Methylation Of Repetitive Elements Research Articles

Repetitive element transcript accumulation is associated with inflammaging in humans.

Chronic, low-grade inflammation increases with aging, contributing to functional declines and diseases that reduce healthspan. Growing evidence suggests that transcripts from repetitive elements (RE) in the genome contribute to this "inflammaging" by stimulating innate immune activation, but evidence of RE-associated inflammation with aging in humans is limited. Here, we present transcriptomic and clinical data showing that RE transcript levels are positively related to gene expression of innate immune sensors, and to serum interleukin 6 (a marker of systemic inflammation), in a large group of middle-aged and older adults. We also: (1) use transcriptomics and whole-genome bisulfite (methylation) sequencing to show that many RE may be hypomethylated with aging, and that aerobic exercise, a healthspan-extending intervention, reduces RE transcript levels and increases RE methylation in older adults; and (2) extend our findings in a secondary dataset demonstrating age-related changes in RE chromatin accessibility. Collectively, our data support the idea that age-related RE transcript accumulation may play a role in inflammaging in humans, and that RE dysregulation with aging may be due in part to upstream epigenetic changes.

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.

Repeat DNA methylation is modulated by adherens junction signaling

Through its involvement in gene transcription and heterochromatin formation, DNA methylation regulates how cells interact with their environment. Nevertheless, the extracellular signaling cues that modulate the distribution of this central chromatin modification are largely unclear. DNA methylation is highly abundant at repetitive elements, but its investigation in live cells has been complicated by methodological challenges. Utilizing a CRISPR/dCas9 biosensor that reads DNA methylation of human α-satellite repeats in live cells, we here uncover a signaling pathway linking the chromatin and transcriptional state of repetitive elements to epithelial adherens junction integrity. Specifically, we find that in confluent breast epithelial cell monolayers, α-satellite repeat methylation is reduced by comparison to low density cultures. This is coupled with increased transcriptional activity at repeats. Through comprehensive perturbation experiments, we identify the junctional protein E-cadherin, which links to the actin cytoskeleton, as a central molecular player for signal relay into the nucleus. Furthermore, we find that this pathway is impaired in cancer cells that lack E-cadherin and are not contact-inhibited. This suggests that the molecular connection between cell density and repetitive element methylation could play a role in the maintenance of epithelial tissue homeostasis.

Abstract A007: DNA methylation analysis comparing high-grade serous ovarian cancer to the normal ovary with adjustment for cell type variation identifies tumor-specific methylation alterations

Abstract A007: DNA methylation analysis comparing high-grade serous ovarian cancer to the normal ovary with adjustment for cell type variation identifies tumor-specific methylation alterations

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.

The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm.

Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.

Particulate matter-induced hypomethylation of Alu and LINE1 in normal human bronchial epithelial cells and epidermal keratinocytes

BackgroundAirborne particulate matter (PM), a complex mixture of organic and inorganic compounds, is a major public health concern due to its adverse health effects. Understanding the biological action of PM is of particular importance in the improvement of public health. Differential methylation of repetitive elements (RE) by PM might have severe consequences for the structural integrity of the genome and on transcriptional activity, thereby affecting human health. This study aimed to evaluate the effect of inhaled and non-inhaled PM (PM2.5, PM10, and PM10-PAH) exposure on DNA methylation. We quantitatively measured the methylation content of Alu and LINE1 in PM-treated normal human bronchial epithelial cells (NHBE) and normal human epidermal keratinocytes (NHEK) by using whole-genome bisulfite sequencing and pyrosequencing.ResultsAll PMs exposure significantly lowered Alu and LINE1 methylation in both cells than in mock-treated controls. Hypomethylation was more prominent in PM10-PAH exposed-NHBE and PM10 exposed-NHEK. Alu and LINE1 methylation change exhibited different sensitivity according to the subfamily evolutionary ages, with stronger effects on the oldest L1-M and Alu J in NHBE, and oldest L1-M and youngest Alu S in NHEK.ConclusionsThese results demonstrate that the differential susceptibility of PM-induced hypomethylation of Alu and LINE1 depends upon RE evolutionary age and PM type.

