More research is needed to identify biomarkers that will allow for the diagnosis and treatment of sleep disorders. Difficulties arise when developing epigenetic treatment due to the highly-sensitive nature of the epigenome in response to the circumstances of its environment. [ 16 ]  For example, SCN promoter regions of mice underwent significant methylation changes in response to slight changes in the mice's length of day. [ 16 ]  Though this points to epigenetic alterations being an adaptive force to deal with day-to-day changes to sleep, it presents problems in treating the behavioral phenotypes of sleep disorders that rely on epigenetic regulation that is easily and quickly altered. [ 16 ]


Structural inheritance  or  cortical inheritance  is the transmission of an  epigenetic  trait in a living  organism  by a self-perpetuating spatial structures. This is in contrast to the transmission of digital information such as is found in  DNA  sequences, which accounts for the vast majority of known  genetic  variation.


Examples of structural inheritance include the propagation of  prions , the infectious proteins of diseases such as  scrapie  (in sheep and goats),  bovine spongiform encephalopathy  ('mad cow disease') and  Creutzfeldt–Jakob disease  (although the protein-only hypothesis of prion transmission has been considered contentious until recently). [ 1 ]  Prions based on heritable protein structure also exist in  yeast . [ 2 ] [ 3 ] [ 4 ]  Structural inheritance has also been seen in the orientation of  cilia  in protozoans such as  Paramecium [ 5 ]   and  Tetrahymena , [ 6 ]  and 'handedness' of the spiral of the cell in  Tetrahymena , [ 6 ]  and shells of snails. Some  organelles  also have structural inheritance, such as the  centriole , and the  cell  itself (defined by the  plasma membrane ) may also be an example of structural inheritance. To emphasize the difference of the molecular mechanism of structural inheritance from the canonical  Watson-Crick base pairing  mechanism of transmission of genetic information, the term 'epigenetic templating' was introduced. [ 7 ] [ 8 ]


Structural inheritance was discovered by  Tracy Sonneborn , and other researchers, during his study on  protozoa  in the late 1930s. Sonneborn demonstrated during his research on  Paramecium  that the structure of the cortex was not dependent on genes, or the liquid cytoplasm, but in the cortical structure of the surface of the ciliates. Preexisting cell surface structures provided a template that was passed on for many generations. [ 9 ]


John R. Preer, Jr., following up on Sonneborn's work, says, "The arrangement of surface structures is inherited, but how is not known, Macronuclei pass on many of their characteristics to new macronuclei, by an unknown and mysterious mechanism." [ 10 ]


Other researchers have come to the conclusion that "the phenomena of cortical inheritance (and related nongenic, epigenetic processes) remind us that the fundamental reproductive unit of life is not a nucleic acid molecule, but the remarkably versatile, intact, living cell." [ citation needed ]


The study of structural inheritance is part of the  extended evolutionary synthesis . [ 11 ]


An article in  Newsweek  mentions research that shows that "Some water fleas sport a spiny helmet that deters predators; others, with identical DNA sequences, have bare heads. What differs between the two is not their genes but their mothers' experiences. If mom had a run-in with predators, her offspring have helmets, an effect one wag called "bite the mother, fight the daughter." If mom lived her life unthreatened, her offspring have no helmets. Same DNA, different traits. Somehow, the experience of the mother, not only her DNA sequences, has been transmitted to her offspring." [ 12 ]


Various additional examples of structural inheritance are presented in the recent book  Origination of Organismal Form .


The  TET enzymes  are a family of ten-eleven translocation (TET)  methylcytosine  dioxygenases. They are instrumental in  DNA demethylation .  5-Methylcytosine (see first Figure) is a  methylated  form of the  DNA  base  cytosine  (C) that often regulates gene  transcription  and has several other functions in the genome. [ 1 ]


Demethylation by TET enzymes (see second Figure), can alter the regulation of transcription.  The TET enzymes catalyze the  hydroxylation  of DNA  5-methylcytosine  (5mC) to  5-hydroxymethylcytosine  (5hmC), and can further catalyse oxidation of 5hmC to  5-formylcytosine  (5fC) and then to 5-carboxycytosine (5caC). [ 2 ]   5fC and 5caC can be removed from the DNA base sequence by  base excision repair  and replaced by  cytosine  in the base sequence.


TET enzymes have central roles in  DNA demethylation  required during embryogenesis, gametogenesis,  memory, learning , addiction and  pain perception . [ 3 ]


The three related  TET  genes,  TET1 ,  TET2  and  TET3  code respectively for three related mammalian proteins TET1, TET2, and TET3.  All three proteins possess 5mC oxidase activity, but they differ in terms of domain architecture. [ 4 ]   TET proteins are large (~180- to 230-kDa) multidomain enzymes.  All TET proteins contain a conserved  double-stranded β-helix  (DSBH) domain, a cysteine-rich domain, and binding sites for the  cofactors   Fe(II)  and  2-oxoglutarate  (2-OG) that together form the core catalytic region in the  C terminus .  In addition to their  catalytic domain , full-length TET1 and TET3 proteins have an  N-terminal  CXXC  zinc finger  domain that can bind DNA. [ 5 ]   The TET2 protein lacks a CXXC domain, but the  IDAX  gene, that's a neighbor of the TET2 gene, encodes a CXXC4 protein.  IDAX is thought to play a role in regulating TET2 activity by facilitating its recruitment to unmethylated CpGs.


The three  TET  genes are expressed as different  isoforms , including at least two isoforms of TET1, three of TET2 and three of TET3. [ 2 ] [ 6 ]   Different isoforms of the  TET  genes are expressed in different cells and tissues.  The full-length canonical TET1 isoform appears virtually restricted to early embryos, embryonic stem cells and  primordial germ cells  (PGCs). The dominant TET1 isoform in most somatic tissues, at least in the mouse, arises from alternative promoter usage which gives rise to a short transcript and a truncated protein designated TET1s. The three isoforms of TET2 arise from different promoters.  They are expressed and active in  embryogenesis and differentiation of  hematopoietic cells .  The isoforms of TET3 are the full length form TET3FL, a short form splice variant TET3s, and a form that occurs in oocytes designated TET3o. TET3o is created by alternative promoter use and contains an additional first N-terminal exon coding for 11 amino acids. TET3o only occurs in oocytes and the one cell stage of the zygote and is not expressed in embryonic stem cells or in any other cell type or adult mouse tissue tested. Whereas TET1 expression can barely be detected in oocytes and zygotes, and TET2 is only moderately expressed, the TET3 variant TET3o shows extremely high levels of expression in oocytes and zygotes, but is nearly absent at the 2-cell stage.  It appears that TET3o, high in oocytes and zygotes at the one cell stage, is the major TET enzyme utilized when almost 100% rapid demethylation occurs in the paternal genome just after fertilization and before DNA replication begins (see  DNA demethylation ).


Many different proteins bind to particular TET enzymes and recruit the TETs to specific genomic locations.  In some studies, further analysis is needed to determine whether the interaction per se mediates the recruitment or instead the interacting partner helps to establish a favourable chromatin environment for TET binding.  TET1‑depleted and TET2‑depleted cells revealed distinct target preferences of these two enzymes, with TET1‑preferring promoters and TET2‑preferring gene bodies of highly expressed genes and enhancers. [ 7 ]


The three mammalian  DNA methyltransferases  (DNMTs) show a strong preference for adding a methyl group to the 5 carbon of a  cytosine  where a cytosine  nucleotide  is followed by a  guanine  nucleotide in the linear  sequence  of  bases  along its  5' → 3' direction  (at  CpG sites ). [ 8 ]   This forms a 5mCpG site.  More than 98% of DNA methylation occurs at CpG sites in mammalian  somatic cells . [ 9 ]   Thus TET enzymes largely initiate demethylation at 5mCpG sites.


Oxoguanine glycosylase  (OGG1) is one example of a protein that recruits a TET enzyme.  TET1 is able to act on 5mCpG if an  ROS  has first acted on the guanine to form   8-hydroxy-2'-deoxyguanosine  (8-OHdG or its tautomer 8-oxo-dG), resulting in a 5mCp-8-OHdG dinucleotide (see Figure). [ 10 ]   After formation of 5mCp-8-OHdG, the  base excision repair  enzyme  OGG1  binds to the 8-OHdG lesion without immediate excision (see Figure).  Adherence of OGG1 to the 5mCp-8-OHdG site recruits  TET1 , allowing TET1 to oxidize the 5mC adjacent to 8-OHdG.  This initiates the demethylation pathway.


EGR1  is another example of a protein that recruits a TET enzyme. [ 11 ]   EGR1 has an important role in learning and memory. [ 12 ] [ 13 ]   When a new event such as  fear conditioning  causes a memory to be formed,  EGR1   messenger RNA   is rapidly and selectively up-regulated in subsets of neurons in specific brain regions associated with learning and memory formation. [ 14 ]   TET1s is the predominant isoform of TET1 that is expressed in neurons. [ 15 ]    When EGR1 proteins are expressed, they appear to bring TET1s to about 600 sites in the neuron genome. [ 11 ]   Then EGR1 and TET1 appear to cooperate in  demethylating  and thereby activating the expression of genes downstream of the EGR1 binding sites in DNA. [ 11 ]


TET processivity can be viewed at three levels, the physical, chemical and genetic levels.  Physical processivity refers to the ability of a TET protein to slide along the DNA from one CpG site to another.  An in vitro study showed that DNA-bound TET does not preferentially oxidize other CpG sites on the same DNA molecule, indicating that TET is not physically processive.  Chemical processivity refers to the ability of TET to catalyze the oxidation of 5mC iteratively to 5caC without releasing its substrate.  It appears that TET can work through both chemically processive and non‑processive mechanisms depending on reaction conditions.  Genetic processivity refers to the genetic outcome of TET‑mediated oxidation in the genome, as shown by mapping of the oxidized bases.  In mouse embryonic stem cells, many genomic regions or  CpG sites  are modified so that 5mC is changed to 5hmC but not to 5fC or 5caC, whereas at many otherCpG sites 5mCs are modified to 5fC or 5caC but not 5hmC, suggesting that 5mC is processed to different states at different genomic regions or CpG sites. [ 7 ]


TET enzymes are  dioxygenases  in the family of  alpha-ketoglutarate-dependent hydroxylases .  A TET enzyme is an  alpha-ketoglutarate  (α-KG) dependent dioxygenase that catalyses an oxidation reaction by incorporating a single oxygen atom from molecular oxygen (O 2 ) into its substrate, 5-methylcytosine in DNA (5mC), to produce the product 5-hydroxymethylcytosine in DNA.  This conversion is coupled with the oxidation of the co-substrate α-KG to  succinate  and carbon dioxide (see Figure).


