Multigenerational epigenetic inheritance: One step forward, two generations back

Neurobiology of Disease 132 (2019) 104591

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Review

Multigenerational epigenetic inheritance: One step forward, two generations back ⁎

Jennifer J. Tuscher , Jeremy J. Day

T

⁎⁎

Department of Neurobiology, Evelyn F. McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Epigenetics Gene expression Heritability Transgenerational DNA methylation Non-coding RNA Histone modifications

Modifications to DNA and histone proteins serve a critical regulatory role in the developing and adult brain, and over a decade of research has established the importance of these “epigenetic” modifications in a wide variety of brain functions across the lifespan. Epigenetic patterns orchestrate gene expression programs that establish the phenotypic diversity of various cellular classes in the central nervous system, play a key role in experiencedependent gene regulation in the adult brain, and are commonly implicated in neurodevelopmental, psychiatric, and neurodegenerative disease states. In addition to these established roles, emerging evidence indicates that epigenetic information can potentially be transmitted to offspring, giving rise to inter- and trans-generational epigenetic inheritance phenotypes. However, our understanding of the cellular events that participate in this information transfer is incomplete, and the ability of this transfer to overcome complete epigenetic reprogramming during embryonic development is highly controversial. This review explores the existing literature on multigenerational epigenetic mechanisms in the central nervous system. First, we focus on the cellular mechanisms that may perpetuate or counteract this type of information transfer, and consider how epigenetic modification in germline and somatic cells regulate important aspects of cellular and organismal development. Next, we review the potential phenotypes resulting from ancestral experiences that impact gene regulatory modifications, including how these changes may give rise to unique metabolic phenotypes. Finally, we discuss several caveats and technical limitations that influence multigenerational epigenetic effects. We argue that studies reporting multigenerational epigenetic changes impacting the central nervous system must be interpreted with caution, and provide suggestions for how epigenetic information transfer can be mechanistically disentangled from genetic and environmental influences on brain function.

1. Introduction Epigenetic modifications provide an essential mechanism for the fine tuning of gene expression in response to extracellular signals and changes in the environment. These alterations can occur in the form of chromatin remodeling, direct covalent modifications to the DNA itself, post-translational modifications to histone proteins, and activity of noncoding RNAs. In the central nervous system, epigenetic mechanisms are responsible for shaping early developmental programming (Fagiolini et al., 2009; Feng et al., 2010; Bale, 2015), responses to stressful or appetitive stimuli (Renthal and Nestler, 2008; McEwen et al., 2012; Day et al., 2013; Stankiewicz et al., 2013; Klengel and Binder, 2015), activity-dependent changes in synaptic plasticity (Cortes-Mendoza et al.,

2013; Karpova et al., 2017; Clayton et al., 2019), and the formation and long-term storage of memory (Miller and Sweatt, 2007; Lubin et al., 2008; Gupta et al., 2010; Day and Sweatt, 2011; Day et al., 2013; Zovkic et al., 2014). Despite the well-established role of epigenetic regulation of a number of these neural processes, whether experiencedependent epigenetic marks acquired in an individual can be inherited by the subsequent generation remains an open topic of debate. Accumulating evidence supports the notion that external stimuli, including environmental exposure to chemicals, extreme heat, psychological stressors, traumatic experience, substances of abuse, and dietary intake, can all shape epigenetic state at the cellular level in the exposed individual (Waddington, 1953; Francis et al., 1999; Anway et al., 2005; Baccarelli and Bollati, 2009; Mathers et al., 2010; Gapp

⁎ Correspondence to: J. J. Tuscher, Department of Neurobiology, UAB The University of Alabama at Birmingham, SHEL 930, 1825 University Blvd, Birmingham, AL 35294-2182, USA. ⁎⁎ Correspondence to: J. J. Day, Department of Neurobiology, Evelyn F. McKnight Brain Institute, UAB The University of Alabama at Birmingham, SHEL 910, 1825 University Blvd, Birmingham, AL 35294-2182, USA. E-mail addresses: [email protected] (J.J. Tuscher), [email protected] (J.J. Day).

https://doi.org/10.1016/j.nbd.2019.104591 Received 2 April 2019; Received in revised form 22 July 2019; Accepted 26 August 2019 Available online 27 August 2019 0969-9961/ © 2019 Elsevier Inc. All rights reserved.

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Fig. 1. Intergenerational vs. Transgenerational Inheritance. A, Intergenerational inheritance involves exposure of the parent generation (F0, male or non-pregnant female) to an environmental factor or external stimuli (e.g., drugs of abuse, high fat diet, stress) that leads to an epigenetic alteration in the parent and parental germline cells (F1; e.g., sperm or oocytes). Epigenetic modifications may be observed in somatic tissues of the F1 adult, but do not persist in the F2 generation. B, For epigenetic effects to be considered transgenerational, modifications acquired in the F0 generation would need to be observed in the F1 and F2 generations, and potentially beyond. For pregnant females that experience environmentally-induced epigenetic changes, the pregnant dam (F0), fetus (F1), and fetal germline cells (F2) would all be exposed to the external stimulus. In this case, intergenerational effects encompass transmission from F0 to F2, and only persistence to the F3 generation would be considered a transgenerational effect.

well documented in plant species, which do not require the passage of epigenetic marks to occur through the germline (Quadrana and Colot, 2016). The intriguing notion that mammalian gene expression programs could similarly be influenced by external stimuli to alter phenotypic traits has more recently become a topic of great interest and debate in biomedical sciences. This is particularly true for instances in which DNA sequence-based inheritance (i.e., Mendelian genetics) cannot fully explain the pattern heritability of certain phenotypic traits across multiple generations. Despite the passage of epigenetic modifications becoming an attractive candidate mechanism for the inheritance of such traits, clear evidence that observable phenotypes are a direct result of germline-dependent transmission of epigenetic alterations has also been severely lacking. This pursuit is complicated by several remarkable features of reproductive biology. In mammals, haploid gamete cells (ovum and sperm) each possess their own unique epigenetic landscapes prior to fertilization. These patterns are dominated by repressive marks, yielding transcriptionally inactive cells and (for sperm) a highly compacted genome architecture in which the majority of histones have been replaced by protamines (Balhorn, 2007). However, upon fertilization these landscapes are rapidly altered, and established epigenetic marks are removed. This reprogramming is critical to establishing a totipotent state in the zygote, but also necessary to ensure the genetic and epigenetic blueprint responsible for determining the fate and function of

et al., 2014; Norouzitallab et al., 2014; Vickers, 2014; Klengel and Binder, 2015; Cadet, 2016; Nilsson et al., 2018; Walker and Nestler, 2018). Alterations in epigenetic profile have also been observed in the offspring of exposed individuals (i.e., intergenerational inheritance (Perez and Lehner, 2019)). However, whether the transfer of such epigenetic marks can occur strictly via germline-dependent transmission, in the absence of any direct exposure to the driving external stimulus, remains controversial. This review will first define the distinction between intergenerational and transgenerational epigenetic inheritance (Skinner, 2008; Perez and Lehner, 2019), as these terms are frequently conflated in the literature. Next, cellular transmission of epigenetic modifications will be discussed, followed by putative mechanisms of heritable, multigenerational epigenetic transmission. Finally, preclinical evidence for multigenerational propagation of epigenetic marks in mammals will be reviewed, as well as the translational implications of such work. For the purposes of this review, multigenerational inheritance encompasses both intergenerational inheritance (from F0 to F1), and transgenerational inheritance (F0 to F2 or beyond; Fig. 1).

2. Germline reprogramming and intergenerational vs. transgenerational epigenetic inheritance Epigenetic inheritance across multiple generations has been most 2

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regarding the putative mechanisms through which such effects would be perpetuated. In addition to direct alterations to the DNA itself, a host of epigenetic factors, including DNA methylation, histone post-translational modifications (PTMs), and small non-coding RNAs, can be passed from one generation to the next via mitosis and meiosis. Below we discuss potential epigenetic routes for cellular inheritance of gene expression profiles, including DNA methylation, post-translation histone modifications, and small non-coding RNAs. Evidence for the transmission of associated heritable phenotypic states will be outlined in later sections of the review.

