Re: Influences of genomic imprinting on brain function and behavior

[문형철]

beyond reason

많은 의학자들은 뇌의 국재이론을 무심코 받아들인다. 국재이론을 진실로 받아들이는 순간 6개월이 지난 중풍환자는 더이상 회복이 불가능한 환자로 규정된다. 치매, 파킨슨 병으로 인한 기억력 저하, 망상은 치료가 불가능한 것으로 받아들여진다. 

하지만 뇌는 중풍으로부터 완전 회복을 할 수 있는 가능성을 가진 가소성 변화기능을 가지고 있다. 

질 볼트 테일러(37세 뇌출혈을 앓고 완전 회복한 천재 뇌신경해부학자)는 그것을 온몸으로 증명하였다. 

아래 논문은 유전제 각인(genomic imprinting)이 뇌의 기능과 행동에 미치는 영향에 관한 이야기...

뇌에 관해서는 메타인지(무엇을 알고 있는지 무엇을 모르고 있는지)조차 안되어 있는 상태라.... 

Influences of genomic imprinting on brain function and behavior

Author links open overlay panelOliviaHo-ShingCatherineDulac

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https://doi.org/10.1016/j.cobeha.2018.08.008Get rights and content

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Highlights

• Imprinted effects in the brain range from monoallelic to parentally-biased expression‎.

• Imprinted expression‎ is highly specific to brain region, cell type, and age.

• Imprinted genes function in critical pathways affecting development and behavior.

• Paternal genes tend to promote growth and reproductive behaviors.

• Maternal genes preferentially promote emotional and cognitive behaviors.

Genomic imprinting is a unique form of epigenetic regulation that is highly preval‎ent in the brain. The complexity of imprinted regulation in the adult and developing brain, and its central roles in neural processes are becoming increasingly appreciated. Here, we outline recent progress in elucidating the influence of imprinted genes on brain function — from neural development to synaptic function, cognition, and social and emotional behaviors. We review how improved sequencing and analytical technology have helped in identifying parental biases in gene expression‎ alongside monoallelic expression‎ in brain tissues, and we discuss the potential roles of imprinted genes in regulating key neural pathways. This review aims to provide a better understanding of the nature and functional significance of genomic imprinting in the normal and pathological brain of mammals, including humans.

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Current Opinion in Behavioral Sciences 2019, 25:66–76

This review comes from a themed issue on Genetic imprinting and behaviour

Edited by William Davies and Lawrence Wilkinson

For a complete overview see the Issue and the Editorial

Available online 27th August 2018

https://doi.org/10.1016/j.cobeha.2018.08.008

2352-1546/© 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Genomic imprinting is a form of epigenetic regulation found in mammals and flowering plants, which leads to the preferential expression‎ from either the maternally or paternally inherited allele of certain genes. Although imprinted genes represent less than one percent of the mammalian genome, imprinted gene expression‎ has been shown to play critical roles in embryonic development and adult tissue function [1,2]. In humans, Prader–Willi and Angelman syndromes were among the first recognized examples of imprinting defects. Prader–Willi syndrome results from disruption of one or more paternally inherited genes on chromosome 15q11–13, and is characterized by mild retardation, impaired satiety, and compulsive behavior [3]. Angelman syndrome results from disruption of the maternally inherited copy of Ube3a (within the 15q11-13 cluster), and is characterized by severe mental retardation, ataxia and frequent laughter. These disorders provide a striking example of how specific parent-of-origin allelic expression‎ is essential for normal behavior control and brain function. In parallel to clinical studies of defects in imprinted genes, studies of chimeric embryos that contain two maternal genomes (gynogenetic) or two paternal genomes (androgenetic) bolstered our understanding of this phenomenon, and suggested that the genome inherited from each parent contributes differently to the development of the brain [4]. In particular it was found that gynogenetic and androgenetic cells populate different regions of the brain throughout development. Gynogenetic cells were found in hippocampal, striatal and higher cortical regions, and embryos developed abnormally large brains. Androgenetic cells on the other hand were found in areas including the hypothalamus and brain stem, and embryos developed abnormally small brains. These early findings suggested that genomic imprinting strongly influences the fate or survival of populations of neuronal cells, and that imprinted gene expression‎ may dramatically alter complex neural networks and brain function.

