Limits...
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.

Show MeSH

Related in: MedlinePlus

Activation of the U2 enhancer requires binding sites for Olig2, whereas repression by Nkx6.2 is independent of binding sites. (A) Sequence of oligonucleotides with wild-type and mutant binding sites for Olig2 and Nkx6.2. (B and C) EMSA was performed with wild-type and mutant binding sites for Olig2 (B) and Nkx6.2 (C). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells which were either untransfected (control) or transfected with expression plasmids for tagged versions of Olig2 or Nkx6.2 ΔNC. The position of the Olig2-specific complex is highlighted by an arrowhead, the position of the Nkx6.2-specific complex by a dot. (D–F) Transient transfections were performed in 33B oligodendroglioma with different versions of the U2 luc reporter and expression plasmids for Olig2, E47 (D), Nkx6.2 (E) or Olig2, E47 and Nkx6.2 (F) as indicated (+). The U2 luc reporter had either wild-type sequences (wt) or carried one or multiple mutations in the Olig2 binding sites (bHLH1m, bHLH2m, bHLH4m, bHLH1/2m, bHLH1/4m, bHLH2/4m, bHLH1/2/4m) (D) or the Nkx6.2 (GTX1m, GTX2m, GTX1/2m) binding sites (E and F). Luciferase activities in extracts from transfected cells were determined in three experiments each performed in duplicates. The luciferase activity obtained for each reporter 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. Statistically significant (P ≤ 0.001) reporter gene activation by Olig2 and E47 (D) was obtained for the wt, 1m, 2m and 1/2m U2 luc reporters as determined by Student’s t-test. Nkx6.2-dependent repression was statistically significant (P ≤ 0.001) for all reporters in E and F.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045606&req=5

Figure 5: Activation of the U2 enhancer requires binding sites for Olig2, whereas repression by Nkx6.2 is independent of binding sites. (A) Sequence of oligonucleotides with wild-type and mutant binding sites for Olig2 and Nkx6.2. (B and C) EMSA was performed with wild-type and mutant binding sites for Olig2 (B) and Nkx6.2 (C). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells which were either untransfected (control) or transfected with expression plasmids for tagged versions of Olig2 or Nkx6.2 ΔNC. The position of the Olig2-specific complex is highlighted by an arrowhead, the position of the Nkx6.2-specific complex by a dot. (D–F) Transient transfections were performed in 33B oligodendroglioma with different versions of the U2 luc reporter and expression plasmids for Olig2, E47 (D), Nkx6.2 (E) or Olig2, E47 and Nkx6.2 (F) as indicated (+). The U2 luc reporter had either wild-type sequences (wt) or carried one or multiple mutations in the Olig2 binding sites (bHLH1m, bHLH2m, bHLH4m, bHLH1/2m, bHLH1/4m, bHLH2/4m, bHLH1/2/4m) (D) or the Nkx6.2 (GTX1m, GTX2m, GTX1/2m) binding sites (E and F). Luciferase activities in extracts from transfected cells were determined in three experiments each performed in duplicates. The luciferase activity obtained for each reporter 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. Statistically significant (P ≤ 0.001) reporter gene activation by Olig2 and E47 (D) was obtained for the wt, 1m, 2m and 1/2m U2 luc reporters as determined by Student’s t-test. Nkx6.2-dependent repression was statistically significant (P ≤ 0.001) for all reporters in E and F.

Mentions: HEK293 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal calf serum (FCS) and transfected by the polyethylenimine (PEI) technique using 10 µg pCMV5-based expression plasmid per 100-mm plate. Cells were harvested 48 h post-transfection for extract preparation (17) and ectopic expression of the respective transcription factor was verified by western blotting using anti-myc tag (1:10 000 dilution, Cell Signaling Technology) or anti-T7 tag (1:10 000 dilution, Novagen) mouse monoclonals. With these extracts, electrophoretic mobility shift analyses (EMSA) were performed as described (18) using 32P-labeled 25-bp double-stranded oligonucleotides containing putative Olig2 or Nkx6.2 binding sites (for sequences, see Figure 5A). In supershift experiments, antibodies were used in a 1:40 dilution and pre-incubated for 30 min with the protein extracts before addition of 32P-labeled oligonucleotide probes.