thousands of distinct cell types cannot simply be undone by parental experience. Without normal reprogramming of the germline, proper cellular differentiation cannot be achieved. However, the erasure of epigenetic marks does not occur with equal efficiency at all sites across the genome (Guibert et al., 2012; Seisenberger et al., 2012; Tang et al., 2015), providing a small window of opportunity for some epigenetic modifications to escape early periods of reprogramming. The matter of which loci are protected during these phases of epigenomic reprogramming, and why some regions escape while others do not, remains an important open question. Another point that merits consideration is why might modifications imposed by the environment be stably maintained across multiple generations? Such a mechanism that allows environmental stimuli to influence gene expression programs in response to external cues without directly altering the DNA has the potential to be adaptive. As such, understanding the complex interactions between the genome, epigenome, and environment may have important implications for the heritability of disease states or adaptive fitness of the species in general. Although the transfer of environmentally induced epigenetic modifications through mitotic and meiotic processes is relatively well established in non-mammalian species (e.g., plants, nematodes, drosophila (Cavalli and Paro, 1998, 1999; Quadrana and Colot, 2016; Serobyan and Sommer, 2017; Minkina and Hunter, 2018; Moore et al., 2019; Posner et al., 2019)), mammalian research on multigenerational epigenetic inheritance has proved considerably more challenging. This is in part due to a number of caveats that must be carefully considered when designing and interpreting experiments. First, it is important to distinguish between intergenerational vs. transgenerational epigenetic inheritance. The importance of this distinction extends beyond semantics, as the mechanisms that govern epigenetic alterations to germ cells as a result of direct exposure to an environmental stimulus may differ from the mechanism through which acquired epigenetic marks are maintained and propagated during cellular division and across generations. Intergenerational inheritance involves exposure of the parent generation (F0, male or non-pregnant female) to an environmental factor (e.g., drugs of abuse, high fat diet, stress, etc.) that leads to an epigenetic alteration in the parent and likely the parental germline cells (F1; e.g., sperm or oocytes). Observation of epigenetic marks or phenotypic traits in offspring through this type of F0 to F1 transmission would be considered an intergenerational effect (see Fig. 1A). Unlike intergenerational inheritance, transgenerational inheritance relies strictly on germline transfer of epigenetic marks in the absence of any direct environmental exposure. As such, for an effect to be considered transgenerational, an inherited epigenetic mark and corresponding trait would need to persist in the F2 generation, and potentially beyond (Fig. 1B). For pregnant females that experience environmentally-induced epigenetic changes, the pregnant dam (F0), fetus (F1), and fetal germline cells (F2) would all be exposed to the external stimulus. In this case, intergenerational effects encompass transmission from F0 to F2 (Fig. 1A), and only persistence to the F3 generation would be considered a transgenerational effect (Fig. 1B). At present, limited evidence exists to support either mode of transgenerational epigenetic inheritance in mammals. Understanding the putative mechanisms through which epigenetic alterations may be passed from generation to generation first requires an understanding of how such marks are maintained at the cellular level during DNA replication and cellular division. Therefore, the cellular transmission of epigenetic modifications will be discussed in the following section, followed by a description of the current evidence in support of transgenerational epigenetic inheritance and its potential underlying mechanisms.

3.1. Cellular transmission of epigenetic marks via DNA methylation To discern how epigenetic inheritance across generations may occur, it is first useful to understand how epigenetic marks are transmitted at the cellular level. DNA methylation involves the covalent addition of a methyl group to cytosines in the 5th position of pyrimidine rings in the DNA backbone, forming 5-methylcytosine (5mC). This predominantly occurs within cytosine-guanine dinucleotides (CpGs) in most cells, and is dynamically regulated by DNA methyltransferases (DNMTs; (Holliday and Pugh, 1975; Bird, 1992)). The stable inheritance of DNA methylation patterns requires not only high fidelity DNA replication, but also faithful maintenance of cytosine methyl marks. DNA replication is incredibly accurate, with an error occurring only once per 107–108 bases copied (Kunkel, 2004). During replication, methylation marks on the parent DNA strand are enzymatically copied to the complimentary daughter strand by maintenance methyltransferase DNMT1, which coordinates the transfer of methyl groups to hemi-methylated CpG sites throughout cellular replication (Bird, 1992). This provides a potential route for methylation marks at hemi-methylated sites to be reestablished and perpetuated across multiple rounds of cell division. Methylation of non-methylated sites can also occur throughout the life cycle of the cell, and this process is coordinated by de novo methyltransferases DNMT3a and DNMT3b (Okano et al., 1999). A key issue regarding the transfer of epigenetic marks is the stability of methylation patterns across multiple rounds of cell division. DNA methylation is considerably less accurate than DNA replication with a ~96% fidelity rate, which roughly translates to 1 error for every 25 methylation marks copied per cell division (Laird et al., 2004). This elevated error rate introduces variation in methylation profiles due to faulty copying during replication and cell division, which in turn yields cell populations with diverse methylation patterns (Silva et al., 1993). Thus, even at the cellular level within a single individual there are multiple opportunities for errors to be introduced into methylation patterns, preventing highly accurate transmission during cell division. Taken together, this suggests that although DNMTs may largely preserve methylation programs across the cell life cycle, this process is inherently less accurate than DNA replication. This issue creates one of several substantial hurdles that must be overcome in order for methylation marks to be stably transmitted not only during cell division, but also across multiple generations. The processes of active and passive DNA demethylation are also key to understanding the cellular maintenance of methylation programs. Cytosines can be actively demethylated by TET (ten eleven translocation) enzymes, which are responsible for the addition of a hydroxyl group to 5-methylcytosines, forming 5hmC (Tahiliani et al., 2009; Ito et al., 2010). This is followed by a series of oxidation steps resulting in several intermediates including 5fC (5-formylcytosine) and 5CaC (5carboxylcytosine), prior to base excision and repair with an unmodified cytosine (He et al., 2011; Ito et al., 2011). It remains to be determined whether these intermediates represent passive transitional stages on the road to demethylation, or if each modification serves its own distinct function. Passive demethylation occurs when DNMT1 activity is not present or inhibited and 5mC marks are not able to be maintained during the replication process, leading to gradual dilution of the methylation profile over time (Rougier et al., 1998; Lee et al., 2014). Both

3. Putative cellular mechanisms for epigenetic inheritance Over the past several decades, much research has focused on the transmission of phenotypic traits or disease states via non-genetic modes of inheritance, and consequently, much debate has ensued 3

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Fig. 2. Methylation levels during epigenetic reprogramming and development. The initial wave of mammalian methylome reprogramming occurs after fertilization in the pronuclei of the paternal and maternal genomes via distinct demethylation mechanisms. Paternal pronuclei undergo active demethylation mediated via TET enzymes, whereas maternal pronuclei are passively demethylated. Global methylation levels remain low in the zygote and blastocyst stage, until widespread reestablishment of methylation by de novo DNMTs 3a and 3b occurs in the embryo after implantation, during the differentiation process. Once methylation programs are established in differentiated cells of the developing embryo, cell-type specific patterns will be maintained in somatic cells during maturation and throughout the lifespan by DNMT1. Methylome reprogramming also occurs in the primordial germline cells of the developing embryo. Unlike the distinct mechanisms experienced by the paternal and maternal pronuclei shortly after fertilization, both active and passive modes of demethylation participate in primordial germ cell reprogramming.

Rougier et al., 1998). These periods of widespread demethylation followed by re-methylation in the post-implantation embryo create a substantial obstacle for the inheritance of any methylation marks that may have been established in the parental epigenome. While this sequence of methylation events appears to be the general rule of thumb, notable examples of persistent DNA methylation after post-fertilization reprogramming have been documented. For example, in mice, DNA methylation persists at specific differentially methylated regions (DMRs) in oocytes during post-fertilization periods that are typically characterized by widespread hypomethylation (Smith et al., 2012). Certain DMRs also remain partially methylated during the preimplantation stage in human oocytes (Guo et al., 2014b), indicating a possible route for epigenetic inheritance via maternally transmitted methylation marks at these sites. Retrotransposable elements known as intracisternal A particles (IAPs) may also be resistant to DNA demethylation during embryonic development, as some of these loci remain mostly hypermethylated while primordial germ cells undergo widespread demethylation (Lane et al., 2003; Guibert et al., 2012; Seisenberger et al., 2012). Persistent DNA methylation within gene bodies at intragenic enhancers has also been reported at the pre-implantation stage in humans. Given that intragenic enhancer elements are also subject to control by DNA methylation state, this represents a potential platform for methylation-based epigenetic inheritance of certain cis-regulatory states (Birnbaum et al., 2012; Blattler et al., 2014). Methylome reprogramming also occurs in the primordial germline cells of the developing embryo (Fig. 2; (Smith et al., 2012). Unlike the distinct mechanisms experienced by the paternal and maternal pronuclei shortly after fertilization, both active and passive modes of demethylation participate in primordial germ cell reprogramming. More specifically, primordial germline demethylation is first exerted through active TET-mediated demethylation, followed by subsequent replication-dependent passive demethylation (Guibert et al., 2012; Seisenberger et al., 2012; Hackett et al., 2013; Hill et al., 2018).

