The Oligodendrocyte Transcription Factor 2 OLIG2 regulates transcriptional repression during myelinogenesis in rodents.

The repressive role of OLIG2 is essential for oligodendrocyte differentiation.a Volcano plot of differential expressed genes (DEGs, log2FC ≥ 1 or ≤ -1; p value <0.05) that are targeted by enhanced OLIG2 binding in rat OPCs to iOL transition. b Representative GO analysis of the significantly downregulated genes in Fig. 1a. c, d Representative phase micrographs(c) and CNP+ and MBP+ immunostaining (d) in rat OPCs transfected with Olig2 or Olig2-VP64 under differentiation condition. n = 3 independent experiments. e Quantitative real-time PCR analysis of oligodendrocyte differentiation-associated genes in rat OPCs transfected with Olig2 or Olig2-VP64 under differentiation condition. n = 3 independent experiments. Error bars indicate SEM (Mag, p = 0.0084; Mbp, p < 0.001; Plp, p < 0.001. two-tailed unpaired Student’s t test). f Heat map representing the expression of OL differentiation–related genes in rat OPC, iOL, iOL transfected with OLIG2 and iOL transfected with OLIG2-VP64. g Schematic diagram for retrovirus injection. h Immunostaining for CC1 on OLIG2 or OLIG2-VP64 retrovirus infected brain at P15. Arrow indicates the cell infected with retrovirus. n = 4 control and 4 Olig2-VP64 mice. i Quantification of the percentage of differentiated oligodendrocytes in infected cells. Error bars indicate SEM (p < 0.001. two-tailed unpaired Student’s t test). Scale bars: 50 μm in (c); 100 μm in (d); 25 μm in (h).

OLIG2 assembles a repressor complex to dominant oligodendrocyte differentiation.a Scatter plot of proteins bound to OLIG2. Enriched epigenetic associated protein especially H3K9m3 modifiers were highlighted. b Representative GO analysis of protein pulled down with OLIG2. c Expression of Enpp6, Mag and Mbp in rat OPCs transfected with indicated shRNA under differentiation condition. Error bars indicate SEM (Enpp6, p = 0.0031; Mag, p < 0.001; Mbp, p < 0.001. two-tailed unpaired Student’s t test). d Immunolabeling of MBP in rat OPCs transfected with indicated siRNA followed by differentiation-induction. n = 3 independent experiments. e Brain at P7 was immunostained with MBP, CC1 and SETDB1. Boxed areas in upper panels are shown at a high magnification in lower panels. Arrow indicates the co-expression of SETDB1 with CC1 but not MBP, and arrowhead indicates the co-expression of SETDB1 with CC1 and MBP. n = 3 different mice. f Co-immunoprecipitation (co-IP) of endogenous OLIG2 with SETDB1 in mouse cortex at P2, P7 and P21, respectively. HC heavy chain. n = 3 independent experiments. g Heatmap of H3K9me3- enrichment signals in rat OPCs and iOLs. h Genomic distribution of H3K9me3 enrichment regions in rat OPCs and specific in iOLs. i Representative GO analysis of genes nearest to iOL-specific H3K9me3 enrichment regions. j Binding motif identified by HOMER with H3K9me3- enrichment peaks. Letter size indicates nucleotide frequency at each position of OLIG2 binding motif. k The percentage of H3K9me3- enrichment peaks with OLIG2 binding motif in rat iOL. Heavy red color means the ratio. l, m Heatmap (l) and enrichment profiles (m) of H3K9me3- enrichment signals at rat OPCs and iOLs surrounding loci of genes which are downregulated and have enhanced OLIG2 binding upon differentiation. n, o Heatmap (n) and enrichment profiles (o) of HA-SETDB1- binding signals at rat OPCs and iOLs surrounding loci of genes which are downregulated and have enhanced OLIG2 binding upon differentiation. Scale bars: 50 μm in (d, e).