Pregnancy exposure to phthalates and placental DNA methylation in the French EDEN cohort

BACKGROUND AND AIM: Foetal growth and development strongly depend upon the health of the placenta. Prenatal exposure to phthalates has been hypothesized to be associated with differential DNA methylation, however epigenome-wide studies on placental tissue are lacking. The aim of this study was to characterize associations between pregnancy urinary concentrations of phthalate metabolites and placental DNA methylation. METHODS: We measured concentrations of 11 phthalate metabolites in maternal spot urine samples collected between 22-29 gestational weeks in 202 mother-son pairs. We analysed DNA methylation levels in term placental tissue using IlluminaHM450 BeadChips. We performed an adjusted epigenome-wide association study (EWAS) and identified differentially methylated regions (DMRs, ≥ 5 CpGs). We also evaluated DNA methylation of repetitive elements Alu and LINE-1. RESULTS:We identified 6 DMRs associated with urinary concentrations of phthalate metabolites. Monoethyl phthalate (MEP) was positively associated with 2 DMRs encompassing imprinted genes (SGCE/PEG10) and a gene involved in the immunological response (GCA). Other positive associations were detected for: monocarboxy-iso-nonyl-phthalate (MCNP) and HSPA1A/HSPA1L genes encoding heat shock proteins; mono(3-carboxypropyl)-phthalate (MCPP) and ART5 encoding protein responsible for protein function regulation; mono-iso-buthyl-phthalate (MiBP) and transcription factor coding REPIN1 gene. The only negative association was identified between mono(2-ethylhexyl)-phthalate (MEHP) exposure and DMR encompassing transcription factor coding ZSCAN16 gene. Finally, we found that pregnancy monobenzyl phthalate concentrations were negatively associated with placental methylation of Alu repeats. CONCLUSIONS:Our study is the first one to describe the genome-wide epigenetic modifications of placental DNA in association with pregnancy exposure to phthalates. Some phthalates may be associated with differential methylation of placental DNA, including changes in repetitive element methylation. Interestingly, 1 of the 5 highlighted phthalate metabolites has been associated with the placental-to-foetal weight ratio in a previous study relying on EDEN cohort. KEYWORDS: Phthalates, Endocrine disrupting chemicals, Chemical exposures, Birth outcomes, Epigenomics

Abstract 76: Global LINE-1 Hypomethylation as Novel Biomarker for Cervical Cancer in Nigerian Women Living with HIV

