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Gene-environment interactions mediated at the epigenetic level may provide an initial step in delivering an appropriate response to environmental changes. 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC), accounts for ~40% of modified cytosine in brain and has been implicated in DNA methylation-related plasticity. To identify the role of 5hmC in gene-environment interactions, we exposed both young (6-week-old) and aged (18-month-old) mice to both an enriched environment and a standard environment. Exposure to EE significantly improves learning and memory in aged mice and reduces 5hmC abundance in mouse hippocampus. Furthermore, we mapped the genome-wide distribution of 5hmC and found that the alteration of 5hmC modification occurred mainly at gene bodies. In particular, genes involved in axon guidance are enriched among the genes with altered 5hmC modification. These results together suggest that environmental enrichment could modulate the dynamics of 5hmC in hippocampus, which could potentially contribute to improved learning and memory in aged animals.
Environmental factors are known to have physiological and behavioral effects on aging and related disease states in mammals [reviewed in [1]]. Prolonged exposure to environmental enrichment (EE), which includes but is not limited to stimuli such as physical exercise, exposure to novel objects, and increased social interactions, are found to improve learning and memory, increase neurogenesis and angiogenesis in the hippocampus of aged mice [2-5], and potentially slow the progress of brain aging in rodents [6-8]. In addition to improving health and cognitive function in humans, voluntary physical exercise can also delay the cognitive deficits associated with aging and related neurodegenerative disorders, such as Alzheimer's disease (AD) [9, 10], mitigate the disease phenotype of fatal neurodegenerative diseases, such as spinocerebellar ataxia type 1 (SCA)[11], and induce dynamic changes in promoter methylation in human skeletal muscle [12].
There is ample evidence that environmental factors, such as physical exercise, nutrient deficiency, pharmacological agents, and pollutants, change DNA methylation states in a gene/promoter-specific manner, while changing the expression of DNA methyl transferases (DNMTs) [12-16]. These findings suggest that the epigenetic landscape of genomic DNAs is responsive to changes in environmental signals during the lifetime of organisms. Gene-environment interactions mediated at the epigenetic level may be an intermediary step to providing an appropriate response of the gene/tissue/organism to the changes in the environment.
5mC has generally been viewed as a stable and long-lasting covalent modification to DNA; however, the fact that ten-eleven translocation (TET) proteins, including TET1, TET2, and TET3, can convert 5mC to 5-hydroxymethylcytosine (5hmC), a hydroxymethylated form of 5mC, gives a new perspective on the previously observed plasticity in 5mC-dependent regulatory processes [17-19]. Furthermore, TET enzymes are also known to further oxidize 5hmC into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which can be readily repaired by DNA repair enzymes (See also the review by Hajkova and colleagues, this issue). 5hmC has been detected in heart and lung tissue, though much higher levels have been found in the central nervous system [17, 20, 21] (See also review by Xuekun Li and colleagues, this issue). Using a specific chemical-labeling method for 5hmC detection, we recently generated the first genome-wide maps of 5hmC in mouse cerebellum and hippocampus during development and aging [22]. Our group and others showed that genomic 5hmC levels are age-specific, involved in active DNA de-methylation, and may be important for on-demand gene regulation [22-26]. Nevertheless, we not know whether 5hmC levels or the genomic distribution of 5hmC are affected by external signals in the environment, including diet, exercise, and social interactions, which are all components of an enriched environment (EE).
As such, a genome-wide analysis of 5hmC distribution in young and aged animals exposed to EE is needed to determine the role of 5hmC in gene-environment interactions. Here we exposed both young (6-week-old) and aged (18-month-old) mice to both an enriched environment and a standard environment, and mapped the dynamics of 5hmC in hippocampus induced by EE. We found that exposure to the EE significantly improves learning and memory in aged mice and reduces 5hmC abundance in mouse hippocampus. Genome-wide profiling of 5hmC suggests that the alteration of 5hmC modification occurs mainly at gene bodies, in particular the genes involved in axon guidance. Together these results suggest that environmental enrichment could modulate the dynamics of 5hmC in hippocampus, which may potentially contribute to the improved learning and memory in aged animals.
