Freeman et al. Epigenetics & Chromatin (2020) 13:17 https://doi.org/10.1186/s13072-020-00338-8 Epigenetics & Chromatin RESEARCH Open Access The conserved DNMT1-dependent methylation regions in human cells are vulnerable to neurotoxicant rotenone exposure Dana M. Freeman1 , Dan Lou1, Yanqiang Li1, Suzanne N. Martos1 and Zhibin Wang1,2,3* Abstract Background: Allele-specifc DNA methylation (ASM) describes genomic loci that maintain CpG methylation at only one inherited allele rather than having coordinated methylation across both alleles. The most prominent of these regions are germline ASMs (gASMs) that control the expression of imprinted genes in a parent of origin-dependent manner and are associated with disease. However, our recent report reveals numerous ASMs at non-imprinted genes. These non-germline ASMs are dependent on DNA methyltransferase 1 (DNMT1) and strikingly show the feature of random, switchable monoallelic methylation patterns in the mouse genome. The signifcance of these ASMs to human health has not been explored. Due to their shared allelicity with gASMs, herein, we propose that non-tradi- tional ASMs are sensitive to exposures in association with human disease. Results: We frst explore their conservancy in the human genome. Our data show that our putative non-germline ASMs were in conserved regions of the human genome and located adjacent to genes vital for neuronal develop- ment and maturation. We next tested the hypothesized vulnerability of these regions by exposing human embryonic kidney cell HEK293 with the neurotoxicant rotenone for 24 h. Indeed,14 genes adjacent to our identifed regions were diferentially expressed from RNA-sequencing. We analyzed the base-resolution methylation patterns of the predicted non-germline ASMs at two neurological genes, HCN2 and NEFM, with potential to increase the risk of neurodegenera- tion. Both regions were signifcantly hypomethylated in response to rotenone. Conclusions: Our data indicate that non-germline ASMs seem conserved between mouse and human genomes, overlap important regulatory factor binding motifs, and regulate the expression of genes vital to neuronal function. These results support the notion that ASMs are sensitive to environmental factors such as rotenone and may alter the risk of neurological disease later in life by disrupting neuronal development. Keywords: DNA methylation, Allele-specifc methylation, Rotenone, Parkinson’s disease, Neurotoxicity Background loci, DNA methylation is coordinated across both inher- DNA methylation refers to the addition of a methyl group ited alleles. However, some loci maintain CpG methyla- (CH3) to the cytosine base of DNA by DNA methyltrans- tion at only one allele and these regions are described to ferases. Tis predominately occurs at cytosine–guanine have allele-specifc methylation (ASM; previously known adjacent sites known as CpG sites. For most genomic as diferentially methylated region DMR) [6]. Te most well-known of these regions are germline ASMs which *Correspondence: [email protected] control the expression of imprinted genes in a parent of 1 Laboratory of Environmental Epigenomes, Department origin-dependent manner. Imprinted genes are crucial in of Environmental Health & Engineering, Bloomberg School of Public development and are commonly associated with genetic Health, Johns Hopkins University, Baltimore, MD, USA Full list of author information is available at the end of the article disorders such as Beckwith–Wiedemann, Angelman, and © The Author(s) 2020. 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://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Freeman et al. Epigenetics & Chromatin (2020) 13:17 Page 2 of 16 Prader-Willi syndromes [10]. In addition to the control of Tese regions regulate the expression of non-imprinted imprinted gene expression, DNA methylation is a key to genes and these genes are hypothesized to have random maintain genome stability via silencing retrotransposons monoallelic expression. Due to their predicted monoal- [11, 74]. lelicity (DNA methylation and transcripts), we hypoth- Investigations demonstrate that two types of genomic esize that these regions are also targets for environmental regions, imprinted germline ASMs and intracisternal factors and associated with disease like germline