Specific DNA Methylation Patterns Research Articles

Abstract P1-06-05: DNA methylation-based classification are mostly concordant with intrinsic subtypes of breast cancer

Abstract Backgrounds: Breast cancer (BC) is a heterogeneous disease and usually divided into 5 subtypes according to clinicopathologic features. These subtypes have concordance with mRNA-based subtypes called as "intrinsic subtypes". Further gene expression analysis categorized triple negative BCs into 7 sub-subtypes which relate to clinical behaviors. DNA methylation is one of the epigenetic systems to regulate gene transcription. DNA methylation in CpG islands (CGIs) associated with the gene promoter region affects gene expression levels. Recently genome-wide analyses suggested that methylation status not only in CGIs, but also in CpG shores (within 2000 bps from CGIs) and CpG shelves (2000-4000 bps from CGIs) contribute to gene silencing. Thus, we hypothesized that intrinsic subtypes may possess specific methylation profiles especially in regions surrounding CGIs (islands, shores and shelves). To date, several reports demonstrated each intrinsic subtype has specific methylation patterns in CGIs. They indicate that DNA methylation is promising markers for cancer detection, prediction of therapeutic response and clinical outcomes. However, there were no report that conducted a genome-wide high-resolution epigenetic analysis for BC. Objectives: This study aims to determine whether specific DNA methylation patterns exist in CGIs, shores and shelves in BC cells, and whether the DNA methylation-based classification correlates with intrinsic subtypes, furthermore, to identify the subtype specific epigenetic markers using high-resolution methylation array. Materials and Methods: DNA was extracted from 31 samples of 28 BC cell lines (8 luminal, 18 basal, 2 unclassified) and 3 samples of 3 non-BC cell lines. We performed genome-wide methylation analysis using Illumina Infinium HumanMethylation450 BeadChip. We did unsupervised hierarchical clustering analysis of cell lines using top 3000 variably methylated (VM) loci among cancer cell lines. We also analyzed top 3000 VM CGI-related loci. Gene functional annotation clustering was performed using DAVID. Results: In top 3000 VM loci, CGIs were most frequently observed (43.0%). According to clustering analysis using top 3000 VM loci, cell lines were roughly classified into 4 clusters. There were trends that cell lines of same subtype fell into the same cluster, but not definitely. In case of clustering using top 3000 VM CGI-related loci, cell lines were clearly divided into 2 clusters, high and low methylated clusters. High methylated cluster contained almost all luminal cell lines and low methylated cluster contained most of basal cell lines and all non-BC cell lines. In low methylated cluster, cell lines of the same sub-subtype, such as Basal-like 1 and 2, were clustered closely. Gene functional annotation clustering suggested that genes related to "transcription" and "differentiation" were more frequently regulated by epigenetics. Conclusions: High-resolution methylation analysis revealed methylation-based clusters were concordant with intrinsic subtypes in BC cells. Transcriptional factors and differentiation-related genes could be differently regulated by epigenetics among different subtypes of BC. Citation Format: Natsue Uehiro, Fumiaki Sato, Sunao Tanaka, Fengling Pu, Junji Itou, Shigehira Saji, Masakazu Toi. DNA methylation-based classification are mostly concordant with intrinsic subtypes of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-06-05.

