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The transcription factor Sox5 modulates Sox10 function during melanocyte development.

Stolt CC, Lommes P, Hillgärtner S, Wegner M - Nucleic Acids Res. (2008)

Bottom Line: The transcription factor Sox5 has previously been shown in chicken to be expressed in early neural crest cells and neural crest-derived peripheral glia.This modulatory activity involved Sox5 binding and recruitment of CtBP2 and HDAC1 to the regulatory regions of melanocytic Sox10 target genes and direct inhibition of Sox10-dependent promoter activation.Both binding site competition and recruitment of corepressors thus help Sox5 to modulate the activity of Sox10 in the melanocyte lineage.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany.

ABSTRACT
The transcription factor Sox5 has previously been shown in chicken to be expressed in early neural crest cells and neural crest-derived peripheral glia. Here, we show in mouse that Sox5 expression also continues after neural crest specification in the melanocyte lineage. Despite its continued expression, Sox5 has little impact on melanocyte development on its own as generation of melanoblasts and melanocytes is unaltered in Sox5-deficient mice. Loss of Sox5, however, partially rescued the strongly reduced melanoblast generation and marker gene expression in Sox10 heterozygous mice arguing that Sox5 functions in the melanocyte lineage by modulating Sox10 activity. This modulatory activity involved Sox5 binding and recruitment of CtBP2 and HDAC1 to the regulatory regions of melanocytic Sox10 target genes and direct inhibition of Sox10-dependent promoter activation. Both binding site competition and recruitment of corepressors thus help Sox5 to modulate the activity of Sox10 in the melanocyte lineage.

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Sox5 recognizes binding sites for Sox10 in the Dct and Mitf promoters. (A) Binding of the long Sox5 isoform to bona fide Sox10 recognition elements was studied in EMSA with the monomeric Sox10 binding site S1 and the dimeric site S4/4′ from the Dct promoter and the binding site 5 from the Mitf promoter as probes (25,32). Extracts from transfected Neuro2a cells served as source for full-length Sox10 and Sox5. Unspecific complexes (ns) were identified as those that also appeared with extract from mock-transfected Neuro2a cells (C). Each of the specific complexes is labeled on the right side of the panel for the presence of Sox5, Sox10 dimers (Sox10 D) or Sox10 monomers (Sox10 M) in the respective complexes. The ‘−’ depicts no extract added. Using the S1 (B), S4/4′ (C) and Mitf (D) probes, EMSA was performed with extracts from transfected Neuro2a cells containing the carboxyterminally truncated MIC variant of Sox10 and/or Sox5. Addition of antibodies directed against Sox10 (α-Sox10) or Sox5 (α-Sox5) during the incubation period was used to identify the complexes containing either Sox protein. Supershifted complexes are marked by asterisks on the right side of the panels. No heteromeric complexes were observed. For titration experiments, a fixed amount of Sox5 and S/S4′ were challenged with increasing amounts of Sox10 (E), or a fixed amount of Sox10 and S/S4′ were reciprocally incubated with increasing amounts of Sox5 (F). As higher amounts of extract have to be used to obtain a Sox5-specific complex than a Sox10-specific complex, unspecific complexes become apparent with increasing Sox5 amounts.
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Figure 4: Sox5 recognizes binding sites for Sox10 in the Dct and Mitf promoters. (A) Binding of the long Sox5 isoform to bona fide Sox10 recognition elements was studied in EMSA with the monomeric Sox10 binding site S1 and the dimeric site S4/4′ from the Dct promoter and the binding site 5 from the Mitf promoter as probes (25,32). Extracts from transfected Neuro2a cells served as source for full-length Sox10 and Sox5. Unspecific complexes (ns) were identified as those that also appeared with extract from mock-transfected Neuro2a cells (C). Each of the specific complexes is labeled on the right side of the panel for the presence of Sox5, Sox10 dimers (Sox10 D) or Sox10 monomers (Sox10 M) in the respective complexes. The ‘−’ depicts no extract added. Using the S1 (B), S4/4′ (C) and Mitf (D) probes, EMSA was performed with extracts from transfected Neuro2a cells containing the carboxyterminally truncated MIC variant of Sox10 and/or Sox5. Addition of antibodies directed against Sox10 (α-Sox10) or Sox5 (α-Sox5) during the incubation period was used to identify the complexes containing either Sox protein. Supershifted complexes are marked by asterisks on the right side of the panels. No heteromeric complexes were observed. For titration experiments, a fixed amount of Sox5 and S/S4′ were challenged with increasing amounts of Sox10 (E), or a fixed amount of Sox10 and S/S4′ were reciprocally incubated with increasing amounts of Sox5 (F). As higher amounts of extract have to be used to obtain a Sox5-specific complex than a Sox10-specific complex, unspecific complexes become apparent with increasing Sox5 amounts.

