Tissue-specific patterns of allelically-skewed DNA methylation

While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ～220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood.     

Validation of a DNA methylation reference panel for the estimation of nucleated cells types in cord blood

Cord blood is widely used as surrogate tissue in epigenome-wide association studies of prenatal conditions. Cell type composition variation across samples can be an important confounder of epigenome-wide association studies in blood that constitute a mixture of cells. We evaluated a newly developed cord blood reference panel to impute cell type composition from DNA methylation levels, including nucleated red blood cells (nRBCs). We estimated cell type composition from 154 unique cord blood samples with available DNA methylation data as well as direct measurements of nucleated cell types. We observed high correlations between the estimated and measured composition for nRBCs (r = 0.92, R² = 0.85), lymphocytes (r = 0.77, R² = 0.58), and granulocytes (r = 0.72, R² = 0.52), and a moderate correlation for monocytes (r = 0.51, R² = 0.25) as well as relatively low root mean square errors from the residuals ranging from 1.4 to 5.4%. These results validate the use of the cord blood reference panel and highlight its utility and limitations for epidemiological studies.     

Key epigenetic changes associated with lung cancer development: Results from dense methylation array profiling

Epigenetic alterations are a common event in lung cancer and their identification can serve to inform on the carcinogenic process and provide clinically relevant biomarkers. Using paired tumor and non-tumor lung tissues from 146 individuals from three independent populations we sought to identify common changes in DNA methylation associated with the development of non-small cell lung cancer. Pathologically normal lung tissue taken at the time of cancer resection was matched to tumorous lung tissue and together were probed for methylation using Illumina GoldenGate arrays in the discovery set (n = 47 pairs) followed by bisulfite pyrosequencing for validation sets (n = 99 pairs). For each matched pair the change in methylation at each CpG was calculated (the odds ratio), and these ratios were averaged across individuals and ranked by magnitude to identify the CpGs with the greatest change in methylation associated with tumor development. We identified the top gene-loci representing an increase in methylation (HOXA9, 10.3-fold and SOX1, 5.9-fold) and decrease in methylation (DDR1, 8.1-fold). In replication testing sets, methylation was higher in tumors for HOXA9 (p < 2.2 × 10⁻¹⁶) and SOX1 (p < 2.2 × 10⁻¹⁶) and lower for DDR1 (p < 2.2 × 10⁻¹⁶). The magnitude and strength of these changes were consistent across squamous cell and adenocarcinoma tumors. Our data indicate that the identified genes consistently have altered methylation in lung tumors. Our identified genes should be included in translational studies that aim to develop screening for early disease detection.     

Leukocyte DNA as Surrogate for the Evaluation of Imprinted Loci Methylation in Mammary Tissue DNA

There is growing interest in identifying surrogate tissues to identify epimutations in cancer patients since primary target tissues are often difficult to obtain. Methylation patterns at imprinted loci are established during gametogenesis and post fertilization and their alterations have been associated with elevated risk of cancer. Methylation at several imprinted differentially methylated regions (GRB10 ICR, H19 ICR, KvDMR, SNRPN/SNURF ICR, IGF2 DMR0, and IGF2 DMR2) were analyzed in DNA from leukocytes and mammary tissue (normal, benign diseases, or malignant tumors) from 87 women with and without breast cancer (average age of cancer patients: 53; range: 31–77). Correlations between genomic variants and DNA methylation at the studied loci could not be assessed, making it impossible to exclude such effects. Methylation levels observed in leukocyte and mammary tissue DNA were close to the 50% expected for monoallellic methylation. While no correlation was observed between leukocyte and mammary tissue DNA methylation for most of the analyzed imprinted genes, Spearman''s correlations were statistically significant for IGF2 DMR0 and IGF2 DMR2, although absolute methylation levels differed. Leukocyte DNA methylation levels of selected imprinted genes may therefore serve as surrogate markers of DNA methylation in cancer tissue.     

Functional annotation of the human brain methylome identifies tissue-specific epigenetic variation across brain and blood

Background

Dynamic changes to the epigenome play a critical role in establishing and maintaining cellular phenotype during differentiation, but little is known about the normal methylomic differences that occur between functionally distinct areas of the brain. We characterized intra- and inter-individual methylomic variation across whole blood and multiple regions of the brain from multiple donors.

