Month: February 2010

This paper started out as a control. My lab is interested in understanding how the enhancers that control gene expression work – focusing on those that control early development in Drosophila. In 2008, we published a paper showing that when we put enhancers from a distantly related family of flies into Drosophila melanogaster embryos, they drive patterns of expression that are identical to the endogenous D. melanogaster enhancers, even though they have almost no conservation of primary DNA sequence. But since they have the same function, they must have something in common – and so we compared the configurations of transcription factor binding sites in orthologous enhancers across different evolutionary timescales looking for something they shared.

What we found is that binding sites in all of these enhancers occur in clusters. They are closer to each other than one would expect if they were scattered randomly in the ~1,000 bp of an enhancer. And, what’s more, sites that were close to each other were far more likely to be conserved. Surely, we thought, this could be no accident. So we proposed that enhancers are organized into compact clusters of sites for one or more factors – and that these “mini modules” are the primary unit of enhancer function.

But as we worked to extend these analyses to whole genomes, we sought a more rigorous, quantitative assessment, of just how improbably different levels of binding site clustering were. Like pretty much everyone in the field, we had used a null model in which binding sites were scattered randomly in an enhancer. But, I’ve been working with genomes long enough to know that nothing is ever truly random – and that all kinds of adaptive and non-adaptive processes create patterns in genome sequences that confound simple analyses. I wanted to come up with a null model for the distribution of sites within in an enhancer that was more realistic.

To do this I turned to my graduate student Rich Lusk, a card-carrying population geneticist trained at the University of Chicago. Rich was proud of his status as one of the few members of the lab who didn’t work on flies – but I convinced him to put aside the abstract models of binding site evolution in yeast and work on developing a real null model for our studies of enhancer evolution.

The idea was to simulate enhancers evolving without any constraint on the organization of transcription factor binding sites they contain, and to see what happens. But this did not mean letting enhancers evolve neutrally – their extreme functional conservation demonstrates that they are under fairly strong constraint. Since it is pretty clear that these enhancers are responding to the same transcription factors in all of these species, Rich’s simulations required that enhancers maintain their binding site composition – but placed no constraints on how the sites were organized relative to each other.

And what we found was striking. Even with no explicit selection on binding site organization – these evolved enhancers had lots of structure! Binding sites were clustered together, and, the closer together sites were, the more conserved they were — just like they were in real enhancers. In made us realize pretty quickly that the patterns we had latched onto – and which many other people were describing in different systems – might not be an evolutionary signature contraint on the organization of sites within in enhancers, but simply a byproduct of selection on binding site composition. If you want details, read the paper! But this has radically altered the way that we look at enhancer evolution.

2. How did you come up with the title.

Rich and I were writing the paper, and we had some really long, hideous, boring title. In writing the paper, the idea that things are not always what they appear to be was at the forefront of my mind. I was thinking about how desperate we and other people in the field were to figure out how enhancers work – it’s a vexing problem that has defied decades of work – and how we all hoped that evolutionary analysis was going to rescue us – and how quickly and eagerly we latched on to the first signs of a signal – and how that was just like a mirage you see in the desert….

3. Any interesting background?

(see 1)

4. When did the work start?

About a year ago. We had been thinking about this for a while, but only when Rich focused on it did things get rolling.

5. Why PLoS Genetics? Did PLoS Biology reject it?

PLoS Genetics was our first choice. PG has become the premier journal for evolutionary genetics – it routinely publishes the most interesting and important work in the field, and everyone reads it. While every paper I’ve sent there has been heavily scrutinized, the editorial process has been fair (though sometimes agonizingly slow….), and each review has been thoughtful and many (including in this case) helped to vastly improve the paper.

Lusk, R., & Eisen, M. (2010). Evolutionary Mirages: Selection on Binding Site Composition Creates the Illusion of Conserved Grammars in Drosophila Enhancers PLoS Genetics, 6 (1) DOI: 10.1371/journal.pgen.1000829