Direct Bisulfite Sequencing Research Articles

Identification of DNA methylation of SOX9 in cervical cancer using methylated-CpG island recovery assay

The aim of the present study was to identify novel methylation markers for cervical cancer screening and to test the clinical application of the most promising biomarker in cervical scrapings. Methylated-CpG island recovery assay-based microarray analysis was carried out on a discovery set consisting of cervical cancer tissue and normal cervical tissue to identify significantly hypermethylated genes. Five hundred and four CpG islands, corresponding to 378 genes, were differentially methylated between cervical cancer tissue and normal cervical tissue. Among them, 30 genes were significantly hypermethylated. Of the 30 genes, SOX9, PKLR and DLX4 were selected for further validation by direct bisulfite sequencing. The SOX9 gene revealed complete methylation in the cervical cancer tissue and complete non-methylation in the normal control tissue. A TaqMan-based real-time PCR assay was performed to detect the methylation levels of the SOX9 gene in 156 cervical scrapings, including 48 normal cervical scrapings, 30 scrapings with cervical intraepithelial neoplasia1 (CIN1), 30 scrapings with CIN2-3 and 48 scrapings with squamous cell carcinoma (SCC). The methylation levels (methylation score) of the SOX9 gene increased significantly with the severity of cervical squamous lesions. The area under the receiver operating characteristic (ROC) curve (AUC) revealed that the methylation score of the SOX9 gene could be used to segregate SCC/CIN2-3 from CIN1/normal (AUC, 0.961; p=0.000). At the optimal cut-off value, a sensitivity of 92.3% and a specificity of 89.7% were obtained. In conclusion, SOX9 methylation is frequently involved in cervical carcinogenesis, and may provide a valuable molecular biomarker for early detection of cervical cancer.

Direct Bisulfite Sequencing for Examination of DNA Methylation with Gene and Nucleotide Resolution from Brain Tissues

DNA methylation is an epigenetic modification that is essential for the development and mature function of the central nervous system. Due to the relevance of this modification to the transcriptional control of gene expression, it is often necessary to examine changes in DNA methylation patterns with both gene and single-nucleotide resolution. Here, we describe an in-depth basic protocol for direct bisulfite sequencing of DNA isolated from brain tissue, which will permit direct assessment of methylation status at individual genes as well as individual cytosine molecules/nucleotides within a genomic region. This method yields analysis of DNA methylation patterns that is robust, accurate, and reproducible, thereby allowing insights into the role of alterations in DNA methylation in brain tissue.

Screening significantly hypermethylated genes in fetal tissues compared with maternal blood using a methylated-CpG island recovery assay-based microarray

BackgroundThe noninvasive prenatal diagnosis procedures that are currently used to detect genetic diseases do not achieve desirable levels of sensitivity and specificity. Recently, fetal methylated DNA biomarkers in maternal peripheral blood have been explored for the noninvasive prenatal detection of genetic disorders. However, such efforts have covered only chromosomal aneuploidy, and fetal methylated DNA biomarkers in maternal whole blood for detecting single-gene diseases remain to be discovered.MethodsTo address this issue, we systematically screened significantly hypermethylated genes in fetal tissues and compared them with maternal peripheral blood potential in an attempt to detect fetal genes in maternal peripheral blood. First, the methylated-CpG island recovery assay combined with a CpG island array was performed for four fetus-toward placental tissues and the corresponding maternal peripheral bloods. Subsequently, direct bisulfite sequencing and combined bisulfite restriction analysis (COBRA) were carried out to validate the methylation status of the hypermethylated genes that were identified by the microarray analysis.ResultsThree hundred and ten significantly hypermethylated genes in the placental tissues were detected by microarray. From the top 15 hypermethylated genes detected by microarray, two were selected for sequencing validation in placental tissue and chorionic villus samples and four were selected for COBRA validation in four placental tissues, ten amniotic fluids and five chorionic villus samples. The six selected genes were confirmed to be hypermethylated in placental tissue and chorionic villus samples, but methylation of the genes could not be detected in the amniotic fluids.ConclusionsOf the many hypermethylated genes and methylation sites that were found in the fetal tissues, some have great potential to be developed into molecular markers for noninvasive prenatal diagnosis of monogenic disorders. Further clinical studies are warranted to confirm these findings.

