Genome-wide DNA Methylation Patterns Research Articles

The CpG island methylator phenotype in breast cancer is associated with the lobular subtype.

Aberrations in DNA methylation patterns are well-described in human malignancies. However, the existence of the 'CpG island methylator phenotype' (CIMP) in human breast cancer is still controversial. Illumina's HumanMethylation 450K BeadChip was used to analyze genome-wide DNA methylation patterns. Chromosomal abnormalities were determined by array-based CGH. Invasive lobular breast carcinomas exhibit the highest number of differentially methylated CpG sites and a strong inverse correlation of aberrant DNA hypermethylation and copy number alterations. Nine differentially methylated regions within seven genes discriminating the investigated subgroups were identified and validated in an independent validation cohort and correlated to a better relapse-free survival. These results depict a clear difference between genetically and epigenetically unstable breast carcinomas indicating different ways of tumor progression and/or initiation, which strongly supports the association of CIMP with the lobular subtype and provide new options for detection and therapy.

MeDIP-on-Chip for methylation profiling.

DNA methylation is an important regulatory step in gene expression. Knowledge of the alterations in DNA methylation at a whole genome scale improves our understanding of gene regulation and potential correlations with biological events or disease progression. Methylated DNA immunoprecipitation (MeDIP) uses an antibody that efficiently enriches methylated DNA fragments for downstream locus-specific or genome-wide analyses. MeDIP-on-Chip uses the MeDIP approach in combination with a tiling array for the investigation of genome-wide DNA methylation patterns (or DNA methylomics). The following protocol describes the application of MeDIP to the hybridization of DNA microarrays and data analysis.

Comparative DNA methylome analysis of endometrial carcinoma reveals complex and distinct deregulation of cancer promoters and enhancers.

BackgroundAberrant DNA methylation is a hallmark of many cancers. Classically there are two types of endometrial cancer, endometrioid adenocarcinoma (EAC), or Type I, and uterine papillary serous carcinoma (UPSC), or Type II. However, the whole genome DNA methylation changes in these two classical types of endometrial cancer is still unknown.ResultsHere we described complete genome-wide DNA methylome maps of EAC, UPSC, and normal endometrium by applying a combined strategy of methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylation-sensitive restriction enzyme digestion sequencing (MRE-seq). We discovered distinct genome-wide DNA methylation patterns in EAC and UPSC: 27,009 and 15,676 recurrent differentially methylated regions (DMRs) were identified respectively, compared with normal endometrium. Over 80% of DMRs were in intergenic and intronic regions. The majority of these DMRs were not interrogated on the commonly used Infinium 450K array platform. Large-scale demethylation of chromosome X was detected in UPSC, accompanied by decreased XIST expression. Importantly, we discovered that the majority of the DMRs harbored promoter or enhancer functions and are specifically associated with genes related to uterine development and disease. Among these, abnormal methylation of transposable elements (TEs) may provide a novel mechanism to deregulate normal endometrium-specific enhancers derived from specific TEs.ConclusionsDNA methylation changes are an important signature of endometrial cancer and regulate gene expression by affecting not only proximal promoters but also distal enhancers.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-868) contains supplementary material, which is available to authorized users.

Genome-wide DNA methylation analysis identifies a metabolic memory profile in patient-derived diabetic foot ulcer fibroblasts

Diabetic foot ulcers (DFUs) are a serious complication of diabetes. Previous exposure to hyperglycemic conditions accelerates a decline in cellular function through metabolic memory despite normalization of glycemic control. Persistent, hyperglycemia-induced epigenetic patterns are considered a central mechanism that activates metabolic memory; however, this has not been investigated in patient-derived fibroblasts from DFUs. We generated a cohort of patient-derived lines from DFU fibroblasts (DFUF), and site- and age-matched diabetic foot fibroblasts (DFF) and non-diabetic foot fibroblasts (NFF) to investigate global and genome-wide DNA methylation patterns using liquid chromatography/mass spectrometry and the Illumina Infinium HumanMethylation450K array. DFFs and DFUFs demonstrated significantly lower global DNA methylation compared to NFFs (p = 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p = 0.05) showed that DFFs and DFUFs cluster together and separately from NFFs. Twenty-five percent of the same probes were identified as DMPs when individually comparing DFF and DFUF to NFF. Functional annotation identified enrichment of DMPs associated with genes critical to wound repair, including angiogenesis (p = 0.07) and extracellular matrix assembly (p = 0.035). Identification of sustained DNA methylation patterns in patient-derived fibroblasts after prolonged passage in normoglycemic conditions demonstrates persistent metabolic memory. These findings suggest that epigenetic-related metabolic memory may also underlie differences in wound healing phenotypes and can potentially identify therapeutic targets.

