Maternal Folic Acid Impacts DNA Methylation Pro le in Male Rat Offspring Associated With Neurodevelopment and Learning/Memory Abilities


 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 FA-N group, 1939 DMGs were identified in the FA-D group, and 1498 DMGs were identified in the FA-S 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 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.


Introduction
Emerging evidence has indicated that early life nutrition in uences brain development, and exerts longterm consequences for memory abilities profoundly [1,2]. Periconceptional folic acid (FA) supplementation not only reduces the incidence of neural tube defects, but also improves cognitive performances in offspring [3,4]. Our previous studies demonstrated that maternal FA supplementation during pregnancy promoted offspring's neurogenesis and synaptogenesis at birth [5], stimulated sensorymotor re ex development in infancy [6,7], and exerted long-term bene cial effects on learning and memory abilities in adolescence and adult in rat offspring [6]. However, the genes or pathways that are epigenetically regulated by FA in neurodevelopment were rarely reported.
Trough one-carbon metabolism, folate provides a labile source of methyl groups for numerous methylation reactions, including DNA methylation [8]. DNA methylation and histone modi cation are the most studied epigenetic modi cations that are reprogrammed considerably in early embryonic development [9]. Epigenetic alterations are thought to module gene expression and silencing [10], and its dysregulation may contribute to numerous neurodevelopment disorders [11,12]. Maternal FA supplementation during pregnancy increased offspring's global DNA methylation level in rats [7], and in uenced offspring's repeat element and imprinted gene methylation in human [13]. A meta-analysis included 1988 newborns from two European birth cohorts revealed that maternal plasma folate during pregnancy impacted epigenome-wide DNA methylation in cord blood, and some of the implicated genes had functional relevance to neurodevelopment [4]. Lambrot et al. [10] provided evidence in mice that lifetime exposure to folate-de ciency may cause altered sperm epigenome and was associated with negative reproductive outcomes. Transient inhibition of DNA methylation by 5-Azacytidine treatment during development could induce neurodegeneration in neonatal mice, and long-lasting de cits in synaptic plasticity and memory abilities in adult mice [14]. Thereby DNA methylation modi cation may play critical roles in neurodevelopment and learning/memory ability in offspring.
In the present study, male offspring derived from dams fed with FA-de cient, FA-normal and FAsupplemented diets throughout pregnancy were sacri ced within 24 h after birth to detect DNA methylation pro les. We hypothesized that maternal FA intake during pregnancy might alter DNA methylation pro les established in utero that subsequently regulated neurodevelopment and long-term learning/memory abilities in male offspring.

Rats and dietary treatment
Three-month-old female Sprague-Dawley rats (Charles River Laboratories, China) were assigned randomly into three dietary groups: (1) FA-de cient diet (FA-D), (2) FA-normal diet (FA-N), and (3) FAsupplemented diet (FA-S). Dietary treatment began from 1 week before mating and throughout the end of pregnancy. Rats were housed in a speci c pathogen-free facility under a controlled 12-h light/dark cycle and were provided with food and water ad libitum. All animal procedures were approved by the Tianjin Medical University Animal Ethics Committee (TMUaEC2015001).
The FA-D and FA-S diet contains 0.1 mg FA/kg diet and 3.5 mg FA/kg diet, respectively. The neonatal male offspring derived from 3 dams were sacri ced within 24 h after birth, and cerebral hemispheres were collected and stored at -80℃ after liquid nitrogen ash-freezing for further methylated DNA immunoprecipitation (MeDIP) chip assay.

MeDIP-Chip assay
Genomic DNA was extracted from brain tissue using a DNeasy Blood & Tissue Kit (Qiagen, USA). The puri ed DNA was quanti ed using nanodrop ND-1000. Then the genomic DNA was sonicated (Bioruptor sonicator, Diagenode, Belgian) to fragments between 200 and 1000 bp. The MeDIP was prepared as follows: 1 μg of sonicated genomic DNA fragments were denatured for 10 min at 94 °C, immunoprecipitated with mouse anti-5-methylcytosine antibody (1:400, Diagenode, Belgian) overnight at 4 °C, and incubated with anti-mouse IgG magnetic beads for 2 hours at 4 °C. After washing, the beads-DNA complex was resuspended in TE buffer with 0.25% SDS and 0.25 mg/mL proteinase K for 2 h at 65°C . Lastly, the MeDIP DNA was puri ed using Qiagen MinElute columns (Qiagen, USA).
MeDIP DNA samples and Input samples were labeled with Cy5-9mer and Cy3-9mer primer, respectively, using a NimbleGen Dual-Color DNA Labeling Kit according to the manufacturer's instruction (Nimblegen Systems, Inc., USA). Then the samples were hybridized to the Arraystar Rat RefSeq Promoter Array, which is designed to investigate the epigenetic modi cations and transcription factor binding sites within 15,987 RefSeq Gene promoter regions totally covered by approximately 180,000 probes. Scanning was performed with the Agilent Scanner G2505C microarray scanner.