Results

Distinct tissue-specific patterns of DNA methylation were identified, with a highly significant over-representation of tissue-specific differentially methylated regions (TS-DMRs) observed at intragenic CpG islands and low CG density promoters. A large proportion of TS-DMRs were located near genes that are differentially expressed across brain regions. TS-DMRs were significantly enriched near genes involved in functional pathways related to neurodevelopment and neuronal differentiation, including BDNF, BMP4, CACNA1A, CACA1AF, EOMES, NGFR, NUMBL, PCDH9, SLIT1, SLITRK1 and SHANK3. Although between-tissue variation in DNA methylation was found to greatly exceed between-individual differences within any one tissue, we found that some inter-individual variation was reflected across brain and blood, indicating that peripheral tissues may have some utility in epidemiological studies of complex neurobiological phenotypes.

Conclusions

This study reinforces the importance of DNA methylation in regulating cellular phenotype across tissues, and highlights genomic patterns of epigenetic variation across functionally distinct regions of the brain, providing a resource for the epigenetics and neuroscience research communities.     

DNA methylation age of human tissues and cell types

Background

It is not yet known whether DNA methylation levels can be used to accurately predict age across a broad spectrum of human tissues and cell types, nor whether the resulting age prediction is a biologically meaningful measure.

Results

I developed a multi-tissue predictor of age that allows one to estimate the DNA methylation age of most tissues and cell types. The predictor, which is freely available, was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. I found that DNA methylation age has the following properties: first, it is close to zero for embryonic and induced pluripotent stem cells; second, it correlates with cell passage number; third, it gives rise to a highly heritable measure of age acceleration; and, fourth, it is applicable to chimpanzee tissues. Analysis of 6,000 cancer samples from 32 datasets showed that all of the considered 20 cancer types exhibit significant age acceleration, with an average of 36 years. Low age-acceleration of cancer tissue is associated with a high number of somatic mutations and TP53 mutations, while mutations in steroid receptors greatly accelerate DNA methylation age in breast cancer. Finally, I characterize the 353 CpG sites that together form an aging clock in terms of chromatin states and tissue variance.

Conclusions

I propose that DNA methylation age measures the cumulative effect of an epigenetic maintenance system. This novel epigenetic clock can be used to address a host of questions in developmental biology, cancer and aging research.     

Epigenome-wide profiling of DNA methylation in paired samples of adipose tissue and blood

Many epigenetic association studies have attempted to identify DNA methylation markers in blood that are able to mirror those in target tissues. Although some have suggested potential utility of surrogate epigenetic markers in blood, few studies have collected data to directly compare DNA methylation across tissues from the same individuals. Here, epigenomic data were collected from adipose tissue and blood in 143 subjects using Illumina HumanMethylation450 BeadChip array. The top axis of epigenome-wide variation differentiates adipose tissue from blood, which is confirmed internally using cross-validation and externally with independent data from the two tissues. We identified 1,285 discordant genes and 1,961 concordant genes between blood and adipose tissue. RNA expression data of the two classes of genes show consistent patterns with those observed in DNA methylation. The discordant genes are enriched in biological functions related to immune response, leukocyte activation or differentiation, and blood coagulation. We distinguish the CpG-specific correlation from the within-subject correlation and emphasize that the magnitude of within-subject correlation does not guarantee the utility of surrogate epigenetic markers. The study reinforces the critical role of DNA methylation in regulating gene expression and cellular phenotypes across tissues, and highlights the caveats of using methylation markers in blood to mirror the corresponding profile in the target tissue.     

An expression microarray approach for the identification of metastable epialleles in the mouse genome

