Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

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

Particulate matter-induced hypomethylation of Alu and LINE1 in normal human bronchial epithelial... Background: Airborne 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 (PM ,PM , and 2.5 10 PM -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. Results: All PMs exposure significantly lowered Alu and LINE1 methylation in both cells than in mock-treated controls. Hypomethylation was more prominent in PM -PAH exposed-NHBE and PM exposed-NHEK. Alu and 10 10 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. Conclusions: These results demonstrate that the differential susceptibility of PM-induced hypomethylation of Alu and LINE1 depends upon RE evolutionary age and PM type. Keywords: Particulate matter, DNA methylation, WGBS, Alu, LINE1, Subfamily Introduction forms of air pollution, is recognized as a major health Air pollution has become an important health concern hazard worldwide, and is associated with respiratory, and ranks as the sixth leading risk factors for premature cardiovascular, and skin diseases [3–5]. However, the death globally [1, 2]. Exposure to air pollution is ubiqui- mechanisms linking PM exposure to adverse health out- tous and typically beyond the control of individuals, and comes have not been completely clarified. The size and the resulting health burden on the population can be composition determine the toxicity of the particle [6]. high. Particulate matter (PM), one of the most toxic PM consists of a mixture of volatile organic compounds, polycyclic aromatic hydrocarbons (PAH) and inorganic chemicals such as heavy metals that, both individually * Correspondence: doskim@knu.ac.kr Department of Anatomy and BK21 Plus KNU Biomedical Convergence and together, cause adverse health effects. PM consti- Program, Daegu, Republic of Korea tutes of microscopic particles of solid or liquid matter Department of Anatomy, School of Medicine, Kyungpook National suspended in the air. Airborne PM is usually classified as University, 2-101 Dongin-dong, Jung-gu, 702-422 Daegu, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lee et al. Genes and Environment (2022) 44:8 Page 2 of 7 coarse PM (<10 mm) and fine PM (<2.5 mm), de- former (PM -PAH) includes several PAHs (benzoan- 10 2.5 10 pending on the aerodynamic diameter of the particles. thracene, benzopyrene, benzofluoranthene, and diben- PM is composed of inhalable particles from dusts, in- zoanthracene, etc.) in ambient PM , the latter (PM ) 10 10 10 dustrial emissions, and traffic emissions. PM is pri- contains heavy metals (arsenic, cadmium, lead, and 2.5 marily composed of organic carbon compounds, nitrates, nickel). They were suspended in phosphate buffered sa- and sulfates. Understanding the biological action of PM line (PBS) at 5-mg/ml concentration. PM was prepared is of particular importance in improvement of public just before cell application and sonicated in an ultrasonic health. bath for 10 min to avoid variability in PM composition Alterations in DNA methylation are associated with and aggregation of particles. various health outcomes, representing an interface be- tween the environment and human disease [7]. Emer- Cell culture and PM treatment ging data indicate that PM exposure modulates DNA NHBE and NHEK were obtained from the American methylation, a major genomic mechanism of gene ex- Type Culture Collection (ATCC, Manassas, VA, USA). pression control, and that these changes might in turn NHEK (ATCC PCS-200-011) was grown in Dermal Cell influence inflammation, disease development, and ex- Basal Media (ATCC PCS-200-030) supplemented with acerbation risk [8]. However, whether such effects are Keratinocyte Growth Kit (ATCC PCS-200-040) to targeted to specific sites or scattered across the genome propagate in serum-free conditions. NHBE (ATCC PCS- globally remain challenging. Alu and long interspersed 300-011) was cultured in serum-free Airway Epithelial nucleotide element 1 (LINE1, L1) are significant compo- Cell Basal Media (ATCC PCS-300-030) supplemented nents of repetitive transposable DNA elements, consti- with Bronchial Epithelial Cell Growth Kit (ATCC PCS- tuting approximately 17% and 11% of the human 300-040). Both cells were grown at 50% confluency and genome, respectively [9], representing as a surrogate were treated with PM ,PM , and PM -PAH for 3 2.5 10 10 marker for genome-wide global methylation levels. Inter- days at a final 50-µg/ml concentration of without a estingly, transposable repeats are considered as a respon- medium change. Cells maintained in culture medium ser to environmental stressors [10, 11] and their with vehicle (0.1% DMSO or 1% PBS) were used as un- reactivation through hypomethylation can increase gen- treated control groups. ome instability, reactivate lowly expressed genes, or dis- rupted gene function, thereby potentially contributing to WGBS library preparation and sequencing disease-related pathological consequences [12, 13] and The cells were washed with PBS, and genomic DNA was provide promising candidate biomarkers for human dis- extracted using a QIAamp DNA Mini Kit (Qiagen, Val- ease including cancer [14]. Unfortunately, PM-induced encia, CA, USA) according to the manufacturer’s in- DNA methylation of repetitive elements (RE) reported struction. The concentration and quality of the DNA in most previous studies is measured in blood cells, were determined using an Agilent Bioanalyzer 2100 representing overall results from the body organs [15– (Agilent Technologies, Santa Clara CA, USA) and agar- 18]. However, methylation in skin and lung, major tar- ose gel electrophoresis. DNAs were fragmented using a gets of air pollution, has not been well studied. In the Bioruptor (Diagenode, Liege, Belgium) to an average size present study, we treated the normal human bronchial of approximately 250 bp, followed by the blunt ending, epithelial cells (NHBE) and normal human epidermal 3’-end addition of dA, and adaptor ligation (in this case keratinocytes (NHEK) with inhaled and non-inhaled PM of methylated adaptors to protect from bisulfite conver- (PM ,PM , and PM -PAH) and then determined the sion). Ligated DNA was bisulfite-converted using the EZ 2.5 10 10 changes in global DNA methylation using whole- DNA Methylation-Gold kit (Zymo Research Corp, Ir- genome bisulfite sequencing (WGBS) and vine, CA, USA). Fragments pf length 200–250 bp were pyrosequencing. excised from a 2% TAE agarose gel, purified using a QIAquick Gel Extraction Kit (Qiagen), and then ampli- Materials and methods fied via PCR. Libraries were constructed from PCR prod- PM preparation ucts with BGI’s DNA nanoball (DNB) technology. The PM , which is a standard diesel PM (SRM1650b) issued qualified libraries were sequenced using the DNBSEQ®- 2.5 by the National Institute of Standard and Technology platform (BGI, Shenzhen, China). Base-calling was per- (Gaithersburg, MD, USA), was bought from Sigma- formed using the BGISEQ-500 software (v 0.3.8.1111). Aldrich (St. Louis, MO, USA). It was dissolved in di- methyl sulfoxide (DMSO) at 50-mg/ml concentration. Data filtering PM -like fine dusts (ERM-CZ100 and ERM-CZ120), Data filtering was conducted using the elimination of which are issued by the European Reference Materials contaminating DNA and low-quality reads. Low-quality (ERM, Belgium), were brought from Sigma-Aldrich. The reads include three types and the reading that accord Lee