Identi cation of differentially methylated regions
The log 2 (MeDIP/Input) getting from raw data was normalized using the Median-centering, quantile normalization and linear smoothing analysis by Bioconductor packages Ringo, limma, and MEDME. Then from the normalized log 2 (MeDIP/Input) data, a sliding-window peak-nding algorithm provided by NimbleScan v2.5 (Roche-NimbleGen) was applied to nd the enriched peaks with speci ed parameters (sliding window width: 1500 bp; minimum probes per peak: 2; P-value minimum cut-off (-log10): 2), and peaks within 500 bp spacing were merged.
To compare the differentially enriched regions between two groups, the log 2 (MeDIP/Input) was averaged for each group (Experiment and Control), and the M' value for each probe was calculated as following: Then the NimbleScan sliding-window peak-nding algorithm was reran on these data to nd the differential enrichment peaks (DEPs) between groups, ltered according to the following criteria: 1) At least one of the two groups had a median (log 2 MeDIP/Input) ≥ 0.3 and a median (M')> 0.
2) At least half of probes in a peak might have coe cient of variability ≤ 0.8 in both two groups.

Promoter classi cation
Promoters were de ned as the regions [-1300 bp, +500 bp] around the transcription start sites (TSS). Promoters were classi ed into 3 categories as follows: 1) High CpG-density promoter (HCP): promoters containing a 500-bp window with GC content ≥ 55%, and CpG observed to expected ratio (O/E) ≥0.6 within -700 bp to +200 bp around TSS.
2) Low CpG-density promoter (LCP): promoters that contained no 500 bp interval and with a CpG O/E ≥0.4.

Functional analysis
Genes containing DEPs in the promoter were considered as the differentially methylated genes (DMGs).
Enrichments in biological process of gene ontology (GO) and pathways that have functional relevant to neurodevelopment and learning/memory abilities were analyzed using DAVID Bioinformatics Resources 6.8 [15] and KEGG pathway analysis [16], respectively.