Parent-of-origin allelic expression‎ in the brain

Early studies of genomic imprinting established the canonical definition of imprinted regulation as the complete epigenetic silencing of one parental allele, causing monoallelic expression‎ from the other parent-of-origin copy [1]. For example Igf2, one of the first identified imprinted gene, was initially observed to be transcriptionally active from the paternal allele while the maternal copy was silent in the developing embryo [5]. However, other imprinted genes, including Gnas and Ube3a displayed a bias in their expression‎ for one of the two parent-of-origin alleles, rather than strict monoallelic expression‎ [6, 7, 8]. More recent genome-wide studies, primarily characterizing expression‎ in genetically tractable hybrid mice, have broadened the understanding of imprinting regulation beyond canonical monoallelic expression‎. Indeed, although some imprinted genes such as Impact are strictly expressed from one parental allele across all tissues, the allelic expression‎ profiles of other imprinted genes, such as Igf2, uncovers newly appreciated and more nuanced imprinted effects across the brain and in non-brain tissues (Figure 1a). Particularly in the postnatal brain, one can identify parentally biased gene expression‎ that changes dynamically across brain regions and developmental stages. Rigorous genome-wide RNA sequencing analyses have uncovered numerous genes with significant parental bias in expression‎ throughout the brain [9, 10, 11, 12, 13,14••,15,16•,17, 18, 19], strongly suggesting that imprinted gene expression‎ may be a common epigenetic mechanism employed by the brain to tightly regulate neural networks. Here we discuss emergent patterns of parentally biased expression‎ identified in the brain.

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Figure 1. (a) Parentally biased imprinted genes in mouse. Spatial regulation of imprinting represented by a hierarchically clustered heat map of the deviation from biallelic gene expression‎. Panel adapted from Perez et al. (2015). L and S subscripts in the gene names indicate long and short isoforms, respectively, and V2 indicates variant 2 isoform. Acronyms of brain regions: AC, anterior cortex; CA, cortical amygdala; Cb, cerebellum; CP, caudate putamen; DM, dorsal midbrain; Hp, hippocampus; Hy, hypothalamus; My, medulla; NA, nucleus accumbens; OB, olfactory bulb; Pa, pallidum; PC, posterior cortex; Po, pons; SA, striatum-like amygdala; Th, thalamus; VM, ventral midbrain. Acronyms of somatic tissues: Hr, heart; Kd, kidney; Lu, lung; Lv, liver; Mu, muscle; Sk, skin; Sp, spleen. (b) The number of coding and non-coding genes among strongly imprinted genes and parentally biased genes in the adult mouse cerebellum. Strongly imprinted genes exhibit a 90:10–100:0 parental bias. Parentally biased genes include genes that exhibit a 60:40–90:10 parental bias. Figure adapted from Perez et al. (2015). (c) The number of clustered and isolated genes among strongly imprinted genes and parentally biased genes in the adult mouse cerebellum. Strongly imprinted genes exhibit a 90:10–100:0 parental bias. Parentally biased genes include genes that exhibit a 60:40–90:10 parental bias. Figure adapted from Perez et al. (2015).