To understand the effects of environmental signals on cognitive function during the aging process, we exposed young and aged mice to either an enriched environment (EE, as described in the materials and methods) for 4 weeks, or kept them in their standard cages (Ctrl) commonly used for housing (Fig. S1). After EE exposure, mice were tested in Morris water maze assay (MWM, as described in the Materials and Methods) for their cognitive function, specifically learning and memory improvements. MWM analysis revealed that, over time, aged mice exposed to EE (hereafter AE) showed a reduced latency to locate a hidden platform in the water maze tank (i.e. learning) compared to aged mice kept in Ctrl (hereafter AC) (Fig. 1A).Furthermore, once the AE mice learned the location of the platform in a quadrant of the MWM during the initial training period, they spent significantly more time in the same quadrant even after the removal of the platform compared to AC mice (Fig. 1B), which points to improved memory retention in AE mice. It was interesting to observe that EE exposure led to aged mice performing as well as the young mice in the MWM (Fig. S2), suggesting that EE treatment made aged mice appear, at least cognitively, to behave more like young mice. As expected, the swim distance of the AE mice also improved, although their swim speed was not changed (Fig. S3) compared to AC mice, suggesting that the cognitive improvements are due to EE exposure alone. There were no statistically significant differences in learning and memory between young mice exposed to EE (YE) and young mice kept in Ctrl (YC) (Fig. 1C and 1D).
Genome-wide distributions of 5hmC in mouse brain tissue (cerebellum and hippocampus) have been mapped previously [24], revealing age- and tissue-specific 5-hmC dynamics [25]. To determine the effect of a prolonged environmental signal on 5hmC, we measured its overall abundance in genomic DNA isolated from the hippocampus, cortex, and cerebellum of all mice, using antibodies specific to 5hmC by dot-blot analysis. When compared to AC mice, we saw a significant reduction in the total genomic 5hmC signal intensity in AE mice (a 2.5-fold decrease, n=10, P<0.05, Student's t-test; means ± S.E.M.) (Fig. 2A). Similar reduction was also observed in young mice (Fig. 2B).
The TET protein family consists of TET1, TET2, and TET3, all of which can convert 5mC to 5hmC. To determine whether EE could alter the expression of Tet1, Tet2 or Tet3, we performed quantitative RT-PCR and did not observe any change of their expressions (data not shown). Recent studies have shown that the loss of Tet1 leads to learning and memory deficits. Given the age-dependent enhancement of learning and memory caused by an enriched environment, we examined the change of 5hmC in response to exposure to EE in Tet1 knockout mice developed previously. We found that Tet1 KO mice showed almost no change in genomic 5hmC level in the hippocampus after they were exposed to EE (Fig. 2B). These results together suggest that the exposure to enriched environment improves memory and learning in aged mice, while altering global 5hmC levels in hippocampus in a Tet1-dependent manner.
To identify EE-induced epigenetic changes in the hippocampus, we examined 5hmC dynamics using a previously developed method, which consists of selective chemical-labeling of 5hmC and biotin-based affinity enrichment of 5hmC-containing DNA fragments followed by deep sequencing [27]. Using this method, we enriched 5hmC-containing genomic DNAs isolated from the hippocampus of young and aged mice previously exposed to either EE or Ctrl (hereafter, YE, young EE-exposed; and YC, young kept in Ctrl cage). Deep sequencing of libraries generated from these 5-hmC-enriched fragments resulted in, on average, > 17 million unique reads per biological replicate (Table S1).
Using these reads, we first identified 5hmC-enriched regions or “peaks” for each biological replicate using the Model-based Analysis of ChIP-Seq (MACS) algorithm [28]. To increase the coverage of the genome, MACS-peaks from two biological replicates for each condition were merged. Using BEDTools [29], we then identified sets of peaks that show distinct distributions specifically associated with EE or aging in major genomic features such as introns, exons, untranslated regions (UTRs), and intergenic regions (Fig. 3A and Table S3). Interestingly EE exposure could induce 5hmC modifications at the loci that are normally marked by 5hmC only in young animals, but not in aged animals (Fig. 3B). Both gain and loss of 5hmC detected by sequencing could be further confirmed by quantitative PCR (Fig. 3C and 3D).
For each condition and age group, we extrapolated the fold change from the expected value for 5hmC distribution based on the genomic distribution of cytosine in the Mus musculus genome (mm9) (Fig. 4A). It was interesting to observe that AE, YC, and YE animals showed a similar magnitude of fold change for any given genomic features compared to AC animals, complementing the behavioural data where the AE, YC, and YE animals showed similar cognitive abilities in the MWM test.Furthermore, we compared and overlapped the peaks that are associated with AC, AE, and YC and found that there are significantly more peaks overlapping between AE and YC. Pearson's Chi-squared test with Yates' continuity correction indicated these correlations were significant (p values were 7.84e-06 and 0.00245, respectively) (Fig. 4B). These findings suggest that 5hmC could be a good epigenetic biomarker reflecting the impact of EE treatment on the epigenome.