MP58-03 IDENTIFICATION OF SPECIFIC METHYLATION-BASED BIOMARKERS FOR THE ASSESSMENT OF THE METASTATIC RISK OF MUSCLE INVASIVE BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Invasive II1 Apr 2015MP58-03 IDENTIFICATION OF SPECIFIC METHYLATION-BASED BIOMARKERS FOR THE ASSESSMENT OF THE METASTATIC RISK OF MUSCLE INVASIVE BLADDER CANCER Beatrice Stubendorff, Kerstin Wilhelm, Kathleen Posselt, James Catto, Elke Schaeffeler, Matthias Schwab, Arndt Hartmann, Susanne Füssel, Vladimir Novotny, Mieczyslaw Gajda, Marc-Oliver Grimm, Michael Stöckle, and Kerstin Junker Beatrice StubendorffBeatrice Stubendorff More articles by this author , Kerstin WilhelmKerstin Wilhelm More articles by this author , Kathleen PosseltKathleen Posselt More articles by this author , James CattoJames Catto More articles by this author , Elke SchaeffelerElke Schaeffeler More articles by this author , Matthias SchwabMatthias Schwab More articles by this author , Arndt HartmannArndt Hartmann More articles by this author , Susanne FüsselSusanne Füssel More articles by this author , Vladimir NovotnyVladimir Novotny More articles by this author , Mieczyslaw GajdaMieczyslaw Gajda More articles by this author , Marc-Oliver GrimmMarc-Oliver Grimm More articles by this author , Michael StöckleMichael Stöckle More articles by this author , and Kerstin JunkerKerstin Junker More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2142AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Adjuvant chemotherapy improves the outcome of bladder cancer patients at risk for the development of metastases. However, there are no parameters available that allow an individual risk assessment. The aim of this project is to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment of muscle invasive bladder tumors and to understand metastasis associated processes regulated by DNA methylation. METHODS we validated differentially methylated genes in two independent cohorts of primary bladder tumors (n=91). Candidate promoter methylation and gene expression were assessed by quantitative analysis of DNA methylation using real-time PCR and qRT-PCR. Quantitative CpG specific methylation level of KISS1R was performed with MALDI-TOF-MS and analyzed using Epityper 1.0 software. Protein expression was determined by immunohistochemical analysis for KISS1R on tissue microarrays from 2 independent patient cohorts comprising 291 muscle-invasive bladder carcinomas. For analyzing the gene function in tumor cell behavior, bladder cancer cells were transfected with siRNA or cDNA clones. Changes in proliferation and migration were measured using real-time cell monitoring. Invasion assay was performed using matrigel coated cell culture inserts. RESULTS Methylation differences were confirmed for KISS1R, SEPT9 (p=0.002) and CSAD (p=0.01). MALDI-TOF analysis provided a tool to validate the DNA methylation at single CpG resolution. The combination of the three genes predicted the metastatic risk of the primary tumors with sensitivity of 90% and specificity of 67% in patient cohort 1 and sensitivity of 75% and specificity of 70% in patient cohort 2. Protein expression of KISS1R was significantly reduced in correlation to positive lymph nodes (p<0.001). Knock down of SEPT9v3 resulted in increased cell migration by 28% (p=0.04) and increased invasion by 22% (p=0.004). Overexpression of KISS1R resulted in decreased cell migration (25%, p=0.1). CONCLUSIONS Muscle-invasive bladder tumors show significant differences in the DNA methylation pattern in correlation to the metastatic potential. KISS1R is characterized by strongest association with the metastatic risk of primary tumors on methylation. Reduced expression of KISS1R and SEPT9 regulated by DNA methylation seems to be involved in metastasis-associated processes. The combination of candidate genes KISS1R, CSAD and SEPT9 could serve as prognostic marker panel for determination of the metastatic risk of primary bladder tumors. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e722 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Beatrice Stubendorff More articles by this author Kerstin Wilhelm More articles by this author Kathleen Posselt More articles by this author James Catto More articles by this author Elke Schaeffeler More articles by this author Matthias Schwab More articles by this author Arndt Hartmann More articles by this author Susanne Füssel More articles by this author Vladimir Novotny More articles by this author Mieczyslaw Gajda More articles by this author Marc-Oliver Grimm More articles by this author Michael Stöckle More articles by this author Kerstin Junker More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

High Resolution Genome Wide DNA Methylation Analysis in a Large Trial Group Reveals a Novel Epigenetically Defined Subgroup of Myeloma Patients Characterized By Developmental Gene Hypermethylation

IntroductionMultiple myeloma is a clinically highly heterogeneous disease, which is reflected by both a complex genome and epigenome. Dynamic epigenetic changes are involved at several stages of myeloma biology, such as transformation and disease progression. Our previous genome wide epigenetic analyses identified prognostically relevant DNA hypermethylation at specific tumor suppressor genes (Kaiser MF et al., Blood 2013), indicating that specific epigenetic programming influences clinical behavior. This clinically relevant finding prompted further investigation of the epigenomic structure of myeloma and its interaction with genetic aberrations.Material and MethodsGenome wide DNA methylation of CD138-purified myeloma cells from 464 patients enrolled in the NCRI Myeloma XI trial at presentation were analyzed using the high resolution 450k DNA methylation array platform (Illumina). In addition, 4 plasma cell leukemia (PCL) cases (two t(11;14) and two (4;14)) and 7 myeloma cell lines (HMCL) carrying different translocations were analysed.Analyses were performed in R Bioconductor packages after filtering and removal of low quality and non-uniquely mapping probes.ResultsVariation in genome wide DNA methylation was analyzed using unsupervised hierarchical clustering of the 10,000 most variable probes, which revealed epigenetically defined subgroups of disease. Presence of recurrent IGH translocations was strongly associated with specific epigenetic profiles. All 60 cases with t(4;14) clustered into two highly similar sub-clusters, confirming that overexpression of the H3K36 methyltransferase MMSET in t(4;14) has a defined and specific effect on the myeloma epigenome. Interestingly, HMCLs KMS-11 and LP-1, which carry t(4;14), MM1.S, a t(14;16) cell line with an E1099K MMSET activating mutation as well as two PCLs with t(4;14) all clustered in one sub-clade. The majority (59/85) of t(11;14) cases showed global DNA hypomethylation compared to t(4;14) cases and clustered in one subclade, indicating a epigenetic programming effect associated with CCND1, with a subgroup of t(11;14) cases showing a variable DNA methylation pattern.In addition to translocation-defined subgroups, a small cluster of samples with a distinct epigenetic profile was identified. In total 7 cases with a shared specific DNA methylation pattern (median inter-sample correlation 0.4) were identified. The group was characterized by DNA hypermethylation (4,341 hypermethylated regions vs. 750 hypomethylated regions) in comparison to all other cases. Intersection of regions hypermethylated in this subgroups with ENCODE datasets revealed mapping to poised enhancers and promoters in H1-hESC, indicating functionally relevant epigenetic changes. Gene set enrichment analysis (KEGG) demonstrated enrichment of developmental pathway genes, e.g. Hedgehog signaling (adj p=5x10exp-13), amongst others and all four HOX clusters were differentially methylated in this group. Of note, three of seven cases in this subgroup carried a t(11;14) and all t(11;14) or t(11;14)-like HMCLs clustered closely together with these patient cases, but not with the cluster carrying the majority of t(11;14) myeloma or t(11;14) PCLs. This potentially indicates that t(11;14) HMCL could be derived from a subgroup of patients with specific epigenetic characteristics.ConclusionOur results indicate that the recurrent IGH translocations are fundamentally involved in shaping the myeloma epigenome through either direct upregulation of epigenetic modifiers (e.g. MMSET) or through insufficiently understood mechanisms. However, developmental epigenetic processes seem to independently contribute to the complexity of the epigenome in some cases. This work provides important insights into the spectrum of epigenetic subgroups of myeloma and helps identify subgroups of disease that may benefit from specific epigenetic therapies currently being developed. DisclosuresWalker:Onyx Pharmaceuticals: Consultancy, Honoraria.

