- Department of Physiology and Pharmacology, Sapienza University of Rome, Rome, Italy
- Department of Psychology and “Daniel Bovet” Center, Sapienza University of Rome, Rome, Italy
- Fondazione Santa Lucia, IRCCS, Rome, Italy
Parents’ stressful experiences can influence an offspring’s vulnerability to many pathological conditions, including psychopathologies, and their effects may even endure for several generations. Nevertheless, the cause of this phenomenon has not been determined, and only recently have scientists turned to epigenetics to answer this question. There is extensive literature on epigenetics, but no consensus exists with regard to how and what can (and must) be considered to study and define epigenetics processes and their inheritance. In this work, we aimed to clarify and systematize these concepts. To this end, we analyzed the dynamics of epigenetic changes over time in detail and defined three types of epigenetics: a direct form of epigenetics (DE) and two indirect epigenetic processes-within (WIE) and across (AIE). DE refers to changes that occur in the lifespan of an inpidual, due to direct experiences with his environment. WIE concerns changes that occur inside of the womb, due to events during gestation. Finally, AIE defines changes that affect the inpidual’s pdecessors (parents, grandparents, etc.), due to events that occur even long before conception and that are somehow (e.g., through gametes, the intrauterine environment setting) transmitted across generations. This distinction allows us to organize the main body of epigenetic evidence according to these categories and then focus on the latter (AIE), referring to it as a faster route of informational transmission across generations-compared with genetic inheritance-that guides human evolution in a Lamarckian (i.e., experience-dependent) manner. Of the molecular processes that are implicated in this phenomenon, well-known (methylation) and novel (non-coding RNA, ncRNA) regulatory mechanisms are converging. Our discussion of the chief methods that are used to study epigenetic inheritance highlights the most compelling technical and theoretical problems of this discipline. Experimental suggestions to expand this field are provided, and their practical and ethical implications are discussed extensively.
Many recent studies have demonstrated that stressful conditions that are experienced by parents can influence the offspring’s vulnerability to many pathological conditions, including psychopathologies-primarily related to a disruption in stress response mechanisms. These effects may even endure for several generations. Nevertheless, the mechanisms of this phenomenon have not been detailed, and only recently have scientists examined epigenetics to answer this question.
In this work, we systematize the concept of epigenetic inheritance, discuss the putative mechanisms, and recapitulate the methods for studying this circumstance, psenting their potentialities and limitations. We focus on the transmission of psychopathologies-especially in relation to disruptions in the stress response-because they have long been the center of the historical debate over the weights of genes and the environment in such processes as inpidual development and inheritance, given their complex nature and clear experience sensitivity. Thus, psychopathologies can be considered one of the most interesting and flourishing fields in the application of epigenetics.
Historical Background: Filling the Gap
Initially, and for a long time, parental influences on an offspring’s development were focused on two possible sources of variance: genes and the environment. Some scientists concentrated on how “slow and still” information could be transmitted to subsequent generations. The phylogenetic perspective was thus a central assumption, more or less implicit. The pmise was that genes themselves carry on blindly: the luckiest genes that are most well suited for the psent environmental conditions “win” and endure ( Dawkins, 1990).
This idea fits well with the Darwinian concept of adaptation as an all-or-nothing process, which can be recalled easily from the collective imaginary through such terms as “survival,” “reproductive power,” and “law of large numbers.” Those who survive live longer, thus theoretically increasing the probability of finding a mate and reproducing. This can be surely the case, but this theory alone is insufficient to explain phylogenetic development.
Yet, for a long time, genes and the environment were considered two separate aspects that interacted at the level of the phenotype. Even epigenetics was conceived of as being able to modify the genetic impact on an inpidual’s organization but remaining inside his existence (i.e., acting only during his lifespan). Until then, there was only one way in which the past could inform the coming new life: genes and parental care. Only when it was demonstrated that epigenetic modifications could be inherited did the ontogenetic (i.e., environmental influences) and phylogenetic (i.e., genetic determinants) worlds-which for years had approached each other in an asymptotic, exhaustive manner-finally merge at a new, theoretical intersection: epigenetic inheritance.
Epigenetics and Inheritance: Some Definitions
1. exposure to an event in generation F0.
2. an effect of the event must be observed in the third or fourth generation-i.e., F2 or F3-depending on whether the mother or father was first affected (F0).
Female exposure to a certain environmental factor during pgnancy might even affect the offspring’s germ cells directly, for which reason only the fourth generation can be considered “event-free” and unsullied. When a certain event produces an epigenetic change in the father, it can only modify his sperm, effecting reliable nongenetic inheritance in the third generation (Figure 1).
Figure 1. Transgenerational epigenetic inheritance. According to the classical definition of transgenerational epigenetic inheritance, environmental triggers that hit pgnant female inpiduals (F0) can affect “directly” not only the first new generation (F1), but also its germ cells that repsent the second generation (F2). For this reason, only changes in F3 can be due “purely” to epigenetic inheritance. The male germline, instead, can be affected only for one generation, allowing observing epigenetic inheritance already at F2.
