Abstract
Maternal care during early-life plays a crucial role in the sculpting of the mammalian brain. Augmented maternal care during the first postnatal week promotes life-long stress resilience and improved memory compared with the outcome of routine rearing conditions. Recent evidence suggests that this potent phenotypic change commences with altered synaptic connectivity of stress sensitive hypothalamic neurons. However, the epigenomic basis of the long-lived consequences is not well understood. Here, we employed whole-genome bisulfite sequencing (WGBS), RNA-sequencing (RNA-seq), and a multiplex microRNA (miRNA) assay to examine the effects of augmented maternal care on DNA cytosine methylation, gene expression, and miRNA expression. The integrated results identify an overlap of 20 prioritized genes impacted by augmented maternal care. These regulatory differences were centered on genes related to neurotransmission, neurodevelopment, protein synthesis and oxidative phosphorylation in addition to stress response genes. Thus, the combined approach enables the discovery of gene-sets that are not identified by each method alone, providing novel means to advance our understanding of the central mechanisms underlying the profound effects of maternal care on the developing brain.
Introduction
Early postnatal care in mammals is critical for survival as well as neurodevelopment. Specifically, early maternal care can epigenetically alter the behavior of offspring for their entire lifetime. Rodent models manipulating maternal care and maternal stress have been used to examine the molecular and cellular mechanisms underlying the long-term behavioral changes observed in the offspring 1–3. In the augmented maternal care (AMC) paradigm, daily separation of rat dams from their pups for 15 minutes from postnatal day 2 (PND2) to postnatal day 8 (PND8) triggers the increased maternal behaviors of arched-back nursing, as well as elevated licking and grooming of the pups by the dam 3,4. Subsequently, it was found that multiple days of pup handling within the first postnatal week of life were required for the AMC response, suggesting AMC initiates changes during this critical period of neurodevelopment in the offspring 5,6. The repeated, daily sessions of enhanced maternal care triggered in the AMC paradigm result in offspring that show reduced plasma corticosterone levels in response to an acute restraint stress 7–11, reduced anxiety related behaviors 11,12, resilience to depressive-like behaviors 11, and enhanced performance on several cognitive tasks as adults12,13.
AMC appears to alter brain function for the lifetime of the animal 14 and as adults AMC offspring show hypo-activation of the HPA axis in response to stress. Previous work has demonstrated that this mechanism depends upon temporally coordinated regulation of both corticotrophin releasing hormone (Crh) in the paraventricular nucleus (PVN) of the hypothalamus 5,13 and glucocorticoid receptor (GR, encoded by Nr3c1) 6 in the hippocampus 13,15. Crh transcripts in the hypothalamus are significantly reduced immediately following AMC at PND9 5,6 and remain repressed throughout adulthood 9. In comparison, increased Nr3c1 expression in the hippocampus was delayed until PND23 9, suggesting that early gene expression changes in the PVN of the hypothalamus drive the initial molecular mechanisms underlying AMC. Together, the elevated hippocampal Nr3c1 expression and reduced hypothalamic Crh expression 2,5,11,16 in AMC adult offspring suggest that increased negative feedback signaling may underlie the observed hypo-responsive HPA axis in the adult AMC offspring.