Abstract IA04: EWS-FLI1, polycomb proteins and stem cells: Clues to the developmental origins of Ewing sarcoma

Abstract IA04: EWS-FLI1, polycomb proteins and stem cells: Clues to the developmental origins of Ewing sarcoma

Abstract 1358: Promoter hypermethylation of KISS1R, KiSS1, SEPT9 and CSAD as a prognostic biomarker panel to assess the metastatic potential of muscle invasive bladder tumors

Abstract Introduction: Adjuvant chemotherapy improves the outcome of bladder cancer patients at risk for the development of metastases. However, there are no parameters available that allow an individual risk assessment. The aim of this project is to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment of muscle invasive bladder tumors and to understand metastasis associated processes regulated by DNA methylation. Materials &amp; methods: Based on our previous microarray analysis we validated differentially methylated genes in a larger cohort of primary bladder tumors (n=100) in correlation to metastasis. Candidate promoter methylation and gene expression were assessed by quantitative analysis of DNA methylation using real-time PCR and qRT-PCR. Protein expression was determined by immunohistochemical analysis for KiSS1 and KISS1R on tissue microarrays from 2 independent patient cohorts comprising 290 muscle-invasive bladder carcinomas. For analyzing the gene function in tumor cell behavior, gene expression was knocked down by siRNA transfection of bladder cancer cells. Changes in proliferation and migration were measured using real-time cell monitoring (xCELLigence, Roche). Invasion assay was performed using matrigel coated cell culture inserts. Results: Methylation differences were confirmed for KISS1R, KiSS1, SEPT9 (p&amp;lt;0.05) and CSAD (p&amp;lt;0.01). mRNA expression differences between patient groups were detected for KISS1R and SEPT9v3. Protein expression of KISS1R was significantly reduced in correlation to positive lymph nodes (p&amp;lt;0.001) and distant metastases (p&amp;lt;0.05). Knock down of SEPT9v3 resulted in increased cell proliferation (77%), migration (15%) and invasion (39%) compared to negative control. Conclusion: Muscle-invasive bladder tumors show significant differences in the DNA methylation pattern in correlation to the metastatic potential. KISS1R is characterized by strongest association with the metastatic risk of primary tumors on methylation as well as RNA and protein expression level. Reduced expression of SEPT9v3 seems to induce cell proliferation, migration and invasion. The combination of candidate genes KISS1R, KiSS1, CSAD and SEPT9 could serve as prognostic marker panel for determination of the metastatic risk of primary bladder tumors. Citation Format: Beatrice Stubendorff, Kerstin Wilhelm, Kathleen Posselt, James Catto, Arndt Harmann, Susanne Fuessel, Vladimir Novotny, Mieczyslaw Gajda, Heiko Wunderlich, Marc-Oliver Grimm, Kerstin Junker. Promoter hypermethylation of KISS1R, KiSS1, SEPT9 and CSAD as a prognostic biomarker panel to assess the metastatic potential of muscle invasive bladder tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1358. doi:10.1158/1538-7445.AM2014-1358

Role for tet in hyperglycemia-induced demethylation: a novel mechanism of diabetic metabolic memory.

During germ cell and early embryonic development—the most sensitive and vulnerable period of epigenetic reprogramming—exposure to an adverse environment leads to abnormal methylation and, possibly, long-term health problems. DNA methylation, a major epigenetic mechanism for gene silencing, is recognized to be responsible for the stability of gene expression status. The majority of cytosine-phosphate-guanine sites (CpGs) in mammalian genomes are methylated. DNA methyltransferase (Dnmt) 3A and 3B are essential for de novo methylation, and Dnmt1 maintains methylation patterns during cell division (1). Establishment and maintenance of cell type–specific DNA methylation patterns are dependent on both methylation and demethylation. DNA demethylation is the process of the removal of a methyl group from nucleotides in DNA, which can be passive or active. It has been generally understood that passive DNA demethylation occurs by a reduction in activity or absence of Dnmts, whereas the mechanism of active DNA demethylation has been controversial in recent decades. Recently, three enzyme families have been implicated in active DNA demethylation via DNA repair. The first is the ten-eleven translocation (Tet) family of enzymes. 5-Methylcytosine (5mC) can be hydroxylated by Tet to form 5-hydroxymethylcytosine (5hmC), which can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The second family is the AID/APOBEC family. 5mC (or 5hmC) can be deaminated by AID/APOBEC family members to form 5-methyluracil (5mU) or 5-hydroxymethyluracil (5hmU). The third is the UDG family of base excision repair glycosylases. TDG and SMUG1 replace these intermediates (i.e., 5mU, 5hmU, or 5caC), culminating in cytosine replacement and DNA demethylation …