Abstract 388: Epigenome-wide profiling identified significant differences in DNA methylation between African-American and European-American men with prostate cancer

Abstract Background: DNA methylation is a dynamic epigenetic mark that is essential for mammalian organismal development. For human prostate cancer (PCa), abundant evidence has accumulated to suggest that somatic epigenetic alterations may appear early during cancer development, as well as more commonly and consistently, than genetic changes. Several cancer-associated genes have been reported to show significant differences in DNA methylation pattern in prostate tissues from African-American (AA) and European-American men, but the genome-wide pattern and extent of these differences are largely unknown. The current study investigated genome-wide DNA methylation differences in AA and EA prostate tissue samples with the aim of assessing the variation of genome-wide patterns of DNA methylation. Methods: To comprehensively examine the DNA methylation pattern in AA and EA samples, we used illumina 450 K methylation platform (Infunium Chip) to conduct genome-wide large scale analysis of DNA methylation changes in 7 normal and 3 PCa tissue samples from AA versus 8 normal and 3 PCa tissue samples from EA. This chip interrogates >485,000 CpG methylation sites per sample at single-nucleotide resolution and assigns each site an average beta value for quantitative methylation level. Results: Pathway analysis of the genes with altered methylation patterns identified top canonical pathways for the involvement of cancer related network for genes involved in axonal guidance, antigen presentation, androgen signaling and protein ubiquitination pathways in prostate cancer tissues compared with normal prostate tissues obtained from AA men. On the other hand, the top canonical pathways identified in prostate cancer tissues compared with normal prostate tissues obtained from EA men are genes involved in epithelial-mesenchymal transition, p53 signaling, nucleotide sugars metabolism and germ cell-sertoli cell junction signaling pathways. Conclusion: Using this quantitative sequencing-based approach, our work uncovers significant global DNA methylation alterations in AA versus EA prostate tissues and provides a mechanistic explanation for the disease disparity. Citation Format: Bernard Kwabi-Addo, Songping Wang, Joseph Devaney. Epigenome-wide profiling identified significant differences in DNA methylation between African-American and European-American men with prostate cancer. [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 388. doi:10.1158/1538-7445.AM2014-388

Abstract 2305: Acute depletion reveals novel co-regulation of DNA methylation at conserved loci by DNMT1 and DNMT3B

Abstract DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, 3B, 3L) and maintaining (DNMT1) DNA methylation genome-wide. Aberrant DNA methylation is frequently observed in cancer; however, little is known about how regulation of this modification goes awry. In this study, we aim to understand how DNA methylation is regulated by the DNMTs throughout the genome by identifying specific and broad changes in methylation patterning upon depletion of the DNMTs. We utilize siRNA technology to acutely deplete NCCIT embryonal carcinoma cells of DNMT mRNA (individually and in combination), and then assay the impact on genome-wide DNA methylation patterns using the HumanMethylation450 Bead Chip (450K array). Depletion of DNMT1 (individual/combination) resulted in widespread hypomethylation, most notably in gene bodies, 3′UTRs, and intergenic sequences. DNMT3 knockdown resulted in more specific changes in DNA methylation, but surprisingly, more hypermethylation (predominately in gene bodies) than hypomethylation events occurred. These specific hypermethylation events, particularly in samples with DNMT3B KD, significantly overlapped with sites hypomethylated in DNMT1 KD conditions, indicating a potential cross-regulatory role for DNMT1 and DNMT3B in regulating DNA methylation across gene bodies. To gain a more comprehensive genome-wide view of DNA methylation in the absence of DNMT3B, we performed Methyl-CpG-Binding-Domain(MBD)-seq on DNMT3B KD cells. MBD-seq revealed dynamic changes in methylation with minor hypermethylation in promoters and subtle hypomethylation across gene bodies; however, analysis of only the most significant methylation changes (> 4-fold) revealed that more hypermethylation events occur in intronic sequences, consistent with results obtained using the 450K array. To further investigate the overlap between DNMT1 hypomethylated and DNMT3B hypermethylated sites, we examined DNA methylation in HCT116 colorectal carcinoma cells lacking (KO) or over-expressing (KI) DNMT1/DNMT3B. Interestingly, a marked number of CpG sites that gained methylation in the DNMT3B KO overlapped significantly with sites that became hypermethylated in DNMT1 and DNMT3B KI, and hypomethylated in DNMT1 KO. Additionally, these HCT116 hypermethylated CpG sites gained methylation in NCCIT DNMT3B KD (individual/combination) and lost methylation in DNMT1 KD. Taken together, these results suggest that DNMT1 and DNMT3B co-regulate DNA methylation at conserved loci across cell types in an opposing fashion, providing novel insight into a potential regulatory mechanism for DNA methylation patterning. Further elucidation of this DNMT1 and DNMT3B co-regulation holds the potential to yield novel therapeutic strategies for correcting aberrant methylation events in cancer. Citation Format: Rochelle Tiedemann, Jeong-Hyeon Choi, Keith Robertson. Acute depletion reveals novel co-regulation of DNA methylation at conserved loci by DNMT1 and DNMT3B. [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 2305. doi:10.1158/1538-7445.AM2014-2305