Mentions: Sox10 has previously been shown to activate Mitf and Dct as its target genes (25–29,32–34). Depending on the studied organism, Mitf gene activation has been proposed to be one of the essential functions or the sole essential task of Sox10 during melanocyte development (23,31). Multiple binding sites for Sox10 have been mapped within the Mitf and Dct promoters. These sites have been found to differentially contribute to overall promoter activation. In the Dct promoter, the S1 and the S4/4′ sites have been reported to be mainly responsible for the Sox10-dependent induction (32). The same holds true for site 5 in case of the Mitf promoter (25). The S1 site from the Dct promoter and the site 5 from the Mitf promoter are furthermore representative of response elements recognized by Sox10 monomers, whereas S4/4′ allows binding of Sox10 dimers as evident from the mobility of the Sox10-containing complexes on these sites in EMSA (Figure 4A) (25,32). We asked whether Sox5 has the capacity to recognize these response elements. When EMSA were performed with the long isoform of Sox5 that we had detected in melanoblasts at 10.5 dpc, binding was detected to all three sites (Figure 4A). From the comparable mobility of the Sox5-containing complex on site 5 and S1 relative to the S4/4′ site, it can furthermore be concluded that the L-Sox5 isoform binds to all sites as a dimer in agreement with its constitutive dimerization in solution (3).Figure 4.


The transcription factor Sox5 modulates Sox10 function during melanocyte development.

Stolt CC, Lommes P, Hillgärtner S, Wegner M - Nucleic Acids Res. (2008)