This definition surely renders the observation of epigenetic inheritance easier, especially in humans, because it pvents the ambiguous interptation of data that are inevitably contaminated by other events that are not transmitted epigenetically through gamete programming. Nevertheless, this approach excludes the possibility of considering faster epigenetic effects, which are certainly more difficult to control experimentally but could still exist and have functions.
In fact, why must epigenetic transmission occur through germ cells and across several generations? The epigenetic modification of certain genes, produced by an environmental trigger, could lead to significant changes in an inpidual’s body that could persist over time and in turn signal the epigenetic reorganization of the subsequent generation. This phenomenon could happen without affecting the germline directly and despite the event that fostered such adaptation no being longer active once the embryo has begun its development. As we will see, experimental manipulation in animal models could overcome these problems. For this reason, we will attempt to unify and organize this potentially confusing terminological flowering in a coherent conceptual framework.
In recent years, many scientists have hypothesized and even demonstrated that certain experiences during the life of an inpidual influence the development of his offspring, even distally. It appears that some experiences modify genetic expssion, influencing:
1. how the organism itself responds to a changeable environment (i.e., more ontogenetic flexibility-direct or synchronous effect) and
2. how his descendants will increase their likelihood of surviving in a specific environment-that is, how information is transmitted to offspring regarding the environment that they will encounter (i.e., more phylogenetic flexibility-indirect and both synchronous and asynchronous effects).
The first aspect, which we will call direct epigenetics (DE), comprises all of the epigenetic changes that occur during an inpidual’s lifespan. Notably, this phenomenon implies even dynamic and short-term regulation of gene expssion, mediated by the action-almost in real time-of regulatory proteins, called transcription factors, such as c-fos, c-jun, ZENK and CREB. The genes that encode for such crucial functional elements are called immediate-early genes, because a change in their expssion is the first event that launches cascades of adaptive events, including the transcriptional aspects of other genes ( Johnson, 2010), ultimately producing even long-lasting effects.
When an epigenetic change produced by a direct experience (DE) is transmitted to the offspring, that same experience becomes an indirect environmental trigger for the ontogenetic development of the new inpidual. Paralleling Crews (2008) and van Otterdijk and Michels (2016), the second form of environmental action (i.e., phylogenetic adaptation) can be pided into two categories of “indirect epigenetics (IE):” within and across. These two aspects can be considered the conceptual product of the historical development of this matter, the latter (across) being a more recent acquisition. Theoretically, these components are related and difficult to distinguish, even operationally.
Within indirect epigenetics (WIE) encompasses all of the epigenetic changes that act synchronously on the developing inpidual. Temporally, it starts at the very moment at which the zygote is formed and the environment begins changing. This category includes all of the factors that, more or less indirectly, can affect the developing inpidual, from the start to end of gestation. The underlying concept is that environmental changes occur when the (proto)-inpidual actually exists, synchronously.
Across indirect epigenetics (AIE) describes what happens from the moment of conception back toward the parents’ earlier life experiences (and even grandparents, as we will discuss), which asynchronously set the composition of germ cells (and possibly that of the intrauterine environment). Some authors have referred to all epigenetic changes that appear to be transmitted across generations as epimutations, in contrast to classical, less frequent genetic mutations ( Bennett-Baker et al., 2003). Notably, in this case, a certain event has consequences that are maintained over time, affecting the offspring’s destiny during gestation and, most importantly, later in life. Clearly, it is reasonable to believe that the closer we are to the moment of conception, the stronger the pdiction power of the variable is, or at least the easier it is to hypothesize a “causal” relationship, because it should be expected in an epistemology of complexity that conceives of development in terms of probabilistic epigenesis (see Gottlieb, 2007 for a theoretical detailed explanation). Nevertheless, as we will see, certain events can act as relevant pdictors even when distal in time.
We can discuss epigenetics only if a modification to gene expssion takes place. This idea, supported by Kovalchuk (2012), renders the function of the intrauterine environment in epigenetic transmission controversial-in cases in which environmental events produce changes that do not affect germ cells directly but persist and affect the newborn in later gestational stages. Nevertheless-and for this same reason-the epigenetic mechanisms that determine the womb cannot be neglected if they are demonstrated to mediate the transmission of information on the genetic expssion of the developing organism (as discussed below). As we will see, of all of the epigenetic mechanisms that are implied in these two indirect forms of transmission, maintenance methylation, de novo methylation and the regulatory and amplifying activities of ncRNA, are the most prominent. Although increasing data strongly suggest transgenerational inheritance of epigenetic information, the non-DNA-based processes by which information is transmitted across generations are largely unknown ( Houri-Zeevi and Rechavi, 2021).
