Wednesday, July 31, 2013

Evolution and Core Processes in Gene Regulation – some “terminal” thoughts (guest post from David Arnosti)

Guest appearance by David Arnosti here; I’m Ian’s colleague at Michigan State University and co-organizer for the ASBMB Special Symposium.

Our symposium last week brought together some folks who don’t regularly appear at the same conferences, including the structural biologists and biochemists who’ve devoted a good part of the last 30 years to unraveling the mechanisms of the central dogma – gene transcription, RNA metabolism, and protein translation. The trend has been to identify the central machinery, publish high-impact papers in single-word-title journals, and then years later discover that things don’t necessarily work the same in all contexts, organisms, developmental settings. Not surprising in eukaryotic transcription, for instance, where the “basal machinery” comprises ~200 proteins. Zach Burton, conference participant, refers to this complexity as a “molecular horror”, but from an evolutionary perspective, it is also an opportunity.

With respect to tracking the changes inherent in functioning of regulatory circuitry, we have more complete understanding of how bacterial systems work. Nice presentations by Saeed Tavazoie and Eduardo Groisman highlighted how easily such systems can transition between regulatory states, with just a few genetic changes in regulatory factors. Robert Landick and Seth Darst, in studies of E. coli RNA polymerase, pointed out that this bacterium actually features a novel outgrowth, an insertion of 188 amino acids that connects directly to the conserved trigger loop in the catalytic site. Nobody knows what it is doing there, but it is lineage specific, and mutations accumulate in this part of the protein when cells are grown under nutrient limitation, suggesting a short-circuit way to globally fiddle with gene expression.

Eukaryotic systems likewise have novel structures to their core machinery; Lawrence Myers described the gene amplification of Mediator subunits in Candida albicans that are linked to pathogenicity, while Jean-Marc Egly pointed out the pervasive effects of mutations in human Mediator and transcription factor TFIIH. With eukaryotes, the importance of variability in core machinery for generating important changes on the population or species level is obscure. Studies such as those presented by Ian Dworkin (host of this blog) and Aviv Regev showed how we are able to identify numerous loci involved in genetic background effects relating to development and immune function – but the overall importance of pervasive impacts generated by changes in an RNA polymerase subunit, for instance, vs. subtle changes in an enhancer is not clear. One mystery I dropped on the conferees was the special features of the RNA Pol II CTD found in Drosophila, but not other eukaryotes – a reflection of their unique developmental gene expression, discussed by Melissa Harrison and Julia Zeitlinger?

Aside from being mistaken once for Bill Gates by a visitor from Shanghai while walking around the University of Chicago, I was able to maintain my identity as a gene regulation specialist who takes to heart Theodosius Dobzhansky’s mantra that “Nothing in Biology Makes Sense Except in the Light of Evolution”. We will see how the combination of biochemical detail and evolutionary perspective can propel us into a future where gene regulation, in all its rich variation, makes sense. Many thanks to Joan Geiling and Barbara Gordon from the ASBMB for making this an outstanding conference!

Friday, July 26, 2013

Wrap-up for day one of: Evolution and Core Processes in Gene Regulation

Yesterday was the first day of the meeting on the "Evolution and core processes in gene regulation". A small conference (~75 people) interested in various aspects of gene regulation. As I mentioned in a previous post, the participants represent a really diverse mix of biologists interested in gene regulation (how, where why genes are turned on and off), including many who do not speak a common "scientific" language.

To start with, Chicago surprised all of us by having truly pleasant weather for late July. Crisp, dry and not too hot. That always helps perk everyone up.

The diversity of talks was represented right from the very first session with talks ranging from using experimental evolution to examine questions relating to evolution of regulatory function (Saeed Tavazoie), to very mechanistic analysis of enhancers that regulate different tissues in different ways (from Scott Barolo).  I did some fairly extensive tweeting (@IanDworkin, #genereg) on what was presented. However there were a few highlights (for me and my own work).