Next generation sequencing approaches have provided powerful new experimental platforms that enabled the detection of a large spectrum of parental biases in RNA transcripts from various biological samples. Initial studies using high-resolution sequencing provided conflicting results. While some highly stringent analyses identified only a few novel imprinted genes and failed to detect some known imprinted genes [20,21]; less conservative approaches [9,10] uncovered a continuum of allelic effects in the expression‎ of specific genes, and even specific isoforms. Both the magnitude and direction of parental bias varied across different brain regions and developmental stages. These results suggested the existence of intricate stage-specific gene regulation of the parental genomes in establishing neural pathways. Interestingly, while many strongly imprinted genes were implicated in neural circuits associated with feeding and motivated behaviors, genes that displayed a parental bias in expression‎ often functioned in cell metabolism and signaling. Further studies strengthened the statistical methods applied to RNA sequencing results, used independent experimental methods to validate the observed parental biases, and corroborate novel imprinted genes [13,14••,16•,19,22]. These parentally biased expression‎ patterns revealed new intricacies of transcriptional regulation in the brain and expanded the scope of parent-of-origin-specific epigenetic regulation. Moreover, the discovery of extensive parent-of-origin expression‎ biases prompted new questions about the range and function of this form of imprinted regulation, particularly throughout the postnatal brain.

While previously known imprinted genes were seen to be mostly expressed at high levels, genes exhibiting a parental bias appeared usually expressed at low to moderate levels [9,14••,16•]. Because of the lower expression‎ levels and spatiotemporal specificity of parentally biased expression‎, some discrepancy remains between studies about the exact number of genes regulated by imprinting [1,11,23,24], highlighting key issues related to the sensitivity of the RNA sequencing technique and statistical analysis [11,24,25,26••,27], and expression‎ variations in different genetic backgrounds [13,19]. Perez et al. (Figure 1a) and other RNA sequencing studies [13,14••,18,19,25,28] characterized and validated imprinted expression‎ patterns of roughly 200 genes (Figure 1a). About one-third of monoallelic imprinted genes are protein-coding, the majority comprised of noncoding microRNAs, small nucleolar RNAs and long non-coding RNAs (lncRNAs), presumably allowing the regulation of a wide network of genes [14••]. On the other hand, almost 95% of parentally biased genes (exhibiting a weak to robust parent-of-origin effect between 50:50 and 90:10) are protein-coding (Figure 1b) [29]. From these studies, consensus has emerged that imprinted effects are more preval‎ent in brain than somatic tissues in mice, rats and humans [18,25,29], supporting the notion that genomic imprinting may constitute a conserved mechanism to instruct neural function.

Multiple studies have characterized higher frequency of imprinted gene expression‎ in the hypothalamus than in other brain regions [9,18,30], supporting the central focus on genomic imprinting in the hypothalamus seen in early studies with chimeric embryos [4,31]. The arcuate nucleus of the hypothalamus expressed 79% more imprinted genes than the dorsal raphe nucleus of the midbrain, and 100–300% more than somatic skeletal tissues [16•]. The cortex also exhibited high frequency of biased expression‎ patterns, with more genes preferentially expressing the maternal allele. By contrast, preference for expression‎ of the paternal allele predominated in mid-brain and hindbrain regions, including the hypothalamus. Thus, imprinted expression‎ in both rodents and humans appears most preval‎ent in the brain, and gene-specific allelic effects are highly tissue-specific, isoform-specific, and age-specific [14••,19,32]. Additionally, some imprinted genes such as Ndn, Grb10 and Ube3a have been shown to be imprinted strictly in neuronal cells, while other genes like Igf2r are imprinted strictly in non-neuronal cells. The dynamic patterns of allelic expression‎ detected in the brain may therefore result from a summation of diverse imprinted effects occurring in different cell types [2,14••,29].

While many studies have found that imprinted expression‎ (i.e. the number of imprinted genes as well as the strength of the parental bias for a given gene) is strongest in embryonic tissues and placenta, a large (though reduced) number of genes display robust allelic effects into adulthood [14••,19]. Interestingly, many genes with a parental bias see changes in the strength of the parental bias, or even switch from a parental bias to a biallelic expression‎ across brain regions and across developmental stages. For example, the growth suppressor gene Grb10 exhibits biallelic expression‎ in the cerebellum at P8 (a pivotal developmental stage for granule cell migration), but switches to monoallelic paternal expression‎ in the adult brain. Moreover, this expression‎ is in sharp contrast to Grb10 in various peripheral tissues, where it exhibits monoallelic maternal expression‎ [9,13,17]. Other genes like Zim1 exhibit even stronger region-specific shifts in allelic bias: in the cerebellum, Zim1 switches from maternally to paternally biased as expression‎ from the maternal allele decreases in adulthood. Expression‎ in other hindbrain and midbrain regions, however remain strongly maternal. Interestingly Igf2, which is exclusively expressed from the paternal allele throughout the body, exhibits varying magnitudes of maternal preference throughout the brain [9,14••] (Figure 1a).