Results
Maternal FA intake during pregnancy altered DNA methylation pro lesin the brain tissue of male offspring To investigate the DNA methylation pro les altered by maternal FA intake during pregnancy, DEPs in promoter region between groups were identi ed using the NimbleScan v2.5 software (Roche-NimbleGen).
Compared with the FA-N group, 1843 DEPs were identi ed in the FA-D group (744 hypermethylated and 1099 hypomethylated), and 1406 DEPs were identi ed in the FA-S group (738 hypermethylated and 668 hypomethylated).
Since one single peak can be assigned to more than one gene promoter, and one promoter may contain more than one peak, the peaks-to-promoters are not always one-to-one correspondence. The DEPs were further analyzed according to its assigned gene promoter. Taken together, the results indicated that maternal FA intake during pregnancy altered DNA methylation pro les in brain tissue of neonatal male offspring, and DEPs localized mostly (approximately 50%) in HCP.
Maternal FA intake during pregnancy altered biological process involved in neurodevelopment and learning/memory abilities in the brain tissue of male offspring We identi ed some identical and distinctive DMGs altered in response to maternal FA de ciency and supplementation during pregnancy. For this reason, data of the maternal FA de ciency (FA-D) or supplementation group (FA-S) are compared with FA normal group (FA-N) separately. Go analysis revealed that DMGs in FA-D/FA-N were enriched in a wide variety of biological process that involved in neural tube development, neural crest cell migration, brain development, neuron differentiation, glia cell differentiation, synapse organization, chemical synaptic transmission, and learning/memory (Table 1).
And DMGs in FA-S/FA-N were enriched in biological process that involved in neural tube development, brain development, neuron differentiation, synapse organization, myelination, chemical synaptic transmission, and memory (Table 2).
Although some biological process involved in neurodevelopment and learning/memory abilities are shared by different FA status, compared with FA-S/FA-N, more DMGs implicated in brain development and neuron differentiation were found in FA-D/FA-N. Additionally, DMGs in FA-D/FA-N distinctively enriched in neural crest cell migration, glia cell differentiation; while DMGs in FA-S/FA-N distinctively enriched in myelination (Table 1, 2).
Taken together, the results indicated that gene implicated in neurodevelopment and learning/memory abilities were differentially methylated in response to maternal FA intake during pregnancy, and there were some identical and distinctive biological process altered under FA de ciency and supplemented conditions.
Maternal FA intake during pregnancy impacting pathways associated with neurodevelopment of male offspring To further discover the potential mechanisms whereby maternal FA intake during pregnancy impacts neurodevelopment through DNA methylation, KEGG pathway analysis was performed on DMGs to identify the enriched pathways associated with neurodevelopment in FA-D/FA-N and FA-S/FA-N. The results showed that DMGs altered by maternal FA de ciency were enriched in pathways including signaling pathways regulating pluripotency of stem cells, autophagy, tight junction, calcium signaling pathway, mammalian/mechanistic target of rapamycin (mTOR) signaling pathway, Notch signaling pathway, neurotrophin signaling pathway, and protein processing in endoplasmic reticulum (ER) (FA-D/FA-N in Table 3). While DMGs altered by maternal FA supplementation were enriched in pathways including signaling pathways regulating pluripotency of stem cells, autophagy, tight junction, calcium signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, transforming growth factor-beta (TGF-β) signaling pathway, and axon guidance (FA-S/FA-N in Table 3).
The results showed that signaling pathways regulating pluripotency of stem cells, autophagy, tight junction, and calcium signaling pathway were overlapped between FA-D/FA-N and FA-S/FA-N, indicating the mechanisms that shared from the FA-de cient status to the FA-normal status and to the FAsupplemented status. Additionally, DMGs in FA-D/FA-N enriched distinctively in mTOR signaling pathway, Notch signaling pathway, neurotrophin signaling pathway, and protein processing in ER; while DMGs in FA-S/FA-N enriched distinctively in MAPK signaling pathway, TGF-β signaling pathway and axon guidance (Table 3), indicating distinctive mechanisms of in uencing neurodevelopment in male offspring under FA de cient or supplemented conditions.
Taken together, the results indicated that pathways associated with neurodevelopment were differentially methylated in response to maternal FA intake during pregnancy, and there some identical and distinctive potential mechanisms impacting neurodevelopment under FA de ciency or supplemented conditions.
Maternal FA intake during pregnancy impacting pathways associated with learning/memory abilities of male offspring To illustrate the potential mechanisms of maternal FA during pregnancy impacting offspring's learning/memory abilities by DNA methylation, enriched pathways associated with synaptic plasticity and learning/memory abilities were identi ed using KEGG pathway analysis. The results showed that DMGs altered by maternal FA de ciency were enriched in pathways including cGMP-PKG signaling pathway, gap junction, calcium signaling pathway, mTOR signaling pathway, Rap1 signaling pathway, Notch signaling pathway, endocytosis, and neurotrophin signaling pathway (FA-D/FA-N in Table 4). While DMGs altered by maternal FA supplementation were enriched in pathways including cGMP-PKG signaling pathway, gap junction, calcium signaling pathway, MAPK signaling pathway, ErbB signaling pathway, glutamatergic synapse, and neuroactive ligand-receptor interaction (FA-S/FA-N in Table 4).
The results showed that cGMP-PKG signaling pathway, gap junction and calcium signaling pathway were overlapped between FA-D/FA-N and FA-S/FA-N, indicating the mechanisms that shared from the FAde cient status to the FA-normal status and to the FA-supplemented status. Additionally, DMGs in FA-D/FA-N enriched distinctively in mTOR signaling pathway, Rap1 signaling pathway, Notch signaling pathway, endocytosis and neurotrophin signaling pathway; while DMGs in FA-S/FA-N enriched distinctively in MAPK signaling pathway, ErbB signaling pathway, glutamatergic synapse, and neuroactive ligand-receptor interaction (Table 4), indicating the distinctive mechanisms of in uencing learning/memory abilities in male offspring under maternal FA de cient or supplemented conditions.
Taken together, the results indicated that pathways associated with learning/memory abilities were differentially methylated in response to maternal FA intake during pregnancy, and there some identical and distinctive potential mechanisms impacting learning/memory abilities under FA de ciency or supplemented conditions.