SETDB1 is required for oligodendrocyte myelination.a Survival curve of Setdb1F/F, Olig1-Cre; Setdb1F/+ and Olig1-Cre; Setdb1F/F mice. b Appearance of optic nerves from Setdb1F/F, Olig1-Cre; Setdb1F/+ and Olig1-Cre; Setdb1F/F littermates at P14. c Electron micrographs of transverse optic nerve sections from Olig1-Cre; Setdb1F/+ and Olig1-Cre; Setdb1F/F mice at P14. n = 3 control and three mutant mice. d Quantification of myelin sheath thickness (g-ratio) in optic nerves. e In situ hybridization of Plp1 in the brain at P14. n = 3 control and three mutant mice (f) Quantification of Plp1+ cells in the brain white matter. Error bars indicate SEM (cortex, p < 0.001; cerebellum, p < 0.001. two-tailed unpaired Student’s t test). g Immunolabeling of MBP in the brain at P14. n = 3 control and three mutant mice. h Immunostaining of CC1 in the corpus callosum at P14. n = 3 control and three mutant mice. i Quantification of CC1+ cells in the corpus callosum. Error bars indicate SEM (p < 0.001. two-tailed unpaired Student’s t test). Scale bars: 2 μm in (c); 100 μm in (e and g); 50 μm in (h).

SETDB1 is critical for proper myelin repair after demyelination.a Schematic diagram showing tamoxifen administration to Pdgfra-CreER; Setdb1 Flox mice in 3 consecutive days at P60, followed by LPC injection and BrdU administration. Brain tissue were collected at 14 Dpl. b Immunolabeling of MBP in the LPC lesions of Pdgfra-CreER; Setdb1F/+ and Pdgfra-CreER; Setdb1F/F mice at 14 Dpl. n = 3 control and three mutant mice. c In situ hybridization of Plp1 in the lesions at 14 Dpl. Boxed areas in left panels are shown at a high magnification in corresponding right panels. n = 3 control and three mutant mice. d Quantification of Plp1+ cells in the LPC lesions. Error bars indicate SEM (p = 0.0023. two-tailed unpaired Student’s t test). e Electron micrographs in corpus callosum of lesions from Pdgfra-CreER; Setdb1F/+ and Pdgfra-CreER; Setdb1F/F mice at 14 Dpl. n = 3 control and three mutant mice. f Scatterplot of g-ratio at 14 Dpl in lesions area. g Percentage of remyelinated axons at 14 Dpl in lesions area. Error bars indicate SEM (p < 0.001. two-tailed unpaired Student’s t test). h Immunostaining for BrdU and CC1 in LPC lesions in corpus callosum from Pdgfra-CreER; Setdb1F/+ and Pdgfra-CreER; Setdb1F/F mice at 14 Dpl. Boxed areas in left panels are shown at a high magnification in corresponding right panels. n = 3 control and three mutant mice. i, j Quantification of CC1+ cells (i) and CC1+ /BrdU+ cells (j) in the LPC lesions area. Error bars indicate SEM (i, p = 0.0061; j, p < 0.001. two-tailed unpaired Student’s t test). Scale bars: 100 μm in (b, c); 2 μm in (e); 50 μm in (h).

