Epistatic relationship between waardenburg syndrome genes mitf and pax3

Epistatic relationship between Waardenburg syndrome genes MITF and PAX3.

Watanabe A, Takeda K, Ploplis B, Tachibana M. Epistatic relationship between Waardenburg syndrome genes MITF and PAX3. Nat Genet ;18(3)– This observation supports an epistatic relationship between MITF and PAX3 and can explain the pigmentary disorders observed in WS1 and 3, because MITF. These results provide evidence that PAX3 directly regulates MITF, and suggest that the failure of this regulation due to PAX3 mutations causes the.

However, SOX10 and other SOX genes have been shown to act on promoters or enhancer sequences which do not completely match this consensus binding site 33 Only the site located between positions — and — fully conformed with the published consensus binding site Fig. This resulted in a dramatic decrease in transcriptional activity identical to that observed with the del mutant promoter Fig.

The same result was obtained when all eight putative SOX10 binding sites were mutated data not shown. This result indicates that, in this context, and for the promoter region tested, SOX10 acts directly by binding to one or several of these sites. To find out whether SOX10 would indeed bind to the six more proximal sites, we next performed a gel shift analysis using extracts from COS cells producing a shortened SOX10 version.

Among the potential sites tested, only one site 5 exhibited strong binding of SOX10 Fig. Assuming that the binding site with the highest affinity would also be the functionally most important one, we next mutated site 5 in the context of the del construct by replacement with a GC-rich element.

This resulted in a decrease in transcriptional activity almost comparable to that observed for the deletion of the MITF promoter in which we mutated the six putative SOX binding sites Fig. The remaining difference in SOX10 responsiveness between both promoter constructs might be due to the contribution of the other weak SOX10 binding sites present in this region. In conclusion, these results indicate that, in this context, and for the promoter region tested, SOX10 acts directly by binding.

Indeed, the effect of PAX3 on transcription activation was abolished in mel cells when the specific binding site that lies between positions — and — Fig. We studied the effects of SOX10 and PAX3, alone or in combination, on the wild-type promoter and on the two most shortened versions of this promoter del and del constructs Fig.

SOXdependent transcription activation from the del mutant promoter was decreased but still present versus fold activation compared with the intact promoter. We expected that the bp deletion would drastically impair both SOX and PAX3-dependent activation, resulting in the loss of the synergistic effect of the two partner factors. Indeed, the PAX3 binding site previously described P1 is removed from this construct.

Surprisingly, in our in vitro model i. To explain the discrepancies between our data and those of Watanabe et al. The two recognition elements for the paired domain and the homeodomain are separated by 6 nucleotides, a proximity that allows PAX3 to interact with high affinity.

In light of these observations, we looked at PAX3-dependent transcription stimulation from the del construct in which each PAX3 binding site, or both, are mutated Fig.

Epistatic relationship between Waardenburg Syndrome genes MITF and PAX3

When the enhancing effect of PAX3 alone was tested on the del mutant promoter with P1 altered, no decrease in activity was observed, as with the del construct. In contrast, when P2 or both binding sites P1 and P2 were destroyed, PAX-dependent stimulation of transcription was abolished. A possible explanation is that the binding site that is mainly used by PAX3 to exert its effect would depend on the cell context mel or HeLa.

As the P2 binding site is close to the TATA box, it was important to prove that its mutation does not disrupt any element critical for the function of the MITF promoter. The fact that SOX10 is able to stimulate transcription from this construct to the same extent as from the intact construct confirmed that the promoter integrity was maintained data not shown.

Epistatic relationship between Waardenburg syndrome genes MITF and PAX3.

Finally, we tested the effects on transcription activation of removing the six SOX10 and the two PAX3 binding sites from the del mutant promoter Fig. The dramatic decrease of SOXdependent stimulation that resulted was similar to that observed with the del promoter mutant, and with the del promoter in which six or eight binding sites for SOX10 were mutated.

PAX3-dependent stimulation was also abolished as expected. In order to test the effect of these reported mutations on the MITF promoter, we analyzed several SOX10 and PAX3 mutant factors for their ability to transactivate this promoter, alone or in synergy with the other partner.

Three PAX3 mutants were generated and tested 36 The induction was reduced from 9- to 3-fold when the insTGA mutant was tested.

Epistatic relationship between Waardenburg Syndrome genes MITF and PAX3 - Semantic Scholar

The seven SOX10 mutants constructed were as follows Fig. Here, the SOX mutants fall into three classes: Although behaving differently with respect to their synergistic effect, all mutants were defective in activation of the MITF promoter, demonstrating the usefulness of our in vitro model. A spontaneous mouse model, the Dom mouse, carries a mutation in the Sox10 gene 25 This mutation, an insertion of an additional G after positionresults in an altered reading frame, which leaves the first amino acids of SOX10 including the HMG domain intact, but replaces the remaining residues by a divergent C-terminus of unrelated amino acids.

For that purpose we performed in situ hybridization studies on the Dom mouse. For simplicity we confined our analysis to homozygous embryos and their wild-type littermates at Using an antisense riboprobe specific for c-Kit, an early marker of the melanocyte lineage, we detected many cells in transverse sections at a position typical of melanocytes Fig.

