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Fig. S1. Related to Figure 1 Fig. S1. Related to Figure 1 20 ● ● Heat map of sample distances PCA Analysis A 20B ● ● ● 20 ● ● ● Stage 10 Genotype 300 Stage ● Stage2D Stage 250 4D OPC 10 Genotype200 ● 2D Genotype 10 150 300 Stage A Stage4D 100 B 2D Stage ● 2D 50 250 4D0 ● OPCOPC2D 0 OPC DIV24D 200 0 DIV4OPC Genotype0 Genotype 150 Thap1A +/+ ● Thap1A +/+ PC2: 14% variance Genotype B -/- ● Thap1 Genotype●● A -/- 100 PC2: 14% variance Thap1B −10 ● ●● AB PC2: 14% variance −10 50 ● −10 ● ● B ● 0 ● mouse_Library_13_D7T34D mouse_Library_14_D3T34D mouse_Library_15_E8T34D mouse_Library_8_D3T32D mouse_Library_7_D7T32D mouse_Library_9_E8T32D mouse_Library_17_D6T34D mouse_Library_16_3FT34D mouse_Library_18_7FT34D mouse_Library_11_D6T32D mouse_Library_10_3FT32D mouse_Library_12_7FT32D mouse_Library_3_E8OPC mouse_Library_1_D7OPC mouse_Library_2_D3OPC mouse_Library_4_3FOPC mouse_Library_5_D6OPC mouse_Library_6_7FOPC ● −20 −20 ● −40 −20 0 20 −20 −40 −20 PC1: 49%0 variance20 PC1: 49% variance −40 −20 0 20 PC1: 49% variance C Overrepresentation of THAP1-dependent pathways - GO Analysis Terms DIV2 heparan sulfate proteoglycan metabolic process DNA damage response mouse_Library_13_D7T34D mouse_Library_14_D3T34D mouse_Library_15_E8T34D mouse_Library_8_D3T32D mouse_Library_7_D7T32D mouse_Library_9_E8T32D mouse_Library_17_D6T34D mouse_Library_16_3FT34D mouse_Library_18_7FT34D mouse_Library_11_D6T32D mouse_Library_10_3FT32D mouse_Library_12_7FT32D mouse_Library_3_E8OPC mouse_Library_1_D7OPC mouse_Library_2_D3OPC mouse_Library_4_3FOPC mouse_Library_5_D6OPC mouse_Library_6_7FOPC membrane biogenesis extracellular matrix organization myelination Analysis - GO negative regulation of supramolecular fiber organization Biological Process (p<0.001) 0 2 4 6 8 10 Fold Enrichment DIV2 & DIV4 cell adhesion biological adhesion anatomical structure development GO Analysis - GO developmental process 0 2 4 6 8 Biological Process (p<0.001) Fold Enrichment Figure S2 - related to Figure 2 A Thap1 +/+ Thap1 -/- B Carbazole Assay 0.15 Thap1+/+ -/- (AU) (AU) Thap1 ** DAPI 0.10 / 525nm O4 * / 0.05 HABP Absorbance Absorbance 0.00 OPC OL C Secreted GAG / Media (µg/mg) D GAG / Homogenate (µg/mg) OPC OL OPC OL Thap1+/+ Thap1-/- Thap1+/+ Thap1-/- Thap1+/+ Thap1-/- Thap1+/+ Thap1-/- CS 73.53 134.07 9.40 23.56 CS 151.64 405.42 169.35 550.80 HS 3.67 7.55 0.44 1.01 HS 46.85 171.20 79.74 121.03 HA 1.40 2.61 0.86 2.42 HA 161.77 186.62 290.16 448.82 F E Secreted Chondroitin Sulfate GAG / Media (µg/mg) Chondroitin Sulfate GAG / Homogenate (µg/mg) OPC OL OPC OL Thap1+/+ Thap1-/- Thap1+/+ Thap1-/- Thap1+/+ Thap1-/- Thap1+/+ Thap1-/- C-4S 63.15 123.88 7.77 19.76 C-4S 134.74 376.49 150.48 472.26 C-6S 7.44 6.34 1.00 2.48 C-6S 13.62 13.40 15.21 48.29 C-2,6S 0.82 1.17 0.13 0.44 C-2,6S 0.25 1.72 0.28 10.14 C-4,6S 0.79 1.05 0.09 0.15 C-4,6S 2.09 5.00 2.33 5.22 Figure S3 - related to Figure 3 Figure S4 - related to Figure 4 β-glucuronidase activity in OPC & OL C β-glucuronidase activity in CNS *** A B *** ns 15 1×10-1 *** *** 10 +/flx 10 Thap1 1×10-2 OPC N-CKO OPC 1×10-3 5 OL OL -4 -Glucuronidase 1×10 β -Glucuronidase 1 (Units / mg lysate) β 0 0 2 4 6 8 (Units / mg lysate) -Glucuronidae (units/Hr) Lysate (mg) CC