Genetic loci displaying environmentally responsive epigenetic marks, termed metastable epialleles, offer a solution to the paradox presented by genetically identical yet phenotypically distinct individuals. The murine viable yellow agouti (A^vy) metastable epiallele exhibits stochastic DNA methylation and histone modifications associated with coat color variation in isogenic individuals. The distribution of A^vy variable expressivity shifts following maternal nutritional and environmental exposures. To characterize additional murine metastable epialleles, we utilized genome-wide expression arrays (N = 10 male individuals, 3 tissues per individual) and identified candidates displaying large variability in gene expression among individuals (V_i = inter-individual variance), concomitant with a low variability in gene expression across tissues from the three germ layers (V_t = inter-tissue variance), two features characteristic of the A^vy metastable epiallele. The CpG island in the promoter of Dnajb1 and two contraoriented ERV class II repeats in Glcci1 were validated to display underlying stochasticity in methylation patterns common to metastable epialleles. Furthermore, liver DNA methylation in mice exposed in utero to 50 mg bisphenol A (BPA)/kg diet (N = 91) or a control diet (N = 79) confirmed environmental lability at validated candidate genes. Significant effects of exposure on mean CpG methylation were observed at the Glcci1 Repeat 1 locus (p < 0.0001). Significant effects of BPA also were observed at the first and fifth CpG sites studied in Glcci1 Repeat 2 (p < 0.0001 and p = 0.004, respectively). BPA did not affect methylation in the promoter of Dnajb1 (p = 0.59). The characterization of metastable epialleles in humans is crucial for the development of novel screening and therapeutic targets for human disease prevention.Key words: epigenetics, metastable epiallele, viable yellow agouti, environmental epigenomics     

Comparative analysis of DNA methylation profiles in peripheral blood leukocytes versus lymphoblastoid cell lines

Previous reports have shown that DNA methylation profiles within primary human malignant tissues are altered when these cells are transformed into cancer cell lines. However, it is unclear if similar differences in DNA methylation profiles exist between DNA derived from peripheral blood leukocytes (PBLs) and corresponding Epstein-Barr Virus transformed lymphoblastoid cell lines (LCLs). To assess the utility of LCLs as a resource for methylation studies we have compared DNA methylation profiles in promoter and 5/ regions of 318 genes in PBL and LCL sample pairs from patients with type 1 diabetes with or without nephropathy. We identified a total of 27 (~8%) genes that revealed different DNA methylation profiles in PBL compared with LCL-derived DNA samples. In conclusion, although the profiles for most promoter regions were similar between PBL-LCL pairs, our results indicate that LCL-derived DNA may not be suitable for DNA methylation studies at least in diabetic nephropathy.     

Fetal lung and placental methylation is associated with in utero nicotine exposure

In utero smoke exposure has been shown to have detrimental effects on lung function and to be associated with persistent wheezing and asthma in children. One potential mechanism of IUS effects could be alterations in DNA methylation, which may have life-long implications. The goal of this study was to examine the association between DNA methylation and nicotine exposure in fetal lung and placental tissue in early development; nicotine exposure in this analysis represents a likely surrogate for in-utero smoke. We performed an epigenome-wide analysis of DNA methylation in fetal lung tissue (n = 85, 41 smoke exposed (48%), 44 controls) and the corresponding placental tissue samples (n = 80, 39 smoke exposed (49%), 41 controls) using the Illumina HumanMethylation450 BeadChip array. Differential methylation analyses were conducted to evaluate the variation associated with nicotine exposure. The most significant CpG sites in the fetal lung analysis mapped to the PKP3 (P = 2.94 × 10⁻⁰³), ANKRD33B (P = 3.12 × 10⁻⁰³), CNTD2 (P = 4.9 × 10⁻⁰³) and DPP10 (P = 5.43 × 10⁻⁰³) genes. In the placental methylome, the most significant CpG sites mapped to the GTF2H2C and GTF2H2D genes (P = 2.87 × 10⁻⁰⁶ − 3.48 × 10⁻⁰⁵). One hundred and one unique CpG sites with P-values < 0.05 were concordant between lung and placental tissue analyses. Gene Set Enrichment Analysis demonstrated enrichment of specific disorders, such as asthma and immune disorders. Our findings demonstrate an association between in utero nicotine exposure and variable DNA methylation in fetal lung and placental tissues, suggesting a role for DNA methylation variation in the fetal origins of chronic diseases.     

Methylation status and protein expression of RASSF1A in breast cancer patients

Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.     