These cells were strongly reduced in number in the homozygous Dom embryos confirming the overall importance of Sox10 for melanocyte development. However, few c-Kit-positive cells remained present. All or some of these cells are probably mast cells. When in situ hybridizations were carried out on wild-type embryos with an Mitf-specific riboprobe, similar numbers of cells were detected in equivalent positions as with the c-Kit probe.

However, contrary to what we observed for c-Kit, we failed to detect any remaining Mitf-positive cells in the homozygous Dom embryos.

This shows that SOX10 influences Mitf expression in vivo during embryonic development. This demonstration of an epistatic relationship between two of the genes whose dysfunction results in Waardenburg syndrome provides a link between the pigmentary—auditory symptoms that are common to the various forms of this syndrome 1. More recently, SOX10 was shown to be another player among the genes defective in this syndrome 9.

Its spatial and temporal pattern of expression supports an important function in early neural crest development in humans and in mice 25— In keeping with this observation, mutations of SOX10 result in a combination of defects affecting neural crest derivatives, such as pigmentation abnormalities, hearing loss and colonic aganglionosis in mice and in humans WS4. The molecular mechanism of SOX10 action during melanogenesis is currently unknown. Although this cell line is physiologically distant from pigment cells, previous molecular studies of genes involved in melanogenesis showed comparable data in HeLa cells and in a melanoma cell line MeWo In contrast, when PAX3 was used as the transcription factor in this assay, a smaller but significant increase in promoter activity fold was observed.

This observation establishes that the two transcription factors act in synergy to transactivate the MITF promoter. Among them, a SOXresponsive region was found lying between positions — and — This discrepancy with the previous report may be related to the cell line used melanoma versus HeLa cellsas other differences in site usage between one cell line and another have already been reported 15 Further experiments are needed to understand the role of this site in melanoma cells.

Eight potential SOX10 binding sites were identified between positions — and — on the basis of their homology with the SOX binding site consensus sequence. Mutation of all these binding sites resulted in a dramatic reduction of SOX10 induction, demonstrating that this transcription factor acts directly on the MITF promoter.

We then tested the affinity of SOX10 for these SOX binding sites by gel shift analysis and found that one site site 5 exhibited strong binding of SOX10 whereas the other sites exhibited weak or no binding. The in vitro analysis mutation of site 5 confirmed these results.

Interestingly, the site that displays strong binding of SOX10 does not fully conform to the consensus for SOX binding sites, thus indicating that binding of SOX proteins to DNA seems to be influenced by additional factors such as the exact flanking sequences or the DNA structure. This latter assumption is also supported by the observation that one of the other potential sites identified in the MITF promoter by sequence inspection binds SOX10 only with low affinity despite the fact that it fully conforms to the consensus.

We then analyzed the behavior of the various deletion mutants to test whether SOX10 and PAX3 had maintained the synergistic effects observed with the wild-type promoter. The synergistic effect was present in the del promoter mutant. With the del construct, and with the del construct in which we mutated the SOX binding sites, synergy was preserved despite the fact that SOX10 induction was dramatically reduced. It is possible that some weak SOXresponsive elements are located in the segment still present in the del construct; alone, they might be unable to strongly activate the MITF promoter in HeLa cells, but they allow synergistic activation in the presence of PAX3.

Another explanation could be that PAX3 and SOX10 can physically interact in such a way that only one or the other needs to bind DNA in order to recruit the entire complex. Nevertheless, this last hypothesis is in disagreement with the results obtained by Kuhlbrodt et al.

Severe alleles in six types of homozygous Sp mice are fatal at the embryonic stage, and even splotch-retarded Spd mice, which have the least severe allele, encoding Pax3 with a substitution mutation at the paired domain, die at birth. The phenotype of Spd mice varies depending on their genetic background, suggesting the existence of modifier genes.

It has been estimated that at least two genes interact with Spd to influence the craniofacial features. In contrast, in humans is transmitted in a dominant manner. WS4 mice as a model for WS4 Homozygous WS4 mice showed pigmentation anomaly white coat color with black eyeaganglionic megacolon and cochlear disorder. Exons 2 and 3, which encode transmembrane domains III and IV of the Ednrb G-protein-coupled receptor protein, were deleted in these mice.

Cochlea of WS4 mice showed endolymphatic collapse, due to the lack of melanocytes intermediate cells in the stria vascularis. JF1 mice as a model for WS2 The JF1 mice are an inbred strain of mice derived from Japanese wild mice, which are often bred by Japanese laymen as fancy panda' mice because of their cute appearance with black eyes and white spotting on a black coat.

JF1 mice are not lethal even in the homozygous state. This non-lethality of JF1 mice is probably due to the fact that the mutation in mice is an insertional mutation in intron 1 that creates a cryptic splicing acceptor site that results in decreased expression of wild-type Ednrb but does not cause aganglionic megacolon. As JF1 mice have pigmentation anomalies and hearing impairment- but not megacolon or dysmorphogenesis- they constitute a mouse model of WS2.

Some of these mice can survive and mate; they are potentially a model for WS4, although cochlear disorders of these mice remain to be examined.