Wider Clarifications and Considerations
The categorization above repsents a mere conceptual distinction that has been conceived simply to elucidate the phenomenon of interest. As a matter of fact, all of these aspects are expected to interact continuously, but we can distinguish, on a case-by-case basis, which conceptual element (ontogenetic vs. phylogenetic, direct vs. indirect, or even within vs. between) has more apparent relevance. Notably, our aim is to indicate that relegating epigenetic transmission only to the moment of gestation is impcise, hindering us from developing a wider and exhaustive understanding of this phenomenon. Moreover, our purpose is to merge and soften all dichotomic types of conceptualization, including those that we have proposed herein.
Wider environmental effectors, such as parental style and cultural aspects, must be considered with caution. They seem to be direct and indirect in their action, as well as synchronous and asynchronous. Certainly, they account for the general setting in which the newborn develops, which in turn begets different and complex forms of information about the past that guides ontogenetic and phylogenetic adaptation. This should be considered the most intuitive means of transgenerational transmission of information-the most naïve but still undeniable Lamarckian addition to Darwinian evolution. Nevertheless, and for this reason, the effect of these two variables is too complicated to account for, and studies that have attempted to demonstrate their function in epigenetic transmission (as we will see in the next section) are not exhaustive. Moreover, a discussion of these wider environmental factors is not pertinent to our discourse, given the level of inquiry that we are considering. Thus, we are setting aside these two aspects from our argumentation, except for pnatal maternal care and the few historical events that have been suggestive objects of study (e.g., the Dutch Famine and the Holocaust).
What does the concept of epigenetic inheritance add to science with respect to the earlier concept of evolution and genetic transmission? This type of communication appears to be faster and more contingent and thus more efficient. For this reason, epigenetics increases our heuristic power through a different concept of evolution, in which the environment has a more proactive role in influencing communication across generations, depending ultimately on two interconnected evolutionary processes: a Darwinian process (slow but steady) and a Lamarckian process (quick but labile). The historical contraposition of these processes is now evolving into a unified theory of evolution ( Skinner, 2021).
Therefore, we can imagine the transmission of information across generations as a succession of cycles, a sequence of light-cones that repsent the multidimensionality of a theoretical wave function that describes the amplitude of indetermination or the potential of the evolving system. Thus, we can picture the “pulsing” of this probabilistic informational mass unfolding across spacetime, merging at the moment of conception (considered our arbitrarily chosen observation point) and then expanding, only to collapse again (Figure 2).
Figure 2. Epigenetics through the Minkowskian cone. Epigenetic changes and related environmental factors visualized in 4D Minkowskian space, assuming conception as our arbitrarily chosen observation point, the zero of the system. Across indirect epigenetics (AIE) includes all those adaptations in parental life that pcede conception; within indirect epigenetics (WIE) describes all those changes that take place during the gestational period and, finally, direct epigenetics (DE) describes all those plastic processes that can occur after birth. Although these processes are strongly interconnected and can overlap on multiple levels in a complex real system, here they are treated as discrete and sequential, for the sake of clarity.
Evidence of Epigenetic Changes
There are several reviews on epigenetic changes ( Jawahar et al., 2021; Jung and Pfeifer, 2021; Conti and Alvares da Silva-Conforti, 2021; Maccari et al., 2021) and their heritability across generations ( Gapp et al., 2014; Skinner, 2014; Babenko et al., 2021; Bohacek and Mansuy, 2021; Szyf, 2021; van Otterdijk and Michels, 2021; Ambeskovic et al., 2021; Pang et al., 2021; Yeshurun and Hannan, 2021). We report only some of the most significant evidence to provide concrete examples of our proposed classification.
Evidence of Direct Epigenetics
Epigenetic changes can be triggered by several environmental factors, such as diet ( Mathers et al., 2010), pollution ( Christensen and Marsit, 2011), smoking ( Talikka et al., 2012), that can be labeled generically as “stressors,” referring to the neutral, adaptive meaning of the term ( Cabib and Puglisi-Allegra, 2012). Epigenetic aberrations have been implicated in many diseases, primarily cancer but also cardiovascular, autoimmune, metabolic and neurodegenerative diseases, often with particular regard to aging ( van Otterdijk et al., 2013; Jung and Pfeifer, 2021).
Several studies have examined the regulatory effects of adult stress on the methylation of the NR3C1 gene as a pathological marker and mediator of pathology, consequent to dysregulation of the hypothalamic-pituitary-adrenal axis (HPA), as exemplified in animal models of social defeat stress ( St-Cyr and McGowan, 2021). In addition, brain-derived neurotrophic factor (BDNF) is downregulated in several areas of the brain in animal models of depssion ( Elfving et al., 2010; Molteni et al., 2010; Qiao et al., 2014). Methylation of the promoter region of BDNF is associated with a reduction in hippocampal volume. In several animal models, hippocampal BDNF levels decline under acute ( Barrientos et al., 2003) and chronic ( Nibuya et al., 1995) stress conditions. Antidepssant treatment upgulates hippocampal BDNF, and knocking out BDNF in animal models impairs the response to treatment ( Khundakar and Zetterström, 2006; Monteggia et al., 2007).