et al. Genes and Environment (2022) 44:8 Page 3 of 7 with one of them will be removed: (1) contain adaptor Statistical analysis sequence; (2) N-base number >10%; (3) the number of Data are presented as the means ± standard error bases with a quality of <20% and >10% was trimmed. (SE) of three independent experiments. One-way Only clean data was used for further analyses. ANOVA was used for the mean difference test be- tween the groups, and Bonferroni’s correction p-value was used for post-hoc comparison the two groups Reads mapping and differentially methylated level when the ANOVA was significant. The statistical analysis analyses were conducted using SAS 9.4 (SAS Inc., Clean reads of each sample were mapped to human UCSC Cary, NC, USA), and the plots were constructed using hg19 reference genome using BSMAP software (v2.90) to R version 4.1.0 (The R Foundation for Statistical obtain BAM file. BAM files were sorted and indexed using Computing, Vienna, Austria). The significance level of Samtools software (v0.1.18). The parameters in the map- the statistical test was set to 5%. ping and the results of mapping were shown in Supple- mentary Table 1. Methylation level was determined by Results dividing the number of reads covering each methylcyto- We comprehensively examined the effect of inhaled and sine by the total reads covering the cytosine. MOABS soft- non-inhaled PM exposure on RE Alu and LINE1 by WGBS. ware (v1.3.2) was used to calculate the methylation level Based on the reference genome and the UCSC RepeatMas- of every cytosine in every sample, and to determine differ- ker, approximately 34.6% of all 28 million CpG sites are in entially methylated cytosine (DMC). After calculating the Alu (25.0%) and LINE1 (9.6%). The RepeatMasker library methylation level, cytosine was considered as “hypomethy- mapped 7,040,695 Alu and 2,651,373 LINE1 loci in the lated” when the methylation level of cytosine ≤0.2, and UCSC hg19 reference genome assembly, corresponding to Fisher’s Exact Test p-value was <0.05. 10.1% and 17.1% of the human genome respectively (Table 1). Because human Alu and LINE1 are heavily meth- Alu and LINE1 methylation analysis ylated in normal tissues, all PMs exposure significantly in- To analyze the methylation level of six evolutionary sub- creased the unmethylated CpGs of Alu and LINE1 in both families in Alu (Alu Y, Alu S, and Alu J) and LINE1 (L1- NHBE and NHEK comparing with mock-treated (0.1% H, L1-P, and L1-M), genomic coordinates of all repeats DMSO or 1% PBS) cells (Table 1), indicating a potentially based on hg19 were extracted and obtained from UCSC defective functionality of these RE. Moreover, PM-induced genome browser using RepeatMasker track as Sae-Lee hypomethylation was prominent in NHBE compared with et al. [19]. All subtypes of 6 target repeats (for example, NHEK as well as in LINE1 compared with Alu (Fig. 1). Al- AluJb, AluJo, AluJr, etc. for AluJ subtypes) were ex- though there was a narrow margin between each PM, tracted, and methyl-cytosine data within 6 repeats were PM -PAH and PM exhibited the strongest effect on RE 10 10 collected from DMC data of MOABS results in each hypomethylation in NHBE and NHEK, respectively (Fig. 1). sample using Python scripts. Data analysis workflow was Furthermore, because CpG content and DNA methylation shown in Supplementary Fig. 1. All CG sites in hg19 and levels dramatically differ across subfamilies, we evaluated DMCs within six repeats were counted through the the sensitivity of DNA methylation in differentially-evolved samples. Alu and LINE1 subfamilies to different types of airborne PM. We subdivided Alu and LINE1 into three evolutionary Pyrosequencing of Alu and LINE1 subfamilies; oldest Alu J and L1-M, intermediate Alu S and Bisulfite-converted DNA was amplified with PCR L1-P, and youngest Alu Y and L1-H. Interestingly, Alu J primers under previously described conditions [20]. The andL1-Mshowedthe strongesthypomethylation in NHBE PCR products were then assayed on the PyroMark Q24 following treatment with three PMs, whereas Alu Y and with PyroMark Gold Q24 Reagents (Qiagen) and then L1-M exhibited the strongest hypomethylation in the analyzed with accompanying software. The degree of NHEK (Fig. 2), indicating the association of differential sus- methylation was expressed for each DNA locus as a per- ceptibility of the RE hypomethylation with evolutionary centage of methylated cytosines over the sum of methyl- ages of subfamilies. In addition, although pyrosequencing is ated and unmethylated cytosines. We used non-CpG not expected to comprehensively reflect DNA methylation cytosine residues as built-in controls to verify the bisul- patterns within individual subfamilies, we have validated fite conversion. In every pyrosequencing run, three con- the WGBS results by pyrosequencing for accuracy and re- trols were included. One well was filled with water to producibility of methylation levels. Significantly reduced ensure no contamination, and two wells were filled with methylation of Alu and LINE1 was detectable in both CpGenome universal methylated and unmethylated NHBE and NHEK following all PMs exposure (Fig. 3). Like- DNA (Chemicon, Temecula, CA, USA) as positive and wise, PM -PAH and PM exhibited the strongest effect 10 10 negative control to weigh the repeatability of the assay. on RE hypomethylation in NHBE and NHEK, respectively Lee et al. Genes and Environment (2022) 44:8 Page 4 of 7 Table 1 PM-induced hypomethylation of Alu and LINE1 in NHBE and NHEK L1-H L1-P L1-M L1 PM PM PM -PAH PM PM PM -PAH PM PM PM -PAH PM PM PM -PAH 2.5 10 10 2.5 10 10 2.5 10 10 2.5 10 10 Unmethylated CpGs 604 622 702 29,409 30,050 34,696 40,765 42,033 49,037 70,778 72,706 84,603 in NHBE Unmethylated CpGs 591 619 592 20,902 22,228 20,774 24,707 26,474 27,270 46,201 49,322 48,637 in NHEK Analyzed total CpGs 43,045 1,282,366 1,325,962 2,651,373 Alu Y Alu S Alu J Alu PM PM PM -PAH PM PM PM -PAH PM PM PM -PAH PM PM PM -PAH 2.5 10 10 2.5 10 10 2.5 10 10 2.5 10 10 Unmethylated CpGs 28,627 26,533 28,674 92,202 85,521 99,032 17,746 17,666 19,296 138,575 129,719 144,627 in NHBE Unmethylated CpGs 17,907 28,937 19,691 40,236 52,578 43,912 6,361 7,565 7,964 64,503 88,747 71,567 in NHEK Analyzed total CpGs 1,784,717 4,454,205 801,773 7,040,695 (Fig. 3), showing that a similar trend might be present be- RE hypomethylation [21]. DNA methylation is a tween WGBS and pyrosequencing methods. common feature of eukaryotic genomes and is a core epigenetic process that influences numerous bio- Discussion logical processes, such as gene repression, control of The first major finding of this study was that Alu and cellular development and differentiation, RE silen- LINE1 methylation was significantly lower after PM ex- cing, and maintenance of genome stability [22]. DNA posures, providing for the first direct experimental evi- methylation mainly changed at locus-specific and dence that PM exposure induces DNA hypomethylation genome-wide levels. A number of methods are avail- in NHBE and NHEK. Recent in vitro experiments have able for the analysis of global DNA methylation shown that oxidative DNA damage by PM can interfere levels [23]. Recently, WGBS has revolutionized the with the ability of DNA methyltransferase, resulting in way of interrogating the methylome to realize Fig. 1 Alu and LINE1 methylation change in NHBE and NHEK after PM exposure. The hypomethylation ratio was calculated as a percentage of CpGs with low methylation to