Abstract 3084: Epigenetic deregulation in H3.3-K27M mutant pediatric high-grade gliomas

Abstract A recurrent mutation substituting lysine 27 with a methionine (K27M) on the amino-terminal tail of histone variant H3.3 was recently identified in about 25% of pediatric high-grade gliomas (pHGGs), the most common malignant brain tumor in childhood. K27M mutant tumors represent a clinically and biologically distinct subgroup of pHGGs, characterized by a unique genome-wide DNA methylation pattern as well as transcriptomic profile. Interestingly, tumors harboring the K27M mutation display a global reduction of the repressive histone modification H3K27me3, which is caused by a dominant-negative effect of K27M mutant H3.3 protein. The purpose of this study was to further elucidate the dominant-negative effect of K27M mutant H3.3 and investigate the connection between K27M-induced reduction of H3K27me3, DNA methylation and gene expression. Furthermore, we aimed to clarify if immunohistochemical (IHC) testing of H3K27me3 represents a reliable tool for clinical application to identify patients suffering from K27M mutant pHGGs, which might benefit from more intense treatment options. Co-immunoprecipitation (CoIP) experiments and histone methyltransferase (HMT) assays were conducted in three different cell lines to elucidate the dominant-negative function of K27M mutant H3.3. The potential impact on different levels of epigenetic gene regulation was analyzed by performing H3K27me3 chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq), whole genome bisulfite sequencing (WGBS) and gene expression profiling on fresh-frozen primary tumors with and without K27M mutations. As a result, we demonstrate that IHC reliably identifies K27M mutant pHGGs with high specificity and sensitivity, and confirm that H3K27me3 levels are globally reduced due to the dominant-negative effect of K27M mutant H3.3. As indicated by our CoIPs, this is a result of an increased interaction of K27M mutant H3.3 with several components of the Polycomb repressive complex 2 (PRC2), including enhancer of zeste homolog 2 (EZH2), which is responsible for di- and trimethylation of H3K27. EZH2 catalytic activity measured by HMT assay is significantly reduced in the presence of K27M mutant H3.3 protein. Genome-wide H3K27me3 ChIP-Seq data of fresh-frozen pHGG samples confirms globally reduced levels of H3K27me3, but also identifies a subset of loci with focal enrichment of this histone modification. By integrating with WGBS data, we show that K27M mutant pHGGs are characterized by reduced H3K27me3 occupancy and concomitant global DNA hypomethylation. Strikingly, 74% of genes found to be specifically upregulated in K27M mutant pHGGs show changes in one or both of these levels of epigenetic regulation. In conclusion, epigenetic deregulation in K27M mutant pHGGs reflected by reduced H3K27me3 occupancy and/or global DNA hypomethylation is a key modulator of gene activity in these highly aggressive brain tumors. Citation Format: Sebastian Bender, Yujie Tang, Anders M. Lindroth, Volker Hovestadt, Marc Zapatka, David T.W. Jones, Marcel Kool, Paul A. Northcott, Dominik Sturm, Peter Lichter, Christoph Plass, Yoon-Jae Cho. Epigenetic deregulation in H3.3-K27M mutant pediatric high-grade gliomas. [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 3084. doi:10.1158/1538-7445.AM2014-3084

