DNA Methylation Of Repetitive Elements Research Articles

Abstract 3485: Global DNA methylation level is associated with the sensitivity of cytotoxic and senotherapeutic drugs in gastrointestinal tumors

Abstract Background: Gastrointestinal cancer has become a severe burden around the world. Marker-guided treatment and clinical trials with sensitive anti-tumor drugs are critical for improving cancer survival. The decreasing DNA methylation levels of repetitive elements, such as LINE-1 and ALU, have been identified as the common biomarkers in cancer and aging. However, it remains unclear whether the methylation levels of repetitive elements can imply the drug sensitivity in gastrointestinal cancer. We investigated the association of LINE-1 and ALU methylations with the sensitivity of cytotoxic and senotherapeutic drugs in gastric cancer (GC) and colorectal cancer (CRC). Methods: The profiles of DNA methylation and gene expression were obtained from GC and CRC patients from the TCGA cohorts. Then, we calculated the averaging methylation level of two repetitive elements (LINE-1 and ALU) to quantify the global methylation levels by using the probes targeting the repetitive elements in 450K methylation array. The sensitivity of anti-tumor drugs, including the cytotoxic (oxaliplatin and fluorouracil) and five senotherapeutic agents (Dasatinib, Nutlin-3a, Navitoclax, Rapamycin, ABT737), were inferred with Oncopredict R package based on the gene expression profile. The Pearson analysis was conducted to test the correlation between DNA methylation and drug sensitivity. Results: The ALU methylation was associated with the sensitivities of cytotoxic (oxaliplatin: r = 0.171, P = 0.001; fluorouracil: r = 0.158, P = 0.002) and senotherapeutic agents (Dasatinib: r = 0.162, P = 0.002, Nutlin-3a: r = 0.109, P = 0.037, Navitoclax: r = 0.007, P = 0.931, Rapamycin: r = 0.144, P = 0.006, ABT737: r = 0.054, P = 0.523) in GC, while the association of LINE-1 methylation with drug sensitivities was observed in less drugs with weak correlation, including the cytotoxic (oxaliplatin: r = 0.132, P = 0.013; fluorouracil: r = 0.122, P = 0.019) and senotherapeutic agents (Dasatinib: r = 0.129, P = 0.014, Nutlin-3a: r = 0.109, P = 0.037, Rapamycin: r = 0.110, P = 0.034). In CRC, there was no correlation between DNA methylation levels of repetitive elements and drug sensitivity except Dasatinib (ALU: r = -0.107, P = 0.043). Conclusion: DNA methylation of repetitive elements could serve as a biomarker for cytotoxic and senotherapeutic drugs in GC. In addition, our findings suggested the promising value of ALU and LINE-1 methylation-guided chemotherapy with senotherapeutics in GC. Keywords: global DNA methylation, repetitive elements, gastrointestinal cancer, drug sensitivity, senotherapeutics Citation Format: Kaixin Lin, Xiaolin Wang, Yanxin Luo, Huichuan Yu. Global DNA methylation level is associated with the sensitivity of cytotoxic and senotherapeutic drugs in gastrointestinal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3485.

Abstract A027: DNA methylation-based fetal niche and repetitive element profiling predict overall survival in clear cell renal cell carcinoma