active and passive demethylation mechanisms contribute to widespread post-fertilization demethylation in the developing zygote, and thus influence the propagation of methylation patterns inherited from the maternal and paternal genomes. Each type of demethylation process will be discussed in greater detail in the following section. 3.2. Multigenerational epigenetic inheritance via DNA methylation Given its relatively stable perpetuation during cell division, DNA methylation has long been considered a putative route through which epigenetic patterns could be transferred from one generation to another. However, the methylome undergoes heavy reprogramming at least twice during development in mammals (Lee et al., 2014), making many skeptical about the likelihood of transgenerational epigenetic inheritance via DNA methylation. The initial wave of mammalian methylome reprogramming occurs in the pronuclei of the paternal and maternal genomes via distinct demethylation mechanisms (Fig. 2; Rougier et al., 1998; Mayer et al., 2000; Oswald et al., 2000; Santos et al., 2002; Iqbal et al., 2011; Smith et al., 2012). Active demethylation in paternal gametes is mediated via TET enzymes, which are responsible for converting 5mC to an unmethylated state through a series of successive oxidation steps (Morgan et al., 2005; Guo et al., 2014a). Concurrently, the maternally inherited genome undergoes passive demethylation prior to implantation, wherein methyl marks fail to be maintained during replication, and are thus gradually diluted in the zygote (Mayer et al., 2000; Guo et al., 2014a). Global methylation levels remain low in the blastocyst stage, until widespread re-establishment of methylation by de novo DNMTs 3a and 3b occurs in the embryo after implantation (Fig. 2; (Santos et al., 2002; Smith et al., 2012). This re-establishment of methylation patterns by DNMT3a and 3b governs cell fate and function during the differentiation process. Once methylation programs are established in differentiated cells of the developing embryo, cell-type specific patterns will be maintained in somatic cells during maturation and throughout the lifespan by DNMT1 (Fig. 2; 4

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modifications would allow for semi-conservative propagation of histone modification patterns. However, it is unclear how parent histone marks are established on daughter histones that have been recently incorporated into nucleosomes of the newly synthesized DNA strand. This may be facilitated in part by the recruitment of histone modifying enzymes to the replication fork during DNA synthesis, or histone modifying enzymes may be able to interact directly with DNA in a sequencespecific manner, allowing the enzyme to remain associated with the DNA during replication (Budhavarapu et al., 2013). In support of this notion, the histone modifying enzyme methyltransferase G9a, which interacts with DNMT1 to coordinate H3K9 dimethylation, localizes to the replication fork during DNA synthesis (Esteve et al., 2006). Similarly, PRC2 (Polycomb Repressive Complex 2), which methylates unmodified H3K27 sites when recruited by previously established trimethylated H3K27 marks, is also found near replication foci (Hansen et al., 2008; Margueron et al., 2009). These examples suggest certain histone modifying enzymes are physically recruited to replication sites, and may participate in the reinstatement of post-translational modifications on daughter histones. Thus, although many details regarding how histone PTMs are maintained during replication remain to be established, this evidence suggests that certain histone modifying enzymes can be recruited to the site of DNA replication and may interact with other epigenetic modifiers (such as DNMTs) to maintain established epigenetic patterns during DNA replication and across cell division.

However, similar to the post-implantation embryo, not all loci appear to be subject to methylation reprogramming. For example, some imprinted genes and retrotransposons are also able escape demethylation in the primordial germline (Hackett et al., 2013). Although imprint control regions typically undergo the initial wave of demethylation during germline development (Brandeis et al., 1993; Hajkova et al., 2002; Guo et al., 2014a), these sites can escape methyl mark erasure during the second period of demethylation (Kafri et al., 1993; Oswald et al., 2000; Sakashita et al., 2014), providing a putative route for methylation patterns to be transmitted from parent to offspring. Despite periods of largescale demethylation during embryonic development, over 115,000 regions remain hypermethylated during methylation reprogramming of the primordial germline in human development, and many of these same regions similarly escape demethylation in mice (Tang et al., 2015). Interestingly, a number of genes that escape methylation reprogramming are expressed in the brain, and are linked to the regulation of neurobiological processes and metabolism (Tang et al., 2015). These gene targets are prime candidates for future investigation of how the transmission of their corresponding epigenetic marks may set the stage for transgenerational inheritance of metabolic disease states or neurological disorders. Collectively, these findings suggest at least some fraction of genomic loci (e.g., particularly imprint control regions, DMRs, IAPs, transposons, and some enhancer elements), may be more resistant to the global methylation reprogramming that occurs during early development. Why and how these particular regions of the genome are able to escape methylation reprogramming remains largely unknown. Nevertheless, such sites provide a putative route for DNA methylation patterns to be conserved and transferred from the parent genome to future generations.

3.4. Multigenerational epigenetic inheritance via histone modifications Similar to global DNA methylation reprogramming, histones and their associated modifications experience chromatin remodeling events that prove challenging for maintaining and transferring epigenetic marks from one generation to the next. For example, during mammalian spermatogenesis, most histones are evacuated from nucleosomes and replaced by small arginine-rich nuclear proteins known as protamines in sperm to facilitate the compaction of paternal DNA (Balhorn, 2007; Casas and Vavouri, 2014). This process serves to protect and stabilize the DNA, but also results in the removal of histones and any corresponding post-translational modification to histone tails. Although this reduces the likelihood of epigenetic transmission of histone modifications, recent studies suggest protamines are also post-translationally modified, and therefore may also be capable of transferring epigenetic information from one generation to the next (Brunner et al., 2014). Whether such modifications can be epigenetically inherited across generations remains to be investigated. Despite the apparent hurdle of widespread histone removal during sperm maturation, a small percentage of histone modifications are preserved in humans and mice (Hammoud et al., 2009; Brykczynska et al., 2010). This has been observed with both active (H3K4me2, H3K4me3) and repressive (H3K27me3, H3K9me3, H4K20me3) histone marks, however specific PTMs and the loci at which they occur may differ by species. In humans, evidence suggests repressive histone marks H3K9me3 and H4K20me3 can be transferred from sperm to zygote, and these alterations are accompanied by highly condensed constitutive heterochromatin (van de Werken et al., 2014). Some retention of histone marks has also been reported near the promoter region of key developmental and housekeeping genes (Erkek et al., 2013). These findings suggest epigenetic transfer of histone modifications may occur via the paternal epigenome, although the opportunity for such contribution is limited given the small percentage of histone PTMs preserved in sperm. Alternatively, paternal chromatin compaction in the developing zygote may be driven by maternal donation of the polycomb repressive complex (Puschendorf et al., 2008). This putative mechanism suggests heritable changes in the paternal epigenome can also be regulated by maternal contribution of repressive complexes that shape the chromatin landscape during embryogenesis. In oocytes, histones are largely retained during development (Gu

3.3. Cellular transmission of epigenetic marks via post-translational histone modifications Histone modifications also alter availability of DNA to the necessary transcriptional machinery required for regulating gene expression. Histones are modified at the N-terminal portion of their tails that extends beyond the nucleosome to interact with regulatory proteins, neighboring histones, and the DNA itself (Marmorstein, 2001; Tsankova et al., 2007). A variety of post-translational modifications (PTMs) to histone tails have been observed in the literature, including methylation, phosphorylation, ubiquitination, SUMOylation, and acetylation, which is perhaps the most well characterized. As with DNA methylation, relatively stable transmission of any of these epigenetic marks during DNA replication and cell division would be required for histone alterations to be heritable. However, the brief lifespan of most histone modifications, and limited knowledge regarding the fidelity of histone modifying enzymes, has dampened enthusiasm for histone PTMs as a plausible route for transgenerational epigenetic inheritance. For example, histone lysine methylation, a relatively stable histone modification, can be maintained on a timescale of hours-to-days (Zee et al., 2010). Histone acetylation and phosphorylation states can fluctuate within minutes (Jackson et al., 1975; Chestier and Yaniv, 1979). Thus, the transient half-life of most histone modifications calls into question the likelihood that such marks could be easily propagated during replication and cell division. For modifications that do survive long enough to be transmitted during replication, the mechanisms through which parent histone marks are established on daughter histones remain poorly understood. Nonetheless, recent work in yeast has demonstrated that certain histone modifications, such as H3K9 methylation, can be stably propagated during mitosis and meiosis in a manner independent of DNA methylation (Ragunathan et al., 2015). One putative mechanism involves the reassembly of two displaced parent histones into the nucleosome of the newly synthesized DNA strand, along with two new daughter histones (Annunziato, 2005). This partial recycling of histones and their corresponding post-translational 5

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across generations in humans, several lines of research suggest that epigenetic factors may also confer risk for the development of certain metabolic conditions. Exposure to high fat or low protein diets in the parent generation, as well as intrauterine exposure to maternal caloric restriction, have all been linked to disruption of metabolic function in offspring, in some cases beyond the F2 generation. To date, the vast majority of multigenerational studies have focused on the inheritance of metabolic phenotypes in offspring, with little characterization of heritable epigenetic changes to somatic cells in the brain regions that regulate energy homeostasis and satiety. Nonetheless, some evidence does suggest metabolic health of future progeny can be impacted by poor nutrition in the parent generation. Specific examples of how high fat or low protein diet in the parent generation can influence metabolic function of offspring will be discussed in the following sections.

et al., 2010). Thus, stable transmission of histone marks from one generation to another maybe be more plausible via a maternal route. In support of this notion, recent examination of the maternal epigenome has uncovered patterns of H3K4me3 and H3K27me3 marks in mouse oocytes that appear to impact embryonic development (Dahl et al., 2016; Zhang et al., 2016; Zheng et al., 2016; Inoue et al., 2017; Xu and Xie, 2018). For example, a reduction in active histone methylation mark H3K4me2 at the promoter regions of genes that shape developmental processes and metabolic function have been linked to altered gene expression in F1 generation embryos. This modification induced by lysine specific demethylase 1 (LSD1) overexpression was associated with developmental defects that were present in F1 offspring and could be transferred across 3 generations (Siklenka et al., 2015). Although limited at present, these studies suggest that both active and repressive histone marks may be perpetuated across generations in mammals, and that these transferable epigenetic marks may yield important implications for embryogenesis and multigenerational inheritance.