analyzed total CpG sites and the means ± SE of three independent WGBS experiments are shown. The mean methylation ratios of PM-treated cells were compared to mock-treated cells using one-way ANOVA, and Bonferroni’s correction p-value was used for post-hoc comparison between two groups where the ANOVA was significant Lee et al. Genes and Environment (2022) 44:8 Page 5 of 7 Fig. 2 Differential hypomethylation of Alu and LINE1 evolutionary subfamilies in NHBE and NHEK following PM treatment. (A) Alu change in NHBE. (B) LINE1 change in NHBE. (C) Alu change in NHEK. (D) LINE1 change in NHEK. The means ± SE of three independent WGBS experiments are shown. Bonferroni-adjusted p-value was the result of comparing between the two groups Fig. 3 Distribution of Alu and LINE1 methylation level in PM-exposed NHBE (A) and NHEK (B). Methylation level was expressed as a percentage of 5-methylcytosine divided by the sum of methylated and unmethylated cytosines. The mean ± 95% confidence interval of three independent pyrosequencing is shown. The comparisons of mean methylation levels were evaluated using ANOVA. Bonferroni-adjusted p-value was the result of a b comparing between the two groups. Cells maintained in culture medium with vehicle were used as control group (CTL , 0.1% DMSO; CTL ,1% PBS) Lee et al. Genes and Environment (2022) 44:8 Page 6 of 7 genome-wide methylation analysis at a single-base dramatically across subfamilies. Interestingly, Byun et al. resolution [24]. The current study determined [32] have demonstrated that the effect of PM exposure uniquely mappableWGBSdatatobethe most re- on DNA methylation depends on the subfamily evolu- producible and accurate measurement of global DNA tionary age, with a stronger negative effect on older methylation levels by comparing with pyrosequencing LINE1 and younger Alu. Recently, older subfamilies of assays of RE, providing WGBS as the gold standard Alu and LINE1 elements (Alu J and L1-M) exhibit great method in methylomics for its unsurpassed reso- hypomethylation in chronic lymphocytic leukemia [33]. lution and coverage. Furthermore, the Alu Y sequence shows remarkable dif- Alu is the largest family of short interspersed nuclear ferences in DNA methylation state across colorectal can- elements in the human genome and LINE1 is a predom- cer drug resistance [34]. Taken together, these results inant member of LINEs [9]. The former is a non- suggest that the evolutionary age of TE subfamilies autonomous, transposable element (TE) to be mobilized might determine differential vulnerability of DNA in trans by LINE1, but the latter is only autonomous TE. methylation to environmental exposures. Moreover, they constitute the critical regulators of gen- etic information expression by providing regulatory se- Conclusions quences or introducing alternative start or stop codons The present study showed PM-induced hypomethylation into functional genes [25], providing to act as global of Alu and LINE1 elements with differential susceptibil- modifiers of gene expression through changes in their ity of the evolutionary subfamily, suggesting that RE hy- own methylation state. Accordingly, growing evidence pomethylation might be a vital mechanism underlying has shown that the altered methylation states of TEs the harmful effects of airborne PM and that monitoring might be associated with aging, autoimmune diseases, of the methylation status for a specific subset of RE cardiovascular disease, or cancer development and pro- could serve as interface sensors between PM and DNA gression [26], suggesting that these changes are not the methylation. Furthermore, these results could provide a simple consequences of the disease, but may often drive better understanding of the effects of PM exposure on the pathogenesis. Interestingly, Alu and LINE1 initiate RE subfamilies and the role of RE in response to envir- the spread of CpG island (CGI) methylation and the onmental risk factors related to human health and dis- CGI length is associates with their distribution [27], in- ease. This study is the first to utilize the WGBS platform dicating the potential centers for de novo methylation in the analysis of the subfamily-specific methylation of events. Unfortunately, current investigations have fo- RE in PM-exposed human skin and lung tissues. How- cused only on analyzing a single common sequence for ever, further work to analyze locus-specific hypomethy- Alu and LINE1 through pyrosequencing assays, which is lation of Alu and LINE1 remains challenging. easier to do than previous methods to quantify total gen- Abbreviations omic 5-methylcytosine [28]. Moreover, recent reports DMC: Differentially methylated cytosine; LINE1: Long interspersed nucleotide have shown that the methylation of a common sequence element 1; NHBE: Normal human bronchial epithelial cells; NHEK: Normal human epidermal keratinocytes; PAH: Polycyclic aromatic hydrocarbons; is not correlated with global methylation content in nor- PM: Particulate matter; RE: Repetitive elements; TE: Transposable element; mal tissues and that CpG content is a primary determin- WGBS: Whole-genome bisulfite sequencing ant of changes over time in DNA methylation at individual CpG sites [29, 30]. Supplementary information The second novel finding of the current study was that The online version contains supplementary material available at https://doi. the evolutionary age of RE subfamilies determined differ- org/10.1186/s41021-022-00235-4. ential susceptibility of DNA hypomethylation to ambient Additional file 1: Supplementary Fig. 1. Graphical overview and PM. Sparse data are available on the effects of environ- representative of Alu and LINE1 data analysis. (A) Workflow chart. mental exposures across different subfamilies of TEs. (B) Analysis of methylation index in every CG-cytosine using MOABS. Based on the peak period of amplification and the level (C) Extraction of repetitive elements including Alu and LINE1. (D) Six re- peats (AluJ, AluS, AluY, LiH, L1M, and L1P) extraction using python script. of nucleotide substitutions, Alu and LINE1 are subdi- (E) CGs extraction within 6 repeats. vided into each subfamily with different evolutionary Additional file 2: Supplementary Table 1. Overview of WGBS data ages; young (Alu Y and L1-H), intermediate (Alu S and analysis. L1-P), and old (Alu J and L1-M) subfamilies [30, 31]. Be- cause of frequent deamination of methylated cytosines Acknowledgements in CpG dinucleotides, older subfamilies remain less rich Authors thank Dr. Yong Chul Boo for his critical reading of this manuscript. in CpG sites and show weaker or no transposon activity, Authors’ contributions whereas young subfamilies are richer in CpGs and still DSK designed the project and draft the manuscript. JYL performed the transcriptionally active in the human genome. Moreover, experiments and data analysis. WKL performed the statistical analysis. All CpG content and DNA methylation levels vary authors approved final manuscript. Lee et al. Genes and Environment (2022) 44:8 Page 7 of 7 Funding 15. Tarantini L, Bonzini M, Apostoli P, Pegoraro V, Bollati V, Marinelli B, et al. This research was supported by Basic Science Research Program through the Effects of particulate matter on genomic DNA methylation content and National Research Foundation of Korea (NRF) funded by the Ministry of iNOS promoter methylation. Environ Health Perspect. 