Plasticity of DNA methylation in a nerve injury model of pain

The response of the peripheral nervous system (PNS) to injury may go together with alterations in epigenetics, a conjecture that has not been subjected to a comprehensive, genome-wide test. Using reduced representation bisulfite sequencing, we report widespread remodeling of DNA methylation in the rat dorsal root ganglion (DRG) occurring within 24 h of peripheral nerve ligation, a neuropathy model of allodynia. Significant (P < 10⁻⁴) cytosine hyper- and hypo-methylation was found at thousands of CpG sites. Remodeling occurred outside of CpG islands. Changes affected genes with known roles in the PNS, yet methylome remodeling also involved genes that were not linked to neuroplasticity by prior evidence. Consistent with emerging models relying on genome-wide methylation and RNA-seq analysis of promoter regions and gene bodies, variation of methylation was not tightly linked with variation of gene expression. Furthermore, approximately 44% of the dynamically changed CpGs were located outside of genes. We compared their positions with the intergenic, tissue-specific differentially methylated CpGs (tDMCs) of an independent experimental set consisting of liver, spleen, L4 control DRG, and muscle. Dynamic changes affected those intergenic CpGs that were different between tissues (P < 10⁻¹⁵) and almost never the invariant portion of the methylome (those CpGs that were identical across all tissues). Our findings—obtained in mixed tissue—show that peripheral nerve injury leads to methylome remodeling in the DRG. Future studies may address which of the cell types found in the DRG, such as specific groups of neurons or non-neuronal cells are affected by which aspect of the observed methylome remodeling.     

Analysis of HLA–ABC locus-specific transcription in normal tissues

We developed a novel human leukocyte antigen HLA–ABC locus-specific quantitative real-time polymerase chain reaction (PCR) to determine the locus-specific gene expression of HLA–ABC in peripheral blood leukocytes (PBLs, n?=?53), colon mucosa (n?=?15), and larynx mucosa (n?=?15). Laser-assisted tissue microdissection allowed us to study the selected cells without interference from surrounding stroma. We report evidence on the specificity of the technique, describing the HLA–ABC locus-specific gene expression patterns found in the PBLs and two solid tissues studied. PBLs showed a higher gene expression of HLA-B than of HLA-A or HLA-C (p?=?4.7?×?10^?10 and p?=?1.6?×?10^?6, respectively). In solid tissue, HLA-A and HLA-B gene expressions were similar and HLA-C expression lower. In particular, in larynx mucosa, significant differences were found between HLA-A and HLA-C expressions and between HLA-B and HLA-C expressions (p?=?6.5?×?10^?4 and p?=?8.1?×?10^?4, respectively). The same differences were observed in colon mucosa, but significance was not reached (p?=?0.08 and p?=?0.06, respectively). Differences in locus-specific regulation may be related to the control of cytotoxic responses of NK and CD8 positive T cells. Gene expression of HLA–ABC specific locus showed no intra-individual variability, but there was a high inter-individual variability. This may result from differences in the expression of common regulatory factors that control HLA–ABC constitutive expression.     

Association between P16INK4a Promoter Methylation and Non-Small Cell Lung Cancer: A Meta-Analysis

Background

Aberrant methylation of CpG islands acquired in tumor cells in promoter regions plays an important role in carcinogenesis. Accumulated evidence demonstrates P^16INK4a gene promoter hypermethylation is involved in non-small cell lung carcinoma (NSCLC), indicating it may be a potential biomarker for this disease. The aim of this study is to evaluate the frequency of P^16INK4a gene promoter methylation between cancer tissue and autologous controls by summarizing published studies.

Methods

By searching Medline, EMBSE and CNKI databases, the open published studies about P^16INK4a gene promoter methylation and NSCLC were identified using a systematic search strategy. The pooled odds of P^16INK4A promoter methylation in lung cancer tissue versus autologous controls were calculated by meta-analysis method.

Results

Thirty-four studies, including 2 652 NSCLC patients with 5 175 samples were included in this meta-analysis. Generally, the frequency of P^16INK4A promoter methylation ranged from 17% to 80% (median 44%) in the lung cancer tissue and 0 to 80% (median 15%) in the autologous controls, which indicated the methylation frequency in cancer tissue was much higher than that in autologous samples. We also find a strong and significant correlation between tumor tissue and autologous controls of P^16INK4A promoter methylation frequency across studies (Correlation coefficient 0.71, 95% CI:0.51–0.83, P<0.0001). And the pooled odds ratio of P^16INK4A promoter methylation in cancer tissue was 3.45 (95% CI: 2.63–4.54) compared to controls under random-effect model.

Conclusion

Frequency of P^16INK4a promoter methylation in cancer tissue was much higher than that in autologous controls, indicating promoter methylation plays an important role in carcinogenesis of the NSCLC. Strong and significant correlation between tumor tissue and autologous samples of P^16INK4A promoter methylation demonstrated a promising biomarker for NSCLC.     