MP28-06 PROMOTER HYPERMETHYLATION OF KISS1R, KISS1, SEPT9 AND CSAD AS A PROGNOSTIC BIOMARKER PANEL TO ASSESS THE METASTATIC POTENTIAL OF MUSCLE INVASIVE BLADDER TUMORS

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2014MP28-06 PROMOTER HYPERMETHYLATION OF KISS1R, KISS1, SEPT9 AND CSAD AS A PROGNOSTIC BIOMARKER PANEL TO ASSESS THE METASTATIC POTENTIAL OF MUSCLE INVASIVE BLADDER TUMORS Beatrice Stubendorff, Kerstin Wilhelm, Kathleen Posselt, James Catto, Arndt Hartmann, Susanne Füssel, Mieczyslaw Gajda, Heiko Wunderlich, Marc-Oliver Grimm, Vladimir Novotny, and Kerstin Junker Beatrice StubendorffBeatrice Stubendorff More articles by this author , Kerstin WilhelmKerstin Wilhelm More articles by this author , Kathleen PosseltKathleen Posselt More articles by this author , James CattoJames Catto More articles by this author , Arndt HartmannArndt Hartmann More articles by this author , Susanne FüsselSusanne Füssel More articles by this author , Mieczyslaw GajdaMieczyslaw Gajda More articles by this author , Heiko WunderlichHeiko Wunderlich More articles by this author , Marc-Oliver GrimmMarc-Oliver Grimm More articles by this author , Vladimir NovotnyVladimir Novotny More articles by this author , and Kerstin JunkerKerstin Junker More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.655AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Adjuvant chemotherapy improves the outcome of bladder cancer patients at risk for the development of metastases. However, there are no parameters available that allow an individual risk assessment. The aim of this project is to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment of muscle invasive bladder tumors and to understand metastasis associated processes regulated by DNA methylation. METHODS Based on our previous microarray analysis we validated differentially methylated genes in a larger cohort (100) of primary bladder tumors in correlation to metastasis. Candidate promoter methylation and gene expression were assessed by quantitative analysis of DNA methylation using real-time PCR and qRT-PCR. Protein expression was determined by immunohistochemical analysis for KiSS1 and KISS1R on tissue microarrays from 2 independent patient cohorts comprising 423 muscle-invasive bladder carcinomas. For analyzing the gene function in tumor cell behavior, gene expression was knocked down by siRNA transfection of bladder cancer cells. Changes in proliferation and migration were measured using real-time cell monitoring (xCELLigence, Roche). Invasion assay was performed using matrigel coated cell culture inserts. RESULTS Methylation differences were confirmed for KISS1R, KiSS1, SEPT9 (p<0.05) and CSAD (p<0.01). mRNA expression differences between patient groups were detected for KISS1R and SEPT9v3. Protein expression of KISS1R was significantly reduced in correlation to positive lymph nodes (p<0.001) and distant metastases (p<0.05). Knock down of SEPT9v3 resulted in increased cell proliferation (77%), migration (15%) and invasion (39%) compared to negative control. CONCLUSIONS Muscle-invasive bladder tumors show significant differences in the DNA methylation pattern in correlation to the metastatic potential. KISS1R is characterized by strongest association with the metastatic risk of primary tumors on methylation as well as RNA and protein expression level. Reduced expression of SEPT9v3 seems to induce cell proliferation, migration and invasion. The combination of candidate genes KISS1R, KiSS1, CSAD and SEPT9 could serve as prognostic marker panel for determination of the metastatic risk of primary bladder tumors. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e298 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Beatrice Stubendorff More articles by this author Kerstin Wilhelm More articles by this author Kathleen Posselt More articles by this author James Catto More articles by this author Arndt Hartmann More articles by this author Susanne Füssel More articles by this author Mieczyslaw Gajda More articles by this author Heiko Wunderlich More articles by this author Marc-Oliver Grimm More articles by this author Vladimir Novotny More articles by this author Kerstin Junker More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Integrated Analysis of DNA Methylation and RNA Transcriptome during In Vitro Differentiation of Human Pluripotent Stem Cells into Retinal Pigment Epithelial Cells

Using the paradigm of in vitro differentiation of hESCs/iPSCs into retinal pigment epithelial (RPE) cells, we have recently profiled mRNA and miRNA transcriptomes to define a set of RPE mRNA and miRNA signature genes implicated in directed RPE differentiation. In this study, in order to understand the role of DNA methylation in RPE differentiation, we profiled genome-scale DNA methylation patterns using the method of reduced representation bisulfite sequencing (RRBS). We found dynamic waves of de novo methylation and demethylation in four stages of RPE differentiation. Integrated analysis of DNA methylation and RPE transcriptomes revealed a reverse-correlation between levels of DNA methylation and expression of a subset of miRNA and mRNA genes that are important for RPE differentiation and function. Gene Ontology (GO) analysis suggested that genes undergoing dynamic methylation changes were related to RPE differentiation and maturation. We further compared methylation patterns among human ESC- and iPSC-derived RPE as well as primary fetal RPE (fRPE) cells, and discovered that specific DNA methylation pattern is useful to classify each of the three types of RPE cells. Our results demonstrate that DNA methylation may serve as biomarkers to characterize the cell differentiation process during the conversion of human pluripotent stem cells into functional RPE cells.