4.2. High fat diet Parental exposure to a diet high in fat content has been linked to phenotypic transmission of impaired metabolic function in offspring. A recent study involving the in vitro transfer of oocytes and sperm from parents both fed a high fat diet to a healthy foster mother reported significant weight gain in F1 offspring relative to offspring from mice fed a normal diet (Huypens et al., 2016). Specifically, female offspring from obese parents gained the most weight, and this pattern of weight gain in the offspring persisted regardless of whether the dam, sire, or both parents received the high fat diet. Although potential epigenetic modifications accompanying the observed phenotypes were not evaluated in this study, the authors did report that obesity in female offspring was accompanied by insulin resistance (Huypens et al., 2016), suggesting future studies should investigate the epigenetic landscape surrounding genes important for insulin regulation. Pregnant female mice exposed to a fatty diet resulted in decreased insulin sensitivity and increased body size in F1 and F2 offspring, an observation authors attributed to both maternal and paternal lineage despite gestational exposure (Dunn and Bale, 2011). Interestingly, in the F3 generation, increased body size was only observed in female offspring, and appeared to occur by a paternal mode of transmission. Although epigenetic modifications were not directly measured, the increased body weight phenotype was accompanied by a disrupted pattern of expression in paternally imprinted genes involved in growth regulation in liver tissue collected from F3 female offspring (Dunn and Bale, 2011). Taken together, the above studies suggest both maternal and paternal modes of transmission may contribute to inheritance of altered metabolic phenotypes. The fact that increased body size and altered gene expression patterns persisted in the F3 generation suggest transgenerational mechanisms of inheritance may be at play, although more definitive characterization of accompanying epigenetic alterations to somatic and germline cells for each generation will be important for substantiating this claim. Maternal transmission of epigenetic inheritance can be particularly challenging to study, as diet-induced phenotypic traits in offspring could arise from nuclear or mitochondrial DNA contributions, effects of maternal diet and metabolism during gestation (e.g., diabetes, obesity), or epigenetic modifications to female germ cells. One benefit of focusing solely on paternal transmission is that sperm carries only genetic and epigenetic material, eliminating some of the potential confounds inherent to studies of this nature. Studies investigating exclusively paternal transmission after exposure to a high fat diet have observed weight gain, hypertension, and glucose intolerance in both F1 and F2 generations (Masuyama et al., 2016). Further, these metabolic impairments were accompanied by H3K9ac and H4K20me1 in the promoter regions of adiponectin and leptin genes in adipose tissue from adult F1 offspring, genes involved in insulin resistance (Fasshauer and Paschke, 2003) and satiety and energy homeostasis (Myers Jr., 2004), respectively. Similar work examining paternal transmission of phenotypic metabolic traits found that sperm from obese male mice exhibited

3.5. Cellular transmission of epigenetic marks by non-coding RNAs Small non-coding RNAs may also act as carriers of heritable epigenetic information. These small RNAs are comprised of a variety of RNA species under 200 nucleotides in length, and include micro RNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and transfer RNA fragments (tRFs). In comparison to DNA methylation and histone modifications, relatively little is known about the role of small non-coding RNAs in germline-dependent epigenetic transfer across generations. Nonetheless, a variety of small non-coding RNA species have been documented in sperm and oocytes, which may be transferred to the zygote at fertilization (Rassoulzadegan et al., 2006; Pauli et al., 2011; Liu et al., 2012; Chen et al., 2016; Cropley et al., 2016; Sharma et al., 2016; Yang et al., 2016). Although the precise mechanisms involved remain to be defined, a host of exogenous factors such as exposure to pathogens, stress, elevated temperature, and dietary composition have been reported to produce alterations in RNA species found in germline cells (Rassoulzadegan et al., 2006; Rechavi et al., 2011; Gapp et al., 2014; Rodgers et al., 2015; Chen et al., 2016; Cropley et al., 2016; Sharma et al., 2016). These alterations may contribute to inheritance of certain metabolic phenotypes linked to high fat diet, which will be discussed further in the following section. Although limited, emerging evidence suggests tRNA fragments, piRNAs, and miRNAs may also contribute to epigenetic silencing at certain transcriptional domains. For example, miRNA inheritance has been linked to altered embryonic development (Tang et al., 2007; Liu et al., 2012), and transmission of stress-induced behavioral phenotypes (Gapp et al., 2014; Rodgers et al., 2015). Finally, research focused on tRNA fragments has begun to identify a role for these non-coding RNAs in the intergenerational transfer of metabolic phenotypes (Chen et al., 2016; Cropley et al., 2016; Sharma et al., 2016). In sum, like DNA methylation and histone modifications, non-coding RNAs also have the potential to convey epigenetic information from parental germline to their offspring and future generations. 4. Effect of epigenetic inheritance on phenotypic traits and brain function 4.1. Nutritional intake and inheritance of metabolic phenotypes The prevalence of metabolic disorders is on the rise in the United States and worldwide (Finucane et al., 2011; Ogden et al., 2014), making the elucidation of cellular and molecular contributions to these diseases particularly relevant. Mendelian inheritance does not adequately account for the apparent heritability of observed phenotypic traits and metabolic disorders. Although environmental exposure to things like shared diet and other sociological and cultural factors most certainly contribute to the passage of impaired metabolic function 6

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increased anxiety and depression-like behavior, as well as elevated plasma levels of the stress hormone corticosterone (Dietz et al., 2011). However, most of these traits could not be recapitulated with in vitro fertilization using sperm from defeated sires, suggesting these effects are likely not due to transmission of epigenetic marks across the germline. However, other studies involving chronic paternal stress resulted in hypoactivation of the hypothalamic-pituitary-adrenal (HPA) axis in offspring, and alterations in glucocorticoid-responsive genes in the bed nucleus of stria terminalis and paraventricular nucleus (Rodgers et al., 2013). Changes in gene expression were also accompanied by an upregulation in 9 different miRNAs in the sperm of stressed vs. nonstressed sires. Given that miRNAs in sperm can impact post-fertilization gene expression during early development (Rassoulzadegan et al., 2006), and that DNMT3a (a key regulator of imprinted gene methylation) was identified as one of the top predicted miRNA targets, these findings provide a potential mode of action through which paternal stress can epigenetically alter germ cells. However, whether changes to stress responsivity and the expression of stress-associated genes extends beyond the F1 generation remains an open question. Paternal transmission of traumatic experience has also been investigated using fear conditioning paired with specific scent cues. Olfactory receptors are selectively expressed by sensory neurons of the olfactory epithelium, with each individual sensory neuron expressing only one of the hundreds (in humans) or thousands (in mice) of genetically encoded olfactory receptors (Godfrey et al., 2004; Monahan et al., 2017). Given the relatively defined nature of olfactory receptor activation by distinct odor stimuli, this system presents an opportunity for elegant investigations into transgenerational epigenetic mechanisms. In a landmark study by Brian Dias and Kerry Ressler, mouse sires were fear conditioned in the presence of a specific odor cue (acetophenone), which is known to activate the M71 odor receptor encoded by the Olfr151 gene (Dias and Ressler, 2014). Surprisingly, this learned association was transferred to F1 and F2 progeny, measured as an elevated “startle” response to the conditioned odor but not to another control odor. These inherited fear responses were accompanied by altered promoter methylation of Olfr151 in F0 sperm, and neuroanatomical differences in the olfactory system of F1 and F2 offspring, including more M71 receptor expressing olfactory sensory neurons and larger M71 glomeruli in the olfactory bulb (Dias and Ressler, 2014; Fig. 3A). Interestingly, in vitro fertilization (IVF) and cross-fostering approaches were incorporated in this study to substantiate the idea that observed phenotypic behavioral responses and molecular changes were not likely due to social transmission from the parent generation. Persistence of behavioral sensitivity to the conditioned scent, and neuroanatomical alterations in the olfactory bulb of IVF-derived F2 progeny, as well as hypomethylation near the odorant receptor gene Olfr151 in sperm from the F0 generation and F1 odor naïve males, provides compelling evidence for epigenetic transmission of these phenotypic traits. Although it is unclear at present how olfactory cues and corresponding alterations in somatic cells of the olfactory system led to methylation changes in sperm of the F0 and F1 generation, this work provides intriguing preliminary evidence that information regarding stressful signals encountered in the environment may be incorporated into the germline and passed on to offspring. Maternal stress during pregnancy leads to elevated production of stress hormones and this heightened activation of the HPA axis has been linked to increased stress sensitivity, cognitive deficits, and dysregulation of stress-related gene expression in offspring (Mueller and Bale, 2007, 2008). When maternal stress is experienced during early pregnancy (i.e., within the first week), male, but not female, offspring display worse performances in tail suspension and forced swim tests, indicative of depression-like behavior (Mueller and Bale, 2007, 2008). Early gestation stress also resulted in elevated production of the stress hormone corticosterone, increased corticotropin-releasing factor in the amygdala, and decreased glucocorticoid receptor expression in the hippocampus of male offspring (Mueller and Bale, 2008). Further,

