Functional diversification of shh paralog enhancers

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).

Molecular biology: RNA in control

Cheah et al.[Nature] show that expression of the NMT1 gene is regulated at the level of pre-mRNA alternative splicing by a riboswitch that binds to thiamine pyrophosphate (TPP). a, At low concentrations of TPP, the TPP-binding (aptamer) region of the riboswitch base-pairs with sequences surrounding a splice site (red blocking line) in a nearby non-coding sequence, and prevents its selection by the splicing machinery. A distal splice site (green arrow) is selected, however, resulting in the generation of a shorter NMT1 mRNA with a coding sequence, or open reading frame (ORF), that translates into a functional NMT1-encoded protein (green signal). b, At high TPP levels, the aptamer undergoes a conformational rearrangement so that the region that was previously bound to the nearby splice site is now used to bind to TPP. This and other conformational changes (not shown) generate a longer mRNA splice variant that contains short, 'decoy' ORFs (red signal), preventing functional NMT1 expression.

300bp intronic regulatory element in hand gene

"Herein we describe the identification of a regulatory region in the hand gene essential and sufficient for the expression in the visceral mesoderm during embryogenesis. We found that hand expression in the circular visceral mesoderm is abolished in embryos mutant for the FoxF domain containing transcription factor Biniou. Furthermore we demonstrate that Biniou regulates hand expression by direct binding to a 300 bp sequence element, located within the 3rd intron of the hand gene. This regulatory element is highly conserved in different Drosophila species. In addition, we provide evidence that Hand is dispensable for the initial differentiation of the embryonic visceral mesoderm." (Hand is a direct target of the forkhead transcription factor Biniou during Drosophila visceral mesoderm differentiation.)