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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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Olig2 and Nkx6.2 interact via their DNA-binding domains. (A) Schematic representation of the domain structure of Olig2 and Nkx6.2 (fl) and the N-terminal (N) and C-terminal (C) parts of each protein used in co-immunoprecipitations. Numbers indicate the amino acid positions that mark end or beginning of the proteins and its domains. (B and C) Co-immunoprecipitations were performed on extracts containing full-length versions of myc-tagged Olig2, T7-tagged Nkx6.2 or both with antibodies directed against the myc-tag (α-myc) (B) and the T7-tag (α-T7) (C) as indicated above the panels. (D) Additionally, co-immunoprecipitations were carried out on extracts containing the myc-tagged full-length Olig2 (fl), its N-terminal (N) or C-terminal (C) parts in the presence of T7-tagged Nkx6.2 and antibodies directed against the myc-tag. (E) In the reverse experiment, T7-tagged full-length Nkx6.2 (fl), as well as its N-terminal (N) or C-terminal (C) parts were tested for their ability to precipitate full-length myc-tagged Olig2 in co-immunoprecipitations with antibodies directed against the T7-tag. Proteins in the input (1/10 in B, C and 1/20 in D, E) and in the precipitate were visualized by western blotting using the two tag-specific antibodies as indicated on the left of each panel in B–E.
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Figure 6: Olig2 and Nkx6.2 interact via their DNA-binding domains. (A) Schematic representation of the domain structure of Olig2 and Nkx6.2 (fl) and the N-terminal (N) and C-terminal (C) parts of each protein used in co-immunoprecipitations. Numbers indicate the amino acid positions that mark end or beginning of the proteins and its domains. (B and C) Co-immunoprecipitations were performed on extracts containing full-length versions of myc-tagged Olig2, T7-tagged Nkx6.2 or both with antibodies directed against the myc-tag (α-myc) (B) and the T7-tag (α-T7) (C) as indicated above the panels. (D) Additionally, co-immunoprecipitations were carried out on extracts containing the myc-tagged full-length Olig2 (fl), its N-terminal (N) or C-terminal (C) parts in the presence of T7-tagged Nkx6.2 and antibodies directed against the myc-tag. (E) In the reverse experiment, T7-tagged full-length Nkx6.2 (fl), as well as its N-terminal (N) or C-terminal (C) parts were tested for their ability to precipitate full-length myc-tagged Olig2 in co-immunoprecipitations with antibodies directed against the T7-tag. Proteins in the input (1/10 in B, C and 1/20 in D, E) and in the precipitate were visualized by western blotting using the two tag-specific antibodies as indicated on the left of each panel in B–E.

Mentions: Within the U2 enhancer we also detected two potential binding sites for Nkx6.2 (Figure 4A). Site GTX1 was present in the U2 core and highly conserved, GTX2 in contrast was in the less conserved 3′-flank. In EMSA, none of the two sites bound significant amounts of full-length Nkx6.2 (Figure 4E). However, even the HoxA5/A6 positive control oligonucleotide with its high-affinity binding site for Nkx6.2 (27) bound very little full-length Nkx6.2. Therefore, we switched to a shortened version of Nkx6.2 that consisted of amino acids 134–216 and essentially corresponded to the homeodomain (Figure 6A). With this shortened Nkx6.2 protein, we obtained protein–DNA complexes both for GTX1 and GTX2 (Figure 4E). Compared to the HoxA5/A6 positive control, less complex was formed with GTX1 and even less with GTX2 suggesting that the three sites bind Nkx6.2 with different avidity. Addition of Nkx6.2-specific antibodies interfered with complex formation thus confirming the presence of the shortened Nkx6.2 version in the respective complexes (Figure 4F).Figure 6.


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)

Olig2 and Nkx6.2 interact via their DNA-binding domains. (A) Schematic representation of the domain structure of Olig2 and Nkx6.2 (fl) and the N-terminal (N) and C-terminal (C) parts of each protein used in co-immunoprecipitations. Numbers indicate the amino acid positions that mark end or beginning of the proteins and its domains. (B and C) Co-immunoprecipitations were performed on extracts containing full-length versions of myc-tagged Olig2, T7-tagged Nkx6.2 or both with antibodies directed against the myc-tag (α-myc) (B) and the T7-tag (α-T7) (C) as indicated above the panels. (D) Additionally, co-immunoprecipitations were carried out on extracts containing the myc-tagged full-length Olig2 (fl), its N-terminal (N) or C-terminal (C) parts in the presence of T7-tagged Nkx6.2 and antibodies directed against the myc-tag. (E) In the reverse experiment, T7-tagged full-length Nkx6.2 (fl), as well as its N-terminal (N) or C-terminal (C) parts were tested for their ability to precipitate full-length myc-tagged Olig2 in co-immunoprecipitations with antibodies directed against the T7-tag. Proteins in the input (1/10 in B, C and 1/20 in D, E) and in the precipitate were visualized by western blotting using the two tag-specific antibodies as indicated on the left of each panel in B–E.
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Figure 6: Olig2 and Nkx6.2 interact via their DNA-binding domains. (A) Schematic representation of the domain structure of Olig2 and Nkx6.2 (fl) and the N-terminal (N) and C-terminal (C) parts of each protein used in co-immunoprecipitations. Numbers indicate the amino acid positions that mark end or beginning of the proteins and its domains. (B and C) Co-immunoprecipitations were performed on extracts containing full-length versions of myc-tagged Olig2, T7-tagged Nkx6.2 or both with antibodies directed against the myc-tag (α-myc) (B) and the T7-tag (α-T7) (C) as indicated above the panels. (D) Additionally, co-immunoprecipitations were carried out on extracts containing the myc-tagged full-length Olig2 (fl), its N-terminal (N) or C-terminal (C) parts in the presence of T7-tagged Nkx6.2 and antibodies directed against the myc-tag. (E) In the reverse experiment, T7-tagged full-length Nkx6.2 (fl), as well as its N-terminal (N) or C-terminal (C) parts were tested for their ability to precipitate full-length myc-tagged Olig2 in co-immunoprecipitations with antibodies directed against the T7-tag. Proteins in the input (1/10 in B, C and 1/20 in D, E) and in the precipitate were visualized by western blotting using the two tag-specific antibodies as indicated on the left of each panel in B–E.
Mentions: Within the U2 enhancer we also detected two potential binding sites for Nkx6.2 (Figure 4A). Site GTX1 was present in the U2 core and highly conserved, GTX2 in contrast was in the less conserved 3′-flank. In EMSA, none of the two sites bound significant amounts of full-length Nkx6.2 (Figure 4E). However, even the HoxA5/A6 positive control oligonucleotide with its high-affinity binding site for Nkx6.2 (27) bound very little full-length Nkx6.2. Therefore, we switched to a shortened version of Nkx6.2 that consisted of amino acids 134–216 and essentially corresponded to the homeodomain (Figure 6A). With this shortened Nkx6.2 protein, we obtained protein–DNA complexes both for GTX1 and GTX2 (Figure 4E). Compared to the HoxA5/A6 positive control, less complex was formed with GTX1 and even less with GTX2 suggesting that the three sites bind Nkx6.2 with different avidity. Addition of Nkx6.2-specific antibodies interfered with complex formation thus confirming the presence of the shortened Nkx6.2 version in the respective complexes (Figure 4F).Figure 6.

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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