These studies have raised an important question about how hypothalamic reprogramming is initiated and maintained by AMC induced early-life gene expression changes. AMC initiates transient structural changes in CRH-expressing cells in the hypothalamus of pups12,16. By PND9 there is decreased excitatory synaptic drive onto hypothalamic Crh expressing neurons in AMC offspring, that returns to baseline by adulthood (PND30-45)16, suggesting that the AMC induced reduction in Crh expression is maintained long-term by additional mechanisms 14. In vitro studies have suggested that the synaptic changes initiate transcriptional regulation in these CRH positive cells12. Specifically, recent work has implicated the neuron restrictive silencing factor (NRSF) in establishing long-term epigenetic gene expression patterns resulting from AMC 14,17. NRSF expression and binding to the Crh gene is increased in hypothalamus of AMC offspring at PND9 but is no longer present in adulthood 12,16. Thus, there is little information about the comprehensive epigenomic processes by which augmented sensory signals from the mother result in enduring, large-scale reprogramming of gene expression in the hypothalamus. In hippocampus, prior studies have described epigenetic DNA methylation alterations and H3K9 acetylation in response to differences in maternal care at Nr3c1 18. The search for additional genes underlying AMC effects on adult hippocampus revealed alterations to gene expression of more than 900 genes related to cellular metabolism and energy production, signal transduction, protein synthesis (mainly ribosomal genes), and neurodevelopment 19. Targeted analysis of a seven megabase (Mb) region of chromosome 18 flanking Nr3c1 revealed that the clustered protocadherins (Pcdh) showed the highest differential response in DNA methylation 20.
Together, these combined results indicate that NRSF binding in PND9 hypothalamus14,15 (13,15) as well as differential gene expression and DNA methylation in adult hippocampus are involved in regulating long-term behavioral changes induced by AMC. However, whereas NRSF ChIP-seq has identified gene sets contributing to AMC-induced cellular changes12, a multi-methodology genome-wide approach has not been undertaken in the hypothalamus. 21. To address this question we performed an integrated, genome-wide examination of epigenetic and gene expression differences in hypothalamus from PND9 pups in response to AMC. These experiments identified that the response to AMC occurs not only in the genes previously identified such as Crh, but in thousands of genes throughout the genome. In addition to a significant global reduction in hypothalamic DNA methylation levels, AMC induces differential methylation and expression in mRNA transcripts related to neurodevelopment, synaptic signaling, ribosome function, and cellular stress. The results provide strong evidence for the role of epigenomic modifications in regulating gene expression profiles related to resilience and offer a suite of prioritized genes and gene pathways for the development of diagnostic biomarkers and therapies for neuropsychiatric disorders in humans.
Materials and Methods
Detailed methods are provided in the Supplementary Materials and Methods section.
Augmented Maternal Care (AMC) was performed as described previously 13,16. Briefly, pups were gathered at birth and five male and five female pups were randomly assigned to dams. Pups were removed for 15 min at 9 AM each day from PND2-PND9 to a separate cage in a different room equipped with a heating pad. Further details can be found in the Supplementry Information (SI) Materials and Methods. For WGBS DNA methylation analysis, genomic DNA was converted by bisulfite treatment using a Zymo kit followed by Illumina sequencing library preparation. WGBS libraries were sequenced on an Illumina Hi-Seq 2500. DMR identification and PMD analysis was performed as detailed in Schroeder 22 and Dunaway 23. The SI Materials and Methods section contain an in depth description of WGBS methods. Bisulfite converted genomic DNA was amplified using custom, gene specific primers and amplicons were analyzed by pyrosequencing on a Biotage PSQ-96MA Pyrosequencer. A detailed description of pyrosequencing methods is contained in the SI Materials and Methods. For RNA-seq total RNA was prepared from tissue using a Qiagen RNeasy Kit (Qiagen). Total RNA was depleted of ribosomal RNA for creation of stranded RNA-seq libraries (See SI Materials and Methods). Bar coded RNA-seq libraries were sequenced on an Illumina Hi-Seq 2500 platform. Analysis of RNA-seq reads is described in SI Materials and Methods. Validation for differential RNA-seq identified genes was performed on samples from the same cohort of PND9 male and female hypothalami (SI Materials and Methods). For miRNA transcript analysis a multiplex miRNA assay of miRNA transcripts was performed using an nCounter Rat miRNA expression assay. A detailed description of miRNA analysis is available in SI Materials and Methods. All sequencing data will be available on the NIH GEO database under accession number GSE99282.