To further explore the biological significance of these identified DhMRs, we used GREAT (Genomic Regions Enrichment of Annotations Tool) to perform gene ontology (GO) analyses for AE-specific DhMRs. Remarkably, several GO biological processes associated with neuronal function and development in brain were identified, particularly genes involved in axon guidance (adjusted p-value = 1.57e-16). Furthermore, we performed additional analyses using WebGestalt, which contains human and mouse protein-protein interaction modules and identifies hierarchical modules. Interestingly, the majority of the genes with altered 5hmC modifications upon exposure to the EE are known to interact based on previous works (Fig. 5) (green-filled dot), which further points to the potentially important role of axon guidance genes in response to EE.
In our study, we show that exposing mice to an enriched environment forms distinct epigenetic signatures (i.e. altered 5hmC distribution profiles) in the genome of animals that showed measurable improvements in memory and learning behaviors as consequences of the exposure. Our findings are in line with previous studies implicating a potential role for epigenetic marks, such as DNA methylation, in gene-environment interactions where exposure to prolonged changes in the environment (i.e. stressors, diet and exercise) leaves epigenetic signatures in the genome with measurable phenotypic outcomes, which in some cases have lasting transgenerational effects [30-32].
In mammalian genome, there are three ten-eleven translocation (TET) proteins, including TET1, TET2, and TET3, which can convert 5mC to 5hmC. We found that the exposure to EE did not alter the expression of any Tet gene. Interestingly we observed that the loss of Tet1 could abolish the induced 5hmC changes by EE exposure, suggesting that Tet1 plays significant role in regulating 5hmC dynamics responding to EE exposure. Indeed this is consistent with the findings that the loss of Tet1 could lead to learning and memory deficits [33-35]. It will be interesting to further determine how quickly following the EE exposure 5hmC signatures form, what is the duration of the 5hmC signatures once formed, and most importantly, what is the role of 5hmC in gene expression.
We exposed mice to EE for four weeks in order to detect measurable changes in memory and learning abilities in mice. However, the onset of the formation of 5hmC signatures and their stability in the mouse hippocampus remain to be determined; this could reveal the epigenetic plasticity of the genome in response to persistent signals in the environment. In our study, EE exposure improved memory and learning behaviors to the levels of young mice regardless of the EE exposure, which was also reflected in the 5hmC signatures. This observation raises the question of whether the epigenome of aged mice is more responsive than that of young mice. We identified an age-dependent decrease in global 5hmC levels in the mouse brain, in contrast to other studies where an increase of 5hmC was detected in the hippocampus of mice with aging [36, 37]. Notably, these studies used different detection methods and age groups to identify 5hmC levels. It is also possible that variations in rearing conditions between the laboratory housing facilities could have contributed to the differences. Our results in this study support the hypothesis that changes in the environment impact the epigenetic landscape in an age- and signal-dependent manner.
Rearing mice in an enriched environment was performed as described previously [38]. All mice were moved to a clean cage (either a standard or enriched chamber) during the “exposure” stage of the experiment. The same individual handled all mice during the exposures to be consistent. Briefly, mice (C57B/6) were randomly assigned to the enriched or the standard environmental condition (Fig. S1) and evaluated in four different groups: i) control young mice (n=14, 6-week-old), ii) enriched young mice (n=14, 6-week-old), iii) control aged-mice (n=10, 18-months-old), and iv) enriched aged-mice (n=10, 18-months-old). For enriched environment sessions (EES), large Rubbermaid clear plastic containers (56.5 cm long X 41.5 cm wide X 22 cm high) were used as enriched environment chambers (EE chambers). For enrichment purposes, the container included a plastic running wheel and an assortment of differently colored and textured plastic toys (balls, tubes, boxes) that were changed every 3 days. To allow proper air circulation, 70-80 small holes (3 mm in diameter) were drilled in the cover and the upper sides where mice were not able to reach. During each EES, the EE chambers contained corncob bedding, food (5-10 pellets of standard rodent diet), and water (in a medium-sized petri dish). Between each EES, the EE chambers and the enrichment apparatus were cleaned with soap and water and disinfected with Virkon-S solution and 70% ethanol. For 4 weeks, mice (groups of 4 from the same cage) were exposed to an enriched environment every day for 3 hours in the same room during the 12-hour light period (lights on 0700–1900 hours), and then returned to their original cages for the remaining 21 h in a temperature-controlled room (22°C). During initial days of EES, the mice were closely monitored for aggressive behaviors, and fighting pairs were separated into different EE cages. Control mice were never exposed to enrichment chambers or the stimulus objects. At the end of the 4 weeks of enrichment sessions, any alterations in hippocampus-dependent spatial memory of mice (both EE and Ctrl) were measured using water maze behavioral testing. Tet1 knockout mice were described previously and obtained from The Jackson Laboratory [39].