Screening of significantly hypermethylated genes in breast cancer using microarray-based methylated-CpG island recovery assay and identification of their expression levels

To screen candidate methylation markers for early detection of breast cancer and to explore the relationship between methylation and gene expression, we performed methylated-CpG island recovery assay (MIRA) combined with CpG island array on 61982 CpG sites across 4162 genes in 10 cancerous and 10 non-cancerous breast tissues. Direct bisulfite sequencing and combined bisulfite restriction analysis (COBRA) were carried out in independent cancerous and non-cancerous samples. Gene expression was analyzed by microarrays and validated using RT-PCR. We detected 70 significantly hypermethylated genes in breast cancer tissues, including many novel hypermethylated genes such as ITGA4, NFIX, OTX2 and FGF12. Direct bisulfite sequencing showed widespread methylation occurring in intragenic regions of the WT1, PAX6 and ITGA4 genes and in the promoter region of the OTX2 gene in breast cancer tissues. COBRA assay confirmed that the WT1, OTX2 and PAX6 genes were hypermethylated in breast cancer tissues. Clustering analysis of the gene expression of 70 significantly hypermethylated genes revealed that most hypermethylated genes in breast cancer were not expressed in breast tissues. RT-PCR assay confirmed that WT1 and PITX2 were only weakly expressed in the breast cancer tissues and were not expressed in most non-cancerous breast tissues. OTX2 and PAX6 were not expressed in either breast cancer or non-cancerous tissues. In conclusion, these results will expand our knowledge of hypermethylated genes and methylation sites for early detection of breast cancer and deepen our understanding of the relationship between methylation and gene expression. The MIRA approach can screen candidate methylated genes for further clinical validation more effectively than gene expression microarray-based strategy.

Identification of novel DNA methylation markers in colorectal cancer using MIRA-based microarrays

To identify novel hypermethylated genes in colorectal cancer (CRC) and to test their potential application in CRC early diagnosis, a genome-wide screening of 57,723 CpG dinucleotides covering 4,010 genes was performed using MIRA-based microarrays in paired DNA samples extracted from 3 fresh frozen CRC tissues and their matching non-cancer tissues from 3 CRC patients undergoing curative surgery. Candidate hypermethylated genes screened by MIRA-based microarrays were further validated in independent CRC samples. A total of 297 CpG dinucleotides covering 211 genes were found to be hypermethylated in CRC tissues. From these 211 candidate methylated genes, three novel hypermethylated genes with more than four probes positive were picked up for validation. Direct bisulfite sequencing revealed that methylations occurred at multiple CpG sites of these three genes in cancer tissues, especially for PHOX2B and FGF12. Combined bisulfite restriction analysis showed that these three genes were methylated in cancer samples but not in non-cancer samples. We also compared the methylation levels of these three novel hypermethylated genes with those of vimentin and SEPT9, well-known hypermethylated genes in CRC, and found that methylated PHOX2B, FGF12 and GAD2 were better than methylated vimentin and SEPT9 in differentiating CRC cancer tissue from non-cancer tissue. Significant enrichment analysis of GO terms of the hypermethylated genes showed that a high proportion of hypermethylated genes in cancer tissues are involved in the regulation of transcription. In conclusion, we found a set of novel hypermethylated genes in CRC, which may have potential to be used as biomarkers for the early diagnosis of CRC.