The first step involves the binding of α-KG and 5-methylcytosine to the TET enzyme active site.  The TET enzymes each harbor a core catalytic domain with a double-stranded β-helix fold that contains the crucial metal-binding residues found in the family of Fe(II)/α-KG- dependent oxygenases. [ 16 ]   α-KG coordinates as a  bidentate ligand  (connected at two points) to  Fe(II)  (see Figure), while the 5mC is held by  a noncovalent force  in close proximity. The TET active site contains a highly conserved triad motif, in which the catalytically-essential Fe(II) is held by two  histidine  residues and one  aspartic acid  residue (see Figure).  The triad binds to one face of the Fe center, leaving three labile sites available for binding α-KG and O 2  (see Figure).  TET then acts to convert 5-methylcytosine to 5-hydroxymethylcytosine while α-ketoglutarate is converted to succinate and CO 2 .


The TET proteins also have activities that are independent of DNA demethylation. [ 17 ]   These include, for instance, TET2 interaction with  O -linked  N -acetylglucosamine ( O-GlcNAc ) transferase to promote  histone  O-GlcN acylation to affect transcription of target genes. [ 18 ]


The mouse  sperm   genome  is 80–90%  methylated  at its  CpG sites  in DNA, amounting to about 20 million methylated sites. [ 19 ]   After  fertilization , early in the first day of  embryogenesis , the paternal chromosomes are almost completely  demethylated  in six hours by an active TET-dependent process, before DNA replication begins (blue line in Figure).


Demethylation of the maternal genome occurs by a different process.  In the mature  oocyte , about 40% of its CpG sites in DNA are methylated.  In the pre- implantation  embryo up to the  blastocyst  stage (see Figure), the only methyltransferase present is an isoform of  DNMT1  designated DNMT1o. [ 20 ]   It appears that demethylation of the maternal chromosomes largely takes place by blockage of the methylating enzyme DNMT1o from entering the nucleus except briefly at the 8 cell stage (see  DNA demethylation ).  The maternal-origin DNA thus undergoes passive demethylation by dilution of the methylated maternal DNA during replication (red line in Figure).  The  morula  (at the 16 cell stage), has only a small amount of  DNA methylation  (black line in Figure).


The newly formed primordial germ cells (PGC) in the implanted embryo devolve from the somatic cells at about day 7 of embryogenesis in the mouse.  At this point the PGCs have high levels of methylation. These cells migrate from the  epiblast  toward the  gonadal ridge . As reviewed by Messerschmidt et al., [ 21 ]  the majority of PGCs are arrested in the  G 2  phase  of the cell cycle while they migrate toward the hindgut during embryo days 7.5 to 8.5. Then demethylation of the PGCs takes place in two waves. [ 21 ]   There is both passive and active, TET-dependent demethylation of the primordial germ cells.  At day 9.5 the primordial germ cells begin to rapidly replicate going from about 200 PGCs at embryo day 9.5 to about 10,000 PGCs at day 12.5. [ 22 ]   During days 9.5 to 12.5  DNMT3a  and  DNMT3b  are repressed and DNMT1 is present in the nucleus at a high level.  But DNMT1 is unable to methylate cytosines during days 9.5 to 12.5 because the  UHRF1  gene (also known as  NP95 ) is repressed and UHRF1 is an essential protein needed to recruit DNMT1 to replication foci where maintenance DNA methylation takes place. [ 22 ]   This is a passive, dilution form of demethylation.


In addition, from embryo day 9.5 to 13.5 there is an active form of demethylation.  As indicated in the Figure of the demethylation pathway above, two enzymes are central to active demethylation.  These are a ten-eleven translocation (TET) methylcytosine dioxygenase and  thymine-DNA glycosylase  (TDG).  One particular TET enzyme, TET1, and TDG are present at high levels from embryo day 9.5 to 13.5, [ 22 ]  and are employed in active TET-dependent demethylation during gametogenesis. [ 21 ]   PGC genomes display the lowest levels of DNA methylation of any cells in the entire life cycle of the mouse by embryonic day 13.5.  [ 23 ]


Learning and memory have levels of permanence, differing from other mental processes such as thought, language, and consciousness, which are temporary in nature. Learning and memory can be either accumulated slowly (multiplication tables) or rapidly (touching a hot stove), but once attained, can be recalled into conscious use for a long time.  Rats subjected to one instance of  contextual fear conditioning  create an especially strong long-term memory. At 24 hours after training, 9.17% of the genes in the genomes of rat hippocampus neurons were found to be  differentially methylated .  This included more than 2,000 differentially methylated genes at 24 hours after training, with over 500 genes being demethylated. [ 24 ]   Similar results to that in the rat hippocampus were also obtained in mice with contextual fear conditioning. [ 25 ]


The hippocampus region of the brain is where contextual fear memories are first stored (see Figure), but this storage is transient and does not remain in the hippocampus.  In rats contextual fear conditioning is abolished when the hippocampus is subjected to hippocampectomy just one day after conditioning, but rats retain a considerable amount of contextual fear when hippocampectomy is delayed by four weeks. [ 26 ]   In mice, examined at 4 weeks after conditioning, the hippocampus methylations and demethylations were reversed (the hippocampus is needed to form memories but memories are not stored there) while substantial differential CpG methylation and demethylation occurred in  cortical  neurons during memory maintenance.  There were 1,223 differentially methylated genes in the anterior cingulate cortex (see Figure) of mice four weeks after contextual fear conditioning. Thus, while there were many methylations in the hippocampus shortly after memory was formed, all these hippocampus methylations were demethylated as soon as four weeks later.


Li et al. [ 27 ]  reported one example of the relationship between expression of a TET protein, demethylation and memory while using  extinction training .  Extinction training is the disappearance of a previously learned behavior when the behavior is not reinforced.


A comparison between  infralimbic prefrontal cortex  (ILPFC) neuron samples derived from mice trained to fear an auditory cue and extinction-trained mice revealed dramatic experience-dependent genome-wide differences in the accumulation of 5-hmC in the ILPFC in response to learning.   Extinction training led to a significant increase in TET3 messenger RNA levels within cortical neurons.  TET3 was selectively activated within the adult  neo-cortex  in an experience-dependent manner.


A  short hairpin RNA  (shRNA) is an artificial  RNA  molecule with a tight hairpin turn that can be used to silence target gene expression via  RNA interference .  Mice trained in the presence of TET3-targeted shRNA showed a significant impairment in fear extinction memory. [ 27 ]


The  nucleus accumbens  (NAc) has a significant role in  addiction .  In the nucleus accumbens of mice, repeated cocaine exposure resulted in reduced  TET1   messenger RNA  (mRNA) and reduced TET1 protein expression. Similarly, there was a ~40% decrease in  TET1  mRNA in the NAc of human cocaine addicts examined postmortem. [ 28 ]


As indicated above in learning and memory, a  short hairpin RNA  (shRNA) is an artificial  RNA  molecule with a tight hairpin turn that can be used to silence target gene expression via  RNA interference .  Feng et al. [ 28 ]  injected shRNA targeted to  TET1  in the NAc of mice.  This could reduce  TET1  expression in the same manner as reduction of  TET1  expression with cocaine exposure.  They then used an indirect measure of addiction,  conditioned place preference .  Conditioned place preference can measure the amount of time an animal spends in an area that has been associated with cocaine exposure, and this can indicate an addiction to cocaine.   Reduced  Tet1  expression caused by shRNA injected into the NAc robustly enhanced cocaine place conditioning.


As described in the article  Nociception , nociception is the  sensory nervous system's  response to harmful stimuli, such as a toxic chemical applied to a tissue. In nociception, chemical stimulation of sensory nerve cells called  nociceptors  produces a signal that travels along a chain of nerve fibers via the  spinal cord  to the  brain . Nociception triggers a variety of physiological and behavioral responses and usually results in a subjective experience, or  perception , of  pain .


Work by Pan et al. [ 3 ]  first showed that TET1 and TET3 proteins are normally present in the spinal cords of mice.  They used a pain inducing model of intra  plantar  injection of 5% formalin into the dorsal surface of the mouse hindpaw and measured time of licking of the hindpaw as a measure of induced pain.  Protein expression of TET1 and TET3 increased by 152% and 160%, respectively, by 2 hours after formalin injection.  Forced reduction of expression of TET1 or TET3 by  spinal  injection of  Tet1-siRNA  or Tet3-siRNA for three consecutive days before formalin injection alleviated the mouse perception of pain.  On the other hand, forced overexpression of TET1 or TET3 for 2 consecutive days significantly produced pain-like behavior as evidenced by a decrease in the mouse of the thermal pain threshold.


They further showed that the nociceptive pain effects occurred through TET mediated conversion of 5-methylcytosine to  5-hydroxymethylcytosine in the promoter of a  microRNA  designated  miR-365-3p , thus increasing its expression.  This microRNA, in turn, ordinarily targets (decreases expression of) the  messenger RNA  of  Kcnh2 , that codes for a protein known as K v 11.1 or KCNH2. KCNH2 is the alpha  subunit  of a  potassium ion channel  in the  central nervous system .  Forced decrease in expression of TET1 or TET3 through pre-injection of siRNA reversed the decrease of KCNH2 protein in formalin-treated mice.


Transgenerational epigenetic inheritance  is the proposed transmission of  epigenetic  markers and modifications from one generation to multiple subsequent generations without altering the  primary structure  of  DNA . [ 1 ]  Thus, the regulation of genes via epigenetic mechanisms can be heritable; the amount of transcripts and proteins produced can be altered by inherited epigenetic changes. In order for epigenetic marks to be heritable, however, they must occur in the gametes in animals, but since plants lack a definitive germline and can propagate, epigenetic marks in any tissue can be heritable. [ 2 ]


The inheritance of epigenetic marks in the immediate generation is referred to as intergenerational inheritance. [ 3 ]  In male mice, the epigenetic signal is maintained through the F1 generation. [ 4 ]  In female mice, the epigenetic signal is maintained through the F2 generation as a result of the exposure of the  germline  in the womb. [ 4 ]  Many epigenetic signals are lost beyond the F2/F3 generation and are no longer inherited, because the subsequent generations were not exposed to the same environment as the parental generations. [ 3 ]  The signals that are maintained beyond the F2/F3 generation are referred to as transgenerational epigenetic inheritance (TEI), because initial environmental stimuli resulted in inheritance of epigenetic modifications. [ 5 ]  There are several mechanisms of TEI that have shown to affect germline reprogramming, such as transgenerational increases in susceptibility to diseases, mutations, and stress inheritance. During germline reprogramming and early embryogenesis in mice, methylation marks are removed to allow for development to commence, but the methylation mark is converted into hydroxymethyl-cytosine so that it is recognized and methylated once that area of the genome is no longer being used, [ 6 ]  which serves as a memory for that TEI mark. Therefore, under lab conditions, inherited methyl marks are removed and restored to ensure TEI still occurs. However, observing TEI in wild populations is still in its infancy, as laboratory studies allow for more tractable systems. [ 7 ]


Environmental factors can induce the epigenetic marks (epigenetic tags) for some epigenetically influenced traits. [ 1 ]  These can include, but are not limited to, changes in temperature, resources availability, exposure to pollutants, chemicals, and endocrine disruptors. [ 8 ]  The dosage and exposure levels can affect the extent of the environmental factors' influence over the epigenome and its effect on later generations. The epigenetic marks can result in a wide range of effects, including minor phenotypic changes to complex diseases and disorders. [ 8 ]  The complex cell signaling pathways of multicellular organisms such as plants and humans can make understanding the mechanisms of this inherited process very difficult. [ 9 ]


There are mechanisms by which environmental exposures induce epigenetic changes by affecting regulation and gene expression. Four general categories of epigenetic modification are known.