Imprinted regulation appears to instruct neural function by modulating the expression‎ levels of genes within imprinted clusters [2]. Studies of specific developmental disorders such as Rett syndrome have demonstrated how even slight changes in gene dosage in brain tissues tightly correlates with the severity of mental illness behavioral phenotypes [33,34]. Therefore even nuanced regulation of imprinted neural genes may be highly relevant to normal brain development and function, as well as disease states. Recently studies have shown that loss of differential methylation alters expression‎ levels within the H19/Igf2 cluster [35], and a twofold increase in imprinted gene Cdkn1c expression‎ mimics neurological and behavioral phenotypes of Silver–Russell syndrome and other associated imprinted disorders [36,37•]. A key question, therefore, is whether parentally-biased expression‎ indicates a similar, though more nuanced coordinated regulation of gene dosage [23]. Indeed, a specific test of this hypothesis showed that for the majority of parentally biased imprinted genes uncovered in the mouse, a positive correlation is observed between the strength of the parental bias and age-regulated changes in gene expression‎ level [14••]. This important result suggested that imprinted regulation may have evolved as a mechanism to provide a tight control of gene dosage, for both monoallelic and parentally biased genes. In humans, RNA profiling of imprinted expression‎ has not demonstrated a clear correlation between imprinted status and gene dosage, though this is possibly due to confounding differences in human genetic backgrounds [38••].

Many genes displaying parental biases are located in imprinted clusters within one megabase of imprinted genes with monoallelic expression‎ (Figure 1c). Clusters often comprise both maternally and paternally biased genes (or gene isoforms) [9,14••,19], with cis-regulatory effects [13], suggesting that imprinted genes within a cluster are likely regulated by shared canonical imprinting mechanisms. Some genes located on different chromosomes, however, exhibit similar patterns of bias across multiple tissues or brain regions; for example, across the brain, Copg2 and Mest located on chromosome 6 exhibit a strikingly similar allelic expression‎ pattern to Ago2 located on chromosome 15 [14••] (Figure 1a). Other imprinted genes like maternally biased Bag3 and paternally biased Bcl-xL (or Bcl2l1) show an anti-correlation in their allelic bias across brain regions. Interestingly, both gene products function in the apoptotic pathway, a common functional target of imprinted expression‎ (Figure 2c). Thus, characterizing allelic expression‎ of the whole transcriptome has caused larger patterns to emerge suggesting that imprinted regulation is orchestrated across wide networks of genes throughout developmental stages and brain regions [14••,19]. Interestingly, knockout of the parentally biased genes Man1a2 and H2-ab1 affects vast networks of genes at imprinted and non-imprinted loci, causing complex phenotypic variations in genetically diverse populations [26••]. Taken together, these observations suggest that parentally biased genes are evolutionarily positioned at critical junctures in gene networks and their nuanced regulation is potentially of high functional significance.