Abstract Background: Fetal cell origin DNA methylation signature (FCO) estimates the proportion of cells in a mixture of cell types that are of fetal origin. Machine learning-based methods can predict DNA methylation of locus-specific repetitive elements (RE) by learning surrounding genetic and epigenetic information. In this study, we applied the FCO signature and RE methylation prediction to clear cell renal cell carcinoma (ccRCC) samples to study FCO and RE alterations in ccRCC carcinogenesis and survival. Methods: The FCO algorithm was applied to 47 ccRCC tumor samples and four adjacent normal samples from the Dartmouth Renal Tumor Biobank. 319 ccRCC tumor samples and 160 adjacent normal samples from The Cancer Genome Atlas (TCGA) were used for external validation. Linear regression models were used to compare the FCO signal between ccRCC tumor and normal samples adjusting for sex and age. Cox proportional hazard models were employed to study FCO with overall survival outcome in ccRCC samples, adjusting for sex, age, and tumor stage. The survival groups were dichotomized to FCO = 0% vs. FCO > 0%. The REMP package from Bioconductor was used to project LINE1 element methylation in 47 ccRCC samples. LINE1 median level methylation was compared by tumor grade. Cox proportional hazard models were employed to study LINE1 methylation level as a continuous variable with overall survival in ccRCC samples, adjusting for sex, age, and tumor stage. Results: The FCO signal is significantly lower in Dartmouth Renal Tumor Biobank archived ccRCC tumor samples compared to adjacent normal samples (Δ = 42.9%, p = 5.7e-11). By comparing the overall survival between FCO = 0% and FCO > 0% groups, we observed significantly worse survival outcomes in ccRCC patients with zero levels of FCO (HR: 2.46, 95% CI: 1.16, 5.18). Similar results were observed in TCGA samples. A significant decrease of FCO was found in ccRCC tumor samples compared to adjacent normal samples (Δ = 32.9%, p = 2.2e-308). A 1% increase in FCO is significantly associated with a better survival outcome (HR: 0.97, 95%CI 0.95, 0.99). For RE methylation prediction, we observed that the median LINE1 methylation level decreases with ccRCC tumor grade. A higher level of LINE1 median methylation is significantly associated with a better survival outcome (p = 0.01). Conclusion: Normal kidney samples possess a fetal niche reflected by DNA methylation, while ccRCC tumorigenesis depletes the fetal cells in normal kidneys. The depletion of FCO is associated with worse survival outcomes in ccRCC. The repetitive element LINE1 methylation level decreases with tumor grade and is related to ccRCC survival outcomes. These findings promise future investigations on the epigenetically targetable fetal niche and repetitive elements in ccRCC carcinogenesis and therapeutics. Citation Format: Ze Zhang, Lucas Salas. DNA methylation-based fetal niche and repetitive element profiling predict overall survival in clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A027.

LINE-1 and Alu methylation signatures in autism spectrum disorder and their associations with the expression of autism-related genes

Long interspersed nucleotide element-1 (LINE-1) and Alu elements are retrotransposons whose abilities cause abnormal gene expression and genomic instability. Several studies have focused on DNA methylation profiling of gene regions, but the locus-specific methylation of LINE-1 and Alu elements has not been identified in autism spectrum disorder (ASD). Here we interrogated locus- and family-specific methylation profiles of LINE-1 and Alu elements in ASD whole blood using publicly-available Illumina Infinium 450 K methylation datasets from heterogeneous ASD and ASD variants (Chromodomain Helicase DNA-binding 8 (CHD8) and 16p11.2del). Total DNA methylation of repetitive elements were notably hypomethylated exclusively in ASD with CHD8 variants. Methylation alteration in a family-specific manner including L1P, L1H, HAL, AluJ, and AluS families were observed in the heterogeneous ASD and ASD with CHD8 variants. Moreover, LINE-1 and Alu methylation within target genes is inversely related to the expression level in each ASD variant. The DNA methylation signatures of the LINE-1 and Alu elements in ASD whole blood, as well as their associations with the expression of ASD-related genes, have been identified. If confirmed in future larger studies, these findings may contribute to the identification of epigenomic biomarkers of ASD.

A unique chromatin profile defines adaptive genomic regions in a fungal plant pathogen.

Genomes store information at scales beyond the linear nucleotide sequence, which impacts genome function at the level of an individual, while influences on populations and long-term genome function remains unclear. Here, we addressed how physical and chemical DNA characteristics influence genome evolution in the plant pathogenic fungus Verticillium dahliae. We identified incomplete DNA methylation of repetitive elements, associated with specific genomic compartments originally defined as Lineage-Specific (LS) regions that contain genes involved in host adaptation. Further chromatin characterization revealed associations with features such as H3 Lys-27 methylated histones (H3K27me3) and accessible DNA. Machine learning trained on chromatin data identified twice as much LS DNA as previously recognized, which was validated through orthogonal analysis, and we propose to refer to this DNA as adaptive genomic regions. Our results provide evidence that specific chromatin profiles define adaptive genomic regions, and highlight how different epigenetic factors contribute to the organization of these regions.

Abstract B58: The role of mutant P53 in repetitive element regulation and the immune response in ovarian cancer

