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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 and Sox10 influence melanocyte-specific gene expression in B16 melanoma cells. Immunocytochemistry was carried out on B16 melanoma cells transfected with an shRNA vector specific for Sox10 (A, B, E, F, I and J), the corresponding scrambled version (C, D, G, H, K and L), an shRNA vector for Sox5 (M, N, Q, R, U and V), its corresponding scrambled version (O, P, S, T, W and X) and a combination of Sox5- and Sox10-specific shRNA vectors (Y and Z). Immunocytochemistry was performed using antibodies against Sox10 (A–D, M–P), Sox5 (E–H, Q–T) and Mitf (I–L, U–Z); and immunolabeling is shown in red. Transfected cells were visualized by GFP autofluorescence (B, D, F, H, J, L, N, P, R, T, V, X and Z) (green color). Arrowheads indicate transfected cells in which expression of endogenous proteins is lost or significantly reduced.
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Figure 8: Sox5 and Sox10 influence melanocyte-specific gene expression in B16 melanoma cells. Immunocytochemistry was carried out on B16 melanoma cells transfected with an shRNA vector specific for Sox10 (A, B, E, F, I and J), the corresponding scrambled version (C, D, G, H, K and L), an shRNA vector for Sox5 (M, N, Q, R, U and V), its corresponding scrambled version (O, P, S, T, W and X) and a combination of Sox5- and Sox10-specific shRNA vectors (Y and Z). Immunocytochemistry was performed using antibodies against Sox10 (A–D, M–P), Sox5 (E–H, Q–T) and Mitf (I–L, U–Z); and immunolabeling is shown in red. Transfected cells were visualized by GFP autofluorescence (B, D, F, H, J, L, N, P, R, T, V, X and Z) (green color). Arrowheads indicate transfected cells in which expression of endogenous proteins is lost or significantly reduced.

Mentions: Despite this complex Sox protein expression pattern, the impact of Sox5 on the expression of Sox10 target genes was assessed in B16 melanoma cells by transfection with shRNA vectors. Using GFP expressed from the IRES-EGFP cassette of the shRNA plasmid, we identified the transfected cells and analyzed the amount of Sox proteins in these cells relative to untransfected neighboring cells. These studies revealed that a Sox10-specific shRNA reduced the amount of Sox10, but not of Sox5 in B16 melanoma cells (Figure 8A, B, E and F), whereas a Sox5-specific shRNA reduced the Sox5, but not the Sox10 amounts (Figure 8M, N, Q and R). The Sox5-specific shRNA likewise had no influence on the Sox6 amounts in transfected cells (Figure 9A and B). Furthermore, scrambled versions of the Sox5-specific or the Sox10-specific shRNAs did not change the level of either Sox protein and served as controls (Figure 8C, D, G, H, O, P, S and T). Concomitant with a reduction of endogenous Sox10 levels, there was also a dramatic reduction of Mitf amounts in >90% of all B16 cells transfected with a Sox10-specific shRNA (Figure 8I and J), thus confirming Mitf as a Sox10 target gene (25–29,33). In contrast, >90% of B16 cells transfected with a Sox5-specific shRNA or the scrambled shRNA versions exhibited unaltered Mitf levels (Figure 8K, L, U, V, W and X). These experiments confirm on a cellular level that on its own endogenous Sox10, but not Sox5 influences Mitf expression in melanocyte-derived cells.Figure 9.


The transcription factor Sox5 modulates Sox10 function during melanocyte development.

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

Sox5 and Sox10 influence melanocyte-specific gene expression in B16 melanoma cells. Immunocytochemistry was carried out on B16 melanoma cells transfected with an shRNA vector specific for Sox10 (A, B, E, F, I and J), the corresponding scrambled version (C, D, G, H, K and L), an shRNA vector for Sox5 (M, N, Q, R, U and V), its corresponding scrambled version (O, P, S, T, W and X) and a combination of Sox5- and Sox10-specific shRNA vectors (Y and Z). Immunocytochemistry was performed using antibodies against Sox10 (A–D, M–P), Sox5 (E–H, Q–T) and Mitf (I–L, U–Z); and immunolabeling is shown in red. Transfected cells were visualized by GFP autofluorescence (B, D, F, H, J, L, N, P, R, T, V, X and Z) (green color). Arrowheads indicate transfected cells in which expression of endogenous proteins is lost or significantly reduced.
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Figure 8: Sox5 and Sox10 influence melanocyte-specific gene expression in B16 melanoma cells. Immunocytochemistry was carried out on B16 melanoma cells transfected with an shRNA vector specific for Sox10 (A, B, E, F, I and J), the corresponding scrambled version (C, D, G, H, K and L), an shRNA vector for Sox5 (M, N, Q, R, U and V), its corresponding scrambled version (O, P, S, T, W and X) and a combination of Sox5- and Sox10-specific shRNA vectors (Y and Z). Immunocytochemistry was performed using antibodies against Sox10 (A–D, M–P), Sox5 (E–H, Q–T) and Mitf (I–L, U–Z); and immunolabeling is shown in red. Transfected cells were visualized by GFP autofluorescence (B, D, F, H, J, L, N, P, R, T, V, X and Z) (green color). Arrowheads indicate transfected cells in which expression of endogenous proteins is lost or significantly reduced.
Mentions: Despite this complex Sox protein expression pattern, the impact of Sox5 on the expression of Sox10 target genes was assessed in B16 melanoma cells by transfection with shRNA vectors. Using GFP expressed from the IRES-EGFP cassette of the shRNA plasmid, we identified the transfected cells and analyzed the amount of Sox proteins in these cells relative to untransfected neighboring cells. These studies revealed that a Sox10-specific shRNA reduced the amount of Sox10, but not of Sox5 in B16 melanoma cells (Figure 8A, B, E and F), whereas a Sox5-specific shRNA reduced the Sox5, but not the Sox10 amounts (Figure 8M, N, Q and R). The Sox5-specific shRNA likewise had no influence on the Sox6 amounts in transfected cells (Figure 9A and B). Furthermore, scrambled versions of the Sox5-specific or the Sox10-specific shRNAs did not change the level of either Sox protein and served as controls (Figure 8C, D, G, H, O, P, S and T). Concomitant with a reduction of endogenous Sox10 levels, there was also a dramatic reduction of Mitf amounts in >90% of all B16 cells transfected with a Sox10-specific shRNA (Figure 8I and J), thus confirming Mitf as a Sox10 target gene (25–29,33). In contrast, >90% of B16 cells transfected with a Sox5-specific shRNA or the scrambled shRNA versions exhibited unaltered Mitf levels (Figure 8K, L, U, V, W and X). These experiments confirm on a cellular level that on its own endogenous Sox10, but not Sox5 influences Mitf expression in melanocyte-derived cells.Figure 9.

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