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The transcription factors Sox10 and Myrf define an essential regulatory network module in differentiating oligodendrocytes.

Hornig J, Fröb F, Vogl MR, Hermans-Borgmeyer I, Tamm ER, Wegner M - PLoS Genet. (2013)

Bottom Line: Once induced, Myrf cooperates with Sox10 to implement the myelination program as evident from the physical interaction between both proteins and the synergistic activation of several myelin-specific genes.This is strongly reminiscent of the situation in Schwann cells where Sox10 first induces and then cooperates with Krox20 during myelination.Our analyses indicate that the regulatory network for myelination in oligodendrocytes is organized along similar general principles as the one in Schwann cells, but is differentially implemented.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen, Germany.

ABSTRACT
Myelin is essential for rapid saltatory conduction and is produced by Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system. In both cell types the transcription factor Sox10 is an essential component of the myelin-specific regulatory network. Here we identify Myrf as an oligodendrocyte-specific target of Sox10 and map a Sox10 responsive enhancer to an evolutionarily conserved element in intron 1 of the Myrf gene. Once induced, Myrf cooperates with Sox10 to implement the myelination program as evident from the physical interaction between both proteins and the synergistic activation of several myelin-specific genes. This is strongly reminiscent of the situation in Schwann cells where Sox10 first induces and then cooperates with Krox20 during myelination. Our analyses indicate that the regulatory network for myelination in oligodendrocytes is organized along similar general principles as the one in Schwann cells, but is differentially implemented.

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Myrf is a Sox10 target gene in OL.(A–C) Primary rat OPC were transfected with expression vectors for scrambled (shSCR) or Sox10-specific shRNAs (shSox10) and GFP, and replated in differentiation medium. One day later, transfected cells were visualized by GFP expression (green) and analyzed for their expression of Sox10 (A), Myrf (B) and Mbp (C) (all in red) as indicated. The yellow color in the merged pictures indicates co-expression. Scale bar, 25 µm. (D,E) Neural tube electroporations were carried out in HH11-stage chicken embryos using expression vectors for GFP (D) and a combination of Sox10 and GFP (E). Analysis was one day after electroporation and the electroporated right side is visualized by GFP expression (green). Sections were simultaneously probed for the occurrence of Sox10 (white) and Myrf (red). Scale bars, 25 µm. (F) Several ECR (ECR1-ECR13) are localized in the Myrf genomic interval on mouse chromosome 19 between the adjacent Dagla and 1810006K21 Rik genes. ECR locations relative to introns and exons of the Myrf gene are shown. ECR1-ECR6 and ECR13 (marked in green) are conserved among mammals, ECR7-ECR12 (marked in pink) additionally in birds. (G, H) The Myrf ECR were tested in 33B cells after transient transfection for their ability to increase expression of a luciferase reporter under control of a minimal promoter (mp). The minimal promoter was taken from the Hsp68 gene (hsp68-luc) (G) or the Myrf gene (myrf-luc) (H). Luciferase activities in extracts from transfected cells were determined 48 hours post-transfection in three experiments each performed in quadruplicates. The luciferase activity obtained for a reporter plasmid containing only the minimal promoter (−) was arbitrarily set to 1. Activities in the presence of ECRs were calculated relative to minimal promoter activity and are presented as mean ± SEM. A reporter in which the minimal promoter was combined with Mbp regulatory regions served as positive control. (I, J) Transfections of the ECR containing hsp68-luc (I) and myrf-luc (J) reporters were carried out in the presence of Sox10-specific shRNA (shSox10) and scrambled (shSCR) shRNA. Luciferase activities were determined and the ratio of activities in the presence of Sox10-specific shRNA versus scrambled shRNA was calculated. Normalized values are presented as mean ± SEM. Experiments were performed three times in quadruplicates. shSox10-dependent downregulation of the activity of ECR9-containing luciferase reporters was statistically significant (P≤0.05, determined by Student's t test).
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pgen-1003907-g004: Myrf is a Sox10 target gene in OL.(A–C) Primary rat OPC were transfected with expression vectors for scrambled (shSCR) or Sox10-specific shRNAs (shSox10) and GFP, and replated in differentiation medium. One day later, transfected cells were visualized by GFP expression (green) and analyzed for their expression of Sox10 (A), Myrf (B) and Mbp (C) (all in red) as indicated. The yellow color in the merged pictures indicates co-expression. Scale bar, 25 µm. (D,E) Neural tube electroporations were carried out in HH11-stage chicken embryos using expression vectors for GFP (D) and a combination of Sox10 and GFP (E). Analysis was one day after electroporation and the electroporated right side is visualized by GFP expression (green). Sections were simultaneously probed for the occurrence of Sox10 (white) and Myrf (red). Scale bars, 25 µm. (F) Several ECR (ECR1-ECR13) are localized in the Myrf genomic interval on mouse chromosome 19 between the adjacent Dagla and 1810006K21 Rik genes. ECR locations relative to introns and exons of the Myrf gene are shown. ECR1-ECR6 and ECR13 (marked in green) are conserved among mammals, ECR7-ECR12 (marked in pink) additionally in birds. (G, H) The Myrf ECR were tested in 33B cells after transient transfection for their ability to increase expression of a luciferase reporter under control of a minimal promoter (mp). The minimal promoter was taken from the Hsp68 gene (hsp68-luc) (G) or the Myrf gene (myrf-luc) (H). Luciferase activities in extracts from transfected cells were determined 48 hours post-transfection in three experiments each performed in quadruplicates. The luciferase activity obtained for a reporter plasmid containing only the minimal promoter (−) was arbitrarily set to 1. Activities in the presence of ECRs were calculated relative to minimal promoter activity and are presented as mean ± SEM. A reporter in which the minimal promoter was combined with Mbp regulatory regions served as positive control. (I, J) Transfections of the ECR containing hsp68-luc (I) and myrf-luc (J) reporters were carried out in the presence of Sox10-specific shRNA (shSox10) and scrambled (shSCR) shRNA. Luciferase activities were determined and the ratio of activities in the presence of Sox10-specific shRNA versus scrambled shRNA was calculated. Normalized values are presented as mean ± SEM. Experiments were performed three times in quadruplicates. shSox10-dependent downregulation of the activity of ECR9-containing luciferase reporters was statistically significant (P≤0.05, determined by Student's t test).