Results
AMC induces large-scale hypo methylation in the PND9 hypothalamus
To investigate genome-wide DNA methylation changes associated with AMC, hypothalami from AMC and control male rat pups were chosen for whole-genome bisulfite sequencing (WGBS) based on significant (p=0.0042) differential AMC (total licking and grooming) at postnatal day 9 (Supplementry Figure 1). Principal component analysis (PCA) was performed on 20,000 base (20-Kb) windows of percent DNA methylation with CpG islands masked (Figure 1A) to explore genome-wide patterns of methylation between treatment conditions in the individual rats. Methylation levels in individual PND9 hypothalami were separated by AMC condition for principal component 1 (30.2% of variance explained), indicating differences in methylation levels between conditions. Unexpectedly, hypothalami from AMC pups displayed significant global hypomethylation (p=0.015) at PND 9 (Figure 1B). Specifically, global CpG methylation levels for augmented hypothalami averaged 76.08% while controls had levels of 79.20% yielding to a 3.12% reduction (SupplementryTable 1). Non-CpG methylation levels in augmented and control hypothalami ranged from 0.86 to 1.46% of total cytosines (Supplementry Table 1) consistent with previous findings of normal neurodevelopment 24. CpG hypomethylation was observed across all chromosomes with the exception of chromosome 13 and X, indicating a genome-wide impact of AMC on CpG methylation levels (Figure 1C). Interestingly, windowing over different genomic features revealed hypomethylation specifically in the region 5 kb upstream of the transcriptional start site (TSS) of CpG island promoters (p=0.0057) in AMC hypothalamus (Supplementry Figure 2). To identify potential hypothalamic genes and pathways contributing to AMC, large-scale differential DNA methylation patterns were identified from the WGBS data sets. Partially methylated domains (PMDs) were mapped from AMC and control WGBS data sets using a hidden Markov model (HMM) previously described for detecting partially methylated domains in neuronal cell lines 22. AMC PMDs averaged 7.3% of the genome and control PMDs averaged 6.7% of the genome. Although consistent with genome-wide hypomethylation the increased PMD coverage with AMC was not significant (p=0.158) (Supplementry Table 2). While PMDs were found throughout the genomes of both groups, only five PMDs were differential by treatment group (Table 1). Consistent with genome-wide hypomethylation shown in Figure 1B, these five PMDs were found in AMC but not control hypothalami. AMC PMDs covered 674,924 bases and overlapped with seven genes. These PMD genes included Dbn1, Prr7, Foxo3, and Cnksr2, which function in neuronal development, and Pak6 which functions in the MAP kinase pathway to regulate cytoskeletal dynamics underlying multiple brain processes 25–29(Table 1). AMC PMDs also contained AY383691 and Ankrd63, which are gene products without well-established functions.
AMC DMRs are linked to neurodevelopmental signaling and neurotransmission
While PMDs highlight broad regions of differential methylation associated with AMC, few genes were found within these loci. Therefore to identify small scale (<3 Kb) methylation changes, differentially methylated regions (DMRs) were identified from WGBS data 23,30. With this bioinformatic approach a total of 9,439 DMRs between AMC and Control PND9 hypothalami were identified (Supplementry Table 3). Of these 9,439 AMC DMRs, 6,228 had reduced methylation levels in the AMC samples, which is consistent with the genome-wide hypomethylation shown in Figure 1. To investigate the relationship between large-scale PMDs and small-scale DMRs, the two genomic features were compared. Surprisingly, of the 9,439 DMRs identified, only one in Pak6 was found within the five AMC PMDs. Visualization of the DMRs using hierarchical clustering analysis (HCA) of significant (p<0.05) DMRs from hypothalami clustered each rat by treatment (Figure 2A). Both PCA of 20kb windows (Figure 1A) and HCA (Figure 2A) independently partitioned the AMC and control hypothalami into separate groups, indicating that distinct methylation patterns distinguish the AMC from control hypothalamic samples at two different resolutions. Motif enrichment analysis of the DMRs revealed a significant (q<0.0001) enrichment only for CTCF binding sites, which were present in 4% of DMRs (Figure 2B). CTCF binds these sites in a methylation sensitive manner and functions as a genomic insulator critical to chromatin architecture 31 Of the 9,439 total DMRs identified, 5,284 were between 5 kb upstream and 1 kb to downstream of 4,023 coding and non-coding RefSeq genes in the rat genome (Rn6) (Supplementry Table 3). Forty-four percent of the DMRs were intergenic and 35.3% were localized to introns. Fourteen percent of DMRs were immediately upstream of genes and only 0.4% and 1.1% were located in the 5’ and 3’ prime untranslated regions (UTRs) respectively (Supplementry Figure 3A). For the DMRs annotated to within genes, the majority (93.6%) were localized to coding genes and 4.1% were located in long non-coding RNAs (lncRNAs) (Supplementry Figure 3B).
