Identification of New Differentially Methylated Genes That Have Potential Functional Consequences in Prostate Cancer

Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.     

Difference Analysis of ClCYC2-Like Genes Expression and DNA Methylation Between the Two Types of Florets in Chrysanthemum lavandulifolium

DNA methylation is an important epigenetic modification, that is involved in the regulation of gene expression and cell differentiation, and plays an important regulatory role in flower development in higher plants. There are two types of florets on the capitulum in the genus Chrysanthemum, the flower symmetry factor CYCLOIDEA (CYC) 2-like genes may be important candidate genes for determining the identity of the two types of florets. In this study, the diploid plant Chrysanthemum lavandulifolium was used as the research material, and qRT-PCR and bisulfite sequencing polymerase chain reaction (BSP) were used to identify the expression and DNA methylation pattern of CYC2-like genes in the two types of florets. Gene expression analysis showed that the six ClCYC2-like genes were significantly different in the two types of florets, and the expression levels of ClCYC2c, ClCYC2d, ClCYC2e and ClCYC2f in the ray florets were significantly higher than those in the disc florets. For the DNA methylation analysis of the three genes ClCYC2c, ClCYC2d, and ClCYC2e, it was found that the DNA methylation levels of these three genes were negative correlated with their expression levels, and the ways in which the three genes were regulated by the DNA methylation were different. It is speculated that the different DNA methylation of ClCYC2-like genes in the two types of florets may affect the differentiation and development of the two types of florets. This study provides new clues about epigenetics for the analysis of capitulum formation in Asteraceae.

     

Cytosine methylation at CG and CNG sites is differential during the development of triploid black poplar

It has been widely shown that polyploidization can result in changes in cytosine methylation. However, little is known regarding how cytosine methylation changes in polyploids development, especially in polyploid trees. In this study, we investigated drifting changes of DNA methylation status at 5′-CCGG sites in the apical bud, young and mature leaf tissues of triploid black poplar (Populus. euramericana) with methylation-sensitive amplification polymorphism (MSAP) and assessed the expression of multiple DNA methyltransferases (MTases) and DNA demethylase during different developmental stages. MSAP analysis detected methylation levels at CG and CNG sites of diploid tissues reduced during development from bud to leaves, while for the triploid, methylation at CNG sites increased during development, but levels of methylation at CG sites first decreased in young leaves before increasing in mature leaves. MTase genes related to CG or CNG methylation were respectively preferential in different triploid tissues with high CG or CNG methylation levels. High expression of DNA demethylase was observed in tissue with high demethylation trends. These finding suggest CG and CNG methylation and their related enzymes are involved with different biological functions and networks of gene regulation in different developmental stages of triploid.     

CNAmet: an R package for integrating copy number, methylation and expression data

SUMMARY: Gene copy number and DNA methylation alterations are key regulators of gene expression in cancer. Accordingly, genes that show simultaneous methylation, copy number and expression alterations are likely to have a key role in tumor progression. We have implemented a novel software package (CNAmet) for integrative analysis of high-throughput copy number, DNA methylation and gene expression data. To demonstrate the utility of CNAmet, we use copy number, DNA methylation and gene expression data from 50 glioblastoma multiforme and 188 ovarian cancer primary tumor samples. Our results reveal a synergistic effect of DNA methylation and copy number alterations on gene expression for several known oncogenes as well as novel candidate oncogenes. AVAILABILITY: CNAmet R-package and user guide are freely available under GNU General Public License at http://csbi.ltdk.helsinki.fi/CNAmet.     

The role of DMDs in the maintenance of epigenetic states

An important aspect of genome reprogramming is the establishment and maintenance of gamete-specific DNA methylation patterns that distinguish the parental alleles of imprinted genes. Disrupting the accurate transmission of genomic imprints by interfering with these methylation patterns causes severe defects in fetal growth and development. The inheritance of sex-specific DNA methylation patterns from both parents is thus a fundamental molecular definition of genomic imprinting. The other cardinal aspect is the regulation of imprinted gene expression over a long genomic distance, spanning a few clustered imprinted genes. There is converging experimental evidence that differentially methylated domains (DMDs), located in non-coding regions of imprinted genes, are involved in both processes. As such, DMDs are the imprinting backbone upon which the fundamental processes of sex-specific methylation and imprinted gene expression are built.     

