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Olig2 regulates Sox10 expression in oligodendrocyte precursors through an evolutionary conserved distal enhancer.

Küspert M, Hammer A, Bösl MR, Wegner M - Nucleic Acids Res. (2010)

Bottom Line: We found that U2 was active in oligodendrocyte precursors, but not in mature oligodendrocytes.U2 activity also did not mediate the initial Sox10 induction after specification arguing that Sox10 expression during oligodendroglial development depends on the activity of multiple regulatory regions.Considering the selective expression of Nkx6.2 at the time of specification and in differentiated oligodendrocytes, Nkx6.2 may be involved in limiting U2 activity to the precursor stage during oligodendrocyte development.

View Article: PubMed Central - PubMed

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

ABSTRACT
The HMG-domain transcription factor Sox10 is expressed throughout oligodendrocyte development and is an important component of the transcriptional regulatory network in these myelin-forming CNS glia. Of the known Sox10 regulatory regions, only the evolutionary conserved U2 enhancer in the distal 5'-flank of the Sox10 gene exhibits oligodendroglial activity. We found that U2 was active in oligodendrocyte precursors, but not in mature oligodendrocytes. U2 activity also did not mediate the initial Sox10 induction after specification arguing that Sox10 expression during oligodendroglial development depends on the activity of multiple regulatory regions. The oligodendroglial bHLH transcription factor Olig2, but not the closely related Olig1 efficiently activated the U2 enhancer. Olig2 bound U2 directly at several sites including a highly conserved one in the U2 core. Inactivation of this site abolished the oligodendroglial activity of U2 in vivo. In contrast to Olig2, the homeodomain transcription factor Nkx6.2 repressed U2 activity. Repression may involve recruitment of Nkx6.2 to U2 and inactivation of Olig2 and other activators by protein-protein interactions. Considering the selective expression of Nkx6.2 at the time of specification and in differentiated oligodendrocytes, Nkx6.2 may be involved in limiting U2 activity to the precursor stage during oligodendrocyte development.

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The Sox10 U2 enhancer contains binding sites for Olig2 and Nkx6.2. (A) Sequence of mouse U2. Asterisks below the sequence indicate positions that are fully conserved among mouse, human and chicken. The 190 bp core region (gray box) is particularly enriched for conserved positions. Putative binding sites are marked by a bar above the sequence. Potential Olig2 binding sites are designated as bHLH1–bHLH6, potential Nkx6.2 binding sites as GTX1 and GTX2. Numbers on the right indicate the exact nucleotide position. (B) Transient transfections were performed in 33B oligodendroglioma with the U2 luc reporter and pCMV-based expression plasmids for Olig1 and E47 as indicated. Increasing amounts of expression plasmid were used as indicated by the triangles (Olig1) and + versus ++ (E47). Luciferase activities in extracts from transfected cells were determined in three experiments each performed in duplicates. The luciferase activity obtained for U2-luc in the absence of ectopic transcription factor was arbitrarily set to 1. Fold inductions in the presence of transcription factors were calculated and are presented as mean ± SD. (C and D) EMSA was performed with radiolabeled double-stranded oligonucleotides encompassing the potential Olig2 binding sites bHLH1, bHLH2 and bHLH4 (for sequence, see Figure 5A). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells that were either untransfected (control) or transfected with expression plasmids for myc-tagged versions of Olig1 or Olig2. Antibodies against the tag (+Ab) were added as indicated. The Olig2-containing complex is marked by an arrowhead, the Olig1-containing complex by a dot and the supershifted complexes by asterisks. All other complexes are unspecific as they were also obtained with the control extracts. (E and F) EMSA was carried out with radiolabeled double-stranded oligonucleotides encompassing GTX1, GTX2 (for sequence, see Figure 5A) and the known high-affinity Nkx6.2 binding sequence HoxA5/A6 (Hox). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells that were either untransfected (control) or transfected with expression plasmids for T7-tagged versions of full-length Nkx6.2 (fl) or amino- and carboxyterminally truncated Nkx6.2 (ΔNC). Antibodies against the tag (+Ab) were added as indicated. The complex containing full-length Nkx6.2 is marked by an arrowhead, the complex containing the ΔNC version by a dot.
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Figure 4: The Sox10 U2 enhancer contains binding sites for Olig2 and Nkx6.2. (A) Sequence of mouse U2. Asterisks below the sequence indicate positions that are fully conserved among mouse, human and chicken. The 190 bp core region (gray box) is particularly enriched for conserved positions. Putative binding sites are marked by a bar above the sequence. Potential Olig2 binding sites are designated as bHLH1–bHLH6, potential Nkx6.2 binding sites as GTX1 and GTX2. Numbers on the right indicate the exact nucleotide position. (B) Transient transfections were performed in 33B oligodendroglioma with the U2 luc reporter and pCMV-based expression plasmids for Olig1 and E47 as indicated. Increasing amounts of expression plasmid were used as indicated by the triangles (Olig1) and + versus ++ (E47). Luciferase activities in extracts from transfected cells were determined in three experiments each performed in duplicates. The luciferase activity obtained for U2-luc in the absence of ectopic transcription factor was arbitrarily set to 1. Fold inductions in the presence of transcription factors were calculated and are presented as mean ± SD. (C and D) EMSA was performed with radiolabeled double-stranded oligonucleotides encompassing the potential Olig2 binding sites bHLH1, bHLH2 and bHLH4 (for sequence, see Figure 5A). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells that were either untransfected (control) or transfected with expression plasmids for myc-tagged versions of Olig1 or Olig2. Antibodies against the tag (+Ab) were added as indicated. The Olig2-containing complex is marked by an arrowhead, the Olig1-containing complex by a dot and the supershifted complexes by asterisks. All other complexes are unspecific as they were also obtained with the control extracts. (E and F) EMSA was carried out with radiolabeled double-stranded oligonucleotides encompassing GTX1, GTX2 (for sequence, see Figure 5A) and the known high-affinity Nkx6.2 binding sequence HoxA5/A6 (Hox). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells that were either untransfected (control) or transfected with expression plasmids for T7-tagged versions of full-length Nkx6.2 (fl) or amino- and carboxyterminally truncated Nkx6.2 (ΔNC). Antibodies against the tag (+Ab) were added as indicated. The complex containing full-length Nkx6.2 is marked by an arrowhead, the complex containing the ΔNC version by a dot.

