Control of myelination in Schwann cells: a Krox20 cis-regulatory element integrates Oct6, Brn2 and Sox10 activities

Abstract

Myelination in Schwann cells is governed by several transcription factors, including the POU proteins Oct6 and Brn2, the high mobility group protein Sox10 and the zinc-finger protein Krox20. How the function of these factors is integrated in the control of myelination has not been established. Previously, we identified an enhancer element controlling Krox20 expression throughout myelination in Schwann cells. In this paper, cell culture experiments were combined with transgenesis to identify transcription factors acting directly upstream of Krox20. The results show that during the promyelin-myelin transition, Krox20 expression is directly activated by Oct6 and Brn2 acting on this enhancer. In addition, the enhancer-dependent synergism between these POU proteins and Sox10 suggests that Krox20 expression requires this combination of factors. These results resolve previous controversy concerning the mechanism of action of Oct6 and Brn2 during myelination and provide an explanation for myelin deficiencies in Waardenberg-Hirschsprung disease patients whereby Sox10 mutations could lead to a loss of Krox20 expression.

Figure 1

Alignment of human and chick sequences orthologous to the mouse myelinating Schwann cell element (MSE) shows several conserved transcription factor-binding sites. The nucleotide sequence of the mouse 1.3 kb MSE is shown (Ghislain et al, 2002). Nucleotide numbering corresponds to the mouse sequence. Restriction sites used in deletion studies are indicated. Conserved residues identified in the human and chick genomes are aligned to the mouse sequence and indicated as dashes. Oct6 footprints are indicated (I–IV; see Fig 2C). In these regions, conserved candidate binding sites for Oct6 and Brn2 are double underlined. These sites are based on the Brn2 binding site matrix (lower left; n can be either 0, 2 or 3 nucleotides) and Oct6 and Brn2 show similar binding characteristics (Li et al, 1993). The AT to GC substitutions introduced to inactivate these sites are shown. Conserved candidate Sox10-binding sites are shown in red with arrows. These sites are based on the Sox9 binding site matrix (lower right; Mertin et al, 1999), and Sox9 and Sox10 have similar DNA-binding characteristics (Peirano et al, 2000). Binding site matrices were generated using the WebLogo program (Crooks et al, 2004). Stack height reflects conservation and symbol height indicates frequency of residue. C, chick; H, human; M, mouse.

Figure 2

Identification of Oct6-binding sites in the myelinating Schwann cell element. (A) 5′ deletions of the 1.3 kb myelinating Schwann cell element (MSE) fused to a minimal β-globin promoter/lacZ reporter were transfected into U138 (left) and HeLa (right) cells, with 300 or 6 ng/well, respectively, of the expression vector, empty (−) or carrying the Oct6 coding sequence (+). The data show the mean β-galactosidase activity of two independent, normalized experiments carried out in duplicate. Values for transfections with the empty promoter/reporter and expression plasmids were set to one. Data from all other transfections are presented as the fold induction over this level. Error bars represent the standard error. (B) The wild-type MSE subfragment, Psp1406I–BanII (left), or a mutant version containing the AT to GC substitutions indicated in Fig 1 (right) were used as probes in bandshift experiments with increasing amounts of Oct6-containing bacterial extracts. As a control, both probes were combined without the bacterial extract (far right). To identify specific complexes, unlabelled competitor oligonucleotides corresponding to a high-affinity Oct6-binding site (wt) or a mutant version unable to bind to Oct6 (mt) were included in the binding reaction at a 200-fold molar excess. Specific complexes are indicated with brackets. FP, free probe. (C) The upper (left) and the upper (centre) and lower (right) strands of the Psp1406I–PstI and the PstI–BanII fragments, respectively, were analysed for Oct6 binding in DNase I footprinting assays using extracts from Oct6-expressing bacteria. Nucleotide numbering corresponds to the mouse 1.3 kb MSE sequence (Fig 1). The positions of the Oct6 footprints are indicated (I–IV).

Figure 3

Oct6-binding sites are essential for the in vivo activity of the myelinating Schwann cell element. (A) Schematic representation of the wild-type (wt) 1.3 kb myelinating Schwann cell element (MSE) and mutant MSE carrying mutations in the Oct6-binding sites II–IV (mt(II–IV); Fig 1) and I–IV (mt(I–IV); Fig 1) fused to a minimal β-globin promoter/lacZ reporter. Results of transgenic experiments are shown. n, the number of transgene positive mice analysed for β-galactosidase activity in the sciatic nerve at postnatal day (P) 2–3 or 30; +, strongly β-galactosidase-positive sciatic nerves with levels similar to those shown in (C,E); +/−, weakly β-galactosidase-positive sciatic nerves with levels similar to those shown in (D,F). (B) The wild-type 1.3 kb MSE and mutant constructs were transfected into HeLa (upper) and U138 (lower), with either 6 or 75 ng/well, respectively, of the expression vector, empty (−) or carrying the murine Oct6 or Brn2 coding sequence (+). Control, promoter/reporter plasmid without MSE. Presentation of transfection data is described in the legend of Fig 2. (C–F) β-Galactosidase-positive sciatic nerves from P2–3 (C,D; transverse section) and P30 (E,F; teased nerve) mice carrying the wild-type 1.3 kb MSE (C,E, (+) in A) or mt(I–IV) mutant construct (D,F, (+/−) in A). Scale bar, 25 μm.

Figure 4

Myelinating Schwann cell element (MSE)-dependent enhancer activity involves synergism between Sox10 and the POU proteins Oct6 and Brn2. The wild-type and mutant 1.3 kb MSE constructs (Fig 3A) and 5′ deletion constructs (Fig 2A) fused to a minimal β-globin promoter/lacZ reporter were co-transfected with the expression vector, empty (−) or carrying the murine Oct6 or Brn2 coding sequence (++, 6 ng/well; +, 0.6 ng/well) and/or Sox10 or Sox2 coding sequence (+, 60 ng/well), as indicated, into U138 (A) and HeLa (B,C) cells. Control, promoter/reporter plasmid without MSE. Presentation of transfection data is described in the legend of Fig 2.