarly, patients with favorable risk cytogenetics [t(8;21) and inv(16)], who achieved a CR, presented with lower ABCB1 levels compared to the ones, who were refractory. Patients with the lowest ABCB1 expression quartile (ABCB1low) showed significantly longer event-free survival (EFS) times than patients in the highest quartile cohort (ABCB1high) (median EFS 322 vs 105 days; p=0.02), while no differences were observed with regard to overall survival. In accordance, there was a significant enrichment of RD cases in the ABCB1high patient group (p=0.03). Next, in order to better understand the regulation of ABCB1 in AML, we specifically evaluated the DNA methylation level of a previously identified GC box important for ABCB1 expression regulation in CML and we performed global analyses of the entire ABCB1 5′ region. While both analyses did not reveal significant differences, further investigation of an ABCB1 associated gene pattern showed a correlation with CD34 and KIT expression (p<0.001). This suggests that like in CML, ABCB1 might be regulated by WNT, and in line, normal CD34+ hematopoietic stem cells also showed high ABCB1 expression levels.Conclusions: In summary, our data provide further evidence for a potential impact of ABCB1 deregulation on the response to AML chemotherapy, especially in more stem cell like leukemia cohorts as well as cytogenetically high risk AML. While we are currently further investigating the involvement of the Wnt/β-catenin pathway in the regulation of ABCB1 transcription in AML, further integration of molecular findings are warranted to better decipher the underlying drug resistance mechanisms. Ultimately, these analyses will improve patient management by adding valuable predictive biomarkers. DisclosuresNo relevant conflicts of interest to declare.

Global alterations of DNA methylation in cholangiocarcinoma target the Wnt signaling pathway

The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled 10 intrahepatic and 8 extrahepatic CCs in comparison to non-neoplastic biliary tissue specimens, using methyl-CpG immunoprecipitation (MCIp) combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments of two CC cell lines using 5-aza-2'deoxycytidine (DAC) treatment. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter-associated regions were nonrandomly distributed and enriched for genes involved in cancer-related pathways including Wnt, transforming growth factor beta (TGF-β), and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2, and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially down-regulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients. We provide a comprehensive analysis to define the genome-wide methylation landscape of human CC. Several candidate genes of cancer-relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the basis for future studies on epigenetic changes in cholangiocarcinogenesis.

Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer

Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

Epo-Induced Erythroid Maturation Is Dependent On Plcγ1 Signalling

Erythropoiesis is a multi-step process in which the development of red blood cells occurs through expansion and differentiation of hematopoietic stem cells (HSCs) into more committed progenitors and finally into erythrocytes. Erythropoietin (Epo) is strictly required for erythropoiesis as it promotes survival and late maturation. In vivo and in vitro studies have pointed out the major role of erythropoietin receptor (EpoR) signalling through JAK2 tyrosine-kinase and STAT5a/b as a central regulator of erythropoiesis. STAT5a/b is essential in regulating early erythroblast survival, however, with regard to differentiation of erythroid progenitors current data are not definitive in establishing a critical, non-redundant role. Phospholipase C gamma 1 (PLCγ1) is known to act as key mediator of calcium-signalling that can substitute for PI3K/AKT in oncogenic models. Interestingly, genetic deletion of murine PLCγ1 in embryonic development using a conventional knockout mouse model resulted in lethality at E9.0 due to generalized growth failure and there was absence of erythrogenesis and vasculogenesis.Here, we revisited the role of Plcγ1 and investigated its function in signalling, differentiation and transcriptomic/epigenetic regulation of erythropoiesis: Upon Epo stimulation, we were able to demonstrate that Plcγ1 is a downstream target of EpoR/Jak2 signalling in lymphoid (Ba/F3) and myeloid (32D) progenitor cell lines (both transfected with EpoR and Jak2-WT) and in a erythroid progenitor (I/11) cell line. In order to specifically assess its role in erythroid development downstream of the EpoR-Jak2 axis, we focused on the murine pro-erythroblast cell line I/11 which is able to differentiate upon dexamethasone-/stem cell factor-withdrawal combined with erythropoietin stimulation. Interestingly, knockdown of Plcγ1 led to a dramatic delay (scr CD44high 21% vs. Plcγ1 shRNA CD44high 64%, p=0.02) in erythroid differentiation and accumulation of immature erythroid progenitors as assessed by flow cytometry technology. Knockdown of Plcγ1 did alter neither proliferation of cells nor the cell cycle distribution and activation of other EpoR downstream molecules as Stat5, Mek and Akt was not impaired. In addition, we analysed the colony-forming potential of Plcγ1-deficient I/11 and fetal liver cells (FLC) compared to controls. Colony formation was dramatically impaired in both - I/11 (scr 138 vs. Plcγ1 shRNA 32, p=0.03) and primary FLC (scr 107 vs. Plcγ1 shRNA 28, p<0.001) - when compared to control cells. Flow cytometry analysis of the colonies revealed a higher amount of immature populations (CD44high, KIT+) in PLCγ1-deficient cells as compared to controls whereas the content of TER119+ cells, reflecting more mature erythroid cells, was higher in controls.To elucidate on the mechanism of Plcγ1-mediated regulation of erythroid development, we performed global gene expression analysis in I/11 cells at various time points of differentiation after knockdown of Plcγ1. Several of the genes that change expression in the absence of Plcγ1 can be classified as transcription/co-transcription factors, epigenetic regulators, metabolic factors or adaptor molecules involved in intracellular signaling. Thus, Plcγ1-deficient cells showed up-regulation of the transcription factor RUNX1 and the adaptor molecule GRAP2 over time compared to controls whereas the epigenetic regulator H2AFY2 was significantly decreased. Stimulated by our observation that profound changes in global gene expression also included the epigenetic machinery (H2afy2), we speculated whether Plcγ1 signalling also modifies the global epigenetic landscape of I/11 pro-erythroblasts. Therefore, we performed genome-wide DNA methylome analysis in I/11 cells upon Plcγ1 knockdown using MCIP-seq (methyl-CpG immunoprecipitation combined with next-generation sequencing). The observed methylation changes were by far dominated by an apparent hypomethylation of differentially methylated regions (DMRs) in Plcγ1 knockdown cells as compared to control cells. In line with this, gene ontology analysis of DMRs revealed a highly significant enrichment of biological terms associated with developmental processes and cell differentiation.Taken together, our findings provide evidence for an essential role of Plcγ1 in regulating erythroid differentiation through alteration of the transcriptomic and epigenetic landscape. Disclosures:No relevant conflicts of interest to declare.

Abstract 2: Genome-wide DNA methylation profiling identifies novel diagnostic and prognostic biomarkers in early stage prostate cancer.

Abstract Prostate cancer (PCa) is the most prevailing cancer in the male population and the second-leading cause of cancer-related death in the Western world. Although genomic alterations during PCa development have been recently characterized [1], substantial evidence shows that genome-wide epigenetic insults complement or even precede genetic alterations [2]. Promoter CpG island methylation frequently correlates with gene silencing of functional genes. Therefore we hypothesized that scanning for early-silenced genes by profiling hypermethylation sites of promoter CpG islands would facilitate identification of novel methylation biomarkers with potential functions during prostate carcinogenesis. DNA from 9 primary pT2a tumors and matched normal tissue was subjected to enrichment of methylated sequences using Methyl CpG Immunoprecipitation (MCIp) [3], followed by array co-hybridization using Agilent CpG island arrays. Candidate genes were selected according to 1) hypermethylation in gene promoter regions in tumor tissue and 2) reduced expression in PCa in comparison to normal prostate tissue based on published gene expression datasets. DNA methylation was quantified by mass spectrometry-based EpiTYPER technology [4]. We identified 245 promoter CpG islands corresponding to 207 genes that were hypermethylated in at least 6/9 early stage PCa. Gene ontology enrichment analyses indicated enrichment of homeobox genes, and of gene functions such as regulation of transcription, cellular biosynthesis and morphogenesis, and neuron development. Quantitative methylation analysis of 9 selected candidate genes confirmed significant hypermethylation in the range of 20 -50% in 6/9 tumors of the initial discovery set in comparison with normal prostate tissue. Further validation in 37 sample triplets of matched normal tissue - prostate intraepithelial neoplasia (PIN) - tumor tissue indicated significant hypermethylation of SPG20 (spastic paraplegia 20, p=0.023, Wilcoxon signed-rank test), SPATA6 (spermatogenesis associated 6, p=0.017), and DUOXA1 (dual oxidase maturation factor 1 alpha, p=0.017) already at the PIN stage. Moreover, methylation analysis of 60 prostate tumor samples with increasing malignancy revealed a significant correlation between DNA hypermethylation patterns and biochemical recurrence of prostate cancer (p=0.008, log-rank test). From this genome-wide study we identified relevant biomarkers for early-detection and prognosis of early stage prostate cancer. The identified genes might represent interesting candidates to further investigate their function in the molecular pathogenesis and development of prostate cancer. Citation Format: Po-Hsien J. Huang, Wolfgang Göring, Thomas Hielscher, Dieter Weichenhan, Wolfgang A. Schulz, Christoph Plass, Clarissa Gerhäuser. Genome-wide DNA methylation profiling identifies novel diagnostic and prognostic biomarkers in early stage prostate cancer. [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 2. doi:10.1158/1538-7445.AM2013-2

