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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 binds to melanocyte-specific promoters and recruits transcriptional corepressors. (A and B) B16 melanoma cells were transiently cotransfected with reporter plasmids in which the luciferase gene was under control of the Dct (A) or the Mitf (B) promoters, and expression plasmids for shRNAs directed against Sox10 and Sox5 or their scrambled versions, as indicated below the bars. Promoter activity in the absence of cotransfected shRNA was arbitrarily set to one and all other activities are expressed relative to this value ± SEM. Luciferase activities were determined in three (A) and five (B) experiments each performed in duplicates. The higher transactivation rates obtained for both promoters in the presence of both Sox5 shRNA and Sox10 shRNA as compared to Sox10 shRNA alone were statistically significant as determined by Student's t-test (**P ⩽ 0.01). (C and D) Immunoprecipitation was performed on formaldehyde-fixed chromatin from B16 melanoma cells in the absence (−) and presence of antibodies. In addition to control IgGs (IgG), antisera specifically directed against Sox5 (α-Sox5), Sox10 (α-Sox10), HDAC1 (α-HDAC1) and CtBP2 (α-CtBP2) were employed. PCR was applied on the immunoprecipitate to detect the Dct promoter region (a) and the Mitf promoter region (c) that contain the previously identified Sox10 response elements. Regions further upstream and more distal to the transcription start sites of the Dct (b) and Mitf (d) genes were additionally amplified by PCR as controls. All fragments were recovered from 1/20 of the material used for immunoprecipitation (input). (H2O), water control. (E) Neuro2a cells were transiently transfected using a reporter plasmid in which the luciferase gene was under control of the 1.5 kb Mitf promoter. Expression plasmids for Sox10, Sox5, HDAC1 and CtBP2 were cotransfected as indicated below the bars. Whereas Sox10 expression plasmids were used at 100 ng, Sox5 expression plasmids were employed at 20 ng and HDAC1 or CtBP2 expression plasmids at 10 ng per transfection reaction. Transactivation rates for each promoter are presented as fold inductions ± SEM. Luciferase activities were determined in three experiments each performed in duplicates. (F) Immunoprecipitation with anti-HA tag antibodies was performed on formaldehyde-fixed chromatin from Neuro2a cells transiently transfected with expression plasmids for the long Sox5 isoform, HA-tagged CtBP2 or their combination as indicated above the lanes. Mock-transfected Neuro2a cells (−) served as control. PCR was applied on the immunoprecipitate to detect the Mitf and the Dct promoter regions under the same conditions as in (C and D). (H2O), water control.
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Figure 10: Sox5 binds to melanocyte-specific promoters and recruits transcriptional corepressors. (A and B) B16 melanoma cells were transiently cotransfected with reporter plasmids in which the luciferase gene was under control of the Dct (A) or the Mitf (B) promoters, and expression plasmids for shRNAs directed against Sox10 and Sox5 or their scrambled versions, as indicated below the bars. Promoter activity in the absence of cotransfected shRNA was arbitrarily set to one and all other activities are expressed relative to this value ± SEM. Luciferase activities were determined in three (A) and five (B) experiments each performed in duplicates. The higher transactivation rates obtained for both promoters in the presence of both Sox5 shRNA and Sox10 shRNA as compared to Sox10 shRNA alone were statistically significant as determined by Student's t-test (**P ⩽ 0.01). (C and D) Immunoprecipitation was performed on formaldehyde-fixed chromatin from B16 melanoma cells in the absence (−) and presence of antibodies. In addition to control IgGs (IgG), antisera specifically directed against Sox5 (α-Sox5), Sox10 (α-Sox10), HDAC1 (α-HDAC1) and CtBP2 (α-CtBP2) were employed. PCR was applied on the immunoprecipitate to detect the Dct promoter region (a) and the Mitf promoter region (c) that contain the previously identified Sox10 response elements. Regions further upstream and more distal to the transcription start sites of the Dct (b) and Mitf (d) genes were additionally amplified by PCR as controls. All fragments were recovered from 1/20 of the material used for immunoprecipitation (input). (H2O), water control. (E) Neuro2a cells were transiently transfected using a reporter plasmid in which the luciferase gene was under control of the 1.5 kb Mitf promoter. Expression plasmids for Sox10, Sox5, HDAC1 and CtBP2 were cotransfected as indicated below the bars. Whereas Sox10 expression plasmids were used at 100 ng, Sox5 expression plasmids were employed at 20 ng and HDAC1 or CtBP2 expression plasmids at 10 ng per transfection reaction. Transactivation rates for each promoter are presented as fold inductions ± SEM. Luciferase activities were determined in three experiments each performed in duplicates. (F) Immunoprecipitation with anti-HA tag antibodies was performed on formaldehyde-fixed chromatin from Neuro2a cells transiently transfected with expression plasmids for the long Sox5 isoform, HA-tagged CtBP2 or their combination as indicated above the lanes. Mock-transfected Neuro2a cells (−) served as control. PCR was applied on the immunoprecipitate to detect the Mitf and the Dct promoter regions under the same conditions as in (C and D). (H2O), water control.

