Long Interspersed Nuclear Element-1 DNA Methylation Research Articles

Skeletal Muscle LINE‐1 Gene Regulation and Inflammatory Response to Chronic, Voluntary Endurance Training in Rodents

ObjectiveWe sought to determine the effects of chronic, voluntary wheel running on Long Interspersed Nuclear Element‐1 (LINE‐1) mRNA expression and promoter methylation, as well as markers of the cGAS‐STING inflammatory pathway, in skeletal muscle.HypothesesLINE‐1 mRNA expression will increase with age, and exercise will mitigate this increase. This difference in expression will be driven through changes in promoter methylation. The cGAS‐STING inflammatory pathway will be upregulated with age and downregulated with rats that exercise‐trained.MethodsPlantaris and soleus skeletal muscles were collected from female Lewis rats that belonged to one of three groups: a young control group (CTL; n=10; 6 mos old), an aged sedentary group (SED; n=12; 15 mos old), and an aged exercise group with access to a running wheel for 9 months (EX; n=12; 15 mos old). RNA, DNA, and protein were isolated from the tissues for analysis. Analysis included RT‐qPCR for LINE‐1 mRNA expression, methylated DNA Immunoprecipitation (MeDIP) to assess the methylation status of the LINE‐1 promoter via RT‐qPCR, and Western blotting for markers of the cGAS‐STING pathway and 4Hydroxynonenal (4HNE). Two primer sets were used for RT‐qPCR: L1‐3 probed for the most active form of LINE‐1 and L1‐Tot probed for all full‐length LINE‐1 elements.ResultsThere was no significant difference in plantaris LINE‐1 mRNA expression or for L1‐3 or L1‐Tot. Plantaris LINE‐1 promoter methylation was undetectable in most samples, so the results are not presented herein. Soleus L1‐3 mRNA was significantly higher in EX compared to CTL (p=0.036) and there was a trend between SED and EX (p=0.053). L1‐Tot also was trending (p=0.059), where EX was higher than CTL (p=0.025). Methylation of the LINE‐1 promoter was significantly different for both L1‐3 and L1‐Tot (p=0.021 and p=0.028, respectively). In both instances, LINE‐1 DNA in SED and EX were significantly more methylated compared to CTL. There were no differences in cGAS protein expression, p‐/pan STING protein expression, or 4HNE expression.ConclusionsContrary to the original hypothesis, LINE‐1 mRNA expression and DNA methylation were higher in the exercise group. Additionally, chronic exercise did not affect the cGAS‐STING inflammatory pathway, highlighting this pathway may be more relevant in extreme age and disease states. Previous skeletal muscle research has focused on using mixed muscle types to look at LINE‐1 expression and regulation, so the differences seen between type I (soleus) and type II (plantaris) fibers is a novel finding that warrants further investigation to understand the underlying mechanisms.

Phyllanthus debilis Methanolic Extract Reduces the Viability of Human Colorectal Adenocarcinoma (HT-29) Cells and Increases LINE-1 and Alu DNA Methylation

Phyllanthus debilis was shown to have a strong anti-proliferative effect on cancer cells with less effect in normal cells. However, its mechanism on the epigenetic mechanism at repeat sequences is unknown. This study was carried out to determine the effect of P. debilis extract on long interspersed nuclear element-1 (LINE-1) and Alu DNA methylation. The anti-proliferative effect of P. debilis methanolic extract on human colorectal adenocarcinoma (HT-29) at 24 hours was done using trypan blue assay. LINE-1 and Alu methylation measurement on the HT-29 cell line was done after 72 hours of treatment using Pyrosequencing. The effect of P. debilis methanolic extract at 24 hours on the viability of HT-29 cells was dose-dependent with the half-maximal inhibitory concentration (IC50) concentration of 0.1 mg/mL. Treatment with P. debilis methanolic extract showed significantly higher Alu DNA methylation when compared with the untreated HT-29 cells (37.0 ± 2.5% vs 32.3 ± 4.3%, p<0.05). Similarly, treatment with 5-aza-2-deoxycytidine also significantly increased the Alu DNA methylation compared with the untreated HT-29 cells (46.0 ± 2.3% vs 37.0 ± 2.5%, p<0.05). For LINE-1, there was a significant increase of LINE-1 methylation when treated with P. debilis extract (80.3 ± 1.3% vs 76.3 ± 2.1%, p<0.05) and with 5-aza-2-deoxycytidine (81.8 ± 4.3% vs 76.3 ± 2.1%, p<0.05) when compared with untreated cells. In conclusion, treatment of P. debilis methanolic extract on HT-29 cell line reduces the viability of HT-29 cells and increases the methylation of Alu and LINE 1. Similar changes in methylation were also seen in the 5-aza treatment. These epigenetic changes by P. debilis methanolic extract may contribute to its anti-cancer properties.

