Wang et al. Genes & Nutrition (2021) 16:1 https://doi.org/10.1186/s12263-020-00681-1 RESEARCH Open Access Maternal folic acid impacts DNA methylation profile in male rat offspring implicated in neurodevelopment and learning/memory abilities Xinyan Wang1, Zhenshu Li1, Yun Zhu2,3, Jing Yan3,4, Huan Liu1,3, Guowei Huang1,3 and Wen Li1,3* Abstract Background: Periconceptional folic acid (FA) supplementation not only reduces the incidence of neural tube defects, but also improves cognitive performances in offspring. However, the genes or pathways that are epigenetically regulated by FA in neurodevelopment were rarely reported. Methods: To elucidate the underlying mechanism, the effect of FA on the methylation profiles in brain tissue of male rat offspring was assessed by methylated DNA immunoprecipitation chip. Differentially methylated genes (DMGs) and gene network analysis were identified using DAVID and KEGG pathway analysis. Results: Compared with the folate-normal diet group, 1939 DMGs were identified in the folate-deficient diet group, and 1498 DMGs were identified in the folate-supplemented diet group, among which 298 DMGs were overlapped. The pathways associated with neurodevelopment and learning/memory abilities were differentially methylated in response to maternal FA intake during pregnancy, and there were some identical and distinctive potential mechanisms under FA deficiency or FA-supplemented conditions. Conclusions: In conclusion, genes and pathways associated with neurodevelopment and learning/memory abilities were differentially methylated in male rat offspring in response to maternal FA deficiency or supplementation during pregnancy. Keywords: Folic acid, Pregnancy, DNA methylation, Learning/memory, Synaptic plasticity Introduction demonstrated that maternal FA supplementation during Emerging evidence has indicated that early life nutrition pregnancy promoted offspring’s neurogenesis and synap- influences brain development and exerts long-term conse- togenesis at birth [5], stimulated sensory-motor reflex de- quences for memory abilities profoundly [1, 2]. Pericon- velopment in infancy [6, 7], and exerted long-term ceptional folic acid (FA) supplementation reduces the beneficial effects on learning and memory abilities in ado- incidence of neural tube defects and improves cognitive lescence and adult in rat offspring [6]. Our previous study performances in offspring [3, 4]. Our previous studies also found maternal folic acid supplementation increased in the offspring’s brains the methylation potential, global DNA methylation level, and DNMT expression and activ- * Correspondence: [email protected] 1Department of Nutrition and Food Science, School of Public Health, Tianjin ity [7]. However, the genes or pathways that are epigeneti- Medical University, Tianjin 300070, China cally regulated by FA in neurodevelopment were rarely 3 Tianjin Key Laboratory of Environment, Nutrition and Public Health, Center reported. for International Collaborative Research on Environment, Nutrition and Public Health, Tianjin 300070, China Through one-carbon metabolism, folate provides a la- Full list of author information is available at the end of the article bile source of methyl groups for numerous methylation © The Author(s). 2021 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/. Wang et al. Genes & Nutrition (2021) 16:1 Page 2 of 11 reactions, including DNA methylation [8]. DNA methy- diet added a 1.4 mg folic acid/kg diet compared to the lation and histone modification are the most studied epi- folate-normal diet, which in rats is equivalent to con- genetic modifications reprogrammed considerably in suming a 400 μg folic acid supplement daily on consum- early embryonic development [9]. Epigenetic alterations ing a healthy diet in human. The FA-S group had the are thought to module gene expression and silencing highest body weight and brain weight of neonatal off- [10], and their dysregulation may contribute to numer- spring; however, there were no differences in brain ous neurodevelopment disorders [11, 12]. Maternal FA weight-to-body weight ratio of neonatal offspring within supplementation during pregnancy increased offspring’s groups. For running methylation analysis, three dams global DNA methylation level in rats [7] and influenced were chosen from each group using random numbers, the offspring’s repeat element