SMBE conference: Vienna July 12-16

Exciting news: I will be giving a talk at the SMBE conference in Vienna in the "Genomic and epigenomic evolution of sex chromosomes: Broad patterns and intriguing cases" Edit: the "Speciation Genomics" symposium. I'll be presenting at 16:30 on Tuesday, 14 July in Festsaal.

Here is the title and abstract:

	Title	Disrupted imprinting, the large X-effect, and extreme hybrid overgrowth in hamsters
	Abstract	Extreme abnormal growth is a recurrent phenotype in mammalian hybrids, indicating that disruption of development may play an important role in mammalian speciation. Disrupted genomic imprinting - the parent-specific epigenetic silencing of one allele - resulting in dosage imbalances between growth factors and repressors has been hypothesized to be the predominant cause of abnormal hybrid growth in mammals. However, genetic imprinting may also expose hybrid incompatibilities through the hemizygous expression of genes. Here we combined genome-wide transcriptomic and quantitative genetic experiments to dissect the genetic underpinnings of extreme overgrowth that manifests in hybrids between two closely related species of dwarf hamsters. Specifically we tested for disrupted imprinting and differences in the expression of growth-related genes in overgrown hybrid placenta. We found that large hybrids show evidence for disrupted paternal imprinting and differential expression of imprinted genes in general. Surprisingly, the disruption of paternal imprinting is associated with reduced gene expression. As paternally imprinted genes tend to repress offspring growth, these data suggest that overgrowth is associated with a reduction in growth repressors rather than an excess of growth factors. Using QTL mapping, we have further identified the X chromosome as the predominant factor explaining hybrid placental overgrowth. However, expression and imprinting status of the X chromosome is not significantly disrupted, indicating that X-linked genetic incompatibilities are not caused by chromosome-wide misregulation. Collectively, our data underscore a central role for both disrupted epigenetic processes and the X chromosome in driving the evolution of abnormal hybrid growth in mammals.

Manuscript II - Planning part 2

In the last post I came up with a number of questions that I an address with my data set, but in the end, I have at couple compelling hypotheses that I can test which should be the focus of the paper.

Here is the first hypothesis:

Many imprinted genes are growth factors that act in a dosage-dependent manner. Abnormal growth is the result of increased expression of genes which promote or repress growth. Abnormal growth manifests in a parent-of-origin manner when one hybrid type exhibits increases in growth promoting genes and the reciprocal hybrid exhibits increases in growth repressing genes.

The common trait to all hybrids, large or small, is that genes which regulate growth show increased expression level. If these genes promote growth then the individual is large, if these genes repress growth then the individual is small. Furthermore, we predict that when imprinting is disrupted, the gene which should be expressed from a single allele is actually expressed from both alleles. This should result in a doubling of expression level.

We can test this hypothesis by looking at expression level in large hybrids and comparing it with small hybrids. If growth promoting genes are expressed from two alleles and show increased expression level in large hybrids then this hypothesis would be supported.

The second hypothesis is this:

There is ample evidence that hemizygosity is bad as it exposes things which are deleterious but recessive. This is probably most clear when we look at the X chromosome. One of the main reasons for Haldane's rule (the disproportionate affliction of incompatibilities in males) is that males have a single copy of the X while females have two X chromosomes. Thus, females are able to mask recessive deleterious things on the X but males are not. As imprinted genes are similarly expressed from a single copy, all recessive incompatabilities involving them will be exposed. Furthermore, in the reciprocal hybrid, the alternate allele is expressed which should ameliorate the incompatibility.

This hypothesis is mute with respect to dosage, and can occur whether or not the dosage of different alleles is disrupted.


I think that the paper will start by addressing these two hypothesis and set up a framework for how to test each using the growth effects in the hamster hybrids.

Manuscript II - planning

I'm about to start writing my next manuscript. It's going to deal with patterns of gene expression in the placenta of F1 hybrids. I'm stalled a bit as I try to figure out exactly what questions I will be addressing and how I will go about discussing them in the manuscript. So, I'm hoping that writing this will help me flesh out my thoughts on what questions I'll be asking, why they are important, and how to address them.

First, all the questions I can think of that I can address with my data:

Is the histology of placentas abnormal in large or small hybrids? males or females?
Is gene expression disrupted in hybrid placentas?
How many genes show differential expression (DE)?
How many genes show DE between P. campbelli and P. sungorus (evolutionary changes)?
How many genes show DE between parents and hybrids (hybrid breakdown)?
How many genes show DE between cxs and SxC hybrids (could explain POE growth)?
How many genes are DE between males and females within each parent species?
How many genes are DE between males and females with the hybrids?
Are sex-specific effects prevalent in hamster hybrids?
For all these categories: what do those genes do? / Is there any functional enrichment for DE genes?
Do imprinted genes show DE?
Is imprinting disrupted (DI) in hybrids? Large, small, or both?
Does DI correlate with POE growth?
How often does disrupted imprinting correlate with differential expression?
Does the simple model of 'disrupted imprinting=2x increase in expression' hold?
Do paternally expressed genes show higher expression in large hybrids than small hybrids?
Do paternally expressed genes show higher expression in large hybrids than the parents?
Do maternally expressed genes show lower expression in large hybrids than small hybrids?
Do maternally expressed genes show lower expression in large hybrids than the parents?
What is happening on the X chromosome in hybrids?
Is X-linked expression disrupted?
Is X-linked expression correlated with abnormal growth?
Is there imprinted X inactivation (XCI) in hybrid placentas?
Is XCI disrupted?
Do any/many genes escape XCI? Which?
Does escape of XCI seem to have anything to do with POE growth?


Ok, that was more than I expected so that's good.
Now some organization.

My first thoughts are that the main question is what are patterns of expression in hybrid placentas and do they reflect a possible basis for parent-of-origin growth? This has three main sub-questions which are (1) what are the patterns of differential expression in hybrid placenta? (2) what is the pattern of imprinting in hybrid placenta? and (3) what is the overlap between DE and DI in hybrids?