Although there are various forms of inheriting epigenetic markers, inheritance of epigenetic markers can be summarized as the dissemination of epigenetic information by means of the germline. [ 19 ]  Furthermore, epigenetic variation typically takes one of four general forms, though there are other forms that have yet to be elucidated. Currently, self-sustaining feedback loops, spatial templating, chromatin marking, and RNA-mediated pathways modify epigenes of individual cells. Epigenetic variation within multicellular organisms is either endogenous or exogenous. [ 20 ]  Endogenous is generated by cell–cell signaling (e.g. during cell differentiation early in development), while exogenous is a cellular response to environmental cues. [ citation needed ]


In sexually reproducing organisms, much of the epigenetic modification within cells is reset during meiosis (e.g. marks at the FLC locus controlling plant vernalization [ 21 ] ), though some epigenetic responses have been shown to be conserved (e.g. transposon methylation in plants [ 21 ] ). Differential inheritance of epigenetic marks due to underlying maternal or paternal biases in removal or retention mechanisms may lead to the assignment of epigenetic causation to some parent of origin effects in animals [ 22 ]  and plants. [ 23 ]


In mammals, epigenetic marks are erased during two phases of the life cycle. Firstly just after fertilization and secondly, in the developing primordial germ cells, the precursors to future gametes. [ 24 ]  During fertilization the male and female gametes join in different cell cycle states and with different configuration of the genome. The epigenetic marks of the male are rapidly diluted. First, the protamines associated with male DNA are replaced with histones from the female's  cytoplasm , most of which are acetylated due to either higher abundance of acetylated histones in the female's cytoplasm or through preferential binding of the male DNA to acetylated histones. [ 25 ] [ 26 ]  Second, male DNA is systematically demethylated in many organisms, [ 27 ] [ 28 ]  possibly through  5-hydroxymethylcytosine . However, some epigenetic marks, particularly maternal DNA methylation, can escape this reprogramming; leading to parental imprinting. [ citation needed ]


In the primordial  germ cells  (PGC) there is a more extensive erasure of epigenetic information. However, some rare sites can also evade erasure of DNA methylation. [ 29 ]  If epigenetic marks evade erasure during both  zygotic  and PGC reprogramming events, this could enable transgenerational epigenetic inheritance. [ citation needed ]


Recognition of the importance of epigenetic programming to the establishment and fixation of cell line identity during early embryogenesis has recently stimulated interest in artificial removal of epigenetic programming. [ 30 ]  Epigenetic manipulations may allow for restoration of  totipotency  in  stem cells  or cells more generally, thus generalizing  regenerative medicine [ citation needed ] .


Cellular mechanisms may allow for co-transmission of some epigenetic marks. During replication, DNA polymerases working on the leading and lagging strands are coupled by the DNA processivity factor  proliferating cell nuclear antigen  (PCNA), which has also been implicated in patterning and strand crosstalk that allows for copy fidelity of epigenetic marks. [ 31 ] [ 32 ]  Work on histone modification copy fidelity has remained in the model phase, but early efforts suggest that modifications of new histones are patterned on those of the old histones and that new and old histones randomly assort between the two daughter DNA strands. [ 33 ]  With respect to transfer to the next generation, many marks are removed as described above. Emerging studies are finding patterns of epigenetic conservation across generations. For instance,  centromeric  satellites resist demethylation. [ 34 ]  The mechanism responsible for this conservation is not known, though some evidence suggests that methylation of histones may contribute. [ 34 ] [ 35 ]  Dysregulation of the promoter methylation timing associated with gene expression dysregulation in the embryo was also identified. [ 36 ]


Whereas the  mutation rate  in a given 100-base gene may be 10 −7  per generation, epigenes may "mutate" several times per generation or may be fixed for many generations. [ 37 ]  This raises the question: do changes in epigene frequencies constitute evolution? Rapidly decaying epigenetic effects on phenotypes (i.e. lasting less than three generations) may explain some of the residual variation in phenotypes after genotype and environment are accounted for. However, distinguishing these short-term effects from the effects of the maternal environment on early  ontogeny  remains a challenge. [ citation needed ]


The relative importance of genetic and epigenetic inheritance is subject to debate. Though hundreds of examples of epigenetic modification of phenotypes have been published, few studies have been conducted outside of the laboratory setting. Therefore, the interactions of genes with the environment cannot be inferred despite the central role of environment in natural selection. Multiple epigenetic factors can influence the state of genes and alter the epigenetic state. Due to the multivariate nature of environmental factors, it is difficult for researchers to pinpoint the exact cause of epigenetic changes outside of a laboratory setting. [ 38 ]


Studies concerning transgenerational epigenetic inheritance in plants have been reported as early as the 1950s. [ 39 ]  One of the earliest and best characterized examples of this is b1  paramutation  in maize. [ 39 ] [ 40 ] [ 41 ] [ 42 ] [ 43 ] [ 44 ] [ 45 ] [ 46 ]  The b1 gene encodes a  basic helix-loop-helix   transcription factor  that is involved in the  anthocyanin  production pathway. When the b1 gene is expressed, the plant accumulates anthocyanin within its tissues, leading to a purple coloration of those tissues. The B-I allele (for B-Intense) has high expression of b1 resulting in the dark pigmentation of the sheath and husk tissues while the B' (pronounced B-prime) allele has low expression of b1 resulting in low pigmentation in those tissues. [ 47 ]  When  homozygous  B-I parents are crossed to homozygous B', the resultant  F1 offspring  all display low pigmentation which is due to  gene silencing  of b1. [ 39 ] [ 47 ]  Unexpectedly, when F1 plants are self-crossed, the resultant F2 generation all display low pigmentation and have low levels of b1 expression. Furthermore, when any F2 plant (including those that are genetically homozygous for B-I) are crossed to homozygous B-I, the offspring will all display low pigmentation and expression of b1. [ 39 ] [ 47 ]  The lack of darkly pigmented individuals in the F2 progeny is an example of  non-Mendelian inheritance  and further research has suggested that the B-I allele is converted to B' via epigenetic mechanisms. [ 41 ] [ 42 ]  The B' and B-I alleles are considered to be  epialleles  because they are identical at the DNA sequence level but differ in the level of  DNA methylation ,  siRNA  production, and chromosomal interactions within the nucleus. [ 45 ] [ 48 ] [ 44 ] [ 43 ]  Additionally, plants defective in components of the  RNA-directed DNA-methylation pathway  show an increased expression of b1 in B' individuals similar to that of B-I, however, once these components are restored, the plant reverts to the low expression state. [ 46 ] [ 49 ] [ 50 ] [ 51 ]  Although spontaneous conversion from B-I to B' has been observed, a reversion from B' to B-I (green to purple) has never been observed over 50 years and thousands of plants in both greenhouse and field experiments. [ 52 ]


Examples of environmentally induced transgenerational epigenetic inheritance in plants has also been reported. In one case, rice plants that were exposed to drought-simulation treatments displayed increased tolerance to drought after 11 generations of exposure and propagation by single-seed descent as compared to non-drought treated plants. Differences in  drought tolerance  was linked to directional changes in DNA-methylation levels throughout the genome, suggesting that stress-induced heritable changes in DNA-methylation patterns may be important in adaptation to recurring stresses. In another study, plants that were exposed to moderate caterpillar herbivory over multiple generations displayed increased resistance to herbivory in subsequent generations (as measured by caterpillar dry mass) compared to plants lacking herbivore pressure. This increase in herbivore resistance persisted after a generation of growth without any herbivore exposure suggesting that the response was transmitted across generations. The report concluded that components of the RNA-directed DNA-methylation pathway are involved in the increased resistance across generations. Transgenerational epigenetic inheritance has also been observed in polyploid plants. Genetically identical reciprocal F1 hybrid triploids have been shown to display transgenerational epigenetic effects on viable F2 seed development. [ citation needed ]


It has been demonstrated in wild radish plants ( Raphanus raphanistrum ) that  TEI can be induced  when the plants are exposed to predators such as  Pieris rapae , the cabbage white caterpillar. The radish plants will increase production of bristly leaf hairs and toxic mustard oil in response to caterpillar predation. The increased levels will also be seen in the next generation. Decreased levels of predation also results in decreased leaf hairs and toxins produced in the current and subsequent generations. [ 53 ]


It is difficult to trace TEI in animals due to the reprogramming of genes during meiosis and embryogenesis, especially in wild populations that are not reared in a lab setting. Further studies must be conducted to strengthen the documentation of TEI in animals. However, a few examples do exist. [ citation needed ]


Induced transgenerational epigenetic inheritance has been demonstrated in animals, such as  Daphnia cucullata .  These tiny crustaceans will develop protective helmets as juveniles if exposed to  kairomones , a type of hormone, secreted by predators while they are in utero. The helmet acts as a method of defense by decreasing the ability of predators to capture the  Daphnia , thus induction of helmet presence will lower mortality rates.  D. cucullata  will develop a small helmet if no kairomones are present. However, depending upon the level of predator kairomones, the length of the helmet will almost double. The next generation of  Daphnia  will display a similar helmet size. If the kairomone levels decrease or disappear, then the third generation will revert to the original helmet size. These organisms display adaptive phenotypes that will affect the phenotype in the subsequent generations. [ 54 ]


Genetic analysis of coral reef fish,  Acanthochromis polyacanthus , has proposed TEI in response to climate change. As climate change occurs, the ocean water temperature increases. When  A. polyacanthus  is exposed to higher water temperatures of up to +3 °C from normal ocean temperatures, the fish express increased DNA methylation levels on 193 genes, resulting in phenotypic changes in the function of oxygen consumption, metabolism, insulin response, energy production, and angiogenesis. The increase in DNA methylation and its phenotypic affects were carried over to multiple subsequent generations. [ 55 ]


