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An evolutionarily conserved intronic region controls the spatiotemporal expression of the transcription factor Sox10.

Dutton JR, Antonellis A, Carney TJ, Rodrigues FS, Pavan WJ, Ward A, Kelsh RN - BMC Dev. Biol. (2008)

Bottom Line: Mutation of the transcription factor SOX10 is associated with several human diseases.A further region, partially required for oligodendrocyte expression, lies in the 5' region of the same intron and contains a putative CSL binding site, consistent with a role for Notch signalling in sox10 regulation.Furthermore, we show that beta-catenin, Notch signalling and Sox9 can induce ectopic sox10 expression in early embryos, consistent with regulatory roles predicted from our transgenic and computational results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK. dutto015@umn.edu

ABSTRACT

Background: A major challenge lies in understanding the complexities of gene regulation. Mutation of the transcription factor SOX10 is associated with several human diseases. The disease phenotypes reflect the function of SOX10 in diverse tissues including the neural crest, central nervous system and otic vesicle. As expected, the SOX10 expression pattern is complex and highly dynamic, but little is known of the underlying mechanisms regulating its spatiotemporal pattern. SOX10 expression is highly conserved between all vertebrates characterised.

Results: We have combined in vivo testing of DNA fragments in zebrafish and computational comparative genomics to identify the first regulatory regions of the zebrafish sox10 gene. Both approaches converged on the 3' end of the conserved 1st intron as being critical for spatial patterning of sox10 in the embryo. Importantly, we have defined a minimal region crucial for this function. We show that this region contains numerous binding sites for transcription factors known to be essential in early neural crest induction, including Tcf/Lef, Sox and FoxD3. We show that the identity and relative position of these binding sites are conserved between zebrafish and mammals. A further region, partially required for oligodendrocyte expression, lies in the 5' region of the same intron and contains a putative CSL binding site, consistent with a role for Notch signalling in sox10 regulation. Furthermore, we show that beta-catenin, Notch signalling and Sox9 can induce ectopic sox10 expression in early embryos, consistent with regulatory roles predicted from our transgenic and computational results.

Conclusion: We have thus identified two major sites of sox10 regulation in vertebrates and provided evidence supporting a role for at least three factors in driving sox10 expression in neural crest, otic epithelium and oligodendrocyte domains.

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Sequence at the 3' end of the zebrafish sox10 intron 1 is essential for sox10:GFP expression. Cohorts of zebrafish injected with constructs containing sox10 regions at the 3' end of intron 1 as shown were used to identify a minimal sequence essential for sox10:GFP expression. Intron 1 fragments were replaced into an inactive sox10:GFP construct and injected embryos examined for GFP expression as previously described in Fig. 4. The transcription factor binding sites identified by TRANSFAC analysis within this region are illustrated by coloured arrows: red (NFKappaB), yellow (Tcf/Lef), green (FoxD3) and blue (Sox family). The transcription factor binding sites within the sequence that are highly conserved in vertebrate sox10 promoter regions are marked (*). m, muscle n, notochord.
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Figure 7: Sequence at the 3' end of the zebrafish sox10 intron 1 is essential for sox10:GFP expression. Cohorts of zebrafish injected with constructs containing sox10 regions at the 3' end of intron 1 as shown were used to identify a minimal sequence essential for sox10:GFP expression. Intron 1 fragments were replaced into an inactive sox10:GFP construct and injected embryos examined for GFP expression as previously described in Fig. 4. The transcription factor binding sites identified by TRANSFAC analysis within this region are illustrated by coloured arrows: red (NFKappaB), yellow (Tcf/Lef), green (FoxD3) and blue (Sox family). The transcription factor binding sites within the sequence that are highly conserved in vertebrate sox10 promoter regions are marked (*). m, muscle n, notochord.

Mentions: Our combined bioinformatics and physical promoter deletion studies had independently converged to indicate that the 3' end of zebrafish sox10 intron 1 might contain regulatory sequences critical for sox10 expression. To characterise functionally the 376 bp region at the 3'end of intron 1 it was initially divided into 3 overlapping regions (Fig. 7). DNA pieces corresponding to these were generated by PCR and ligated into the psox10-1252Δ(+1862–+2220):GFP vector. This vector lacks the entire 376 bp piece and both transient and germline transgenic fish made with this vector do not express GFP in a sox10-like pattern. Re-introduction of fragment A into this vector did not restore any GFP expression. Insertion of fragment B restored the GFP expression, albeit only weakly (in 12% of scored fish; n = 33). Notably, the distribution of these GFP positive cells was consistent with the sox10-like expression pattern (see +B in Table 2). Re-introduction of fragment C into the inactive vector also restored GFP to 23% of the fish scored and again the distribution of GFP positive cells largely corresponded to that expected for sox10 expression (see +C, Table 2). Significantly, a construct (psox10-1252Δ(+1862–+2220)+BC) containing a fragment (BC) spanning both the B and C regions was able to restore GFP expression in appropriate cell types and with a distribution not significantly different (p > 0.05) to the parent psox10-1252:GFP construct (see +BC, Table 2). Thus, adding the BC fragment, which spans all six conserved, biologically relevant sequence predictions identified bioinformatically, to the psox10-1252Δ(+1862–+2220) vector is sufficient to restore most aspects of the sox10-like pattern. The positions of these putative transcription factor binding sites in the B and C regions are shown in Fig. 7.