Bovine large luteal cells show increasing de novo DNA methylation of the main ovarian CYP19A1 promoter P2

Transformation of the estrogen producing large dominant follicle into a functional progesterone producing corpus luteum involves profound and well-orchestrated changes in cell type-specific gene expression profiles, possibly involving epigenetic mechanisms of gene silencing. As an experimental paradigm to examine the involvement of de novo DNA methylation in the process of luteinization, the transcript abundance and promoter-specific DNA methylation levels of CYP19A1, which encodes the key enzyme for estrogen biosynthesis, were analyzed in enzymatically dispersed and purified large granulosa luteal cells of early- to mid-cycle bovine corpora lutea. To characterize the morphology and physiology of isolated corpora lutea, their weights and the respective plasma progesterone levels were analyzed. Transcript abundance of CYP19A1, HSD3B1, GHR, and of LHGCR was quantified by real-time PCR. Methylation levels were analyzed by bisulfite direct sequencing. The data indicated that corpora lutea weights and plasma progesterone levels significantly increased during the early luteal phase (days 3–6 of the cycle). The growth of small and large luteal cells was particularly pronounced between days 3 and 4. Large luteal cells are characterized by high HSD3B1 and GHR, but low LHCGR transcript abundance, whereas CYP19A1 expression was very low or undetectable. The DNA methylation levels of the main ovarian CYP19A1 promoter P2 significantly increased from day 5. In conclusion, the data indicated de novo DNA methylation approximately five days after the luteinizing hormone-induced down-regulation of CYP19A1 expression, suggesting that DNA methylation during the early luteal phase might play a role for permanent silencing of previously down-regulated genes.

Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA

The complex relationship between DNA methylation, chromatin modification, and underlying DNA sequence is often difficult to unravel with existing technologies. Here, we describe a novel technique based on high-throughput sequencing of bisulfite-treated chromatin immunoprecipitated DNA (BisChIP-seq), which can directly interrogate genetic and epigenetic processes that occur in normal and diseased cells. Unlike most previous reports based on correlative techniques, we found using direct bisulfite sequencing of Polycomb H3K27me3-enriched DNA from normal and prostate cancer cells that DNA methylation and H3K27me3-marked histones are not always mutually exclusive, but can co-occur in a genomic region-dependent manner. Notably, in cancer, the co-dependency of marks is largely redistributed with an increase of the dual repressive marks at CpG islands and transcription start sites of silent genes. In contrast, there is a loss of DNA methylation in intergenic H3K27me3-marked regions. Allele-specific methylation status derived from the BisChIP-seq data clearly showed that both methylated and unmethylated alleles can simultaneously be associated with H3K27me3 histones, highlighting that DNA methylation status in these regions is not dependent on Polycomb chromatin status. BisChIP-seq is a novel approach that can be widely applied to directly interrogate the genomic relationship between allele-specific DNA methylation, histone modification, or other important epigenetic regulators.

Genome-Wide Screen for Differential DNA Methylation Associated with Neural Cell Differentiation in Mouse

Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in in-vitro induced neural stem cells (NSCs) and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score>1.96) enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.

Systematic assessment of reduced representation bisulfite sequencing to human blood samples: A promising method for large-sample-scale epigenomic studies

Complementary to the time- and cost-intensive direct bisulfite sequencing, we applied reduced representation bisulfite sequencing (RRBS) to the human peripheral blood mononuclear cells (PBMC) from YH, the Asian individual whose genome and epigenome has been deciphered in the YH project and systematically assessed the genomic coverage, coverage depth and reproducibility of this technology as well as the concordance of DNA methylation levels measured by RRBS and direct bisulfite sequencing for the detected CpG sites. Our result suggests that RRBS can cover more than half of CpG islands and promoter regions with a good coverage depth and the proportion of the CpG sites covered by the biological replicates reaches 80-90%, indicating good reproducibility. Given a smaller data quantity, RRBS enjoys much better coverage depth than direct bisulfite sequencing and the concordance of DNA methylation levels between the two methods is high. It can be concluded that RRBS is a time and cost-effective sequencing method for unbiased DNA methylation profiling of CpG islands and promoter regions in a genome-wide scale and it is the method of choice to assay certain genomic regions for multiple samples in a rapid way.