To comprehensively identify the functions associated with the 4,023 genes mapped to AMC DMRs, gene ontology (GO) and pathways analyses were performed (Figure 2C). Gene ontology analysis revealed a significant (q<0.05) enrichment for the biological processes of axon guidance, behavior, and protein auto-phosphorylation. Membrane raft, adherens junction, and synapse part were the significantly (q<0.05) enriched cellular components. Protein serine/threonine kinase activity, receptor signaling protein activity, and kinase binding were the most unique significant (q<0.05) molecular functions. Pathway analyses revealed a significant (q<0.05) enrichment for protein kinase A signaling, axonal guidance signaling, epithelial adherens junction signaling, Wnt/β-catenin signaling, and molecular mechanisms of cancer. Notably, the molecular mechanisms of cancer pathway are a collection of developmental signaling pathways. Overall, all of the above GO terms and pathways are related to neurodevelopment as well as synaptic and cellular signaling (Figure 2C).
To further examine the DMRs for relevance to AMC, the 9,439 DMRs were subject to permutation testing, which yielded seven high confidence or “gold” DMRs (family-wise error rate (FWER) < 0.05) (Supplementry Table 4). These gold DMRs were annotated to LOC00911370, Tuba3b, Glb1l, Papolb, Grb10, Tenm3, and Zfp332a (Supplementry Table 4). Zfp322a and Tenm3 both exhibited hypomethylation and increased gene expression in AMC pups. Interestingly, Tenm3 plays a critical role in axon guidance and Zfp322a is a transcription factor that inhibits cellular differentiation 32,33. The hypomethylation of the Tenm3 associated DMR was validated (p=0.01) by an independent pyrosequencing assay (Supplementry Figure 4).
Differential AMC gene expression influences translation and oxidative phosphorylation
To investigate the regulatory relationship between DMRs and gene expression, RNA-seq analysis was performed on postnatal day 9 (PND9) male hypothalami (Figures 3A and B & Supplementry Table 5). Examination of the top 500 most variably expressed genes revealed clustering by AMC condition (Figure 3A). For differential analysis, a total of 2,464 significant (q<0.05) differentially expressed (DE) transcripts were identified between AMC and control hypothalami (Figure 3B, 3C & Supplementry Table 6A & 6B). There were 1,411 genes with reduced expression and 1,053 with increased expression following AMC. Log2 fold changes in gene transcripts ranged from 2.67 to - 2.08 (Supplementry Table 6A & 6B) and the top 20 significant (q<0.05) genes ranked by fold change are shown in Table 2.
Consistent with previous studies that have established a key role for stress-induced neuropeptide Crh 5,11, we also found that Gal, Oxt, and Ucn3, transcripts encoding Galanin, Oxytocin and the Crh homologue Urocortin, respectively, were significantly reduced in handled pup hypothalamus as were transcripts for the Crh1 receptor (p<0.05)(Table 2 & Figure 3B). Pomc, which encodes the protein precursor for the hormone ACTH, a key mediator of stress responses in the periphery was also reduced (reviewed in 34. Interestingly, the Galaninreceptor Gpr151 was the top most up-regulated gene. Also of interest was the upregulation of the serotonin transporter gene, Slc6a4, since its protein SERT is the target of selective serotonin reuptake inhibitor (SSRI) and tricyclic antidepressant drug classes (Table 2).
