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).