Mentions: To explore the relationship between Sox10 and Myrf, we first used primary oligodendroglial cultures. We transfected OPC with a Sox10-specific shRNA or a scrambled version in the presence of GFP, and analyzed the cells after one day in culture under differentiating conditions. While nearly all cells (98±2%) transfected with a scrambled shRNA exhibited Sox10 expression, only very few (16±5%) continued to be Sox10-positive in the presence of a Sox10-specific shRNA (Figure 4A). At the same time, cells transfected with the Sox10-specific shRNA failed to induce Myrf or Mbp expression in contrast to untransfected cells and cells transfected with the scrambled shRNA version (Figure 4B,C). This confirms that Myrf expression depends on Sox10.


The transcription factors Sox10 and Myrf define an essential regulatory network module in differentiating oligodendrocytes.

Hornig J, Fröb F, Vogl MR, Hermans-Borgmeyer I, Tamm ER, Wegner M - PLoS Genet. (2013)

Myrf is a Sox10 target gene in OL.(A–C) Primary rat OPC were transfected with expression vectors for scrambled (shSCR) or Sox10-specific shRNAs (shSox10) and GFP, and replated in differentiation medium. One day later, transfected cells were visualized by GFP expression (green) and analyzed for their expression of Sox10 (A), Myrf (B) and Mbp (C) (all in red) as indicated. The yellow color in the merged pictures indicates co-expression. Scale bar, 25 µm. (D,E) Neural tube electroporations were carried out in HH11-stage chicken embryos using expression vectors for GFP (D) and a combination of Sox10 and GFP (E). Analysis was one day after electroporation and the electroporated right side is visualized by GFP expression (green). Sections were simultaneously probed for the occurrence of Sox10 (white) and Myrf (red). Scale bars, 25 µm. (F) Several ECR (ECR1-ECR13) are localized in the Myrf genomic interval on mouse chromosome 19 between the adjacent Dagla and 1810006K21 Rik genes. ECR locations relative to introns and exons of the Myrf gene are shown. ECR1-ECR6 and ECR13 (marked in green) are conserved among mammals, ECR7-ECR12 (marked in pink) additionally in birds. (G, H) The Myrf ECR were tested in 33B cells after transient transfection for their ability to increase expression of a luciferase reporter under control of a minimal promoter (mp). The minimal promoter was taken from the Hsp68 gene (hsp68-luc) (G) or the Myrf gene (myrf-luc) (H). Luciferase activities in extracts from transfected cells were determined 48 hours post-transfection in three experiments each performed in quadruplicates. The luciferase activity obtained for a reporter plasmid containing only the minimal promoter (−) was arbitrarily set to 1. Activities in the presence of ECRs were calculated relative to minimal promoter activity and are presented as mean ± SEM. A reporter in which the minimal promoter was combined with Mbp regulatory regions served as positive control. (I, J) Transfections of the ECR containing hsp68-luc (I) and myrf-luc (J) reporters were carried out in the presence of Sox10-specific shRNA (shSox10) and scrambled (shSCR) shRNA. Luciferase activities were determined and the ratio of activities in the presence of Sox10-specific shRNA versus scrambled shRNA was calculated. Normalized values are presented as mean ± SEM. Experiments were performed three times in quadruplicates. shSox10-dependent downregulation of the activity of ECR9-containing luciferase reporters was statistically significant (P≤0.05, determined by Student's t test).
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pgen-1003907-g004: Myrf is a Sox10 target gene in OL.