Fusion of TTYH1 with the C19MC microRNA cluster drives expression of a brain-specific DNMT3B isoform in the embryonal brain tumor ETMR

Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.

LSD1 Influences Both Histone and DNA Methylation During Terminal Erythroid Maturation

The molecular mechanisms that drive the maturation of a committed erythroid progenitor to a functional red blood cell are incompletely understood. LSD1 (Lysine-Specific Histone Demethylase 1) is a widely expressed histone demethylase that plays an important role in erythroid maturation (Kereyni, elife, 2013). Although LSD1 is important for a number of biologic processes ranging from embryonic development to leukemogenesis, the molecular mechanisms underlying the influence of LSD1 on gene expression are incompletely understood. The goal of our study is to elucidate the molecular mechanisms by which LSD1 regulates erythroid gene expression and influences erythroid maturation. We hypothesize that LSD1 promotes specific patterns of histone and DNA methylation that facilitate gene expression changes necessary for normal erythroid maturation to occur. To address this hypothesis, the functional and molecular consequences of LSD1 knockdown were assessed in Extensively Self Renewing Erythroblasts (ESREs), a non-transformed, karyotypically normal model of terminal erythroid maturation (England, Blood, 2011). Primary fetal liver was cultured in the presence of EPO, SCF, IGF1 and dexamethasone to derive ESREs. The ESREs were capable of extensive ex-vivo expansion, doubling daily at the proerythroblast phase, however when matured, >90% of cells became benzidine positive and >65% enucleated within 3 days. Lentiviral-mediated shRNA was used to knock down LSD1 in expanding ESREs. Imaging flow cytometry done on maturation day 3 demonstrated that the knockdown cells had impairments in multiple facets of maturation, with larger cell and nuclear areas, higher kit expression, and lower rates of enucleation than the scramble control. LSD1 knockdown was also associated with impaired hemoglobin accumulation (78% vs. 95% benzidine positive; p<0.005). Treatment of ESREs with an inhibitor to LSD1 (Tranylcypromine; TCP) resulted in similar abnormalities in cell and nuclear size, kit expression, hemoglobin accumulation, and enucleation (40% vehicle vs.1% TCP). The functional deficits in maturation, including abnormal kit expression and low rates of enucleation, persisted on maturation day 4.To delineate the molecular mechanisms underlying this maturation impairment, RNA-seq was done in LSD1 knockdown and scramble control samples, and 230 differentially expressed genes (FDR<0.01) were identified using cuffdiff (Trapnell, Nat Biotech, 2013). Consistent with LSD1's role in erythroid maturation, Ingenuity Pathway Analysis identified multiple networks involving hemoglobin synthesis, and GATA1, EPO, and KLF1 were all predicted as upstream regulators (p-values of 8.24e10-11, 7.25 e10-6, and 3.86e10-4, respectively). To better understand how LSD1 influences gene expression, chromatin immunoprecipitation coupled with high throughput sequencing was used to identify sites of H3K4me2 binding in the differentially expressed genes. 214/230 differentially expressed genes were associated with sites of H3K4me2 occupancy. Quantitative ChIP demonstrated that LSD1 inhibition was associated with increases in H3K4me2 levels at a subset of these sites, however consistent with previous studies, global levels of H3K4me2, determined by Enzyme Linked Immunosorbent Assay (ELIZA), did not change significantly.Although it is known that LSD1 demethylates and stabilizes the maintenance DNA methyltransferase DNMT1 (Wang, Nat Genet 2009), the consequences of LSD1 loss on DNA methylation (5-methyl cytosine; 5-mC) have yet to be investigated. To gain a comprehensive understanding of how LSD1 regulates erythroid gene expression, changes in the level of 5-mC were assessed after knockdown or inhibition of LSD1. Global 5-mC levels, determined by ELIZA assay, were ∼30% lower in TCP treated samples than vehicle treated control (p<0.02) and western blot demonstrated a 3-fold decrease in DNMT1 protein in the TCP treated samples. Both methyl binding domain pull-down coupled with quantitative PCR and genome-wide bisulfite sequencing were utilized to assess changes in 5-mC levels in the differentially expressed genes. Loss of LSD1 was associated with significantly lower levels of 5-mC at several differentially expressed, erythroid-specific genes, such as bh1. Taken together, these data support the hypothesis that LSD1 influences both histone and DNA methylation at genes important for erythroid maturation. Disclosures:No relevant conflicts of interest to declare.

Influence of DNMT Genotype on Global and Site Specific DNA Methylation Patterns in Neonates and Pregnant Women

This study examines the relationship between common genetic variation within DNA methyltransferase genes and inter-individual variation in DNA methylation. Eleven polymorphisms spanning DNMT1 and DNMT3B were genotyped. Global and gene specific (IGF2, IGFBP3, ZNT5) DNA methylation was quantified by LUMA and bisulfite Pyrosequencing assays, respectively, in neonatal cord blood and in maternal peripheral blood. Associations between maternal genotype and maternal methylation (n ≈ 333), neonatal genotype and neonatal methylation (n ≈ 454), and maternal genotype and neonatal methylation (n ≈ 137) were assessed. The findings of this study provide some support to the hypothesis that genetic variation in DNA methylating enzymes influence DNA methylation at global and gene-specific levels; however observations were not robust to correction for multiple testing. More comprehensive analysis of the influence of genetic variation on global and site specific DNA methylation is warranted.

