Epigenetics and the renaissance of heresy

MINIREVIEW / MINISYNTHÈSE

Epigenetics and the renaissance of heresy1,2

Susannah Varmuza

Abstract: Classic neo-Darwinian theory is predicated on the notion that all heritable phenotypic change is mediated byalterations of the DNA sequence in genomes. However, evidence is accumulating that stably heritable phenotypes canalso have an epigenetic basis, lending support to the long-discarded notion of inheritance of acquired traits. As manyof the examples of epigenetic inheritance are mediated by position effects, the possibility exists that chromosome rear-rangements may be one of the driving forces behind evolutionary change by exerting position effect alterations in geneactivity, an idea articulated by Richard Goldschmidt. The emerging evidence suggests that Goldschmidt’s controversialhypothesis deserves a serious reevaluation.

Key words: epigenetics, position effects, inheritance of acquired traits.

Résumé : La théorie néo-darwinienne classique est fondée sur la notion que les changements phénotypiques héréditai-res sont dus à des altérations de la séquence d’ADN des génomes. Cependant, des évidences s’accumulent à l’effet quedes phénotypes héréditaires stables peuvent avoir un fondement épigénétique. De telles observations viennent raviver leconcept, depuis longtemps abandonné, d’hérédité des caractères acquis. Puisque plusieurs des exemples d’hérédité épi-génétique sont le résultat d’effets de position, il existe une possibilité que les réarrangements chromosomiques soientune des forces motrices de l’évolution en modifiant l’activité des gène via une altération des effets de position, cetteidée ayant été articulée par Richard Goldschmidt. Les évidences récentes suggèrent que l’hypothèse controversée deGoldschmidt mérite une réévaluation sérieuse.

Mots clés : épigénétique, effets de position, hérédité des caractères acquis.

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According to Dobzhansky, “…in biology nothing makessense except in the light of evolution” (1970). Evolution isan idea that inspires huge emotional responses, in part be-cause it speaks to our very identities. The religious overtonesassociated with debates about evolution are not restricted tothose between evolutionary biologists and creationists (theinspiration for the quote above). Among evolutionary biolo-gists, there is an aura of deification of Darwin that tends tostifle discourse on ideas that are construed by the main-stream to be anti-Darwinian, perhaps, as pointed out byGould (1981), to counteract the political machinations of thecreationist movement. Over the decades, attempts bynon-traditionalists to introduce new thinking into the study

of evolution have met with either stony silence or rancorousderision. Goldschmidt, Gould, and proponents ofLamarckian inheritance can still raise hackles, even posthu-mously (“Goldschmidt is a bum!” echoed around the lecturetheatre at a recent scientific meeting, 44 years after hisdeath. See Stephen Jay Gould’s essay The Uses of Heresy:An Introduction to Richard Goldschmidt’s “The MaterialBasis of Evolution” at the beginning of the 1982 reprint ofGoldschmidt’s book for descriptions of similar sentimentsexpressed in an earlier era.) Where insight and logical analy-sis failed in the past, hard data may just succeed in breakingthrough the intellectual rigidity. Genome projects are yield-ing evidence that is forcing us to re-evaluate theneo-Darwinian dogma. The coding sequences in mouse andhuman genomes are 99% identical. Why do we look so dif-ferent?

Fifty years after the publication of the structure of DNA,we are just beginning to realise how intriguing genomes re-ally are. DNA is not a complicated molecule. The structureis remarkably uniform, even boring. Yet the level of infor-mation integration is awe inspiring. One need only watch adish of developing embryos to appreciate the enormouscomputational power and fidelity of the genome in action.

How does a simple molecular structure composed of onlyfour different components generate so much complexity?The simple answer is that the DNA does not operate alone.It is intimately bound up in proteins and other molecules

Genome 46: 963–967 (2003) doi: 10.1139/G03-115 © 2003 NRC Canada

963

Received 30 July 2003. Accepted 6 October 2003. Publishedon the NRC Research Press Web site at http://genome.nrc.caon 12 November 2003.

Corresponding Editor: R.S. Singh.

S. Varmuza. Department of Zoology, University of Toronto,25 Harbord St., Toronto, ON M5S 3G5, Canada (email:[email protected]).