1221Baseline Global DNA Hypermethylation Increase the Risk of Cerebrovascular Disease Mortality in Japanese Population

Abstract Background DNA methylation plays an important role in progression of cardiovascular disease (CVD). The methylation status of long interspersed nuclear element-1 (LINE-1) reflects the global DNA methylation level and is associated with lipid profiles and glycemia. We conducted a longitudinal study to examine the association between LINE-1 DNA methylation and CVD mortality in a Japanese elderly population. Methods We targeted for 357 subjects (143 men and 214 women) who were more than 60 years, participated in the health checkup in 1990, and had no clinical history of cancer, stroke, or ischemic heart disease. During 29 years of follow-up period, total 69 subjects were died from CVD, and died of cerebrovascular disease (CBD) and ischemic heart disease (IHD) were 25 and 13, respectively. LINE-1 DNA methylation was measured using PBMCs. We defined the hypermethylation group as greater than median of the LINE-1 DNA methylation levels and hypomethylation group as others. Multivariable Hazard ratios (HRs) for each disease mortality were calculated by the Cox proportional hazard model. We adjusted for age, sex, smoking habits, and alcohol consumption. Results Significantly higher HRs for CVD and CBD mortality were observed in the hypermethylation group compared to the hypomethylation group (CVD: HR 1.67 [95%CI 1.02-2.73], CBD: HR 3.29 [95%CI 1.29-8.40]). However, IHD mortality did not associated with LINE-1 DNA methylation. Conclusions We found that LINE-1 DNA hypermethylation in PBMCs was associated with high CVD mortality, especially CBD mortality. Key messages Higher levels of LINE-1 methylation in PBMCs can be a predictive marker for CBD risk

Global DNA hypermethylation in peripheral blood mononuclear cells and cardiovascular disease risk: a population-based propensity score-matched cohort study

BackgroundDNA methylation plays an important role in the pathogenesis and progression of cardiovascular disease (CVD) but the prospective association of DNA methylation with CVD has not been evaluated. Here, we...

Using Differential Threshold Effects of Individual and Combined Periconceptional Methyl Donor Status on Maternal Genomic LINE-1 and Imprinted H19 DNA Methylation to Predict Birth Weight Variance in the Taiwan Pregnancy-Newborn Epigenetics (TPNE) Cohort Study

Few studies have comprehensively examined the effect of methyl donor status on maternal DNA methylation and birth outcomes. This study examined associations between periconceptional methyl donor status and genome-wide and specific imprinted gene methylation and fetal growth indices in the Taiwan Pregnancy-Newborn Epigenetics cohort. Plasma folate, choline (free form), and betaine concentrations of the participants enrolled at 7-10 weeks of gestation were analyzed. DNA methylation at regulatory sequences of the imprinted H19 gene and genomic long interspersed nuclear element 1 (LINE-1) were measured in maternal lymphocytes using bisulfite/high-resolution melt polymerase chain reaction. Associations with birth weight (BW) were estimated through multiple regressions from 112 mother-newborn pairs. A nonlinear "L-shaped" relation and an inverse association between maternal plasma folate in T1 (mean ± SE: 17.6±5.1 nmol/L) and lymphocytic LINE-1 methylation (β:-0.49, P=0.027) were characterized. After adjusting for LINE-1 methylation, individual maternal folate concentrations were positively associated with BW variance (β=0.24, P=0.035), and the association was more pronounced in mothers with choline in T1 (mean ± SE: 5.4±0.6 μmol/L; β: 0.40, P=0.039). Choline status of the mothers in T2 (mean ± SE: 7.2±0.6 μmol/L) was inversely associated with LINE-1 methylation (β: -0.43, P=0.035), and a positive association was evident between T1 choline and H19 methylation (β: 0.48, P=0.011). After adjusting for epigenetic modification, maternal choline status predicted a positive association with BW (β: 0.56, P=0.005), but the effect was limited to mothers with high betaine concentrations in T3 (mean ± SE: 36.4±8.8 μmol/L), depending on folate status. Our data highlight the differential threshold effects of periconceptional folate, choline, and betaine status on genomic LINE-1 and H19 DNA methylation and how their interplay has a long-term effect on BW variance.

Lower S-adenosylmethionine levels and DNA hypomethylation of placental growth factor (PlGF) in placental tissue of early-onset preeclampsia-complicated pregnancies.