Genome‐wide methylation screen in low‐grade breast cancer identifies novel epigenetically altered genes as potential biomarkers for tumor diagnosis

Aberrant DNA methylation constitutes a well-established epigenetic marker for breast cancer. Changes in methylation early in cancer development may be clinically relevant for cancer detection and prognosis-based therapeutic decisions. In the present study, a combination of methyl-CpG immunoprecipitation (MCIp) and human CpG island (CGI) arrays was applied to compare genome-wide DNA methylation profiles in 10 low-grade in situ and invasive breast cancers against 10 normal breast samples. In total, 214 CGIs were found to be hypermethylated in ≥6 of 10 tumors. Functional term enrichment analyses revealed an overrepresentation of homeobox genes and genes involved in transcription and regulation of transcription. Significant hypermethylation of 11 selected genes in tumor vs. normal tissue was validated in two independent sample sets (45 tumors and 11 controls, 43 tumors and 8 controls) using quantitative EpiTyper technology. In tumors, median methylation levels of BCAN, HOXD1, KCTD8, KLF11, NXPH1, POU4F1, SIM1, and TCF7L1 were ≥30% higher than in normal samples, representing potential biomarkers for tumor diagnosis. Using the 90th percentile of methylation levels in normal tissue as cutoff value, 62-92% of in situ samples (n=13), 72-97% of invasive samples from the first validation set (n=32), and 86-100% of invasive samples from the second validation set (n=43) were classified as hypermethylated. Hypermethylation of KLF11 and SIM1 might also be associated with increased risk of developing metastases. In summary, early methylation changes are frequent in the low-grade pathway of breast cancer and may be useful in the development of differential diagnostic and possibly also prognostic markers.

