Using admixed populations to separate cis and trans effects

A new article by Price et al. looks at the effects of cis and trans acting variation on gene expression. A number of studies have approached the genetics of gene expression in humans by doing GWA mapping of the genetic determinants of the expression of a gene (usually many genes measured on a microarray). However, this approach is strongly biased towards finding signals in cis. The cis region around a gene represents a small fraction of the SNPs in the genome, and so testing for cis effects suffers a much smaller multiple testing correction than trans variation, which must be significant beyond a multiple testing threshold for the entire genome.

Price et al. cleverly circumvent this by looking in the recently admixed African American population. African Americans have on average 20% European ancestry and 80% African ancestry. Because this admixture is recent, there are only a few generations of recombination and so the genome of an African American can be thought of as a mosaic made up of big blocks of alternating African and European ancestry. So at any location in the genome African Americans differ in whether they have locally inherited 0, 1 or 2 chromosomes from African ancestors. African Americans also vary in their genome-wide admixture proportion. Price et al. use this fact, to look at trans effects ancestry, by looking at the correlation between genome-wide ancestry proportion and the expression level of genes. They contrast this to the effect of cis ancestry (i.e. 0,1, or 2 African alleles at a site), to obtain an estimate of the variance explained by cis and trans effects. Somewhat surprisingly (at least if you read mostly human genetics papers) they find that only 12% of heritable variation in gene expression level is explained by cis effects. This kind of result has also been seen in Drosophila where trans effects make up the bulk of within species variation, but contribute less to between species differences (see evolgens post on this topic: Slightly Deleterious in Trans).

See also Gene expression

Would a gene by any other name smell as sweet?

I was thinking about blogging about the paper on metal tolerance evolution in Arabidopsis halleri via a cis-regulatory change (Hanikenne et al), but I see that gnxp has already done so here. So I thought in the vein of the 'cis-regulatory vs protein' evolution debate, I would point people towards a recent paper (Scalliet et al and here for a commentary) looking at a phenotypic change involving a coding change in roses. The plant in question is the Chinese rose, which apparently is where the garden variety hybrid tea rose (chinese x european) gets its scent from. The final part of the pathway which underlies the rose's scent involves two genes (OOMT1 and OOMT2), and the authors identify a single residual underlying the crucial difference in the specificity of the two proteins. OOMT1 appears to have arisen by a gene duplication in Chinese roses, as it is absent in other roses. So here is a case of recent gene duplication followed by protein divergence underlying a novel phenotype.

Interesting the Arabidopsis halleri paper identifies both a change in copy number and cis regulatory mutation underlying a phenotype. Changes in copy number can be considered regulatory mutations (as they can change the expression level of a protein), and can be selected for because of this; a recent example of this is the amalyse copy number variation (Perry et al, see the commentary on this paper by Coyne and Hoekstra). Subsequent selection pressures may favour the functionality of the duplicates to change, by amino-acid substitutions, or changes in where the two duplicates are expressed. Thus it is very likely that evolution proceeds by a combination of regulatory (cis and copy number) and protein changes (including mutations in trans factors etc) .


References:
Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4
Hanikenne et al. Nature 2008

Evolution of Protein Expression: New Genes for a New Diet
Jerry A. Coyne, Hopi E. Hoekstra. Current Biology 2007

Diet and the evolution of human amylase gene copy number variation
Perry et al 2007. Nature Genetics

Plant biology: Scent of a rose
Shadan S. Nature (News and Views) 2008

Scent evolution in Chinese roses
Scalliet et al. PNAS 2008

"Exons, Schmexons"

A summary by PZ Myers of Coyne and Wray's keynote speeches on evodevo. It sounds like it would have been fun to see, particularly the dueling t-shits (one is quoted in the title of this post). I think that Coyne is right that the only real way to know where selected changes occur and what type of mutations they are is to do very detailed follow-up work.

I thought I would give a link to a relatively recent paper by Wray's group looking for positive selection in promoters using the human, chimp and macaque sequences (Haygood et al). Their main point (at least in the coding vs noncoding debate) is that many promoters seem to undergo positive selection compared to exons (especially in interesting categories of genes). The paper looks for positive selection by looking for promoter regions that have significantly more substitutions than nearby intronic regions.

I've not read it in a while so I'll avoid commenting on the technical details. However, a lack of genes with d_N/d_S>1 is not proof that genes do not often experience positive selection, just that d_N/d_S>1 is a pretty crappy measure of positive selection. The problem is that d_N incorporates all of the selection against amino-acid changes plus any weak signal of positive selection. For a gene to meet the d_N/d_S>1 criteria it has to have had a whole bunch of amino-acid changes, if positive selection on a gene often involves just a few amino-acid changes it will not satisfy d_N/d_S>1. Promoter sequences on the other had could be made up of a mixture of near-neutrally evolving regions plus a small number of more constrained regions. A few additional substitutions in the promoter due to positive selection could easily tip the balance to make the promoter be 'rapidly evolving' (i.e. faster than the nearby intron) because the promoter's rate of substitution was not that different from the intronic rate anyway. That's not to say that the promoters found to be rapidly evolving are not interesting, just that the results should not be taken to mean that there is more positive selection on promoters than exons, as this is like comparing apples to oranges.

