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.

heritability_methylation_fig4b

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.

Literature

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

Annonser

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:

dna_demethylation_fig1

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.

journal.pone.0030687.g008

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?

Literature

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

kaffe_tryffel

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.

Literature

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

Journal club of one: ”Parental olfactory experience influences behavior and neural structure in subsequent generations”

Okay, neither chickens nor genetics, really, but a little epigenetic inheritance. Dias & Ressler in Nature neuroscience:

When an odor (acetophenone) that activates a known odorant receptor (Olfr151) was used to condition F0 mice, the behavioral sensitivity of the F1 and F2 generations to acetophenone was complemented by an enhanced neuroanatomical representation of the Olfr151 pathway.

Meaning that the offspring of conditioned mice score higher in an odour potentiated startle test (more about that below), avoid the odour at a lower concentration in an aversion test and have more neurons expressing that odorant receptor in their olfactory epithelium and bulb, counted by betagalactosidase staining in transgenic mice expressing M71, the product of Olfr151, coupled to LacZ.

Furthermore,

Bisulfite sequencing of sperm DNA from conditioned F0 males and F1 naive offspring revealed CpG   hypomethylation in the Olfr151 gene. In addition, in vitro fertilization, F2 inheritance and cross-fostering revealed that these transgenerational effects are inherited via parental gametes.

That is, they detect a difference in methylation in one CpG dinucleotide in the 3′ region of the gene.

Comments:

First, I love how the journal does exactly the thing I like to see with figures: below each figure is a link that leads to a data file with the underlying data!

Olfactory behaviour is not my thing, so the tests are new to me, but I’m a bit puzzled by the way they calculate the results from the odour potentiated startle tests. The point is to test whether the presence of the odour make the mice react stronger to a noise. After buzzing the sound 15 times without odour, they perform ten trials with odour plus sound and ten trials with sound only. But in calculating the score, they use only the difference between the first trial with odour and the last trial with sound only divided by how much the mouse reacted to the last of the first 15 sounds. Maybe this is standard, but why throw away the trials in between?

It is not only the olfactory potentiated startle and the sensitivity test, but the staining results. Again, this is not my area, but the results all seem to point to increased sensitivity in the offspring of the treated animals. They react stronger in the startle test, react at lower concentration in the avoidance test and they (in this case, the transgenic mice) have more neurons expressing M71. The cross fostering and the fact that the males were treated but not the females points to genuine inheritance. So, how does the treatment get into the germline? It has to cross that boundary and enter the sperm somehow. Unless there is some mysterious way for information from the central nervous system to travel to the testis, acetophenone must affect the spermatogenesis as well as the olfactory neurons.

All this is very hypothetical, so a little skepticism is not surprising. Gonzalo Otazu wrote in a comment on the Nature news webpage:

The statistical tests in the paper, both for the behavioral measurements as well as for the size of the M71 glomeruli , use as n, number of samples, the number of F1 and F2 individuals. This would be fine if the individuals were actually independent samples. However, they arise from a presumably small number of FO males. The numbers of FO males are not given in the paper. This is a major concern given that there is a lot of variability in the levels of expression of olfactory receptors in these mice that might be inheritable …

I think this is a good point but it will not be solved, as the comment later suggests, by adjusting the degrees of freedom of the test. From the F1 generation and on, genetic differences between the treatment groups, if they do exist, will amplify into a bias issue. That is, it is a systematic difference that might be bigger or smaller than the treatment effect and go in the same or opposite direction — we don’t know. However, the bias should not be there all the time, and not in the same direction, so it strengthens the authors’ case that they’ve done the treatment at least twice (with C57B/6J and with M17-LacZ mice, if not more times).

Maybe my preference for genetics is showing, but I feel the big unadressed alternative hypothesis in most transgenerational effects experiments is cryptic heritability. If you divide individuals into two groups, treat one of them and look for treatment effects in the offspring, you need to be sure that there are not genetc differences between the founders of the two groups. In the subsequent generations, genetic and non-genetic inheritance will be counfounded by design.

Again, randomisation and replication will help, but to be really sure, maybe one can use founders of known relatedness to create a mixed population — say take founders from full-sibships and split them equally between treatment groups, allowing segregation to randomise the genotypes of the next generation. It doesn’t say in the methods — the authors might even have done something like this. One could even use a genetic mixed model that includes relatedness as to estimate treatment effects over in the prescence of a genetic effect. I have a suspicion this experiment would require a much larger sample size, which means more time, work and animals — but I also believe that many would find confounding genetic variation more plausible than transgenerational epigenetic effects of unknown mechanism.

Literature

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