Abstract B10: Genome-wide profiling reveals hyper- and hypomethylation at microRNA promoters in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Dysregulation of microRNA (miRNA) expression contributes to CLL pathogenesis. Abnormal promoter methylation is involved in aberrant gene expression in leukemic cells. Here, we investigate the role of epigenetic mechanisms in the global regulation of miRNA expression in CLL. In order to characterize disease-specific aberrant methylation upstream of miRNAs, methylated DNA from 24 CLL patients and 10 healthy age-matched controls was enriched by Methyl-CpG immunoprecipitation. Differentially methylated genomic loci were identified using a custom tiling array covering the genomic sequences 35 kb upstream and 5 kb downstream of 939 annotated miRNAs. We identified putative promoter sequences for 781 miRNAs by determining regions of enriched trimethylated histone 3 lysine 4 (H3K4me3) in CLL samples, healthy B cells or cancer cell lines. The methylation patterns upstream of miRNAs clearly distinguished CLL cells from healthy B cells. Differentially methylated sequences are enriched in H3K4me3, suggesting that miRNA promoters are non-randomly targeted for epigenetic dysregulation. 458 miRNAs display differential methylation in at least 5 out of the 24 CLL samples. Noteworthy, hypomethylation accounted for two thirds of all differentially methylated regions at putative miRNA promoters. Hypomethylated regions are limited to strictly defined sequence stretches, predominantly found outside of CpG islands (89%). Correlating promoter methylation with miRNA expression, we observed loss of methylation and strong transcriptional upregulation of mir-155 in CLL. Similarly, the promoter of mir-1204 showed significantly reduced methylation in CLL associated with increased expression. Mir-129-2, a well known tumor suppressor in solid tumors, could be detected among the epigenetically silenced miRNAs. Furthermore mir-551b, which has not yet been described in the context of CLL, was found to be downregulated and a target of promoter hypermethylation in CLL cells. In conclusion, a combination of DNA methylation profiling and comprehensive miRNA promoter identification has allowed us to identified novel aberrantly regulated miRNAs in CLL. Hypomethylation, which has previously been underestimated, is determined to be a major mechanism for miRNA activation in CLL. Citation Format: Constance Baer, Michael Rehli, John C. Byrd, Clemens-Martin Wendtner, Christoph Plass, Rainer Claus, Lukas Frenzel, Manuela Zucknick, Yoon Jung Park, Lei Gu, Dieter Weichenhan, Martina Fischer, Christian Pallasch. Genome-wide profiling reveals hyper- and hypomethylation at microRNA promoters in chronic lymphocytic leukemia [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B10.

DNA methylation changes in cells regrowing after fractioned ionizing radiation

Background and purposeRepeated exposure to ionizing radiation (IR) can result in adaptive reactions. While DNA methylation changes in adaption to repeated stress exposure are established for a variety of drugs, their role in fractioned ionizing radiation is largely unknown. Material and methodsMCF7 breast cancer cells were treated 5 times a week with IR in fractions of 2Gy, resulting in total doses of 10 and 20Gy. Cells were harvested 48 and 72h after the last irradiation, as well as after a recovery period of at least 14 d. To identify genes differentially methylated in irradiated versus non-irradiated cells, we used methyl-CpG immunoprecipitation (MCIp) followed by global methylation profiling on CpG island microarrays. ResultsMCIp profiling revealed methylation changes in several CpG islands 48h after FIR with 10 and 20Gy. Cells receiving a total dose of 10Gy started regrowing after 14 d and exhibited similar radioresistance as mock-treated cells. Differential methylation of the CpG units associated with FOXC1 (p<0.001) and TRAPPC9 (p<0.001) could be confirmed by time-of-flight mass spectrometry (Sequenom). ConclusionsIn summary, these data indicate that regrowth of MCF7 cells after 10Gy FIR is associated with locus-specific alterations in DNA methylation.

Profiling of Aberrant DNA Methylation In AML Reveals Subclasses of CpG Islands with Epigenetic or Genetic Association

AbstractAbstract 2498CpG islands show frequent and often disease-specific epigenetic alterations during malignant transformation, however, the underlying mechanisms are poorly understood. We used methyl-CpG immunoprecipitation (MCIp) to generate comparative DNA methylation profiles of 30 patients with acute myeloid leukemia for human CpG islands across the genome. DNA methylation profiles across 23.000 CpG islands revealed highly heterogeneous methylation patterns in AML with over 6000 CpG islands showing aberrant de novo methylation in AML. Based on these profiles we selected a subset of 380 CpG islands (covering 15.000 individual CpGs) for detailed fine-mapping analyses of aberrant DNA methylation in 185 patients with AML (50% normal karyotype). We found that a proportion of patients (5/185) displayed a concerted hypermethylation at almost all studied loci, representing the rare CpG island methylator phenotype (CIMP) in AML. Meta analysis of methylation profiling and published ChIP sequencing data separated CpG islands in two groups. A highly correlated subgroup of CpG island regions was strongly associated with histone H3 lysine 27 trimethylation in human hematopoietic progenitor cells, suggesting that disease-related de novo DNA methylation at these CpG islands is linked with polycomb group protein (PcG)-mediated repression. The group of mainly non-PcG target CpG islands showed heterogeneous methylation patterns across patients and unsupervised hierarchical clustering revealed a correlation of methylation profiles with genetic disease markers, including oncofusion proteins as well as CEBPA- and NPM1-mutations. Our study suggests that both epigenetic as well as genetic aberrations may underlay AML-related changes in CpG island DNA methylation states. Disclosures:No relevant conflicts of interest to declare.