To comprehensively identify the functions associated with the 2,464 transcripts, gene ontology (GO) and pathways analyses were performed (Figure 3D). Gene ontology analysis revealed a significant (q<0.05) enrichment for the biological processes of translation, protein localization to organelle, and response to oxidative stress. Mitochondrial membrane, focal adhesion, and synapse part were the significantly (q<0.05) enriched cellular components. Structural constituent of ribosome, peptidase activator activity, and oxidoreductase activity acting on NAD(P)H were the most unique significant (q<0.05) molecular functions. Pathway analyses revealed a significant (q<0.05) enrichment for eIF2 signaling, regulation of eIF4 and p760K signaling, oxidative phosphorylation, mitochondrial dysfunction, and mTOR signaling (Figure 3D). Notably, most of the ontologies and pathways are related to the output of PI3K/AKT/mTOR/PTEN signaling 35, particularly translation by the ribosome (eIF2 and eIF4 signaling) and oxidative phosphorylation in the mitochondria. Motif enrichment analysis identified several transcription factor binding sites, with the top significant (q<0.0001) enrichment representing binding sites for the transcription factor Elk1 that were present in 31% of transcripts (Supplementry Table 7). Elk1 is part of an insulin signaling network that cross-talks with PI3K/AKT/mTOR/PTEN signaling 35 and is also involved in synaptic plasticity, learning, and neurodegenerative disease 36. Together these results detail differential regulation of a variety of cellular processes, and signaling pathways not previously associated with AMC, which are consistent with neurodevelopmental events occurring in PND9 hypothalamus during AMC 35.
To validate and expand on DE genes identified by genome-wide RNA-seq analysis, reverse transcriptase quantitative PCR (RT-qPCR) analysis was performed on select genes of interest in both males and females as well as a second, independent cohort of AMC offspring. RT-qPCR analysis confirmed that Crh (Supplementry Figure 5A) was significantly reduced in AMC hypothalamus (p=0.0006). There was a trend (p=0.07) for an increased expression of Ube3a ligase domain containing isoforms (Supplementry Figure 5B), which were discovered using RNA-seq. There was also a significant (p<0.0001) reduction in transcripts containing the alternative 3’UTR of Ube3a1 (Supplementry Figure 5C), which has the non-coding function of a competing endogenous RNA (ceRNA) or “miRNA sponge” that is critical to neurodevelopment 37.
Reciprocal differences in miRNA and target gene expression
One of the top gene expression changes was decreased expression of the Dgcr8 microprocessor complex subunit gene (Table 2). Dgcr8, in combination with Drosha, is involved in the processing of primary miRNA (pri-miRNA) transcripts into precursor miRNAs (pre-miRNA) 38. The decreased expression of Dgcr8 suggests alterations to miRNA processing. Also suggestive of alterations to miRNA regulation was the decrease in the Ube3a1 ceRNA. Since the RNA-seq data from PND9 hypothalamus did not accurately profile miRNA transcripts, we next evaluated the impact of AMC on miRNA expression using a multiplex miRNA expression assay on PND9 hypothalamus from handled and control pups. A total of 156 miRNAs were expressed at levels above background, with five showing significant (p<0.05) differential expression between AMC and control samples. Of these five, transcript levels for rno-miR-488, rno-miR-144, and rno-miR-542-5p were increased while transcript levels for rno-miR-421 and rno-miR-376b-5p were reduced (Figure 4A). The five significant (p<0.05) DE miRNAs were then analyzed alongside the significant (q<0.05) DE mRNAs for both a bioinformatically predicted match and reciprocal DE relationship in order to produce a list of 127 putative target genes (Supplementry Table 8). Among the 127 putative mRNA targets of DE miRNAs are the stress response genes Gal (rno-miR-144), Pomc (rno-miR-488), and Pvalb (rno-miR-488)34,39,40. Other miRNA targets include the sialidase Neu1 (rno-miR-542-5p) and the Amyloid beta precursor protein (APP) protease Adam10 (rno-miR-421), and transcripts for the neuronal regulators Pcdh9 (rno-miR-376b-5p) and Avpr1a (rno-miR-488) (Supplementry Table 8)41–43. Finally, rno-mir-542-5p was predicted to target the Ube3a1 ceRNA and the two showed a reciprocal relationship in expression (Figure 4B).