Abstract 74: Genome-wide DNA methylation analysis of pT1a renal cell carcinoma and renal oncocytomas

Abstract Nephron sparing surgery (partial nephrectomy) is currently performed on small renal masses (4cm or less in size) confined to the kidney (pT1a). Up to 25% of small renal masses are benign, the majority being oncocytomas. Benign oncocytomas lack distinct radiographic findings on computerized tomography (CT) and cannot be diagnosed accurately using imaging alone. No difference in tumor size at presentation or tumor growth rate was noted between oncocytoma and RCC in a review of the literature. A needle biopsy or non-invasive urine based differential diagnosis of a benign lesion potentially would allow patients with equivocal renal lesions to avoid unnecessary surgery as well as anxiety if under active surveillance. The epigenetic alteration of aberrant DNA methylation has potential benefits as a choice of a biomarker because aberrant methylation occurs frequently and early in tumorgenesis and can be detected at extremely sensitive and specific levels by currently available technology. It is known that aberrant hypermethylation of the VHL gene occurs in a subset of clear cell RCC but is absent in oncocytoma. We hypothesized that additonal genes with a differential methylation status between benign oncocytoma and RCC could be identified. In this study, we performed genome-wide detection of DNA methylation in 25 RCC (pT1a), 25 oncocytomas of 4cm or less in size, and 4 age-matched histologically normal kidney tissues using the Infinium HumanMethylation27 BeadChip. To date, we have evidence that the profile of aberrant gene methylation is different between benign and malignant small renal masses matched for median size. Infinium-identified differentially methylated genes are validated using direct bisulfite sequencing, then a qMSP assay designed. If the panel of differentially methylated genes we have identified are verified in an independent set of small renal masses, we can then determine the predictive value of methylation-based detection of benign oncocytoma versus malignant RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 74. doi:10.1158/1538-7445.AM2011-74

Epigenetic profile of developmentally important genes in bovine oocytes

Assisted reproductive technologies are associated with an increased incidence of epigenetic aberrations, specifically in imprinted genes. Here, we used the bovine oocyte as a model to determine putative epigenetic mutations at three imprinted gene loci caused by the type of maturation, either in vitro maturation (IVM) in Tissue Culture Medium 199 (TCM) or modified synthetic oviduct fluid (mSOF) medium, or in vivo maturation. We applied a limiting dilution approach and direct bisulfite sequencing to analyze the methylation profiles of individual alleles (DNA molecules) for H19/IGF2, PEG3, and SNRPN, which are each associated with imprinting defects in humans and/or the mouse model, and are known to be differentially methylated in bovine embryos. Altogether, we obtained the methylation patterns of 203 alleles containing 4,512 CpG sites from immature oocytes, 213 alleles with 4,779 CpG sites from TCM-matured oocytes, 215 alleles/4,725 CpGs in mSOF-matured oocytes, and 78 alleles/1,672 CpGs from in vivo-matured oocytes. The total rate of individual CpGs and entire allele methylation errors did not differ significantly between the two IVM and the in vivo group, indicating that current IVM protocols have no or only marginal effects on these critical epigenetic marks. Furthermore, the mRNA expression profiles of the three imprinted genes and a panel of eight other genes indicative of oocyte competence were determined by quantitative real-time PCR. We found different mRNA expression profiles between in vivo-matured oocytes versus their in vitro-matured counterparts, suggesting an influence on regulatory mechanisms other than DNA methylation.

Possible down regulation of the p16 gene promoter in individuals with hepatocellular carcinoma.