Mentions: U2mut-lacZ resembled the previously described U2-lacZ transgene except for the presence of an inactivating mutation in the Olig2 binding site bHLH4 (Figure 4A). After separation from the vector backbone and purification, the U2mut-lacZ transgene was microinjected as a SpeI/KpnI fragment into the male pronucleus of fertilized FVB oocytes. Transgenic mice were generated from injected oocytes according to standard techniques. Founder mice and transgenic offspring were identified and genotyped by PCR analysis of DNA prepared from tail biopsies using the U2-specific forward primer 5′-GGCACAGAAAGGTCTCTTTG-3′ and the lacZ-specific reverse primer 5′-AGTAGCTGTCAGCGTCTGGT-3′ as described previously (12).


Olig2 regulates Sox10 expression in oligodendrocyte precursors through an evolutionary conserved distal enhancer.

Küspert M, Hammer A, Bösl MR, Wegner M - Nucleic Acids Res. (2010)

The Sox10 U2 enhancer contains binding sites for Olig2 and Nkx6.2. (A) Sequence of mouse U2. Asterisks below the sequence indicate positions that are fully conserved among mouse, human and chicken. The 190 bp core region (gray box) is particularly enriched for conserved positions. Putative binding sites are marked by a bar above the sequence. Potential Olig2 binding sites are designated as bHLH1–bHLH6, potential Nkx6.2 binding sites as GTX1 and GTX2. Numbers on the right indicate the exact nucleotide position. (B) Transient transfections were performed in 33B oligodendroglioma with the U2 luc reporter and pCMV-based expression plasmids for Olig1 and E47 as indicated. Increasing amounts of expression plasmid were used as indicated by the triangles (Olig1) and + versus ++ (E47). Luciferase activities in extracts from transfected cells were determined in three experiments each performed in duplicates. The luciferase activity obtained for U2-luc in the absence of ectopic transcription factor was arbitrarily set to 1. Fold inductions in the presence of transcription factors were calculated and are presented as mean ± SD. (C and D) EMSA was performed with radiolabeled double-stranded oligonucleotides encompassing the potential Olig2 binding sites bHLH1, bHLH2 and bHLH4 (for sequence, see Figure 5A). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells that were either untransfected (control) or transfected with expression plasmids for myc-tagged versions of Olig1 or Olig2. Antibodies against the tag (+Ab) were added as indicated. The Olig2-containing complex is marked by an arrowhead, the Olig1-containing complex by a dot and the supershifted complexes by asterisks. All other complexes are unspecific as they were also obtained with the control extracts. (E and F) EMSA was carried out with radiolabeled double-stranded oligonucleotides encompassing GTX1, GTX2 (for sequence, see Figure 5A) and the known high-affinity Nkx6.2 binding sequence HoxA5/A6 (Hox). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells that were either untransfected (control) or transfected with expression plasmids for T7-tagged versions of full-length Nkx6.2 (fl) or amino- and carboxyterminally truncated Nkx6.2 (ΔNC). Antibodies against the tag (+Ab) were added as indicated. The complex containing full-length Nkx6.2 is marked by an arrowhead, the complex containing the ΔNC version by a dot.
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Figure 4: The Sox10 U2 enhancer contains binding sites for Olig2 and Nkx6.2. (A) Sequence of mouse U2. Asterisks below the sequence indicate positions that are fully conserved among mouse, human and chicken. The 190 bp core region (gray box) is particularly enriched for conserved positions. Putative binding sites are marked by a bar above the sequence. Potential Olig2 binding sites are designated as bHLH1–bHLH6, potential Nkx6.2 binding sites as GTX1 and GTX2. Numbers on the right indicate the exact nucleotide position. (B) Transient transfections were performed in 33B oligodendroglioma with the U2 luc reporter and pCMV-based expression plasmids for Olig1 and E47 as indicated. Increasing amounts of expression plasmid were used as indicated by the triangles (Olig1) and + versus ++ (E47). Luciferase activities in extracts from transfected cells were determined in three experiments each performed in duplicates. The luciferase activity obtained for U2-luc in the absence of ectopic transcription factor was arbitrarily set to 1. Fold inductions in the presence of transcription factors were calculated and are presented as mean ± SD. (C and D) EMSA was performed with radiolabeled double-stranded oligonucleotides encompassing the potential Olig2 binding sites bHLH1, bHLH2 and bHLH4 (for sequence, see Figure 5A). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells that were either untransfected (control) or transfected with expression plasmids for myc-tagged versions of Olig1 or Olig2. Antibodies against the tag (+Ab) were added as indicated. The Olig2-containing complex is marked by an arrowhead, the Olig1-containing complex by a dot and the supershifted complexes by asterisks. All other complexes are unspecific as they were also obtained with the control extracts. (E and F) EMSA was carried out with radiolabeled double-stranded oligonucleotides encompassing GTX1, GTX2 (for sequence, see Figure 5A) and the known high-affinity Nkx6.2 binding sequence HoxA5/A6 (Hox). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells that were either untransfected (control) or transfected with expression plasmids for T7-tagged versions of full-length Nkx6.2 (fl) or amino- and carboxyterminally truncated Nkx6.2 (ΔNC). Antibodies against the tag (+Ab) were added as indicated. The complex containing full-length Nkx6.2 is marked by an arrowhead, the complex containing the ΔNC version by a dot.
Mentions: U2mut-lacZ resembled the previously described U2-lacZ transgene except for the presence of an inactivating mutation in the Olig2 binding site bHLH4 (Figure 4A). After separation from the vector backbone and purification, the U2mut-lacZ transgene was microinjected as a SpeI/KpnI fragment into the male pronucleus of fertilized FVB oocytes. Transgenic mice were generated from injected oocytes according to standard techniques. Founder mice and transgenic offspring were identified and genotyped by PCR analysis of DNA prepared from tail biopsies using the U2-specific forward primer 5′-GGCACAGAAAGGTCTCTTTG-3′ and the lacZ-specific reverse primer 5′-AGTAGCTGTCAGCGTCTGGT-3′ as described previously (12).

Bottom Line: We found that U2 was active in oligodendrocyte precursors, but not in mature oligodendrocytes.U2 activity also did not mediate the initial Sox10 induction after specification arguing that Sox10 expression during oligodendroglial development depends on the activity of multiple regulatory regions.Considering the selective expression of Nkx6.2 at the time of specification and in differentiated oligodendrocytes, Nkx6.2 may be involved in limiting U2 activity to the precursor stage during oligodendrocyte development.

View Article: PubMed Central - PubMed

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

ABSTRACT
The HMG-domain transcription factor Sox10 is expressed throughout oligodendrocyte development and is an important component of the transcriptional regulatory network in these myelin-forming CNS glia. Of the known Sox10 regulatory regions, only the evolutionary conserved U2 enhancer in the distal 5'-flank of the Sox10 gene exhibits oligodendroglial activity. We found that U2 was active in oligodendrocyte precursors, but not in mature oligodendrocytes. U2 activity also did not mediate the initial Sox10 induction after specification arguing that Sox10 expression during oligodendroglial development depends on the activity of multiple regulatory regions. The oligodendroglial bHLH transcription factor Olig2, but not the closely related Olig1 efficiently activated the U2 enhancer. Olig2 bound U2 directly at several sites including a highly conserved one in the U2 core. Inactivation of this site abolished the oligodendroglial activity of U2 in vivo. In contrast to Olig2, the homeodomain transcription factor Nkx6.2 repressed U2 activity. Repression may involve recruitment of Nkx6.2 to U2 and inactivation of Olig2 and other activators by protein-protein interactions. Considering the selective expression of Nkx6.2 at the time of specification and in differentiated oligodendrocytes, Nkx6.2 may be involved in limiting U2 activity to the precursor stage during oligodendrocyte development.

Show MeSH
Related in: MedlinePlus