1This article is one of a seclection of papers published in thisSpecial Issue on The Impact of the DNA Double HelixModel on Cytogenetics and Evolutionary Mechanisms.

2This paper has undergone the Journal’s usual peer reviewprocess.

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that impose a strict order of activity on the sequences in theDNA molecules in our chromsomes. Naked DNA is chaotic,but chromatin represents order and continuity. How does itwork?

One can find numerous reviews outlining the biochemistryand biology of chromatin structure (see for exampleFelsenfeld and Groudine 2003; Orlando 2003; Jenuwein andAllis 2001; Turner 2000; the March 2003 issue of the Annalsof the New York Academy of Science). I will focus instead onhow our growing knowledge of chromatin and epigenetics(using the modern meaning of the word) is changing ourviews of how genomes work from an initial orgy ofreductionist thinking in the 1960s and 1970s, to a more inte-grated view in the early 21st century. I will do this from theperspective of rehabilitation of two ideas that have been (andcontinue to be) scorned in some quarters for many decades.Those ideas are the inheritance of acquired traits, attributedto Lamarck, and the notion of systemic macromutationalmutations described by Goldschmidt (1940).

Jean Baptiste Pierre Antoine de Monet, Chevalier deLamarck, 1744–1829, was a poor academic in the MuséeNational d’Histoire Naturelle. He invented the terms “inver-tebrate”and “biology” and reorganized invertebrate taxon-omy (see The Zoological Philosophy of J.B Lamarck by R.Burkhardt in the introduction to the 1984 English translationof Lamarck’s book Zoological Philosophy). His idea that useor disuse could lead to adaptive morphological change wasthe basis for the notion that evolution is directed towards in-creased complexity, necessitating the continuous appearanceof new simple species at the bottom end of the line(Lamarck 1809). The mechanism by which use and disusecould lead to adaptive change was the inheritance of ac-quired traits, a commonly held idea in the 18th and 19thcenturies.

While the hypothesis of the inheritance of acquired traitswas not invented by Lamarck, nor was it the main focus ofhis somewhat odd ideas, it became associated with his nameto distinguish one mechanism of evolution from anothermechanism — natural selection. Neo-Darwinists were ableto reject Lamarckism (the non-Darwinian mechanism) whenMendelian genetics provided a scientific basis for rejectionof inheritance of acquired traits.

Goldschmidt (1879–1958) was an extremely productivegeneticist, but started out as a developmental biologist work-ing on nematodes and trematodes. Much of his research dur-ing the first part of his career focused on geographicvariation and sex determination in gypsy moths (Lymantria).His extensive work on the population genetics of geographicvariation led him to believe that microevolution workswithin species, but cannot explain speciation itself. This hebelieved was caused by macroevolution, for which he postu-lated two mechanisms: mutations in genes of large effect(eg. Homeotic mutations in fruit flies), and “systemic muta-tions”, by which he meant large-scale chromosome rear-rangements that led to a change in the developmentalprogram, and thus in the morphology of the animal, througha process poorly understood at the time — position effects(Goldschmidt 1940).

Recent advances in our understanding of epigenetic phe-nomena make it possible to link both of these ideas. There isevidence from a wide range of phyla that inheritance of ac-

quired traits occurs readily in nature, and that it is oftenlinked to position effects. I will describe some recent experi-ments from mice, yeast, plants, and fruit flies to illustratehow position effects and inheritance of acquired traits canalter the activity of the genome to effect morphologicalchange.

Emma Whitelaw’s group has done a wonderful study ofalleles at two different loci that demonstrate epigeneticinheritance — Avy and Axinfu (formerly Fused kinky). Bothmutant alleles are caused by a nearby insertion of an intra-cisternal type A particle (IAP) retrotransposon. The unusualcharacteristics of these alleles are mediated by variablemethylation patterns of the IAP provirus (Rakyan et al.2003; Morgan et al. 1999).

First, a little perspective. Both maternal and paternalgenomes come together in the zygote with huge amounts ofmethylation baggage — there is a lot of differentialmethylation between the two genomes at fertilization. How-ever, just after fertilization there is a massive demethylationof the paternal genome within the male pronucleus. The ma-ternal genome becomes passively demethylated duringpreimplantation cleavage divisions (Mayer et al. 2000;Oswald et al. 2000).