Possible TEI has been studied in guinea pigs ( Cavia aperea ) by exposing males to increased ambient temperature for two months. In the lab, the males were allowed to mate with the same female before and after the heat exposure to determine if the high temperatures affected the offspring. Since it serves as a thermoregulatory organ, samples of the liver were studied in the father guinea pigs (F0 generation) and liver and testes of the male offspring (F1 generation). The F0 males experienced an immediate epigenetic response to the increase in temperature; the levels of hormones in the liver responsible for thermoregulation increased. The F1 generation also displayed the different methylated epigenetic response in their liver and testes, indicating that they could potentially pass on the epigenetic marks to the F2 generation. [ 56 ]


Although genetic inheritance is important when describing  phenotypic  outcomes, it cannot entirely explain why offspring resemble their parents. Aside from genes, offspring come to inherit similar environmental conditions established by previous generations. One environment that human offspring commonly share with their maternal parent for nine months is the  womb.  Considering the duration of the  fetal stages of development , the environment of the mother's womb can have long lasting effects on the health of offspring. [ citation needed ]


An example of how the environment within the womb can affect the health of an offspring is the  Dutch hunger winter  of 1944–45 and its causal effect on induced transgenerational epigenetic inherited diseases. During the Dutch hunger winter, the offspring exposed to famine conditions during the third trimester of development were smaller than those born the year before the famine. Moreover, the offspring born during the famine and their subsequent offspring were found to have an increased risk of  metabolic diseases ,  cardiovascular diseases ,  glucose intolerance , diabetes, and obesity in adulthood. The effects of this famine on development lasted up to two generations. [ 9 ] [ 57 ]  The increased risk factors to the health of F1 and F2 generations during the Dutch hunger winter is a known phenomenon called " fetal programming ", which is caused by exposure to harmful environmental factors in utero. [ 57 ]


The loss of genetic expression which results in  Prader–Willi syndrome  or  Angelman syndrome  has in some cases been found to be caused by epigenetic changes (or "epimutations") on both the  alleles , rather than involving any genetic  mutation . In all 19 informative cases, the epimutations that, together with physiological imprinting and therefore silencing of the other  allele , were causing these syndromes were localized on a chromosome with a specific parental and grandparental origin. Specifically, the paternally derived chromosome carried an abnormal maternal mark at the  SNURF-SNRPN , and this abnormal mark was inherited from the paternal grandmother. [ 58 ]


Several cancers have been found to be influenced by transgenerational epigenetics. Epimutations on the  MLH1  gene has been found in two individuals with a phenotype of  hereditary nonpolyposis colorectal cancer , and without any frank MLH1 mutation which otherwise causes the disease. The same epimutations were also found on the spermatozoa of one of the individuals, indicating the potential to be transmitted to offspring. [ 58 ]  In addition to  epimutations  to the  MLH1  gene, it has been determined that certain cancers, such as  breast cancer , can originate during the  fetal  stages within the  uterus . [ 59 ]  Furthermore, evidence collected in various studies utilizing  model systems  (i.e. animals) have found that exposure during  parental generations  can result in multigenerational and transgenerational inheritance of breast cancer. [ 59 ]  More recently, studies have discovered a connection between the adaptation of  male germinal cells  via  pre-conception   paternal  diets and the regulation of breast cancer in developing offspring. [ 59 ]  More specifically, studies have begun to uncover new data that underscores a relationship between transgenerational epigenetic inheritance of breast cancer and  ancestral  alimentary components or associated  markers , such as birth weight. [ 59 ]  By utilizing model systems, such as mice, studies have shown that stimulated paternal obesity at the time of conception can epigenetically alter the paternal germ-line. The paternal germ-line is responsible for regulating their daughters' weight at birth and the potential for their daughter to develop breast cancer. [ 60 ]  Furthermore, it was found that modifications to the  miRNA   expression  profile of the male germline is coupled with elevated body weight. [ 60 ]  Additionally, paternal obesity resulted in an increase in the percentage of female offspring developing  carcinogen -induced  mammary tumors , which is caused by changes to mammary miRNA expression. [ 60 ]


Aside from cancer related afflictions associated with the effects of transgenerational epigenetic inheritance, transgenerational epigenetic inheritance has recently been implicated in the progression of  pulmonary arterial hypertension  (PAH). [ 61 ]  Recent studies have found that transgenerational epigenetic inheritance is likely to be involved in the progression of PAH because current therapies for PAH do not repair the irregular phenotypes associated with this disease. [ 61 ]  Current treatments for PAH have attempted to correct symptoms of PAH with  vasodilators  and  antithrombotic protectors , but neither has effectively alleviated the complications related to the impaired phenotypes associated with PAH. [ 61 ]  The inability of vasodilators and antithrombotic protectants to correct PAH suggests that the progression of PAH is dependent upon multiple variables, which is likely to be consequent of transgenerational epigenetic inheritance. [ 61 ]  Specifically, it is thought that transgenerational epigenetics is linked to the phenotypic changes associated with  vascular remodeling . [ 61 ]  For example,  hypoxia  during  gestation  may induce transgenerational epigenetic alterations that could prove to be detrimental during the early phases of  fetal development  and increase the possibility of developing PAH as an adult. [ 61 ]  Though hypoxic states could induce the transgenerational epigenetic variance associated with PAH, there is strong evidence to support that a variety of maternal risk factors are linked to the eventual progression of PAH. [ 61 ]  Such maternal risk factors linked to late-onset PAH includes placental dysfunction, hypertension, obesity, and preeclampsia. [ 61 ]  These maternal risk factors and environmental stressors coupled with transgenerational epigenetic changes can result in prolonged insult to the signaling pathways associated with the vascular development during fetal stages, thus increasing the likelihood of having PAH. [ 61 ]


One study has shown childhood abuse, which is defined as "sexual contact, severe physical abuse and/or severe neglect", leads to epigenetic modifications of glucocorticoid receptor expression. [ 62 ] [ non-primary source needed ] [ 63 ] [ non-primary source needed ]  Glucocorticoid receptor expression plays a vital role in hypothalamic-pituitary-adrenal (HPA) activity. Additionally, animal experiments have shown that epigenetic changes can depend on mother–infant interactions after birth. [ 64 ] [ non-primary source needed ]  Furthermore, a recent study investigating the correlations between  maternal stress in pregnancy  and methylation in teenagers/their mothers has found that children of women who were abused during pregnancy were more likely to have methylated glucocorticoid-receptor genes. [ 65 ] [ non-primary source needed ]  Thus, children with methylated glucocorticoid-receptor genes experience an altered response to stress, ultimately leading to a higher susceptibility of experiencing anxiety. [ 65 ] [ non-primary source needed ]


Additional studies examining the effects of  diethylstilbestrol  (DES), which is an  endocrine disruptor , have found that the grandchildren (third-generation) of women exposed to DES significantly increased the probability of their grandchildren developing  attention-deficit/hyperactivity disorder  (ADHD). [ 66 ]  This is because women exposed to  endocrine disruptors , such as DES, during  gestation  may be linked to multigenerational  neurodevelopmental  deficits. [ 66 ]  Furthermore, animal studies indicate that endocrine disruptors have a profound impact on germline cells and neurodevelopment. [ 66 ]  The cause of DES's multigenerational impact is postulated to be the result of biological processes associated with  epigenetic reprogramming  of the  germline , though this has yet to be determined. [ 66 ]


Epigenetic inheritance may only affect  fitness  if it predictably alters a trait under selection. Evidence has been forwarded that environmental stimuli are important agents in the alteration of epigenes. Ironically, Darwinian evolution may act on these neo-Lamarckian acquired characteristics as well as the cellular mechanisms producing them (e.g. methyltransferase genes).
Epigenetic inheritance may confer a fitness benefit to organisms that deal with environmental changes at intermediate timescales. [ 67 ]  Short-cycling changes are likely to have DNA-encoded regulatory processes, as the probability of the offspring needing to respond to changes multiple times during their lifespans is high. On the other end, natural selection will act on populations experiencing changes on longer-cycling environmental changes. In these cases, if  epigenetic priming  of the next generation is deleterious to fitness over most of the interval (e.g. misinformation about the environment), these genotypes and epigenotypes will be lost. For intermediate time cycles, the probability of the offspring encountering a similar environment is sufficiently high without substantial selective pressure on individuals lacking a genetic architecture capable of responding to the environment. Naturally, the absolute lengths of short, intermediate, and long environmental cycles will depend on the trait, the length of epigenetic memory, and the generation time of the organism.
Much of the interpretation of epigenetic fitness effects centers on the hypothesis that epigenes are important contributors to phenotypes, which remains to be resolved. [ citation needed ]


Inherited epigenetic marks may be important for regulating important components of fitness. In plants, for instance, the  Lcyc  gene in  Linaria vulgaris  controls the symmetry of the flower.  Linnaeus  first described radially symmetric mutants, which arise when  Lcyc  is heavily methylated. [ 68 ]  Given the importance of floral shape to pollinators, [ 69 ]  methylation of  Lcyc  homologues (e.g.  CYCLOIDEA ) may have deleterious effects on plant fitness. In animals, numerous studies have shown that inherited epigenetic marks can increase susceptibility to disease. Transgenerational epigenetic influences are also suggested to contribute to disease, especially cancer, in humans. [ 70 ]  Tumor methylation patterns in gene promoters have been shown to correlate positively with familial history of cancer. [ 71 ]  Furthermore, methylation of the  MSH2  gene is correlated with early-onset colorectal and endometrial cancers. [ 72 ]


Experimentally demethylated seeds of the  model organism   Arabidopsis thaliana  have significantly higher mortality, stunted growth, delayed flowering, and lower fruit set, [ 73 ]  indicating that epigenes may increase fitness. Furthermore, environmentally induced epigenetic responses to stress have been shown to be inherited and positively correlated with fitness. [ 74 ]  In animals, communal nesting changes mouse behavior increasing parental care regimes [ 75 ]  and social abilities [ 76 ]  that are hypothesized to increase offspring survival and access to resources (such as food and mates), respectively. [ citation needed ]


Epigenetics play a crucial role in regulation and development of the immune system. [ 77 ]  In 2021, evidence of inheritance of trained immunity across generations to progeny of mice with a systemic infection of  Candida albicans  was provided. [ 78 ]  The progeny of mice survived the  Candida albicans  infection via functional, transcriptional, and epigenetic changes linked to the immune gene loci. [ 78 ]  The responsiveness of myeloid cells to the  Candida albicans  infection increased in inflammatory pathways, and resistance was increased to infections in the next generations. [ 78 ]  Immunity in vertebrates can also be transferred from maternal through the passing of hormones, nutrients and antibodies. [ 79 ]  In mammals, the maternal factors can be transferred via lactation or the placenta. [ 79 ]  The transgenerational transmission of immune-related traits are also described in plants and invertebrates. Plants have a defense priming system which enables them to have an alternate defense response that can be accelerated upon exposure to stress actions or pathogens. [ 80 ]  After the event of priming, priming stress clue information is stored, and the memory may be inherited in the offspring (intergenerational or transgenerational). [ 80 ]  In studies, the progeny of  Pseudomonas syringae  infected  Arabidopsis  were primed during the expression of systemic acquired resistance (SAR). [ 81 ]  The progeny showed to have resistance against (hemi)-biotrophic pathogens which is associated with salicylic dependent genes and the defense regulatory gene, non expressor of PR genes (NPR1). [ 81 ]  Transgenerational SAR in the progeny was associated with increased acetylation of histone 3 at lysine 9, hypomethylation of genes, and chromatin marks on promoter regions of salicylic dependent genes. [ 81 ]  Similarly in insects, the red flour beetle  Tribolium castaneum  is primed through the exposure of the pathogen  Bacillus thuringiensis . [ 79 ]  Double-mating experiments with the red flour beetle demonstrated that paternal transgenerational immune priming is mediated by sperm or seminal fluid which enhances survival upon exposure to pathogens and contribute to epigenetic changes. [ 79 ]