Sox5 recognizes binding sites for Sox10 in the Dct and Mitf promoters. (A) Binding of the long Sox5 isoform to bona fide Sox10 recognition elements was studied in EMSA with the monomeric Sox10 binding site S1 and the dimeric site S4/4′ from the Dct promoter and the binding site 5 from the Mitf promoter as probes (25,32). Extracts from transfected Neuro2a cells served as source for full-length Sox10 and Sox5. Unspecific complexes (ns) were identified as those that also appeared with extract from mock-transfected Neuro2a cells (C). Each of the specific complexes is labeled on the right side of the panel for the presence of Sox5, Sox10 dimers (Sox10 D) or Sox10 monomers (Sox10 M) in the respective complexes. The ‘−’ depicts no extract added. Using the S1 (B), S4/4′ (C) and Mitf (D) probes, EMSA was performed with extracts from transfected Neuro2a cells containing the carboxyterminally truncated MIC variant of Sox10 and/or Sox5. Addition of antibodies directed against Sox10 (α-Sox10) or Sox5 (α-Sox5) during the incubation period was used to identify the complexes containing either Sox protein. Supershifted complexes are marked by asterisks on the right side of the panels. No heteromeric complexes were observed. For titration experiments, a fixed amount of Sox5 and S/S4′ were challenged with increasing amounts of Sox10 (E), or a fixed amount of Sox10 and S/S4′ were reciprocally incubated with increasing amounts of Sox5 (F). As higher amounts of extract have to be used to obtain a Sox5-specific complex than a Sox10-specific complex, unspecific complexes become apparent with increasing Sox5 amounts.
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Figure 4: Sox5 recognizes binding sites for Sox10 in the Dct and Mitf promoters. (A) Binding of the long Sox5 isoform to bona fide Sox10 recognition elements was studied in EMSA with the monomeric Sox10 binding site S1 and the dimeric site S4/4′ from the Dct promoter and the binding site 5 from the Mitf promoter as probes (25,32). Extracts from transfected Neuro2a cells served as source for full-length Sox10 and Sox5. Unspecific complexes (ns) were identified as those that also appeared with extract from mock-transfected Neuro2a cells (C). Each of the specific complexes is labeled on the right side of the panel for the presence of Sox5, Sox10 dimers (Sox10 D) or Sox10 monomers (Sox10 M) in the respective complexes. The ‘−’ depicts no extract added. Using the S1 (B), S4/4′ (C) and Mitf (D) probes, EMSA was performed with extracts from transfected Neuro2a cells containing the carboxyterminally truncated MIC variant of Sox10 and/or Sox5. Addition of antibodies directed against Sox10 (α-Sox10) or Sox5 (α-Sox5) during the incubation period was used to identify the complexes containing either Sox protein. Supershifted complexes are marked by asterisks on the right side of the panels. No heteromeric complexes were observed. For titration experiments, a fixed amount of Sox5 and S/S4′ were challenged with increasing amounts of Sox10 (E), or a fixed amount of Sox10 and S/S4′ were reciprocally incubated with increasing amounts of Sox5 (F). As higher amounts of extract have to be used to obtain a Sox5-specific complex than a Sox10-specific complex, unspecific complexes become apparent with increasing Sox5 amounts.
Mentions: Sox10 has previously been shown to activate Mitf and Dct as its target genes (25–29,32–34). Depending on the studied organism, Mitf gene activation has been proposed to be one of the essential functions or the sole essential task of Sox10 during melanocyte development (23,31). Multiple binding sites for Sox10 have been mapped within the Mitf and Dct promoters. These sites have been found to differentially contribute to overall promoter activation. In the Dct promoter, the S1 and the S4/4′ sites have been reported to be mainly responsible for the Sox10-dependent induction (32). The same holds true for site 5 in case of the Mitf promoter (25). The S1 site from the Dct promoter and the site 5 from the Mitf promoter are furthermore representative of response elements recognized by Sox10 monomers, whereas S4/4′ allows binding of Sox10 dimers as evident from the mobility of the Sox10-containing complexes on these sites in EMSA (Figure 4A) (25,32). We asked whether Sox5 has the capacity to recognize these response elements. When EMSA were performed with the long isoform of Sox5 that we had detected in melanoblasts at 10.5 dpc, binding was detected to all three sites (Figure 4A). From the comparable mobility of the Sox5-containing complex on site 5 and S1 relative to the S4/4′ site, it can furthermore be concluded that the L-Sox5 isoform binds to all sites as a dimer in agreement with its constitutive dimerization in solution (3).Figure 4.

Bottom Line: The transcription factor Sox5 has previously been shown in chicken to be expressed in early neural crest cells and neural crest-derived peripheral glia.This modulatory activity involved Sox5 binding and recruitment of CtBP2 and HDAC1 to the regulatory regions of melanocytic Sox10 target genes and direct inhibition of Sox10-dependent promoter activation.Both binding site competition and recruitment of corepressors thus help Sox5 to modulate the activity of Sox10 in the melanocyte lineage.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen, Fahrstrasse 17, D-91054 Erlangen, Germany.

ABSTRACT
The transcription factor Sox5 has previously been shown in chicken to be expressed in early neural crest cells and neural crest-derived peripheral glia. Here, we show in mouse that Sox5 expression also continues after neural crest specification in the melanocyte lineage. Despite its continued expression, Sox5 has little impact on melanocyte development on its own as generation of melanoblasts and melanocytes is unaltered in Sox5-deficient mice. Loss of Sox5, however, partially rescued the strongly reduced melanoblast generation and marker gene expression in Sox10 heterozygous mice arguing that Sox5 functions in the melanocyte lineage by modulating Sox10 activity. This modulatory activity involved Sox5 binding and recruitment of CtBP2 and HDAC1 to the regulatory regions of melanocytic Sox10 target genes and direct inhibition of Sox10-dependent promoter activation. Both binding site competition and recruitment of corepressors thus help Sox5 to modulate the activity of Sox10 in the melanocyte lineage.

Show MeSH
Related in: MedlinePlus