Abstract While therapies that activate the host immune system have shown tremendous promise for a wide variety of solid tumors, less than 10% of ovarian cancer (OC) patients have responded. Previous work from our group showed that DNA methyltransferase inhibitors (DNMTis) activate the interferon response in tumors through upregulation of repetitive elements (REs) including endogenous retroviruses (ERVs), remnants of exogenous retroviruses that integrated into the germline several million years ago. DNMTis increase ERVs in a mouse model of ovarian cancer, activating interferon signaling and recruiting CD8+ T cells to kill the tumors. Importantly, nearly all high-grade serous OCs harbor a mutation in the TP53 gene, encoding the tumor suppressor protein P53. p53 may transcriptionally repress ERVs to ensure genome stability; 30% of all p53 binding sites are in ERV-derived sequences. We treated seven human cancer cell lines, including four OC lines (3 TP53 wild-type, 4 TP53 mutant) with DNMTi and analyzed repetitive element expression (RNA-Seq), open chromatin (ATAC-Seq), and DNA methylation (MeDIP + MRE). Interestingly, RE upregulation was significantly higher in the P53 mutant cell lines. This was true for the SINE, LINE, and ERV groups of repetitive elements. The DNA methylation of these elements was not significantly different between TP53 wild-type and mutant cell lines, but the open chromatin peaks (ATAC-Seq) were significantly increased in the TP53 mutant cell lines. We thus hypothesize that wild-type TP53 may partner with DNA methylation to transcriptionally repress RE transcription. Treating TP53 wild-type cells with Nutlin-3A, which activates P53, represses transcription of some ERVs. When we overexpressed R175H or R273H mutant P53 constructs in the SKOV3 P53-null cell line and treated with DNMTi, we observed much higher upregulation of ERVs after DNMTi treatment than when we overexpressed the control wild type P53 vector. We predicted that higher ERVs would induce a higher interferon response and perhaps change the microenvironment in P53 mutant tumors. We used the ID8 MOSE mouse ovarian cancer model modified by the MacNeish laboratory to compare p53+/+ and p53-/- tumor response to DNMTi treatment. Characterization of the tumor microenvironment indicated a significantly higher frequency of lymphocytes, Treg cells, MDSCs, and myeloid cells in the p53-/- model in comparison to the p53 wild-type model. In current work, we are creating isogenic cell lines of human and mouse wild-type, mutant, and null p53 and comparing the response to DNMTi in vitro and in vivo. We will then determine whether P53 binding directly regulates repetitive elements transcriptionally, and more broadly, whether mutant P53 cells are more sensitive to DNMTi upregulation of repetitive elements and induction of an immune response. Results of this work will be particularly relevant for high-grade serous OC, which is driven by mutant P53 and is generally unresponsive to immunotherapies. Citation Format: Stephanie Gomez, James I. McDonald, Elisa Arthofer, Noor Diab, Aneil Srivastava, Paul Austin, Katherine B. Chiappinelli. The role of mutant P53 in repetitive element regulation and the immune response in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B58.

DNA methylation of individual repetitive elements in hepatitis C virus infection-induced hepatocellular carcinoma

BackgroundThe two most common repetitive elements (REs) in humans, long interspersed nuclear element-1 (LINE-1) and Alu element (Alu), have been linked to various cancers. Hepatitis C virus (HCV) may cause hepatocellular carcinoma (HCC) by suppressing host defenses, through DNA methylation that controls the mobilization of REs. We aimed to investigate the role of RE methylation in HCV-induced HCC (HCV-HCC).ResultsWe studied methylation of over 30,000 locus-specific REs across the genome in HCC, cirrhotic, and healthy liver tissues obtained by surgical resection. Relative to normal liver tissue, we observed the largest number of differentially methylated REs in HCV-HCC followed by alcohol-induced HCC (EtOH-HCC). After excluding EtOH-HCC-associated RE methylation (FDR < 0.001) and those unable to be validated in The Cancer Genome Atlas (TCGA), we identified 13 hypomethylated REs (11 LINE-1 and 2 Alu) and 2 hypermethylated REs (1 LINE-1 and 1 Alu) in HCV-HCC (FDR < 0.001). A majority of these REs were located in non-coding regions, preferentially enriched with chromatin repressive marks H3K27me3, and positively associated with gene expression (median correlation r = 0.32 across REs). We further constructed an HCV-HCC RE methylation score that distinguished HCV-HCC (lowest score), HCV-cirrhosis, and normal liver (highest score) in a dose-responsive manner (p for trend < 0.001). HCV-cirrhosis had a lower score than EtOH-cirrhosis (p = 0.038) and HCV-HCC had a lower score than EtOH-HCC in TCGA (p = 0.024).ConclusionsOur findings indicate that HCV infection is associated with loss of DNA methylation in specific REs, which could implicate molecular mechanisms in liver cancer development. If our findings are validated in larger sample sizes, methylation of these REs may be useful as an early detection biomarker for HCV-HCC and/or a target for prevention of HCC in HCV-positive individuals.