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5'' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.     

Gene and enhancer trap tagging of vascular-expressed genes in poplar trees

We report a gene discovery system for poplar trees based on gene and enhancer traps. Gene and enhancer trap vectors carrying the beta-glucuronidase (GUS) reporter gene were inserted into the poplar genome via Agrobacterium tumefaciens transformation, where they reveal the expression pattern of genes at or near the insertion sites. Because GUS expression phenotypes are dominant and are scored in primary transformants, this system does not require rounds of sexual recombination, a typical barrier to developmental genetic studies in trees. Gene and enhancer trap lines defining genes expressed during primary and secondary vascular development were identified and characterized. Collectively, the vascular gene expression patterns revealed that approximately 40% of genes expressed in leaves were expressed exclusively in the veins, indicating that a large set of genes is required for vascular development and function. Also, significant overlap was found between the sets of genes responsible for development and function of secondary vascular tissues of stems and primary vascular tissues in other organs of the plant, likely reflecting the common evolutionary origin of these tissues. Chromosomal DNA flanking insertion sites was amplified by thermal asymmetric interlaced PCR and sequenced and used to identify insertion sites by reference to the nascent Populus trichocarpa genome sequence. Extension of the system was demonstrated through isolation of full-length cDNAs for five genes of interest, including a new class of vascular-expressed gene tagged by enhancer trap line cET-1-pop1-145. Poplar gene and enhancer traps provide a new resource that allows plant biologists to directly reference the poplar genome sequence and identify novel genes of interest in forest biology.     

Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

Background

A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.

Results

Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.

Conclusions

We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.     

癌症DNA甲基化调控位点的识别

DNA甲基化是一种重要的表观遗传学修饰,在基因的转录调控方面具有重要的作用。异常的DNA甲基化可以导致癌症等复杂疾病发生,癌基因相关的DNA甲基化调控位点的识别对于解析癌症的发生发展机制及识别新的癌症标记具有重要意义。本研究通过整合The Cancer Genome Atlas(TCGA)的泛癌症基因组的高通量甲基化谱和基因表达谱,识别癌基因相关的DNA甲基化调控位点。对于每种癌症分批次计算Cp G位点甲基化与相关基因表达之间的相关性,并筛选调控下游基因的Cp G位点(包括强调控位点、弱调控位点和不调控位点),结果表明仅有一半的Cp G位点对下游基因具有调控作用;对癌症间共享的调控位点的分析发现不同癌症间共享的调控位点不尽相同,表明癌症特异的甲基化调控位点的存在。进一步地,对差异甲基化和差异表达基因的功能富集分析揭示了受甲基化调控的基因确实参与了癌症发生发展相关的功能。本研究的结果是对当前甲基化调控位点集的重要补充,也是识别癌症新型分子标记特征的重要资源。     

Integrated epigenome profiling of repressive histone modifications, DNA methylation and gene expression in normal and malignant urothelial cells

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20-30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5-10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer.     

Identification of tumor suppressors and oncogenes from genomic and epigenetic features in ovarian cancer