The p16 tumor suppressor gene is an important negative regulator of the cell cycle. Inactivation of p16, especially via promoter hypermethylation, has been found in numerous human cancers such as breast, lung, colorectal, and liver. To determine the role of epigenetic methylation in p16 regulation in Iranian patients with hepatocellular carcinoma (HCC). The methylation pattern in the p16 gene promoter was analyzed by bisulfite direct sequencing in 43 paraffin-embedded formalin-fixed tissues from patients with HCC. In addition, normal specimens from liver graft donors were used as the control group. The bisulfite direct sequencing showed heterozygous hypermethylation in 13.9% of individuals with HCC. Homozygous methylation within the GC-box IV was detected in another 58.1% of the patients. It is proposed that methylation, but not necessarily hypermethylation, may play a role in the down-regulation of the p16 gene promoter at least in some Iranian patients with HCC.

Genome Wide Study of DNA Methylation In AML

AbstractAbstract 3618DNA methylation is the most stable epigenetic modification and has a major role in cancer initiation and progression. The two main aims for this research were, firstly, to use the genome wide analysis of DNA methylation to better understand the development of acute myeloid leukemia (AML). The second aim was to detect differentially methylated genes/regions between certain subtypes of AML and normal bone marrow (NBM). We used the methylated DNA immunoprecipitation technique followed by high-throughput sequencing by Illumina Genome Analyser II (MeDIP -seq) for 9 AML samples for which ethical approval has been obtained. The selected leukemias included three with the t(8; 21), three with the t(15; 17) translocations and three with normal karyotypes (NK). The control samples were 3 normal bone marrows (NBMs) from healthy donors. The number of reads generated from Illumina ranged between 18– 20 million paired-end reads/lane with a good base quality from both ends (base quality > 30 represented 75%-85% of reads). The reads were aligned using 2 algorithms (Maq and Bowtie) and the methylation analysis was performed by Batman software (Bayesian Tool for Methylation Analysis).The creation of this genome-wide methylation map for AML permits the examination of the patterns for key genetic elements. Investigation of the 35,072 promoter regions identified 80 genes, which showed a significant differential methylation levels in leukemic cases in comparison to NBM; consistently high methylation levels in leukaemia were detected in the promoters of 70 genes e.g. DPP6, ID4, DCC, whereas high methylation levels in NBM, lost in leukaemia was observed in 10 genes e.g. ATF4. For each AML subtype, we also identified significant differentially methylated promoter regions e.g. PAX1 for t(8; 21), GRM7 for t(15; 17), NPM2 for NK.An analysis of gene body methylation identified 49 genes with significantly higher methylation in AML in comparison to NBM e.g. MYOD1 and 31 genes with a higher methylation in NBMs than AML e.g. GNG8. A similar analysis of 23,600 CpG islands identified 400 CpG islands with significant differential methylation levels between leukaemia and NBMs (212 CpG islands were found to have significantly increased methylation in leukaemia and 188 CpG islands had significantly higher methylation in NBMs). The pattern of methylation in CpG island “shores” (2 KB from either side of each CpG island) has been investigated and 312 CpG island shores showed a higher methylation in leukaemia and 88 CpG shores had a significant increase methylation levels in NBMs. This genome wide methylation map has been validated by using direct bisulfite sequencing of the regions identified above (Spearman r= 0.8, P <0.0001) and also by using Illumina Infinium assay (Spearman r= 0.7 P <0.0001) which interrogates regions at single representative CpGs. Comparison of previous array based gene expression data with this methylation map revealed a significant negative correlation between promoter methylation and gene expression (Pearson r= -0.9, P< 0.0001) while, gene body methylation showed a small negative correlation with gene expression, that was found in genes of CpG density >3% (Pearson r= -0.3, P< 0.0001). Conclusion:we have established a high-resolution (100bp) map of DNA methylation in AML and thus identified a novel list of genes, which have significantly differential methylation levels in AML. Disclosures:No relevant conflicts of interest to declare.

