Interactions between genetic and epigenetic

More speculation about epigenetics and ways that epigenetic mechanisms of gene regulation could contribute to differences between individuals. Many cases, both in plants and animals, have to do with transposable elements, which makes a lot of sense since DNA methylation is involved in silencing the expression of transposable elements. Think about genetical genomics studies such as Gibbs & al (2010), where gene expression and DNA methylation is mapped to genomic regions. First, when expression QTL and methylation QTL coincide, it might be a good idea to start looking for transposable element insertions. Finding them are not as easy as finding SNPs, but hopefully, there will be SNPs tagging the actual variant and DNA methylation will spread outside of the inserted element to CpGs that are being typed. The element itself could of course work as a promoter, but it could also spread methylation into regulatory sequences of the gene, suppressing expression, or increase expression by changing the effect of an insulator.

Second, apparently the DNA methylation of transposable elements can sometimes be variable. This is the case with axin fused, Cabp-IAP and the agouti epialleles (Druker & al 2004; Vasicek & al 1997; Morgan & al 1999); among mice that carry the insertion there is DNA methylation variation causing phenotypic differences. This means that in populations where the insertion segregates, there should be a DNA methylation by gene interaction in the effect on the phenotype. I think that is fun, and I’d like to see someone find that in a mapping study. It might make things more difficult, though. The methylation–gene expression association might be hard to detect because it only exists in one of the alleles.

Third, maybe that is actually how a DNA methylation variant might escape reprogramming. Since some transposable elements are among the sequences that are not demethylated after fertilisation, and if that effect also applies to the newly inserted copy of the transposable element, our hypothetical regulatory methylation difference might be preserved through meiosis that way.


Gibbs, J. R., van der Brug, M. P., Hernandez, D. G., Traynor, B. J., Nalls, M. A., Lai, S. L., … & Singleton, A. B. (2010). Abundant quantitative trait loci exist for DNA methylation and gene expression in human brain. PLoS genetics, 6(5), e1000952.

Morgan, H. D., Sutherland, H. G., Martin, D. I., & Whitelaw, E. (1999). Epigenetic inheritance at the agouti locus in the mouse. Nature genetics, 23(3), 314-318.

Vasicek, T. J., Zeng, L. I., Guan, X. J., Zhang, T., Costantini, F., & Tilghman, S. M. (1997). Two dominant mutations in the mouse fused gene are the result of transposon insertions. Genetics, 147(2), 777-786.

Druker, R., Bruxner, T. J., Lehrbach, N. J., & Whitelaw, E. (2004). Complex patterns of transcription at the insertion site of a retrotransposon in the mouse. Nucleic acids research, 32(19), 5800-5808.

Morning coffe: ”epigenetics” is also ambiguous


I believe there is an analogy between the dual meaning of the word ”gene” and two senses of epigenetics, that this distinction is easy to get wrong and that it contributes to the confusion about the meaning of epigenetics. Gene can mean a sequence that has a name and a function, or it can mean a genetic variant. I sometimes, half-jokingly, call this genetics(1) and genetics(2). The order is wrong from a historical perspective, since the study of heritable variation predates the discovery of molecular genes. The first deals with the function of sequences and their products. The second deals with differences between individuals carrying different variants.

The same can be said about epigenetics. On one hand there is epigenetics(1), aiming to understand the normal function of certain molecular features, i.e. gene regulatory states that can be passed on through cell division. On the other hand, epigenetics(2) aims to explain individual variation between individuals that differ not in their DNA sequence but in other types of heritable states. And the recurring reader knows that I think that, since a lot of genetics(2) makes no assumptions about the molecular nature of the variation it studies, it will mostly work even if some of these states turn out to be epigenetic. In that sense, epigenetics(2) is a part of genetics.

Paper: ”Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens”

Since I love author blog posts about papers, I thought I’d write a little about papers I’ve contributed too. So far, they’re not that many, but maybe it can be a habit.

Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens” was published in BMC Genomics in 2012. The title says it very well: the paper looks at differential expression and DNA methylation of a subset of genes in the hypothalamus of Red Junglefowl and domestic White Leghorn chickens. My contribution was during my MSc project in the group. Previously (Lindqvist & al 2007; Nätt & al 2009) Daniel Nätt, Pelle Jensen and others found a transgenerational effect of unpredictable light stress on domestic chickens. After that, and being interested in chicken domestication, a DNA methylation comparison of wild and domestic seems like a natural thing to do. And it turns out Red Junglefowl and White Leghorns differ in expression of a bunch of genes and in methylation of certain promoters (where promoter is operationally defined as a region around the start of the gene model). And when looking at two generations, the contrasts are correlated between parent and offspring. There is some heritable basis of the differences in gene expression and  DNA methylation.

In Red Junglefowl, ancestor of domestic chickens, gene expression and methylation profiles in thalamus/hypothalamus differed substantially from that of a domesticated egg laying breed. Expression as well as methylation differences were largely maintained in the offspring, demonstrating reliable inheritance of epigenetic variation.

What I did was methylation sensitive high resolution melting. HRM is a typing method based on real time PCR. After PCR you often make a melting curve by ramping up the temperature, denaturing the PCR product. The melting characteristics depend on the sequence, so you can use melting to check that you get the expected PCR product, and it turns out that the difference can be big enough to type SNPs. And if you can type SNPs, you can analyse DNA methylation. So we treat the DNA with bisulfite, which deaminates cytosines to uracil unless they are protected by methylation, and get a converted sequence where an unmethylated C is like a C>T SNP. We set up standard curves with a mixture of whole-genome amplified and in vitro methylated DNA and measured the degree of methylation.

That is averaging over the population of DNA molecules in the sample; I’ve been wondering how HRM performs when the CpGs in the amplicon have heterogenous methylation differences. We’ve used HRM for genotyping as well, and it works, but we’ve switched to pyrosequencing, which gives cleaner results and where the assay design is much easier to get right the first time. I don’t know whether the same applies for methylation analysis with pyro.


My favourite part of the paper is figure 4b (licence: cc:by 2.0) which shows methylation analysis in the advanced intercross of Red Junglefowl and White Leghorns, which immediately leads to, as mentioned in the paper, the thought of DNA methylation QTL mapping.


Nätt, D., Rubin, C. J., Wright, D., Johnsson, M., Beltéky, J., Andersson, L., & Jensen, P. (2012). Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens. BMC genomics, 13(1), 59.

Lindqvist C, Janczak AM, Nätt D, Baranowska I, Lindqvist N, et al. (2007) Transmission of Stress-Induced Learning Impairment and Associated Brain Gene Expression from Parents to Offspring in Chickens. PLoS ONE 2(4): e364. doi:10.1371/journal.pone.0000364

Nätt D, Lindqvist N, Stranneheim H, Lundeberg J, Torjesen PA, et al. (2009) Inheritance of Acquired Behaviour Adaptations and Brain Gene Expression in Chickens. PLoS ONE 4(7): e6405. doi:10.1371/journal.pone.0006405

Also: the spectre of epigenetic inheritance

What is is that is so scandalous about epigenetic inheritance? Not much, in my opinion. Some of the points on the spectrum clearly happen in the wild: stable and fluctuating epigenetic inheritance in plants, parental effects in animals and genomic imprinting in both. Widespread epigenetic inheritance in animals would change a lot of things, of course, but even if epigenetic inheritance turns out to be really important and common, genetics and evolution as we know them will not break. The tools to study and understand them are there.