(A–C) Primary rat OPC were transfected with expression vectors for scrambled (shSCR) or Sox10-specific shRNAs (shSox10) and GFP, and replated in differentiation medium. One day later, transfected cells were visualized by GFP expression (green) and analyzed for their expression of Sox10 (A), Myrf (B) and Mbp (C) (all in red) as indicated. The yellow color in the merged pictures indicates co-expression. Scale bar, 25 µm. (D,E) Neural tube electroporations were carried out in HH11-stage chicken embryos using expression vectors for GFP (D) and a combination of Sox10 and GFP (E). Analysis was one day after electroporation and the electroporated right side is visualized by GFP expression (green). Sections were simultaneously probed for the occurrence of Sox10 (white) and Myrf (red). Scale bars, 25 µm. (F) Several ECR (ECR1-ECR13) are localized in the Myrf genomic interval on mouse chromosome 19 between the adjacent Dagla and 1810006K21 Rik genes. ECR locations relative to introns and exons of the Myrf gene are shown. ECR1-ECR6 and ECR13 (marked in green) are conserved among mammals, ECR7-ECR12 (marked in pink) additionally in birds. (G, H) The Myrf ECR were tested in 33B cells after transient transfection for their ability to increase expression of a luciferase reporter under control of a minimal promoter (mp). The minimal promoter was taken from the Hsp68 gene (hsp68-luc) (G) or the Myrf gene (myrf-luc) (H). Luciferase activities in extracts from transfected cells were determined 48 hours post-transfection in three experiments each performed in quadruplicates. The luciferase activity obtained for a reporter plasmid containing only the minimal promoter (−) was arbitrarily set to 1. Activities in the presence of ECRs were calculated relative to minimal promoter activity and are presented as mean ± SEM. A reporter in which the minimal promoter was combined with Mbp regulatory regions served as positive control. (I, J) Transfections of the ECR containing hsp68-luc (I) and myrf-luc (J) reporters were carried out in the presence of Sox10-specific shRNA (shSox10) and scrambled (shSCR) shRNA. Luciferase activities were determined and the ratio of activities in the presence of Sox10-specific shRNA versus scrambled shRNA was calculated. Normalized values are presented as mean ± SEM. Experiments were performed three times in quadruplicates. shSox10-dependent downregulation of the activity of ECR9-containing luciferase reporters was statistically significant (P≤0.05, determined by Student's t test).
Mentions: To explore the relationship between Sox10 and Myrf, we first used primary oligodendroglial cultures. We transfected OPC with a Sox10-specific shRNA or a scrambled version in the presence of GFP, and analyzed the cells after one day in culture under differentiating conditions. While nearly all cells (98±2%) transfected with a scrambled shRNA exhibited Sox10 expression, only very few (16±5%) continued to be Sox10-positive in the presence of a Sox10-specific shRNA (Figure 4A). At the same time, cells transfected with the Sox10-specific shRNA failed to induce Myrf or Mbp expression in contrast to untransfected cells and cells transfected with the scrambled shRNA version (Figure 4B,C). This confirms that Myrf expression depends on Sox10.

Bottom Line: Once induced, Myrf cooperates with Sox10 to implement the myelination program as evident from the physical interaction between both proteins and the synergistic activation of several myelin-specific genes.This is strongly reminiscent of the situation in Schwann cells where Sox10 first induces and then cooperates with Krox20 during myelination.Our analyses indicate that the regulatory network for myelination in oligodendrocytes is organized along similar general principles as the one in Schwann cells, but is differentially implemented.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen, Germany.

ABSTRACT
Myelin is essential for rapid saltatory conduction and is produced by Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system. In both cell types the transcription factor Sox10 is an essential component of the myelin-specific regulatory network. Here we identify Myrf as an oligodendrocyte-specific target of Sox10 and map a Sox10 responsive enhancer to an evolutionarily conserved element in intron 1 of the Myrf gene. Once induced, Myrf cooperates with Sox10 to implement the myelination program as evident from the physical interaction between both proteins and the synergistic activation of several myelin-specific genes. This is strongly reminiscent of the situation in Schwann cells where Sox10 first induces and then cooperates with Krox20 during myelination. Our analyses indicate that the regulatory network for myelination in oligodendrocytes is organized along similar general principles as the one in Schwann cells, but is differentially implemented.

Show MeSH
Related in: MedlinePlus