The identification of genetic and epigenetic alterations from primary tumor cells has become a common method to identify genes critical to the development and progression of cancer. We seek to identify those genetic and epigenetic aberrations that have the most impact on gene function within the tumor. First, we perform a bioinformatic analysis of copy number variation (CNV) and DNA methylation covering the genetic landscape of ovarian cancer tumor cells. We separately examined CNV and DNA methylation for 42 primary serous ovarian cancer samples using MOMA-ROMA assays and 379 tumor samples analyzed by The Cancer Genome Atlas. We have identified 346 genes with significant deletions or amplifications among the tumor samples. Utilizing associated gene expression data we predict 156 genes with altered copy number and correlated changes in expression. Among these genes CCNE1, POP4, UQCRB, PHF20L1 and C19orf2 were identified within both data sets. We were specifically interested in copy number variation as our base genomic property in the prediction of tumor suppressors and oncogenes in the altered ovarian tumor. We therefore identify changes in DNA methylation and expression for all amplified and deleted genes. We statistically define tumor suppressor and oncogenic features for these modalities and perform a correlation analysis with expression. We predicted 611 potential oncogenes and tumor suppressors candidates by integrating these data types. Genes with a strong correlation for methylation dependent expression changes exhibited at varying copy number aberrations include CDCA8, ATAD2, CDKN2A, RAB25, AURKA, BOP1 and EIF2C3. We provide copy number variation and DNA methylation analysis for over 11,500 individual genes covering the genetic landscape of ovarian cancer tumors. We show the extent of genomic and epigenetic alterations for known tumor suppressors and oncogenes and also use these defined features to identify potential ovarian cancer gene candidates.     

Hormonally mediated epigenetic changes to steroid receptors in the developing brain: Implications for sexual differentiation

The establishment of sex-specific neural morphology, which underlies sex-specific behaviors, occurs during a perinatal sensitive window in which brief exposure to gonadal steroid hormones produces permanent masculinization of the brain. In the rodent, estradiol derived from testicular androgens is a principal organizational hormone. The mechanism by which transient estradiol exposure induces permanent differences in neuronal anatomy has been widely investigated, but remains elusive. Epigenetic changes, such as DNA methylation, allow environmental influences to alter long-term gene expression patterns and therefore may be a potential mediator of estradiol-induced organization of the neonatal brain. Here we review data that demonstrate sex and estradiol-induced differences in DNA methylation on the estrogen receptor α (ERα), estrogen receptor β (ERβ), and progesterone receptor (PR) promoters in sexually dimorphic brain regions across development. Contrary to the overarching view of DNA methylation as a permanent modification directly tied to gene expression, these data demonstrate that methylation patterns on steroid hormone receptors change across the life span and do not necessarily predict expression. Although further exploration into the mechanism and significance of estradiol-induced alterations in DNA methylation patterns in the neonatal brain is necessary, these results provide preliminary evidence that epigenetic alterations can occur in response to early hormone exposure and may mediate estradiol-induced organization of sex differences in the neonatal brain.     

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.)

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

Cell specific patterns of methylation in the human placenta

Epigenetic processes, such as DNA methylation, are known to regulate tissue specific gene expression. We explored this concept in the placenta to define whether DNA methylation is cell-type specific. Cytotrophoblasts and fibroblasts were isolated from normal midtrimester placentas. Using immunocytochemistry, we demonstrated 95% purity for cytotrophoblasts and 60-70% for fibroblasts. We compared DNA methylation profiles from cytotrophoblasts, fibroblasts and whole placental villi using bisulfite modified genomic DNA hybridized to the Illumina Methylation27 array. Euclidean cluster analysis of the DNA methylation profiles showed 2 main clusters, one containing cytotrophoblasts and placenta, the other fibroblasts. Differential methylation analysis identified 442 autosomal CpG sites that differed between cytotrophoblasts and fibroblasts, 315 between placenta and fibroblasts and 61 between placenta and cytotrophoblasts. Three candidate methylation differences were validated by targeted pyrosequencing assays. Pyrosequencing assays were developed for CpG sites less methylated in cytotrophoblasts than fibroblasts mapping to the promoter region of the beta subunit of human chorionic gonadotropin 5 (CGB5), as well as 2 CpG sites mapping to each of 2 tumor suppressor genes. Our data suggest that epigenetic regulation of gene expression is likely to be a key factor in the functional specificity of cytotrophoblasts. These data are proof of principle for cell-type specific epigenetic regulation in placenta and demonstrate that the methylation profile of placenta is mainly driven by cytotrophoblasts.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.