Key epigenetic changes associated with lung cancer development

Epigenetic alterations are a common event in lung cancer and their identification can serve to inform on the carcinogenic process and provide clinically relevant biomarkers. Using paired tumor and non-tumor lung tissues from 146 individuals from three independent populations we sought to identify common changes in DNA methylation associated with the development of non-small cell lung cancer. Pathologically normal lung tissue taken at the time of cancer resection was matched to tumorous lung tissue and together were probed for methylation using Illumina GoldenGate arrays in the discovery set (n = 47 pairs) followed by bisulfite pyrosequencing for validation sets (n = 99 pairs). For each matched pair the change in methylation at each CpG was calculated (the odds ratio), and these ratios were averaged across individuals and ranked by magnitude to identify the CpGs with the greatest change in methylation associated with tumor development. We identified the top gene-loci representing an increase in methylation (HOXA9, 10.3-fold and SOX1, 5.9-fold) and decrease in methylation (DDR1, 8.1-fold). In replication testing sets, methylation was higher in tumors for HOXA9 (p < 2.2 × 10^?16) and SOX1 (p < 2.2 × 10^?16) and lower for DDR1 (p < 2.2 × 10^?16). The magnitude and strength of these changes were consistent across squamous cell and adenocarcinoma tumors. Our data indicate that the identified genes consistently have altered methylation in lung tumors. Our identified genes should be included in translational studies that aim to develop screening for early disease detection.     

Genome-Wide Analysis of DNA Methylation in Five Tissues of Zhikong Scallop,Chlamys farreri

DNA methylation plays a vital role in tissue development and differentiation in eukaryotes. Epigenetic studies have been seldom conducted in the extremely diverse and evolutionarily highly successful bilaterian lineage Mollusca. In the present study, we conducted the genome-wide profiling of DNA methylation for five tissues of a bivalve mollusc, Chlamys farreri using the methylation-sensitive amplification polymorphism (MSAP) technique. The methylation levels were quite similar among tissues, ranging from 20.9% to 21.7%. CG methylation was the dominant type (14.9%–16.5%) in the C. farreri genome, but CHG methylation also accounted for a substantial fraction of total methylation (5.1%–6.3%). Relatively high methylation diversity was observed within tissues. Methylation differentiation between tissues was evaluated and 460 tissue-specific epiloci were identified. Kidney differs from the other tissues in DNA methylation profiles. Our study presents the first look at the tissue-specific DNA methylation patterns in a bivalve mollusc and represents an initial step towards understanding of epigenetic regulatory mechanism underlying tissue development and differentiation in bivalves.     

基于双层BiGRU网络的哺乳动物组织m6A甲基化位点预测

目的 N⁶-甲基化腺苷（N⁶-methyladenosine，m⁶A）是RNA中最常见、最丰富的化学修饰，在很多生物过程中发挥着重要作用。目前已经发展了一些预测m⁶A甲基化位点的计算方法。然而，这些方法在针对不同物种或不同组织时，缺乏稳健性。为了提升对不同组织中m⁶A甲基化位点预测的稳健性，本文提出一种能结合序列反向信息来提取数据更高级特征的双层双向门控循环单元（bidirectional gated recurrent unit，BiGRU）网络模型。方法 本文选取具有代表性的哺乳动物组织m⁶A甲基化位点数据集作为训练数据，通过对模型网络、网络结构、层数和优化器等进行搭配，构建双层BiGRU网络。结果 将模型应用于人类、小鼠和大鼠共11个组织的m⁶A甲基化位点预测上，并与其他方法在这11个组织上的预测能力进行了全面的比较。结果表明，本文构建的模型平均预测接受者操作特征曲线下面积（area under the receiver operating characteristic curve，AUC）达到93.72%，与目前最好的预测方法持平，而预测准确率（accuracy，ACC）、敏感性（sensitivity，SN）、特异性（specificity，SP）和马修斯相关系数（Matthews correlation coefficient，MCC）分别为90.07%、90.30%、89.84%和80.17%，均高于目前的m⁶A甲基化位点预测方法。结论 和已有研究方法相比，本文方法对11个哺乳动物组织的m⁶A甲基化位点的预测准确性均达到最高，说明本文方法具有较好的泛化能力。