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Figure 2. (a) Roles of imprinted genes during cortical neurogenesis. Genes preferentially expressed from the maternal and paternal allele appear in red and blue, respectively. In black are imprinted genes that are biallelically expressed in this context. Strongly biased and monoallelically expressed imprinted genes are in bold. Lines with arrowheads indicate enhancement, and lines with notched ends indicate reduction of the biological function. The asterisks by Mir379-410 indicate that the corresponding biological functions are regulated by three miRNAs of this cluster: Mir369-3p, Mir496, and Mir543. (b) Regulation of apoptosis. Within the circle are products of imprinted genes that interact directly with the apoptosis machinery, and outside the circle are products of imprinted genes that influence pro-apoptosis or anti-apoptosis. L and S subscripts in the gene names indicate long and short isoforms, respectively. (c) Functions of imprinted genes in synaptic transmission and plasticity. Products of genes preferentially expressed from the maternal and paternal allele appear in red and blue, respectively. Products of biallelically expressed genes appear in dark gray. Products of strongly biased and monoallelically expressed imprinted genes are in bold. Lines with arrowheads represent stimulatory molecular interactions, whereas lines with notched ends represent inhibitory molecular interactions. The overall role of imprinted genes in synaptic plasticity is shown in green to indicate synaptic potentiation or activation, in orange to indicate synaptic depression or inhibition, in violet to indicate the induction of structural changes, and in gray when the contribution to synaptic transmission or plasticity remains unclear. White-filled circles with beige borders represent vesicles containing membrane receptors or channels.

Roles of imprinted genes in the brain

Extensive studies have shown that imprinted genes regulate essential neurodevelopmental processes (Figure 2a), including neural differentiation, migration and cell survival [29]. Key examples of the involvement of imprinted genes in these processes are reviewed here.

Neural stem cells (NSCs) originate in the ventricular zone of the developing cortex and asymmetrically proliferate to produce glia or intermediate progenitor cells, and regenerate the stem cell pool. Progenitor cells give rise to immature, then differentiated neurons [39]. NSCs highly express the paternal Plagl1, a zinc-finger protein that induces the expression‎ of the maternal cyclin-dependent kinase inhibitor Cdkn1c. Cdkn1c inhibits cyclin-dependent kinases in order to prevent NSC mitosis, and thereby activates a shift from proneural to proglial differentiation [29,40]. At a later stage, Cdkn1c promotes neuronal migration within the cortical plate by inducing actin polymerization [41]. Igf2 and paternal Dlk1 in the embryonic brain regulate self-renewal of NSCs and intermediate progenitors in the subgranular zone and promote fetal cortical neurogenesis in mice and humans [42]. Interestingly, Igf2 is biallelically expressed in the choroid plexus and subventricular vasculature, maternally biased in other brain regions and paternally expressed in somatic tissues [5,14••,43]. Biallelic expression‎ of Igf2 acts as a paracrine factor that regulates NSC homeostasis in the subventricular zone, but paternal expression‎ of Igf2 in the hippocampus acts as an autocrine factor for neurogenesis in the subgranular zone [44••]. Igf2 expression‎ demonstrates highly spatiotemporal imprinted regulation of transcriptional dosage to instruct adult neurogenesis. In the cerebellum, paternally biased Dio3 regulates thyroid hormone signaling, inhibiting premature differentiation of granule cells, their migration from the external to internal layer, and the dendritic arborization of Purkinje cells [45]. Loss of imprinted expression‎ at the Dlk1-Dio3 locus leads to reduced neural differentiation in human stem cells [46]. Loss of Dio3 in mice leads to increased social aggression and reduced maternal behavior [47]. Similarly, a two-fold increase in Cdkn1c expression‎ — mimicking a loss of imprinting — caused in mice an increased motivation for food, increased aggression and an unstable social hierarchy [36,37•].

In the developing nervous system, apoptosis is a mechanism to control the neuronal pool and synaptic matching between neurons. Internal and external stimuli regulate apoptosis by shifting the balance between pro-apoptotic and anti-apoptotic proteins. Interestingly, many genes of the apoptotic pathway are affected by imprinted regulation (Figure 2b). Paternally expressed genes seem to play mostly anti-apoptotic roles, while maternally expressed genes are more pleiotropic [14••,48]. The maternally expressed Kcnk9 plays a role in mediating neuronal excitability [49], and promotes apoptosis in granule cells [50]. Loss of the paternally expressed Peg3 or Magel2 causes a reduction in hypothalamic oxytocin neurons. Loss of Peg3 also increases apoptosis in forebrain, striatal and amygdalar regions [48]. Bcl-xL is a major anti-apoptotic regulator in both mouse and human brains, the deletion of which causes massive apoptosis in postmitotic immature neurons [51]. Remarkably, although Bcl-xL exhibits a modest paternal bias in expression‎ throughout the brain (Figure 1a) [9,11], the deletion of its paternal, but not maternal allele causes severe cell loss in the cortex, that specifically affects excitatory but not inhibitory neurons or glia [14••]. Thus, like previously uncovered imprinted genes including Ube3a, Gnas and Phlda2 [6,7,52]; newly uncovered biased Bcl-xL demonstrates cell type specificity in imprinted regulation. Therefore, the discovery through transcriptome analyses of other parentally biased genes open interesting avenues for further study — to understand the function of these allelic effects on complex traits and the underlying mechanisms by which they are established.