altered DNA methylation patterns and corresponding changes in insulin signaling genes, effects also observed in the pancreatic islets of F1 and F2 offspring (Wei et al., 2014). In addition to DNA methylation and histone PTMs, small non-coding RNAs have also been implicated in the epigenetic transfer of metabolic phenotypes. For example, injection of total RNA extracted from the sperm of mice on a high fat diet into zygotes yielded impaired metabolic function in F1 offspring (Zhang et al., 2018). This effect was not observed when sperm was donated from obese mice with genetic deletion of Dnmt2, a methyltransferase that alters the stability of tRNAs. Such findings suggest that high fatdiet induced changes in metabolic function may be transferred to the F1 generation via paternal donation of non-coding RNAs in sperm. Nevertheless, it currently remains unclear whether high fat diet-induced epigenetic alterations also occur in brain tissue, and if such marks are heritable across multiple generations. 4.3. Caloric restriction and low protein diets Caloric restriction has also been shown to alter the epigenome in a heritable way. Male offspring of calorically restricted dams have low birth weights and suffer from several metabolic defects (Radford et al., 2012). When dietary restrictions were in place during primordial germline reprogramming, this led to DNA hypomethylation at 111 regions in F1 sperm (Radford et al., 2012). However, changes in methylation patterns at specific loci in F1 sperm were not observable in brain or liver cells of F2 embryos, suggesting this particular epigenetic effect is only intergenerational in nature. As with high fat diet studies, small non-coding RNA species such as miRNAs and tRNA fragments are likely also involved in the transmission of phenotypic traits related to metabolism. For example, low protein diets have been linked to altered tRNA fragment abundance, and this effect can be paternally transmitted via sperm to alter embryogenesis (Chen et al., 2016; Cropley et al., 2016; Sharma et al., 2016). Other work examining the role of non-coding RNAs in epigenetic transmission of metabolic traits reported elevated cholesterol synthesis in the liver of IVF-derived offspring from sires on a low protein diet (Sharma et al., 2016). Phenotypic changes in metabolic function were accompanied by dysregulation of hepatic cholesterol biosynthesis genes in the F1 generation. These effects appear to be mediated in part by diet-induced changes in the abundance of tRNAGly-GCC fragments in F0 sperm, which repress genes normally regulated by the endogenous retroelement MERVL (Mouse endogenous retrovirus with leucine tRNA primer). Collectively, such work suggests low protein diet-induced alterations in non-coding RNAs can be paternally transmitted to subsequent generations via germ cells. At present, these effects appear to be mostly intergenerational in nature. Notably, non-coding RNA species may prove an attractive candidate for future research on transgenerational epigenetic inheritance of metabolic traits, as they may not be subject to the same type of epigenetic reprogramming events experienced by DNA methylation and histone modifications during early development. Taken together, the above studies provide evidence for intergenerational inheritance of metabolic phenotypes that occur as consequence of diet-induced changes in the F0 generation. Present findings suggest intergenerational alterations in metabolic function are mediated in part by non-coding RNA species transferred to offspring via paternal germline cells. However, whether diet-induced epigenetic alterations observed in germ cells are also present in somatic cells of the CNS (i.e., in the brain), and the heritability of such marks across multiple generations, remain open questions. 5. Stress exposure and inheritance of altered stress responsivity Activation of the endocrine system by maternal or paternal exposure to stressful events can alter gene expression and have long-term behavioral consequences in offspring. Male mice that experienced chronic social defeat or variable stress prior to breeding yielded progeny with 7

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Fig. 3. Putative mechanisms for transgenerational epigenetic inheritance of neuronal and behavioral phenotypes. A, Multigenerational epigenetic transmission of a startle response after paternal olfactory fear conditioning. Fear conditioning in the presence of acetophenone odorant leads to reduced methylation of the Olfr151 gene in the olfactory bulb of the paternal F0 generation (left) and in sperm (F1; right). Hypomethylation of Olfr151 (a gene that encodes M71 odor receptors) is associated with an increase in the number of olfactory sensory neurons expressing M71 receptors, and larger corresponding glomeruli in the olfactory bulb of the F0 generation (left). More M71-expressing olfactory sensory neurons and larger M71 glomeruli are also observed in adult F1 and F2 progeny without any direct exposure to the acetophenone odor cue. Neuroanatomical adaptations observed in the F1/F2 generation are thought to increase sensitivity to acetophenone when offspring are later exposed to this specific odorant, resulting in elevated startle to the odor cue in the absence of direct pairing with fear conditioning. B, Multigenerational epigenetic transmission of a cocaine resistant phenotype after paternal cocaine exposure. Cocaine self-administration leads to increased H3K9K14ac2 at Bdnf promoter IV and elevated BDNF protein and mRNA transcripts in somatic cells of the mPFC in the F0 generation (left), as well as increased H3K9K14ac2 at all Bdnf promoters in F0 sperm (F1 generation; right). During adulthood, parallel changes in BDNF protein, mRNA, and H3 acetylation are observed in the mPFC of F1 offspring. Elevated BDNF levels in the mPFC have previously been linked to a reduction in the reinforcing effects of cocaine, and subsequently, a decline in drug-seeking behavior. Thus, elevated Bdnf mRNA and protein levels in the mPFC of F1 progeny prior to drug exposure are thought to confer a protective phenotype by blunting the rewarding properties of cocaine and reducing the propensity for drug seeking. 8

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sensitization (Crozatier et al., 2003), and impaired synapse maturation during development (Bellone et al., 2011). Although such work provides valuable translational information regarding the deleterious effects of cocaine exposure during pregnancy, numerous studies focused on epigenetic transmission of behavioral traits in the context of addiction have opted to focus on paternal transmission, as this circumvents potential interpretational confounds that can arise in transgenerational inheritance studies. For example, past drug experience in female rats can negatively impact maternal care for pups later in life (Johnson et al., 2011), and a single instance of maternal separation has been shown to influence drug intake in pups (Martini and Valverde, 2012). Thus, investigation of paternal drug use on neurobiological processes and behavioral phenotypes in offspring from drug naïve dams evades these potential confounds. This approach also rules out the potential for transfer of epigenetic marks by direct drug exposure to the F1 or primordial germline of F2 generations in utero. At present, a majority of the evidence for transmission of phenotypic traits across generations as a consequence of paternal or maternal drug use is behavioral and correlational in nature. Nonetheless, a variety of behavioral phenotypes have been observed in offspring from cocaine experienced sires, including hyperlocomotion, impaired spatial memory, and elevated anxiety levels (Abel et al., 1989; Fischer et al., 2017). Some of the earliest work to report intergenerational transmission of addiction-related phenotypic traits found that both male and female adolescents (P16) from cocaine-sired offspring displayed hyperlocomotive activity (Abel et al., 1989). More recently, sex-specific effects on locomotion have been reported in male, but not female, offspring of cocaine-sired mice (Fischer et al., 2017). Anxiety-like behaviors have also commonly been observed in the progeny of cocaineexperienced rodents. For example, male offspring from cocaine-sired rats reportedly display defensive burying and novelty-induced hypophagia, which are considered measures of anxiety-like behavior (White et al., 2016). Similarly, male offspring of rats receiving chronic cocaine injections also demonstrated increased anxiety levels relative to saline controls, as measured by amount of time in the open arm of the elevated plus maze (Fischer et al., 2017). Despite these intriguing observations, it should be noted that at least one study measuring locomotion was not able to recapitulate a similar hyperactive phenotype in adult mouse progeny (P60) sired by cocaineexperienced fathers (Killinger et al., 2012). Similarly, this same study also observed no impact on paternally transmitted anxiety-like traits after cocaine experience in the elevated plus maze or open field test (Killinger et al., 2012). One potential difference that could account for these discrepancies is that sex-specific statistical analyses were used for some studies (Fischer et al., 2017), while others collapsed sex during analysis (Killinger et al., 2012), which would alter power for detecting behavioral effects within each sex. Differences in amount of handling, behavioral protocols, and total amount of cocaine exposure may have also contributed to these incongruent observations. Despite these discrepancies, at least some research suggests that paternal exposure to cocaine can lead to elevated locomotor activity and other anxiety-like traits in F1 offspring, and that male offspring may be particularly susceptible to these behavioral abnormalities. In addition to hyperactivity effects, paternal exposure to cocaine may also disrupt normal memory function in F1 progeny, as impairments have been observed in the performance of certain memory tasks in cocaine-sired rodents. For example, adolescent (P35) offspring from cocaine-sired rats demonstrated perseverative behavior in a T-Maze task (Abel et al., 1989), and similar impairments were observed in cocaine-sired CD1 mouse offspring in the 5-Arm Maze, a spatial working memory task (He et al., 2006). In a self-administration paradigm, adult male offspring also exhibited impaired spatial memory in the object location task (Wimmer et al., 2017). Although most of this evidence suggests spatial memory tasks are particularly susceptible to disruption by paternal drug use, others have reported no impairment in the Morris Water Maze, another type of hippocampus-dependent spatial