Positive and negative feedback loops are commonly observed in molecular mechanisms and regulation of homeostatic processes. There is evidence that feedback loops interact to maintain epigenetic modifications within one generation, as well as contributing to TEI in various organisms, and these feedback loops can showcase putative adaptations to environmental perturbances. Feedback loops are truly a repercussion of any epigenetic modification, since it results in changes in expression. Even more so, the feedback loops seen across multiple generations because of TEI showcases a spatio-temporal dynamic that is associated with TEI alone. For example, elevated temperatures during embryogenesis and PIWI RNA (piRNA) establishment are directly proportional, providing a heritable outcome for repressing transposable elements via piRNA clusters. [ 82 ]  Furthermore, subsequent generations retain an active locus to continue establishing piRNA, which its formation was previously enigmatic. [ 82 ]  In another case, it was suggested that endocrine disruption had a feedback loop interaction with methylation of varying genomic sites in  Menidia beryllina , which may have been a function of TEI. [ 83 ]  When exposure was removed, and  M. beryllina  F2 offspring still retained these methylation marks, which caused a  negative feedback  loop on expression of various genes. [ 83 ]  In another example, hybridization of eels can lead to feedback loops contributing to transposon demethylation and transposable element activation. [ 84 ]  Because TE's are typically silenced in the genome, their presence and potential expression creates a feedback loop to prevent hybrids from reproducing with other hybrids or non-hybrid species, which eliminates the proliferation of TE expression and prevents TEI in this context. This phenomenon is known as a form of post-zygotic reproductive isolation.


Inherited epigenetic effects on phenotypes have been well documented in bacteria, protists, fungi, plants, nematodes, and fruit flies. [ 85 ] [ 19 ]  Though no systematic study of epigenetic inheritance has been conducted (most focus on model organisms), there is preliminary evidence that this mode of inheritance is more important in plants than in animals. [ 85 ]  The early differentiation of animal  germlines  is likely to preclude epigenetic marking occurring later in development, while in plants and fungi  somatic  cells may be incorporated into the germ line. [ 86 ] [ 87 ]


It is thought that transgenerational epigenetic inheritance can enable certain populations to readily adapt to variable environments. [ 19 ]  Though there are well documented cases of transgenerational epigenetic inheritance in certain populations, there are questions to whether this same form of adaptability is applicable to mammals. [ 19 ]  More specifically, it is questioned if it applies to humans. [ 19 ]  As of late, most of the experimental models utilizing mice and limited observations in humans have only found epigenetically inherited traits that are detrimental to the health of both organisms. [ 19 ]  These harmful traits range from increased risk of disease, such as  cardiovascular disease , to premature death. [ 19 ]  However, this may be based on the premise of limited reporting bias because it is easier to detect negative experimental effects, opposed to positive experimental effects. [ 19 ]  Furthermore, considerable  epigenetic reprogramming  necessary for the  evolutionary success of germlines  and the initial phases of  embryogenesis  in mammals may be the potential cause limiting transgenerational inheritance of chromatin marks in mammals. [ 19 ]


Life history  patterns may also contribute to the occurrence of epigenetic inheritance.  Sessile  organisms, those with low dispersal capability, and those with simple behavior may benefit most from conveying information to their offspring via epigenetic pathways. Geographic patterns may also emerge, where highly variable and highly conserved environments might host fewer species with important epigenetic inheritance. [ citation needed ]


Humans have long recognized that traits of the parents are often seen in offspring. This insight led to the practical application of selective breeding of plants and animals, but did not address the central question of inheritance: how are these traits conserved between generations, and what causes variation? Several positions have been held in the history of evolutionary thought. [ citation needed ]


Addressing these related questions, scientists during the time of the Enlightenment largely argued for the blending hypothesis, in which parental traits were homogenized in the offspring much like buckets of different colored paint being mixed together. [ 88 ]  Critics of Charles Darwin's  On the Origin of Species , pointed out that under this scheme of inheritance, variation would quickly be swamped by the majority phenotype. [ 89 ]  In the paint bucket analogy, this would be seen by mixing two colors together and then mixing the resulting color with only one of the parent colors 20 times; the rare variant color would quickly fade. [ citation needed ]


Unknown to most of the European scientific community, the monk  Gregor Mendel  had resolved the question of  how traits are conserved between generations  through breeding experiments with pea plants. [ 90 ]   Charles Darwin  thus did not know of Mendel's proposed "particulate inheritance" in which traits were not blended but passed to offspring in discrete units that we now call genes. Darwin came to reject the blending hypothesis even though his ideas and Mendel's were not unified until the 1930s, a period referred to as the  modern synthesis . [ citation needed ]


In his 1809 book,  Philosophie Zoologique , [ 91 ]   Jean-Baptiste Lamarck  recognized that each species experiences a unique set of challenges due to its form and environment. Thus, he proposed that the characters used most often would accumulate a "nervous fluid". Such acquired accumulations would then be transmitted to the individual's offspring. In modern terms, a nervous fluid transmitted to offspring would be a form of epigenetic inheritance. [ citation needed ]


Lamarckism , as this body of thought became known, was the standard explanation for change in species over time when Charles Darwin and  Alfred Russel Wallace  co-proposed a theory of evolution by natural selection in 1859. Responding to Darwin and Wallace's theory, a revised  neo-Lamarckism  attracted a small following of biologists, [ 92 ]  though the Lamarckian zeal was quenched in large part due to  Weismann's [ 93 ]  famous experiment in which he cut off the tails of mice over several successive generations without having any effect on tail length. Thus the emergent consensus that acquired characteristics could not be inherited became canon. [ 24 ]


Non-genetic variation and inheritance, however, proved to be quite common. Concurrent with the 20th-century development of the  modern evolutionary synthesis  (unifying Mendelian genetics and natural selection),  C. H. Waddington  (1905–1975) was working to unify  developmental biology  and genetics. In so doing, he adopted the word "epigenetic" [ 94 ]  to represent the ordered differentiation of embryonic cells into functionally distinct cell types despite having identical primary structure of their DNA. [ 95 ]  Researchers discussed Waddington's epigenetics sporadically - it became more of a catch-all for puzzling non-genetic heritable characters rather than a concept advancing the body of inquiry. [ 96 ] [ 97 ]  Consequently, the definition of Waddington's word has itself evolved, broadening beyond the subset of developmentally signaled, inherited cell specialization.


Some scientists have questioned whether epigenetic inheritance compromises the foundation of the modern synthesis. Outlining the  central dogma of molecular biology ,  Francis Crick [ 98 ]  succinctly stated, "DNA is held in a configuration by histone[s] so that it can act as a passive template for the simultaneous synthesis of RNA and protein[s].  None  of the detailed 'information' is in the histone." However, he closes the article stating, "this scheme  explains the majority  of the present experimental results!" Indeed, the emergence of epigenetic inheritance (in addition to advances in the study of evolutionary-development,  phenotypic plasticity , evolvability, and systems biology) has strained the current framework of the modern evolutionary synthesis, and prompted the re-examination of previously dismissed evolutionary mechanisms. [ 99 ]


Furthermore, patterns in epigenetic inheritance and the evolutionary implications of the  epigenetic codes  in living organisms are connected to both  Lamarck's  and  Darwin's  theories of evolution. [ 100 ]  For example, Lamarck postulated that environmental factors were responsible for modifying phenotypes  hereditarily , which supports the constructs that exposure to environmental factors during critical stages of development can result in  epimutations  in  germlines , thus augmenting phenotypic variance. [ 100 ]  In contrast, Darwin's theory claimed that natural selection strengthened a populations ability to survive and remain  reproductively fit  by favoring populations that are able to readily adapt. [ 100 ]  This theory is consistent with intergenerational plasticity and phenotypic variance resulting from heritable adaptivity. [ 100 ]


In addition, some epigenetic variability may provide beneficial  plasticity , so that certain organisms can adapt to fluctuating environmental conditions. However, the exchange of epigenetic information between generations can result in epigenetic aberrations, which are epigenetic traits that deviate from the norm. Therefore, the offspring of the  parental generations  may be predisposed to specific diseases and reduced plasticity due to epigenetic aberrations. Though the ability to readily adapt when faced with a new environment may be beneficial to certain populations of species that can quickly reproduce, species with long generational gaps may not benefit from such an ability. If a species with a longer generational gap does not appropriately adapt to the anticipated environment, then the  reproductive fitness  of the offspring of that species will be diminished. [ citation needed ]


There has been critical discussion of mainstream evolutionary theory by  Edward J Steele , Robyn A Lindley and colleagues, [ 101 ] [ 102 ] [ 103 ] [ 104 ] [ 105 ]   Fred Hoyle  and  N. Chandra Wickramasinghe , [ 106 ] [ 107 ] [ 108 ]  Yongsheng Liu [ 109 ] [ 110 ]   Denis Noble , [ 111 ] [ 112 ]  John Mattick [ 113 ]  and others that the logical inconsistencies as well as Lamarckian Inheritance effects involving direct DNA modifications, as well as the just described indirect, viz. epigenetic, transmissions, challenge conventional thinking in evolutionary biology and adjacent fields. [ citation needed ]


Transgenerational epigenetic inheritance in plants  involves mechanisms for  the passing of epigenetic marks from parent to offspring  that differ from those reported in animals. [ 1 ]  There are several kinds of epigenetic markers, but they all provide a mechanism to facilitate greater  phenotypic plasticity  by influencing the expression of genes without altering the DNA code. These modifications represent responses to environmental input and are reversible changes to gene expression patterns that can be passed down through generations. In plants, transgenerational epigenetic inheritance could potentially represent an evolutionary adaptation for sessile organisms to quickly adapt to their changing environment.


In eukaryotes, negatively-charged DNA is wrapped extremely tightly around positively-charged proteins, called histones, to form chromatin. [ 2 ]  Since the DNA is wrapped so tightly, it is inaccessible to transcription enzymes that function to copy the DNA into RNA (See:  Central dogma of molecular biology ). The inaccessible DNA must be unwound to be transcribed into RNA. The mechanism by which the DNA can be unwound is called chromatin remodeling. Chromatin remodeling is one apparatus through which epigenetics acts.