Integrative epigenomic prioritization of differentially regulated genes
To identify a convergent set of genes regulated by AMC in hypothalamus we integrated genes identified by the DNA methylation, RNA-seq, and multiplex miRNA assay analyses. For this combined analysis the 127 unique genes potentially regulated by the five differential miRNAs from Supplementry Table 8 were compared with the 4,023 unique genes in proximity to DMRs shown in Figure 2. The overlap between these three gene lists resulted in 20 unique genes (Figure 5A). The presence of factors from diverse processes suggests that many pathways contribute to the AMC response in hypothalamus (Table 3). For example, Avrp1a functions in the stress pathway and regulates social behaviors, Kcnh5 is an ion channel that regulates hormone and neurotransmitter release, and Ring1 is representative of several factors on this list that repress gene expression levels (Table 3)44–46. Ring1, a component of the Polycomb Repressive Complex 1 (PRC1) complex, together with Ube3a, another ubiquitin E3 ligase, has been recently shown to regulate genes associated with autism 23. The integration of genome-wide RNA-seq, DMRs, and miRNA target data sets produces a refined set of gene transcripts (Figure 5A), while also demonstrating the genome-wide nature of the differential expression and epigenetic modification that follows AMC (Figure 5B).
Discussion
Early life environment has tremendous power to shape neurodevelopment with long-term consequences 1–3. In humans, disjointed or reduced maternal care that occurs in cases of poverty, maternal depression, or addiction is associated with increased risk for developing cognitive and emotional deficits, neuropsychiatric disorders, and cognitive decline later in life 47,48. On the opposite end of the spectrum, studies in humans suggest that a positive early-life environment can produce resilience to affective disorders 49–51. In animal models, enhancements in maternal care can positively impact the developing brain leading to reduced anxiety, resilience to depression-like behaviors, and enhanced learning and memory 10,11,15. The re-programming of the stress response in offspring receiving augmented maternal care can last a lifetime 52 and help offset age-related cognitive deficits 14. Much of the previous work on AMC has focused on regulation of stress hormones such as Crh and the glucocorticoid receptor. While critical for the AMC phenotype 11 these two components of the stress system are likely not the only important factors for such large-scale and diverse reprogramming of the developing brain.
Together, our results provide the first integrative, genome-wide overview of the genes and pathways that contribute to the “reprogrammed” cognitive and emotional phenotype resulting from AMC. The results offer five groundbreaking observations. First, we identified a significant (p=0.015) brain region specific reduction of global DNA methylation in the hypothalamus as well as 5 large-scale PMDs. Second, we identified 9,439 significant (p<0.05) small-scale DMRs associated with 4,023 unique genes, which function in neurodevelopment and neurotransmission and may contribute to the establishment and maintenance of the behavioral phenotype. Third, we identified 2,464 significant (q<0.05) DE transcripts by RNA-seq analysis that extends our understanding of the gene expression differences to neurotransmission and other crucial neurologic functions by highlighting a role for translation and oxidative phosphorylation. Fourth, we identified the predicted targets of 5 significantly (p<0.05) altered miRNAs and also a putative interaction between mir-542-5p and the Ube3a1 3’UTR. Finally, we integrated these diverse data sets and identified a list of 20 genes with three lines of evidence (methylation, expression, and altered miRNA target) that are highly relevant to the stress resiliency phenotype resulting from AMC.