A (agouti) encodes the ASP inhibitory ligand for themelanocortin 1 receptor, although it can also bind othermelanocortin receptors (notably the melanocortin receptor 4on many cell types that is linked to a signaling pathway in-volving cellular responses to insulin). A is normally ex-pressed in hair follicles in a cyclical manner, resulting in ayellow band (A “ON”) on a black background (A “OFF”).Dominant yellow alleles express A ectopically, resulting incompletely yellow hair shafts, as well as other defects (eg.obesity). Avy, which contains an IAP provirus upstream ofthe A hair follicle promoters, is expressed ectopically whenthe IAP LTR promoter is hypomethylated, but is expressednormally (i.e., in the yellow band pattern) when the IAPLTR is hypermethylated. Thus, some mice are completelyyellow and fat, whereas others with the same genotype areagouti and slim. The proportion of pups with the yellowphenotype depends on the phenotype of the mother — yel-low Avy moms produce a higher proportion of yellow pupsthan agouti Avy moms. Avy dads produce the same proportionof yellow and agouti pups, regardless of their own pheno-type. In other words, it looks like a maternal effect. The Avy

phenotype can be enhanced by supplementation of the ma-ternal diet with compounds that stimulate methylation bio-chemistry (folic acid, vitamin B12, methionine, etc.),supporting the idea that Avy displays maternal effects, possi-bly through the uterine environment.

Axin is a wnt signaling pathway component, and involvedin axis determination during development. The IAP provirusin Axinfu is inserted into the sixth intron and, whenhypomethylated, yields an aberrant transcript that encodes atruncated form of the Axin protein that has been shown inother organisms to act as a dominant negative signaling mol-ecule during axis development. Hypermethylation of the IAPshuts off this aberrant transcript.

Whitelaw’s group tested a number of ideas regarding thetransgenerational inheritance of both Avy and Axinfu. First,they demonstrated that maternal environment did not includeeither uterine environment, or even oocyte composition. Avy

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pups from yellow moms transferred into agouti moms stilldeveloped the high proportion of yellow phenotype. Avy/Avy

pups derived by mating yellow Avy moms with agouti Avy

dads produced some agouti pups, indicating that the agoutiepiallele survives in an Avy egg. Moreover, yellow Avy/Avy

moms whose fathers were agouti Avy produced agouti pupswhen outcrossed to black (a/a) tester males, indicating thatthe grandpaternal Avy epiallele survives as is in the femalegermline.

The story with Axinfu is a bit different in that epigeneticinheritance was observed for both maternal and paternal al-leles. In both cases, hypomethylated (affected) moms anddads gave rise to a higher proportion of hypomethylatedpups, whereas hypermethylated (unaffected) moms and dadsproduced hypermethylated pups.

An interesting twist on this story came about when theWhitelaw group examined the effect of genetic backgroundon epigenetic inheritance patterns for both Avy and Axinfu.Avy was tested in a C57BL/6 strain background, and dis-played maternal transgenerational inheritance only. More-over, the methylation pattern of the sperm reflected thesomatic methylation pattern, i.e., it was either hypermethyl-ated or hypomethylated. Yet the offspring did not reflect thepaternal sperm methylation pattern, suggesting that it did notsurvive after fertilization. However, when Avy males werecrossed to 129 females, the paternal phenotype was reflectedin the offspring. The Axinfu allele was originally tested in a129-strain background, and displayed epigenetic inheritancefor both maternal and paternal alleles. Yet when Axinfu maleswere crossed to C57BL/6 females, the paternal inheritancepattern was eliminated. Thus, 129 eggs allowed both mater-nal and paternal transmission of inherited methylationmarks, whereas C57BL/6 eggs were more likely to changepaternal, but not maternal, methylation marks.

Another example of epigenetic inheritance in mice waspublished recently (Herman et al. 2003). In this case, an en-gineered allele of the Rasgrf1 locus caused altered methyl-ation in trans of the wild type allele that was bothmitotically and meiotically stable. These observations arereminiscent of paramutation in plants.