There are three mechanisms of chromatin remodeling that are well-supported by research:


DNA methylation refers to the addition of a methyl group (CH 3 ) to a cytosine (C)  nucleotide  in DNA. [ 2 ]  In plants, methylation occurs in every cytosine sequence context (CG, CHG and CHH where H represents either A, T, or C). [ 3 ]  The addition of a methyl group to a C nucleotide manipulates DNA expression patterns by changing the availability of genes for transcription. DNA methylation has also been shown to prevent disruption in the DNA sequence by repressing  transposable elements .


DNA methylation patterns in plants are more complex than in animals and these patterns must be maintained to ensure their successful transfer to progeny. [ 3 ]  There are three pathways for maintenance of DNA methylation patterns in plants: the maintenance of CG by the enzyme MET1 (DNA Methyltransferase 1), the CMT3/SUVH pathway, and the RNA-dependent DNA methylation pathway (RdDM).


The first is the maintenance of CG by MET1. In plants, CG methylation is the most common context in which DNA methylation appears, and it is faced with the most scrutiny during maintenance. [ 3 ]  After DNA replication, the previously methylated CG sites become hemi-methylated – the term given to the asymmetric status of a newly-synthesized strand of DNA in which the parent strand is methylated and the daughter strand is not. [ 3 ] [ 4 ]  MET1 is recruited to the site of hemi-methylated CG and, with the help of enzymes, copies the methylation of the parent strand onto the daughter strand. [ 3 ]


The second pathway – called the CMT3/SUVH pathway – methylates CHG contexts using the enzyme CMT3 (Chromomethylase 3) in conjunction with the histone H3K9me which is maintained by enzymes called SUVH histone methyltransferases (Mtases). [ 3 ] [ 5 ]  The SUVH enzymes contain a binding domain for methylated cytosines and CMT3 has a binding domain for H3K9me. [ 5 ]  The existence of these binding domains suggests that among DNA methylation and H3K9me, a self-reinforcing loop – a metabolic cycle – maintains the repression of transposable elements and repeats. [ 3 ] [ 5 ]


The third pathway for maintenance of DNA methylation patterns in plants is a pathway called RNA-dependent DNA Methylation (RdDM). [ 3 ]  The enzyme CMT2 maintains cytosine methylation in CHH sequence contexts. The methylation of CHH is asymmetric, meaning that the normal degradation of methylation that occurs during DNA replication will be maintained and the methylation will be lost in one daughter strand.


Although these three pathways are distinct, there is cross-talk among them. [ 3 ]  Some non-CG contexts require MET1 and some CHH contexts require the CMT3/SUVH pathway.


Demethylation is the removal of methyl groups from DNA. Plants are unique in their use of active DNA demethylation. [ 6 ]  They use the Base Excision Repair (BER) pathway to remove methylated cytosines and replace them with unmodified ones. Glycosylases catalyze the removal of the single methylated cytosines and DNA repair enzymes clean the ends and add new cytosines.


Histone modification refers to the addition or subtraction of certain chemical groups from the  amino acids  of histones that change transcription activity by initiating a change in the structure of the chromatin or by recruiting transcription enzymes. [ 2 ] [ 7 ]  Mediation of chromatin occurs most frequently through  post-translational modifications  like methylation, acetylation, ubiquitination, and phosphorylation, which are the main histone modifications occurring in plants. [ 7 ] [ 8 ]   Usually, the addition of a histone modification increases transcription by neutralizing the positive charge of the histones and, thereby, unwinding the negatively-charged DNA. [ 9 ]  Histone writers, readers, and erasers are the regulatory machinery that recognize and modify the histone modifications. [ 8 ]  Writers add, erasers remove, and readers recognize the post-translational modifications of histones. Each writer, reader and eraser is further subdivided based on the type of post-translational modification they act on or recognize.


Chromatin-remodeling complexes are protein complexes that change the composition of and interactions among  nucleosomes , thus changing chromatin structure and DNA expression patterns. [ 10 ]  These ATP-dependent protein complexes are divided into four subfamilies: the SWI/SNF Subfamily, the Imitation Switch (ISWI) Subfamily, the Chromodomain Helicase DNA-Binding (CHD) Subfamily, and the Inositol Requiring 80 (INO80) Subfamily. SWI/SNF chromatin remodelers work to promote transcription by ejecting nucleosomes at specific positions on the chromosome but they are thought to have a minimal role in eukaryotic chromatin structure. ISWI Subfamily remodelers inhibit transcription by regulating the spacing of nucleosomes. CDH Subfamily remodelers are very diverse and vary in their structure and function. Some CDH subfamily remodelers repress transcription while others promote it. Lastly, INO80 Subfamily remodelers are highly conserved meaning that they have similar structures and functions across many species. Not only do they function to promote transcription, INO80s also play a role in DNA double-strand break repair.


Plants are immobile, so they are under especially strong pressure to adapt quickly and effectively to their environment. [ 1 ]  Seed dormancy and germination is an example of a plant function that is heavily mediated by epigenetics – by histone modification. [ 7 ]  When histones are deacetylated, the transcription of photosynthesis genes is ‘turned off’ and germination is arrested. Stressful events like drought, exposure to UV radiation, cold, and pathogens have been seen to trigger a heritable increase in the frequency of  recombination . [ 11 ] [ 12 ] [ 13 ] [ 14 ] [ 1 ]  The increase in recombination frequency allows for an increase in genetic variation. The more variation within a population, the greater the possibility that a well-adapted  genotype  will arise for selection to act on during stress-inducing environmental conditions. [ 1 ]


DNA methylation is a refined regulator of gene expression; controlling which genes to express and how much to express them. [ 15 ]  Consequently,  phenotypes  induced by methylation are continuous, rather than being discrete. For example, crosses of wildtype and methylated mutant  Linaria vulgaris  plants resulted in offspring that varied in their methylation of the gene controlling floral symmetry:  Lcyc . [ 16 ]  By proxy, their expression of  Lcyc  was varied and their floral symmetry phenotypes were a continuous distribution ranging from radially symmetrical to bilaterally symmetrical and anywhere in between. Cubas et al.’s (1999) experiment characterized the first natural morphological mutant. This mutation of the  Lcyc  gene in  Linaria vulgaris  causes the flower to become radially symmetrical as opposed to bilaterally symmetrical. The authors found that, in the mutant,  Lcyc  is highly methylated which prohibits the transcription of the gene; effectively silencing it. Consequently, in wildtypes, growth of the dorsal stamen (the male organ of the flower) is arrested early-on in its development but, in mutants, the development of the dorsal stamen persists. Additionally, the dorsal petal develops similarly to neighboring petals in mutants, but in wildtypes, the dorsal petal is unique. The naturally-occurring methylation of  Lcyc  was found to be heritable and reversible.


An influential experiment by Jacobsen and Meyerowitz (1997) characterized seven epigenetic alleles (epialleles) of the SUPERMAN (SUP) gene in  Arabidopsis thaliana . [ 17 ]  These seven epialleles are referred to as the clark kent alleles (clk 1-7) and they are associated with hypermethylation in SUP. The SUP gene controls flower development in  Arabidopsis  and the presence of a clk allele is correlated with an increase in the number of stamens and carpels (the female organ of the flower) in the first ten flowers of each plant. Additionally, the presence of clk alleles is associated with a reduction in SUP RNA expression which, in wild type plants, occurs within developing stamens during the establishment of  floral meristems .


These examples of epigenetics in nature demonstrate the ability of epialleles to drastically influence phenotypes and increase genetic variability within populations. The stable heritability of epigenetic marks across generations suggests that resulting phenotypes would have substantial fitness effects and may respond, as DNA sequence variation does, to natural selection. [ 15 ]  The changes that epialleles induce can be quickly turned on and just as quickly degraded resulting in phenotypic plasticity. The heritability of epigenetic marks can serve as a generational memory to better prepare progeny for local environmental conditions.


Transvection  is an  epigenetic  phenomenon that results from an interaction between an  allele  on one  chromosome  and the corresponding allele on the  homologous chromosome . Transvection can lead to either  gene  activation or repression. [ 1 ]  It can also occur between nonallelic regions of the genome as well as regions of the genome that are not transcribed.


The first observation of  mitotic  (i.e. non- meiotic ) chromosome pairing was discovered via microscopy in 1908 by  Nettie Stevens . [ 2 ]   Edward B. Lewis  at  Caltech  discovered transvection at the  bithorax  complex in  Drosophila  in the 1950s. [ 3 ]  Since then, transvection has been observed at a number of additional loci in  Drosophila , including the genes known as  white ,  decapentaplegic ,  eyes absent ,  vestigial , and  yellow . [ 4 ] [ 5 ]
 [ 6 ] [ 7 ] [ 8 ]  As defined by Lewis, "Operationally, transvection is occurring if the  phenotype  of a given  genotype  can be altered solely by disruption of somatic (or meiotic) pairing. Such disruption can generally be accomplished by introduction of a heterozygous rearrangement that disrupts pairing in the relevant region but has no position effect of its own on the phenotype" (cited by  Ting Wu  and Jim Morris, 1999 [ 9 ] ). Recently, pairing-mediated phenomena have been observed in species other than  Drosophila , including mice, humans, plants, nematodes, insects, and fungi. In light of these findings, transvection may represent a potent and widespread form of  gene regulation . [ 10 ] [ 11 ]


Transvection appears to be dependent upon chromosome pairing. In some cases, if one allele is placed on a different chromosome by a  translocation , transvection does not occur. Transvection can sometimes be restored in a translocation homozygote, where both alleles may once again be able to pair. Restoration of phenotype has been observed at  bithorax ,  decapentaplegic ,  eyes absent , and  vestigial , and with transgenes of  white . In some cases, transvection between two alleles leads to intragenic  complementation  while disruption of transvection disrupts the complementation.


Transvection is believed to occur through a variety of mechanisms. In one mechanism, the  enhancers  of one allele activate the  promoter  of a paired second allele. Other mechanisms include pairing-sensitive silencing and enhancer bypass of a  chromatin insulator  through pairing-mediated changes in gene structure. [ 12 ] [ 13 ]


The  physiological relevance  of transvection has recently been documented in the context of sex-biased gene expression. In  Drosophila , transvection acts on the female X-linked gene  yellow , which is homozygous in females (XX) versus  hemizygous  in males (XY). [ 14 ]


X chromosome reactivation (XCR)  is the process by which the inactive X chromosome (the Xi) is re-activated in the cells of  eutherian   female   mammals .  Therian  female mammalian cells have two X chromosomes, while males have only one, requiring  X-chromosome inactivation  (XCI) for  sex-chromosome dosage compensation . In eutherians, XCI is the random inactivation of one of the X chromosomes, silencing its expression. Much of the scientific knowledge currently known about XCR comes from research limited to mouse models or stem cells.