 The work from the Barolo lab tearing apart the enhancers of the Patched gene (regulated by the transcription factor Gli  mediating hedgehog signaling pathway) was really neat and I am still mulling around the findings in my head.  We know that some proteins bind to DNA, and some of these proteins (called transcription factors) help to regulate gene expression. Sometimes turning genes on, sometimes turning them off. In this case the transcription factor Gli (or as we call it in Drosophila, cubitus interruptus) can take on two forms, one called an activator, the other as a repressor. It turns out that while both of these forms bind to the same general binding sites (DNA words  that sometimes can be spelled a bit differently analogous to "colour" and "color"), how tightly they bind (depending on the spelling) matters. More importantly it matters that some of the sites allow the protein to bind tightly, and others weakly. Having the weak binding sites matters (and it matters that they are weak).

I also really enjoyed the approach that Nir Yakoby was taking to understand the evolution of developmental mechanisms underlying how the shell of the egg for fruit flies was patterned. His lab is taking both a cool approach to thinking about regions of gene expression (gene X is expressed here, here and here, but not here), and how it relates to evolution of the eggshell.

Justin Fay (one of the organizers), also talked about one of his model systems using yeast, and how variation throughout one particular gene (FZF1) was important for yeast to grow in the presence of sulfite (think wine - yeast - sulfites....). The take home message (for me anyways) was that the whole gene had a signature of natural selection (between species), and that the differences in the DNA in many places across the gene seem to contribute to the effects with respect to how the yeast can grow with sulfite.

 There was far more to the first day to the meeting (check the twitter feed for details) and I can not do it all justice, but hopefully this gives you a small flavour for the meeting. If you attending, post about what got you excited.

Wednesday, July 24, 2013

Genetics really is hard (to interpret)

I am sure this will not surprise most of you, but genetics research can be really hard. I don't simply mean that doing genetics experiments is hard (which it can be), but interpreting the results from genetic analysis can be difficult.  This post is about an interesting story involving the analysis of a a gene called I'm not dead yet (Indy) in the fruitfly Drosophila (one of the geneticists favorite organisms) and its role in extending lifespan. This story, that has taken place over the past decade has taken a number of interesting twist and turns involving many of the subjects that I like to discuss in this blog and my own work, including trying to make sense of the results from genetic studies, the influence of factors like genetic background and environment on mutational effects, and of course Drosophila itself. While I do not study lifespan (longevity), I have been interested, and following the story for this research over the past 5-6 years because of the implications of the influence of genetic background effects (which I do work on).  I should also mention that other than being a geneticist I do not claim to have any great knowledge of the study of aging, but I will do my best on that.

I hope in this (and future) posts to accomplish a few things, so I thought I would lay them all out first (in case I start to ramble off in strange directions).

  1. Describe a cool story about something important to just about everyone (who does not want to find out how to live longer).
  2. Discuss the means and logic of how genetic analysis. That is how we (geneticists) go about figuring out whether a particular gene (or variant of a gene) influences something we care about (like how long we live).
  3. Context matters a lot for genetic analysis. Factors like the food used to feed your critters (among many others factors), and the genetic background (of the critters) that the mutation is studied in can profoundly change what you see (the results).
  4. Scientists, even when making honest efforts to perform good, reproducible research can get different results because of seemingly subtle differences in 2&3. 

Not surprisingly, many scientists are interested in the biology of aging, and in particular in what factors influence longevity. In addition to it being very cool, and of obvious importance to many people on the planet, it is also important for aspects of evolutionary theory. The point being that many scientists are interested, and approach questions of aging from many different perspectives, which is great. It is also not surprising that geneticists (and again the general public) are interested in finding genes that influence the aging process (why do some people live longer than others). So in the year 2000 (you know, when all of our computers did not shut down) when a paper entitled "Extended life-span conferred by cotransporter gene mutations in Drosophila" came out, there was a lot of buzz. The basic results suggested that reducing the function or expression of a particular gene, Indy increased how long fruit flies lived. While we (the people) are not fruit flies, by the year 2000 research had already clearly demonstrated that there were many shared genes in all animals (including people and flies), and many seemed to have pretty similar functions. Thus explaining the excitement and buzz. By the way, Indy is short for "I'm not dead yet", and if you do not get the reference check this out (start at 0:58 if the two minutes is too long), or here if you prefer it in musical form, or here as a cartoon.