Looking back at the post from yesterday, there are different flavours of epigenetic inheritance. At the most heritable end of the spectrum, epigenetic variants behave pretty much like genetic variants. Because quantitative genetics is agnostic to the molecular nature of the variants, as long as they behave like an inheritance system, most high-level genetic analysis will work the same. It’s just that on the molecular level, one would have to look to epigenetic marks, not to sequence changes, for the causal variant. Even if a substantial proportion of the genetic variance is caused by epigenetic variants rather than DNA sequence variants, this would not be a revolution that changes genetics or evolution into something incommensurable with previous thought.

The most revolutionary potential lies somewhere in the middle of the scale, in parental effects with really high fidelity of transmission that are potentially responsive to the environment, but in principle these things can still be dealt with by the same theoretical tools. Most people just didn’t think they were that important. How about soft inheritance? It seems dramatic, but all examples deal with specific programmed mechanisms: soft inheritance of the sensitivity to a particular odour or of the DNA methylation and expression state of a particular locus. No-one has yet suggested a generalised Lamarckian mechanism; that is still out of the question. DNA mutations are still unable to pass from somatic cells to gametes. Whatever tricks transgenerational mechanisms use to skip over the soma–germline distinction, they must be pretty exceptional. Discoveries of widespread soft inheritance in nature would be surprising, a cause for rethinking certain things and great fun. But conceptually, it is parental effects writ large. We can understand that. We have the technology.

Morning coffee: the spectrum of epigenetic inheritance


Let us think aloud about the different possible meanings of epigenetic inheritance. I don’t want to contribute to unnecessary proliferation of terminology — people have already coined molar/molecular epigenetics (Crews 2009), intergenerational/transgenerational effects (Heard & Martienssen 2014), and probably several more dichotomies. But I thought it could be instructive to try to think about epigenetic inheritance in terms of the contribution it could make to variance components of a quantitative genetic model. After all, quantitative genetics is mostly agnostic about the molecular nature of the heritable variation.

At one end of the spectrum we find molecular epigenetic marks such as DNA methylation, as they feature in the normal development of the organism. Regardless of how faithfully they are transmitted through mitosis, or even if they pass through meiosis, they only contribute to individual variation if they are perturbed in different ways between individuals. If they do vary between individuals, though, in a fashion that is not passed on to the offspring, they will end up in the environmental variance component.

What about transmissible variation? There are multiple non-genetic ways for information to be passed a single generation: maternal or paternal effects need not be epigenetic in the molecular sense. They could be, like genomic imprinting, but they could also be caused by some biomolecule in the sperm, something that passes the blood–placenta barrier or something deposited by the mother into the egg. Transgenerational effects of this kind make related individuals more similar, they will affect the genetic variance component unless they are controlled. And in the best possible world of experimental design, parental effects can be controlled and modelled, and we can in principle separate out the maternal, paternal and genetic component. Think of effects like in Weaver & al (2004) that are perpetuated by maternal behaviour. If the behavioural transmission is strong enough they might form a pretty stable heritable effect that would appear in the genetic variance component if it’s not broken up by cross-fostering.

However, if the variation behaves like germ-line variation it will be irreversible by cross-fostering, inseparable from the genetic variance component, and it will have the potential to form a genuine parallel inheritance system. The question is: how stable will it be? Animals seem to be very good at resetting the epigenetic germline each generation. The most provocative suggestion is probably some type of variation that is both faithfully transmitted and sometimes responsive to the environment. Responsiveness means less fidelity of transmission, though, and it seems (Slatkin 2009) like epigenetic variants need to be stable for many generations to make any lasting impact on heritability. Then, at the heritable end of the spectrum, we find epigenetic variants that arise from some type of random mutation event and are transmitted faithfully through the germline. If they exist, they will behave just like any genetic variants and even have a genomic locus.

Epigenetics: what happened with this?

In 2012, Yan Li & Chris O’Neill published a paper about DNA methylation in the early mouse embryo, claiming that the first wave of demethylation following fertilisation in the mouse embryo doesn’t happen.