So far, this sounds really descriptive and one of the strengths of my project is that I have a couple really strong a priori predictions about disrupted expression and imprinting and the size of the hybrids. Therefore, I think I should be able to motivate this in a more hypothesis-driven manner, rather than just as a simple description of expression patterns.

To do that, I need to lay out the prediction of gene expression.

To be continued...

Denver Museum of Nature and Science

K~ and I visited the Denver Museum today and were given a private tour of the collections. J. D~ was kind enough to spend his entire Friday afternoon showing us around and letting us see all the cool stuff. Fortunately, we got the grand tour despite the fact that museum is currently moving it's entire collection to a brand new housing area with fancy state-of-the-art rolling cabinets.

We spent most of the time with the bird collection and we got to see about 40 passenger pigeons (extinct), an ossified goose trachea (shout out to M. J~), and articulated skeletons of a bunch of birds including a kestrel and three different owls (which K~ identified on sight as a saw-whet, burrowing, and pygmy).

They also had two complete elephant bird eggs (these are watermelon size, the hand for scale doesn't actually do it justice). Elephant birds are from Madagascar and went extinct a couple hundred years ago. The fact that any eggs stil exist is amazing - not to mention two complete, unbroken ones.

 The Denver Museum had a collection trip to the Galapagos in 1960, one year before they stopped allowing collections, and returned with a number of Geospiza finches.

Most excitingly, they had a male and two female speciments of the infamous Ivory-Billed Woodpecker (unfortunately, the photo is a bit blurry*):

















We also got to see a bunch of the beetles, scorpions, spiders, camel scorpions (which I've never even heard of before but looked bad-ass - apparently the mouthparts for predator in the movie Predator were based on these guys) and whip scorpions (which gained fame in the movie Harry Potter and the Goblet of Fire as the spider that Moody used to demonstrate the unforgivable curses to the defense agains the dark arts class). Also we were able to see some more beetles, butterflies and moths as they were being moved to the new shelves (the staff were fantastic and took a break from moving specimens to let us look at the cool beetles).

After the birds and bugs we headed over to the fossils - there were two dire wolves (smaller than I expected), an irish elk skull (with antlers spanning further than I could reach from toe to fingertip), a bunch of mammoth and mastodon skulls, leg bones, and vertebrae, a petrified tree stump we helped move, and a Stockoceros (which is like a pronghorn with two horns).

One really cool specimen we saw was a giant ground sloth.

Then we got to the mammal cabinets and looked mainly at the chipmunks, but we got to see some lions, tigers, a gorilla, and bats too. Didn't see any Phodopus but I guess I've seen plenty of them.

All in all it was a great afternoon. Major thanks to J. D~ for showing us around and answering all our questions. Also to the staff of the Denver Museum for taking time to show us some cool biology!

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*I'm told all ivory-billed photos are blurry - it's a tradition. 

Parent-of-Origin Effects Paper is out!

http://authorservices.wiley.com/bauthor/onlineLibraryTPS.asp?DOI=10.1111/evo.12500&ArticleID=1354661

Capture design

One project that I'm working on is to map the genetic factors that cause parent-of-origin growth in dwarf hamsters. This project will end up being the final chapter in my dissertation and has a number of angles that hopefully will all come together in the end to forma  complete story. One of these angles is a mapping panel made out of ~200 backcrossed hamster individuals. I'm using a RAD-seq approach which has recently been sent for sequencing. This will use anonymous markers across the genome to find regions associated with growth. The shortcoming of this approach is that the markers are anonymous and not necessarily associated with known genes. In order to place known genes on the map I am designing a capture.

The capture will pull out ~10,000 gene regions (parts of exons) that are known to contain snps from each individual. I will be able to sequence those specific regions in a subset of individuals and then place those genes on the genetic map. In the end I will have actual genes that associate with growth.

I've never designed a custom capture before but J~ has and so with some guidance I'm figuring it out. My goal is to have a list of sequences, each around 200bp long (I should check that length - it needs to be long enough to sequence on Illumina) that have a diagnostic snp in the middle somewhere.

The first step is to find genes that have snps in them and I have done this earlier for my F1 placental transcriptome project.
Now that I have a set of genes with known snps, I need to make sure that none of these fall near exon boundaries. To do this I am using a Blat approach. Blat is nice because it will align my transcriptome to the Mesocricetus auratus genome and split up the genes into different exons. The output of a blat gives me the exact breakpoints in each gene so that I can choose snps that are far from those areas.

Then, once I have the list of genes that have snps far from breakpoints, we send that in to agilent (I think we're using agilent) who will actually design the capture for me. Then it just remains to do the labwork and sequence it.

Parent-of-Origin Effect paper was just accepted!!

My very first first-author paper has just been accepted by the journal Evolution!

It's now going through the typesetting process. They publish the pre-formated articles here and while it's not up now, it should appear soon.

They said expect ~12 weeks for the final proofs to be ready and then I expect there is a queue before it shows up in print.

Generally, it's a description of the parent-of-origin growth effects in dwarf hamsters and the first dalliances of mine to understand the genetic basis and answer the question: is imprinting involved or not?

I'm really excited. Now on to the writing of my second paper...

Parent-of-Orign Growth Effects Paper

J~ and I recently submitted my first first-author manuscript to Evolution. The reviews came back fairly positive, but there were some things that they would like changed. In total, I thought the reviews were really reasonable and I've already started collecting some extra data.