Many imprinted genes (both monoallelic and biased) play further roles in the adult brain by regulating synaptic transmission and plasticity, thus ultimately modulating the function of neural circuits (Figure 2c). Deletion of maternally expressed Kcnk9 in mice reduces membrane resting potential, altering the firing patterns in cerebellar granule cells [53]. Ube3a maternal deletion causes increased amplitudes of action potentials in the hippocampus, due to increased levels of α1-NaKA channels, which help maintain the membrane resting potential [54,55]. Likewise, glutamatergic neurons derived from patients with Angelman syndrome show reduced action potential firing and synaptic activity; these are likely secondary consequences of altered resting potential [56]. Ube3a maternal deletions also show impaired capacity to replenish presynaptic vesicles in L4 interneurons, causing weaker inhibitory postsynaptic currents [57]. Most imprinted genes known to regulate synaptic plasticity are maternally expressed, typically promoting synaptic potentiation (Figure 2c). UBE3A promotes long-term potentiation (LTP) in excitatory synapses by preventing the inhibition of N-methyl-d-aspartate receptors (NMDARs), and the activity-dependent internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) [58,59]. Apoptotic genes including Bcl-xL have also recently been discovered to play an essential role in induction of long-term depression (LTD), by activating modest levels of caspases in the post-synapse and thereby inducing AMPAR internalization [60,61].

The extensive roles of imprinted genes in neural development, synaptic function, and plasticity implicates them further in learning and memory. Kcnk9 null mutants, for example, exhibit impaired contextual memory and fragmented sleep with reduced rapid eye movement [62,63]. Ube3a maternal deletions also perform poorly in contextual memory tasks [59]. Monocular deprivation in Ube3a maternal deletion mutants fails to induce ocular dominance in the visual cortex, indicating a role in experience-dependent plasticity [64]. Recently, RNA sequencing of the visual system in dark-reared mice during the visual critical period uncovered up-regulation of three imprinted microRNAs: miR882 in the retina, and miR329 and miR453 in the visual cortex [65]. Although there were no significant changes in the expression‎ levels of imprinted genes identified in the visual cortex (isoform-specific Herc3, Trappc9 and H13; and maternal Rtl1 and maternally-biased Ago2), interestingly, in the suprachiasmic nucleus of dark-reared mice one maternal isoform of H13 altered in imprinted status from 70:30 percent maternal:paternal transcripts to 60:40. These findings underline the complex tissue-specific and isoform-specific nature of imprinted regulation; and implicate imprinted genes in modulating plasticity during critical periods in the brain.