Abstract Purpose: Nigeria faces a high health burden of cervical cancer (CC), which is worsened by high prevalence of human immunodeficiency virus (HIV) infection. Repetitive elements (RE) are DNA sequences that occur in multiple copies throughout the human genome. HIV infection can lead to RE hypomethylation that causes genome instability, an event often seen in the early phase of tumorigenesis. We aim to examine global RE hypomethylation as a novel epigenetic biomarker for CC among HIV-positive women in Nigeria. Methods: This study involved three groups of women: a) HIV-positive with CC (n=39); b) HIV-positive and cancer-free (n=52); and c) HIV-negative with CC (n=23). We estimated three types of global RE methylation in cervical tissue using genome-wide methylation data: long interspersed nuclear elements (LINE-1), Alu, and human endogenous retrovirus (HERV). We used multiple linear regression adjusting for age, education, parity, employment, cancer stage, body mass index, and sample batch to compare the biomarkers across the HIV/ICC groups and paired t-test to compare 26 pairs of tumor vs. surrounding normal tissues, stratified by HIV status. Receiver operating characteristic curve (ROC) and area under the ROC (AUC) were used to examine the diagnostic value. Results: Among HIV-positive women, all 3 global RE methylation biomarkers were hypomethylated in CC compared to cancer-free (LINE-1: mean difference [MD]=-0.049, p-value=2.9e-8; Alu: MD=-0.011, p-value=2.0e-4; HERV: MD=-0.013, p-value=1.7e-6). Paired analyses showed a larger, more significant MD in HIV-positive stratum than HIV-negative, especially LINE-1 (MD=-0.048 vs. -0.020, p-value=0.004 vs. 0.254). LINE-1 achieved the highest AUC (0.85, 95% CI: 0.76-0.95) in distinguishing tumor tissue from normal tissue among HIV-positive women, followed by HERV (0.82, 95% CI: 0.73-0.92) and Alu (0.60, 95% CI: 0.47-0.72). Conclusion: Global LINE-1 hypomethylation may serve as a novel biomarker for CC screening and early detection for women living with HIV in low- and middle-income countries. Citation Format: Yinan Zheng, Jonah Musa, Kyeezu Kim, Yishu Qu, Brian Joyce, Jun Wang, Demirkan Gursel, Olugbenga Silas, Abdulkareem Fatimah, Godwin Imade, Alani Akanmu, Jian-Jun Wei, Masha Kocherginsky, Kwang-Youn Kim, Firas Wehbe, Chad Achenbach, Rose Anorlu, Melissa Simon, Atiene Sagay, Folasade Ogunsola, Robert Murphy, Lifang Hou. Global LINE-1 Hypomethylation as Novel Biomarker for Cervical Cancer in Nigerian Women Living with HIV [abstract]. In: Proceedings of the 9th Annual Symposium on Global Cancer Research; Global Cancer Research and Control: Looking Back and Charting a Path Forward; 2021 Mar 10-11. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2021;30(7 Suppl):Abstract nr 76.

Global line-1 hypomethylation as novel biomarker for cervical cancer in Nigerian women living with HIV.

e17513 Background: Nigeria faces a high health burden of cervical cancer (CC), which is worsened by high prevalence of human immunodeficiency virus (HIV) infection. Repetitive elements (RE) are DNA sequences that occur in multiple copies throughout the human genome. HIV infection can lead to RE hypomethylation that causes genome instability, an event often seen in the early phase of tumorigenesis. We aim to examine global RE hypomethylation as a novel epigenetic biomarker for CC among HIV-positive women in Nigeria. Methods: This study involved three groups of women: a) HIV-positive with CC (n=39); b) HIV-positive and cancer-free (n=52); and c) HIV-negative with CC (n=23). We estimated three types of global RE methylation in cervical tissue using genome-wide methylation data: long interspersed nuclear elements (LINE-1), Alu, and human endogenous retrovirus (HERV). We used multiple linear regression adjusting for age, education, parity, employment, cancer stage, body mass index, and sample batch to compare the biomarkers across the HIV/ICC groups and paired t-test to compare 26 pairs of tumor vs. surrounding normal tissues, stratified by HIV status. Receiver operating characteristic curve (ROC) and area under the ROC (AUC) were used to examine the diagnostic value. Results: Among HIV-positive women, all 3 global RE methylation biomarkers were hypomethylated in CC compared to cancer-free (LINE-1: mean difference [MD]=-0.049, p-value=2.9e-8; Alu: MD=-0.011, p-value=2.0e-4; HERV: MD=-0.013, p-value=1.7e-6). Paired analyses showed a larger, more significant MD in HIV-positive stratum than HIV-negative, especially LINE-1 (MD=-0.048 vs. -0.020, p-value=0.004 vs. 0.254). LINE-1 achieved the highest AUC (0.85, 95% CI: 0.76-0.95) in distinguishing tumor tissue from normal tissue among HIV-positive women, followed by HERV (0.82, 95% CI: 0.73-0.92) and Alu (0.60, 95% CI: 0.47-0.72). Conclusions: Global LINE-1 hypomethylation may serve as a novel biomarker for CC screening and early detection for women living with HIV in low- and middle-income countries.

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.