Abstract 3089: (Epi)genetic profiling enables molecular re-classification of CNS-primitive neuroectodermal tumors

Abstract According to the current WHO classification of CNS tumors, childhood CNS primitive neuro-ectodermal tumors (CNS-PNETs; WHO °IV) are poorly differentiated embryonal tumors with early onset and aggressive clinical behavior. Histological diagnosis can be complicated by morphological heterogeneity and divergent differentiation. Recent studies suggest the existence of molecular subgroups of CNS-PNETs sharing biological characteristics with other childhood CNS tumors. Here, we aimed at a comprehensive molecular characterization of CNS-PNETs and compared our results to profiles of other brain tumor classes in order to define the biological nature of tumors diagnosed as CNS-PNETs. A collective of 197 fresh-frozen or paraffin-embedded tumor samples with an institutional diagnosis “CNS-PNET” was profiled for genome-wide DNA methylation patterns and copy-number alterations, complemented by transcriptomic profiling of a subset (n=63). (Epi-)genetic profiles of CNS-PNETs were compared to those of >1.000 other childhood brain tumors including embryonal, astrocytic, and ependymal entities, and their respective molecular subgroups. We screened selected groups of tumors for recurrent mutations and expression of established molecular markers. Five experienced neuropathologists independently revisited the histology of 48 CNS-PNETs. Bioinformatic analysis of DNA methylation and gene expression profiles using clustering methods and class prediction algorithms resulted in a clear segregation of pediatric brain tumors by histological entities and molecular subgroups. Exceptionally, profiles of tumors classified as “CNS-PNET” suggested significant overlap with various well-defined entities, including AT/RT, ETMR, high-grade glioma, medulloblastoma, and ependymoma. When screening CNS-PNETs with DNA methylation profiles highly resembling other entities, hallmark genetic alterations of these, such as amplification of 19q13.42, mutations in IDH1 or H3F3A, or mutations/deletions of the SMARCB1 locus, were frequently detected. Molecularly distinct tumor subsets were associated with differential protein expression patterns of previously established subgroup markers INI-1, LIN28A, and OLIG2. Blinded histopathological evaluation resulted in a large proportion of CNS-PNETs being re-classified in line with affiliations suggested by molecular diagnostic tools. The correct classification of CNS-PNET remains challenging. Based on the detection of recurrent genetic aberrations, many cases can be reliably re-classified, indicating that a significant proportion of CNS-PNETs may comprise a variety of other tumor subtypes. These findings suggest that the use of established and novel subgroups markers is needed in order to assist the histopathological evaluation of these tumors. The molecular biology underlying distinct subsets of CNS-PNETs only remains to be elucidated. Citation Format: Dominik Sturm, Paul A. Northcott, David T.W. Jones, Andrey Korshunov, Daniel Picard, Peter Lichter, Annie Huang, Stefan M. Pfister, Marcel Kool. (Epi)genetic profiling enables molecular re-classification of CNS-primitive neuroectodermal 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 3089. doi:10.1158/1538-7445.AM2014-3089

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

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

Altered imprinted gene expression and methylation patterns in mid-gestation aborted cloned porcine fetuses and placentas.

To determine the expression patterns of imprinted genes and their methylation status in aborted cloned porcine fetuses and placentas. RNA and DNA were prepared from fetuses and placentas that were produced by SCNT and controls from artificial insemination. The expression of 18 imprinted genes was determined by quantitative real-time PCR (q-PCR). Bisulfite sequencing PCR (BSP) was conducted to determine the methylation status of PRE-1 short interspersed repetitive element (SINE), satellite DNA and H19 differentially methylated region 3 (DMR3). The weight, imprinted gene expression and genome-wide DNA methylation patterns were compared between the mid-gestation aborted and normal control samples. The results showed hypermethylation of PRE-1 and satellite sequences, the aberrant expression of imprinted genes, and the hypomethylation of H19 DMR3 occurred in mid-gestation aborted fetuses and placentas. Cloned pigs generated by somatic cell nuclear transfer (SCNT) showed a greater ratio of early abortion during mid-gestation than did normal controls because of the incomplete epigenetic reprogramming of the donor cells. Altered expression of imprinted genes and the hypermethylation profile of the repetitive regions (PRE-1 and satellite DNA) may be associated with defective development and early abortion of cloned pigs, emphasizing the importance of epigenetics during pregnancy and implications thereof for patient-specific embryonic stem cells for human therapeutic cloning and improvement of human assisted reproduction.

Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways.

Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells−/− (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation.

Prediction of CpG island methylation status by integrating DNA physicochemical properties

As an inheritable epigenetic modification, DNA methylation plays important roles in many biological processes. The non-uniform distribution of DNA methylation across the genome implies that characterizing genome-wide DNA methylation patterns is necessary to better understand the regulatory mechanisms of DNA methylation. Although a series of experimental technologies have been proposed, they are cost-ineffective for DNA methylation status detection. As complements to experimental techniques, computational methods will facilitate the identification of DNA methylation status. In the present study, we proposed a Naïve Bayes model to predict CpG island methylation status. In this model, DNA sequences are formulated by “pseudo trinucleotide composition” into which three DNA physicochemical properties were incorporated. It was observed by the jack-knife test that the overall success rate achieved by the proposed model in predicting the DNA methylation status was 88.22%. This result indicates that the proposed model is a useful tool for DNA methylation status prediction.

Genome-wide DNA methylation changes in skeletal muscle between young and middle-aged pigs

BackgroundAge-related physiological, biochemical and functional changes in mammalian skeletal muscle have been shown to begin at the mid-point of the lifespan. However, the underlying changes in DNA methylation that occur during this turning point of the muscle aging process have not been clarified. To explore age-related genomic methylation changes in skeletal muscle, we employed young (0.5 years old) and middle-aged (7 years old) pigs as models to survey genome-wide DNA methylation in the longissimus dorsi muscle using a methylated DNA immunoprecipitation sequencing approach.ResultsWe observed a tendency toward a global loss of DNA methylation in the gene-body region of the skeletal muscle of the middle-aged pigs compared with the young group. We determined the genome-wide gene expression pattern in the longissimus dorsi muscle using microarray analysis and performed a correlation analysis using DMR (differentially methylated region)-mRNA pairs, and we found a significant negative correlation between the changes in methylation levels within gene bodies and gene expression. Furthermore, we identified numerous genes that show age-related methylation changes that are potentially involved in the aging process. The methylation status of these genes was confirmed using bisulfite sequencing PCR. The genes that exhibited a hypomethylated gene body in middle-aged pigs were over-represented in various proteolysis and protein catabolic processes, suggesting an important role for these genes in age-related muscle atrophy. In addition, genes associated with tumorigenesis exhibited aged-related differences in methylation and expression levels, suggesting an increased risk of disease associated with increased age.ConclusionsThis study provides a comprehensive analysis of genome-wide DNA methylation patterns in aging pig skeletal muscle. Our findings will serve as a valuable resource in aging studies, promoting the pig as a model organism for human aging research and accelerating the development of comparative animal models in aging research.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-653) contains supplementary material, which is available to authorized users.

Dietary fat quality impacts genome-wide DNA methylation patterns in a cross-sectional study of Greek preadolescents.

The type and the amount of dietary fat have a significant influence on the metabolic pathways involved in the development of obesity, metabolic syndrome, diabetes type 2 and cardiovascular diseases. However, it is unknown to what extent this modulation is achieved through DNA methylation. We assessed the effects of cholesterol intake, the proportion of energy intake derived from fat, the ratio of polyunsaturated fatty acids (PUFA) to saturated fatty acids (SFA), the ratio of monounsaturated fatty acids (MUFA) to SFA, and the ratio of MUFA+PUFA to SFA on genome-wide DNA methylation patterns in normal-weight and obese children. We determined the genome-wide methylation profile in the blood of 69 Greek preadolescents (∼10 years old) as well as their dietary intake for two consecutive weekdays and one weekend day. The methylation levels of one CpG island shore and four sites were significantly correlated with total fat intake. The methylation levels of 2 islands, 11 island shores and 16 sites were significantly correlated with PUFA/SFA; of 9 islands, 26 island shores and 158 sites with MUFA/SFA; and of 10 islands, 40 island shores and 130 sites with (MUFA+PUFA)/SFA. We found significant gene enrichment in 34 pathways for PUFA/SFA, including the leptin pathway, and a significant enrichment in 5 pathways for (MUFA+PUFA)/SFA. Our results suggest that specific changes in DNA methylation may have an important role in the mechanisms involved in the physiological responses to different types of dietary fat.