methylation of the glucocorticoid receptor promoter was also elevated in hippocampal tissue, suggesting epigenetic mechanisms are likely involved, although most likely as a result of direct exposure to stress hormones in utero, and not through germline inheritance of such modifications. In guinea pigs, maternal stress induced by repeated exposure to flashing light similarly increased anxiety-related behaviors and basal corticosterone levels in offspring while reducing glucocorticoid receptor expression in the hippocampus when the stressor occurred during mid, but not late, pregnancy (Kapoor and Matthews, 2005; Kapoor et al., 2009). Taken together, these studies suggest timing of perturbation during pregnancy may play a key role in the extent to which developmental processes will be disrupted or induce long-term epigenetic changes in the neural tissues of their progeny. At any rate, the vast majority of studies examining the effects of maternal trauma on stress vulnerability in offspring to date involve direct in utero exposure to the F1 (developing embryo), and F2 (embryo primordial germline) generations, and as such are intergenerational in nature. Additional research will be required to determine whether epigenetic modifications that occur as a consequence of maternal stress are transgenerational, extending out to the F3 offspring. Another important consideration for research examining the consequence of parental stress on offspring is that maternal stress can also alter the level of maternal care dams provide to their pups after birth (Ivy et al., 2008), in addition to affecting the hormonal milieu of the placenta. Poor maternal care during this early postnatal period is traumatic in its own right (Weaver et al., 2004; Rice et al., 2008; Ivy et al., 2010; Korosi and Baram, 2010; McClelland et al., 2011), and can thus act as its own direct exposure to stress early in life, making interpretation of transgenerational vs. intergenerational social transmission of such effects on behavior and gene expression challenging. For example, stressed mothers provide poor maternal care, and the stress of poor maternal care acts as its own direct insult to the developing pup. This elevates stress sensitivity in adult F1 progeny, likely affecting their own ability to provide adequate maternal care, thus perpetuating the cycle. An important mechanistic distinction is that this continued passage of a stress sensitive phenotype and any accompanying epigenetic alterations likely occurs from direct exposure to a stressful postnatal experience in each generation, making this a repeating cycle of intergenerational effects, rather than transgenerational effects, which require transfer of epigenetic modifications via the germline in the absence of direct exposure. To avoid these type of experimental confounds, some investigators have opted to focus on patrilineal inheritance, as male rodents are not typically involved in rearing offspring in a laboratory setting. Exposure to paternal stress prior to breeding is thought to minimize potential social or behavioral confounds that can be imposed during early postnatal periods. However, it is worth noting that mate quality can impact maternal investment of female rodents in caring for their offspring (Drickamer et al., 2000; Mashoodh et al., 2012). Therefore, even subtle interactions between the sire and dam during or after mating could theoretically impact maternal behavior and postnatal care of offspring, and thus it should not be assumed that separation of an exposed sire from the dam after mating will guarantee avoidance of any potential behavioral confounds. 6. Substance abuse and inheritance of behavioral phenotypes Exposure to drugs of abuse can modify the epigenome and alter neural plasticity, and each of these phenomena is thought to establish and maintain the addicted state (Renthal and Nestler, 2008; Heller et al., 2014). The evidence for epigenetic modifications and changes in gene expression in reward circuitry of the drug-exposed individual is well established, but more recently epigenetic modifications have also been documented in the sperm and oocytes after drug exposure (Vassoler and Sadri-Vakili, 2014). Prenatal (i.e., in utero) exposure to cocaine in rodent addiction models can result in elevated self-administration of cocaine (Keller Jr. et al., 1996), altered stimulant-induced 9

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protein levels (Vassoler et al., 2013; Collo et al., 2014). Interestingly, Bdnf levels in the offspring of drug-exposed sires is reported to positively correlate with amount of cocaine intake in the parent generation (Le et al., 2017). This aligns with previous research suggesting elevated mPFC BDNF levels coincide with reduced drug-seeking behavior, and may play a key role in mediating the protective effects some have observed in the offspring of cocaine-sired rodents (Berglind et al., 2007; Vassoler et al., 2013;Fig. 3B). Elevated DNA methylation has also been observed in the sperm of highly motivated “addict” vs. non-addict cocaine-exposed F0 rats, a pattern which persisted in their F1 descendants (Le et al., 2017). Consistent with these distinct methylation patterns, transcriptome sequencing of the Nucleus Accumbens (NAc) in the F1 generation revealed that reduced global methylation in F0 sperm coincided with significantly lower expression levels of genes important for developmental regulation, gap junction formation, and axon guidance in the addict-like phenotype vs. non-addict F1 progeny (Le et al., 2017). Overall, these findings suggest highly motivated drug-seeking behavior may alter DNA methylation in a way that is distinct from methylation that occurs as a result of drug exposure alone, and that these epigenetic modifications may be transmitted to F1 offspring. Additional research will be required to determine if heritability of the epigenetic patterns observed in the F1 generation extends to F2 progeny. Collectively, this body of work provides a preliminary glimpse into the generational effects of cocaine exposure on anxiety, memory, sensitivity to drugs of abuse, and corresponding alterations to the epigenome and gene expression. Although converging evidence does suggest transmission of behavioral phenotypes, much work remains to be done to see if these observations extend beyond the F1 generation, making them true transgenerational rather than intergenerational effects. Further, characterization of which epigenetic marks are transferred from drug experienced parent to offspring, the brain regions and cell types they occur in, how such modifications survive epigenetic reprogramming during development, and how such alterations would manifest as the observed behavioral phenotype remain to be explained. Thus, future studies will be important to further dissect the multigenerational transmission of these phenotypic traits, and the epigenetic remodeling events that may subserve them.

task (Killinger et al., 2012; Fischer et al., 2017). Nonetheless, the balance of studies to date suggest tasks that recruit the hippocampus may be more heavily influenced by the consequences of paternal exposure to cocaine. However, several factors may have contributed to the observed differences in memory task performance. For example, Killinger and Fischer used experimenter administered cocaine, whereas Wimmer and He utilized self-administration models, allowing the subject to lever press for cocaine reward. Differences in these experimental approaches should therefore be considered when interpreting the data, as each approach would likely engage distinct brain regions to a different extent. Self-administration relies on learning about contingencies between behavioral responses, environmental cues, and the rewarding properties of the drug, whereas experimenter administration is more passive. Further, self-administration studies likely resulted in more overall cocaine experience vs. the experimenter delivered studies, and this could also be an important factor in paternal transmission of certain druginduced phenotypic behaviors. Perhaps not surprisingly, cocaine-sired offspring of both sexes display behavioral differences in response to their own experience with cocaine. One example of this is stimulant-induced hyperlocomotion, suggesting increased sensitivity to cocaine experience (Fischer et al., 2017). Cocaine exposure in the parent generation may shift the doseresponse curve for female mice, which show reduced magnitude in their conditioned place preferences, and in response to a lower dose of cocaine than usual (5 mg/kg vs. 10 mg/kg; Fischer et al., 2017). Male offspring appear to experience the effects of parental exposure to cocaine slightly differently. For example, cocaine-sired male rats acquire self-administration more slowly, and took less total drug overall (Vassoler et al., 2013), suggesting a sex-specific protective effect against drug seeking in male progeny. In a model that divided rats into groups based on motivation level to voluntarily self-administer cocaine, researchers found that the highly motivated behavioral phenotype (termed “Addict” rats in the study) could be transmitted to F1 and F2 generations (Le et al., 2017). Interestingly, the heritable addict-like behavioral phenotype was linked to high motivation to drug seek, rather than higher levels of cocaine intake alone, as transmission of this behavioral phenotype was not observed in offspring when the F0 generation received passive experimenter-administered cocaine injections (Le et al., 2017). This addict-like phenotype was also not observed when the F0 generation sired offspring prior to drug experience, suggesting the phenotype is acquired as a consequence of contingent drug experience, and is not likely a result of direct genetic inheritance. Cocaine use is known to shape changes in gene expression and plasticity in the reward circuitry that underlies addiction-related behaviors. However, currently evidence for which corresponding epigenetic modifications are also observed in the brains of the F1 generation after paternal drug experience, and the potential mechanisms through which this might occur, are quite limited. One of the earliest studies to link cocaine-induced epigenetic alterations to heritable changes in behavior found a significant decrease in Dnmt1 and increased Dnmt3a in the seminiferous tubules of the testes in male mice trained to self-administer cocaine (He et al., 2006). Although epigenetic modifications were not measured in adult offspring, several morphological and behavioral abnormalities were noted, including decreased cerebral volume, reduced head diameter, impaired attention and working memory. This study was one of the first to show drug experience alters not only the self-administering rodent, but also their germline cells, hinting at a potential mechanism through which cocaine-induced changes to the epigenome could be transferred to the next generation. A more recent study also observed epigenetic alterations to the sperm of cocaine-experienced sires, this time in the form of elevated H3 acetylation at Bdnf promoters I, IV, and VI (Vassoler et al., 2013). These effects in the F0 generation were accompanied by sex-specific increases in H3 acetylation at Bdnf promoter IV in the medial prefrontal cortex (mPFC) of cocaine-sired male offspring, as well as increased Bdnf mRNA and