The main issue that they had was that I present evidence that some genes are properly imprinted in hamsters, some are polymorphic in their imprinting status within cross types, and some are expressed from two alleles in both hybrids. None of these patterns can explain POE growth as in all cases the two reciprocal hybrids show the same patterns. The reviewers were concerned that as I don't know the imprinting status of these alleles within each species, it is hard ("impossible" was the word they used) to interpret whether imprinting is breaking down in the hybrids. To address this apparent shortcoming, I'm planning on doing two things. First, making the writing clearer. I need to make it clear that if we see the same pattern of expression in hybrids, it cannot explain POE growth, regardless of whether the gene is imprinted in the parentals or not. While I think that should be sufficient, I am also planning on collecting more data in the form of a qPCR experiment. If I can show that the expression level is the same between parentals and hybrids, that is good evidence that the gene is not disrupted in the hybrids, and is therefore not imprinted in the parents. This should be easy(-ish) data to collect and so I'm going forth with some qPCR experiments.

There were other minor things, but all can be addressed in the text fairly easily. Hopefully I can turn this around and get it back to the reviewers soon.

DDIG

It's been a while since my last post. I've been working on a couple things recently that I should start writing about, but first, I should announce that I was awarded the NSF DDIG. The award money will go towards mapping the maternally inherited factors that result in POE overgrowth in dwarf hamsters.
The crossing scheme is this: make a small F1 (cam x sun) and then back cross those females to a campbelli male. See the figure.

I am focusing only maternally inherited factors because F1 hybrid males are sterile so I cannot do an F2 cross or a sun x F1 male backcross. As should be clear in the figure, the variation occurs due to recombination in the F1 female between P. campbelli and P. sungorus chromosomes, but that variation is only inherited through the mother, I cannot send variation through the father.

While it's kind of a bummer that I will be blind to anything that causes POE overgrowth when inherited from the paternal line, it's not the end of the world. We expect that maternally inherited things that are silenced are growth factors and their failure to be silenced is what causes overgrowth. In other words, we don't have an a priori expectation that paternally inherited factors are involved.

The grant money will support a RAD digest (Peterson et al. 2012) and Illumina sequencing in order to build the genetic map and map the causative factors. The shortcoming of using RADs is that they are anonymous markers - I don't know what genes are near them in the genome. In order to actually localize genes, I will do two things: (1) use a custom-designed array capture to sequence exons from the mapping panel and associate them with the anonymous markers  on the genetic map and (2) use RNAseq on the placentas of small and large backcross offspring to associate genes that are mis-expressed in those individuals with the genetic map.

I'm pretty excited about this whole approach as I think that it will give me the ability to get almost down to the gene level without fine-mapping. We'll see if I'm right or not, but it's pretty exciting that NSF is impressed enough to give me monies for it.

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Peterson, B. K., J. N. Weber, E. H. Kay, H. S. Fisher, and H. E. Hoekstra. 2012. Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLOS ONE 7:e37135.

DDIG and the Government Shutdown

I'm currently writing my DDIG (Doctoral Dissertation Improvement Grant) which will be submitted to NSF. It's going pretty slowly, but hopefully I'll have something worthwhile here shortly. Fortunately for me, the House Republicans were kind enough to shut the government down and give me a couple extra days to write. Thanks John Boehner! Thanks Ted Cruz! You guys are the best!

New Hamsters!

We just received a new strain of Siberian dwarf hamsters. They are an inbred line from Bruce Goldman at UMASS Amherst. They are reported to change color reliably and, as they are a different collection from the stocks that we've been using so far, they should be genetically distinct from ours to some degree.

I'm hoping to use them to assay imprinting within P. sungorus if they are different enough. Of the projects that I've done so far, the main shortcoming is that I do not know which genes are imprinted in hamsters and which are not. I have to guess based on the genes that are imprinted in house mice, deer mice, and humans. This is a problem because we know that different species imprint different genes. For example, deer mice do not imprint Mash2 while house mice do. Of the genes I surveyed in our original strains of dwarf hamster, there were three that appeared to not be imprinted, but that may instead be due to imprinting breaking down in both reciprocal hybrids. Using this new strain of P. sungorus I should be able to determine which genes are actually imprinted within that species.

This will of course depend on whether there are an appreciable number of nucleotide differences between the two strains. In order to determine whether an allele is imprinted, I have to find at least one diagnostic SNP within the expressed region. If these two strains are very closely related, they may not have such diagnostic SNPs. However, I think that they likely will as between P. campbelli and P. sungorus there are 3-6 diagnostic sites per kilobase and those two are relatively recently diverged. The standing polymorphism within one species should be less, but likely still high enough for there to be at least 1 SNP in each exon. I am assuming that these two independent collections will have sampled different subsets of the standing species-level polymorphism and so will have enough differences to answer my question.

candidate imprinted gene sequencing experiment

I have a paper that I'm working on in which I have sequenced the cDNA from 8 candidate imprinted genes in both species and the hybrids. I did this in order to determine whether imprinting is breaking down at these loci. I found that there is no signature of imprinting breakdown in 7 of these genes (the eighth had no diagnostic differences) in 6 hybrids (3 P. campbelli x P. sungorus and 3 P. sungorus x P. campbelli). However, we have since realized that there may be sex-specific issues and as the original 6 ended up being 4 males and 2 females (we had no way to sex-type the offspring at the time) we have decided to repeat this experiment with 3 males and 3 females.

Fortunately I have all the RNA extracted for the next-gen project and so all I had to do was synthesize cDNA and amplify then sequence the genes of interest. I'll update with results.

So far 6 of the 7 candidates that have diagnostic fixed differences have been sequenced. The results remain the same as before: no breakdown of imprinting in one hybrid: there are some genes where breakdown occurs in both hybrids and some where imprinting is correctly maintained. Furthermore, there are no differences between the two sexes in the pattern of expression.

The 7th gene, Peg3, is not amplifying and so I can't sequence it. It is an important gene to assay because it is one of the main partners in the deer mice that cause parent-of-origin growth (Vrana et al, 2000). It is frustrating that it won't amplify reliably as samples I ran a year ago were great. The primers do not include an intron and so the first thing I will do is to try the primers on gDNA instead of cDNA - maybe the gene is not expressed highly in the placenta of hamsters and that is why the PCR won't work.

update 7-15-13: The PCR with gDNA worked fine, not a brilliantly blazing as the first time I did it, but not bad. This leaves me with the question of whether the original PCRs had gDNA contamination with the RNA. I think that this is not the case as Peg3 showed imprinted expression in those samples. If gDNA was a contaminate, the sequences would have been heterozygous and shown what I would have interpreted as biallelic expression. The only way imprinted expression could be found is if the gene is truly imprinted and there was no gDNA contamination.