Genome-wide DNA methylation and gene expression patterns provide insight into polycystic ovary syndrome development.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. However, the epigenetic mechanism involved in PCOS progression remains largely unknown. Here, combining the DNA methylation profiling together with transcriptome analysis, we showed that (i) there were 7929 differentially methylated CpG sites (β > 0.1, P < 0.05) and 650 differential transcripts (fold change > 1.5, P < 0.005) in PCOS compared to normal ovaries; (ii) 54 genes were identified with methylated levels that were correlated with gene transcription in PCOS; and (iii) there were less hypermethylated sites, but many more hypomethylated sites residing in CpG islands and N_Shore in PCOS. Among these genes, we identified that several significant pathways, including the type I diabetes mellitus pathway, p53 signaling pathway and NOD-like receptor signaling pathway, and some immune and inflammatory diseases may be highly involved in PCOS development. These results suggested that differences in genome-wide DNA methylation and expression patterns exist between PCOS ovaries and normal ovaries; epigenetic mechanisms may in part be responsible for the different gene expression and PCOS phenotype. All of this may improve our understanding of the basic molecular mechanism underlying the development of PCOS.

Genome-wide DNA methylation patterns and transcription analysis in sheep muscle.

DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS). While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.

High-throughput targeted repeat element bisulfite sequencing (HT-TREBS): genome-wide DNA methylation analysis of IAP LTR retrotransposon.

In vertebrates, DNA methylation-mediated repression of retrotransposons is essential for the maintenance of genomic integrity. In the current study, we developed a technique termed HT-TREBS (High-Throughput Targeted Repeat Element Bisulfite Sequencing). This technique is designed to measure the DNA methylation levels of individual loci of any repeat families with next-generation sequencing approaches. To test the feasibility of HT-TREBS, we analyzed the DNA methylation levels of the IAP LTR family using a set of 12 different genomic DNA isolated from the brain, liver and kidney of 4 one-week-old littermates of the mouse strain C57BL/6N. This technique has successfully generated the CpG methylation data of 5,233 loci common in all the samples, representing more than 80% of the individual loci of the five targeted subtypes of the IAP LTR family. According to the results, approximately 5% of the IAP LTR loci have less than 80% CpG methylation levels with no genomic position preference. Further analyses of the IAP LTR loci also revealed the presence of extensive DNA methylation variations between different tissues and individuals. Overall, these data demonstrate the efficiency and robustness of the new technique, HT-TREBS, and also provide new insights regarding the genome-wide DNA methylation patterns of the IAP LTR repeat elements.

Infinium monkeys: Infinium 450K array for the Cynomolgus macaque (Macaca fascicularis).

The Infinium Human Methylation450 BeadChip Array (Infinium 450K) is a robust and cost-efficient survey of genome-wide DNA methylation patterns. Macaca fascicularis (Cynomolgus macaque) is an important disease model; however, its genome sequence is only recently published, and few tools exist to interrogate the molecular state of Cynomolgus macaque tissues. Although the Infinium 450K is a hybridization array designed to the human genome, the relative conservation between the macaque and human genomes makes its use in macaques feasible. Here, we used the Infinium 450K array to assay DNA methylation in 11 macaque muscle biopsies. We showed that probe hybridization efficiency was related to the degree of sequence identity between the human probes and the macaque genome sequence. Approximately 61% of the Human Infinium 450K probes could be reliably mapped to the Cynomolgus macaque genome and contain a CpG site of interest. We also compared the Infinium 450K data to reduced representation bisulfite sequencing data generated on the same samples and found a high level of concordance between the two independent methodologies, which can be further improved by filtering for probe sequence identity and mismatch location. We conclude that the Infinium 450K array can be used to measure the DNA methylome of Cynomolgus macaque tissues using the provided filters. We also provide a pipeline for validation of the array in other species using a simple BLAST-based sequence identify filter.