References:
Haygood R, Fedrigo O, Hanson B, Yokoyama KD, Wray GA.
Promoter regions of many neural- and nutrition-related genes have experienced positive selection during human evolution.
Nat Genet. 2007

Tan is the new black

There's a really interesting paper by Sean Carroll's group in Cell ( Jeong et al. ) on pigmentation differences between two closely related species of Drosophila (D. santomea and D yakuba). D santomea has a small range and is restricted high altitudes on the island of São Tomé, while Yakuba is more widespread and lives at lower altitudes on São Tomé. The species can hybridize, and form a natural hybrid zone, in fact the mtDNA has introgressed between species (Bachtrog et al. Llopart A et al). D. santomea unlike the rest of the Melanogaster clade lacks abdominal pigmentation. Carroll's group looks at one of the genes involved in this change in detail.

A couple of previous QTLs studies looking at abdominal pigmentation differences between these two species had identified a QTL close to a candidate gene tan on the X chromosome. While another small QTL maps near the Yellow gene also on the X chromosome. The authors show that Tan in combination with the Yellow gene produces the abdominal pigmentation in D. melanogaster. No coding differences are found between the tan gene of D santomea and D yakuba, suggesting that changes in the regulation are prime candidates for the difference in pigmentation. They then show that Yellow and Tan expression is present in the abdominal region of D Yakuba but absent in D Santomea. Replacing the X chromosome of D yakuba with that of D santomea removes the Tan expression pattern but not the yellow pattern, suggesting that the difference in Tan expression is controlled in cis (and yellow is controlled in trans). The authors identify a cis regulatory module for tan that controls the abdominal expression in D. melanogaster. They then show that this D. melanogater cis regulatory module can create the pigmentation pattern in male D. Santomea. A couple of changes have occured in the D. Santomea sequence in the regulatory module at otherwise conserved sites. The authors show that these sites are responsible for the reduced abdominal expression of tan.

At this point the authors decided to look at polymorphism and divergence data in D. santomea in this module. This is when the story gets really interesting. The authors I guess hoped to find the signal of a sweep in D. santomea around this region, and that all individuals would be fixed for the mutations that inactivated the cis module. What they found however was that the changes were not fixed in the population, but that there appear to be three distinct inactivation mutations at the cis module. They confirmed that the two newly discovered mutations (both deletions) removed the abdominal expression of tan, and so are likely to remove the pigmentation as well. Thus D. santomea has three different mutations at the same locus resulting in the same phenotype, which is pretty incredible case of parallel mutation. The authors argue that this is likely the result of selection rather than simply neutrality following relaxed constraint, and I find that pretty convincing. There is no observed polymorphism for pigmentation in D. santomea suggesting that the combined effect of these three mutations combined have removed pigmentation. It seems unlikely that this small mutational target (the module) experienced three neutral mutations that have essentially removed pigmentation.

I wonder if one of the mating choice QTLs (between D Santomea and D Yakuba) maps to the same location as the QTL that lead to the indentification of tan (I've not checked the co-ordinates of the QTLs in Moehring et al ). Interestingly, the yellow gene (the other pigmentation gene with reduced expression) seems to show a signal of introgression between D Santomea and D Yakuba (Llopart et al. ), I wonder if the
introgression at yellow gene has prevented the accumulation of strong cis mutations at yellow, meaning that its expression had to be reduced by a trans effect.

My only minor quibble with this otherwise great paper was the stridency about cis regulatory evolution. This paper is another really pretty example of cis regulatory evolution, but to my mind it in no way seals the debate about protein versus cis regulatory evolution ( Hoekstra and Coyne ). This case is another loss of function mutation, I would like to see more gain of function mutations in cis before making up my mind that cis regulatory evolution predominants. I also feel that the follow up of these cases is somewhat biased towards following up cis regulatory changes. The authors do not follow up the reduction in yellow expression which operates in trans, thus the paper has ignored a trans effect (which admittedly may be a cis effect at another upstream gene). Also the authors do not seem to show that the cis regulatory module of tan (a pleiotropic gene) is itself free of pleiotropic effects (a prerequisite for freeing up cis regulatory evolution).

References
Jeong S, Rebeiz M, Andolfatto P, Werner T, True J, Carroll SB. The evolution of gene regulation underlies a morphological difference between two Drosophila sister species. Cell. 2008 Mar 7;132(5):783-93.

Moehring AJ, Llopart A, Elwyn S, Coyne JA, Mackay TF. The genetic basis of prezygotic reproductive isolation between Drosophila santomea and D. yakuba due to mating preference.Genetics. 2006 May;173(1):215-23.

Llopart A, Lachaise D, Coyne JA.Multilocus analysis of introgression between two sympatric sister species of Drosophila: Drosophila yakuba and D. santomea. Genetics. 2005 Sep;171(1):197-210.

Bachtrog D, Thornton K, Clark A, Andolfatto P.
Extensive introgression of mitochondrial DNA relative to nuclear genes in the Drosophila yakuba species group.
Evolution Int J Org Evolution. 2006 Feb;60(2)