Mentions: We next addressed the ability of both Sox proteins to influence the activities of the Mitf and Dct promoters in luciferase reporter assays. Whereas Sox10 robustly activated both promoters in transiently transfected Neuro2a cells, no such activation was observed for the long Sox5 isoform (L-Sox5) over a broad range of concentrations (Figure 5A and B and data not shown). This agrees with previous findings that all Sox5 isoforms lack a classical transactivation domain (3). When reporter genes were cotransfected simultaneously with both Sox proteins, Sox10 furthermore lost its ability to efficiently activate either the Dct promoter (Figure 5A) or the Mitf promoter (Figure 5B). We thus conclude that the long Sox5 isoform counteracts the transcriptional activity of Sox10 on its melanocytic target gene promoters in heterologous cell lines. Qualitatively similar results were also obtained in transiently transfected B16 melanoma cells (Figure 5C and D), although Sox10-dependent activation rates were lower, likely because of the presence of endogenous Sox10 in these cells (47) (see also Figure10A and B).Figure 5.


The transcription factor Sox5 modulates Sox10 function during melanocyte development.

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

Sox5 binds to melanocyte-specific promoters and recruits transcriptional corepressors. (A and B) B16 melanoma cells were transiently cotransfected with reporter plasmids in which the luciferase gene was under control of the Dct (A) or the Mitf (B) promoters, and expression plasmids for shRNAs directed against Sox10 and Sox5 or their scrambled versions, as indicated below the bars. Promoter activity in the absence of cotransfected shRNA was arbitrarily set to one and all other activities are expressed relative to this value ± SEM. Luciferase activities were determined in three (A) and five (B) experiments each performed in duplicates. The higher transactivation rates obtained for both promoters in the presence of both Sox5 shRNA and Sox10 shRNA as compared to Sox10 shRNA alone were statistically significant as determined by Student's t-test (**P ⩽ 0.01). (C and D) Immunoprecipitation was performed on formaldehyde-fixed chromatin from B16 melanoma cells in the absence (−) and presence of antibodies. In addition to control IgGs (IgG), antisera specifically directed against Sox5 (α-Sox5), Sox10 (α-Sox10), HDAC1 (α-HDAC1) and CtBP2 (α-CtBP2) were employed. PCR was applied on the immunoprecipitate to detect the Dct promoter region (a) and the Mitf promoter region (c) that contain the previously identified Sox10 response elements. Regions further upstream and more distal to the transcription start sites of the Dct (b) and Mitf (d) genes were additionally amplified by PCR as controls. All fragments were recovered from 1/20 of the material used for immunoprecipitation (input). (H2O), water control. (E) Neuro2a cells were transiently transfected using a reporter plasmid in which the luciferase gene was under control of the 1.5 kb Mitf promoter. Expression plasmids for Sox10, Sox5, HDAC1 and CtBP2 were cotransfected as indicated below the bars. Whereas Sox10 expression plasmids were used at 100 ng, Sox5 expression plasmids were employed at 20 ng and HDAC1 or CtBP2 expression plasmids at 10 ng per transfection reaction. Transactivation rates for each promoter are presented as fold inductions ± SEM. Luciferase activities were determined in three experiments each performed in duplicates. (F) Immunoprecipitation with anti-HA tag antibodies was performed on formaldehyde-fixed chromatin from Neuro2a cells transiently transfected with expression plasmids for the long Sox5 isoform, HA-tagged CtBP2 or their combination as indicated above the lanes. Mock-transfected Neuro2a cells (−) served as control. PCR was applied on the immunoprecipitate to detect the Mitf and the Dct promoter regions under the same conditions as in (C and D). (H2O), water control.