7. Current limitations and challenges, and approaches to move forward Many gaps remain in our understanding of how epigenetic modifications acquired in the parent generation might contribute to the transmission of phenotypic traits in their offspring. Despite evidence that certain behaviors and disease states seemingly persist across generations, the idea that these traits are caused by germline-dependent inheritance of epigenetic marks remains controversial. This is in part due to a number of issues that remain unresolved, including unambiguous proof of heritability, demonstration of survival of epigenetic marks during epigenome reprogramming, their selective maintenance in specific somatic tissues, and the extent to which these inherited modifications are causal of an observable phenotype. The following section highlights current limitations and key issues in transgenerational epigenetic inheritance research, and proposes guidelines for how future research might address these existing gaps. 7.1. Technical limitations and challenges in experimental design Epigenetic factors help shape cell fate and function, and accordingly, methylation patterns likely differ between distinct subpopulations of cells both within the brain and throughout the periphery. As such, the tissue collected to generate bulk homogenate for most EWAS studies likely differs in composition, with some cell types being overrepresented while others remain underrepresented (Jaffe and Irizarry, 2014). Thus, observations made from methylation patterns consisting largely of one cell type may not generalize to other cell populations. 10

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7.4. Unambiguous demonstration of heritability

Single-cell level resolution of methylation and histone profiles in future studies will be necessary to overcome this technical limitation, and provide a more accurate reflection of the heterogeneity of epigenetic patterns across unique cell populations. In addition to the technical limitations of currently available approaches, careful attention must be paid to experimental design in order to reduce potential variability and contribution of non-epigenetic factors to studies of this nature. For example, preclinical research should consider using inbred rodent strains with tightly regulated environments to reduce the potential for genetic and environmental confounds. Further, if transgenerational effects are to be evaluated, studies must be extended long enough to allow for evaluation of epigenetic modifications and phenotypic traits in the appropriate generation (i.e., F2 for male or non-pregnant female exposure, F3 for exposure to pregnant females; see Fig. 1). For some studies, an attractive alternative may involve the use of in vitro fertilization and transfer to foster mothers, which allows for strict observation of transmission of epigenetic marks via the gametes and reduces the possible influence of altered behavioral effects in the parent generation (e.g., changes in maternal care) indirectly altering behavior or epigenetic changes in progeny.

One challenge transgenerational epigenetic inheritance studies have yet to overcome is definitively demonstrating that epigenetic marks present in the germline of the parent generation are truly inherited by the F1 generation, rather than removed and re-established in offspring. During early prenatal development, epigenetic marks present in the parent germline (e.g., DNA methylation and histone PTMs) are largely removed in the developing zygote after fertilization. At present, it remains unclear how specific epigenetic modifications in the maternal and paternal gametes would survive this largescale removal of epigenetic marks. Further, it is not clear how epigenetic modifications that do survive this period of reprogramming would be transferred from undifferentiated embryonic cells to specific differentiated somatic tissues (i.e., neurons) in the adult offspring. Although DNMTs assist in preserving methylation marks during cellular differentiation and in somatic tissues, the accuracy of DNA methylation during replication is imperfect (~96% fidelity rate). Although this may sound relatively high, in reality it allows for errors to be introduced into methylation patterns across multiple rounds of DNA replication and cell division. Therefore, accurate sustained transmission of such epigenetic modifications, which would be necessary for the transgenerational passage of such marks, would be difficult to achieve. Even less is known about how post-translational modifications of histones are maintained during DNA replication and cell division, so this would also be an issue for studies focused on a histone-mediated mode of epigenetic inheritance. Given the high probability for the loss of both histone PTMs and DNA methylation modifications over multiple rounds of replication and cell division, future studies should focus on providing convincing evidence that epigenetic marks observed in the parent germline are also observed at several other key developmental stages (gametes > zygote > blastocyst > post-implantation embryo > F1 offspring) with parallel evidence of the heritable mark across these same stages in the F2 generation. Bisulfite sequencing, methylated DNA immunoprecipitation (MeDIP), or chromatin immunoprecipitation (ChIP)-based approaches with antibodies against specific histone marks may be best suited to track histone PTM or DNA methylation profiles across generations. Sequencing data (e.g., RNA-seq, ChIP-seq) demonstrating gene expression profiles and phenotypic traits expected to accompany the observed pattern of epigenetically inherited marks would further strengthen such work, and provide a plausible proximal mechanism linking inheritance of these marks to biological functions in the affected cell population.

7.2. Crosstalk between different epigenetic modifications The choreography of various epigenetic factors that act in concert to initiate transcriptional programs and define cellular fate are complex and interconnected. Combinatorial approaches will be required to more accurately define methylation state in conjunction with histone modifications by using approaches that incorporate bisulfite sequencing information with ChIP data from the same cellular populations (Brinkman et al., 2012; Statham et al., 2012). Because epigenetic modifications are dynamic and reversible, intermediate states of DNA methylation (e.g., 5fC and 5caC) may also need to be taken into consideration in EWAS studies to capture a deeper level of resolution on these dynamic states. At present, it is unclear whether these are truly intermediate states, or if they serve unique functions of their own.

7.3. Contribution of genetic influence on epigenetic landscape In addition to the complex interplay amongst different epigenetic factors, there is also the potential for indirect genetic contribution to the transmission of epigenetic marks. That is, normal variation in DNA sequence between individuals may underlie some DNA methylation differences observed between individuals, making it particularly challenging to disentangle genetic vs. epigenetic transmission of epigenetic marks and corresponding effects on phenotypic traits (Gibbs et al., 2010; Bell et al., 2011; Gertz et al., 2011). For this reason, epigenetic inheritance studies should include DNA sequencing controls whenever possible to ensure observed effects are not an indirect result of genetic rather than epigenetic differences between individuals (Lappalainen and Greally, 2017). This consideration is also important for monitoring gene variants that could lead to an indirect epimutation that impacts transcription patterns in the sense or antisense orientation at the region of interest. This phenomenon was recently reported to accompany a rare disease across several generations, wherein genetic mutation in one gene resulted in elimination of its usual transcription termination signal, and subsequently led to atypical methylation at a gene neighboring the region of interest (Chan et al., 2006; Ligtenberg et al., 2009; Gueant et al., 2018). Although ultimately methylation marks were altered at the disease-linked gene, this change was an indirect consequence of direct mutation in the DNA sequence, rather than through gamete inheritance of the methylation mark, and as such is not truly representative of epigenetic inheritance.

7.5. Transfer of germline modifications to target somatic tissues In order for epigenetic modifications laid down in the germline to influence brain function, it is likely that these modifications would have to survive through early development in order to alter gene expression in target neurons that control the phenotype in question. However, it is presently unclear what determines which somatic tissues will maintain the epigenetic marks originating from undifferentiated stem cells. It seems unlikely that such modifications would be transferred from the germline to all somatic tissues, so how are such marks passed on only to specific cell populations of cells but not others? For example, if substance abuse leads to an epigenetic alteration at gene X only in nucleus accumbens neuron populations, how does this mark 1) get transferred from parental neural tissue to the germline, and 2) from the germline containing undifferentiated cells back to just a distinct subpopulation of neurons in a specific brain region (but not in other somatic tissues)? It is easier to imagine how this might occur for phenotypes that are encoded by highly specific expression of specific genes in specific cell populations (e.g., olfactory receptors), but it is far more challenging to understand how this selectivity could occur for more complex polygenic or omnigenic phenotypes.