MetMap Enables Genome-Scale Methyltyping for Determining Methylation States in Populations

The ability to assay genome-scale methylation patterns using high-throughput sequencing makes it possible to carry out association studies to determine the relationship between epigenetic variation and phenotype. While bisulfite sequencing can determine a methylome at high resolution, cost inhibits its use in comparative and population studies. MethylSeq, based on sequencing of fragment ends produced by a methylation-sensitive restriction enzyme, is a method for methyltyping (survey of methylation states) and is a site-specific and cost-effective alternative to whole-genome bisulfite sequencing. Despite its advantages, the use of MethylSeq has been restricted by biases in MethylSeq data that complicate the determination of methyltypes. Here we introduce a statistical method, MetMap, that produces corrected site-specific methylation states from MethylSeq experiments and annotates unmethylated islands across the genome. MetMap integrates genome sequence information with experimental data, in a statistically sound and cohesive Bayesian Network. It infers the extent of methylation at individual CGs and across regions, and serves as a framework for comparative methylation analysis within and among species. We validated MetMap's inferences with direct bisulfite sequencing, showing that the methylation status of sites and islands is accurately inferred. We used MetMap to analyze MethylSeq data from four human neutrophil samples, identifying novel, highly unmethylated islands that are invisible to sequence-based annotation strategies. The combination of MethylSeq and MetMap is a powerful and cost-effective tool for determining genome-scale methyltypes suitable for comparative and association studies.

Abstract LB-221: Differential aberrant DNA methylation predicts for Tarceva response in NSCLC

Abstract Recent phase III clinical trials have shown that some non-small cell lung cancer (NSCLC) patients without epidermal growth factor receptor mutations derive clinical benefit from erlotnib, a small molecule EGFR inhibitor. Currently there is no way to identify this important patient subset. In an effort to explore whether aberrant DNA methylation profiles could be used to better define the subset of patients with NSCLC who might respond to erlotinib, we have undertaken an integrated genomics approach employing both gene expression profiling and methylated DNA immunoprecipitation (meDIP). In the initial phase of this study, we used a series of twelve NSCLC cell lines without EGFR mutations, which we defined in a previous report as either sensitive or resistant to erlotinib, based on an in vitro microtiter assay (cell titer glow). We treated each cell line with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) qod for 6 days and confirmed gene induction using quantitative RT-PCR (QPCR). We then performed gene expression profiling on the mock treated (DMSO) and 5-azadC (1 M) treated cell lines using the Affymetrix U133P 2.0 chip. Consistent with previous reports we found that the expression levels of large numbers of genes are affected by 5-azadC treatment, and in both directions. In addition, since it is known that 3′ biased gene expression arrays can miss expression changes in alternatively spliced (and methylated) variants of genes (such as RASSF1A and p16), we sought to improve our ability to discover differentially methylated regions (DMR) by using meDIP in parallel with gene expression profiling. We performed meDIP on DNA prepared from cell pellets made at the same time as we did the 5-azadC treatments. We confirmed successful immunoprecipitation and enrichment of methylated DNA using QPCR for several targets including the imprinted H19 locus, as well as several others. Analysis of these data suggests that thousands of loci are differentially methylated between the twelve cell lines, and that there are several hundred DMRs which differentiate between Tarceva sensitive and resistant NSCLC lines. While there was overlap between the meDIP and gene expression data, there were many genes that appeared only on one or the other platform. However, we confirmed that both approaches identify loci that are differentially methylated by direct sodium bisulfite sequencing between the sensitive and resistant cell lines. Subsequent validation of the DMRs in a broader panel of NSCLC lines shows that our approach has successfully identified markers which differentiate between Tarceva sensitive and resistant cell lines. Future studies will determine whether these DMRs can distinguish between tumors that respond to erlotnib and those that are resistant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-221.

Hypermethylation and transcriptional downregulation of the TIMP3 gene is associated with allelic loss on 22q12.3 and malignancy in meningiomas.