Now the problem remains of how to get the Peg3 primers to work on cDNA like they did before. I have increased the extension time by 20 seconds in case the full 1000bps weren't being copied and I increased the cycle number from 35 to 40. Although I'm a little skeptical of amping anything 40 cycles, if there are faint bands there, 40 cycles should make them show up.

update 7-16-13: All the cDNA bands are really faint if they're there at all. I'm pretty sure the positive is present, but it's kinda shitty all the way around.  E~ suggested that I should do a comparison between the gDNA of the original sequences and the gDNA of the one's I'm struggling with now to tell if there has been a problem with the primers (all gDNA's fail) or perhaps a SNP in the priming site (new gDNAs fail, old ones work). Although this is going against the Prime Directive (Science's General Order no. 1: only change one variable at a time) I am using new Taq too. This is partly because we are out of the old, and partly because I think the old may not be working. Here is my  predictions/interpretation table:

IF:                                               Then:
Both old and new gDNA fail     primers are bad, reorder primers
Old gDNA works, new fails      SNP in the priming site, redesign primers
Old gDNA fails, new works      WTF, I have no idea what this would mean, I hope it doesn't happen...
Both old and new gDNA work  old Taq was bad, the new is good, re-try with cDNA

update 7-30-13: here is the gel:

Land 1 is the ladder, lanes 2-7 are the samples, the first three are the old gDNA, the second three are the new gDNA, lanes 8 and 9 are the positive and negative control, lane 10 is another ladder. 

All the gDNAs worked, so I will assume that the issue was bad Taq and tomorrow I will run out the cDNA with the good Taq.

update 8-1-13: Didn't work. The old cDNA still doesn't amplify. Try making new cDNA

update 8-14-13: I figured it out! I had cleaned the cDNA in the previous trials and since I had started with so little, I lost most of it. This time I did not clean the cDNA and have beautiful bands. Now for sequencing!

Lane 1 is the ladder, 2-18 are Peg3 from cDNA, 19 is the positive control and 20 is the negative control.

Such a ton of work for so simple a problem. I'm just glad it's working.

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Vrana, P. B., J. A. Fossella, P. Matteson, T. del Rio, M. J. O'Neill, and S. M. Tilghman. 2000. Genetic and epigenetic incompatibilities underlie hybrid dysgenesis in Peromyscus. Nat Genet 25:120–124.

RNAseq Libraries sent!

I finished pooling my samples (lesson learned: don't use small volumes it evaporates as you pipette) a couple weeks ago and they've been sitting in the fridge ready to ship off just waiting for the finances to come through with the University of Utah. All the paperwork was finalized yesterday and we just missed the shipping time (dropped the package off at 4:05 for a 4pm pickup). We had to pick it back up and hold onto it until today. We'll add some more dry ice and send it off this afternoon.

It has been a ton of work to get that 30ul ready for shipping. Now we cross our fingers and hope that it comes out all right. In the mean time, I think I'll have a beer.

Evolution Conference 2013 - Tuesday

Many more talks today, here's some notes on the ones that stood out:

Jennifer Kovacs told us about a cool ability of the endosymbionts of aphids and how they defend against ladybugs. When a female ladybug eats the infected aphids, she suffers no immediate consequences, but her offspring show a reduced ability to successfully pupate. Those that do pupate are on average larger than normal which may be due to a “filter” effect where the smaller, weaker ones were weeded out by the bacteria. As aphids reproduce clonally, the relatedness between two aphids on the same leaf is 1. This means there is strong selection to stop the adult ladybug’s offspring from consuming other aphid clones. The only hole in the story so far is that no one has been able to show that the aphid’s bacteria survives the adult ladybug’s stomach and gets into her ovaries. Somehow it must get into her eggs, but they haven’t been able to show that yet. Still a really cool story of host defense. 

Taichi’s talk was fun too. He’s looking at Bergman’s Rule (body size increases with latitude) and how it correlates with the gut microbiome. He’s showed that especially in the east coast there is a strong correlation between composition of microbes and latitude that also correlates with average body size in house mice. There is less of a pattern on the west coast, but that is likely because the colonization was much more recent and there has been subsequent gene flow from Mus spretus that may be confounding the pattern. 

Jamie Zuniga-Vega talked about superfetation in live-bearing fish. Superfetation is where one female carries many broods at different developmental timepoints concurrently. There are three main hypotheses for superfetation which are: (1)  it lowers the peak cost of reproduction - at any given time, female must invest less per unit time. (2) It results from morphological constraint - many offspring take up a lot of space forcing the female to not be hydrodynamic anymore, if the offspring are at different stages, they take up less space. And (3) it compensates for high adult mortality. If there is a high probablility of adult mortality, high fecundity may be able to comensate. Thus superfetation may increase rate of offspring production rate. They found that hypothesis 2 is best model for two species where fast water causes higher rates of superfetation, but the third species doesn't have anything that shows preference of one hypothesis over another.

Keenan Morrison then talked about anamniotic eggs and how they may predispose species for the evolution of matrotrophy.  Amniotic eggs are impermeable and cannot absorb nutrients, but anamniotic eggs can assimilate resources across the “shell.” As these eggs already can accept nutrients from the environment, it is easy to make the next step to accepting nutrients from the mother’s uterus.