2009;117:217–22. Education, Science and Technology (No. NRF-2020R1I1A3067217) and by a 16. Madrigano J, Baccarelli A, Mittleman MA, Wright RO, Sparrow D, Vokonas PS, grant of the Korea Health Technology R&D Project through the Korea Health et al. Prolonged exposure to particulate pollution, genes associated with Industry Development Institute (KHIDI) funded by the Ministry of Health & glutathione pathways, and DNA methylation in a cohort of older men. Welfare, Republic of Korea (grant number: HP20C0004). Environ Health Perspect. 2011;119:977–82. 17. De Prins S, Koppen G, Jacobs G, Dons E, Van de Mieroop E, Nelen V, et al. Influence of ambient air pollution on global DNA methylation in healthy Availability of data and materials adults: A seasonal follow-up. Environ Int. 2013;59:418–24. All data generated or analyzed during this study are included in this 18. Ding R, Jin Y, Liu X, Zhu Z, Zhang Y, Wang T, et al. Characteristics of DNA published article. methylation changes induced traffic-related air pollution. Mutat Res Genet Toxicol Environ Mutagen. 2016;796:46–53. 19. Sae-Lee C, Biasi JD, Robinson N, Barrow TM, Mathers JC, Koutsidis G, et al. Declarations DNA methylation patterns LINE-1 and Alu for pre-symptomatic dementia in type 2 diabetes. PLoS One. 2020;15:e0234578. Ethics approval and consent to participate 20. Kim KY, Kim DS, Lee SK, Lee IK, Kang JH, Chang YS, et al. Association of low- Not applicable. dose exposure to persistent organic pollutants with global DNA hypomethylation in healthy Koreans. Environ Health Perspect. 2010;118:370–4. Consent for publication 21. Valinluck V, Tsai HH, Rogstad DK, Burdzy A, Bird A, Sowers LC. Oxidative Not applicable. damage to methyl-CpG sequence inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acids Res. 2004;32:4100–8. Competing interests 22. Li E, Zhang Y. DNA methylation in mammals. Cold Spring Harb Perspect The authors declare that they have no conflict of interests. Biol. 2014;6:a019133. 23. Terry MB, Delgado-Cruzatak L, Vin-Raviv N, Wu HC, Santella RM. DNA Author details methylation in white blood cells: Association with risk factors in Department of Anatomy and BK21 Plus KNU Biomedical Convergence epidemiologic studies. Epigenetics. 2011;6:828–37. Program, Daegu, Republic of Korea. Preventive Medicine, School of 24. Li Q, Hermanson PJ, Springer NM. Detection of DNA methylation by whole- Medicine, Kyungpook National University, Daegu, Republic of Korea. genome bisulfite sequencing. Methods Mol Biol. 2018;1676:185–96. Department of Anatomy, School of Medicine, Kyungpook National 25. Ali A, Han K, Liang P. Role of transposable elements in gene regulation in University, 2-101 Dongin-dong, Jung-gu, 702-422 Daegu, Republic of Korea. the human genome. Life (Basel). 2021;11:118. 26. Wilson AS, Power BE, Molloy PL. DNA hypomethylation and human Received: 29 August 2021 Accepted: 20 January 2022 diseases. Biochim Biophys Acta. 2007;1775:138–62. 27. Kang MI, Rhyu MG, Kim YH, Jung YC, Hong SJ, Cho CS, et al. The length of CpG islands is associated with the distribution of Alu and L1 retroelements. References Genomics. 2006;87:580–90. 1. Brunekreef B, Holgate ST. Air pollution and health. Lancet. 2002;360:1233–42. 28. Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method 2. Schraufnagel DE, Balmes JR, Cowl CT, De Matteis S, Jung SH, Mortimer K, for estimating global DNA methylation using bisulfite PCR of repetitive DNA et al. Air pollution and noncommunicable diseases: A review by the forum elements. Nucleic Acids Res. 2004;32:e38. of International Respiratory Societies’ Environmental Committee, Part 2: Air 29. Wolff EM, Byun HM, Han HF, Sharma S, Nichols PW, Siegmund KD, et al. pollution and organ systems. Chest. 2019;155:417–26. Hypomethylation of a LINE-1 promoter activates an alternate transcript of 3. Samet J, Krewski D. Health effects associated with exposure to ambient air the MET oncogene in bladders with cancer. PLoS Genet. 2010;6:e1000917. pollution. J Toxicol Environ Health A. 2007;70:227–42. 30. Byun HM, Nordio F, Coull BA, Tarantini L, Hou L, Bonzini M, et al. Temporal 4. Ruckerl R, Schneider A, Breitner S, Peters A. Health effects of particulate air stability of epigenetic markers: Sequence characteristics and predictors of pollution: A review of epidemiological evidence. Inhal Toxicol. 2011;23:555–92. short-term DNA methylation variations. PLoS One. 2012;7:e39220. 5. Kim KE, Cho D, Park HJ. Air pollution and skin disease: Adverse effects of 31. Ohshima K, Hattori M, Yada T, Gojobori T, Sakaki Y, Okada N. Whole-genome airborne particulate matter on various skin diseases. Life Sci. 2016;152:126–34. screening indicates a possible burst of formation of processed pseudogenes 6. Valavanidis A, Fiotakis K, Vlachogianni T. Airborne particulate matter and and Alu repeats by particular L1 subfamilies in ancestral primates. Molecular human health: Toxicological assessment and importance of size and reconstitution of extinct LINE-1 elements. Genome Biol. 2003;4:R74. composition of particles for oxidative damage and carcinogenic 32. Giordano J, Ge Y, Gelfand Y, Abrusan G, Benson G, Warburton PE. mechanisms. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2008; Evolutionary history of mammalian transposons determined by genome- 26:339–62. wide defragmentation. PLoS Comput Biol. 2007;3:e137. 7. Ozanne SE, Constancia M. Mechanisms of disease: the development origins 33. Byun HM, Motta V, Panni T, Bertazzi PA, Apostoli P, Hou L, et al. Evolutionary of disease and the role of the epigenotype. Nat Clin Prat Endocrinol Metab. age of repetitive element subfamilies and sensitivity of DNA methylation to 2007;3:539–46. airborne pollutants. Part Fibre Toxicol. 2013;10:28. 8. Rider CF, Carlsten C. Air pollution and DNA methylation: effects of exposure 34. Barrow TM, Doo NW, Milne RL, Giles GG, Willmore E, Strathdee G, et al. in humans. Clin Epigenetics. 2019;11:131. Analysis of retrotransposon subfamily DNA methylation reveals novel early 9. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial epigenetic changes in chronic lymphocytic leukemia. Haematologica. 2021; sequencing and analysis of the human genome. Nature. 2001;409:860–921. 106:98–110. 10. Mioussea IR, Chalbota MG, Lumenb A, Fergusona A, Kavourasa IG, 35. Lin X, Stenvang J, Rasmussen MH, Zhu S, Jensen NF, Tarpgaard LS, et al. The Koturbasha I. Transposable elements in response to environmental stressors. potential role of Alu Y in the development of resistance to SN38 Mutat Res Rev Mutat Res. 2015;765:19–39. (Irinotecan) or oxaliplatin in colorectal cancer. BMC Genom. 2015;16:404. 11. Mirouze M, Lanciano S. Transposable elements: All mobile, all different, some stress responsive, some adaptive. Curr Opin Genet Dev. 2018;49:106–14. 12. Kazazian HH, Moran JV. Mobile DNA in health and disease. N Engl J Med. Publisher’sNote 2017;377:361–70. Springer Nature remains neutral with regard to jurisdictional claims in 13. Burns KH. Our conflict with transposable elements and its implications for published maps and institutional affiliations. human disease. Annu Rev Pathol. 2020;15:51–70. 14. Ponomaryova AA, Rykova EY, Gervas PA, Cherdyntseva NV, Mamedov IZ, Azhikina TL. Aberrant methylation of LINE-1 transposable elements: A search for cancer biomarker. Cells. 2020;9:2017. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes and Environment Springer Journals

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

Lee, Ji Yun; Lee, Won Kee; Kim, Dong Sun
Genes and Environment , Volume 44 (1) – Feb 16, 2022
Download PDF
7 pages

Loading next page...