Partial XCR may derepress one or more genes on the Xi, and the level of restored gene expression may not be as high as it would normally be on the active X chromosome (the Xa). Complete XCR restores the Xi to Xa and erases the  epigenetic  memory of XCI, meaning that inducing X-inactivation again will randomly select an X chromosome to silence, rather than deterministically silencing the original Xi. [ 1 ]


XCR is an emerging topic of interest for multiple reasons: [ 1 ]


In 1959  Susumu Ohno  showed that the two X chromosomes of mammals were different: one appeared similar to the  autosomes ; the other was condensed and heterochromatic. [ 3 ]  This finding suggested, independently to two groups of investigators, that one of the X chromosomes underwent inactivation.


Early in the study of XCI in  early embryonic development , the original state of the two X chromosomes and the mechanism by which they differentiated into the Xa and Xi was unknown. One hypothesis was that the chromosomes were inactive until one was activated. The other, favored hypothesis that was eventually validated was that both maternal and paternal X chromosomes are active until some mechanism inactivates one chromosome. [ 7 ]


As early as 1981, the concept of X reactivation appears in the literature, with a paper on induced XCR in somatic cell hybrid clones by 5- azacitidine  treatment, [ 8 ]  and a paper on XCR in mouse oocytes. [ 9 ]


Experiments originally showed that eliminating the X-inactive specific transcript ( Xist ) gene, a gene that encodes a lncRNA that mediates XCI, in post-XCI  somatic cells  did not result in detectable Xi reactivation. Therefore, it was believed that XCI, once established in somatic cells, was irreversible and that Xist, while required to initiate XCI, was not required to sustain it. [ 10 ]  However, newer investigations using modern technologies have found evidence that Xist RNA is involved in the maintenance of XCI. For example, Zhang, et al. found that “Deleting Xist results in a loss of  nucleolar  association and an inability to maintain Xi  heterochromatin , leading to Xi reactivation at the single gene level.” [ 11 ]


Unlike XCI, XCR lacks an appropriate  in vitro  system for study, limiting current research to mouse embryos and  in vitro  stem cell reprogramming. [ 12 ]


In mice, Xi reactivation is known to occur in three developmental stages: [ 13 ]


It is known that in the inner cell mass (the embryo proper) of mice, XCI occurs randomly, while in the  extra-embryonic tissues , imprinted XCI deterministically inactivates the paternal X. In early mouse embryos, the paternal X chromosome is already partially silenced at the  zygote  stage by imprinted XCI, suggesting that sex-chromosome dosage compensation exists from conception. Following re-activation of the paternal X in the ICM, complete and random XCI occurs post-implantation. [ 14 ] [ 15 ]


Regarding female mouse germ cells, mature mammalian oocytes were observed to have two active X chromosomes by studies that recorded twice the expression levels from the oocytes from XX female mice as opposed to XO mice in the 1960s. [ 16 ]  To determine whether XCI never occurred in these cells, or if the Xi was reactivated post-XCI, Kratzer and Chapman found that  oogonia  have an Xi and an Xa, but the Xi is gradually reactivated shortly before entering the meiotic cycle on the eleventh day. [ 9 ]


In human development, X chromosome reactivation also occurs in PGCs. [ 17 ]  In the ICM of human embryos, however, X reactivation does not occur, because humans do not have imprinted XCI, so XCI has yet to happen in this stage of development. [ 18 ]


Because only one X chromosome is expressed, genetic mutations that lead to cancer are  dominant  when they occur on the Xa, and silent on the Xi. Increased dosage of X-linked genes, by whole or partial replication of the Xa or by whole or partial loss of XCI, is linked to  oncogenesis . Translocations between the Xi and  autosomal chromosomes  can result in the silencing of autosomal tumor suppressors or reactivation of X-linked oncogenes. X-linked genes may escape inactivation, which potential oncogenic consequences. Impaired XCI and  skewed X-inactivation  may also be associated with oncogenesis. [ 19 ]


Gain of an X chromosome, [ 20 ]   Klinefelter syndrome , [ 21 ]  and  XX male syndrome [ 22 ]  are risk factors linked to  male breast cancer . In females, there is no evidence that  trisomy X  or  tetrasomy X  females are at higher risk of cancer. [ 19 ]


Breast cancer  and  ovarian cancer , particularly more aggressive strains, commonly lack an Xi and have two Xa's. [ 23 ]  Breast cancer cells commonly have two identical Xa's, possibly from duplication of the original Xa and loss of the original Xi. [ 24 ]  Gains of X chromosomes have been observed in many other cancers, including  leukemia , [ 25 ]   prostate cancer , [ 26 ] [ 27 ]  and intracranial germ cell cancers. [ 28 ]  Potential mechanisms leading to this overexpression of X-linked genes include  chromosome segregation  errors, defects in general heterochromatin maintenance, and defects in Xi-specific silencing factors, the latter two of which would partially reactivate the Xi. [ 23 ]


The presence of two Xa's is a measure of the  pluripotency , or ability to differentiate into many different cell types, of  embryonic stem cells . [ 29 ]   Cell hybrid  experiments fusing somatic cells and stem cells in vitro resulted in reactivation of the entire Xi. [ 30 ] [ 31 ] [ 32 ] [ 33 ]   Reprogramming  of mouse and human  fibroblast  cells to  induced pluripotent stem cells  (iPSCs) reactivates the Xi found in the original fibroblasts. If re-differentiated and XCI occurs again, the selection of the Xi is not randomized, i.e. the X that was originally the Xi in the starting fibroblast will be deterministically inactivated again, evidencing the incompleteness of X reactivation. [ 34 ]  Reprogramming can be accomplished by nuclear cloning, cell fusion with pluripotent cells, or expression of pluripotency factors. [ 13 ]  Factors implicated in XCI maintenance include  origin recognition complex 2  ( Orc2 ),  heterochromatin protein 1  (HP1a), [ 35 ]   macroH2A1 , [ 36 ]  and  Bmil. [ 13 ]  Disruption of  Orc2  or HP1a function have both been shown to lead to partial Xi reactivation. [ 35 ]


The mechanisms of Xi reactivation remains unknown, including whether they share a common pathway or if different contexts leverage completely different pathways. [ 13 ]  For XCR to be repressed, silencing of  Xist  expression is required but not sufficient. [ 37 ]  The erasure of chromatin modifications that maintain the heterochromatin of the Xi significantly contributes to XCR.  H3K27me3 , macroH2A, and their associated mechanisms of  DNA methylation  and  histone   deacetylation , act as a barriers to XCR in the ICM. [ 18 ]  Deleting Xist RNA, demethylating DNA, and inhibiting histone hypoacetylation together increases reactivation in primary mouse embryonic fibroblasts on the gene level in a stochastic manner. [ 38 ] [ 39 ]


X reactivation can be induced and tolerated in mouse extra-embryonic tissues. [ 13 ]  For example, a mutation of the  Polycomb protein EED  leads to a lack of  Xist  RNA coating on the Xi and X reactivation in differentiated  trophoblast  stem cells. [ 40 ]


Xist  repression in XCR is induced by  Tsix  RNA. [ 41 ]   Tsix  deletion appears to decrease the efficiency of XCR in the ICM  in vivo,  but not in iPSC reprogramming. [ 42 ]


Studies have elucidated some of the progression of XCR in the reversal of imprinted XCI in the mouse ICM, [ 43 ]  of random XCI in iPSC reprogramming, [ 44 ]  and in germ cell development. [ 45 ]  XCR proceeds gradually, with sets of genes regaining expression at the early, mid, or late stage of XCR. At the initiation of X reactivation, early genes are transcriptionally activated and  Xist  repression starts. The reactivation of these early genes happens before complete  Xist  silencing, suggesting the latter is not necessary for the reactivation of some genes. [ 44 ]  As XCR progresses, intermediate and late genes are transcriptionally activated,  Xist  RNA is lost, H3K27me3 is lost, and  Tsix  is reactivated. During this process, in genome topology there may possibly be acquisition of  topologically associating domains  (TADs), which are units of genome organization in 3D space, and loss of mega-domains. At completion, there is complete transcriptional reactivation of genes on the Xi, loss of epigenetic memory, DNA hypomethylation, fully established TADs, and if XCI is re-instated, the choice of Xi will be random. [ 1 ]


The human X chromosome contains a disproportionate number of genes associated with  intellectual disability . [ 46 ]  X-linked disorders in heterozygous female patients are particularly relevant here because these patients carry an X chromosome with the mutated disorder allele and an X chromosome with a healthy wild-type allele. [ 47 ]  XCI randomly selects one X chromosome as the Xi, leaving the genes on the other X, the Xa, to be expressed. [ 48 ]  Therefore, heterozygous female patients are a  mosaic  of healthy and diseased cells. In non-cell-autonomous X-linked disorders, such as  hemophilia A , [ 49 ]  the healthy cells can compensate for the diseased cells. In other disorders such as  Kabuki syndrome , the mutation affects the cell such that cells carrying the healthy allele are more common (a skewed XCI pattern), reducing the severity of the disease. [ 50 ]  However, in X-linked disorders where the healthy cells are insufficient to restore wild-type function, X chromosome reactivation may be a potential therapy. By reactivating the Xi in diseased cells, the previously unexpressed wild-type allele can regain some level of expression and restore function. Disorders that could hypothetically be treated by X reactivation include Rett syndrome, [ 51 ] [ 52 ]   CDKL5 deficiency disorder , [ 53 ]   Fragile X syndrome , etc. [ 54 ] [ 55 ] [ 56 ]


XCI serves a central function in sex-chromosome dosage compensation. Xi reactivation currently doesn't target specific genes, but rather targets the entire chromosome, running the risk of overexpression of other X-linked genes and subsequent side effects. Treatments also may not be able to specifically target the Xi chromosome, instead causing genome-wide disruption of epigenetic patterns. [ 13 ]


XCI is essential for early embryonic development. For example, female mouse embryos that inherited a paternal germline Xist deletion had trophoblast cells where both X chromosomes were fully expressed, resulting in a lethal phenotype. [ 57 ]  Pre-XCI Xist deletion in mouse zygotes can be tolerated to the extent that the embryos can be carried to term, but none survive past weaning as a result of issues with organ development. [ 58 ]  However, these concerns may be mitigated by the fact that applications of X reactivation are more focused on post-XCI cells, past the early developmental stages.