David Reznick followed the previous two talks and discussed how placental or matrotrophic fish should have greater male-female conflict. Furthermore he argued that placentas should correlate with long gonopodium (penises), small body size, lack of courtship, and sneaker mating strategies, non-placentals on the other hand should show strong sexual dimorphism, elaborate mating rituals etc. I was unclear on his logic as to why this is true (can’t placentals have pre-mating sexual selection as well as post-mating conflict?) and am looking forward to reading a paper he will hopefully publish ont eh topic (I felt better about my confusion as Doug was also unsure of his logic here). Surprisingly, he has found that there is an elevated speciation rate in non-placentas compared to placental fish. This is opposite of what I would have expected given the opportunity for maternal-offspring conflict in placental species. The species that do not have placentas instead show elaborate mating rituals and are characterized by strong premating sexual selection. This may then go hand-in-hand with the observation that birds, where strong premating sexual selection/sexual dimorphism is common, speciate twice as fast as mammals, where developmental conflict is more pervasive. Reznick also found that there is no support for “adaptive hypotheses” of the origin of placentas, but I’ll need to see his paper to remember/understand why he thinks so. Hopefully the paper with come out soon. 

I was lost with Turelli’s talk. He went fast and had a ton of words on his slides. It sounded impressive, but I couldn’t follow anything he was saying. 

Rob Unckless’ talk was a great conclusion for the day. He talked about the drive system in Drosophila affinis. He and I had spent many hours talking about this system back in the Jaenike lab and creating recursion equations to model it. the equations we made then were comparatively quite simple - one driver, one suppressor. What he talked about today was how this system may maintain polymorphism of the Y chromosome given many drivers and many suppressors. Apparently he had to make a perl script to generate the recursion equations as they got so complicated. I am going to invite him out for a talk in the spring if I can manage it. 

The last talk of the evening was Jack Sullivan’s SSB presidential address. It was really fun and he showed great pictures of bacula and was quite entertaining. Much of his talk was work done by J~ either as his masters student or recently in collaboration. He ended with the statement that you should always treat your students well so that you can exploit them years into the future. 

I got to hang out with Rob and Yaniv, Erica, Mike Shapiro, Yasir, Daniel Matute and a bunch of others in the evening which was a blast.  All in all it was a superb conference.  

Evolution Conference 2013 - Monday

Lynda Delph talked about how she found Haldane’s rule in a species of plant that has sex chromosomes. I got a little lost with all of her logic so I should probably read her paper, but I think she was arguing that the dominance theory does not explain male rarity while both dominance theory and the faster male theory serve to explain pollen sterility. I’ll have to look up her paper though to see the actual data again before I make too many claims about what she said.

Yaniv’s talk went well too, he described some ngs data he has on M. nasutus and M. gutattus and some patterns of introgression that he found.

The highlight of my day was a talk by Matt Brandley who was looking at placentation in squamates, specifically skinks. He was trying to figure out how the evolution of viviparity occurs and used a species of skink that shows polymorphism for viviparity to compare uterine gene expression. He found that there was not much different at all between the two reproductive modes but thought that due to pretty stringent penalties against high variance and a large sample size that had high variance in expression levels that he may have missed a bunch of true differences. He said that he had just gotten the data back and hadn’t had time to really play with it much yet. In a previous paper he’s described some differences in uterine gene expression in another skink and compared it to mammals and found that many of the same genes are used to facilitate live birth. For example both systems must repress the mother’s immune system to keep her from rejecting the offspring. 

Lila’s talk was fun too, it was good to hear about Margaret’s old project looking at thermal adaptation in mimulus in Yellowstone park. 

There were no talks in the afternoon and I headed up the tram with Taichi and Katya to hike down the mountain, ended up meeting Rob up there and eventually Kris. We spotted a marmot and a mountain goat which was pretty fun. Below are photos of the view from the top and a golden manteled ground squirrel who was interested in some peanuts.

My poster session was in the evening and I was pretty worried that no-one would show up after the afternoon’s break, but it was packed. I had about 10 minutes in the beginning free and after that it was one person after another. Turelli came and chated as did Kristi Montooth and Nitin as well as a number of other folks. Had a couple really good chats about imprinting and ligers and how the dominance theory may explain growth disorders involving imprinting. Afterwards Kris and I went and hung out with Doug and Matt and Paulo in the bar at the hotel and ended up heading home early as we were pretty exhausted after 3 hours on the mountain and 2 hours of hard poster-sessioning.

Evolution Conference 2013 - Sunday

I saw a long-tailed weasel near the main tent later in the day which was pretty exciting and Matt saw a moose in the parking lot that Kris and I ran down to try and find but couldn’t.

Talks-

Amy Dapper just presented some theory that the pattern of evolution of male reproductive genes is more consistent with a null of relaxed constraint rather than positive selection. It was quite interesting and based on a couple observations such as that sex-specific gene expression causes the selection coefficient to be reduced by half and that genes that select for highly competitive sperm are only actually beneficial when the female mates multiply (and with males who have different alleles at those loci). This causes the strength of selection to be scaled down even further by the harmonic mean of the number of mates. Her main result was to show that recently published dN/dS ratios that seem so high in male reproductive genes fall exactly on the null expectation of dN/dS that she calculated. It was interesting and she said that it should be published by the end of the summer. It may make a fun lab meeting paper to discuss once it's out.

David Gokhman presented new data on the epigenome (methylome specifically) of the neandertal and denisovan. He used the natural degradation of C->U and Cmeth->T to determine which bases in ancient dna was methylated. found that of the regions differentially methylated between humans and neandertals (Hox8,9,10) all control bone growth and expression correlates with patterns of neandertal morphology and known human pathology. Really cool.

Went to Zach Gompert’s talk - way over my head. I followed the selection equation parts and then once he actually got into the model I lost it.

Also attended Joan Roughgarden’s talk. Was not impressed. 

Matt Hahn discussed genomic islands of speciation and described how the divergence in these islands - when measured in an absolute manner, is incredibly low. The point is that while there is likely something - selection possibly -  driving the patterns of Fst that we identify as “islands” it does not mean that there is speciation with gene flow occurring. 