MOLECULAR (RE-)CLASSIFICATION OF CNS-PRIMITIVE NEUROECTODERMAL TUMORS

BACKGROUND: According to the current WHO classification of CNS tumors, childhood CNS primitive neuro-ectodermal tumors (CNS-PNETs; WHO °IV) are poorly differentiated embryonal tumors with early onset and aggressive clinical behavior. Histological diagnosis can be complicated by morphological heterogeneity and divergent differentiation. Recent studies suggest the existence of molecular subgroups of CNS-PNETs sharing biological characteristics with other childhood CNS tumors. Here, we aimed at a comprehensive molecular characterization of CNS-PNETs and compared our results to profiles of other brain tumor entities in order to define the biological nature of tumors diagnosed as CNS-PNETs. METHODS: A collection of 211 fresh-frozen or paraffin-embedded tumor samples with an institutional diagnosis “CNS-PNET” was profiled for genome-wide DNA methylation patterns and copy-number alterations, complemented by transcriptomic profiling of a subset (n = 71). (Epi-)genetic profiles of CNS-PNETs were compared to those of >3000 other childhood brain tumors including embryonal, astrocytic, and ependymal entities, and their respective molecular subgroups. We screened selected groups of tumors for recurrent mutations and expression of established molecular markers. RESULTS: DNA methylation and gene expression profiles showed a clear segregation of pediatric brain tumors by histological entities and molecular subgroups. Interestingly, CNS-PNET profiles showed a significant overlap with various well-defined entities, including AT/RT, ETMR, high-grade glioma, medulloblastoma, and ependymoma. When screening CNS-PNETs with profiles highly resembling other entities, hallmark genetic alterations of these, such as amplification of 19q13.42, mutations in IDH1 or H3F3A, or mutations/deletions of the SMARCB1 locus, were frequently detected. Also, established protein markers, such as INI-1, LIN28A, and OLIG2, confirmed the reclassification of these CNS-PNETs. Strikingly, a subset (∼25%) of CNS-PNETs which could not be reclassified segregated into 3-4 distinct molecular subgroups, each with its own characteristic pattern of copy-number aberrations, DNA-methylation and gene expression. Currently, whole genome and RNA-sequencing of these distinct subgroups of CNS-PNETs is ongoing to reveal their underlying genetics. CONCLUSIONS: The correct classification of CNS-PNETs remains challenging. Based on the detection of recurrent genetic aberrations, many cases can be reliably re-classified, indicating that a significant proportion of CNS-PNETs may comprise a variety of other tumor subtypes. These findings suggest that the use of established and novel subgroups markers is needed in order to assist the histopathological evaluation of these tumors. In addition, we have identified a number of true CNS-PNET subtypes and are currently analyzing them in more detail in order to elucidate the genetics of these distinct groups. SECONDARY CATEGORY: Tumor Biology.

Altered Histone Mark Deposition and DNA Methylation at Homeobox Genes in Human Oral Squamous Cell Carcinoma

We recently reported a role of polycomb repressive complex 2 (PRC2) and PRC2 trimethylation of histone 3 lysine 27 (H3K27me3) in the regulation of homeobox (HOX) (Marcinkiewicz and Gudas, 2013, Exp Cell Res) gene transcript levels in human oral keratinocytes (OKF6-TERT1R) and tongue squamous cell carcinoma (SCC) cells. Here, we assessed both the levels of various histone modifications at a subset of homeobox genes and genome wide DNA methylation patterns in OKF6-TERT1R and SCC-9 cells by using ERRBS (enhanced reduced representation bisulfite sequencing). We detected the H3K9me3 mark at HOXB7, HOXC10, HOXC13, and HOXD8 at levels higher in OKF6-TERT1R than in SCC-9 cells; at IRX1 and SIX2 the H3K9me3 levels were conversely higher in SCC-9 than in OKF6-TERT1R. The H3K79me3 mark was detectable only at IRX1 in OKF6-TERT1R and at IRX4 in SCC-9 cells. The levels of H3K4me3 and H3K36me3 marks correlate with the transcript levels of the assessed homeobox genes in both OKF6-TERT1R and SCC-9. We detected generally lower CpG methylation levels on DNA in SCC-9 cells at annotated genomic regions which were differentially methylated between OKF6-TERT1R and SCC-9 cells; however, some genomic regions, including the HOX gene clusters, showed DNA methylation at higher levels in SCC-9 than OKF6-TERT1R. Thus, both altered histone modification patterns and changes in DNA methylation are associated with dysregulation of homeobox gene expression in human oral cavity SCC cells, and this dysregulation potentially plays a role in the neoplastic phenotype of oral keratinocytes.