 
/lp/springer-journals/particulate-matter-induced-hypomethylation-of-alu-and-line1-in-normal-H0DCnqg0sP

References (39)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2022
eISSN
1880-7062
DOI
10.1186/s41021-022-00235-4
Publisher site
See Article on Publisher Site

Abstract

Background: Airborne 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 (PM ,PM , and 2.5 10 PM -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. Results: All PMs exposure significantly lowered Alu and LINE1 methylation in both cells than in mock-treated controls. Hypomethylation was more prominent in PM -PAH exposed-NHBE and PM exposed-NHEK. Alu and 10 10 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. Conclusions: These results demonstrate that the differential susceptibility of PM-induced hypomethylation of Alu and LINE1 depends upon RE evolutionary age and PM type. Keywords: Particulate matter, DNA methylation, WGBS, Alu, LINE1, Subfamily Introduction forms of air pollution, is recognized as a major health Air pollution has become an important health concern hazard worldwide, and is associated with respiratory, and ranks as the sixth leading risk factors for premature cardiovascular, and skin diseases [3–5]. However, the death globally [1, 2]. Exposure to air pollution is ubiqui- mechanisms linking PM exposure to adverse health out- tous and typically beyond the control of individuals, and comes have not been completely clarified. The size and the resulting health burden on the population can be composition determine the toxicity of the particle [6]. high. Particulate matter (PM), one of the most toxic PM consists of a mixture of volatile organic compounds, polycyclic aromatic hydrocarbons (PAH) and inorganic chemicals such as heavy metals that, both individually * Correspondence: doskim@knu.ac.kr Department of Anatomy and BK21 Plus KNU Biomedical Convergence and together, cause adverse health effects. PM consti- Program, Daegu, Republic of Korea tutes of microscopic particles of solid or liquid matter Department of Anatomy, School of Medicine, Kyungpook National suspended in the air. Airborne PM is usually classified as University, 2-101 Dongin-dong, Jung-gu, 702-422 Daegu, Republic of Korea Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lee et al. Genes and Environment (2022) 44:8 Page 2 of 7 coarse PM (<10 mm) and fine PM (<2.5 mm), de- former (PM -PAH) includes several PAHs (benzoan- 10 2.5 10 pending on the aerodynamic diameter of the particles. thracene, benzopyrene, benzofluoranthene, and diben- PM is composed of inhalable particles from dusts, in- zoanthracene, etc.) in ambient PM , the latter (PM ) 10 10 10 dustrial emissions, and traffic emissions. PM is pri- contains heavy metals (arsenic, cadmium, lead, and 2.5 marily composed of organic carbon compounds, nitrates, nickel). They were suspended in phosphate buffered sa- and sulfates. Understanding the biological action of PM line (PBS) at 5-mg/ml concentration. PM was prepared is of particular importance in improvement of public just before cell application and sonicated in an ultrasonic health. bath for 10 min to avoid variability in PM composition Alterations in DNA methylation are associated with and aggregation of particles. various health outcomes, representing an interface be- tween the environment and human disease [7]. Emer- Cell culture and PM treatment ging data indicate that PM exposure modulates DNA NHBE and NHEK were obtained from the American methylation, a major genomic mechanism of gene ex- Type Culture Collection (ATCC, Manassas, VA, USA). pression control, and that these changes might in turn NHEK (ATCC PCS-200-011) was grown in Dermal Cell influence inflammation, disease development, and ex- Basal Media (ATCC PCS-200-030) supplemented with acerbation risk [8]. However, whether such effects are Keratinocyte Growth Kit (ATCC PCS-200-040) to targeted to specific sites or scattered across the genome propagate in serum-free conditions. NHBE (ATCC PCS- globally remain challenging. Alu and long interspersed 300-011) was cultured in serum-free Airway Epithelial nucleotide element 1 (LINE1, L1) are significant compo- Cell Basal Media (ATCC PCS-300-030) supplemented nents of repetitive transposable DNA elements, consti- with Bronchial Epithelial Cell Growth Kit (ATCC PCS- tuting approximately 17% and 11% of the human 300-040). Both cells were grown at 50% confluency and genome, respectively [9], representing as a surrogate were treated with PM ,PM , and PM -PAH for 3 2.5 10 10 marker for genome-wide global methylation levels. Inter- days at a final 50-µg/ml concentration of without a estingly, transposable repeats are considered as a respon- medium change. Cells maintained in culture medium ser to environmental stressors [10, 11] and their with vehicle (0.1% DMSO or 1% PBS) were used as un- reactivation through hypomethylation can increase gen- treated control groups. ome instability, reactivate lowly expressed genes, or dis- rupted gene function, thereby potentially contributing to WGBS library preparation and sequencing disease-related pathological consequences [12, 13] and The cells were washed with PBS, and genomic DNA was provide promising candidate biomarkers for human dis- extracted using a QIAamp DNA Mini Kit (Qiagen, Val- ease including cancer [14]. Unfortunately, PM-induced encia, CA, USA) according to the manufacturer’s in- DNA methylation of repetitive elements (RE) reported struction. The concentration and quality of the DNA in most previous studies is measured in blood cells, were determined using an Agilent Bioanalyzer 2100 representing overall results from the body organs [15– (Agilent Technologies, Santa Clara CA, USA) and agar- 18]. However, methylation in skin and lung, major tar- ose gel electrophoresis. DNAs were fragmented using a gets of air pollution, has not been well studied. In the Bioruptor (Diagenode, Liege, Belgium) to an average size present study, we treated the normal human bronchial of approximately 250 bp, followed by the blunt ending, epithelial cells (NHBE) and normal human epidermal 3’-end addition of dA, and adaptor ligation (in this case keratinocytes (NHEK) with inhaled and non-inhaled PM of methylated adaptors to protect from bisulfite conver- (PM ,PM , and PM -PAH) and then determined the sion). Ligated DNA was bisulfite-converted using the EZ 2.5 10 10 changes in global DNA methylation using whole- DNA Methylation-Gold kit (Zymo Research Corp, Ir- genome bisulfite sequencing (WGBS) and vine, CA, USA). Fragments pf length 200–250 bp were pyrosequencing. excised from a 2% TAE agarose gel, purified using a QIAquick Gel Extraction Kit (Qiagen), and then ampli- Materials and methods fied via PCR. Libraries were constructed from PCR prod- PM preparation ucts with BGI’s DNA nanoball (DNB) technology. The PM , which is a standard diesel PM (SRM1650b) issued qualified libraries were sequenced using the DNBSEQ®- 2.5 by the National Institute of Standard and Technology platform (BGI, Shenzhen, China). Base-calling was per- (Gaithersburg, MD, USA), was bought from Sigma- formed using the BGISEQ-500 software (v 0.3.8.1111). Aldrich (St. Louis, MO, USA). It was dissolved in di- methyl sulfoxide (DMSO) at 50-mg/ml concentration. Data filtering PM -like fine dusts (ERM-CZ100 and ERM-CZ120), Data filtering was conducted using the elimination of which are issued by the European Reference Materials contaminating DNA and low-quality reads. Low-quality (ERM, Belgium), were