Genome Biology | Abstract | gb-2007-8-6-r106 | Functional diversification of sonic hedgehog paralog enhancers identified by phylogenomic reconstruction: "We demonstrate that the sonic hedgehog a (shha) paralogs sonic hedgehog b (tiggy winkle hedgehog; shhb) genes of fishes have a modified ar-C enhancer which specifies a diverged function at the embryonic midline. We have identified several conserved motifs indicative of putative transcription factor binding sites by a local alignment of ar-C enhancers of numerous vertebrate sequences. To trace the evolutionary changes among paralog enhancers, phylogenomic reconstruction was carried out and lineage-specific motif changes were identified. The relevance of the motif composition to observed developmental differences was studied through transgenic functional analyses.
Altering and exchanging motifs between paralog enhancers resulted in the reversal of enhancer specificity in the floor plate and notochord. A model reconstructing enhancer divergence during vertebrate evolution was developed."
Comments (by sterding):1. Enhancers ar-C in two paralogs of zebrafish shh gene are diverse in sequence conservation (fig.1) and in functionality (fig. 3); ar-C in shha enhances the expression of reporter gene in notochord, while reporter with ar-C in sshb shows expression in floor plate, additionally.
2. Local alignment of ar-C shows that 4 conserved motifs(C1,C2,C3,C4) in shha, while only 2 of them(C1,C3) present in sshb branch (fig. 4).
3. Experiment with mutation in specific motif shows that C1 are critical for notochord specificity, C3 is not so important from current result; C2 and C4 are floor plate repressor (fig. 5,6).
1 comment:
Five questions for the author:
1. From the UCSC genome browser or EnsEMBL, there are 3 homologs in zebrafish(1 shhb on chr2, 2 shha on chr7 and chr15). But in the paper, it seems that you only considered the shha paralog on chr7, but not the other shha(chr15:35368743-35379673 in zebrafish). I can see any explanation for that. Maybe I did not read carefully enough, could you explain here again?
2. How can you DEFINE the range/border of the C1-C4 motifs? Doing local alignment is not enough to define the sequence block as you highlight in Figure 4.
3. From the UCSC Gene track (see the attached picture with the email), we could see that there are four different transcription isoforms for SHH gene. I am guessing you are using one of them (also the RefSeq/EnsEMBL one). Did you ever consider the rest ones? or, did you ever test your conclusion on the rest transcripts?
4. I created the CNEs track and shown it in the attached picture. As you may see, there are two CNEs within intron1, and 1 in intron2 (for the transcript you chose). I'm not sure you did notice the rest CNEs(CNS in your paper) around that transcript; obviously there are two close CNEs downstream(of 3'UTR) the transcript (in contrast, I did not see any upstream CNEs that you stated as ar-D in the paper). Interestingly, unlike the rest CNEs with both copies on shha, there is a CNEs with an extra copy on shhb(the olive color one, chr2:27277k). And this CNE occurs intronially in two transcripts. I am not sure it's possible to test that the CNE contributes to the different expression patter for the two paralogs, or different transcript isoforms.
5. Last question maybe unrelated with the content; how can you get the idea to test SHH? and how can you choose ar-C as the target? Did you ever test or you get negative result for the rest CNEs? It becomes interesting to test the regulatory function of CNEs; but to see the subfunctionality of CNEs, it's my first time.
attached picture:
http://www.ii.uib.no/~xianjund/CRUNS/SHH.gif
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