There are many papers on epigenetic inheritance in engi-neered plants. The field of RNA interference (RNAi) reallygot its start in plants with studies of cosuppression. Plantsseem to be able to pass on modifications more readily as aresult of the flexibility of the germline. Perhaps the most sat-isfying story, however, comes from nature. Enrico Coen andcolleagues looked at the molecular basis for one of the veryfew morphological variants in a plant found in the wild, theLinaria (toadflax) variant that produces radially symmetricalflowers (Cubas et al. 1999). In this case, the variant is a re-sult of heritable methylation of the Lcyc gene. As this vari-ant has been around for a long time (it was described byLinnaeus), it is clearly quite stable. (Cubas et al. 1999).

The modern study of epigenetics owes a huge debt to thefruit fly Drosophila melanogaster. Position effect variegation(PEV), a form of non-genetic (i.e., not encoded in the DNA)variation has allowed us to find both cis and trans elementsof chromatin modification through genetic and molecularmanipulation. Some of the trans elements share structuralsimilarity with proteins that modulate developmentalswitches. For example, a well characterized chromatin pro-

tein that plays a major role in PEV, HP-1, possesses a pro-tein motif called a chromodomain, as does the polycomb(Pc) protein that locks repressed chromatin states in placeafter the initialization of active and silent gene expressionpatterns during early development. Pc belongs to a class ofproteins encoded by the Polycomb – trithorax group(PcG/TrxG) genes whose roles are to maintain cellular mem-ory of transcriptional ON/OFF states. Pc mediates its effectson the AbdA and AbdB homeotic selector genes through a ciselement called fab7. Chromatin immunoprecipitation (ChIP)experiments have demonstrated that Pc binds to fab7. How-ever, it is not alone in binding to fab7. Cavalli and Paro(1998) demonstrated that the fab7 element can confer partialsilencing on nearby genes: a fab7–UAS–lacZ transgene, witha linked mini-white marker gene is expressed at low levels(patchy lacZ owing to leaky expression of an unlinkedhs-GAL4, yellow eyes). The repression of lacZ andmini-white is increased at higher temperatures (28 °C). Inone case, a nearby endogenous gene, scalloped, is also re-pressed at higher temperatures. A transient burst of GAL4expression during embryogenesis, mediated by a heat shock,derepresses both lacZ and mini-white and in the transgenicline in which scalloped is affected, that gene is alsoderepressed. Both the highly repressed state (pale yelloweyes) and the derepressed state (red eyes) can be passed onthrough several generations in the absence of either inducingenvironmental condition. The effect on the endogenous scal-loped gene is also epigenetically heritable.

Fruit flies do not methylate their DNA. While there maybe some kind of covalent DNA modification that we don’tknow about operating in fruit flies, the best evidence sug-gests that epigenetic stability, both mitotic and meiotic, ismediated by protein complexes that bind to the DNA and arereformed at each cell division. A similar kind of mechanisminvolving specific kinds of histones is thought to distinguishcentromeres from other regions of the chromosome(Henikoff et al. 2001).

Mitotic and meiotic inheritance of an epigenetic state hasbeen demonstrated in yeast (Grewal and Klar 1996).Schizosaccharomyces pombe mating type interconversion in-volves a gene conversion event in which coding sequencefrom one of two silent loci replaces the coding sequence atthe active mat locus. The two silent loci, p and m, are sepa-rated by a 15-kb region known as the K region that is re-quired in cis for silencing of p and m. Marker genes, forexample the ura4 gene, inserted into the K region are silent.However, a small proportion (1%) become activated. Onceactivated, this state is stable enough to allow selection ofphenotypic variants. These metastable states are also pre-served during meiosis, and behave like genetic variants, eventhough they are epigenetic variants (Grewal and Klar 1996.)The “mark” may be mediated by binding of a chromo-domain protein, Swi6, to the K region (Nakayama et al.2000).