IntroductionThe pathophysiology of preeclampsia is largely unknown. Serum placental induced growth factor (PlGF) levels are decreased during second trimester pregnancy. Aberrant DNA methylation is suggested to be involved in the etiology of preeclampsia (PE). We hypothesize that DNA methylation is altered in PE placentas determined the methylation index by measuring placental S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels. In addition, we assessed global DNA methylation status by long-interspersed nuclear element-1 (LINE-1) and DNA methylation status of the PlGF gene.MethodsPlacental tissue of 11 early onset PE (EOPE), 11 late onset PE (LOPE) and 60 controls consisting of 25 uncomplicated controls 20 fetal growth restriction (FGR) and 15 preterm births (PTB) controls was collected from a nested case-control study of The Rotterdam Periconceptional Cohort. RNA and DNA was isolated from placental tissue and DNA was treated with sodium bisulfite. SAM and SAH levels were measured by LC-ESI-MS/MS. Methylation of LINE-1 and PlGF genes was analyzed by Sequenom Epityper and. mRNA expression of PlGF was assessed with qPCR. Differences were assessed by analysis of covariance (ANCOVA) corrected for gestational age and birth weight.ResultsPlacental SAM levels were significantly lower in placental tissue of EOPE pregnancies compared to PTB controls (mean difference -240 ± 71.4 nmol/g protein, P = 0.01). PlGF DNA methylation was decreased in placental tissue of EOPE cases versus LOPE (mean difference -17.4 ± 5.1%, P = 0.01), uncomplicated controls (mean difference -23.4 ± 5.4%%, P <0.001), FGR controls (mean difference -17.9 ± 4.6%, P = 0.002) and PTB controls (mean difference -11.3 ± 3.8% P = 0.04). No significant differences were observed in SAH, SAM:SAH ratio, LINE-1 DNA methylation and PlGF mRNA expression between groups.DiscussionThe hypomethylation state of the placenta in EOPE, which is reflected by lower SAM and PlGF DNA hypomethylation underlines the possible role of placental DNA hypomethylation in the pathophysiology of EOPE, which needs further investigation.

Positive association of low-level environmental phthalate exposure with sperm motility was mediated by DNA methylation: A pilot study

Accumulating evidence indicates that phthalate exposures may affect human semen quality. Epigenetic modifications such as DNA methylation might be linked chemical exposure and spermatogenesis epigenetic reprogramming. In the present study, we investigated associations between phthalate exposures, DNA methylation and sperm quality in undergoing fertility assessment male population. Urine was used for phthalate exposures monitoring, six selected metabolites (i.e., monomethyl phthalate (MMP), monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), mono-(2-ethylhexyl) phthalate (MEHP) and mono (2-ethyl-5-oxohexyl) phthalate (MEOHP)) were measured by using HPLC−MS/MS. Sperm quality parameters were determined by computer-assisted semen analysis (CASA). Sperm DNA methylation patterns (long interspersed nuclear element-1(LINE-1), H19 and LIT1) were analysed employing high-melting resolution (HRM) PCR. Urinary MMP, MEHP, MEOHP, sum of DEHP metabolites (∑DEHP) and sum of selected phthalates metabolites (∑PAEs) were significantly positively associated with sperm motility. Sperm LINE-1 DNA methylation were found to be negatively associated with ∑DEHP exposure and sperm quality (ejaculate volume, total sperm number and motility). Epigenetic modification LINE-1 DNA methylation demonstrated mediating effects in association between DEHP exposure and sperm motility, and 20.7% of the association was mediated by serum LIEN-1 DNA methylation. These results extend the previous studies in association between phthalate exposures and classical semen parameters, mainly of inverse association, and sperm DNA methylation may be linked phthalate exposures and male reproductive health outcome.

The effect of inflammation-related lifestyle exposures and interactions with gene variants on long interspersed nuclear element-1 DNA methylation.

To examine the relationship between inflammation-related lifestyle factors and long interspersed nuclear element-1 (LINE-1) DNA methylation, and test for interaction by gene variants involved in one-carbon metabolism. The study population consisted of 280 individuals undergoing colonoscopy screening. Multivariable linear regression was employed to examine associations of physical activity, BMI and NSAID use with LINE-1 DNA methylation and interactions with MTR and MTHFR gene variants. The highest quartile of physical activity compared with the lowest was associated with higher LINE-1 DNA methylation (p=0.005). Long-term NSAID use and a normal BMI were associated with increased LINE-1 DNA methylation among individuals with the variant MTR allele (p=0.02; p=0.03). This study provides evidence that inflammation-related exposures may influence LINE-1 DNA methylation.

LINE-1 in response to exposure to ionizing radiation.

