Sox10 binds to intron 1 of the <i>Myrf</i> gene both in vivo and in vitro.

(A) Schematic representation of the location of regions from the Myrf locus probed by PCR in ChIP studies including the Myrf minimal promoter (myrf), ECR9, ECR11/12 and additional control regions from the 5′ (fl5) and 3′ (3fl) flanking regions of the Myrf gene and from within the adjacent Dagla gene (dagla). (B–E) ChIP was performed on 33B cells (B,C), rat primary oligodendroglial cells kept in proliferation medium (OPC) or differentiation medium (OL) (D) and P14 spinal cord from wildtype (wt) and Sox10ΔCNS (ko) mice (E) using antibodies directed against Sox10 (α-Sox10) (B,D,E) or Sox8 (α-Sox8) (C) and control preimmune serum (PI). Quantitative PCR was applied on the immunoprecipitate. Values for each fragment correspond to the percentage of material precipitated from the input and represent the mean ± SEM of at least 3 biological replicates. (F) EMSA was performed with radiolabelled double-stranded oligonucleotides 3 and 4 in wildtype (3, 4) and mutant (3a, 3b, 3c, 3bc, 4a) versions as indicated below the gels. Oligonucleotides were incubated in the absence (−), or presence (control, Sox10) of protein extracts before gel electrophoresis as indicated above the lanes. Extracts were from mock-transfected HEK293 cells (control) or HEK293 cells expressing full length Sox10 (Sox10). Oligonucleotides with site B and site C/C′ from the Mpz promoter [29] served as positive control for Sox10 binding and as marker for the mobility of complexes containing either Sox10 monomers (m) or dimers (d). (G) ECR9_myrf-luc reporter plasmids were co-transfected in wildtype (wt) or mutant (3bc, 4a, mt) version in 33B cells with empty shRNA expression vector or vectors coding for Sox10-specific shRNA. The mt version corresponds to a combination of the 3bc and 4a mutations. Luciferase activities were determined and the activity of the wildtype ECR9_myrf-luc reporter in the presence of empty shRNA expression vector was arbitrarily set to 1. All other activities were calculated relative to this value and are presented as mean ± SEM. All experiments were performed three times in quadruplicates.