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about epigenetic mechanisms in somatic and dividing cells over the past several decades, the mechanisms that drive heritable, germline-dependent transmission of epigenetic marks remain poorly understood. At present, germline-dependent transmission appears to be the exception rather than the rule for mammalian species. However, while currently limited, these exceptions have provided dramatic evidence for the potential implications for transgenerational epigenetic contributions to a number of global public health issues, including obesity, substance use disorders, and stress response in the face of traumatic experience. Future research will be required not only for providing more conclusive evidence that epigenetic alterations acquired in one generation can be stably inherited and propagated to future generations, but also for unravelling the molecular mechanisms that make this type of information transfer possible.

7.6. Evidence demonstrating the causal nature of epigenetic alterations In addition to the limited proof concerning unambiguous heritability, evidence demonstrating causality is also currently lacking. Future studies should include rigorous evidence that epigenetic alterations passed from one generation to the next are the root cause of the observed phenotype, rather than a co-occurrence. Recent advances in epigenetic editing techniques will be particularly important for demonstrating both necessity and sufficiency of key epigenetic modifications in the manifestation of observable phenotypic traits. For example, CRISPR/dCas9-based strategies allow for epigenetic modifying enzymes, including DNMTs and HATs, to be tethered to a catalytically inactive dCas9 protein (Liu et al., 2016; Vojta et al., 2016; Kwon et al., 2017; Pflueger et al., 2018; Savell et al., 2019). This dCas9/epigenetic modifier construct can then be directly targeted to specific loci throughout the genome. These type of directed epigenetic alterations will allow for subsequent changes in gene expression, behavior, and phenotypic traits to be observed in response to specific epigenetic modifications, which can then be monitored across multiple generations. Such work will be essential for providing more rigorous and compelling evidence regarding the causal nature of transgenerational epigenetic inheritance.

Acknowledgements This work was supported by NIH grants DA039650, DA034681, and MH114990 (J.J.D.). Illustrations were generated using BioRender. References Abel, E.L., Moore, C., Waselewsky, D., Zajac, C., Russell, L.D., 1989. Effects of cocaine hydrochloride on reproductive function and sexual behavior of male rats and on the behavior of their offspring. J. Androl. 10, 17–27. Annunziato, A.T., 2005. Split decision: what happens to nucleosomes during DNA replication? J. Biol. Chem. 280, 12065–12068. Anway, M.D., Cupp, A.S., Uzumcu, M., Skinner, M.K., 2005. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308, 1466–1469. Baccarelli, A., Bollati, V., 2009. Epigenetics and environmental chemicals. Curr. Opin. Pediatr. 21, 243–251. Bale, T.L., 2015. Epigenetic and transgenerational reprogramming of brain development. Nat. Rev. Neurosci. 16, 332–344. Balhorn, R., 2007. The protamine family of sperm nuclear proteins. Genome Biol. 8, 227. Bell, J.T., Pai, A.A., Pickrell, J.K., Gaffney, D.J., Pique-Regi, R., Degner, J.F., Gilad, Y., Pritchard, J.K., 2011. DNA methylation patterns associate with genetic and gene expression variation in HapMap cell lines. Genome Biol. 12, R10. Bellone, C., Mameli, M., Luscher, C., 2011. In utero exposure to cocaine delays postnatal synaptic maturation of glutamatergic transmission in the VTA. Nat. Neurosci. 14, 1439–1446. Berglind, W.J., See, R.E., Fuchs, R.A., Ghee, S.M., Whitfield Jr., T.W., Miller, S.W., McGinty, J.F., 2007. A BDNF infusion into the medial prefrontal cortex suppresses cocaine seeking in rats. Eur. J. Neurosci. 26, 757–766. Bierut, L.J., Dinwiddie, S.H., Begleiter, H., Crowe, R.R., Hesselbrock, V., Nurnberger Jr., J.I., Porjesz, B., Schuckit, M.A., Reich, T., 1998. Familial transmission of substance dependence: alcohol, marijuana, cocaine, and habitual smoking: a report from the collaborative study on the genetics of alcoholism. Arch. Gen. Psychiatry 55, 982–988. Bird, A., 1992. The essentials of DNA methylation. Cell 70, 5–8. Birnbaum, R.Y., Clowney, E.J., Agamy, O., Kim, M.J., Zhao, J., Yamanaka, T., Pappalardo, Z., Clarke, S.L., Wenger, A.M., Nguyen, L., Gurrieri, F., Everman, D.B., Schwartz, C.E., Birk, O.S., Bejerano, G., Lomvardas, S., Ahituv, N., 2012. Coding exons function as tissue-specific enhancers of nearby genes. Genome Res. 22, 1059–1068. Blattler, A., Yao, L., Witt, H., Guo, Y., Nicolet, C.M., Berman, B.P., Farnham, P.J., 2014. Global loss of DNA methylation uncovers intronic enhancers in genes showing expression changes. Genome Biol. 15, 469. Brandeis, M., Kafri, T., Ariel, M., Chaillet, J.R., McCarrey, J., Razin, A., Cedar, H., 1993. The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBO J. 12, 3669–3677. Brinkman, A.B., Gu, H., Bartels, S.J., Zhang, Y., Matarese, F., Simmer, F., Marks, H., Bock, C., Gnirke, A., Meissner, A., Stunnenberg, H.G., 2012. Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk. Genome Res. 22, 1128–1138. Brunner, A.M., Nanni, P., Mansuy, I.M., 2014. Epigenetic marking of sperm by posttranslational modification of histones and protamines. Epigenetics Chromatin 7, 2. Brykczynska, U., Hisano, M., Erkek, S., Ramos, L., Oakeley, E.J., Roloff, T.C., Beisel, C., Schubeler, D., Stadler, M.B., Peters, A.H., 2010. Repressive and active histone methylation mark distinct promoters in human and mouse spermatozoa. Nat. Struct. Mol. Biol. 17, 679–687. Budhavarapu, V.N., Chavez, M., Tyler, J.K., 2013. How is epigenetic information maintained through DNA replication? Epigenetics Chromatin 6, 32. Cadet, J.L., 2016. Epigenetics of stress, addiction, and resilience: therapeutic implications. Mol. Neurobiol. 53, 545–560. Casas, E., Vavouri, T., 2014. Sperm epigenomics: challenges and opportunities. Front. Genet. 5, 330. Cavalli, G., Paro, R., 1998. The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis. Cell 93, 505–518. Cavalli, G., Paro, R., 1999. Epigenetic inheritance of active chromatin after removal of the main transactivator. Science 286, 955–958. Chan, T.L., Yuen, S.T., Kong, C.K., Chan, Y.W., Chan, A.S., Ng, W.F., Tsui, W.Y., Lo, M.W.,

8. Conclusions Although it has quickly captured the attention of the neuroscience community, research on multigenerational epigenetic inheritance in mammalian species remains in its infancy. Despite evidence of intergenerational and transgenerational phenotypes in many different model systems, a number of critical questions remain to be answered. Perhaps most importantly, it is currently unclear what biological problem this form of inheritance solves. Here, it is worth considering whether generational passage of epigenetic changes make sense from an evolutionary biology perspective. For example, if an external stimulus (e.g., drug use, stress, poor nutrition) results in overwhelming negative consequences for the individual and their ability to create optimally fit progeny, why might such epigenetic modifications and consequent phenotypes be allowed to persist for multiple generations? Why would the same malleable system that allowed for epigenetic marks to be deposited in the first place not respond in a manner more adaptive for the species? Although it is worth noting that some evidence in the rodent literature indicates drug experience may confer a protective phenotype in offspring, this is not necessarily reflective of what happens in humans. If anything, parental drug experiences confer more – not less – vulnerability for substance use disorders in offspring generations (Bierut et al., 1998; Goldman et al., 2005; Vink, 2016). This problem is inherent to all epigenetics studies, as the nature of the system is somehow both stable (i.e., transmissible across generations) yet plastic in response to external stimuli. A second concept that is valuable to consider is the relative weight of epigenetic changes in comparison to genetic influences. Genetic contributions to observable phenotypes in mammals are powerful, numerous, and extremely reproducible. Key examples include primary sex determination by Y-linked genes, severe neurodevelopmental disorders arising from de novo mutations, and the highly penetrant nature of familial forms of neuropsychiatric or neurodegenerative disease states. Are epigenetic modifications simply occurring on top of the large set of robust genetic factors that contribute to development and adult functions of the nervous system, providing only slight tweaks or temporary changes? Or are epigenetic modifications able to overturn or usurp genetically defined programs, establishing new qualitatively different transcriptional landscapes and behavioral phenotypes? Addressing this question will be essential to determining the position of epigenetic mechanisms in the hierarchy of molecular mechanisms that contribute to brain development, function, and disease. Although we have gleaned a considerable amount of information 12

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