ASMs A-particle (IAP)-like retrotransposons, seem vulnerable [34, 70]. to environmental factors. Terefore, these two regions Te goal of this study was to determine whether our are proposed to be pivotal for understanding human dis- identifed candidate ASMs in the mouse genome were in ease in response to exposure and popularly pursued in conserved regions of the human genome and to explore animal and epidemiological studies [32, 51]. Te former the possible adverse efects of diferential methylation is attractive because exposure altered ASMs are antici- in these regions by examining their tissue expressivity pated to be faithfully transmitted to somatic cells dur- and functional enrichment. Last, we tested our hypoth- ing rounds of global demethylation and remethylation in esis that genes adjacent to non-germline ASMs would be early embryos [5, 33]. As a result, parental exposure can vulnerable to environmental factors by exposing human be epigenetically inherited to modify ofspring phenotype embryonic kidney HEK293 cells to the pesticide rotenone [22]. Te latter is exemplifed in mice by the bisphenol for 24 h. We used whole transcriptome RNA-sequencing A-hypomethylated IAP at the agouti gene for variations and targeted bisulfte-amplicon sequencing to evalu- of coat color and obesity, as well as by altered methyla- ate changes in expression of adjacent genes and DNA tion of IAPs at AxinFu for tail kinkiness [17, 61, 83]. methylation at candidate ASMs in response to rotenone. In our recent work, we developed two approaches, Indeed, our data demonstrate the vulnerability of these no-rescued DMRs (NORED) and methylation mosaicity new non-traditional ASMs to environmental exposure analyses (MethylMosaic), to identify numerous genomic [23]. loci bearing potential ASMs [48]. Both the known imprinted germline ASMs and newly identifed ASMs Results are dependent on DNA methyltransferase 1 (DNMT1) DNMT1‑dependent regions in the mouse and human [44]. Many of these novel ASMs are presumably sequence genome (single nucleotide polymorphism; SNP)-infuenced Two approaches, NORED and MethylMosaic, inde- ASMs [35]. For example, in a reciprocal cross between pendently identifed over 2000 regions with DNMT1 129S1/SvlmJ and Cast/EiJ or between C57BL/6NJ and dependency and allele-specifc methylation. To simplify Cast/EiJ, the Cast allele with SNP C of Hcn2 ASM is future interpretation, we focused on 207 overlapped always hypomethylated (i.e., independent of parental ori- regions (i.e., ‘NORED + MethylMosaic’ regions) to initi- gin), whereas the 129 allele or the C57 allele with SNP A ate our investigation. We compared these 207 regions is always hypermethylated. Standing out of the previously from mouse and observed 145 of these regions were con- appreciated sequence-dependent ASMs, a new paradigm served in the human genome. Most regions identifed in of switchable ASMs that shows equal chances of either the human genome were highly conserved with > 70% paternal or maternal allele to be methylated was revealed matched bases for more than 90% of the entire span of by our report [48]. Importantly, the switchable feature the region (Fig. 1a, b). Analyzing these regions in the seems also conserved in the human genome. At the genome browser, we noted that 70% of the conserved DLGAP2 locus, independent evidence confrms a mater- regions in the human genome were located in the gene nally imprinted ASM during pre-implantation switched body and approximately 50% of them had transcrip- to a random ASM in somatic tissues during gestation tion factor binding sites in human embryonic stem cells [50]. Collectively, the mouse genome or human genome (Fig. 1c, d). Te two transcription factors that were the contains more ASMs (including both sequence-depend- most signifcantly enriched were POL2RA and TAF1 ent and switchable ASMs) than previously appreciated with binding sites at 19% of conserved DNMT1 regions. [15, 48, 50, 55]. Te newly revealed random, switchable Both were concentrated around transcription start sites ASMs remind us of features in X chromosome inactiva- and regulate RNA polymerase II binding and processiv- tion, leading to a proposed hypothesis of regional autoso- ity in gene transcription. Te third most enriched tran- mal chromosome inactivation [76]. scription factor