Dolph Schulter’s ASN presidential talk was interesting. He talked about the latitude-speciation correlation and if it exists or not, decided that ecological opportunity (which we can’t really measure) is really what drives everything. Talked about how the stickleback fish species are the result of repeated fixation of standing variation and how that doesn’t align with the standard thoughts about DMI’s - he has wide-scale additivity across many genes that seems to isolate species. Claimed that it was much different than the classic DMI model.

Evolution Conference 2013 - Saturday

Kris and I arrived late Friday night. It’s good that we had Siri to navigate for us as we would have been completely lost otherwise. Turns out that Brice got one hell of an awesome mansion for us to live in for the next couple days.  

Driving in in the morning, we saw a coyote - great way to start a biology conference. 

We got a bit lost on the way in - it was not immediately obvious where the conference actually was, but we finally found it and got registered.

Here’s some talks that I went to that really stood out:

Daniel Matute - great, talked fast and I am not 100% familiar with all the molecular techniques that you can use in drosophila, so I got a little lost in the end. He is using deficiency mapping technique to find the genes that isolate melanogaster/yakuba (I think it was yakuba, but now that I write it I’m not really sure). Inviability depends on x-x interactions as well as a 3rd locus somewhere else. Should have typed this yesterday directly after the talk as I can’t remember the specific details. I talked with him later and it turns out that he’ll be starting as a professor soon and is looking for grad students.

Robin Hopkins discussed reinforcement among flowers in texas: blue in most of the range but red in the area of overlap. She had me pretty convinced that it was reinforcement: hybrids show really low fitness, but are common when flower color is the same between the two species.  Also got into the mechanism of reinforcement: the butterfly pollinators have a preference for flowers of the same color - some like red, some like blue, but the ones that like red always go to red and the ones that like blue always go to blue. The one thing that I was confused about is that the preferences I just described operate in the area of sympatry, but not in the area of allopatry where pollinators have a distinct preference for blue. I’m not sure why it would be different as it’s the same species of butterfly that does the all pollinating. She also got into the genetics of flower color change from light blue to dark red, it’s a 2-locus, each with complete dominance (the standard 9:3:3:1 ratio). One loci controls brightness, one does red/blue. Yaniv asked afterward it it may be collapse rather than reinforcement though, but I had to run to the next talk and didn’t hear what she said. I’ll try to talk to Yaniv about it.

Matt Jones’ talk was great, it will be fantastic to have him in the lab.

Amanda Moehring claimed to have found the first speciation gene for a behavioral trait in melanogaster but she didn't actually present any data, only asserted that it had an effect. I found it really frustrating  as she showed images of her deletion mapping, but not any actual data about what the region she found actually does. Many of the speciation people were there (Matute, Noor, Yasir) just shaking their heads at her the whole time. It was one of these  extraordinary  claims that have no evidence supporting it. I talked to both Daniel Matute and Yasir about it later and neither were impressed. I’ll be looking up the papers and possibly writing more about it later.

I realized that I printed the wrong version of my poster and had to skip the poster session to re-print it, but I ended up getting together with Yasir and Erica and Ryan and Matt for some beers later.

Great first day.

Diluting and pooling - last step

I have now prepared and amplified all my samples. I used 12 cycles and 8ul of product so that I wouldn't blow through the entire 20ul of library if something went wrong. Most of these had plenty of product, the couple that didn't I re-amplified for 14 cycles. For the dilution, I used the values from the nanodrop rather than the bioanalyzer as Sara has had some issues with pooling based on bioanalyzer values.

Here are the bioanalyzer traces of the completed pools:

The final concentration is a little low, but as the sequencing facility only needs ~10ul of 10nmol/L it should be fine. I actually ended up making 4 pools as the first two didn't stack up well on the qPCR. I think this was because I was pipetting such small volumes that the liquid was evaporating and changing my concentration. For the second set (Pools 3 and 4, seen above) I made sure that my sample volumes were never less than ~10ul. It seemed to work much better. Here's the qPCR trace of 5 of my samples. They should all be right on top of each other (except for the light blue one which is the no-template-control:

There is only about 1 cycle spread from the first one to the last, whereas the pools 1 and 2 had at least a 4 cycle spread. 

S~ is now setting up the account at Utah and I'll be sending the samples as soon as all the paperwork goes through. (We've had some issues with sequencing at Berkeley and so we're gonna try a new facility.)

Epigenetics and a new direction

I have been keeping an eye on a couple other science blogs - homologus and Judge Starling among them, and I have decided that I should include among my descriptions of experiments an occasional review of some bit of literature that I find interesting. And so:

PNAS recently published two papers: Epigenetics, a "Core Concepts" piece by Sarah Williams (2013) and Epigenetics: Core Misconcept by Mark Ptashne (2013) where he argues that Williams is perpetuating some of the main misconceptions about epigenetics. In order to analyze this little spat, I will first define epigenetics and then analyze what each of the authors say and whether Williams is indeed perpetuating misconceptions. This disagreement boils down to another fundamental argument over whether DNA methylation and/or histone placement should be considered "epigenetic mechanisms" and I will conclude by pitching in my two cents in this debate.

Here are a couple of current definitions of epigenetics from the literature:

From Bird (2007): "... epigenetic events... [are] the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states."

From Eccleston et al (2007): "Epigenetics is typically defined as the study of heritable changes in gene expression that are not due to changes in DNA sequence."

From Sasaki et al (2008): "Epigenetics refers to a collection of mechanisms and phenomena that define the phenotype of a cell without affecting the genotype"

From Bonasio et al (2010): "Epigenetic signals are responsible for the establishment, maintenance, and reversal of metastable transcriptional states that are fundamental for the cell’s ability to “remember” past events, such as changes in the external environment or developmental cues."

These definitions all agree that epigenetics does not include changes to the base sequence of DNA, but rather changes in the expression level of different genes. Said another way, epigenetic changes must be reversible: coding changes are not reversible (a back mutation is quite rare) while expression changes are (it's relatively easy to turn genes on and off). These definitions also all agree that these expression changes must be heritable from one generation to the next (it should be noted that this is most often cell generations i.e.: heritable through mitosis, rather than organism generations, though both are applicable).