All of these examples (excepting the Lcyc gene inLinaria) have been demonstrated to exhibit epigenetic inher-itance through position effects conferred by linkage of targetgenes to DNA elements that cause heritable silencing or her-itable ectopic expression of nearby genes. In other words,these elements can cause genes to be turned on or off at ab-normal times or locations, with dramatic effects on resulting

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phenotype. The notion that the timing and location of geneactivity can have a more profound effect on morphologythan the sequence of amino acids of specific proteins is notnew to developmental biologists. We have known for de-cades that loss and gain of function of gene activity is amore likely source of variation at the morphological levelthan alteration in protein sequence. For example, the murinePax6 gene is quite capable of substituting for the Drosophilaeyeless gene, as long as it is expressed in the right cells.When it does so, it makes a Drosophila eye, not a mouseeye (Halder et al. 1995). On the other hand, both loss offunction mutations (off state) and ectopic expression oftransgenes (on state) are well known mechanisms for gener-ating morphological change. Moreover, isogenic lines ofboth fruit flies and plants exhibit a wide range of heritablephenotypic variation when Hsp90 expression is disruptedtemporarily (Sollars et al. 2003; Queltch et al. 2002). Thus,Goldschmidt’s idea that a change in the morphological pat-tern can be caused by rearrangements of chromosomes thatalter the positions of genes, and therefore their activity dur-ing development, is no longer laughable, but a serious, andtestable, hypothesis. The other side of the macromutationalcoin requires that such chromosome rearrangements shouldbe able to erect reproductive barriers. This has recently beendemonstrated in isogenic lines of Saccharomyces cerevisiaewith engineered chromosome rearrangements (Delneri et al.2003). Not all reproductive isolation can be attributed togenic incompatibilities.

The dependence of neo-Darwinian theory on geneticchange (i.e., DNA sequence change) is tantamount to beingpainted into a corner. Is it really necessary to invoke DNAsequence alterations if epigenetic on/off switches are botheffective and stable enough to do the job? As with point mu-tations, most epigenetic changes from the “norm” will bedeleterious and subject to rapid elimination. On the otherhand, a system that is responsive to environmental changeshas the capacity to provide the necessary developmentalflexibility to ensure survival under difficult conditions. Forexample, many organisms adopt different life styles whenfaced with extremes of food availability, water, and tempera-ture (yeast change from single cells to filamentous hyphae;nematodes adopt a dauer life style). These changes are de-velopmental, i.e., mediated by epigenetic alterations in the“reading” of the genetic program. In other words, epigeneticmechanisms are fundamental, not specialized. One does notneed to invoke the evolution of epigenetic inheritance sys-tems (EIS) as a specialized mechanism for driving evolution,just as one does not need to invoke the evolution of the ge-netic code, or bilaminar cell membranes. The really interest-ing question is “what constrains epigenetic mechanismsfrom running amok?”. Some of these issues are discussed indepth in Epigenetic Inheritance and Evolution, TheLamarckian Dimension, by Jablonka and Lamb (1995).

Neo-Darwinism is predicated on the notion that gradualchange accumulating in the DNA sequence and subjected toselective pressure is sufficiently powerful to account for evo-lution. This is the current dogma that drives much of popula-tion genetics. Heretics like Lamarck, Goldschmidt, andGould thought there was more to it than that. We are nowgathering evidence that phenotypic variation need not reflectmutations in the genome, that induced phenotypic

non-genetic changes can be heritable, and that, indeed, sig-nificant variation can be induced in the absence of geneticvariation. Much of this phenotypic variation is likely to bethe result of altered timing and location of gene expressionduring development. And finally, the chromosomal milieucan have profound effects on the timing and location of geneexpression, and on the ability of eukaryotic life forms tomate and breed successfully.

If all organisms share the same suite of genes, with a fewnovel inventions here and there, then phenotypic changefrom one species to another must be mediated by some othermechanism. This was the crux of the problem as viewed byGoldschmidt. He articulated the notion that species share thePOSSIBILITIES of alternate developmental pathways (reac-tion norms), but make use of only one at a time. While heknew that position effects must play some kind of role, thestate of molecular knowledge in the 1930s and 1940s wassuch that no obvious explanation clarified these issues. Hadhe been aware of the pervasiveness of gene silencing mecha-nisms, he would immediately have understood its implica-tions with respect to both morphology and evolution, andmight not have made the logical leap that so offended hiscolleagues (rejection of the corpuscular gene). He knew hewas right about the bigger issue. It has only taken the rest ofus 60 years to realize it.

Acknowledgements

I would like to thank the editors of Genome for giving mea forum for discussing the ideas presented here. Most of thisessay is the result of many long and entertaining conversa-tions with my colleague and fellow subversive, Ellen Larsen.

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Epigenetics and the renaissance of heresy