Investigation of Gene Expression and DNA Methylation From Seven Different Brain Regions of a Crab-Eating Monkey as Determined by RNA-Seq and Whole-Genome Bisulfite Sequencing.

The crab-eating monkey is widely used in biomedical research for pharmacological experiments. Epigenetic regulation in the brain regions of primates involves complex patterns of DNA methylation. Previous studies of methylated CpG-binding domains using microarray technology or peak identification of sequence reads mostly focused on developmental stages or disease, rather than normal brains. To identify correlations between gene expression and DNA methylation levels that may be related to transcriptional regulation, we generated RNA-seq and whole-genome bisulfite sequencing data from seven different brain regions from a single crab-eating monkey. We identified 92 genes whose expression levels were significantly correlated, positively or negatively, with DNA methylation levels. Among them, 11 genes exhibited brain region-specific characteristics, and their expression patterns were strongly correlated with DNA methylation level. Nine genes (SLC2A5, MCM5, DRAM1, TTC12, DHX40, COR01A, LRAT, FLVCR2, and PTER) had effects on brain and eye function and development, and two (LHX6 and MEST) were previously identified as genes in which DNA methylation levels change significantly in the promoter region and are therefore considered brain epigenetic markers. Furthermore, we characterized DNA methylation of repetitive elements at the whole genome through repeat annotation at single-base resolution. Our results reveal the diverse roles of DNA methylation at single-base resolution throughout the genome and reflect the epigenetic variations in adult brain tissues.

Effects of Physical Exercise on Endothelial Function and DNA Methylation

Essential hypertension is the leading preventable cause of death in the world. Epidemiological studies have shown that physical training can reduce blood pressure (BP), both in hypertensive and healthy individuals. Increasing evidence is emerging that DNA methylation is involved in alteration of the phenotype and of vascular function in response to environmental stimuli. We evaluated repetitive element and gene-specific DNA methylation in peripheral blood leukocytes of 68 volunteers, taken before (T0) and after (T1) a three-month intervention protocol of continuative aerobic physical exercise. DNA methylation was assessed by bisulfite-PCR and pyrosequencing. Comparing T0 and T1 measurements, we found an increase in oxygen consumption at peak of exercise (VO2peak) and a decrease in diastolic BP at rest. Exercise increased the levels of ALU and Long Interspersed Nuclear Element 1 (LINE-1) repetitive elements methylation, and of Endothelin-1 (EDN1), Inducible Nitric Oxide Synthase (NOS2), and Tumour Necrosis Factor Alpha (TNF) gene-specific methylation. VO2peak was positively associated with methylation of ALU, EDN1, NOS2, and TNF; systolic BP at rest was inversely associated with LINE-1, EDN1, and NOS2 methylation; diastolic BP was inversely associated with EDN1 and NOS2 methylation. Our findings suggest a possible role of DNA methylation for lowering systemic BP induced by the continuative aerobic physical training program.

Changes in one-carbon metabolism and DNA methylation in the hearts of mice exposed to space environment-relevant doses of oxygen ions (16O)

Cardiovascular disease constitutes an important threat to humans after space missions beyond the Earth's magnetosphere. Epigenetic alterations have an important role in the etiology and pathogenesis of cardiovascular disease. Previous research in animal models has shown that protons and 56Fe ions cause long-term changes in DNA methylation and expression of repetitive elements in the heart. However, astronauts will be exposed to a variety of ions, including the smaller fragmented products of heavy ions after they interact with the walls of the space craft. Here, we investigated the effects of 16O on the cardiac methylome and one-carbon metabolism in male C57BL/6 J mice. Left ventricles were examined 14 and 90 days after exposure to space-relevant doses of 0.1, 0.25, or 1 Gy of 16O (600 MeV/n). At 14 days, the two higher radiation doses elicited global DNA hypomethylation in the 5′-UTR of Long Interspersed Nuclear Elements 1 (LINE-1) compared to unirradiated, sham-treated mice, whereas specific LINE-1 elements exhibited hypermethylation at day 90. The pericentromeric major satellites were affected both at the DNA methylation and expression levels at the lowest radiation dose. DNA methylation was elevated, particularly after 90 days, while expression showed first a decrease followed by an increase in transcript abundance. Metabolomics analysis revealed that metabolites involved in homocysteine remethylation, central to DNA methylation, were unaffected by radiation, but the transsulfuration pathway was impacted after 90 days, with a large increase in cystathione levels at the lowest dose. In summary, we observed dynamic changes in the cardiac epigenome and metabolome three months after exposure to a single low dose of oxygen ions.