So what did they do in this study? The punchline is that using multiple, independently generated mutations they demonstrated that as you reduced Indy expression and function, the fruit flies lived for a longer time (increased longevity) when compared to the fruit flies with normal (wild-type) copies of the Indy gene. Seems straightforward enough, and by using multiple independent mutations they demonstrate (at one level) the repeatability of the results. That is, there results are not some strange one-off random results, but can be reproduced, which provides some degree of generality to these results.

Of course, results are rarely so simple and clear, and with additional investigations layers of complexities are often demonstrated.  Studying longevity can be particularly difficult, and not only because you will have to wait a long time to see when something dies of natural causes.

So does Indy actually influence lifespan? The short answer is that the results from follow up studies have been pretty mixed, so it is perhaps not as clear as hoped from the original study. More on that soon in subsequent posts!

References and links if you want more information from the original studies
Rogina B, et al. (2000) Extended life-span conferred by cotransporter gene mutations in Drosophila. Science 290:2137–2140.

Toivonen, et al. 2007. No influence of Indy on lifespan in Drosophila after correction for genetic and cytoplasmic background effects. PLoS Genetics. 3(6):e95

Wang, et al. 2009. Long-lived Indy and calorie restriction interact to extend life span. PNAS USA. 106(23):9262-7. doi: 10.1073/pnas.0904115106

Toivonen JM, Gems D, Partridge L. Longevity of Indy mutant Drosophila not attributable to Indy mutation. Proc Natl Acad Sci USA. doi: 10.1073/pnas.0902462106.

Helfand, SL. et al., 2009. Reply to Partridge et al.: Longevity of Drosophila Indy mutant is influenced by caloric intake and genetic background. 106(21): E54. doi:  10.1073/pnas.0902947106.

Frankel. S. & B. Rogina. 2012. Indy mutants: live long and prosper. Frontiers in Genetics. 3(13). doi: 10.3389/fgene.2012.00013

Rogina B, Helfand SL. 2013. Indy mutations and Drosophila longevity. Front Genet. 4:47. 
  doi: 10.3389/fgene.2013.00047

From the meeting ASBMB:Evolution and Core Processes in Gene Regulation - Chicago (July 25th-28th)

Well this will be something new for me. I will be both blogging and tweeting from the meeting on the evolution and core processes in Gene Regulation from Chicago over the next few days. I will also be giving a talk there as well.  Understanding how and why genes are turned on (or off) both in space (different parts of your body), and time (when you are growing, or as you age) remains an important question that spans a great deal of biology. It turns out that even when asking seemingly similar questions related to gene regulation, the approaches (and language) that say a biochemist or an evolutionary geneticist use can be completely different (and mutually incomprehensible). So meetings like this are really important to getting us all on the same pages and interacting with one another.

This meeting is being organized by David Arnosti, Justin Fay and Ilya Ruvinsky. The three of them (all great scientists and fun to talk with) are bringing together a very diverse group of scientists from hardcore biochemists to straight up evolutionary biologists. We did a related symposium back in 2011, and also in 2008 (at Michigan State University). It was a lot of fun then. Should be more fun (for me anyways) now, as I am not one of the organizers this time. In any case the program for the meeting can be found here.

David, Justin and Ilya also sent this to all of the speakers (to think about for their talk):

We understand that the DNA-RNA-protein dogma is sculpted by evolutionary forces, but where have experimental insights shown HOW particular features of the central machinery are shaped? Some aspects of the process of gene expression have been tightly linked to evolutionary innovations, such as variable HOX expression and limb morphology. 

In your system, which aspects, if any, of the process studied are known to reflect selective pressure? For example, for central components of gene expression machinery, are there particular features that can be linked to specific conditions or attributes of the species/developmental stage/tissue? Alternative tissue-specific or species-specific components, activities?

If you were founding a new research institute dedicated to understand how evolutionary selection acts at all levels of gene expression, how would you combine nuts-and-bolts mechanistic studies with Big Picture evolution research?

What technical breakthroughs would make the biggest impacts on such efforts? Let's think big; e.g. mosquito blood meals preserved in amber (OK, that was a joke).

Tweets start tomorrow afternoon (Thursday July 25th at around 2PM).  I also hope to be doing a daily summary of the meeting on this blog.  Time to get my talk ready!