OLIG2-SETDB1 repressor complex functions at the onset of mouse OPC differentiation.a Quantification of BrdU+ and PDGFRα+ cells in the corpus callosum of Olig1-Cre; Setdb1F/+ and Olig1-Cre; Setdb1F/F mice at P14. b In situ hybridization of Enpp6+ in the corpus callosum at P14. n = 3 control and three mutant mice. c Quantification of Enpp6+ cells in the corpus callosum at P14. Error bars indicate SEM (p < 0.001. two-tailed unpaired Student’s t test). d Expression of Enpp6 in spinal cords of Setdb1 control and mutant mice (left panel) and rat iOLs transfected with Olig2 or Olig2-VP64 (right panel). Error bars indicate SEM (p < 0.001. two-tailed unpaired Student’s t test). e Immunostaining of MBP in the corpus callosum from NG2-Cre; Setdb1F/+ and NG2-Cre; Setdb1F/F mice at P14. n = 3 control and three mutant mice. f Quantification of CC1+ cell in the corpus callosum and spinal cord from NG2-Cre; Setdb1F/+ and NG2-Cre; Setdb1F/F mice at P14. Error bars indicate SEM (p < 0.001. two-tailed unpaired Student’s t test). g Schematic diagram showing markers of each stage in oligodendrocyte lineage. h Survival curve of Cnp-Cre; Setdb1F/+ and Cnp-Cre; Setdb1F/F mice. i Quantification of myelin sheath thickness (g-ratio) in optic nerves from Cnp-Cre; Setdb1F/+ and Cnp-Cre; Setdb1F/F mice at P14. n = 3 control and three mutant mice. j Quantification of CC1+ cells in the spinal cord of Cnp-Cre; Setdb1F/+ and Cnp-Cre; Setdb1F/F mice at P14. n = 3 control and three mutant mice. k Schematic diagram showing tamoxifen administration to Sox10-CreER; Setdb1 Flox mice in two consecutive days at P3, followed by tissue collection at P15. n = 3 control and three mutant mice. l–o Immunostaining of MBP and CC1 in the corpus callosum (l, m) and spinal cord (n, o) from Sox10-CreER; Setdb1F/+ and Sox10-CreER; Setdb1F/F mice at P14. n = 3 control and three mutant mice. p Quantification of CC1+ cells in the white matter or gray matter of spinal cord. Error bars indicate SEM (p < 0.001. two-tailed unpaired Student’s t test). Scale bars: 100 μm in (b); 250 μm in (e and l–o).

Olig2 recruits SETDB1 to silent inhibitors of oligodendrocyte differentiation.a, b Scatterplot showing differentially expressed transcripts between iOL from Olig1-Cre; Setdb1F/+ and Olig1-Cre; Setdb1F/F brain cortex (a) and between rat OPCs transfected with Olig2 and Olig2-VP64 under differentiation condition (b). c, d Expression of well-known inhibition factors to rat OPC differentiation. Error bars indicate SEM (***p < 0.001; Hes1 in (d), p = 0.0029. two-tailed unpaired Student’s t test). e Efficiency of Sox11 knockdown in rat OPCs. Error bars indicate SEM (***p < 0.001; Hes1 in Olig2-VP64, p = 0.0029. one-way ANOVA). f Expression of Enpp6, Mbp and Plp1 was measured in rat OPCs under differentiation condition following transfection with indicated siRNA. Error bars indicate SEM (p < 0.001. one-way ANOVA). g Immunolabeling of CNP and OLIG2 in OPCs under differentiation condition following transfection with indicated siRNA. n = 3 independent experiments. h Genome browser visualization of OLIG2 or H3K9me3-targeting regions on the gene loci of Sox11 during rat OPC differentiation. i Luciferase activity of Sox11 promoter-driven reporters in control (Scramble) or Setdb1 knockdown (Setdb1 K/D) 293T cells transfected with indicated plasmids. Error bars indicate SEM (***p < 0.001; OLIG2+ in Scramble, p = 0.0078; SETDB1+ in Scramble, p = 0.0095. one-way ANOVA). j Immunolabeling for MBP and CC1 on the corpus callosum from Olig1-Cre; Setdb1F/+ and Olig1-Cre; Setdb1F/+; Sox11 F/F mice at P14. n = 3 control and three mutant mice. k, l Quantification of CC1+ oligodendrocytes density (k) and the percentage of MBP+ area (l) in corpus callosum of control and Sox11 mutant mice. Error bars indicate SEM (k, p = 0.0064; l, p = 0.0052. two-tailed unpaired Student’s t test). Scale bars: 50 μm in (g, j).