Altered immune phenotype and DNA methylation in panic disorder

BackgroundMultiple studies have related psychiatric disorders and immune alterations. Panic disorder (PD) has been linked with changes in leukocytes distributions in several small studies using different methods for immune characterization. Additionally, alterations in the methylation of repetitive DNA elements, such as LINE-1, have been associated with mental disorders. Here, we use peripheral blood DNA methylation data from two studies and an updated DNA methylation deconvolution library to investigate the relation of leukocyte proportions and methylation status of repetitive elements in 133 patients with panic disorder compared with 118 controls.Methods and resultsWe used DNA methylation data to deconvolute leukocyte cell-type proportions and to infer LINE-1 element methylation comparing PD cases and controls. We also identified differentially methylated CpGs associated with PD using an epigenome-wide association study approach (EWAS), with models adjusting for sex, age, and cell-type proportions. Individuals with PD had a lower proportion of CD8T cells (OR: 0.86, 95% CI: 0.78–0.96, P-adj = 0.030) when adjusting for age, sex, and study compared with controls. Also, PD cases had significantly lower LINE-1 repetitive element methylation than controls (P < 0.001). The EWAS identified 61 differentially methylated CpGs (58 hypo- and 3 hypermethylated) in PD (Bonferroni adjusted P < 1.33 × 10–7).ConclusionsThese results suggest that those with panic disorder have changes to their immune system and dysregulation of repeat elements relative to controls.

Abstract 1265: The effects of ancestry and histology on the lung tumor methylome

Abstract Methylation-based biomarkers can provide molecular insight on the contribution of ancestry and histology to lung tumor biology. Loss of global methylation is a benchmark of aging and a signal of declining biological maintenance. In addition to aging, global loss of methylation occurs in response to some cancer-promoting exposures, including tobacco use. Reduced levels of global DNA methylation are associated with increased risk of several cancers, including lung. In lung tumors, global DNA hypomethylation is often associated with a more aggressive diseases and shorter survival time. Several studies have identified population-level differences in global methylation present at birth. Additionally, gender and ancestry influence DNA methylation age (DNAm) and may predispose some individuals to accelerated aging. We hypothesized that differences in global hypomethylation and accelerated aging may contribute to differences in lung tumor biology by genetic ancestry. Therefore, we investigated methylome differences between European Americans (EA) and African Americans (AA) from the NCI-Maryland Case Control Study, which is overrepresented with AA patients (~40%) from the Baltimore area. We assessed lung tumor and paired non-involved adjacent tissue (NAT) methylation (n = 77 EA, n = 39 AA) using the Illumina EPIC methylation array. We used the Repetitive Element Methylation Prediction (REMP) package in R to identify roughly 20,000 predicted LINE-1 CpG's. We observed significantly decreased LINE-1 methylation in tumor tissue compared with NAT in both AA and EA. Additionally, among tumor samples we observed significantly decreased LINE-1 methylation in AA LUSC samples compared to EA LUSC, with only modest differences between AA LUAD and EA LUAD. In addition to decreased levels, LINE-1 methylation in AA LUSC tumors also had the greatest heterogeneity within CpG's of the same sample and between other AA LUSC samples. We observed accelerated aging (increased DNAm relative to chronological age) in 57/67 EA NAT samples and 28/38 AA NAT samples with no significant differences in age acceleration between ancestry or histology. Our results show global hypomethylation of LINE-1 is a distinctive feature of LUSC in AA compared to EA, which is consistent with the enhanced genomic instability observed in AA LUSC relative to EA LUSC. We also observed accelerated aging in the vast majority of the NAT samples, which may implicate accelerated age with lung cancer risk. Future analysis will work towards completing a comprehensive analysis of the epigenetic landscape of lung cancer in AA and EA patients. We plan to study the relationship between global hypomethylation and genetic variants, smoking behaviors, and patient survival. Citation Format: Emily L. Rossi, Isaac Zhao, Rony F. Arauz, Adriana Zingone, Bríd M. Ryan. The effects of ancestry and histology on the lung tumor methylome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1265.