This picture, figure 1 from Seisenberger & al (2013; license: cc:by 3.0), shows what it is about. The curves represent DNA methylation level, and first time the curves drop represents the demethylation in question:


Li & O’Neill used a variation of immunostaining for methylated cytosine. Figures 8 and 3 summarise the results: eight shows embryos stained for methylated cytosine with two different preparation methods. The main claim of the paper is that the added trypsin treatment in the preparation helps unmask DNA methylation. So maybe the cytosine methylations are not removed, but temporarily hidden by something else. Figure 3 shows a Western blot for methyl-binding domain protein 1. The claim here is that if MBD1 is expressed, DNA methylation is also there. The obvious alternative hypothesis is that their variation on the protocol creates some kind of artefact and that MBD1 expression doesn’t matter.


Figure 8, Li & O’Neill (cc:by 3.0).

The paper has been cited mostly by review papers, and I haven’t seen any further news on the subject. Does anyone know if anything more has happened?


Li Y, O’Neill C (2012) Persistence of Cytosine Methylation of DNA following Fertilisation in the Mouse. PLoS ONE 7(1) e30687. doi:10.1371/journal.pone.0030687

Seisenberger, S., Peat, J. R., Hore, T. A., Santos, F., Dean, W., & Reik, W. (2013). Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers. Philosophical Transactions of the Royal Society B: Biological Sciences 368(1609), 20110330.

Morning coffee: epigenetic inheritance of odour sensitivity


A while ago I wrote a bit about the recent paper on epigenetic inheritance of acetophenone sensitivity and odorant receptor expression. I spent most of the post talking about potential problems, but actually I’m not that negative. There is quite a literature building up about these transgenerational effects, that is quite inspiring if a little overhyped. I for one do not think epigenetic inheritance is particularly outrageous or disrupting to genetics and evolution as we know it. Take this paper: even if it means inheritance of an acquired trait, it is probably not very stable over the generations, and it is nothing like a general Lamarckian transmission mechanism that can work for any trait. It is probably very specific for odourant receptors. It might allow for genetic assimilation of fear of odours though, which would be cool, but probably not at all easy to demonstrate. But no-one knows how it works, if it does — there are even multiple unknown steps. How does fear conditioning translate to DNA methylation differences sperm that translates to olfactory receptor expression in the brain of the offspring?

A while after the transgenerational effects paper I saw this one in PNAS: Rare event of histone demethylation can initiate singular gene expression of olfactory receptors (Tan, Song & Xie 2013). I had no idea olfactory receptor expression was that fascinating! (As is often the case when you scratch the surface of another problem in biology, there turns out to be interesting stuff there …) Mice have lots and lots of odorant receptor genes, but each olfactory neuron only expresses one of them. Apparently the expression is regulated by histone 3 lycine 9 methylation. The genes start out methylated and suppressed, but once one of them is expressed it will keep all other down by downregulating a histone demethylase. This is a modeling paper that shows that if random demethylation happens slowly enough and the feedback to shut down further demethylation is fast enough, these steps are sufficient to explain the specificity of expression. There are some connections between histone methylation and DNA methylation: it seems that DNA methylation binds proteins that bring histone methylases to the gene (review Cedar & Bergman 2009). Dias & Ressler saw hypomethylation near the olfactory receptor gene in question, Olfr151. Maybe that difference, if it survives through to the developing brain of the offspring, can make demethylation of the locus more likely and give Olfr151 a head start in the race to become the first expressed receptor gene.


Brian G Dias & Kerry J Ressler (2013) Parental olfactory experience influences behavior and neural structure in subsequent generations Nature neuroscience doi:10.1038/nn.3594

Longzhi Tan, Chenghang Zong, X. Sunney Xie (2013) Rare event of histone demethylation can initiate singular gene expression of olfactory receptors. PNAS 10.1073/pnas.1321511111

Howard Cedar, Yehudit Bergman (2009) Linking DNA methylation and histone modification: patterns and paradigms. Nature reviews genetics doi:10.1038/nrg2540