Illustrating the unique functions of immediate-early genes (IEGs; cited above), CREB-mediated transient plasticity mediates the transition to an irreversible phenotype of addiction through the accumulation of delta-FosB, which mediates structural synaptic readaptations that strengthen themselves in a positive-feedback process ( Koob and Volkow, 2010).
The levels of certain stress-related proteins have been analyzed. CRFR1 levels are elevated in total KO (TKO) mice, as reported by Haramati et al. (2011). Similarly, Andolina et al. (2018) examined the effects of the absence of miR-34a/b/c (i.e., a TKO model, or TKO) on coping behavior. MiR-34 levels were assessed in all of the main areas that are involved in stress responses, peaking in DRN. TKO mice tended to resort to active coping behaviors when challenged by the forced swim test (FST). Accordingly, TKO mice overexpssed CRFR1 in DRN, compared with WT mice, and their “resilient” behavioral phenotype could be reverted through the injection of a CRFR1 antagonist, confirming its function in regulating stress response strategies.
Evidence of Within Indirect Epigenetics
Animal models have been used to demonstrate the indirect effects of environmental factors during pgnancy on the offspring’s development. For example, maternal fat diet increases the susceptibility of male offspring to liver disease through epigenetic reprogramming of lipid metabolism and inflammatory responses ( Pruis et al., 2014). Further, pnatal undernutrition, for example, can permanently alter DNA methylation in the sperm of adult offspring in regions that are resistant to zygotic reprogramming, potentiating transgenerational transmission of metabolic disorders ( Radford et al., 2014).
Fetal programming also depends on miRNAs, although there is limited evidence in WIE. Placental miRNAs have been implicated by several groups ( Maccani et al., 2013; Morales-Prieto et al., 2014), but there is still scarce proof of their actual involvement and there is no direct evidence of the epigenetic changes that consequently occur in the developing fetus.
Evidence of Across Indirect Epigenetics and Transgenerational Epigenetic Inheritance
Across indirect epigenetic changes, per se, define only intergenerational epigenetic inheritance, which is inheritance from one generation to the next ( Pang et al., 2021). AIE can be and has been considered, instead, a necessary but insufficient condition for transgenerational epigenetic inheritance, at least per its canonical definition ( Skinner, 2008). Many experiments have been performed to prove some form of epigenetic inheritance in the past two decades. We report several examples below.
Rat malnutrition impairs cognition in offspring ( Galler and Seelig, 1981). A low-protein diet over 10 generations produces even more severe cognitive deficits, which are evident after two generations, on returning to a regular diet ( Stewart et al., 1980). Dunn and Bale (2009) have demonstrated that a maternal high-fat diet in mice increases body size and insulin sensitivity, which endure until the second generation; these effects nearly vanish in the F3 generation, despite the alterations in body size being observed solely in female offspring, suggesting an imprinting mechanism. Parental addiction in rodents alter the sensitivity of offspring to drugs, eliciting adaptive counterregulatory responses ( Byrnes et al., 2011; Vassoler et al., 2013; Finegersh and Homanics, 2014).
Direct proof of transgenerational epigenetic inheritance in humans remains lacking ( van Otterdijk and Michels, 2021). Nevertheless, there is notable indirect evidence (i.e., longitudinal studies with no or few insights into putative epigenetic mechanisms).
There is copious evidence of epigenetic changes in animal models, but this field must improve to generate stronger evidence and implement new techniques that could apply to human studies, in which direct and robust proof remains lacking. We have compiled many studies and pided them by epigenetic type and research model (Table 1). As discussed, this review’s aim is not to report all existing studies in the field but to provide some examples that can help us better understand epigenetic changes and their inheritance.
Table 1. Evidences of the three defined forms of epigenetic changes: direct epigenetics (DE), within indirect epigenetics (WIE) and across indirect epigenetics (AIE).
How does epigenetic inheritance occur concretely? Although several epigenetic processes have been considered to answer this question, given the wide range of this work, we will focus on two of the more extensively studied mechanisms: methylation and ncRNA.
First-Generation Epigenetic Mechanisms
Methylation and Demethylation
DNA methylation is an enzymatic process by which a methyl group (CH 3) is covalently bound to the fifth position of a cytosine residue (5-methylcytosine, 5mC) to alter gene expssion. In mammalian DNA, this regulatory activity acts on CpG palindromes (i.e., diagonally symmetric couples of guanine-cytosine pairs), whereas asymmetric methylation is rare ( Chen and Li, 2004). When methylation affects the promoter region, it is associated with gene silencing-the most well-known function of this mechanism; however, when it involves the transcribed region, it increases transcriptional activity ( Jones, 2012). DNA methylation is involved in many processes, particularly those that are important for early development, such as genomic imprinting, X-chromosome inactivation, and transposon silencing ( Smith and Meissner, 2013).
