Something new under the sun

May 31, 2011 § Leave a comment

One of the great surprises of the genomic era has been how similar the coding regions of genes are between species.  It seems that we have been underestimating the evolutionary role of altered regulation — increasing or decreasing the expression of a gene, in different places, at different times — relative to protein sequence changes.  So the question of how evolution produces novel patterns of expression of existing genes has become one of the hot topics of the day.  There are at least 4 ways you can imagine this happening via changes to the DNA near your gene of interest.  First, an enhancer that drives the expression of your gene could be created out of nothing by mutation, in a region of DNA that previously had nothing to do with regulating your gene.  Second, a pre-formed enhancer may “jump” into the region near your gene, carried by a transposon.  Third, an enhancer that was originally driving the expression of a neighboring gene may switch its activity to the promoter of your gene.  And finally, an existing enhancer that drives the expression of your gene at a particular time in development, or in a particular site in the body, may be modified by mutation such that it now causes expression in a different time and place.  This is called co-option.  The idea is that every functional enhancer involves many transcription factor binding sites: if an enhancer has binding sites for transcriptional activators A, B and C and repressor D, it will be active at times and places when A, B and C are present and D is not.  If evolution now adds activator site E, through a random mutation of a sequence that was quite similar to E anyway, then perhaps the enhancer can activate transcription at any time or place where you have three out of the four activating transcription factors: ABC still works, but so do ABE, ACE and BCE… and perhaps if you have all four, you can over-ride the presence of D.  I’m making this up, you understand, but that’s the general idea: you co-opt some of the pre-existing binding sites, add one or more new ones, and the result is that your gene of interest is expressed somewhere novel in time or space.

As usual for events that happened millions of years ago, specific examples of novelty are not that easy to identify with confidence.  But Sean Carroll and colleagues now think that they’ve spotted a new and interesting example of co-option (Rebeiz et al. 2011.  Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences.  PNAS doi/01.1073/pnas.1105937108).  What they did was to take a group of several closely related species of Drosophila and identified 20 genes that might be expected to evolve relatively rapidly.  They then carefully examined the expression of these genes in several larval stages of each of the Drosophila species.  They saw many changes in expression, most of which turned out not to meet their definition of a novel expression pattern — for example, some changes that initially looked as if something new was happening turned out to be merely shifts of the timing of an expression pattern in one species relative to another.  But they did find one gene that had a unique pattern of expression in one species and no others, the Ned-1 gene.  In most of the Drosophila species studied, this gene is expressed in wing, leg and central nervous system tissues.  In one species, D. santomea, it’s also expressed in the developing optic lobe. Rebeiz et al. checked exhaustively that this pattern was neither a timing shift nor a remnant of an older expression pattern that had been lost in all of D. santomea‘s relatives.  It really does look novel.

Now that we’ve found a novel pattern, can we figure out where it comes from? The authors first checked that the change in expression was encoded in the regulatory sequence of the Nep1 gene, and not, for example, due to changes in the levels of the transcription factors that bind to the Nep1 enhancer: in other words, the pattern was driven by changes in the cis-regulatory regions, and not by changes in trans.  They found that the novel expression pattern could be recapitulated by a GFP reporter under the control of the regulatory sequences in the first intron of the Nep1 gene: definitely a pattern that’s controlled by changes in local DNA sequence, therefore.  There’s no sign of an enhancer jumping into this region, and no sign of a nearby promoter that drives optic lobe expression of something else being seduced into switching its affections from one gene to another.  What’s more, the sequences responsible for the wing/leg/central nervous system expression (which is maintained in D. santomea) overlap with the sequence responsible for optic lobe expression, providing the first hint that this might be an example of co-option.

The hallmark of co-option would be that some elements of the enhancer would be required for both the old and the new expression pattern. To test for this, the authors scanned for blocks of sequence that are conserved between the enhancer region in D. santomea and the same region in another Drosophila species.  They found 7 blocks of 10 base pairs or more, and systematically went through and scrambled the sequence of each block individually.  They then checked whether the D. santomea enhancer with scrambled blocks still drives both the old expression pattern and the new one.  In 6 of the 7 cases, optic lobe expression was significantly reduced; at the same time, other features of the Nep1 expression pattern were also altered.  For example, scrambling the block they labeled block 7 takes out the novel expression pattern completely, and at the same time reduces central nervous system expression to about half of normal levels.  So, the novel feature — optic lobe expression — clearly depends on conserved sequences that are also important for the ancestral expression pattern.  This is exactly what you would expect from the co-option scenario.

What changed in the D. santomea enhancer to create this new pattern?  You might think that this question is trivial to answer now that sequencing is so easy: sequence the enhancer from D. santomea and compare it with the sequence of the enhancer from a different species.  But if you did that you would be comparing sequences from two individuals, and so differences between individuals would be mixed in with the differences between species.  Rebeiz et al. therefore sequenced enhancers from 16 individual lines of D. santomea and a total of 33 lines from two other species, coming to the conclusion that there are just 4 fixed mutations in the D. santomea enhancer sequence.  Each of these contributes to the optic lobe expression of Nep1.  So there are at least 10 sites in total — the 6 conserved blocks plus the 4 new mutations — that are required for this new gene expression pattern.

Because there are so many closely-related Drosophila species, it’s possible to reconstruct the enhancer sequence of the last common ancestor of D. santomea and its closest relative, D. yakuba. Rebeiz et al. did this, and found that this ancestral enhancer actually has significant optic lobe activity, about 40% of the D. santomea level.  In fact there are signs that there was trace activity even further back.  So, the conserved sequences of the Nep1 enhancer have hidden potential: with a few changes, they can drive expression in the optic lobe.  In most Drosophila species, this potential activity remains latent.  In the common ancestor of D. santomea and D. yakuba, mutations led to modest expression; in D. santomea, 4 additional mutations led to increased expression, while in D. yakuba other mutations shut the modest ancestral level of expression down.

One would assume that co-option of existing enhancer elements is often a faster route to a given expression pattern than starting from scratch and evolving an enhancer out of nowhere.  [Actually I find the distinction between them almost a matter of semantics: the difference between an enhancer that comes out of nowhere and one that is co-opted is partly a matter of the time frame you’re considering (everything looks like recent tinkering if you don’t look back in time very far) and partly a matter of whether the “out of nowhere” enhancer found another function before it found the function that you happen to be interested in. Unless you’re looking at the very first function for a gene — which seems unlikely without an evolutionary TARDIS — nothing is going to look as if it’s de novo.  Am I missing something?]  The authors estimate that a minimum of 8 inputs, more likely 11, are required to generate the Nep1 optic lobe activity — obviously it’s easier to get to 11 if you start with 6 or 7.  Insertion of a fully-fledged enhancer would be faster yet, perhaps, but there’s only a certain number of mobile elements in any given genome.  With more studies like this, perhaps we can get a sense of how often new functions arise via co-option versus transposon insertion and promoter switching.

Rebeiz M, Jikomes N, Kassner VA, & Carroll SB (2011). Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences. Proceedings of the National Academy of Sciences of the United States of America PMID: 21593416

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