General Transcription Factor Binding at CpG Islands in Normal Cells Correlates with Resistance to De novo DNA Methylation in Cancer Cells

Aberrant DNA methylation at CpG islands is thought to contribute to cancer initiation and progression, but mechanisms that establish and maintain DNA methylation status during tumorigenesis or normal development remain poorly understood. In this study, we used methyl-CpG immunoprecipitation to generate comparative DNA methylation profiles of healthy and malignant cells (acute leukemia and colorectal carcinoma) for human CpG islands across the genome. While searching for sequence patterns that characterize DNA methylation states, we discovered several nonredundant sequences in CpG islands that were resistant to aberrant de novo methylation in cancer and that resembled consensus binding sites for general transcription factors (TF). Comparing methylation profiles with global CpG island binding data for specific protein 1, nuclear respiratory factor 1, and yin-yang 1 revealed that their DNA binding activity in normal blood cells correlated strictly with an absence of de novo methylation in cancer. In addition, global evidence showed that binding of any of these TFs to their consensus motif depended on their co-occurrence with neighboring consensus motifs. In summary, our results had two major implications. First, they pointed to a major role for cooperative binding of TFs in maintaining the unmethylated status of CpG islands in health and disease. Second, our results suggest that the majority of de novo methylated CpG islands are characterized by the lack of sequence motif combinations and the absence of activating TF binding.

Abstract B49: Epigenetic changes during disease progression in a murine model of human breast cancer

Abstract It is now well accepted that epigenetic alterations contribute to tumorigenesis. DNA methylation has been recognized as an early and possibly initiating event in the development of breast cancer and thus represents a potentially valuable marker for early detection and chemoprevention. In the present study, we aimed to characterize DNA methylation changes associated with the development of breast cancer in the C3(1)/SV40 large T-antigen transgenic mouse model of mammary carcinogenesis. This model has been utilized in various studies to demonstrate chemopreventive efficacy of both natural products as well as pharmacological agents such as green and black tea or the COX-2 inhibitor celecoxib. For DNA methylation analyses, wildtype (wt) and transgenic (tg) mice were sacrificed at 4–24 weeks of age to collect mammary and tumor tissue, respectively. Initially, we selected eight candidate genes know to be silenced by hypermethlytion in human breast cancer: Cadherin1, Cyclin D2, RASSF1a, Timp3, Twist, GSTπ, SMPD3, and GHSR1a. DNA methylation of CpG islands associated with these genes was quantified by EpiTYPER MassARRAY technology based on mass spectrometry. Interestingly, none of these genes was hypermethylated in murine tumors. On average, we detected less than 16% methylation, which was not significantly different between wt and tg tissue and did not change with age. Consequently, we next decided to analyze methylation changes at a genome-wide level, using methylation-specific DNA array analysis after methyl-CpG immunoprecipitation (MCIp). Hypermethylated DNA of age-matched wt and tg animals was enriched by binding to recombinant methyl-CpG-binding domain protein MBD2. Dual-color labeled samples were then hybridized to Agilent murine CpG island microarrays containing oligonucleotide probes for &amp;gt;16.000 CpG islands. Based on data obtained with tissue derived of animals aged 16–24 weeks, we selected genes commonly hypermethylated in tg mice for subsequent validation by MassARRAY analysis. As an example, five genes, Mab21l2, Lyl1, Atp6v1b1, Espn, A930037G, showed significant hypermethylation in a range of 52 to 69% in tumor tissue of mice aged 24 weeks, whereas an average methylation of 6 to 15% was detected in mammary tissue of age-matched wt animals. Interestingly, when we analyzed mammary tissue of mice at increasing age, we observed a gradual increase in hypermethylation in tg vs. wt animals for all selected genes, starting at 12–16 weeks of age even before tumors were detected. These data indicate a potential early role of deregulated DNA methylation in C3(1)/SV40 large T-antigen-induced mammary carcinogenesis. The newly identified genes, which have rarely been investigated in relation to human breast cancer so far, may serve as interesting targets for chemoprevention studies and will be further characterized. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B49.