Several reviews have described the key roles imprinted genes play in regulating metabolism and growth in early life into adulthood [66,67]. Moreover, studies of knockout mice and patients with imprinted disorders further highlight that appropriate regulation of imprinted genes directs various neuronal pathways that influence social behavior, particularly interactions between the offspring and mother or conspecifics. Targeted mutation of paternal Peg3 causes a severe lack of maternal care in mothers. Peg3 loss in the prenatal pup and placenta, though, demonstrates reduced ultrasonic vocalizations in Peg3− pups, leading to reduced maternal behavior even in wild-type mothers [68,69]. Furthermore, an recent study demonstrated that loss of maternal expression‎ of Phlda2 in offspring led to an increase in maternal care behaviors, including nursing and grooming in wild-type mothers. Interestingly, the Phlda2 dosage from prenatal pups influences in utero gene expression‎ in the mother’s hypothalamus and hippocampus, showing a remarkable instance where imprinted regulation modulates through gene dosage the level of maternal investment in offspring [70]. Studies using more elaborate crosses of inbred mice may begin to disentangle the influences of genetic background, imprinted effects and the perinatal environment on behavior [71]. An interesting recent study compared mice lacking maternal Nesp to those lacking paternal Grb10, two imprinted genes exhibiting similar patterns of expression‎ in the brain. While Nespm− mice make more impulsive choices than wild-type littermates, Grb10p− mice make fewer ones, uncovering how imprinted genes with different allelic expression‎ profiles may affect opposing behaviors [72•]. In humans, a genome-wide association analysis of children with language impairments found significant parent-of-origin effects at two loci that were also previously associated with schizophrenia and autism [73,74]. Finally, a study of children with Prader–Willi syndrome reports that the loss of paternal expression‎ at the Snrpn/Ube3a locus may cause the subjects’ observed deficit in pitch discrimination but increased behavioral response to hearing music [75].

Cellular basis and regulation of parent-of-origin effects

The growing evidence for functional effects of allele-specific expression‎ in the brain has prompted in-depth investigations of the mechanisms underlying imprinted regulation [2,23]. The majority of imprinted genes reside in roughly 1-megabase clusters that have parent-of-origin-specific epigenetic modifications, including primarily DNA methylation and histone modifications. Most imprinted control regions are differentially methylated during germline development by de novo DNA methyltransferases including DNMT1. After fertilization, the differential allelic methylation must be maintained by DNMTs throughout adulthood. The differential methylation of imprinted control regions directs allele-specific binding of enhancer or repressor elements — the most well studied example being CTCF insulation of the Igf2-H19 cluster [44••,76]. Methylation at promoter regions also directs allelic expression‎ of lncRNAs that often repress expression‎ from the opposite allele [1,77•]. Thirty percent of uncovered parentally biased genes are not clustered however, and few are located near a germline-derived differentially methylated region [1,78]. Thus, for both clustered and non-clustered imprinted genes questions emerge about what regulatory factors are affecting biased expression‎ in the postnatal brain; and how are methylation marks propagated in different brain regions?

By combining approaches to profile allelic expression‎ as well as methylation and quantitative traits, recent and future studies of genomic imprinting can illuminate more direct correlations between the mechanisms and functional phenotypes of parentally biased regulation [79,80]. One recent study examined the association of early childhood temperament and socio-emotional behavior with the DNA methylation status of nine extensively studied imprinted loci, in umbilical blood cells. Results suggest a significant association between the MEG3 (maternally expressed gene 3) DMR methylation levels and children’s inhibition to novelty. By contrast, the PEG3 (paternally expressed gene 3) methylation levels were significantly associated with impulsivity, demonstrating another instance, in humans, where differential maternal and paternal regulations evoke opposing behaviors in offspring. These results suggest possible correlations between methylation patterns at imprinted loci and human behavioral phenotypes, opening interesting avenues for future risk assessments of certain child psychiatric disorders [81].