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Figure 10: Sox5 binds to melanocyte-specific promoters and recruits transcriptional corepressors. (A and B) B16 melanoma cells were transiently cotransfected with reporter plasmids in which the luciferase gene was under control of the Dct (A) or the Mitf (B) promoters, and expression plasmids for shRNAs directed against Sox10 and Sox5 or their scrambled versions, as indicated below the bars. Promoter activity in the absence of cotransfected shRNA was arbitrarily set to one and all other activities are expressed relative to this value ± SEM. Luciferase activities were determined in three (A) and five (B) experiments each performed in duplicates. The higher transactivation rates obtained for both promoters in the presence of both Sox5 shRNA and Sox10 shRNA as compared to Sox10 shRNA alone were statistically significant as determined by Student's t-test (**P ⩽ 0.01). (C and D) Immunoprecipitation was performed on formaldehyde-fixed chromatin from B16 melanoma cells in the absence (−) and presence of antibodies. In addition to control IgGs (IgG), antisera specifically directed against Sox5 (α-Sox5), Sox10 (α-Sox10), HDAC1 (α-HDAC1) and CtBP2 (α-CtBP2) were employed. PCR was applied on the immunoprecipitate to detect the Dct promoter region (a) and the Mitf promoter region (c) that contain the previously identified Sox10 response elements. Regions further upstream and more distal to the transcription start sites of the Dct (b) and Mitf (d) genes were additionally amplified by PCR as controls. All fragments were recovered from 1/20 of the material used for immunoprecipitation (input). (H2O), water control. (E) Neuro2a cells were transiently transfected using a reporter plasmid in which the luciferase gene was under control of the 1.5 kb Mitf promoter. Expression plasmids for Sox10, Sox5, HDAC1 and CtBP2 were cotransfected as indicated below the bars. Whereas Sox10 expression plasmids were used at 100 ng, Sox5 expression plasmids were employed at 20 ng and HDAC1 or CtBP2 expression plasmids at 10 ng per transfection reaction. Transactivation rates for each promoter are presented as fold inductions ± SEM. Luciferase activities were determined in three experiments each performed in duplicates. (F) Immunoprecipitation with anti-HA tag antibodies was performed on formaldehyde-fixed chromatin from Neuro2a cells transiently transfected with expression plasmids for the long Sox5 isoform, HA-tagged CtBP2 or their combination as indicated above the lanes. Mock-transfected Neuro2a cells (−) served as control. PCR was applied on the immunoprecipitate to detect the Mitf and the Dct promoter regions under the same conditions as in (C and D). (H2O), water control.
Mentions: We next addressed the ability of both Sox proteins to influence the activities of the Mitf and Dct promoters in luciferase reporter assays. Whereas Sox10 robustly activated both promoters in transiently transfected Neuro2a cells, no such activation was observed for the long Sox5 isoform (L-Sox5) over a broad range of concentrations (Figure 5A and B and data not shown). This agrees with previous findings that all Sox5 isoforms lack a classical transactivation domain (3). When reporter genes were cotransfected simultaneously with both Sox proteins, Sox10 furthermore lost its ability to efficiently activate either the Dct promoter (Figure 5A) or the Mitf promoter (Figure 5B). We thus conclude that the long Sox5 isoform counteracts the transcriptional activity of Sox10 on its melanocytic target gene promoters in heterologous cell lines. Qualitatively similar results were also obtained in transiently transfected B16 melanoma cells (Figure 5C and D), although Sox10-dependent activation rates were lower, likely because of the presence of endogenous Sox10 in these cells (47) (see also Figure10A and B).Figure 5.

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