Discussion
In present study, the results showed that maternal FA intake during pregnancy altered DNA methylation pro les in brain tissue of neonatal male offspring, and pathways associated with neurodevelopment and learning/memory were differentially methylated. DMGs altered by maternal FA de ciency (FA-D/FA-N) were enriched in eight pathways associated with neurodevelopment and eight pathways associated with learning/memory abilities. While DMGs altered by maternal FA supplementation (FA-S/FA-N) were enriched in seven pathways associated with neurodevelopment and seven pathways associated with learning/memory abilities. There were some identical and distinctive potential mechanisms impacting neurodevelopment or learning/memory abilities under FA de ciency or supplemented conditions. Potential mechanisms of maternal FA intake during pregnancy impacting offspring's neurodevelopment through DNA methylation DNA methyltransferases (DNMT) 3a-null mice showed impairments in postnatal neurogenesis [17], and DNMT3b knockdown in human embryonic stem cells altered the timing of neuronal differentiation and maturation [18], supporting vital roles of DNA methylation in neurodevelopment. Consistently, the results in present study illustrated that the DMGs altered by maternal FA de ciency and supplementation were both enriched in pathways of signaling pathways regulating pluripotency of stem cells, autophagy, tight junction, and calcium signaling pathway, all of which are associated with neurodevelopment. Signaling pathways regulating pluripotency of stem cells activates a core transcriptional network including Oct4, Nanog and Sox2. These three transcription factors coordinate with their downstream target genes promotes self-renewal and pluripotency of stem cells, thereby impacting embryonic development and neurodevelopment. Autophagy, a self-degradative process that acts as a pro-survival or pro-death mechanism depending on different physiological or pathological conditions [19], is important for embryonic development, neural proliferation and differentiation during brain development [20,21]. Tight junctions are essential for establishing the selectively permeable barrier between neighboring cells, tight junctions formed between brain capillary endothelial cells help creating blood-brain barrier that act to protect the brain from harmful substances and maintain brain homeostasis, modi ed tight junction protein expression and disrupted blood-brain barrier structure are observed in cerebral pathologies [22,23]. Calcium signaling regulates nearly every aspect of neural development, including neural induction, neural rosettes formation, neural progenitor cells proliferation, neuronal migration and differentiation [24][25][26], speci c synaptic connections development, and impairments in calcium-dependent network maturation are associated with neurodevelopment disorders [27].
Additionally, DMGs altered by maternal FA de ciency were enriched distinctively in mTOR signaling pathway, Notch signaling pathway, neurotrophin signaling pathway and protein processing in ER. The mTOR signaling plays vital roles in dendritic formation and axon guidance during normal brain development [28], and aberrant mTOR signaling has been implicated in neurodevelopment disorders that characterized by cognitive de cits, such as tuberous sclerosis and fragile X syndrome [29]. Notch signaling maintains the neural stem cells pool during brain development [30], regulates cortical cell-type differentiation in an orderly progression [31], and interacts with Reelin signaling regulating cortical neuronal migration [32]. Neurotrophins are growth factors consisting of nerve growth factor, brain derived neurotrophic factor, neurotrophin (NT) 3, and NT-4, that participated in a wide aspects of neurodevelopment, including neural survival, proliferation, differentiation, myelination, apoptosis, and axonal growth [33]. The ER is a subcellular organelle where proteins are folded with the help of lumenal chaperones. Proteins that correctly folded are packaged into transport vesicles that shuttle to Golgi complex; while accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress and unfolded protein response that may induce aberrant neuronal differentiation and inhibition of dendrite outgrowth [34].
While DMGs altered by maternal FA supplementation were enriched distinctively in MAPK signaling pathway, TGF-β signaling pathway and axon guidance. MAPKs have been involved in modulation of hippocampal development. Extracellular signal-regulated kinase (ERK) signaling regulates maintenance of neural progenitor cells in dentate gyrus [35]. ERK and p38 activities are dynamically regulated during postnatal development that may correlates with the occurrence of hippocampal programmed cell death and synaptogenesis [36]. TGF-β signaling has been implicated in axonal formation and speci cation, and neuronal migration during brain development [37,38]. Axon guidance including axon outgrowth, axon repulsion and axon attraction, represent key stages in the formation of neuronal network.