The addition of CH 3 groups to CpG islands is catalyzed by DNA methyltransferases (DNMTs). DNMT1 primarily maintains DNA methylation patterns during replication, whereas DNMT3A, DNMT3B and DNMT3L (a noncatalytic isoform of DNMT3, termed DNMT3-like) are principally involved in establishing new DNA methylation patterns-a mechanism that is called de novo methylation-that characterize embryo development, in particular ( Chen and Li, 2004).
The maintenance of methylation is crucial for ensuring the continuity of the structural and functional identities of somatic cells throughout cell pision. During the S phase of the cell cycle, DNMT1 reaches hemimethylated CpGs with the aid of ubiquitin-like with PHD and RING finger domains 1 (UHRF1) proteins such that each newly synthesized DNA strand can be methylated per its complementary strand. Thus, after each replication, the symmetry of the methylation pattern is restored ( Zhang et al., 2011; Wu and Zhang, 2014).
Although methylation patterns are stable, they can be erased by two mechanisms: active and passive demethylation. Passive demethylation repsents a failure in maintenance (so-called replication-dependent dilution) and occurs primarily in the absence of functional DNMT1/UHRF1: if the symmetry of methylation is not reestablished, methylation is lost through replications ( Smith and Meissner, 2013; Wu and Zhang, 2014).
Figure 3. Methylation and demethylation. Methylation is a regulatory process of gene expssion, catalyzed by DNA methyltransferase enzymes, owing to the addition of a methyl group to the fifth position of a cytosine. DNA methyltransferases 1 (DNM1) is mainly involved in maintenance methylation that restores symmetric DNA methylation patterns after DNA replication. DNM3A, DNMTB and DNMTL, instead, are involved in the catalytic process that produces de novo methylation by adding methyl groups to unmethylated DNA strands. Methylation processes can be reverted by two mechanisms: passive demethylation due to loss of methylation across consecutive DNA replications; active demethylation mediated by ten-eleven translocation (TET) proteins.
Methylation and Epigenetic Inheritance
Maintenance and de novo methylation and active and passive demethylation are crucial for embryonic development and epigenetic inheritance. Gametes are completely demethylated and are remethylated after fertilization to erase all epigenetic marks that an inpidual accumulates over his lifespan. However, this resetting process is impeded during early development, perhaps accounting for transgenerational transmission of these epigenetic footprints ( van Otterdijk and Michels, 2021).
Elimination and restoration of methylation markers occurs in two steps (Figure 4). Immediately after fertilization, global demethylation is observed that erases methylation marks of the parental gametes through two sex-dependent mechanisms. First, the DNA in paternal pronuclei undergoes rapid, active demethylation that is mediated by TET3 proteins, which spare only imprinting control regions (ICRs) and certain retrotransposons, such as intracisternal A particles. This process takes place at approximately the time of DNA replication and ends before the first cell pision is completed. Then, the maternal genome is progressively demethylated through passive demethylation across subsequent cleavage steps ( Seisenberger et al., 2013). Consequently, the totipotency of the zygote is established and maintained across the first several cell pisions.
Figure 4. Biomarker reset. The elimination and restoration of methylation markers happen in two steps. A first, active demethylation takes place in parental gametes, right after fertilization. This process is mostly active-and therefore faster: it is completed by the first cell pision-for paternally inherited genome, while maternal pronucleus is slowly demethylated by passive diffusion across replications. This first global erasure of methylation marks spares only imprinted loci and some retrotransposons, and it is deemed to establish cellular totipotency. After the implantation of the developing blastocyst, a first de novo methylation wave begins, driving the crucial process of cellular differentiation. At the beginning of gametogenesis, when primordial germ cells start to migrate, a second demethylation takes place: gametes’ chromatin is globally demethylated, also including imprinted loci. After sex-determination, gametogonia are remethylated by a second wave of de novo methylation, which is higher (90%) and faster (it is mostly complete before birth) for male gametes and slower (40%) and lower (it does not end until puberty) for female gametes. Imprinting patterns are usually reestablished during this phase. The established patterns can be altered by direct or indirect experiences, particularly during gestation and right after birth. These processes depend on the activity of several epigenetic enzymes, among which DNA methyltransferases (DNMTs) and TETs are prominent. The regulation of these processes by non-coding RNA (ncRNA), has also been established.