brought from Sigma-Aldrich. The reads include three types and the reading that accord Lee et al. Genes and Environment (2022) 44:8 Page 3 of 7 with one of them will be removed: (1) contain adaptor Statistical analysis sequence; (2) N-base number >10%; (3) the number of Data are presented as the means ± standard error bases with a quality of <20% and >10% was trimmed. (SE) of three independent experiments. One-way Only clean data was used for further analyses. ANOVA was used for the mean difference test be- tween the groups, and Bonferroni’s correction p-value was used for post-hoc comparison the two groups Reads mapping and differentially methylated level when the ANOVA was significant. The statistical analysis analyses were conducted using SAS 9.4 (SAS Inc., Clean reads of each sample were mapped to human UCSC Cary, NC, USA), and the plots were constructed using hg19 reference genome using BSMAP software (v2.90) to R version 4.1.0 (The R Foundation for Statistical obtain BAM file. BAM files were sorted and indexed using Computing, Vienna, Austria). The significance level of Samtools software (v0.1.18). The parameters in the map- the statistical test was set to 5%. ping and the results of mapping were shown in Supple- mentary Table 1. Methylation level was determined by Results dividing the number of reads covering each methylcyto- We comprehensively examined the effect of inhaled and sine by the total reads covering the cytosine. MOABS soft- non-inhaled PM exposure on RE Alu and LINE1 by WGBS. ware (v1.3.2) was used to calculate the methylation level Based on the reference genome and the UCSC RepeatMas- of every cytosine in every sample, and to determine differ- ker, approximately 34.6% of all 28 million CpG sites are in entially methylated cytosine (DMC). After calculating the Alu (25.0%) and LINE1 (9.6%). The RepeatMasker library methylation level, cytosine was considered as “hypomethy- mapped 7,040,695 Alu and 2,651,373 LINE1 loci in the lated” when the methylation level of cytosine ≤0.2, and UCSC hg19 reference genome assembly, corresponding to Fisher’s Exact Test p-value was <0.05. 10.1% and 17.1% of the human genome respectively (Table 1). Because human Alu and LINE1 are heavily meth- Alu and LINE1 methylation analysis ylated in normal tissues, all PMs exposure significantly in- To analyze the methylation level of six evolutionary sub- creased the unmethylated CpGs of Alu and LINE1 in both families in Alu (Alu Y, Alu S, and Alu J) and LINE1 (L1- NHBE and NHEK comparing with mock-treated (0.1% H, L1-P, and L1-M), genomic coordinates of all repeats DMSO or 1% PBS) cells (Table 1), indicating a potentially based on hg19 were extracted and obtained from UCSC defective functionality of these RE. Moreover, PM-induced genome browser using RepeatMasker track as Sae-Lee hypomethylation was prominent in NHBE compared with et al. [19]. All subtypes of 6 target repeats (for example, NHEK as well as in LINE1 compared with Alu (Fig. 1). Al- AluJb, AluJo, AluJr, etc. for AluJ subtypes) were ex- though there was a narrow margin between each PM, tracted, and methyl-cytosine data within 6 repeats were PM -PAH and PM exhibited the strongest effect on RE 10 10 collected from DMC data of MOABS results in each hypomethylation in NHBE and NHEK, respectively (Fig. 1). sample using Python scripts. Data analysis workflow was Furthermore, because CpG content and DNA methylation shown in Supplementary Fig. 1. All CG sites in hg19 and levels dramatically differ across subfamilies, we evaluated DMCs within six repeats were counted through the the sensitivity of DNA methylation in differentially-evolved samples. Alu and LINE1 subfamilies to different types of airborne PM. We subdivided Alu and LINE1 into three evolutionary Pyrosequencing of Alu and LINE1 subfamilies; oldest Alu J and L1-M, intermediate Alu S and Bisulfite-converted DNA was amplified with PCR L1-P, and youngest Alu Y and L1-H. Interestingly, Alu J primers under previously described conditions [20]. The andL1-Mshowedthe strongesthypomethylation in NHBE PCR products were then assayed on the PyroMark Q24 following treatment with three PMs, whereas Alu Y and with PyroMark Gold Q24 Reagents (Qiagen) and then L1-M exhibited the strongest hypomethylation in the analyzed with accompanying software. The degree of NHEK (Fig. 2), indicating the association of differential sus- methylation was expressed for each DNA locus as a per- ceptibility of the RE hypomethylation with evolutionary centage of methylated cytosines over the sum of methyl- ages of subfamilies. In addition, although pyrosequencing is ated and unmethylated cytosines. We used non-CpG not expected to comprehensively reflect DNA methylation cytosine residues as built-in controls to verify the bisul- patterns within individual subfamilies, we have validated fite conversion. In every pyrosequencing run, three con- the WGBS results by pyrosequencing for accuracy and re- trols were included. One well was filled with water to producibility of methylation levels. Significantly reduced ensure no contamination, and two wells were filled with methylation of Alu and LINE1 was detectable in both CpGenome universal methylated and unmethylated NHBE and NHEK following all PMs exposure (Fig. 3). Like- DNA (Chemicon, Temecula, CA, USA) as positive and wise, PM -PAH and PM exhibited the strongest effect 10 10 negative control to weigh the repeatability of the assay. on RE hypomethylation in NHBE and NHEK, respectively Lee et al. Genes and Environment (2022) 44:8 Page 4 of 7 Table 1 PM-induced hypomethylation of Alu and LINE1 in NHBE and NHEK L1-H L1-P L1-M L1 PM PM PM -PAH PM PM PM -PAH PM PM PM -PAH PM PM PM -PAH 2.5 10 10 2.5 10 10 2.5 10 10 2.5 10 10 Unmethylated CpGs 604 622 702 29,409 30,050 34,696 40,765 42,033 49,037 70,778 72,706 84,603 in NHBE Unmethylated CpGs 591 619 592 20,902 22,228 20,774 24,707 26,474 27,270 46,201 49,322 48,637 in NHEK Analyzed total CpGs 43,045 1,282,366 1,325,962 2,651,373 Alu Y Alu S Alu J Alu PM PM PM -PAH PM PM PM -PAH PM PM PM -PAH PM PM PM -PAH 2.5 10 10 2.5 10 10 2.5 10 10 2.5 10 10 Unmethylated CpGs 28,627 26,533 28,674 92,202 85,521 99,032 17,746 17,666 19,296 138,575 129,719 144,627 in NHBE Unmethylated CpGs 17,907 28,937 19,691 40,236 52,578 43,912 6,361 7,565 7,964 64,503 88,747 71,567 in NHEK Analyzed total CpGs 1,784,717 4,454,205 801,773 7,040,695 (Fig. 3), showing that a similar trend might be present be- RE hypomethylation [21]. DNA methylation is a tween WGBS and pyrosequencing methods. common feature of eukaryotic genomes and is a core epigenetic process that influences numerous bio- Discussion logical processes, such as gene repression, control of The first major finding of this study was that Alu and cellular development and differentiation, RE silen- LINE1 methylation was significantly lower after PM ex- cing, and maintenance of genome stability [22]. DNA posures, providing for the first direct experimental evi- methylation mainly changed at locus-specific and dence that PM exposure induces DNA hypomethylation genome-wide levels. A number of methods are avail- in NHBE and NHEK. Recent in vitro experiments have able for the analysis of global DNA methylation shown that oxidative DNA damage by PM can interfere levels [23]. Recently, WGBS has revolutionized the with the ability of DNA methyltransferase, resulting in way of interrogating the methylome to realize Fig. 1 Alu and LINE1 methylation change in NHBE and NHEK after PM exposure. The