MYRF Is a Membrane-Associated Transcription Factor That Autoproteolytically Cleaves to Directly Activate Myelin Genes
(A) Mutational analysis of the CTGGYAC motif in six luciferase reporters containing MYRF-bound DNA regions from near the Trf, Mag, Cntn2, Rffl, and Nfasc genes in CG-4 cells. In four out of the six sequences, mutation of a single CTGGYAC sequence (Δ) was sufficient to abolish the effect of MYRF. Fold inductions for all conditions are expressed relative to the pGL3-Promoter and pCS6 co-transfected control cells. Data are shown as means and SEMs from three independent experiments. ***p<0.001. (B) DNA pulldowns using double-stranded oligonucleotides corresponding to the predicted MYRF binding site in the Rffl intronic enhancer, or equivalent oligonucleotides with the seven base pair motif mutated, conjugated to magnetic beads. The wild-type Rffl sequence efficiently captured Myc-MYRF330–1139 from cell lysates, whereas no interaction was detected for the mutated sequence or beads without DNA (C). (D) Sequence alignment between MYRF, Dictyostelium MrfA (Uniprot Q54PT9), and S. cerevisiae Ndt80 (Uniprot P38830) showing conservation of basic amino acids required for DNA binding by Ndt80 (highlighted). (E) DNA pulldown assay measuring interaction between the DNA sequence from the Rffl enhancer and the DNA binding domain of wild-type or mutant MYRFs. All detections performed with anti-Myc.