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)

Activation of the U2 enhancer requires binding sites for Olig2, whereas repression by Nkx6.2 is independent of binding sites. (A) Sequence of oligonucleotides with wild-type and mutant binding sites for Olig2 and Nkx6.2. (B and C) EMSA was performed with wild-type and mutant binding sites for Olig2 (B) and Nkx6.2 (C). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells which were either untransfected (control) or transfected with expression plasmids for tagged versions of Olig2 or Nkx6.2 ΔNC. The position of the Olig2-specific complex is highlighted by an arrowhead, the position of the Nkx6.2-specific complex by a dot. (D–F) Transient transfections were performed in 33B oligodendroglioma with different versions of the U2 luc reporter and expression plasmids for Olig2, E47 (D), Nkx6.2 (E) or Olig2, E47 and Nkx6.2 (F) as indicated (+). The U2 luc reporter had either wild-type sequences (wt) or carried one or multiple mutations in the Olig2 binding sites (bHLH1m, bHLH2m, bHLH4m, bHLH1/2m, bHLH1/4m, bHLH2/4m, bHLH1/2/4m) (D) or the Nkx6.2 (GTX1m, GTX2m, GTX1/2m) binding sites (E and F). Luciferase activities in extracts from transfected cells were determined in three experiments each performed in duplicates. The luciferase activity obtained for each reporter 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. Statistically significant (P ≤ 0.001) reporter gene activation by Olig2 and E47 (D) was obtained for the wt, 1m, 2m and 1/2m U2 luc reporters as determined by Student’s t-test. Nkx6.2-dependent repression was statistically significant (P ≤ 0.001) for all reporters in E and F.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045606&req=5

Figure 5: Activation of the U2 enhancer requires binding sites for Olig2, whereas repression by Nkx6.2 is independent of binding sites. (A) Sequence of oligonucleotides with wild-type and mutant binding sites for Olig2 and Nkx6.2. (B and C) EMSA was performed with wild-type and mutant binding sites for Olig2 (B) and Nkx6.2 (C). Oligonucleotides were incubated in the absence (−) or presence of extracts from 293 cells which were either untransfected (control) or transfected with expression plasmids for tagged versions of Olig2 or Nkx6.2 ΔNC. The position of the Olig2-specific complex is highlighted by an arrowhead, the position of the Nkx6.2-specific complex by a dot. (D–F) Transient transfections were performed in 33B oligodendroglioma with different versions of the U2 luc reporter and expression plasmids for Olig2, E47 (D), Nkx6.2 (E) or Olig2, E47 and Nkx6.2 (F) as indicated (+). The U2 luc reporter had either wild-type sequences (wt) or carried one or multiple mutations in the Olig2 binding sites (bHLH1m, bHLH2m, bHLH4m, bHLH1/2m, bHLH1/4m, bHLH2/4m, bHLH1/2/4m) (D) or the Nkx6.2 (GTX1m, GTX2m, GTX1/2m) binding sites (E and F). Luciferase activities in extracts from transfected cells were determined in three experiments each performed in duplicates. The luciferase activity obtained for each reporter 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. Statistically significant (P ≤ 0.001) reporter gene activation by Olig2 and E47 (D) was obtained for the wt, 1m, 2m and 1/2m U2 luc reporters as determined by Student’s t-test. Nkx6.2-dependent repression was statistically significant (P ≤ 0.001) for all reporters in E and F.
Mentions: HEK293 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal calf serum (FCS) and transfected by the polyethylenimine (PEI) technique using 10 µg pCMV5-based expression plasmid per 100-mm plate. Cells were harvested 48 h post-transfection for extract preparation (17) and ectopic expression of the respective transcription factor was verified by western blotting using anti-myc tag (1:10 000 dilution, Cell Signaling Technology) or anti-T7 tag (1:10 000 dilution, Novagen) mouse monoclonals. With these extracts, electrophoretic mobility shift analyses (EMSA) were performed as described (18) using 32P-labeled 25-bp double-stranded oligonucleotides containing putative Olig2 or Nkx6.2 binding sites (for sequences, see Figure 5A). In supershift experiments, antibodies were used in a 1:40 dilution and pre-incubated for 30 min with the protein extracts before addition of 32P-labeled oligonucleotide probes.

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