The gene for the tissue inhibitor of metalloproteinase 3 (TIMP3) on 22q12.3 had been reported to be inactivated by promoter methylation in various types of cancers, with controversial findings in meningiomas. We performed direct sodium bisulfite sequencing in a series of 50 meningiomas, including 27 benign meningiomas [World Health Organization (WHO) grade I], 11 atypical meningiomas (WHO grade II) and 12 anaplastic meningiomas (WHO grade III), and found hypermethylation of TIMP3 in 67% of anaplastic meningiomas, but only 22% of atypical and 17% of benign meningiomas. Moreover, TIMP3 methylation scores were significantly inversely correlated with TIMP3 mRNA expression levels (P = 0.0123), and treatment of the meningioma cell line Ben-Men-1 with demethylating agents induced an increased TIMP3 mRNA expression. TIMP3 is located in the chromosomal band 22q12, the allelic loss of which occurs early in meningioma tumorigenesis and preferentially targets the NF2 tumor suppressor gene. In our tumor panel, all meningiomas with TIMP3 hypermethylation--except for a single case--exhibited allelic losses on 22q12.3. Thus, TIMP3 inactivation by methylation seems fairly exclusive to meningiomas with allelic losses on 22q12 but--in contrast to NF2 mutation--appears to be involved in meningioma progression as it is associated with a more aggressive, high-grade meningioma phenotype.

Specific CpG Dinucleotides Within Genes Encoding Key Proteins of Steroid Biosynthesis Show Tissue-Specific Methylation Levels That Negatively Correlate with Transcript Abundance in Estrogen-Active Dominant and Pre-Ovulatory Bovine Follicles.

The profound morphological reorganization of the somatic cell layers, granulosa and theca, during the transformation from the follicular to luteal stage, is preceded by a well orchestrated modulation of cell type-specific gene expression. In vertebrates, methylation of cytosines in the context of CpG dinucleotides plays an important role in the local organisation of the chromatin and thus the long-term regulation of gene expression via epigenetic mechanisms. The goal of this study was to elucidate possible epigenetic mechanisms behind the LH induced transition from the large dominant estrogen-active follicle to the preovulatory follicle, which is an intermediate stage towards full luteinization. For this, the methylation status and transcript abundance of key genes of steroid biosynthesis, Star encoding steroidogenic acute regulatory protein, Cyp11a1 encoding cytochrome P450 cholesterol side-chain cleavage enzyme, Hsd3b encoding 3-beta-hydroxysteroid dehydrogenase, Cyp17 encoding steroid 17-alpha-hydroxylase, Cyp19 encoding aromatase cytochrome P450 were measured by bisulfite direct sequencing and real-time PCR, respectively, in the granulosa and theca of large dominant and preovulatory bovine follicles. The abundance of several other key transcripts was also quantified: Fshr transcripts, encoding follicle stimulating hormone receptor, Lhr transcripts, encoding LH receptor, and Ghr transcripts, encoding growth hormone receptor. Pcna transcripts encoding the proliferating cell nuclear antigen, were measured as a marker for proliferation activity. The appearance of Ptgs2 transcripts, encoding COX-2 in the granulosa was used as an indicator for the start of prostaglandin biosynthesis prior to ovulation. Gapdh transcripts, encoding glyceraldehyde 3-phosphate dehydrogenase, were measured as a housekeeping control to assess the fundamental metabolic activity of cells. The steroid hormones progesterone and 17-beta-estradiol were monitored to distinguish estrogen-active and estrogen-inactive follicles. Expression analysis revealed that (i) independently of the follicular stage, the gene expression profile was very different in granulosa and theca; (ii) the abundance of several key transcripts was significantly lower in estrogen-inactive, compared with estrogen-active, dominant follicles; (iii) in the granulosa of preovulatory follicles, transcripts encoding steroidogenic enzymes and hormone receptors were largely down regulated, whereas (iv) progesterone and estradiol were found at high concentrations in the follicular fluid. Methylation analysis revealed that (v) neighboring CpGs can be methylated at very different levels; (vi) specific CpGs within the Hsd3b, Cyp11a1, and particularly within the Cyp19 gene show different methylation levels in granulosa and theca that negatively correlate with the respective transcript abundance levels; (vii) methylation levels of CpGs investigated did not significantly change during the transition from the estrogen-active dominant to preovulatory follicular stage. Collectively, our data show that preovulatory follicles have a transient and unique gene expression profile and are clearly different from both the preceding and subsequent (follicular and luteal, respectively) stages. The methylation data show site and tissue-specific, but not differentiation-specific methylation levels and suggest that the methylation of specific CpGs, particularly within Cyp19, precludes transcription. (poster)