Increasing methylation of sperm rDNA and other repetitive elements in the aging male mammalian germline.

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age‐related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α‐satellite and interspersed LINE1 repeats, marmoset α‐satellite, bovine alpha‐ and testis satellite I, mouse minor and major satellite, and LINE1‐T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age‐related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo.

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.

Effects of Prenatal Air Pollution on Family-Specific, Genome-Wide, Repetitive Element Methylation in Cord Blood

OPS 48: Air pollution and epigenetics, Room 417, Floor 4, August 26, 2019, 4:30 PM - 5:30 PM Background/Objective: Repetitive elements (REs) comprise more than 50% of the genome and methylation suppresses their transpositions (insertions into other genomic regions) that can damage DNA. Global average RE methylation is inversely associated with particulate matter ≤2.5µm (PM2.5) exposure, however, averaging all RE methylation may overlook their biological activity, as RE subfamilies and individual REs vary in methylation. We employed a method predicting genome-wide, RE subfamily-specific, methylation in cord blood and investigated their associations with prenatal PM2.5 exposure. Methods: Trimester average PM2.5 was estimated using a satellite-based spatio-temporally resolved prediction model and residence location of 443 women from the PROGRESS birth-cohort in Mexico City. We measured cord blood DNA methylation with the Illumina EPICArray using a random forest-based algorithm to predict genome-wide, locus-specific CpG methylation of long interspersed elements-1 (L1) and a short interspersed element (Alu) using nearby CpG sites. We modeled associations between PM2.5 with: 1) methylation of subfamilies within the most recent L1PA, L1PB, and Alu lineages averaged across the genome; and 2) genomic locus-specific aggregates of 2 or more adjacent RE CpGs from these subfamilies. Results: We imputed methylation of 1,183 CpG aggregates and 25 subfamilies within the L1PA and L1PB lineages and 69,596 aggregates and 36 Alu subfamilies with mean methylation of 92%. Average methylation of 20 L1 and 35 Alu subfamilies was inversely associated with 2nd trimester PM2.5. Restricting analysis to full-length L1, methylation of L1PA2, L1PA3, L1PA5, and the active element, L1HS, were inversely associated with PM2.5 in the 2nd and 3rd trimester. Investigations of individual CpGs yielded two aggregates associated with PM2.5 after multiple comparison corrections. Conclusions: Reduced RE subfamily methylation may promote transpositions and cell toxicity that could be related to health endpoints such as cancer. Future work will investigate RE expression and whether RE methylation/expression mediate relationships between PM2.5 exposure and health.

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.

Night Shift Work, DNA Methylation and Telomere Length: An Investigation on Hospital Female Nurses.

Increased breast cancer risk has been reported in some night shift (NS) workers but underlying biological mechanisms are still unclear. We assessed the association between NS work and DNA methylation of tumor suppressor (TP53, CDKN2A, BRCA1, BRCA2) and estrogen receptor (ESR1, ESR2) genes, methylation of repetitive elements (LINE-1, Alu), and telomere length (TL). Forty six female nurses employed in NS for at least two years were matched by age (30–45 years) and length of service (≥1 year) with 51 female colleagues not working in NS. Each subject underwent a semi-structured interview and gave a blood sample. We applied linear regression and spline models adjusted for age, BMI, smoking habit, oral contraceptive use, parity and marital status/age at marriage. Currently working in NS was associated with ESR1 hypomethylation (β: −1.85 (95%CI: −3.03; −0.67), p = 0.003). In current and former NS workers we observed TP53 (−0.93 (−1.73; −0.12), p = 0.03) and BRCA1 (−1.14 (−1.71; −0.58), p <0.001) hypomethylation. We found an increase between TL and number of years in NS in subjects employed in NS <12 years (0.06 (0.03; 0.09), p <0.001), while a decrease if employed in NS ≥12 years (−0.07 −0.10; −0.04), p <0.001). Our findings show NS-associated markers potentially involved in cellular aging, genomic instability, and cancer development.