Consistent with prior descriptions for reduced methylation together with reduced CRH expression in the hypothalamus of AMC rats 21, the connection between DNA methylation and gene expression in our genome-wide study was complex, and more apparent at the level of pathways than individual genes. At the level of gene ontology, the cellular component term “synapse part” is significantly (q<0.05) enriched in both datasets. Reactive oxygen species (ROS) generated by oxidative phosphorylation in the mitochondria are not only a key signaling molecule behind synaptic plasticity and memory formation 53, but are also critical to neurodevelopment since they regulate PI3K/AKT/mTOR/PTEN signaling 54. Furthermore, the hypomethylated Grb10 gold DMR (FWER < 0.05) belongs to a genomically-imprinted gene encoding for a receptor-binding protein that interacts with insulin and insulin-like growth factor receptors of the PI3K/AKT/mTOR/PTEN signaling pathway 55. Translational control is achieved via eukaryotic initiation factors (eIFs) that interact with the ribosome to drive the protein synthesis needed for synaptic plasticity 56. Furthermore, PI3K/AKT/mTOR/PTEN signaling, Wnt/β-catenin signaling, and protein kinase A (PKA) signaling converge to shape neuroplasticity, neurogenesis, cell survival, and resilience 57. Finally, protein kinase A signaling is activated by G-protein-coupled receptors (GPCRs) that have neurotransmitter and hormone ligands, which include Crh and Urn3 58. The GPCRs of PKA signaling utilize cyclic adenosine monophosphate (cAMP) as a secondary messenger to activate PKA, which is also affected by handling to alter relevant gene expression profiles, potentially including the glucocorticoid receptor gene Nr3c1 52. Ultimately, the overlapping pathways between the methylation and gene expression suggest a broader and potentially long-term impact on numerous cellular and molecular functions important for hypothalamic function.
The genes and pathways identified from our unbiased analysis of AMC-induced hypothalamic changes may be used towards the development of biomarkers and therapeutics. Suderman et al. have shown that epigenetic programming of the HPA axis by maternal care is conserved in humans at a select locus 59. Given the potential conservation, the suite of new candidate loci identified may be examined in humans towards the development of novel epigenetic biomarkers of resiliency 60. Such a diagnostic may then be used to identify individuals that would benefit from corrective measures, which include environmental enrichment 61 and pharmacologic agents. The suite of genetic loci identified may also be used towards the development of inventive pharmacologic agents. Other studies have shown that the transcriptomic profile and epigenetic modifications of altered maternal care can be reprogrammed at adulthood by the global epigenetic modifiers, specifically the methyl donor L-methionine or the histone deacetylase inhibitor trichostatin A (TSA) 18,19,62,63. More recently, it has been shown that pharmacologic agents can mimic some of the effects of AMC in vitro 12.
Therefore, some of the epigenomic profile established by maternal care is plastic and can be manipulated later in life. However, these previously examined modifiers result in attenuations that are global in nature and may have off-target effects. Our results could be used to overcome this limitation since the suite of therapeutic candidates identified can be utilized as prioritized targets for somatic epigenetic editing studies 64–67. Ultimately, our results could form the molecular foundation for developing effective interventions for deficient early maternal care in humans or be developed as new treatments for psychiatric disorders67.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
This work used the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH [S10 OD018174] Instrumentation Grant. B.I.L. was supported by a postdoctoral fellowship award from the Canadian Institutes of Health Research (CIHR). A.V.C .was supported by National Institutes of Health [T32MH073124-06]. A.S-T and T.Z.B. were supported by NIH MH73136 and P50 096889
Supplementary Information accompanies the paper on the BiorXiv website