Efficient molecular screening of Lynch syndrome by specific 3' promoter methylation of the MLH1 or BRAF mutation in colorectal cancer with high-frequency microsatellite instability

It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes. Thus, some practical and efficient screening strategy to select highly possible Lynch syndrome patients is exceedingly desirable. We performed a comprehensive study to evaluate the methylation status of whole MLH1 promoter region by direct bisulfite sequencing of the entire MLH1 promoter regions on Lynch and non-Lynch colorectal cancers (CRCs). Then, we established a convenient assay to detect methylation in key CpG islands responsible for the silencing of MLH1 expression. We studied the methylation status of MLH1 as well as the CpG island methylator phenotype (CIMP) and immunohistochemical analysis of mismatch repair proteins on 16 cases of Lynch CRC and 19 cases of sporadic CRCs with high-frequency microsatellite instability (MSI-H). Sensitivity to detect Lynch syndrome by MLH1 (CCAAT) methylation was 88% and the specificity was 84%. Positive likelihood ratio (PLR) was 5.5 and negative likelihood ratio (NLR) was 0.15. Sensitivity by mutational analysis of BRAF was 100%, specificity was 84%, PLR was 6.3 and NLR was zero. By CIMP analysis; sensitivity was 88%, specificity was 79%, PLR was 4.2, and NLR was 0.16. BRAF mutation or MLH1 methylation analysis combined with MSI testing could be a good alternative to screen Lynch syndrome patients in a cost effective manner. Although the assay for CIMP status also showed acceptable sensitivity and specificity, it may not be practical because of its rather complicated assay.

Quantitative correlation between promoter methylation and messenger RNA levels of the reduced folate carrier

BackgroundMethotrexate (MTX) uptake is mediated by the reduced folate carrier (RFC). Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied.MethodsIn the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8), including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines.ResultsA partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p < 0.05) was identified between the promoter methylation and RFC mRNA levels in this a panel of malignant cell lines.ConclusionThis study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.

DNA Methylation of Placenta-specific Cyp19 Promoters of Cattle and Sheep

Locally produced estrogens are important paracrine regulators of placental growth and differentiation in cattle and sheep. The key enzyme of estrogen biosynthesis, aromatase cytochrome P450, is encoded by CYP19. Transcription of this gene in fetal cotyledons is the first regulatory step of placental estrogen production. The aim of the present study was to comparatively investigate if epigenetic mechanisms as tissue- and differentiation-specific DNA methylation might be involved in the regulation of placental CYP19 expression. Earlier investigations demonstrated that cattle and sheep use different promoters, P1.1 and P1.5 for placental CYP19 expression, respectively. During the present investigation, methylation of individual CpG dinucleotides within these promoter regions was analyzed with bisulfite direct sequencing in placental and luteal tissue. Transcript abundance in sheep was determined with qPCR. Both promoters contain only few CpGs and can therefore be classified as "low CpG densitiy regions". The average methylation levels of both promoter regions were significantly reduced in cotyledons compared to caruncles and corpus luteum of both species, which inversely coincides with high and low expression levels, respectively. It was evident however that even neighbouring CpGs can show very different, individual methylation levels. From the data we conclude that (1) CYP19 promoters are differentially methylated in ovine and bovine placental tissues of foetal and maternal origin; (2) DNA methylation is suggestively involved in the regulation of CYP19 expression; (3) the DNA methylation status on its own is not sufficient for the selection of tissue-and species-specific CYP19 promoters.