Epigenetics in an ecotoxicological context

Epigenetics can play a role in interactions between chemicals and exposed species, between species and abiotic ecosystem components or between species of the same or another population in a community. Technological progress and advanced insights into epigenetic processes have led to the description of epigenetic features (mainly DNA methylation) in many ecologically relevant species: algae, plants, several invertebrates and fish. Epigenetic changes in plants, insects and cladocerans have been reported to be induced by various environmental stress factors including nutrition or water deficiency, grazing, light or temperature alterations, social environment, and dissolved organic matter concentrations. As regards chemicals, studies in rats and mice exposed to specific pesticides, hydrocarbons, dioxins, and endocrine disrupting chemicals demonstrated the induction of epigenetic changes, suggesting the need for further research with these substances in an ecotoxicological context. In fish and plants, exposure to polyaromatic hydrocarbons, metals, and soluble fractions of solid waste affected the epigenetic status. A novel concept in ecotoxicological epigenetics is the induction of transgenerational stress resistance upon chemical exposure, as demonstrated in rice exposed to metals. Evaluating epigenetics in ecotoxicological field studies is a second relatively new approach. A cryptic lineage of earthworms had developed arsenic tolerance in the field, concurrent with specific DNA methylation patterns. Flatfish caught in the framework of environmental monitoring had developed tumours, exhibiting specific DNA methylation patterns. Two main potential implications of epigenetics in an ecotoxicological context are (1) the possibility of transgenerationally inherited, chemical stress-induced epigenetic changes with associated phenotypes and (2) epigenetically induced adaptation to stress upon long-term chemical exposure. Key knowledge gaps are concerned with the causality of the relation between epigenetic and phenotypic changes, the persistence of transgenerational effects, the implications at population level and the costs of tolerance. Epigenetic changes following exposure to multiple stressors constitute another promising area of further research.

DNA Methylation Regulated Nucleosome Dynamics

A strong correlation between nucleosome positioning and DNA methylation patterns has been reported in literature. However, the mechanistic model accounting for the correlation remains elusive. In this study, we evaluated the effects of specific DNA methylation patterns on modulating nucleosome conformation and stability using FRET and SAXS. CpG dinucleotide repeats at 10 bp intervals were found to play different roles in nucleosome stability dependent on their methylation states and their relative nucleosomal locations. An additional (CpG)5 stretch located in the nucleosomal central dyad does not alter the nucleosome conformation, but significant conformational differences were observed between the unmethylated and methylated nucleosomes. These findings suggest that the correlation between nucleosome positioning and DNA methylation patterns can arise from the variations in nucleosome stability dependent on their sequence and epigenetic content. This knowledge will help to reveal the detailed role of DNA methylation in regulating chromatin packaging and gene transcription.

Constitutional trisomy 8 mosaicism as a model for epigenetic studies of aneuploidy

BackgroundTo investigate epigenetic patterns associated with aneuploidy we used constitutional trisomy 8 mosaicism (CT8M) as a model, enabling analyses of single cell clones, harboring either trisomy or disomy 8, from the same patient; this circumvents any bias introduced by using cells from unrelated, healthy individuals as controls. We profiled gene and miRNA expression as well as genome-wide and promoter specific DNA methylation and hydroxymethylation patterns in trisomic and disomic fibroblasts, using microarrays and methylated DNA immunoprecipitation.ResultsTrisomy 8-positive fibroblasts displayed a characteristic expression and methylation phenotype distinct from disomic fibroblasts, with the majority (65%) of chromosome 8 genes in the trisomic cells being overexpressed. However, 69% of all deregulated genes and non-coding RNAs were not located on this chromosome. Pathway analysis of the deregulated genes revealed that cancer, genetic disorder, and hematopoiesis were top ranked. The trisomy 8-positive cells displayed depletion of 5-hydroxymethylcytosine and global hypomethylation of gene-poor regions on chromosome 8, thus partly mimicking the inactivated X chromosome in females.ConclusionsTrisomy 8 affects genes situated also on other chromosomes which, in cooperation with the observed chromosome 8 gene dosage effect, has an impact on the clinical features of CT8M, as demonstrated by the pathway analysis revealing key features that might explain the increased incidence of hematologic malignancies in CT8M patients. Furthermore, we hypothesize that the general depletion of hydroxymethylation and global hypomethylation of chromosome 8 may be unrelated to gene expression regulation, instead being associated with a general mechanism of chromatin processing and compartmentalization of additional chromosomes.

An alternative splice process renders MLL either into an transcriptional activator or repressor

The biological process of differentiation - from a fertilized egg to a human being - is a consecutive mechanism that leads to the establishment of tissue-specific gene expression, but also to a coordinated shut-down of all those genes that are not necessary for a given cell type. This process is accompanied by posttranslational modifications of the chromatin (DNA methylation and covalent histone modifications), also termed the "epigenetic layer". All epigenetic processes are mediated by protein complexes that either mediate specific DNA methylation patterns, or modify nucleosomal proteins in a covalent fashion (acetylation, methylation, phosphorylation and ubiquitinylation). One important player involved in epigenetics is the MLL protein which represents a histone H3 methyltransferase. The MLL gene gained much attention because of its frequent genetic rearrangements, thereby creating oncogenic MLL fusion genes that cause acute leukemia in pediatric and adult patients. This article is summarizing certain functional aspects about MLL, but is mainly emphasizing on an alternative splice event within the PHD domain. This changes the biological properties of the MLL protein, thereby influencing its ability of being either a transcriptional activator or repressor.