Allele-specific DNA methylation in mouse strains is mainly determined by cis-acting sequences

DNA methylation is a vital epigenetic mark that participates in establishing and maintaining chromatin structures and regulates gene transcription during mammalian development and cellular differentiation. Differences in epigenetic patterns between individuals may contribute to phenotypic variation and disease susceptibility; however, little is known about the extent of such variation or how different epigenetic patterns are established. Here we have compared DNA methylation profiles of macrophages from two inbred mouse strains (C57BL/6 and BALB/c) at 181 large genomic intervals that were selected based on differential gene expression patterns. Using a DNA methylation-dependent fractionation approach based on a combination of methyl-CpG immunoprecipitation and locus-wide tiling arrays, we identified several hundred differentially methylated regions, and simultaneously uncovered previously unrecognized genetic variability between both mouse strains at the studied loci. DNA sequence and methylation differences were validated by DNA sequencing and mass spectrometry analysis of bisulfite-treated DNA for a subset of regions. Importantly, we show that in F1 hybrids, the majority of strain-specific methylation patterns in somatic cells were maintained on the parental allele, regardless of their status in the male germ line. The common association of differentially methylated regions with sequence polymorphisms suggests that the genomic context determines the developmentally regulated epigenetic status at most nonimprinted regions of mammalian genomes.

Comparative methylation profiling of tumor samples using methyl-CpG-immuno precipitation (MCIp) and CpG island microarrays

Comparative methylation profiling of tumor samples using methyl-CpG-immuno precipitation (MCIp) and CpG island microarrays

Locus-wide detection of cell type specific DNA methylation patterns using comparative methyl-CpG-Immunoprecipitation (MCIp)

Locus-wide detection of cell type specific DNA methylation patterns using comparative methyl-CpG-Immunoprecipitation (MCIp)

Global, comparative analysis of tissue-specific promoter CpG methylation

Understanding cell-type-specific epigenetic codes on a global level is a major challenge after the sequencing of the human genome has been completed. Here we applied methyl-CpG immunoprecipitation (MCIp) to obtain comparative methylation profiles of coding and noncoding genes in three human tissues, testis, brain, and monocytes. Forty-four mainly testis-specific promoters were independently validated using bisulfite sequencing or single-gene MCIp, confirming the results obtained by the MCIp microarray approach. We demonstrate the previously unknown somatic hypermethylation at many CpG-rich, testis-specific gene promoters, in particular in ampliconic areas of the Y chromosome. We also identify a number of miRNA genes showing tissue-specific methylation patterns. The comparison of the obtained tissue methylation profiles with corresponding gene expression data indicates a significant association between tissue-specific promoter methylation and gene expression, not only in CpG-rich promoters. In summary, our study highlights the exceptional epigenetic status of germ-line cells in testis and provides a global insight into tissue-specific DNA methylation patterns.

Genome-Wide Profiling of CpG Methylation Identifies Novel Targets of Aberrant Hypermethylation in Myeloid Leukemia

The methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. Because aberrant hypermethylation may be used as a marker for disease, a sensitive method for the global detection of DNA methylation events is of particular importance. We describe a novel and robust technique, called methyl-CpG immunoprecipitation, which allows the unbiased genome-wide profiling of CpG methylation in limited DNA samples. The approach is based on a recombinant, antibody-like protein that efficiently binds native CpG-methylated DNA. In combination with CpG island microarrays, the technique was used to identify >100 genes with aberrantly methylated CpG islands in three myeloid leukemia cell lines. Interestingly, within all hypermethylation targets, genes involved in transcriptional regulation were significantly overrepresented. More than half of the identified genes were absent in microarray expression studies in either leukemia or normal monocytes, indicating that hypermethylation in cancer may be largely independent of the transcriptional status of the affected gene. Most individually tested genes were also hypermethylated in primary blast cells from acute myeloid leukemia patients, suggesting that our approach can identify novel potential disease markers. The technique may prove useful for genome-wide comparative methylation analysis not only in malignancies.