Potential mechanisms of maternal FA intake during pregnancy impacting offspring's long-term learning/memory abilities through DNA methylation DNA methylation on cytosine residues in CpG sequences is a mitotically stable epigenetic modi cation that can produce long-term changes in gene expression [11,39], and transient inhibition of DNA methylation during development exerts long-term deleterious effects on synaptic plasticity and memory abilities in adult mice [14]. Consistently, the results in present study showed that the DMGs altered by maternal FA de cient and supplementation were both enriched in pathways of cGMP-PKG signaling pathway, gap junction and calcium signaling pathway. cGMP-PKG signaling mediates early memory consolidation and early-phase LTP [40], in addition, NO-cGMP-PKG signaling pathway regulates fear memory consolidation by promoting both presynaptic and postsynaptic alterations following fear conditioning at lateral amygdala synapses via NO-driven "retrograde signaling" [41]. Gap junctions in mammalian brain act to synchronize neuronal activity and connect glial cells participating in the regulation of brain metabolism and homeostasis, and suggested to contribute to fear learning and memory [42,43]. Postsynaptic calcium signaling is suggested to induce activity-dependent LTP in CA1 and CA2 hippocampal neurons; calcium/calmodulin may activates many plasticity-inducing pathways, such as CaMK, Ras/ERK, and PKA [44], and astroglial calcium signaling displays short-term plasticity and down-regulates synaptic transmission [45].
Additionally, DMGs altered by maternal FA de ciency were enriched distinctively in mTOR signaling pathway, Rap1 signaling pathway, Notch signaling pathway, endocytosis and neurotrophin signaling pathway. mTOR regulates protein translation in response to neuronal activity, thereby modulating synaptic plasticity and long-term memory formation, and has been implicated in fear memories acquisition/consolidation in the hippocampus/amygdala [46]. Small GTPase Rap1 signals synaptic glutamate receptor (GluR)-2/3 AMPARs removes by activating Rap1-p38MAPK signaling during LTD [47]. Notch signaling plays a critical role in brain development. Recently, emerging evidence has suggested that Notch signaling also plays an important role in synaptic plasticity and spatial memory formation [48,49]. Neurotrophins are rstly described as growth factors that support neuronal differentiation and survival. Later studies have indicated their roles in synapse formation and plasticity, including increasing neurotransmitter output, membrane excitability, and synaptic contacting size locally at synapse, and affecting gene expression and phenotypic enhancement distally at the neuronal soma [50].
While DMGs altered by maternal FA supplementation were enriched distinctively in MAPK signaling pathway, ErbB signaling pathway, Glutamatergic synapse, and Neuroactive ligand-receptor interaction. MAPK is activated after learning or LTP induction in hippocampus and amygdala, and pharmacological inhibition of MAPK activation impairs LTP, learning, and long-term memory [51]. ErbB signaling modulates hippocampal metabotropic GluRI-dependent LTD and object recognition memory [52], and pharmacological inhibition of ErbB signaling during adolescence impairs reference memory in adult mice [53]. In glutamatergic synapse, glutamate released from neuron presynaptic terminal acts on postsynaptic ionotropic or metabotropic GluRs to regulate synaptic plasticity, and afterwards, are removed from the synaptic cleft by EAATs located either on the presynaptic terminal, postsynaptic neuron, or neighboring glial cells. Signals resulting from ligand-receptor interactions trigger intracellular signaling that ultimately modulate gene expression, which could be the underlying basis of learning and memory [54].

Conclusion
In conclusion, the present study found that maternal FA de ciency and supplementation during pregnancy altered DNA methylation pro les in brain tissue of male offspring, and those DMGs were associated with neurodevelopment and learning/memory abilities. These ndings provide novel insights into the mechanism studies of maternal FA intake impacting offspring's neurodevelopment and learning/memory abilities with DNA methylation involved. Availability of data and materials

Abbreviations
All of the data are available with reasonable request from the corresponding author.
Ethics approval and consent to participate The MeDIP-Chip assay was performed on brain tissue from folic acid-de cient (FA-D) and folic acidnormal (FA-N) male offspring (n=3/group). Enriched biological processes (BP) of GO terms by DAVID were used to identify the differentially methylated genes (DMGs) associated with neurodevelopment and learning/memory abilities. Table 2 Gene implicated in neurodevelopment and learning/memory abilities that were differentially methylated in FA-S/FA-N