Reprint of “Neuronal cell-type specific DNA methylation patterns of the Cacna1c gene”

Gene expression of the alpha-1 subunit of the L-type voltage-gated calcium channel, CACNA1C, is known to be complexly regulated. Because CACNA1C is not only a crucial gene in normal brain function but also a promising candidate risk gene for psychiatric disorders such as bipolar disorder and schizophrenia, elucidating the molecular basis of transcriptional regulatory mechanism will be critically important. Here we examined DNA methylation status of CpG islands and a CpG island shore on mouse Cacna1c in neuronal and non-neuronal nuclei, which were separated with a fluorescent activated cell sorting technique. We found that neurons and non-neurons showed differential DNA methylation profile on a CpG island shore. This difference was evolutionarily conserved in human neuronal and non-neuronal nuclei in the prefrontal cortex, suggesting that DNA methylation status on the CpG island shore of Cacna1c may have an important role in transcript regulation.

Abstract 2889: Identification of estrogen receptor modulated gene methylation network in breast cancer.

Abstract While cancer as a complex disease is known to arise from a series of aberrant genetic and epigenetic profiles, its growth and disease-specific outcome can often be characterized by status of one oncogene, such as Estrogen Receptor (ER) of breast cancer. Positive ER appears in around 70% of human breast cancer cases and recent studies dissected the Estrogen-mediated gene regulation through specific DNA methylation patterns by comparison between ER+ and ER- tumor samples. However, this simple approach did not take into consideration the influence of other confounding regulation factors due to the heterogeneity of breast cancer and may be limited in uncovering complex underlying mechanism. To address this problem, we first hypothesize that DNA methylation status and its inter-gene similarity are modulated by multiple factors including ER. The disruption of ER will disrupt association of methylation patterns between two genes which are correlated otherwise. The epigenetic signaling cascade maintained by specific ER status can be further interpreted from the “co-methylation” relationship among genes. Here we proposed a novel algorithm to identify the modulated gene methylation based on the “modulator of regulation” concept. Setting ER as the modulator, an ER modulated gene pair (ER-GP) was defined as pair of genes with differential correlation between ER+ and ER- samples. Fisher transformation on Pearson correlation coefficients was adopted for statistical power adjustment on sample sizes and the differences of absolute correlation coefficients were tested for statistical significance in the Fisher transform domain. By applying the algorithm to the MBDCap-Seq data of 76 breast tumors (47 ER+ and 29 ER-), we identified 5,673 putative ER-GPs of co-methylation (co-methyl. ER-GPs) involving 1,657 unique genes. In order to delineate a comprehensive and systematic view of interaction between identified ER-GPs, the co-methyl. ER-GPs were combined to construct the ER modulated gene methylation network. With an overall connectivity (average number of first-order neighbors of each gene in a network) of 6.85, genes in the methylation network were highly linked to each other and formed a branched signaling cascade. The nucleosome assembly protein 1-like 5 NAP1L5, previously reported as regulated by both estradiol and tamoxifen treatment, was identified as the top hub gene, indicating that it may serve as an important regulator in the ER modulation. Further Gene Ontology analysis revealed that NAP1L5 and its co-methylated genes were involved in nucleotide biosynthetic process, chromosome organization, and cell proliferation. In the present study, we proposed a novel algorithm to identify the ER modulated methylation regulatory network. The findings provided biological insights into the underlying mechanism of epigenetic signaling governed by ER and may contribute to improvement of breast cancer treatment. Citation Format: Yu-Chiao Chiu, Tzu-Hung Hsiao, Fei Gu, Yidong Chen, Tim H-M Huang, Eric Y. Chuang. Identification of estrogen receptor modulated gene methylation network in breast 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 2889. doi:10.1158/1538-7445.AM2013-2889 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

1139 SPECIFIC CHANGES IN THE DNA METHYLATION PATTERN ENABLE AN EARLY ASSESSMENT OF THE METASTATIC RISK OF PRIMARY BLADDER TUMOURS