It is becoming increasingly recognized that Long Interspersed Nuclear Element, 1 (LINE-1), the most ubiquitous repetitive element in the mammalian genomes, plays an important role in the pathogenesis of disease and in the response to exposure to environmental stressors. Ionizing radiation is a known genotoxic stressor, but it is capable of targeting the cellular epigenome as well. Radiation-induced alterations in LINE-1 DNA methylation are the most frequently observed epigenetic effects of exposure. The extent of this aberrant DNA methylation, however, strongly depends on a number of factors, including the type and dose of radiation. Two other factors are being discussed in this commentary - the evolutionary age and type of the LINE-1 promoter, as well as the type of irradiated cell. This knowledge will further aid in elucidating the mechanisms of response to ionizing radiation exposure, as well in understanding the pathogenesis of the negative health effects associated with exposure.

Inter-Strain Differences in LINE-1 DNA Methylation in the Mouse Hematopoietic System in Response to Exposure to Ionizing Radiation

Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons are the major repetitive elements in mammalian genomes. LINE-1s are well-accepted as driving forces of evolution and critical regulators of the expression of genetic information. Alterations in LINE-1 DNA methylation may lead to its aberrant activity and are reported in virtually all human cancers and in experimental carcinogenesis. In this study, we investigated the endogenous DNA methylation status of the 5′ untranslated region (UTR) of LINE-1 elements in the bone marrow hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and mononuclear cells (MNCs) in radioresistant C57BL/6J and radiosensitive CBA/J mice and in response to ionizing radiation (IR). We demonstrated that basal levels of DNA methylation within the 5′-UTRs of LINE-1 elements did not differ significantly between the two mouse strains and were negatively correlated with the evolutionary age of LINE-1 elements. Meanwhile, the expression of LINE-1 elements was higher in CBA/J mice. At two months after irradiation to 0.1 or 1 Gy of 137Cs (dose rate 1.21 Gy/min), significant decreases in LINE-1 DNA methylation in HSCs were observed in prone to radiation-induced carcinogenesis CBA/J, but not C57BL/6J mice. At the same time, no residual DNA damage, increased ROS, or changes in the cell cycle were detected in HSCs of CBA/J mice. These results suggest that epigenetic alterations may potentially serve as driving forces of radiation-induced carcinogenesis; however, future studies are needed to demonstrate the direct link between the LINE-1 DNA hypomethylation and radiation carcinogenesis.

Prospective study of DNA methylation at LINE-1 and Alu in peripheral blood and the risk of prostate cancer.

Evidence suggests that global blood DNA methylation levels may be associated with the risk of various cancers, but no studies have evaluated this relationship for prostate cancer. We used pyrosequencing to quantify DNA methylation levels at the long interspersed nuclear element 1 (LINE-1) and Alu repetitive elements in pre-diagnostic blood samples from 694 prostate cancer cases and 703 controls from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. We evaluated prostate cancer risk associated with the mean methylation level for each element using logistic regression, adjusting for potential confounders. We did not observe a significant association with prostate cancer for LINE-1 [odds ratio (OR) for the highest compared to the lowest quartile = 1.01, 95% confidence interval (CI): 0.73-1.39, Ptrend = 0.99] or Alu (OR = 0.94, 95% CI: 0.68-1.29, Ptrend = 0.69) methylation levels overall. However, for Alu, we observed that higher DNA methylation levels were associated with a significant increased risk for those diagnosed 4 or more years after blood draw (OR = 2.26, 95% CI: 1.27-4.00, Ptrend = 4.4 × 10(-3) ). In contrast, there was no association for those diagnosed 2 (OR = 1.13, 95% CI: 0.67-1.90, Ptrend = 0.64) or 3 years after draw (OR = 1.22, 95% CI: 0.71-2.07, Ptrend = 0.32), and a decreased risk for those diagnosed less than 2 years after draw (OR = 0.40, 95% CI: 0.25-0.65, Ptrend = 3.8 × 10(-5) ; Pheterogeneity = 5.3 × 10(-6) ). Although LINE-1 DNA methylation levels were not associated with prostate cancer, we observed an association for Alu that varied by time from blood draw to diagnosis. Our study suggests that elevated Alu blood DNA methylation levels several years before diagnosis may be associated with an increased prostate cancer risk.

A cross-sectional study of global DNA methylation and risk of colorectal adenoma.

