Fig. 4

SOX10 binds to RNLTR12-int encoded transcript, and this is required for SOX10 binding to Mbp promoter (A) RT-qPCR after cross-linking RIP (from P7 rat brains) with SOX10 or control immunoglobulin G1, showing SOX10 association with RNLTR12-int transcript. n = 4 independent experiments (each time 5–6 brains were pooled). Mean ± SEM, ∗p < 0.05, ratio paired t test (two sided). U1: U1 spliceosomal small nuclear RNA. (B) SOX10 binds RNLTR12-int as determined by EMSA. Binding reactions contained RNLTR12-int transcript (3.4 nM) and increasing concentrations (0.125–4 μM) of SOX10. Complexes were separated on a 0.6% native agarose gel and detected by GelRed. (C) Binding kinetics of RNLTR12-int transcript and SOX10, analyzed by surface plasmon resonance (SPR). SOX10 protein was sequentially injected at five different concentrations (0.1–1.6 μM) over the streptavidin-coated flow cell sensor surface immobilized with 3′ end biotin-labeled RNLTR12-int or kept blank. Sensorgram of the single-cycle kinetics (SCK) represents the data corrected for non-specific binding to the flow cell surface (gray). The curve fit with 1:1 binding is shown (dark pink). (D) ChIP analyses with SOX10 or control immunoglobulin G1, 4 days after siRNA transfection, showing SOX10 occupancy to Mbp promoter is affected in absence of RNLTR12-int transcript. Left: (top) schematic representation of Mbp promoter. Rat genome assembly: rn6, FP, forward primer; RP, reverse primer for PCR amplification after ChIP. SOX10 binding conserved elements22: S2 (“AACAAT”) and S1 (“TTCAAA”). Black right arrow after S1: transcription start site (TSS). (Bottom) PCR-amplified immunoprecipitated DNA, run on 2% agarose gel. Right: qPCR analysis after ChIP. n = 3 independent experiments. Mean ± SEM, ∗∗p < 0.01, ratio paired t test (two sided). (E) Illustration represents SOX10-mediated transcription of Mbp is RNLTR12-int transcript dependent. Copies of RNLTR12-int exists in the rat genome. For simplicity reason, only one is drawn. RNLTR12-int is transcribed. Association of the transcription factor SOX10 to Mbp promoter requires a direct binding interaction between SOX10 and RNLTR12-int transcript. Green arrow: TSS. (F) In OL, only a fraction (0.8%) of SOX10 targets is downregulated upon RNLTR12-int inhibition. Venn diagram represents the overlap between SOX10 targets (ChIP-seq data: GEO: GSE64703) and the genes downregulated due to RNLTR12-int inhibition (RNA-seq data: GEO: GSE237744). ∗∗p < 0.0001, hypergeometric test. See also Figure S5.