Abstract LB-235: DNA methylation of individual repetitive elements in HCV infection-induced HCC

Abstract Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. Hepatitis C virus (HCV) infection is a major risk factor for HCC in the US. HCV infection may cause cancer by suppressing host defense that controls the propagation of repetitive element (RE) sequences, including LINE-1 and Alu, the most representative REs in the human genome. DNA methylation of RE helps stabilize the genome, while reduced RE methylation, also referred as global hypomethylation, leads to genomic instability and is known as a hallmark of cancer. However, current available high-throughput technology for DNA methylation profiling only enables genomewide RE methylation study on a global scale. The role of individual RE methylation in cancer is largely understudied. In this study, using our recently developed and published machine learning algorithm to enhance the RE coverage of the Illumina methylation array platform, we examined the role of individual RE methylation in HCV-induced HCC across the entire genome. We conducted an epigenome-wide association study on 22 HCC, 20 cirrhotic, and 44 normal liver tissues. We included equal numbers of HCV-induced and alcohol-induced HCC/cirrhosis cases. The Illumina 450k array profiled 1939 REs and we computationally predicted 33184 REs; correlations between the overlapping 1541 profiled and predicted RE methylation were &amp;gt; 0.91. Sex, tumor differentiation, and tumor stage were similarly distributed across the HCV and alcohol groups. We adjusted for batch effects and used Benjamini-Hochberg adjusted false discovery rate (FDR) to account for multiple testing. Relative to normal liver tissue, we observed over twice as many differentially methylated (FDR &amp;lt; 0.05) REs in HCV-HCC compared to alcohol-HCC tissue (2911 vs 1304, 749 in common). We validated our results in The Cancer Genome Atlas (TCGA) (325 vs 129, 74 in common) with high correlation (r = 0.90). We also observed that 169 REs were significantly altered in HCV-cirrhosis tissue (compared to none in alcohol-cirrhosis tissue), of which, 17 REs in 13 genes, including PIK3CD, LY9, GCK, NLRC3, and PRKCZ, were consistently hypomethylated in both HCC and cirrhotic liver tissue induced by HCV (r = 0.87). Using TCGA RNA sequencing data, we found that these REs were located in gene body and their hypomethylation was associated with lower expression of their host genes (r &amp;gt; 0.3, p &amp;lt; 0.05). Gene Ontology Enrichment analysis showed that these genes were enriched in T-cell activation &amp; differentiation and cytokine production (FDR &amp;lt; 0.05). Our findings indicate that HCV infection has a more extensive impact on the DNA methylation landscape in RE than alcohol consumption, and that HCV infection may also induce similar hypomethylation in cirrhotic liver. Hypomethylated RE may alter local chromatin environment and disrupt expression of genes involved in immune and inflammatory responses, both of which may promote HCC development. RE hypomethylation in specific genomic locus may serve as early detection biomarker of HCC in patients with HCV-induced cirrhosis. Citation Format: Yinan Zheng, Jimmy Ren, Ryan A. Hlady, Keith D. Robertson, Robert L. Murphy, Lewis R. Roberts, Lifang Hou. DNA methylation of individual repetitive elements in HCV infection-induced HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-235.

Prenatal phthalate exposure, birth outcomes and DNA methylation of Alu and LINE-1 repetitive elements: A pilot study in China

BackgroundEpigenetic mechanisms, such as altered DNA methylation, may participate in the relationship between prenatal phthalate exposure and adverse birth outcomes. ObjectiveTo explore the mediation effect of DNA methylation in the associations of phthalate exposure before delivery with birth outcomes in a Chinese cohort. MethodsEight phthalate metabolites in maternal urine before delivery and DNA methylation of Alu and long interspersed nucleotide elements (LINE-1) in cord blood were determined among 106 mother-infant pairs. General additive models were used to assess the associations of maternal urinary phthalate metabolites with birth outcomes and DNA methylation; the mediating role of DNA methylation in cord blood was evaluated by mediation analysis. ResultsWe found sex-specific associations between prenatal phthalate exposure and birth outcomes and DNA methylation of cord blood. For example, the molar sum of di-2-(ethylhexyl) phthalate (∑DEHPm) metabolites in maternal urine was positively associated with gestational age among male newborns only (P < 0.05); maternal urinary monobenzyl phthalate (MBzP) was negatively associated with Alu methylation among female newborns only (P < 0.05). Mediation analysis did not find that methylation of Alu and LINE-1 to be a direct mediator in the relationships between maternal urinary phthalate metabolites before delivery and birth outcomes. ConclusionPrenatal exposure to certain phthalates was associated with altered birth outcomes and decreased repetitive element methylation of newborns. However, the altered birth outcomes exerted by prenatal phthalate exposure does not seem to be directly mediated through repetitive element methylation in cord blood.