Understanding the correlations between methylation patterns and the effects on gene dosage has proven to be more complex. Imprinted gene dosage can be modulated through canonical, CTCF-mediated mechanisms, or through the relaxation of imprinted marks, potentially activating the less dominant allele [76]. Interestingly, a recent study of mice deficient in TET1, a dioxygenase that initiates DNA demethylation, found that TET1 demethylation is sporadic, leading to variable DNA methylation at a subset of imprinted loci [82••]. Cell-specific methylation analyses have the potential to illuminate regulation patterns that instruct parentally biased expression‎. A study in the mouse visual system found that tissue-specific imprinting does not always correlate with all methylation patterns in imprinted control regions. Although the Peg13/Ago2 imprinted control region is methylated in the brain, retina and cardiac tissues, the genes within the cluster are not imprinted in all the tissues. Ago2, for example, is imprinted in the visual cortex but not in the retina [65]. These new data showcase the need to understand the full networks of epigenetic regulation associated with parental bias in expression‎, in order to be able to specifically target disordered imprinting. Other recent studies investigate secondary DMRs, which unlike primary DMRs, acquire differential methylation on the promoter and gene body after fertilization and implantation. Acquiring parent-of-origin specific methylation depends on the methylation status of the primary DMR, forming a wider-reaching hierarchy of epigenetic regulation [83]. While methylation at primary DMRs is quite stable, secondary DMRs are more labile. Guntrum et al. [84•] found at the Dlk1 locus nearly 90% methylation of the primary control region, but only around 70% methylation at the secondary DMR. Their findings argue that variability of secondary methylation could be the mechanism behind the variability observed in imprinted methylation patterns. Such variability could be key in understanding how particularly parentally biased imprinted expression‎ arises.

With improved genetic techniques, investigations can address the expression‎ of imprinted loci in specific neuronal populations. Imaging coupled with microdissection is one new approach to profile parental expression‎ of specific neuronal cell types, reducing some of the heterogeneity seen in whole tissue expression‎ studies [85]. High-resolution in situ methods offer another attractive approach, which avoids any genetic manipulation and maintains spatiotemporal dynamics while profiling allelic expression‎ in cells [35,86,87]. Bonthuis et al. [28] targeted nascent RNAs of genes exhibiting non-canonical biased expression‎, and showed data suggesting that tissue-level biases in expression‎ represent allele-specific expression‎ effects in subpopulations of neurons. Single-cell RNA and methylation techniques have also become more widely used and are particularly appealing to disentangle the heterogeneous environment of the brain [81,88, 89, 90, 91,92•]. One of these studies reanalyzed single-cell RNA sequencing data from postmortem human brains, and observed a high frequency of monoallelic expression‎ in single cells, the majority of events attributed to random monoallelic expression‎. Multiple genes detected as monoallelic across individuals, however, were expressed in neuronal cells, and not oligodendrocytes. Like imprinted genes uncovered in the mouse, genes uncovered by single-cell RNA-seq in humans including Syt1, Stmn1 and Ngfrap1 have roles in synaptic transmission, plasticity, and neuronal differentiation [92•]. A single-cell study pharmacologically inhibited methylation of the H19/Igf2 imprinted locus, leading to two distinct subpopulations of monoallelic and biallelic cells in mouse cardiac tissue. These results emphasize that misregulation of imprinted clusters can elicit striking heterogeneity at the level of single cells. This type of phenotypic mosaicism likely plays a role in the presentation of human imprinted disorders [35]. As single-cell methods become more sensitive, we will be able to validate and uncover more nuances of imprinted expression‎ in specific neuronal circuits. Furthermore, a clear understanding of the cellular mechanisms of imprinted regulation have enabled authors to pharmacologically unsilence the repressed parental allele, effectively ‘curing’ disordered expression‎ in specific neuronal populations [56,93••].

The emergent theme from recent studies is that imprinted genes exhibit various degrees of parentally biased expression‎ in different regions of the brain. These genes form extensive regulatory networks that influence the physiology of neural circuits and affect behavioral phenotypes in the adult. High-resolution studies have introduced unexpected nuances in our canonical perception of imprinted expression‎ and regulation, uncovering novel paradigms to study behavior control in healthy brains. Moreover, studies of imprinted regulation at the cellular level is opening new avenues for therapeutic advancements, and may inspire targeted treatments of neural injuries in adults [94], and imprinted disorders like Angelman syndrome [93••].

Disclosure and acknowledgements

We thank Julio Perez for critically reading this manuscript, and Harvard Molecular and Cellular Biology Graphics team for assistance in producing the figures. Some of the work described in this review was supported by the Howard Hughes Medical Institute.

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as

• of special interest

•• of outstanding interest

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