An evolutionarily conserved intronic region controls the spatiotemporal expression of the transcription factor Sox10.

Dutton JR, Antonellis A, Carney TJ, Rodrigues FS, Pavan WJ, Ward A, Kelsh RN - BMC Dev. Biol. (2008)

Sequence at the 3' end of the zebrafish sox10 intron 1 is essential for sox10:GFP expression. Cohorts of zebrafish injected with constructs containing sox10 regions at the 3' end of intron 1 as shown were used to identify a minimal sequence essential for sox10:GFP expression. Intron 1 fragments were replaced into an inactive sox10:GFP construct and injected embryos examined for GFP expression as previously described in Fig. 4. The transcription factor binding sites identified by TRANSFAC analysis within this region are illustrated by coloured arrows: red (NFKappaB), yellow (Tcf/Lef), green (FoxD3) and blue (Sox family). The transcription factor binding sites within the sequence that are highly conserved in vertebrate sox10 promoter regions are marked (*). m, muscle n, notochord.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2601039&req=5

Figure 7: Sequence at the 3' end of the zebrafish sox10 intron 1 is essential for sox10:GFP expression. Cohorts of zebrafish injected with constructs containing sox10 regions at the 3' end of intron 1 as shown were used to identify a minimal sequence essential for sox10:GFP expression. Intron 1 fragments were replaced into an inactive sox10:GFP construct and injected embryos examined for GFP expression as previously described in Fig. 4. The transcription factor binding sites identified by TRANSFAC analysis within this region are illustrated by coloured arrows: red (NFKappaB), yellow (Tcf/Lef), green (FoxD3) and blue (Sox family). The transcription factor binding sites within the sequence that are highly conserved in vertebrate sox10 promoter regions are marked (*). m, muscle n, notochord.
Mentions: Our combined bioinformatics and physical promoter deletion studies had independently converged to indicate that the 3' end of zebrafish sox10 intron 1 might contain regulatory sequences critical for sox10 expression. To characterise functionally the 376 bp region at the 3'end of intron 1 it was initially divided into 3 overlapping regions (Fig. 7). DNA pieces corresponding to these were generated by PCR and ligated into the psox10-1252Δ(+1862–+2220):GFP vector. This vector lacks the entire 376 bp piece and both transient and germline transgenic fish made with this vector do not express GFP in a sox10-like pattern. Re-introduction of fragment A into this vector did not restore any GFP expression. Insertion of fragment B restored the GFP expression, albeit only weakly (in 12% of scored fish; n = 33). Notably, the distribution of these GFP positive cells was consistent with the sox10-like expression pattern (see +B in Table 2). Re-introduction of fragment C into the inactive vector also restored GFP to 23% of the fish scored and again the distribution of GFP positive cells largely corresponded to that expected for sox10 expression (see +C, Table 2). Significantly, a construct (psox10-1252Δ(+1862–+2220)+BC) containing a fragment (BC) spanning both the B and C regions was able to restore GFP expression in appropriate cell types and with a distribution not significantly different (p > 0.05) to the parent psox10-1252:GFP construct (see +BC, Table 2). Thus, adding the BC fragment, which spans all six conserved, biologically relevant sequence predictions identified bioinformatically, to the psox10-1252Δ(+1862–+2220) vector is sufficient to restore most aspects of the sox10-like pattern. The positions of these putative transcription factor binding sites in the B and C regions are shown in Fig. 7.

Bottom Line: Mutation of the transcription factor SOX10 is associated with several human diseases.A further region, partially required for oligodendrocyte expression, lies in the 5' region of the same intron and contains a putative CSL binding site, consistent with a role for Notch signalling in sox10 regulation.Furthermore, we show that beta-catenin, Notch signalling and Sox9 can induce ectopic sox10 expression in early embryos, consistent with regulatory roles predicted from our transgenic and computational results.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK. dutto015@umn.edu

ABSTRACT

Background: A major challenge lies in understanding the complexities of gene regulation. Mutation of the transcription factor SOX10 is associated with several human diseases. The disease phenotypes reflect the function of SOX10 in diverse tissues including the neural crest, central nervous system and otic vesicle. As expected, the SOX10 expression pattern is complex and highly dynamic, but little is known of the underlying mechanisms regulating its spatiotemporal pattern. SOX10 expression is highly conserved between all vertebrates characterised.

Results: We have combined in vivo testing of DNA fragments in zebrafish and computational comparative genomics to identify the first regulatory regions of the zebrafish sox10 gene. Both approaches converged on the 3' end of the conserved 1st intron as being critical for spatial patterning of sox10 in the embryo. Importantly, we have defined a minimal region crucial for this function. We show that this region contains numerous binding sites for transcription factors known to be essential in early neural crest induction, including Tcf/Lef, Sox and FoxD3. We show that the identity and relative position of these binding sites are conserved between zebrafish and mammals. A further region, partially required for oligodendrocyte expression, lies in the 5' region of the same intron and contains a putative CSL binding site, consistent with a role for Notch signalling in sox10 regulation. Furthermore, we show that beta-catenin, Notch signalling and Sox9 can induce ectopic sox10 expression in early embryos, consistent with regulatory roles predicted from our transgenic and computational results.

Conclusion: We have thus identified two major sites of sox10 regulation in vertebrates and provided evidence supporting a role for at least three factors in driving sox10 expression in neural crest, otic epithelium and oligodendrocyte domains.

Show MeSH
Related in: MedlinePlus