Repetitive Element DNA Methylation is Associated with Menopausal Age.

To investigate associations between the age of menopause and the DNA methylation levels of two repetitive elements, Alu and LINE-1, we performed plasma DNA extraction on 161 subjects and serum cell-free DNA extraction on 120 subjects. We grouped women by menopausal age as follows: ≤ 48 years (earlier menopause), ≥ 52 years (later menopause), and 48-52 years (control). The DNA methylation levels of Alu and LINE-1 were measured by MethyLight PCR. The results showed that the DNA methylation levels of both Alu and LINE-1 were inversely correlated with menopausal age in the plasma DNA cohort (r = 0.079, P < 0.001 for Alu; r = 0.045, P = 0.007 for LINE-1) as well as in the serum DNA cohort (r = 0.087, P = 0.001 for Alu; r = 0.041, P = 0.026 for LINE-1). Alu methylation levels in both the plasma and serum DNA cohorts and LINE-1 methylation levels in the plasma cohort were remarkably higher in the earlier menopause group than in the later menopause and control groups (P < 0.01 and P < 0.05, respectively). In the serum DNA cohort, the LINE-1 methylation levels in the later menopause group were significantly lower than that in the earlier menopause group and control group (P < 0.05). Therefore, methylation levels of Alu and LINE-1 were significantly associated with menopausal age. Women with earlier menopause showed hypermethylation in both repetitive elements, while women with later menopause showed hypomethylation. These findings suggest that altered DNA methylation in leukocytes and serum cell-free DNA may represent a biomarker of menopausal age.

Methylation status of alu repetitive elements in children with tuberculosis disease

Investigation of DNA methylation in Alu repetitive elements (REs) was shown to be a promising field to explore transcriptional changes in human genome under disease condition. To scrutinize the association between Alu methylation and tuberculosis (TB) disease in children, the difference in Alu DNA methylation level was compared with healthy controls. Whole-blood genomic DNA from 36 TB-infected children and 32 healthy controls was isolated, and the level of Alu repeat DNA methylation was examined by methylation-specific polymerase chain reaction. The median Alu methylation level in TB patients was 30% (Interquartile range [IQR], 25-30%), whereas in healthy controls, it was 75% (IQR, 50-75%) (P < 0.0001). The median level of DNA methylation of Alu RE in TB cases was significantly lower than healthy controls. Receiver operating characteristic curve analysis showed that the area under the curve for diagnosis was 0.969 (95% confidence interval, 0.936-1) (P < 0.0001), with 100% sensitivity and 84% specificity. Our results point out that detection of Alu DNA methylation in whole-blood DNA may be clinically useful tool for the diagnosis and prognosis of TB disease in children.

Prenatal urinary polycyclic aromatic hydrocarbon metabolites, global DNA methylation in cord blood, and birth outcomes: A cohort study in China.

Prenatal exposure to polycyclic aromatic hydrocarbons (PAHs) is a potential risk factor for adverse birth outcomes. Epigenetic mechanisms may play a key role in which PAHs exert its effects. Our study aimed to examine whether prenatal PAH exposure was associated with adverse birth outcomes and altered DNA methylation and to explore potential mediating roles of DNA methylation. Ten urinary PAH metabolites were measured from 106 pregnant women during late pregnancy in a Chinese cohort study. Cord blood DNA methylation in long interspersed nucleotide element-1 (LINE-1) and Alu repetitive elements as surrogates of global DNA methylation was analyzed by bisulfite pyrosequencing. Multivariable linear regression was used to estimate the associations of urinary PAH metabolites with birth outcomes and DNA methylation, and a mediation analysis was also conducted. Prenatal urinary 2-hydroxynaphthalene (2-OHNa), ∑OHNa (sum of 1- and 2-OHNa), and sum of monohydroxy-PAH (∑OH-PAHs) were associated with lower birth length (e.g.,-0.80%, 95% CI:-1.39%,-0.20% for the third vs. first tertile of 2-OHNa; p for trend=0.01). Prenatal urinary 2-OHNa and 1-hydroxyphenanthrene (1-OHPh) were associated with lower Alu and LINE-1 methylation (e.g.,-1.88%, 95% CI:-3.73%,-0.10% for the third vs. first tertile tertile of 2-OHNa in Alu methylation; p for trend=0.04). Mediation analysis failed to show a mediator effect of global DNA methylation in the association between prenatal urinary OH-PAHs and birth outcomes. Prenatal specific PAH exposures are associated with decreased birth length and global DNA methylation. However, global DNA methylation does not mediate the associations of prenatal PAH exposure with birth outcomes. Further studies are needed to confirm the results.

