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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 is coexpressed with other Sox genes in B16 melanoma cells. (A) PCR experiments were performed on DNaseI-treated, reverse transcribed RNAs from B16 melanoma cells (B16) and spinal cords of 7-day old mice (P7), the latter serving as a positive control as most Sox genes are known to be expressed in this tissue. Transcripts were detected with mouse-specific primers for Sox5, Sox6, Sox13, Sox9, Sox10, Dct, Mitf and β-actin. Template-free control reactions were performed in parallel (−). (B) Equal amounts of extracts from B16 melanoma cells were subjected to SDS–PAGE and western blotting using antibodies directed against Sox5, Sox10 and acetylated α-tubulin. On the right side, the size of coelectrophoresed molecular weight markers are given. (C) EMSA was performed with the binding site from the Mitf promoter as probe and extracts from B16 melanoma cells. Addition of antibodies directed against Sox10 (α-Sox10) or Sox5 (α-Sox5) to the reactions during the incubation period was used to identify the Sox5- and Sox10-containing complexes, respectively. The ‘−’ depicts no extract added.
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Figure 6: Sox5 is coexpressed with other Sox genes in B16 melanoma cells. (A) PCR experiments were performed on DNaseI-treated, reverse transcribed RNAs from B16 melanoma cells (B16) and spinal cords of 7-day old mice (P7), the latter serving as a positive control as most Sox genes are known to be expressed in this tissue. Transcripts were detected with mouse-specific primers for Sox5, Sox6, Sox13, Sox9, Sox10, Dct, Mitf and β-actin. Template-free control reactions were performed in parallel (−). (B) Equal amounts of extracts from B16 melanoma cells were subjected to SDS–PAGE and western blotting using antibodies directed against Sox5, Sox10 and acetylated α-tubulin. On the right side, the size of coelectrophoresed molecular weight markers are given. (C) EMSA was performed with the binding site from the Mitf promoter as probe and extracts from B16 melanoma cells. Addition of antibodies directed against Sox10 (α-Sox10) or Sox5 (α-Sox5) to the reactions during the incubation period was used to identify the Sox5- and Sox10-containing complexes, respectively. The ‘−’ depicts no extract added.

Mentions: Using the B16 cell line, we next investigated whether Sox5 is also expressed in melanoma cells. First, we searched for the presence of Sox5 transcripts. RT–PCR studies revealed that B16 cells not only contained Sox10, Dct and Mitf transcripts, but also significant levels of Sox5 transcripts (Figure 6A). Sox6 and Sox9 transcripts were also detected (Figure 6A) in agreement with previous studies (48,49). Sox6 expression is thus clearly higher in B16 melanoma than in melanoblasts at 11.5 dpc. The same discrepant expression was also observed for Sox9 which is strongly expressed in B16 melanoma, but virtually absent from melanoblasts in mouse embryos (Figure 1U–X), again in good agreement with previously published data (49). The presence of Sox6 and Sox9 in B16 melanoma constitutes a significant complication as functional compensation between closely related Sox proteins is possible in this cell line. In contrast, such a functional compensation is unlikely in vivo, as melanoblasts only express Sox5 and Sox10 in significant amounts. Sox13 was similarly absent from 11.5 dpc melanoblasts and B16 melanoma cells (Figure 1Q–T and Figure 6A).Figure 6.


The transcription factor Sox5 modulates Sox10 function during melanocyte development.

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

Sox5 is coexpressed with other Sox genes in B16 melanoma cells. (A) PCR experiments were performed on DNaseI-treated, reverse transcribed RNAs from B16 melanoma cells (B16) and spinal cords of 7-day old mice (P7), the latter serving as a positive control as most Sox genes are known to be expressed in this tissue. Transcripts were detected with mouse-specific primers for Sox5, Sox6, Sox13, Sox9, Sox10, Dct, Mitf and β-actin. Template-free control reactions were performed in parallel (−). (B) Equal amounts of extracts from B16 melanoma cells were subjected to SDS–PAGE and western blotting using antibodies directed against Sox5, Sox10 and acetylated α-tubulin. On the right side, the size of coelectrophoresed molecular weight markers are given. (C) EMSA was performed with the binding site from the Mitf promoter as probe and extracts from B16 melanoma cells. Addition of antibodies directed against Sox10 (α-Sox10) or Sox5 (α-Sox5) to the reactions during the incubation period was used to identify the Sox5- and Sox10-containing complexes, respectively. The ‘−’ depicts no extract added.
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Figure 6: Sox5 is coexpressed with other Sox genes in B16 melanoma cells. (A) PCR experiments were performed on DNaseI-treated, reverse transcribed RNAs from B16 melanoma cells (B16) and spinal cords of 7-day old mice (P7), the latter serving as a positive control as most Sox genes are known to be expressed in this tissue. Transcripts were detected with mouse-specific primers for Sox5, Sox6, Sox13, Sox9, Sox10, Dct, Mitf and β-actin. Template-free control reactions were performed in parallel (−). (B) Equal amounts of extracts from B16 melanoma cells were subjected to SDS–PAGE and western blotting using antibodies directed against Sox5, Sox10 and acetylated α-tubulin. On the right side, the size of coelectrophoresed molecular weight markers are given. (C) EMSA was performed with the binding site from the Mitf promoter as probe and extracts from B16 melanoma cells. Addition of antibodies directed against Sox10 (α-Sox10) or Sox5 (α-Sox5) to the reactions during the incubation period was used to identify the Sox5- and Sox10-containing complexes, respectively. The ‘−’ depicts no extract added.
Mentions: Using the B16 cell line, we next investigated whether Sox5 is also expressed in melanoma cells. First, we searched for the presence of Sox5 transcripts. RT–PCR studies revealed that B16 cells not only contained Sox10, Dct and Mitf transcripts, but also significant levels of Sox5 transcripts (Figure 6A). Sox6 and Sox9 transcripts were also detected (Figure 6A) in agreement with previous studies (48,49). Sox6 expression is thus clearly higher in B16 melanoma than in melanoblasts at 11.5 dpc. The same discrepant expression was also observed for Sox9 which is strongly expressed in B16 melanoma, but virtually absent from melanoblasts in mouse embryos (Figure 1U–X), again in good agreement with previously published data (49). The presence of Sox6 and Sox9 in B16 melanoma constitutes a significant complication as functional compensation between closely related Sox proteins is possible in this cell line. In contrast, such a functional compensation is unlikely in vivo, as melanoblasts only express Sox5 and Sox10 in significant amounts. Sox13 was similarly absent from 11.5 dpc melanoblasts and B16 melanoma cells (Figure 1Q–T and Figure 6A).Figure 6.

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