The maternal factor Stella has been suggested to protect the maternal genome and paternal ICRs and intracisternal A particles from active demethylation. These regions undergo H3K9 (a Stella binding site) demethylation. Moreover, inside of the oocyte and zygote, the DNMT1o isoform pdominates and is more concentrated in their cytoplasm. In contrast, DNMT1 is the chief isoform in somatic cell nuclei but is scarce in the zygote. These differences in nuclear and cytoplasmic concentrations of DNMT1 isoforms account for global passive demethylation and might explain the maintenance of maternal ICRs ( Cardoso and Leonhardt, 1999; Seisenberger et al., 2013). Nevertheless, recent studies suggest that active and passive processes govern the demethylation of the maternal and paternal genomes ( van Otterdijk and Michels, 2021). After the implantation of the developing blastocyst, the inner mass cells (IMCs) undergo a wave of de novo methylation, which drives their differentiation. This process is mediated by DNMT3 ( Chen and Li, 2004; Seisenberger et al., 2013).
It appears that epigenetic transmission might be possible when the second demethylation step is pvented, as in the case of genomic imprinting, which constitutes the strongest evidence for transgenerational epigenetic inheritance in mammals ( van Otterdijk and Michels, 2021). Correct repssion of transcription of certain genes is crucial for a good developmental outcome. A glitch during genomic imprinting, for example, can cause severe pathologies, such as Prader-Willi and Angelman syndromes, which are derived from the loss of nonimprinted paternal and maternal genes, respectively ( Cassidy et al., 2000).
New-Generation Epigenetic Mechanisms
Non Coding RNA and Epigenetic Regulation
ncRNAs that are less than 200 nucleotides are labeled “short” or “small,” whereas those that exceed this length are defined as “long” (lncRNAs). These two groups can be subpided, depending on their genomic origin and biogenic activity.
lncRNAs are pided into five subgroups:
* natural antisense transcript (NAT), a complementary sequence to a coding RNA at the same locus (cis-NAT) or a distal genomic locus (trans-NAT).
* long intergenic ncRNA (lincRNA), which is encoded from the introns of intergenic regions (macroRNA or vlincRNA).
* sense overlapping, which is transcribed from the same DNA strand as another transcript.
* sense intronic, originating from the introns of coding genes.
* processed transcript, an RNA transcript that is spliced or polyadenylated.
Whereas NATs primarily regulate the expssion of the sense partner transcript, the activities of the other four classes remain unknown, but they are likely to include transcriptional regulation, RNA stability, and the recruitment of protein complexes and other subcellular elements. lncRNAs are usually transcribed and processed similarly to coding mRNAs ( Peschansky and Wahlestedt, 2014).
Small RNAs are grouped into five clusters: PIWI-interacting RNAs (piRNAs), endogenous short interfering RNAs (endo-siRNAs), miRNAs (or miRs), transfer-derived RNAs (tDRs or tsRNAs) and small nucleolar RNAs (snoRNAs). PiRNAs are usually composed of 26-30 nucleotides and can silence the transcription of target RNAs, promoting the trimethylation of histone 3 lysine 9 (H3K9me3), a marker of inactive chromatin, by a histone methyltransferase ( Luteijn and Ketting, 2013).
Our understanding of the processes that generate mature small ncRNAs is patchy. Only the biogenesis of miRNAs has been determined. The formation of miRNAs begins in the nucleus with the transcription of a primary miRNA (pri-miRNA) by RNA polymerase II (RNA Pol II). Pri-miRNAs are attacked by the microprocessor complex, composed of RNase III (Drosha) and DGCR8 (Pasha). Drosha cleaves pri-miRNA into a shorter transcript, whereas Pasha stabilizes the interaction between Drosha and pri-mRNA. This catalytic event produces a stem-loop structure, the pcursor miRNA (p-miRNA). The p-miRNA is then exported to the cytoplasm by Ran-GTP, which energizes the transport system, and exportin-5 (EXP5), which interacts directly with the stem-loop structure. Here, the p-miRNA associates with Dicer (another RNase III), which cleaves it into two molecules of approximately 22 nucleotides: guide strand (or mature miRNA) and passenger strand (or miRNA*). These two species are then loaded into argonaute (Ago) proteins, which select the mature miRNA (while miRNA* is degraded) and deliver it to the RNA-induced silencing complex, through which it arrives at its targets, destabilizing mRNA and inhibiting transcription ( Blahna and Hata, 2012).
ncRNAs can be epigenetic targets and epigenetic effectors. Their genetic loci can be subject to epigenetic regulation, like protein-coding genes, becoming susceptible of environmental influences; further, they govern gene expssion ( Peschansky and Wahlestedt, 2014; Szyf, 2021). This dual nature of ncRNAs implicates them as “change amplifiers.” In this sense, ncRNAs are similar to transcription factors.