Endogenous sex hormone exposure and repetitive element DNA methylation in healthy postmenopausal women.

Epigenetic mechanisms may help to explain the complex and heterogeneous relation between sex hormones and cancer. Few studies have investigated the effects of sex hormones on epigenetic markers related to cancer risk such as levels of methylation within repetitive DNA elements. Our objective was to describe the association between endogenous sex hormone exposure and levels of LINE-1 and Alu methylation in healthy postmenopausal women. We nested a cross-sectional study within the Alberta Physical Activity and Breast Cancer Prevention Trial (2003-2006). Study participants consisted of healthy postmenopausal women who had never been diagnosed with cancer (n = 289). Sex hormone exposures included serum concentrations of estradiol, estrone, testosterone, androstenedione, and sex hormone-binding globulin. We estimated the participants' lifetime number of menstrual cycles (LNMC) as a proxy for cumulative exposure to ovarian sex hormones. Buffy coat samples were assessed for DNA methylation. Linear regression was used to model the associations of interest and to control for confounding. Both estradiol and estrone had a significant positive dose-response association with LINE-1 methylation. LNMC was associated with both LINE-1 and Alu methylation. Specifically, LNMC had a non-linear "U-shaped" association with LINE-1 methylation regardless of folate intake and a negative linear association with Alu methylation, but only amongst low folate consumers. Androgen exposure was not associated with either outcome. Current and cumulative estrogen exposure was associated with repetitive element DNA methylation in a group of healthy postmenopausal women. LINE-1 and Alu methylation may be epigenetic mechanisms through which estrogen exposure impacts cancer risk.

Associations between adiposity and repetitive element DNA methylation in healthy postmenopausal women.

To describe the association between adiposity and repetitive element DNA methylation in healthy postmenopausal women. A cross-sectional study was conducted using baseline information from 289 women who participated in the Alberta Physical Activity and Breast Cancer Prevention trial. After adjusting for important confounders, long interspersed nuclear element-1 methylation was positively associated with intra-abdominal fat area (p=0.03), body fat percent (p=0.048), fat mass (p=0.01), waist circumference (p=0.03), hip circumference (p=0.001), BMI (p=0.03), current weight (p=0.002), weight at age 20 (p=0.02) and adulthood weight gain (p=0.03). No significant associations were found between any of the adiposity measures and Alu methylation. Current and historical adiposity measures are positively associated with long interspersed nuclear element-1 methylation in healthy postmenopausal women.

Prediction of genome-wide DNA methylation in repetitive elements.

DNA methylation in repetitive elements (RE) suppresses their mobility and maintains genomic stability, and decreases in it are frequently observed in tumor and/or surrogate tissues. Averaging methylation across RE in genome is widely used to quantify global methylation. However, methylation may vary in specific RE and play diverse roles in disease development, thus averaging methylation across RE may lose significant biological information. The ambiguous mapping of short reads by and high cost of current bisulfite sequencing platforms make them impractical for quantifying locus-specific RE methylation. Although microarray-based approaches (particularly Illumina's Infinium methylation arrays) provide cost-effective and robust genome-wide methylation quantification, the number of interrogated CpGs in RE remains limited. We report a random forest-based algorithm (and corresponding R package, REMP) that can accurately predict genome-wide locus-specific RE methylation based on Infinium array profiling data. We validated its prediction performance using alternative sequencing and microarray data. Testing its clinical utility with The Cancer Genome Atlas data demonstrated that our algorithm offers more comprehensively extended locus-specific RE methylation information that can be readily applied to large human studies in a cost-effective manner. Our work has the potential to improve our understanding of the role of global methylation in human diseases, especially cancer.

Review: DNA methylation and alcohol use disorders: Progress and challenges.