and imprinted gene and then three neonatal male offspring derived from methylation in humans [13]. A meta-analysis that in- each dam also used random numbers. There were no cluded 1988 newborns from two European birth cohorts between-group differences in conception rate, live birth revealed that maternal plasma folate during pregnancy rate, stillbirth rate, absorbed embryo rate, or sex impacted epigenome-wide DNA methylation in the cord distribution. blood. And some of the implicated genes had functional The neonatal male offspring were sacrificed within 24 h relevance to neurodevelopment [4]. Lambrot et al. [10] after birth, and cerebral hemispheres were collected and provided evidence in mice that lifetime exposure to fol- stored at − 80 °C after liquid nitrogen flash-freezing for ate deficiency may cause an altered sperm epigenome further methylated DNA immunoprecipitation (MeDIP) and was associated with adverse reproductive outcomes. chip assay. Transient inhibition of DNA methylation by 5- Azacytidine treatment during development could induce MeDIP-Chip assay neurodegeneration in neonatal mice and long-lasting Genomic DNA was extracted from brain tissue using a deficits in synaptic plasticity and memory abilities in DNeasy Blood & Tissue Kit (Qiagen, USA). The purified adult mice [14]. Thereby DNA methylation modification DNA was quantified using nanodrop ND-1000. Then may play critical roles in neurodevelopment and learn- the genomic DNA was sonicated (Bioruptor sonicator, ing/memory ability in offspring. Diagenode, Belgian) to fragments between 200 and 1000 In the present study, male offspring derived from dams bp. The MeDIP was prepared as follows: 1 μg of soni- fed with FA-deficient, FA-normal, and FA-supplemented cated genomic DNA fragments was denatured for 10 diets throughout pregnancy were sacrificed within 24 h min at 94 °C, immunoprecipitated with mouse anti-5- after birth to detect DNA methylation profiles. We hy- methylcytosine antibody (1:400, Diagenode, Belgian) pothesized that maternal FA intake during pregnancy overnight at 4 °C, and incubated with anti-mouse IgG might alter DNA methylation profiles established in magnetic beads for 2 h at 4 °C. After washing, the beads- utero that subsequently regulated neurodevelopment DNA complex was resuspended in TE buffer with 0.25% and long-term learning/memory abilities in male SDS and 0.25 mg/mL proteinase K for 2 h at 65 °C. offspring. Lastly, the MeDIP DNA was purified using Qiagen MinElute columns (Qiagen, USA). Methods MeDIP DNA samples and Input samples were labeled Rats and dietary treatment with Cy5-9mer and Cy3-9mer primer, respectively, using As our previous study [6], 3-month-old female Sprague- a NimbleGen Dual-Color DNA Labeling Kit according Dawley rats (Charles River Laboratories, China) were to the manufacturer’s instruction (Nimblegen Systems, assigned randomly into three dietary groups (12 rats/ Inc., USA). Then the samples were hybridized to the group): (1) FA-deficient diet (FA-D), (2) FA-normal diet Arraystar Rat RefSeq Promoter Array, which is designed (FA-N), and (3) FA-supplemented diet (FA-S). Dietary to investigate the epigenetic modifications and transcrip- treatment began from 1 week before mating and tion factor binding sites within 15,987 RefSeq Gene pro- throughout the end of pregnancy. Rats were housed in a moter regions covered by approximately 180,000 probes. specific pathogen-free facility under a controlled 12-h Scanning was performed with the Agilent Scanner light/dark cycle and were provided with food and water G2505C microarray scanner. ad libitum. All animal procedures were approved by the Tianjin Medical University Animal Ethics Committee Identification of differentially methylated regions (TMUaEC2015001). The log2 (MeDIP/Input) getting from raw data was normal- Diet compositions were modified from the AIN-93G ized using the Median-centering, quantile normalization, and diet (TestDiet, USA), which contains 2.1 mg FA/kg diet. linear smoothing analysis by Bioconductor packages Ringo, The FA-D and FA-S diets contain 0.1 mg FA/kg diet and limma, and MEDME. Then from the normalized log2 3.5 mg FA/kg diet, respectively. The folate-supplemented (MeDIP/Input) data, a sliding-window peak-finding Wang et al. Genes & Nutrition (2021) 16:1 Page 3 of 11 algorithm provided by NimbleScan v2.5 (Roche-NimbleGen) Since one single peak can