BackgroundThe methylation of DNA is recognized as a key epigenetic mechanism and evidence for its role in the development of several malignancies is accumulating. We evaluated the relationship between global methylation in DNA derived from normal appearing colon mucosal tissue and blood leukocytes, and colorectal adenoma risk.MethodsPatients, aged 40 to 65, scheduled for a screening colonoscopy were recruited. During the colonoscopy, two pinch biopsies of healthy, normal appearing mucosa were obtained from the descending colon. A fasting blood sample was also collected. The methylation status of LINE-1 (long interspersed nuclear element-1) repetitive sequences, as a surrogate measure of global methylation, was quantified in DNA extracted from normal colon mucosa and blood leukocytes. Statistical analysis of the relationship between global DNA methylation and adenoma risk was conducted on 317 participants, 108 subjects with at least one pathologically confirmed adenoma and 209 subjects with a normal colonoscopy.ResultsA statistically significant inverse relationship was observed between LINE-1 methylation in colon tissue DNA and adenoma risk for males and for both sexes combined for the lowest methylation quartile compared to the highest (adjusted ORs = 2.94 and 2.26 respectively). For blood, although the overall pattern of odds ratio estimates was towards an increase in risk for lower methylation quartiles compared to the highest methylation quartile, there were no statistically significant relationships observed. A moderate correlation was found between LINE-1 methylation levels measured in tissue and blood (Pearson correlation 0.36).ConclusionsWe observed that lower levels of LINE-1 DNA methylation in normal appearing background colon mucosa were associated with increased adenoma risk for males, and for both sexes combined. Though these findings provide some support for a relationship between LINE-1 DNA methylation in colon mucosal tissue and adenoma risk, large prospective cohort studies are needed to confirm results. Until such investigations are done, the clinical usefulness of LINE-1 methylation as a biomarker of increased adenoma risk is uncertain. Regardless, this study contributes to a better understanding of the role of global DNA methylation as an early event in CR carcinogenesis with implications for future etiologic research.

Global long interspersed nuclear element 1 DNA methylation in a Colombian sample of patients with late-onset Alzheimer's disease.

Alterations in DNA methylation have implicated as an epigenetic event in the pathogenesis of late-onset Alzheimer's disease (LOAD). The objective of this work was to evaluate global DNA methylation levels for long interspersed nuclear element 1 (LINE-1) repetitive sequences in Colombian patients with LOAD and controls. The LINE-1 DNA methylation levels in peripheral blood samples from 28 Colombian patients with LOAD and 30 healthy participants were assessed using a methylation-sensitive high-resolution melting (MS-HRM) quantitative assay. We did not find differences in LINE-1 methylation levels between patients with Alzheimer's disease (AD; median 76.2%, interquartile range [IQR]: 69.8-81.9) and control participants (median 79.8%, IQR: 73.2-83.8; P = .3). Additional stratified analyses did not show differences in LINE-1 methylation levels for male or female patients versus controls nor for apolipoprotein E4 carriers and noncarriers. This is the first report of LINE-1 methylation levels in patients with LOAD using the cost-effective MS-HRM technique, and this is the first global DNA methylation study in Latin American patients with AD.

Gestational intake of methyl donors and global LINE-1 DNA methylation in maternal and cord blood: Prospective results from a folate-replete population

Maternal diet affects offspring DNA methylation in animal models, but evidence from humans is limited. We investigated the extent to which gestational intake of methyl donor nutrients affects global DNA methylation in maternal and umbilical cord blood. Among mother-infant pairs in Project Viva, a folate-replete US population, we estimated maternal intakes of vitamin B12, betaine, choline, folate, cadmium, zinc and iron periconceptionally and during the second trimester. We examined associations of these nutrients with DNA methylation, measured as %5-methyl cytosines (%5mC) in Long Interspersed Nuclear Element-1 (LINE-1), in first trimester (n = 830) and second trimester (n = 671) maternal blood and in cord blood at delivery (n = 516). Cord blood methylation was higher for male than female infants {mean [standard deviation (SD)] 84.8 [0.6] vs. 84.4 [0.7]%}. In the multivariable-adjusted model, maternal intake of methyl donor nutrients periconceptionally and during the second trimester of pregnancy was not positively associated with first trimester, second trimester or cord blood LINE-1 methylation. Periconceptional betaine intake was inversely associated with cord blood methylation [regression coefficient = -0.08% (95% confidence interval (CI): -0.14,-0.01)] but this association was attenuated after adjustment for dietary cadmium, which itself was directly associated with first trimester methylation and inversely associated with cord blood methylation. We also found an inverse association between periconceptional choline [-0.10%, 95% CI: -0.17,-0.03 for each SD (~63 mg/d)] and cord blood methylation in males only. In this folate-replete population, we did not find positive associations between intake of methyl donor nutrients during pregnancy and DNA methylation overall, but among males, higher early pregnancy intakes of choline were associated with lower cord blood methylation.