β Cortex Striatum D Cerebellum Pearson Correlation = 0.9407 ± 0.009 LAMP1 Intensity CS56 Intensity Figure S5- related to Figure 5 10000 *** *** *** *** 1000 +/flx 100 Thap1 Thap1+/flx;GusTg 10 (Units / mg lysate) 1 -Glucuronidase activity β CC Cortex Striatum Cerebellum SUPPLEMENTAL FIGURE LEGENDS Figure S1 related figure 1. THAP1 loss disrupts the extracellular matrix transcriptome (A) Heatmap for sample distances and (B) principal component analysis (PCA) showing the variance in the samples used for RNAseq analysis: 3 clonal lines for each genotype (Thap1+/+ and Thap1-/-) from 3 stages of OL cultures: OPC, DIV2 & DIV4. (C) Thap1+/+ vs Thap1-/- differentially expressed genes (DEG) selective for either DIV2 (703) or present at both DIV2 & DIV4 (277) used for enrichment analysis of GO (Gene Ontology) terms to identify overrepresented biological pathways. Graphs show the most significantly overrepresented GO terms (p<0.001, sorted as fold overrepresented) in OL from DIV2 (GENEONTOLOGY, GO - Biological Process), and from the DIV2 & DIV4 overlapping group (DAVID, GO - GO - Biological process) Figure S2 related figure 2. Thap1 null oligodendrocytes accumulate and secrete excess glycosaminoglycans (A) Representative images demonstrating accumulation of HA-GAG (stained using hyaluronan binding protein, HABP; red) in Thap1-/- OL cultures (+T3-DIV4). Arrows indicate surrounding regions of select OL with HA-GAG accumulation. (B) Carbazole assay (demonstrating GAG content) of total cell homogenate extracted from Thap1+/+ and Thap1-/- OPC and OL (+T3-DIV4). Absorbance at 525 nm for OPC; Thap1+/+ = 0.066 AU ± 0.0006; Thap1-/- = 0.094 AU ± 0.0022; t-test: t(2) = 12.70; p = 0.006 & OL - Thap1+/+ = 0.036 AU ± 0.0023; Thap1-/- = 0.059 AU ± 0.0019; t-test: t(2) = 7.96; p = 0.015. Table displaying the estimated amount of (C-D) CS, HS and HA GAGs (�g/mg) and (E- F) composition of mono (C-4S, C-6S) and bi-sulfated (C-2,6S and C-4,6S) CS-GAGs (�g/mg) (C,E) secreted in the media or (D,F) from cell homogenate from Thap1+/+ and Thap1-/- OPC and OL (+T3-DIV4) using GRIL LC/MS analysis. Figure S3 related figure 3. Excess glycosaminoglycan secretion by Thap1 null cells impairs oligodendroglial maturation via a non-cell autonomous mechanism Schematic illustrating co-culture experimental paradigm to test for paracrine effects of GAGs secreted by the OL lineage. Thap1 null (Thap1-/-) OL were co-cultured with LV- GFP labelled control (Thap1+/+; GFP) OPC at 10:1 ratio in differentiation media for four days (+T3). The two possible outcomes from this experimental paradigm are depicted in the illustration. Figure S4 related figure 4. β-glucuronidase activity during oligodendrocyte differentiation. (A) β-glucuronidase activity (Units / Hr) for Thap1+/+ OPC and OL (+T3-DIV4) lysate (0 - 8 μg; x-axis; STAR Methods). (B) Normalized β-glucuronidase activity (Units/mg Lysate) for OPC = 8.71 U / mg ± 0.27 and OL = 7.97 U / mg ± 0.18; t-test: t(2) = 2.18; p = 0.16). (C) β-glucuronidase activity in multiple brain regions (cerebral cortex, corpus callosum, striatum and cerebellum) of P21 THAP1 N-CKO (Thap1flx/-; nestin-Cre+) and control (Thap1+/flx) mice. Bar graph shows mean ± SEM values of normalized β-glucuronidase activity Units/Lysate (mg). Cerebral cortex - Thap1+/flx = 10.32 U / mg ± 0.43 ; N-CKO = 4.76 U / mg ± 0.106; t-test: p < 0.0001; corpus callosum - Thap1+/flx = 13.49 U / mg ± 0.33 ; N-CKO = 5.38 U / mg ± 0.129; t-test: p < 0.0001; striatum - Thap1+/flx = 9.98 U / mg ± 0.183 ; N-CKO = 5.10 U / mg ± 0.10; t-test: p < 0.0001 and cerebellum - Thap1+/flx = 14.97 U / mg ± 0.552 ; N-CKO = 5.58 U / mg ± 0.117; t-test: p < 0.0001) from P21 mice. (D) Pearson’s coefficient value shows ~ 95% colocalization (R = 0.94 ± 0.009; multiple region of interest (ROI) for N=50 cells) for CS-GAG (CS-56) and lysosomes (LAMP1) in OL (DIV4). Graph demonstrating co-localization of CS-56 and LAMP1 pixels for the image represented in FIg. 5N calculated using Image J. Figure S5 related figure 5. β-glucuronidase overexpression in CNS tissue. β-glucuronidase activity in multiple brain regions (cerebral cortex, corpus callosum, striatum and cerebellum) of P21 Thap1+/flx and Thap1+/flx;TgGUS mice. Bar graph shows mean ± SEM values of normalized β-glucuronidase activity Units/Lysate (mg). Cerebral cortex - Thap1+/flx = 10.32 U / mg ± 0.43 ; Thap1+/flx;TgGUS = 2402 U / mg ± 120.6; t-test: p < 0.0001; corpus callosum - Thap1+/flx = 13.49 U / mg ± 0.33 ; Thap1+/flx;TgGUS = 2208 U / mg ± 140.2; t-test: p < 0.0001; striatum - Thap1+/flx = 9.98 U / mg ± 0.183 ; Thap1+/ flx;TgGUS = 2056 U / mg ± 110.3; t-test: p < 0.0001 and cerebellum - Thap1+/flx = 14.97 U / mg ± 0.552 ; Thap1+/flx;TgGUS = 2078 U / mg ± 724.9; t-test: p < 0.0001. Table S1: Thap1 +/+ Vs Thap1 -/- DEG - OPC Symbol Entrez Gene IDMGI Gene/Markerlog2FoldChange.OPC ID Gm13841 665032 MGI:3650890 6.23655312 Anxa6 11749 MGI:88255 5.53098858 Pttg1 30939 MGI:1353578 5.10951903 Gls 14660 MGI:95752 5.07292798 Cyb5 109672 MGI:1926952 4.71999895 Becn1 56208 MGI:1891828 4.65986585 Rnu3a 19850 MGI:97977 4.60437764 BC002163 170658 MGI:3612445 4.58577533 Gm24119 MGI:5453896 4.54253579 Cast 12380 MGI:1098236 4.5420729 Grb10 14783 MGI:103232 4.34730574 Mtx2 53375 MGI:1859652 4.3130571 Mpg 268395 MGI:97073 4.30652836 Chek1 12649 MGI:1202065 4.26920775 Zmym3 56364 MGI:1927231 4.19365533 Gm23946 MGI:5453723 4.17892506 Ikbkg 16151 MGI:1338074 4.14781631 Gm26316 MGI:5456093 4.00594565 Mob1b 68473 MGI:1915723 3.99843807 Gm9923 100040505 MGI:3704365 3.98440042 Ddx5 13207 MGI:105037 3.93903572 4933434E20Rik 99650 MGI:1914027 3.80517724 Gm24407 MGI:5454184 3.79625284 Igsf1 209268 MGI:2147913 3.76136572 Wnk1 232341 MGI:2442092 3.76009824 Gm22973 MGI:5452750 3.75193685 Gm12663 105274305 MGI:3652168 3.73161149 Urm1 68205 MGI:1915455 3.6503159 Gm15093 100039890 MGI:3712496 3.64071013 Gm10439 382243 MGI:3710525 3.63068496 Vax1 22326 MGI:1277163 3.61313008 Ift20 55978 MGI:1915585 3.61197371 Tuba1c 22146 MGI:1095409 3.60134524 Mtch2 56428 MGI:1929260 3.59670124 Gm22614 MGI:5452391 3.57908954 Fndc3c1 333564 MGI:2685630 3.57634117 Snora23 100379145 MGI:3819494 3.57421696 Snrpb2 20639 MGI:104805 3.55768887 Abca6 76184 MGI:1923434 3.55346446 Gm21738 MGI:5433902 3.54830341 A730017C20Rik 225583 MGI:2442934 3.52225805 Luzp4 434865 MGI:3708816 3.50201774 Gm25890 MGI:5455667 3.46201989 Terc 21748 MGI:109558 3.44328045 Kdm5a 214899 MGI:2136980 3.43607442 ACEA_U3 No associated
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