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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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U2 activity in transiently transfected oligodendroglioma cells is modulated by the presence of Olig2 and Nkx6.2. Transient transfections were performed in 33B cells with a luciferase reporter under control of the Sox10 U2 enhancer and the β-globin minimal promoter (U2-luc). Empty pCMV expression plasmids (−) or expression plasmids for Olig2 (A and C), E47 (A), Nkx6.2 (B and C), Nkx6.1 (B), Nkx2.2 (B) and various combinations thereof (A and C) were co-transfected as indicated below the bars. Increasing amounts of expression plasmid were used as indicated by the triangles (Olig2, Nkx6.2, Nkx6.1 and Nkx2.2) 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. High statistical significance (P ≤ 0.001) was obtained for activation rates obtained by Olig2 and E47 and for repression rates obtained by Nkx6.2 or Nkx6.1 as determined by Student’s t-test.
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Figure 3: U2 activity in transiently transfected oligodendroglioma cells is modulated by the presence of Olig2 and Nkx6.2. Transient transfections were performed in 33B cells with a luciferase reporter under control of the Sox10 U2 enhancer and the β-globin minimal promoter (U2-luc). Empty pCMV expression plasmids (−) or expression plasmids for Olig2 (A and C), E47 (A), Nkx6.2 (B and C), Nkx6.1 (B), Nkx2.2 (B) and various combinations thereof (A and C) were co-transfected as indicated below the bars. Increasing amounts of expression plasmid were used as indicated by the triangles (Olig2, Nkx6.2, Nkx6.1 and Nkx2.2) 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. High statistical significance (P ≤ 0.001) was obtained for activation rates obtained by Olig2 and E47 and for repression rates obtained by Nkx6.2 or Nkx6.1 as determined by Student’s t-test.

Mentions: To analyze whether Olig2 is able to influence U2 activity, we performed transient transfections with a U2-luc reporter in 33B oligodendroglioma cells which endogenously express Sox10 and Olig2 (data not shown). Whereas co-transfection of the U2-luc reporter with increasing amounts of an empty expression plasmid did not change luciferase activities, a small but reproducible increase in luciferase activity was observed after co-transfection of Olig2 (Figure 3A). Similar slight increases of luciferase reporter gene activity were obtained in co-transfection with the Class A bHLH protein E47. As Olig2 and E47 have been shown to physically interact and as class A and Class B bHLH proteins often function as heterodimers in vivo (24,25), we also analyzed the effect of combined Olig2 and E47 on U2 enhancer activity. Intriguingly, we now obtained a robust induction of U2 activity that ranged from 8- to 20-fold depending on the amounts of co-transfected expression plasmids (Figure 3A).Figure 3.


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)

U2 activity in transiently transfected oligodendroglioma cells is modulated by the presence of Olig2 and Nkx6.2. Transient transfections were performed in 33B cells with a luciferase reporter under control of the Sox10 U2 enhancer and the β-globin minimal promoter (U2-luc). Empty pCMV expression plasmids (−) or expression plasmids for Olig2 (A and C), E47 (A), Nkx6.2 (B and C), Nkx6.1 (B), Nkx2.2 (B) and various combinations thereof (A and C) were co-transfected as indicated below the bars. Increasing amounts of expression plasmid were used as indicated by the triangles (Olig2, Nkx6.2, Nkx6.1 and Nkx2.2) 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. High statistical significance (P ≤ 0.001) was obtained for activation rates obtained by Olig2 and E47 and for repression rates obtained by Nkx6.2 or Nkx6.1 as determined by Student’s t-test.
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Figure 3: U2 activity in transiently transfected oligodendroglioma cells is modulated by the presence of Olig2 and Nkx6.2. Transient transfections were performed in 33B cells with a luciferase reporter under control of the Sox10 U2 enhancer and the β-globin minimal promoter (U2-luc). Empty pCMV expression plasmids (−) or expression plasmids for Olig2 (A and C), E47 (A), Nkx6.2 (B and C), Nkx6.1 (B), Nkx2.2 (B) and various combinations thereof (A and C) were co-transfected as indicated below the bars. Increasing amounts of expression plasmid were used as indicated by the triangles (Olig2, Nkx6.2, Nkx6.1 and Nkx2.2) 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. High statistical significance (P ≤ 0.001) was obtained for activation rates obtained by Olig2 and E47 and for repression rates obtained by Nkx6.2 or Nkx6.1 as determined by Student’s t-test.
Mentions: To analyze whether Olig2 is able to influence U2 activity, we performed transient transfections with a U2-luc reporter in 33B oligodendroglioma cells which endogenously express Sox10 and Olig2 (data not shown). Whereas co-transfection of the U2-luc reporter with increasing amounts of an empty expression plasmid did not change luciferase activities, a small but reproducible increase in luciferase activity was observed after co-transfection of Olig2 (Figure 3A). Similar slight increases of luciferase reporter gene activity were obtained in co-transfection with the Class A bHLH protein E47. As Olig2 and E47 have been shown to physically interact and as class A and Class B bHLH proteins often function as heterodimers in vivo (24,25), we also analyzed the effect of combined Olig2 and E47 on U2 enhancer activity. Intriguingly, we now obtained a robust induction of U2 activity that ranged from 8- to 20-fold depending on the amounts of co-transfected expression plasmids (Figure 3A).Figure 3.

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