Small RNAs and Epigenetic Inheritance
As discussed, fetal programming alone does not account for epigenetic transmission, unless we include the effect of pvious environmental factors (i.e., AIE) in its definition. As pointed out by Bohacek and Mansuy (2015), germ cell reprogramming could be a key mechanism of transgenerational epigenetic inheritance. Notably, miRNAs control de novo DNA methylation by regulating transcriptional repssors ( Sinkkonen et al., 2008). Epigenetic changes in germ cells arise and are maintained throughout methylation and acetylation, but miRNAs, particularly those in sperm, appear to have important functions (e.g., Bohacek and Mansuy, 2021; Rodgers et al., 2021; Fraser and Lin, 2021; Pang et al., 2021; Yeshurun and Hannan, 2021).
Conversely, global suppssion of miRNA (paired with the functional pdominance of endo-siRNAs) has been observed in mature oocytes and during early embryonic development ( Ma et al., 2010; Suh et al., 2010). Consistent with these data, oocytes lack DGCR8 (Pasha), which is necessary for miRNA but not endo-siRNA pathways ( Ma et al., 2010). miRNAs could be important mediators of placental development through their regulation of genetic expssion ( Babenko et al., 2021). Their function in the latter phases of zygote development remains unknown, but as we will discuss, there is evidence of the role of miRNAs in the regulation of oocyte function ( Tang et al., 2007; Soni et al., 2013). piRNAs are another class of small RNAs that are important in epigenetic inheritance and are highly expssed in sperm and oocytes; tsRNAs, which are enriched in mature mouse sperm, are critical in epigenetic inheritance ( Peng et al., 2012; Roovers et al., 2021; Chen Q. et al., 2021; Sharma et al., 2021). However, much work is needed to determine their functions.
Mechanisms of Epigenetic Inheritance: An Overview
NcRNAs might mediate the establishment of new patterns of gene expssion by regulating DNMT1 and TET in adult somatic cells (DE). Following fertilization, synchronous alterations to the intrauterine environment could define new expssion patterns (WIE), particularly through the activities of small ncRNAs on DNMT1, DNMT3A/B/L and TET, interfering with the maintenance of pexisting epigenetic hallmarks. Depending on when an environmental change occurs, the influence on the offspring might depend on the offspring’s sex and materialize using a sex-specific cluster of enzymes (see Figure 4).
Methodological Matters: Maternal vs. Paternal Contribution
The first studies on epigenetic forms of transmission focused on the effects of maternal care on the early stages of life, later considering nongenetic forms of developmental programming of fetal development during pgnancy. A practical problem arose, however: because mothers carry their children for 9 months and then care for them, it was difficult to distinguish between p-, peri- and postnatal epigenetic effects. Thus, several groups concluded that the paternal contribution should be considered. In many species, the only contribution of males is their sperm, which does not interfere with the gestational and postnatal periods.
This approach has been useful in demonstrating epigenetic inheritance, but it does not allow one to frame the entire landscape of mechanisms of epigenetic transmission: excluding maternal pgestational function because it is intractable for study fails to demonstrate that it does not exist or that it is irrelevant. Most of the literature has focused on the paternal role in mediating AIE (see Yeshurun and Hannan, 2021), whereas maternal function has been neglected. The drawback of many models of epigenetic inheritance is that they do not allow one to distinguish and define paternal and maternal contributions simultaneously for every effector that mediates the transmission of a certain property, such as stress reactivity. Stress vulnerability could result from the co-occurrence of maternal and paternal factors or show maternal or paternal pference, depending on the effector (e.g., which miRNA or group of miRNAs). Further, the pvalence of maternal and paternal contributions could depend on environmental conditions that could bring about, for example, paternal pvalence when the father is stressed or the pdominance of maternal contribution under baseline conditions.
Methodological Insights and Technical Niceties
Table 2. Some useful techniques that can be used to study and control for some crucial developmental variables.
Defining the Spacetime of Epigenetic Inheritance: Ideal Models
As reported above, several experiments have been conducted to demonstrate the existence of epigenetic inheritance. The results remain incomplete and sometimes conflicting, perhaps because only one route of transmission is usually considered at a time (e.g., maternal stress during pgnancy, paternal stress before mating). Moreover, the same type of event can occur in disparate moments and contexts, targeting subsequent generation through different routes.
This possibility implies that it would be better to apply several types of environmental conditions on all possible levels. For example, male and female mice could be stressed immediately prior to or long before fertilization-mildly or robustly and acutely or chronically-but also during gestation or after delivery (the latter two with regard to mothers only). It would then be interesting to study how a certain transmitted vulnerability interacts with an environmental condition that is similar to the causative factor throughout the offspring’s life. This approach is consistent with the model that, as in genetic inheritance, epigenetic inheritance can mediate the transmission of vulnerability (considered a type of epigenetic diathesis), which could remain silent and unexpssed unless-or until, depending on one’s degree of fatalism-certain environmental events take place ( Godfrey et al., 2007). Once epigenetic inheritance has been detected, the next crucial step is to determine the underlying molecular mechanisms.