hypomethylation ratio was calculated as a percentage of CpGs with low methylation to analyzed total CpG sites and the means ± SE of three independent WGBS experiments are shown. The mean methylation ratios of PM-treated cells were compared to mock-treated cells using one-way ANOVA, and Bonferroni’s correction p-value was used for post-hoc comparison between two groups where the ANOVA was significant Lee et al. Genes and Environment (2022) 44:8 Page 5 of 7 Fig. 2 Differential hypomethylation of Alu and LINE1 evolutionary subfamilies in NHBE and NHEK following PM treatment. (A) Alu change in NHBE. (B) LINE1 change in NHBE. (C) Alu change in NHEK. (D) LINE1 change in NHEK. The means ± SE of three independent WGBS experiments are shown. Bonferroni-adjusted p-value was the result of comparing between the two groups Fig. 3 Distribution of Alu and LINE1 methylation level in PM-exposed NHBE (A) and NHEK (B). Methylation level was expressed as a percentage of 5-methylcytosine divided by the sum of methylated and unmethylated cytosines. The mean ± 95% confidence interval of three independent pyrosequencing is shown. The comparisons of mean methylation levels were evaluated using ANOVA. Bonferroni-adjusted p-value was the result of a b comparing between the two groups. Cells maintained in culture medium with vehicle were used as control group (CTL , 0.1% DMSO; CTL ,1% PBS) Lee et al. Genes and Environment (2022) 44:8 Page 6 of 7 genome-wide methylation analysis at a single-base dramatically across subfamilies. Interestingly, Byun et al. resolution [24]. The current study determined [32] have demonstrated that the effect of PM exposure uniquely mappableWGBSdatatobethe most re- on DNA methylation depends on the subfamily evolu- producible and accurate measurement of global DNA tionary age, with a stronger negative effect on older methylation levels by comparing with pyrosequencing LINE1 and younger Alu. Recently, older subfamilies of assays of RE, providing WGBS as the gold standard Alu and LINE1 elements (Alu J and L1-M) exhibit great method in methylomics for its unsurpassed reso- hypomethylation in chronic lymphocytic leukemia [33]. lution and coverage. Furthermore, the Alu Y sequence shows remarkable dif- Alu is the largest family of short interspersed nuclear ferences in DNA methylation state across colorectal can- elements in the human genome and LINE1 is a predom- cer drug resistance [34]. Taken together, these results inant member of LINEs [9]. The former is a non- suggest that the evolutionary age of TE subfamilies autonomous, transposable element (TE) to be mobilized might determine differential vulnerability of DNA in trans by LINE1, but the latter is only autonomous TE. methylation to environmental exposures. Moreover, they constitute the critical regulators of gen- etic information expression by providing regulatory se- Conclusions quences or introducing alternative start or stop codons The present study showed PM-induced hypomethylation into functional genes [25], providing to act as global of Alu and LINE1 elements with differential susceptibil- modifiers of gene expression through changes in their ity of the evolutionary subfamily, suggesting that RE hy- own methylation state. Accordingly, growing evidence pomethylation might be a vital mechanism underlying has shown that the altered methylation states of TEs the harmful effects of airborne PM and that monitoring might be associated with aging, autoimmune diseases, of the methylation status for a specific subset of RE cardiovascular disease, or cancer development and pro- could serve as interface sensors between PM and DNA gression [26], suggesting that these changes are not the methylation. Furthermore, these results could provide a simple consequences of the disease, but may often drive better understanding of the effects of PM exposure on the pathogenesis. Interestingly, Alu and LINE1 initiate RE subfamilies and the role of RE in response to envir- the spread of CpG island (CGI) methylation and the onmental risk factors related to human health and dis- CGI length is associates with their distribution [27], in- ease. This study is the first to utilize the WGBS platform dicating the potential centers for de novo methylation in the analysis of the subfamily-specific methylation of events. Unfortunately, current investigations have fo- RE in PM-exposed human skin and lung tissues. How- cused only on analyzing a single common sequence for ever, further work to analyze locus-specific hypomethy- Alu and LINE1 through pyrosequencing assays, which is lation of Alu and LINE1 remains challenging. easier to do than previous methods to quantify total gen- Abbreviations omic 5-methylcytosine [28]. Moreover, recent reports DMC: Differentially methylated cytosine; LINE1: Long interspersed nucleotide have shown that the methylation of a common sequence element 1; NHBE: Normal human bronchial epithelial cells; NHEK: Normal human epidermal keratinocytes; PAH: Polycyclic aromatic hydrocarbons; is not correlated with global methylation content in nor- PM: Particulate matter; RE: Repetitive elements; TE: Transposable element; mal tissues and that CpG content is a primary determin- WGBS: Whole-genome bisulfite sequencing ant of changes over time in DNA methylation at individual CpG sites [29, 30]. Supplementary information The second novel finding of the current study was that The online version contains supplementary material available at https://doi. the evolutionary age of RE subfamilies determined differ- org/10.1186/s41021-022-00235-4. ential susceptibility of DNA hypomethylation to ambient Additional file 1: Supplementary Fig. 1. Graphical overview and PM. Sparse data are available on the effects of environ- representative of Alu and LINE1 data analysis. (A) Workflow chart. mental exposures across different subfamilies of TEs. (B) Analysis of methylation index in every CG-cytosine using MOABS. Based on the peak period of amplification and the level (C) Extraction of repetitive elements including Alu and LINE1. (D) Six re- peats (AluJ, AluS, AluY, LiH, L1M, and L1P) extraction using python script. of nucleotide substitutions, Alu and LINE1 are subdi- (E) CGs extraction within 6 repeats. vided into each subfamily with different evolutionary Additional file 2: Supplementary Table 1. Overview of WGBS data ages; young (Alu Y and L1-H), intermediate (Alu S and analysis. L1-P), and old (Alu J and L1-M) subfamilies [30, 31]. Be- cause of frequent deamination of methylated cytosines Acknowledgements in CpG dinucleotides, older subfamilies remain less rich Authors thank Dr. Yong Chul Boo for his critical reading of this manuscript. in CpG sites and show weaker or no transposon activity, Authors’ contributions whereas young subfamilies are richer in CpGs and still DSK designed the project and draft the manuscript. JYL performed the transcriptionally active in the human genome. Moreover, experiments and data analysis. WKL performed the statistical analysis. All CpG content and DNA methylation levels vary authors approved final manuscript. Lee et al. Genes and Environment (2022) 44:8 Page 7 of 7 Funding 15. Tarantini L, Bonzini M, Apostoli P, Pegoraro V, Bollati V, Marinelli B, et al. This research was supported by Basic Science Research Program through the Effects of particulate matter on genomic DNA methylation content and National Research Foundation of Korea (NRF) funded by the Ministry of iNOS promoter methylation. Environ Health Perspect. 