Risk for alcohol use disorders (AUDs) is influenced by gene-environment interactions. Environmental factors can affect gene expression through epigenetic mechanisms such as DNA methylation. This review outlines the findings regarding the association of DNA methylation and AUDs. We searched PubMed (by April 2016) and identified 29 studies that examined the association of DNA methylation and AUDs. We also evaluated the methods used in these studies. Two studies demonstrated elevated global (repetitive element) DNA methylation levels in AUD subjects. Fifteen candidate gene studies showed hypermethylation of promoter regions of six genes (AVP, DNMT3B, HERP, HTR3A, OPRM1, and SNCA) or hypomethylation of the GDAP1 promoter region in AUD subjects. Five genome-wide DNA methylation studies demonstrated widespread DNA methylation changes across the genome in AUD subjects. Six studies showed significant correlations of DNA methylation with gene expression in AUD subjects. Three studies revealed interactive effects of genetic variation and DNA methylation on susceptibility to AUDs. Most studies analyzed AUD-associated DNA methylation changes in the peripheral blood; a few studies examined DNA methylation changes in postmortem brains of AUD subjects. Chronic alcohol consumption may result in DNA methylation changes, leading to neuroadaptations that may underlie some of the mechanisms of AUD risk and persistence. Future studies are needed to confirm the few existing results, and then to elucidate whether DNA methylation changes are the cause or consequence of AUDs. DNA methylation profiles may be used to assess AUD status or monitor AUD treatment response. (Am J Addict 2017;26:502-515).

DNA hypomethylation and its mediation in the effects of fine particulate air pollution on cardiovascular biomarkers: A randomized crossover trial

BackgroundShort-term exposure to fine particulate matter (PM2.5) air pollution has been associated with altered DNA methylation in observational studies, but it remains unclear whether this change mediates the effects on cardiovascular biomarkers. ObjectiveTo examine the impact of ambient PM2.5 on gene-specific DNA methylation and its potential mediation in the acute effects of PM2.5 on cardiovascular biomarkers. MethodsWe designed a randomized, double-blind crossover trial using true or sham air purifiers for 48h among 35 healthy college students in Shanghai, China, in 2014. We measured blood global methylation estimated in long interspersed nucleotide element-1 (LINE‑1) and Alu repetitive elements, methylation in ten specific genes, and ten cardiovascular biomarkers. We used linear mixed-effect models to examine the associations between PM2.5 and methylation. We also performed causal mediation analyses to evaluate the potential mediation of methylation in the associations between PM2.5 and biomarkers. ResultsAir purification increased DNA methylation in repetitive elements and all candidate genes. An IQR increase (64μg/m3) in PM2.5 was significantly associated with reduction of methylation in LINE-1 (1.44%), one pro-inflammatory gene (CD40LG, 9.13%), two pro-coagulant genes (F3, 15.20%; SERPINE1, 3.69%), and two pro-vasoconstriction genes (ACE, 4.64%; EDN1, 9.74%). There was a significant mediated effect (17.82%, P=0.03) of PM2.5 on sCD40L protein through CD40LG hypomethylation. Hypomethylation in other candidate genes generally showed positive but non-significant mediation. ConclusionsThis intervention study provided robust human evidence that ambient PM2.5 could induce rapid decreases in DNA methylation and consequently partly mediate its effects on cardiovascular biomarkers.

Maternal residential proximity to major roadways, birth weight, and placental DNA methylation

BackgroundExposure to traffic pollution during fetal development has been associated with reduced fetal growth, and there is evidence to suggest that epigenetic mechanisms in the placenta in the form of variant DNA methylation may be a potential mechanism underlying this effect. ObjectivesTo examine the association between residential proximity to nearest major roadway, as a marker of traffic-related pollution, fetal growth and placental DNA methylation. MethodsWe obtained residential addresses, placenta samples, and demographic data from 471 women following delivery of term infants. Using generalized linear models we evaluated the association between living close to a major roadway (defined as living ≤150m from a primary highway or primary road or ≤50m from a secondary road) and fetal growth and DNA methylation of repetitive elements (LINE-1 and AluYb8). We evaluated epigenome-wide methylation in a subset of 215 women to further investigate specific variation in DNA methylation associated with proximity to major roadways. ResultsLiving close to a major roadway was associated with a 175.9g (95% CI: −319.4, −32.5; p=0.016) lower birth weight, 1.8 (95% CI: 0.9, 3.8; p=0.09) times the odds of being small for gestational age, and 0.82 percentage points (95% CI: −1.57, −0.07; p=0.03) lower mean placental LINE-1 methylation levels in fully adjusted models. In epigenome-wide analyses, 7 CpG sites were significantly associated with residential proximity to major roadways. Additional adjustment for placental methylation did not attenuate the association between roadway proximity and birth weight. ConclusionsLiving close to major roadways was associated with both lower fetal growth and significant placental epigenetic changes. However, the observed epigenetic changes appear insufficient to explain the observed association between roadway proximity and fetal growth.