One other condition is also commonly found in the literature and is necessary for an epigenetic system: it must be self-perpetuating (Riddihough and Zahn 2010; Bonasio et al 2010). Self-perpetuating means that there is a positive feedback loop somewhere such that once it is initiated and even in the absence of the initial signal, the new expression level will persist.

In summary: there are three characteristics that are necessary for a system to be labeled "epigenetic:"
1.) It must be reversible by dealing with changes in expression level not sequence identity.
2.) It must be heritable, at least through mitosis, possibly through meiosis and fertilization.
3.) It must be self-perpetuating in the absence of the initial signal.

The predominate biological process that this refers to is development where a single cell divides and differentiates into a large number of distinct tissue types. At the end of the process, each cell in every tissue has the complete genome of the organism, but each cell type has it's own specific set of genes that is expressed. This is one of the most incredible - but rarely-recognized - cases of plasticity: a single set of genes gives rise to a vast number of different cell types and functions. Epigenetics is concerned with how, for example, a liver cells divides into two new liver cells. Certain genes are on in each liver cell that, when present in the daughter cells, cue those to also express the suite of "liver genes." Thus, this aspect of development complies with the heritable and self-perpetuating requirements of epigenetics. It is also reversible as no coding changes have occurred - only expression levels change, not the DNA sequence. We know this is true because the entire genome is present in every cell - it is certainly NOT the case the all of the "non-liver genes" are destroyed or excluded from future liver cells. This is depicted in the following diagram (from Ptashne 2013):

The blue circle protein (a transcription factor) activates the gene for the red oval protein (also a transcription factor) by binding to a cis-regulatory element (blue box). The red protein activates itself by binding to the red cis-regulatory element and also activates a suite of other green genes by binding to their cis-regulatory elements (red boxes). 

So what are some concrete examples of epigenetics? The prime example is a transcription factor that activates the expression of itself (self-perpetuating) and also activates the expression (reversible) of a suite of other genes as in the above figure. When this transcription factor is in the cytoplasm of a cell undergoing the cytokinesis phase of mitosis, it will be allocated into the two daughter cells (heritable) and will thus cause them to express a similar suite of genes as the parent cell. Another common example is prions. Prions are poteins with a specific tertiary conformation (reversible) that cause other proteins (self-perpetuating) to assume the same confirmation (heritable). Though prions seem to me to be in a slightly different class of epigenetics than transcription factors as prions don't involve DNA at all. This is not to say that prions are not epigenetic - they most certainly are - but rather that the term epigenetic is quite broad. I will from here on concentrate on the subset of epigenetics that deals directly with gene expression.

Moving on to Williams' (2013) Core Concept article, I found it to be a bit brief but not horribly off the mark. Williams focused on only two types of epigenetic inheritance: DNA methylation and histone placement and she did not go into detail about the mechanism by which these epigenetic changes occur. Her argument mainly concerned the reversible aspect of epigenetics: the fact that differential methylation (or differential histone placement) results in differential gene expression. She also asserted that these 'flags' on a bit of DNA are copied along with the rest of the genome during the S phase of the cell cycle and are therefore heritable. She did not directly address whether methylation or histone modifications are self-perpetuating, though the argument is that due to the action of methyltransferases the methylated sites are indeed perpetuated, and hence the expression profile of that site is maintained.  Specifically, De Novo Methyltrasnferase 1 (DNMT1) finds DNA where a methyl group is on one strand but not the other (as woud occur durring DNA synthesis) and it methylates the second strand. This results in two daughter cells each with accurately-reproduced methylation patterns and hence similar expression profiles as the parent cell.

Ptashne (2013) takes issue however with Williams' (2013) description of epigenetics. In doing so he puts forth two criteria which together are both necessary and sufficient to call something "epigenetic": these are memory and specificity. Ptashne's "memory" is a combination of the earlier "self-perpetuating" and "heritable" - a distinction that often becomes blurred (for instance: see the preceding paragraph - my argument for heritable and self-perpetuating are actually the same). As such I agree with Ptashne (2013) that the more general term of "memory" is useful. Technically, memory is the continuation of a specific expression profile of a given gene. Under this criterion DNA methylation is an epigenetic mechanism as DNMT1 faithfully transmits the signal to new molecules. On the other hand, histone placements are not known to be heritable or self-perpetuate, even though they do seem to correlate with gene expression, and so should not be considered "epigenetic" according to Ptashne (2007, 2013).

Both DNA methylation and histone placement also fails Ptashne's (2013) second test of specificity. Specificity has to do with which genes a give transcription factor acts upon: for instance in the earlier figure, the red circle only acts on the blue oval's regulatory region, NOT on any of the geen genes' regulatory regions. The opposite of something being specific is for it to be general in which case it acts ubiquitously across the genome. This is the sense in which DNMT1 fails the test: any time it finds a bit of hemi-methylated DNA, it methylates the unmethlyated strand regardless of location. Histone placement also fails this test, as according to Ptashne (2007) we have no evidence that histones are placed specifically. Ptashne states: "... it is said [that] chemical modifications to DNA... drive gene regulation. This obviously cannot be true because the enzymes that impose such modifications lack the essential specificity: All nucleosomes, for example, “look alike,” and so these enzymes would have no way, on their own, of specifying which genes to regulate under any given set of conditions." (Ptashne 2013). Clearly Ptashne takes issue with Williams' claim that DNA methylation and histone modifications are epigenetic mechanisms.