2009;117:217–22. Education, Science and Technology (No. NRF-2020R1I1A3067217) and by a 16. Madrigano J, Baccarelli A, Mittleman MA, Wright RO, Sparrow D, Vokonas PS, grant of the Korea Health Technology R&D Project through the Korea Health et al. Prolonged exposure to particulate pollution, genes associated with Industry Development Institute (KHIDI) funded by the Ministry of Health & glutathione pathways, and DNA methylation in a cohort of older men. Welfare, Republic of Korea (grant number: HP20C0004). Environ Health Perspect. 2011;119:977–82. 17. De Prins S, Koppen G, Jacobs G, Dons E, Van de Mieroop E, Nelen V, et al. Influence of ambient air pollution on global DNA methylation in healthy Availability of data and materials adults: A seasonal follow-up. Environ Int. 2013;59:418–24. All data generated or analyzed during this study are included in this 18. Ding R, Jin Y, Liu X, Zhu Z, Zhang Y, Wang T, et al. Characteristics of DNA published article. methylation changes induced traffic-related air pollution. Mutat Res Genet Toxicol Environ Mutagen. 2016;796:46–53. 19. Sae-Lee C, Biasi JD, Robinson N, Barrow TM, Mathers JC, Koutsidis G, et al. Declarations DNA methylation patterns LINE-1 and Alu for pre-symptomatic dementia in type 2 diabetes. PLoS One. 2020;15:e0234578. Ethics approval and consent to participate 20. Kim KY, Kim DS, Lee SK, Lee IK, Kang JH, Chang YS, et al. Association of low- Not applicable. dose exposure to persistent organic pollutants with global DNA hypomethylation in healthy Koreans. Environ Health Perspect. 2010;118:370–4. Consent for publication 21. Valinluck V, Tsai HH, Rogstad DK, Burdzy A, Bird A, Sowers LC. Oxidative Not applicable. damage to methyl-CpG sequence inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acids Res. 2004;32:4100–8. Competing interests 22. Li E, Zhang Y. DNA methylation in mammals. Cold Spring Harb Perspect The authors declare that they have no conflict of interests. Biol. 2014;6:a019133. 23. Terry MB, Delgado-Cruzatak L, Vin-Raviv N, Wu HC, Santella RM. DNA Author details methylation in white blood cells: Association with risk factors in Department of Anatomy and BK21 Plus KNU Biomedical Convergence epidemiologic studies. Epigenetics. 2011;6:828–37. Program, Daegu, Republic of Korea. Preventive Medicine, School of 24. Li Q, Hermanson PJ, Springer NM. Detection of DNA methylation by whole- Medicine, Kyungpook National University, Daegu, Republic of Korea. genome bisulfite sequencing. Methods Mol Biol. 2018;1676:185–96. Department of Anatomy, School of Medicine, Kyungpook National 25. Ali A, Han K, Liang P. Role of transposable elements in gene regulation in University, 2-101 Dongin-dong, Jung-gu, 702-422 Daegu, Republic of Korea. the human genome. Life (Basel). 2021;11:118. 26. Wilson AS, Power BE, Molloy PL. DNA hypomethylation and human Received: 29 August 2021 Accepted: 20 January 2022 diseases. Biochim Biophys Acta. 2007;1775:138–62. 27. Kang MI, Rhyu MG, Kim YH, Jung YC, Hong SJ, Cho CS, et al. The length of CpG islands is associated with the distribution of Alu and L1 retroelements. References Genomics. 2006;87:580–90. 1. Brunekreef B, Holgate ST. Air pollution and health. Lancet. 2002;360:1233–42. 28. Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP. A simple method 2. Schraufnagel DE, Balmes JR, Cowl CT, De Matteis S, Jung SH, Mortimer K, for estimating global DNA methylation using bisulfite PCR of repetitive DNA et al. Air pollution and noncommunicable diseases: A review by the forum elements. Nucleic Acids Res. 2004;32:e38. of International Respiratory Societies’ Environmental Committee, Part 2: Air 29. Wolff EM, Byun HM, Han HF, Sharma S, Nichols PW, Siegmund KD, et al. pollution and organ systems. Chest. 2019;155:417–26. Hypomethylation of a LINE-1 promoter activates an alternate transcript of 3. Samet J, Krewski D. Health effects associated with exposure to ambient air the MET oncogene in bladders with cancer. PLoS Genet. 2010;6:e1000917. pollution. J Toxicol Environ Health A. 2007;70:227–42. 30. Byun HM, Nordio F, Coull BA, Tarantini L, Hou L, Bonzini M, et al. Temporal 4. Ruckerl R, Schneider A, Breitner S, Peters A. Health effects of particulate air stability of epigenetic markers: Sequence characteristics and predictors of pollution: A review of epidemiological evidence. Inhal Toxicol. 2011;23:555–92. short-term DNA methylation variations. PLoS One. 2012;7:e39220. 5. Kim KE, Cho D, Park HJ. Air pollution and skin disease: Adverse effects of 31. Ohshima K, Hattori M, Yada T, Gojobori T, Sakaki Y, Okada N. Whole-genome airborne particulate matter on various skin diseases. Life Sci. 2016;152:126–34. screening indicates a possible burst of formation of processed pseudogenes 6. Valavanidis A, Fiotakis K, Vlachogianni T. Airborne particulate matter and and Alu repeats by particular L1 subfamilies in ancestral primates. Molecular human health: Toxicological assessment and importance of size and reconstitution of extinct LINE-1 elements. Genome Biol. 2003;4:R74. composition of particles for oxidative damage and carcinogenic 32. Giordano J, Ge Y, Gelfand Y, Abrusan G, Benson G, Warburton PE. mechanisms. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2008; Evolutionary history of mammalian transposons determined by genome- 26:339–62. wide defragmentation. PLoS Comput Biol. 2007;3:e137. 7. Ozanne SE, Constancia M. Mechanisms of disease: the development origins 33. Byun HM, Motta V, Panni T, Bertazzi PA, Apostoli P, Hou L, et al. Evolutionary of disease and the role of the epigenotype. Nat Clin Prat Endocrinol Metab. age of repetitive element subfamilies and sensitivity of DNA methylation to 2007;3:539–46. airborne pollutants. Part Fibre Toxicol. 2013;10:28. 8. Rider CF, Carlsten C. Air pollution and DNA methylation: effects of exposure 34. Barrow TM, Doo NW, Milne RL, Giles GG, Willmore E, Strathdee G, et al. in humans. Clin Epigenetics. 2019;11:131. Analysis of retrotransposon subfamily DNA methylation reveals novel early 9. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial epigenetic changes in chronic lymphocytic leukemia. Haematologica. 2021; sequencing and analysis of the human genome. Nature. 2001;409:860–921. 106:98–110. 10. Mioussea IR, Chalbota MG, Lumenb A, Fergusona A, Kavourasa IG, 35. Lin X, Stenvang J, Rasmussen MH, Zhu S, Jensen NF, Tarpgaard LS, et al. The Koturbasha I. Transposable elements in response to environmental stressors. potential role of Alu Y in the development of resistance to SN38 Mutat Res Rev Mutat Res. 2015;765:19–39. (Irinotecan) or oxaliplatin in colorectal cancer. BMC Genom. 2015;16:404. 11. Mirouze M, Lanciano S. Transposable elements: All mobile, all different, some stress responsive, some adaptive. Curr Opin Genet Dev. 2018;49:106–14. 12. Kazazian HH, Moran JV. Mobile DNA in health and disease. N Engl J Med. Publisher’sNote 2017;377:361–70. Springer Nature remains neutral with regard to jurisdictional claims in 13. Burns KH. Our conflict with transposable elements and its implications for published maps and institutional affiliations. human disease. Annu Rev Pathol. 2020;15:51–70. 14. Ponomaryova AA, Rykova EY, Gervas PA, Cherdyntseva NV, Mamedov IZ, Azhikina TL. Aberrant methylation of LINE-1 transposable elements: A search for cancer biomarker. Cells. 2020;9:2017.

Journal

Genes and Environment – Springer Journals

Published: Feb 16, 2022

Keywords: Particulate matter; DNA methylation; WGBS; Alu; LINE1; Subfamily

There are no references for this article.