Morris water maze (MWM) was performed as previously described [40] with the following modifications. MWM training took place in a round, water-filled tub (52 inch diameter) in an environment rich with extra maze cues. Mice were placed in the water maze with their paws touching the wall from 4 different starting positions (N, S, E, W) in water that began at 25°C and typically declined to 22°C by the time a whole group of mice were tested. At the end of each day of testing, water was drained, and the tank was cleaned with quatracide. An invisible escape platform was located in the same spatial location 1 cm below the water surface independent of a subject's start position on a particular trial. In this way subjects would be able to use extra maze cues to determine the platform's location. Each subject was given 4 trials/day for 6 days with a 15-min inter-trial interval. The maximum trial length was 60 s, and if subjects did not reach the platform in the allotted time, they were manually guided to it. Upon reaching the platform, subjects were left on it for an additional 5 s to allow for survey of the spatial cues in the environment to guide future navigation. After each trial, subjects were dried and kept in a dry plastic holding cage filled with paper towels to allow them to dry off. The holding cage was placed half-on, half-off a heating pad. Animals on the heating pad were not left unattended. Following the 6 days of task acquisition, a probe trial was presented, during which time the platform was removed and the amount of time swam in the quadrant that previously contained the escape platform during task acquisition was measured over 60 s. All trials were videotaped and analyzed by means of MazeScan (Clever Sys, Inc.).
Tissue dissection and DNA isolation from young and aged mice (mice (C57B/6) were performed as previously described (22). To perform dot blot and deep sequencing, genomic DNA samples were sonicated into 100–500 bp by Misonix sonicator 3000. We performed the dot blot analysis of the genomic DNA using a Bio-Dot Apparatus (#170-6545, BIO-RAD) as previously described [22].
5hmC enrichment was performed using a previously described procedure with an improved selective chemical labeling method (22). DNA libraries were generated following the Illumina protocol for “Preparing Samples for ChIP Sequencing of DNA” (Part# 111257047 Rev. A) using 25-50 ng of input genomic DNA or 5hmC-captured DNA to initiate the protocol.
Processing of sequencing data was performed as previously described (22). Briefly,FASTQ sequence files from biological replicates were concatenated and aligned to the Mus musculus reference genome (NCBI37v1/ mm9) using Bowtie 0.12.6, keeping only unique non-duplicate genomic matches with no more than 2 mismatches within the first 25 bp. Unique, non-duplicate reads from non-enriched input genomic DNA of hippocampus and each 5hmC-enriched sequence set were counted in 100-, 1000-, and 10,000-bp bins genome-wide and subsequently normalized to the total number of non-duplicate reads in millions. Input-normalized values were then subtracted from 5hmC-enriched values per bin to generate normalized 5hmC signals. We determined chromosome-wide densities as reads per chromosome divided by the total number of reads in millions. Expected values were determined by dividing the total NCBI37v1/mm9 length by 106, and then by multiplying with chromosome length. For chromosomes X and Y in the male samples, the expected values were divided by 2. For comparison between autosomes and ChrX, chromosome-wide 5-hmC read densities were divided by input read densities to assess 5hmC enrichment.
To determine genomic regions that present altered 5hmC profiles due to EE treatment (EE-DhmRs), we first identified true 5hmC-enriched regions or “peaks” using Model-based Analysis of ChIP-Seq (MACS) algorithm (26).
Input genomic DNA and 5hmC-enriched DNA were diluted to 100 pg/μL, and 1 μL was used in triplicate 20-uL qPCR reactions, each with 1X PowerSYBR Green PCR Master Mix (ABI), 0.5 uM forward and reverse primers, and water. Reactions were run on an SDS 7500 Fast Instrument using standard cycling conditions. Primers are listed in Table S2. Fold enrichment was calculated as 2^-dCt, where dCt = Ct (5-hmC-enriched) – Ct (Input).
The authors would like to thank J. Schroeder at Emory Rodent Behavioral Core for the help with behavioral assay, and C. Strauss for critical reading of the manuscript. This work was supported in part by NIH grants (NS051630 and MH102690) and the Simons Foundation Autism Research Initiative to P.J.
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Sequencing data have been deposited to GEO with accession number GSE61194.
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