I disagree with Ptashne, and think that histone modifications and DNA methylation should be considered epigenetic. I will discuss each of these in turn, first DNA methylation. To be fair, Ptashne (2013) does opt-out of making a call on the methylation side of things, citing that its role in development remains unclear because it doesn't occur in the flies or worms - which he seems to consider the two "real" models for development. I can only conclude that Ptashne is slightly ignorant of the current findings in mammal models (yes mammal models for development do exist, even if Ptashne doesn't consider them "real").  Sasaki et al published an entire review in Nature Reviews Genetics five years ago (Sasaki 2008) that describes most of how trans-generational epigenetic events are established in the germ-line. One of the main things that happens during germ cell development is the erasure and reestablishment of the parental imprint. Imprinting is regulated by DNA methylation in the imprinting control region and fits both of Ptashne's requirements: memory because the methylation established at this point in development regulates gene expression throughout life, and specificity because there are only a subset of genes that are imprinted - it's not a ubiquitous genome-wide process. So contrary to what Ptashne (2013) claims, we do know plenty about DNA methylation in mammalian development and furthermore it does fit both of his requirements. That being said, we still do not have a solid understanding of exactly how the specificity is achieved - there is not a DNMT specific for each region that needs to be methylated (indeed there are only 3 DNMTs that actively methylate DNA). Presumably there is some type of factor that is specific to certain regions and recruits the DNMTs to specific areas. DNMT3L may fill this role as it does not actually methylate DNA, but instead complexes with DNMT3a and facilitates proper methylation (Sasaki 2008). This DNMT3a-DNMT3L complex also relies upon a specific histone (H3K4 allows methylation while H3K4me does not)(Sasaki 2008), and so specificity may be achieved due to an interaction between local histone type and the DNMT complex. In short DNA methylation certainly counts as an epigenetic mechanism of transcription control.

Ptashne (2007, 2013) also claims that we have no evidence that the pattern of histones is heritable and so should not be considered epigenetic. On the contrary, it is true that allele-specific histone patterns are conserved within tissues and so must be heritable. For example, histones (not DNA methylation in this case) are the primary drivers of imprinted expression of the Kcnq1 region of Chromosome 7 in mice (Umlauf et al 2004; Lewis et al 2004). This shows that histones do fit the specificity criterion - they are allele specific, as well as the memory criterion - imprinting is tissue-specific (and *naturally* allele-specific). As far as I can tell, we do not know how this specificity is achieved, but just because we don't  know how it's achieved, doesn't mean that it is not indeed specific.

In conclusion, I consider both differential DNA methylation and differential histone placement to be true epigenetic mechanisms of gene regulation as both of these processes are heritable, reversible, self-perpetuating, and specific.

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Bird, A. 2007. Perceptions of epigenetics. Nature 447:396–398.

Bonasio, R., S. Tu, and D. Reinberg. 2010. Molecular Signals of Epigenetic States. Science 330:612–616.

Eccleston, A., N. DeWitt, C. Gunter, B. Marte, and D. Nath. 2007. Epigenetics. Nature 447:395.

Lewis, A., K. Mitsuya, D. Umlauf, P. Smith, W. Dean, J. Walter, M. Higgins, R. Feil, and W. Reik. 2004. Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation. Nature Genetics 36:1291–1295.

Ptashne, M. 2007. On the use of the word “epigenetic.” Current Biology 17:R233–R236.

Ptashne, M. 2013. Epigenetics: Core misconcept. Proceedings of the National Academy of Sciences 110:7101–7103.

Riddihough, G., and L. M. Zahn. 2010. What is epigenetics? Science 330:611.

Sasaki, H., and Y. Matsui. 2008. Epigenetic events in mammalian germ-cell development: reprogramming and beyond. Nature Reviews Genetics 2008:129–140.

Umlauf, D., Y. Goto, R. Cao, F. Cerqueira, A. Wagschal, Y. Zhang, and R. Feil. 2004. Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes. Nature Genetics 36:1296–1300.

Williams, S. 2013. Epigenetics. Proceedings of the National Academy of Sciences 110:3209.

Placental Histology

Another experiment that I'm pursuing in collaboration with L~ (an undergraduate here at UM) is to analyze placental histology of hybrid hamsters. She found that there seems to be some striking abnormalities in the layering of the hybrids' placentas. Placentas are very complex and formed from three main embryonic tissue layers (labyrinthine trophoblast, spongiotrophoblast, and trophoblast giant cells) as well as a maternal layer (decidua).

***This figure is taken from Wagschal and Feil (2006) - a great paper on imprinting and the placenta***

So far L~ has found that there seems to be a sex-specific placental abnormalities in the hybrids - only the female hybrids show major disruptions of the boundaries between the layers, especially between the labyrinthine trophoblast and spongiotrophoblast. This is quite exciting as I can find nowhere in the published literature that reports sex-specific hybrid phenotypes. It also complicates the over/under-growth story because overgrowth assorts by cross type, not sex while undergrowth is only found in males, not females. If we assume that abnormal layering of the placenta results in abnormal nutrient transfer, then L~'s defects do not explain the patterns of over/under-growth.

However, the issue we have is that the sample size is quite low - 7 males and 2 females total from both hybrid types. We are now curious if the pattern that both females show abnormal morphology is true or only an artifact of small sample size. To address this, I have just set up 10 more crosses (expectation is ~50 offspring/placentas) that L~ and I will be dissecting and doing histology to next week.

I hope to be able to present the findings at the Evolution Conference in Salt Lake in a couple months.

6-4-13 update: So far I have dissected 5 Sun x Cam (overgrown) crosses and 1 Cam x Sun (undergrown) cross resulting in 10 placentas (7 Sun x Cam and 3 Cam x Sun). I fixed these in 4%PFA overnight and used the histology lab's tissue processer to embed them in parafin. Yesterday I sectioned them and put them on slides. Now I need to stain them and then measure them.  I had forgotten how difficult and touchy sectioning is. Major props to L~ for all the sectioning she did on the earlier samples.

6-18-13 update: I have dissected everything, embedded them, sectioned them, stained them, and imaged them. I'm going to present the results at Evolution this weekend and I'll type up a post afterwards.

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Wagschal, A. and R. Feil. 2006. Genomic imprinting in the placenta. Cytogenetic Genome Research 113:90–98.