Xi loss or Xi reactivation is associated with certain cancers. Xist deletion in the blood compartment of mice has been shown to cause  hematologic cancers , suggesting an important role for Xist RNA and for XCI in cancer suppression. [ 59 ]  In addition, while Xist deletion can be tolerated in the epithelial cells and the gut, the mouse becomes more susceptible to stress-induced gut cancer, suggesting Xist and XCI are protective against chronic stress. [ 60 ]  Xist deletion in mouse brains have seen no negative effects in some studies. [ 61 ]  The variation among organs and cell-types for tolerance of partial X reactivation currently appears to recommend a local approach targeting individual organs for X reactivation. For example, in  neurodevelopmental disorders , X reactivation may be targeted to the brain and central nervous system. [ 62 ]


In developing a treatment to reactivate the Xi, screens to identify single factors that inhibit XCI have listed many potential pharmacological targets to consider for further study. [ 63 ] [ 64 ]  However, XCI is complex and maintained by a diverse set of proteins [ 35 ]  and multiple mechanisms, such as DNA methylation, histone hypoacetylation, and the role of Xist RNA. [ 38 ]  More effective X reactivation may require a synergistic combination of factors, and studies have considered combinations of  decitabine  with  Aurora kinase inhibitors , [ 65 ]  Xist  antisense oligonucleotides  (ASOs), [ 66 ]  and DNA  TOP2A / TOP2B  knockdown. [ 67 ]  Some experimental synergistic combinations of drugs have outperformed any single factor for levels of X reactivation. [ 62 ]


X-inactivation  (also called  Lyonization , after English geneticist  Mary Lyon ) is a process by which one of the copies of the  X chromosome  is inactivated in  therian  female  mammals . The inactive X chromosome is silenced by being packaged into a transcriptionally inactive structure called  heterochromatin . As nearly all female mammals have two X chromosomes, X-inactivation prevents them from having twice as many X chromosome  gene products  as males, who only possess a single copy of the X chromosome (see  dosage compensation ).


The choice of which X chromosome will be inactivated in a particular embryonic cell is random in  placental mammals  such as humans, but once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell and its descendants in the organism (its cell line). The result is that the choice of inactivated X chromosome in all the cells of the organism is a random distribution, often with about half the cells having the paternal X chromosome inactivated and half with an inactivated maternal X chromosome; but commonly, X-inactivation is unevenly distributed across the cell lines within one organism ( skewed X-inactivation ).


Unlike the random X-inactivation in placental mammals, inactivation in  marsupials  applies exclusively to the paternally-derived X chromosome.


The paragraphs below have to do only with rodents and do not reflect XI in the majority of mammals.
X-inactivation is part of the activation cycle of the X chromosome throughout the female life. The egg and the fertilized zygote initially use maternal transcripts, and the whole embryonic genome is silenced until  zygotic genome activation . Thereafter, all mouse cells undergo an early,  imprinted  inactivation of the paternally-derived X chromosome in  4–8 cell stage   embryos . [ 3 ] [ 4 ] [ 5 ] [ 6 ]  The  extraembryonic tissues  (which give rise to the  placenta  and other tissues supporting the embryo) retain this early imprinted inactivation, and thus only the maternal X chromosome is active in these tissues.


In the early  blastocyst , this initial, imprinted X-inactivation is  reversed  in the cells of the  inner cell mass  (which give rise to the embryo), and in these cells both X chromosomes become active again. Each of these cells then independently and randomly inactivates one copy of the X chromosome. [ 5 ]  This inactivation event is irreversible during the lifetime of the individual, with the exception of the germline. In the female  germline  before meiotic entry, X-inactivation is reversed, so that after meiosis all haploid  oocytes  contain a single active X chromosome.


The  Xi  marks the inactive,  Xa  the active X chromosome.  X P  denotes the paternal, and  X M  to denotes the maternal X chromosome. When the egg (carrying  X M ), is fertilized by a sperm (carrying a Y or an  X P ) a diploid zygote forms. From zygote, through adult stage, to the next generation of eggs, the X chromosome undergoes the following changes:


The X activation cycle has been best studied in mice, but there are multiple studies in humans. As most of the evidence is coming from mice, the above scheme represents the events in mice. The completion of the meiosis is simplified here for clarity. Steps 1–4 can be studied in in vitro fertilized embryos, and in differentiating stem cells; X-reactivation happens in the developing embryo, and subsequent (6–7) steps inside the female body, therefore much harder to study.


The timing of each process depends on the species, and in many cases the precise time is actively debated. [The whole part of the human timing of X-inactivation in this table is highly questionable and should be removed until properly substantiated by empirical data]


The descendants of each cell which inactivated a particular X chromosome will also inactivate that same chromosome. This phenomenon, which can be observed in the coloration of  tortoiseshell cats  when females are  heterozygous  for the  X-linked  pigment gene, should not be confused with  mosaicism , which is a term that specifically refers to differences in the  genotype  of various cell populations in the same individual; X-inactivation, which is an  epigenetic  change that results in a different phenotype, is  not  a change at the  genotypic  level. For an individual cell or lineage the inactivation is therefore  skewed  or ' non-random ', and this can give rise to mild symptoms in female 'carriers' of  X-linked  genetic disorders. [ 12 ]


Typical females possess two X chromosomes, and in any given cell one chromosome will be active (designated as Xa) and one will be inactive (Xi). However, studies of individuals with  extra copies of the X chromosome  show that in cells with more than two X chromosomes there is still only one Xa, and all the remaining X chromosomes are inactivated. This indicates that the default state of the X chromosome in females is inactivation, but one X chromosome is always selected to remain active.


It is understood that X-chromosome inactivation is a random process, occurring at about the time of  gastrulation  in the  epiblast  (cells that will give rise to the embryo). The maternal and paternal X chromosomes have an equal probability of inactivation. This would suggest that women would be expected to suffer from X-linked disorders approximately 50% as often as men (because women have two X chromosomes, while men have only one); however, in actuality, the occurrence of these disorders in females is much lower than that. One explanation for this disparity is that 12–20%  [ 13 ]  of genes on the inactivated X chromosome remain expressed, thus providing women with added protection against defective genes coded by the X-chromosome. Some [ who? ]  suggest that this disparity must be evidence of preferential (non-random) inactivation. Preferential inactivation of the paternal X-chromosome occurs in both marsupials and in cell lineages that form the membranes surrounding the embryo, [ 14 ]  whereas in placental mammals either the maternally or the paternally derived X-chromosome may be inactivated in different cell lines. [ 15 ]


The time period for X-chromosome inactivation explains this disparity. Inactivation occurs in the epiblast during gastrulation, which gives rise to the embryo. [ 16 ]  Inactivation occurs on a cellular level, resulting in a mosaic expression, in which patches of cells have an inactive maternal X-chromosome, while other patches have an inactive paternal X-chromosome. For example, a female heterozygous for haemophilia (an X-linked disease) would have about half of her liver cells functioning properly, which is typically enough to ensure normal blood clotting. [ 17 ] [ 18 ]  Chance could result in significantly more dysfunctional cells; however, such statistical extremes are unlikely. Genetic differences on the chromosome may also render one X-chromosome more likely to undergo inactivation. Also, if one X-chromosome has a mutation hindering its growth or rendering it non viable, cells which randomly inactivated that X will have a selective advantage over cells which randomly inactivated the normal allele. Thus, although inactivation is initially random, cells that inactivate a normal allele (leaving the mutated allele active) will eventually be overgrown and replaced by functionally normal cells in which nearly all have the same X-chromosome activated. [ 17 ]


It is hypothesized that there is an autosomally-encoded 'blocking factor' which binds to the X chromosome and prevents its inactivation. [ 19 ]  The model postulates that there is a limiting blocking factor, so once the available blocking factor molecule binds to one X chromosome the remaining X chromosome(s) are not protected from inactivation. This model is supported by the existence of a single Xa in cells with many X chromosomes and by the existence of two active X chromosomes in cell lines with twice the normal number of autosomes. [ 20 ]


Sequences at the  X inactivation center  ( XIC ), present on the X chromosome, control the silencing of the X chromosome. The hypothetical blocking factor is predicted to bind to sequences within the XIC.


The effect of female X heterozygosity is apparent in some localized traits, such as the unique coat pattern of a  calico cat . It can be more difficult, however, to fully understand the expression of un-localized traits in these females, such as the expression of disease.


Since males only have one copy of the X chromosome, all expressed X-chromosomal  genes  (or  alleles , in the case of multiple variant forms for a given gene in the population) are located on that copy of the chromosome. Females, however, will primarily express the genes or alleles located on the X-chromosomal copy that remains active. Considering the situation for one gene or multiple genes causing individual differences in a particular  phenotype  (i.e., causing variation observed in the population for that phenotype), in homozygous females it does not particularly matter which copy of the chromosome is inactivated, as the alleles on both copies are the same. However, in females that are heterozygous at the causal genes, the inactivation of one copy of the chromosome over the other can have a direct impact on their phenotypic value. Because of this phenomenon, there is an observed increase in phenotypic variation in females that are heterozygous at the involved gene or genes than in females that are homozygous at that gene or those genes. [ 21 ]  There are many different ways in which the phenotypic variation can play out. In many cases, heterozygous females may be asymptomatic or only present minor symptoms of a given disorder, such as with  X-linked adrenoleukodystrophy. [ 22 ]


The differentiation of phenotype in heterozygous females is furthered by the presence of X-inactivation skewing. Typically, each X-chromosome is silenced in half of the cells, but this process is skewed when preferential inactivation of a chromosome occurs. It is thought that skewing happens either by chance or by a physical characteristic of a chromosome that may cause it to be silenced more or less often, such as an unfavorable mutation. [ 23 ] [ 24 ]


On average, each X chromosome is inactivated in half of the cells, although 5-20% of women display X-inactivation skewing. [ 23 ]  In cases where skewing is present, a broad range of symptom expression can occur, resulting in expression varying from minor to severe depending on the skewing proportion. An extreme case of this was seen where monozygotic female twins had extreme variance in expression of  Menkes disease  (an X-linked disorder) resulting in the death of one twin while the other remained asymptomatic. [ 25 ]


It is thought that X-inactivation skewing could be caused by issues in the mechanism that causes inactivation, or by issues in the chromosome itself. [ 23 ] [ 24 ]  However, the link between phenotype and skewing is still being questioned, and should be examined on a case-by-case basis. A study looking at both symptomatic and asymptomatic females who were heterozygous for  Duchenne  and Becker muscular dystrophies (DMD) found no apparent link between transcript expression and skewed X-Inactivation. The study suggests that both mechanisms are independently regulated, and there are other unknown factors at play. [ 26 ]


The X-inactivation center (or simply XIC) on the X chromosome is  necessary and sufficient  to cause X-inactivation.  Chromosomal translocations  which place the XIC on an autosome lead to inactivation of the autosome, and X chromosomes lacking the XIC are not inactivated. [ 27 ] [ 28 ]


The XIC contains four non- translated   RNA  genes,  Xist ,  Tsix ,  Jpx  and  Ftx , which are involved in X-inactivation. The XIC also contains binding sites for both known and unknown  regulatory proteins . [ 29 ]