You have accessJournal of UrologyBladder Cancer: Basic Research (III)1 Apr 20131139 SPECIFIC CHANGES IN THE DNA METHYLATION PATTERN ENABLE AN EARLY ASSESSMENT OF THE METASTATIC RISK OF PRIMARY BLADDER TUMOURS Beatrice Stubendorff, Kerstin Wilhelm, Supriya Dubey, Ewa Dudziec, James Catto, Mieczyslaw Gajda, Heiko Wunderlich, Marc-Oliver Grimm, and Kerstin Junker Beatrice StubendorffBeatrice Stubendorff Jena, Germany More articles by this author , Kerstin WilhelmKerstin Wilhelm Jena, Germany More articles by this author , Supriya DubeySupriya Dubey Jena, Germany More articles by this author , Ewa DudziecEwa Dudziec Oxford, United Kingdom More articles by this author , James CattoJames Catto Sheffield, United Kingdom More articles by this author , Mieczyslaw GajdaMieczyslaw Gajda Jena, Germany More articles by this author , Heiko WunderlichHeiko Wunderlich Eisenach, Germany More articles by this author , Marc-Oliver GrimmMarc-Oliver Grimm Jena, Germany More articles by this author , and Kerstin JunkerKerstin Junker Homburg, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.754AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Adjuvant chemotherapy improves the outcome of bladder cancer patients at risk for the development of metastases. However, there are no parameters available that allow an individual risk assessment. Therefore, identification of reliable prognostic markers for early prediction of tumour spread is required. The aim of this project is to determine whether changes in DNA methylation correlate with the metastasis risk and to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment. METHODS In total, 38 muscle-invasive primary bladder tumours were analysed. Mean follow-up was 2 years. Differences in the methylation pattern were detected by CpG Island (CpGI) microarray including 9 metastatic and 14 non-metastatic samples. Cluster analysis enabled selection of differentially methylated genes. Promoter methylation and gene expression were assessed by quantitative analysis of DNA methylation using real-time PCR (n=38) and qRT-PCR (n=12). For in vitro re-expression T24 cells were exposed to DNMT inhibitor 5-aza-dC. Bladder cancer cells RT112 were transfected using siRNA. Proliferation and migration was measured using real-time cell monitoring. Invasion assay was performed using matrigel coated cell culture inserts. RESULTS Microarray analysis resulted in 201 promoter CpGIs with significant differences (p<0.05) in DNA methylation between metastatic and non-metastatic primary bladder tumours. Validation of methylation status showed differences between patient groups for candidate genes KISS1R, SEPT9 (p<0.05) and CSAD (p<0.01). KISS1R expression differs between the patient groups (p=0.03). Correlation between promoter methylation and mRNA expression was confirmed for all 3 genes by treatment of cell line with 5-aza-dC. SEPT9 transcript variant 3 (SEPT9v3) showed a trend to lower expression in metastatic tumours. Knock down of SEPT9v3 has highest influence on cell proliferation (increased by 77% in compared to non-transfected control). Cell migration and invasion are increased by 15% and 39%, respectively. CONCLUSIONS Primary bladder tumours show significant differences in the DNA methylation pattern in correlation to the metastatic potential. KISS1R shows strongest association with the metastatic risk on methylation and expression level. Reduced expression of SEPT9v3 induces cell proliferation, migration and invasion. The combination of candidate genes KISS1R, CSAD, SEPT9 could serve as prognostic marker panel for determination of the metastatic risk of primary bladder tumours. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e465 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Beatrice Stubendorff Jena, Germany More articles by this author Kerstin Wilhelm Jena, Germany More articles by this author Supriya Dubey Jena, Germany More articles by this author Ewa Dudziec Oxford, United Kingdom More articles by this author James Catto Sheffield, United Kingdom More articles by this author Mieczyslaw Gajda Jena, Germany More articles by this author Heiko Wunderlich Eisenach, Germany More articles by this author Marc-Oliver Grimm Jena, Germany More articles by this author Kerstin Junker Homburg, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

DNA-methylation effect on cotranscriptional splicing is dependent on GC architecture of the exon–intron structure

DNA methylation is known to regulate transcription and was recently found to be involved in exon recognition via cotranscriptional splicing. We recently observed that exon–intron architectures can be grouped into two classes: one with higher GC content in exons compared to the flanking introns, and the other with similar GC content in exons and introns. The first group has higher nucleosome occupancy on exons than introns, whereas the second group exhibits weak nucleosome marking of exons, suggesting another type of epigenetic marker distinguishes exons from introns when GC content is similar. We find different and specific patterns of DNA methylation in each of the GC architectures; yet in both groups, DNA methylation clearly marks the exons. Exons of the leveled GC architecture exhibit a significantly stronger DNA methylation signal in relation to their flanking introns compared to exons of the differential GC architecture. This is accentuated by a reduction of the DNA methylation level in the intronic sequences in proximity to the splice sites and shows that different epigenetic modifications mark the location of exons already at the DNA level. Also, lower levels of methylated CpGs on alternative exons can successfully distinguish alternative exons from constitutive ones. Three positions at the splice sites show high CpG abundance and accompany elevated nucleosome occupancy in a leveled GC architecture. Overall, these results suggest that DNA methylation affects exon recognition and is influenced by the GC architecture of the exon and flanking introns.

A cell epigenotype specific model for the correction of brain cellular heterogeneity bias and its application to age, brain region and major depression

Brain cellular heterogeneity may bias cell type specific DNA methylation patterns, influencing findings in psychiatric epigenetic studies. We performed fluorescence activated cell sorting (FACS) of neuronal nuclei and Illumina HM450 DNA methylation profiling in post mortem frontal cortex of 29 major depression and 29 matched controls. We identify genomic features and ontologies enriched for cell type specific epigenetic variation. Using the top cell epigenotype specific (CETS) marks, we generated a publically available R package, “CETS,” capable of quantifying neuronal proportions and generating in silico neuronal profiles from DNA methylation data. We demonstrate a significant overlap in major depression DNA methylation associations between FACS separated and CETS model generated neuronal profiles relative to bulk profiles. CETS derived neuronal proportions correlated significantly with age in the frontal cortex and cerebellum and accounted for epigenetic variation between brain regions. CETS based control of cellular heterogeneity will enable more robust hypothesis testing in the brain.