The specific spacetime of an action of an epigenetic effector that is suspected to mediate transgenerational epigenetic transmission (for example, a miRNA) should be identified using the following experimental design. In a murine model, WT and manipulated (M)-i.e., KO, OE, or KD of the gene that encodes the epigenetic effector-oocytes could be fertilized with WT or M sperm in all possible combinations through IVF or natural breeding that is paired with embryo explants and implantation. The four possible types of zygotes that are produced could be implanted in WT or M dams-the latter of which allows one to control the effects of the intrauterine environment (including the placenta).
Figure 5. From in vitro fertilization (IVF) to fostering. Here, we schematize the suggested ideal model that could help define with great pcision the spacetime of a given epigenetic factor’s action. Once its role in fetal programming has been established, investigating its possible play in transgenerational epigenetic inheritance processes might be easier. See the text for more details.
Conditional models are pferred when defining the weight of a specific effector in a specific place and time (e.g., during paternal or maternal gametogenesis, zygote formation, the third week of gestation throughout the placenta, right after birth). In contrast, developmental models should allow one to observe the final, complex outcome of a certain alteration of a gene (such as polymorphisms and genes that encode epigenetic elements) in a more complex, systemic manner. The latter approach is not conducive to gaining a pcise understanding of mechanisms but still has ecological value that cannot be ignored.
Another noteworthy issue concerns whether to use IVF or natural breeding, followed by embryo extraction and implantation. IVF requires superovulation and the use of an artificial culture, which could alter the programing of gametes ( Bohacek and Mansuy, 2021). The use of natural breeding, conversely, fails to control for the effects of the manipulation of male and female reproductive fluids ( Bohacek and Mansuy, 2021), warranting further comparison with offspring that result from natural breeding.
These considerations are pivotal to correctly interpt data, despite the manipulation of a factor and the breeding procedure (e.g., conditional vs. developmental and artificial vs. natural). Moreover, the proposed model is only theoretical and does not impose its complete application, although it would likely produce the strongest evidence possible, whatever results emerge. Once the activity of a certain effector has been described, a more specific molecular analysis can be conducted to link the steps of the underlying mechanism of the specific process of epigenetic inheritance.
Conclusions and Future Perspectives
In this review article, we have introduced the concept of epigenetics, defining its spatial and temporal properties, allowing us to distinguish between types of epigenetics: a direct form of epigenetics (DE) and two forms of indirect epigenetics-within (WIE) and across (AIE). We have organized the main body of epigenetic evidence according to these three categories and focused on the latter (AIE), referring to it as a more rapid means of transmitting information across generations-compared with genetic inheritance-that guides human evolution in a Lamarckian (i.e., experience-dependent) manner. We have thus defined epigenetic inheritance in terms of AIE and illustrated the putative molecular mechanisms of this phenomenon.
Finally, we have discussed the main methodological matters regarding the study of epigenetic inheritance and have suggested strategies to solve some of the most compelling technical and theoretical problems that plague this field. The experimental models that we have proposed are inapplicable to human research, for obvious ethical reasons, but if we detail the mechanisms that underlie epigenetic inheritance, thus isolating key effectors to examine, we could study the “natural experiments” that we have (and probably will) occasionally encountered in history. There is no doubt that translational research could benefit from this scientific effort. Epigenetic inheritance, when maladaptive, can have a silent, unseen, but dramatic impact on health, perpetrating detrimental adaptations across generations.
Thus, there is no reason why a similar therapeutic approach should be overlooked for epigenetic abnormalities that affect an inpidual at early age and even during fetal development.
The environment is another level that confirms its well-established function as an epigenetic regulator and is also thus a potentially invaluable therapeutic “tool” ( Maccari et al., 2021). To strengthen the therapeutic power of the environment, paradoxically, we must understand the specific mechanisms that are altered by epigenetic adaptations following certain experiences. Yeshurun and Hannan (2018) have suggested a therapeutic/pventive approach, called “enviromimetics,” that aims to ameliorate paternal psychophysical conditions before conception to revert or pvent epigenetic alterations in sperm, thus reducing the transgenerational impact of stress.
Attaining this ideal therapeutic power will require new studies on AIE-particularly on the gap between two generations. These studies could ensure greater “spacetime resolution” of such a complex phenomenon, thus facilitating the development of a prompt and effective intervention. Although we have detailed how epigenetic factors can lead to many pathologies, we must be reminded that they are usually crucial in all of the adaptive processes that ensure the survival of the inpidual and species ( van Otterdijk and Michels, 2021). For this reason, the decision to interfere with their activity should be strongly supported by a profound understanding of the specific case in question and applied with great caution.
IL conceived the general theoretical framework, collected most of the bibliography, wrote the first draft of the article and designed and drew the ps. Both authors developed, refined and carefully reviewed the final version of the article.
This work was supported by the Research Projects of Sapienza University of Rome grants ATENEO AA 2021 (C351BDB6) and ATENEO AA 2021 (RG11715C7E0A7187).
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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