Volume 29, Issue 1, Pages 5-16 (January 2016) The Interaction of Myc with Miz1 Defines Medulloblastoma Subgroup Identity  BaoHan T. Vo, Elmar Wolf, Daisuke Kawauchi, Anneli Gebhardt, Jerold E. Rehg, David Finkelstein, Susanne Walz, Brian L. Murphy, Yong Ha Youn, Young-Goo Han, Martin Eilers, Martine F. Roussel  Cancer Cell  Volume 29, Issue 1, Pages 5-16 (January 2016) DOI: 10.1016/j.ccell.2015.12.003 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cancer Cell 2016 29, 5-16DOI: (10.1016/j.ccell.2015.12.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of Myc, MycVD, and Myc/ΔPOZ Engineered Tumors (A) Kaplan-Meier survival curves of mice transplanted with cerebellar cells purified from (1) Trp53−/−;Cdkn2c−/− mice infected with Myc (red line), MycVD (blue line), or empty vector (black line) and (2) Myc/ΔPOZ mice infected with Myc (green line). ∗∗∗p < 0.0001 MycVD or Myc/ΔPOZ versus Myc. (B) H&E staining of sections from mouse Myc, MycVD, and Myc/ΔPOZ tumors. (a) An example of Myc tumors showing anaplastic morphology with cell molding and abundant mitotic figures and apoptotic bodies. (b, c) Examples of MycVD tumors showing large pleomorphic nuclei with abundant cytoplasm. (d–f) Examples of Myc/ΔPOZ tumors showing a proliferating cell population with anaplastic/large cell features (d) or a population of cells composed of anaplastic cells with pleomorphic nuclei with a scattering of large and bizarre nuclei and abundant cytoplasm (e, f). Morphology of the cell population in (e, f) shows an unclassifiable type. Scale bar represents 100 μm. (C) Microarray analysis of gene expression induced by MycVD versus Myc and Myc/ΔPOZ versus Myc. The plot shows values of all genes by grouping them into 30 equally sized bins. The x axis shows the change in expression between tumors that arise in a Myc/ΔPOZ background in comparison with control G3 MB, both of which are induced by overexpression of wild-type Myc. The y axis shows changes in expression between MycVD-induced tumors in comparison with Myc/G3 MB. Slope, regression coefficient; r, Pearson correlation coefficient; p value (two-tailed t test). (D) Principal component analysis for GNPs (n = 3), SHH MB (Ptch1+/−;Cdkn2c−/−) (n = 3), Myc (n = 3), MycVD (n = 3), and Myc/ΔPOZ (n = 3) tumors. Percentages represent the proportion of variance in each vector. (E) Gene set enrichment analysis of gene expression in four Myc/ΔPOZ tumors and four MycVD tumors in comparison with three Myc tumors. List and examples of selected gene sets of upregulated genes are shown. NES, normalized enrichment score. See also Figure S1 and Table S1. Cancer Cell 2016 29, 5-16DOI: (10.1016/j.ccell.2015.12.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Myc/Miz1 Interaction Is Required for Proliferation of Tumorspheres (A) Representative images of Myc, MycVD, and Myc/ΔPOZ tumorspheres. Scale bar represents 200 μm. (B) Proliferation of tumorsphere cells passaged in vitro. (C) Immunofluorescence of RFP marked Myc, MycVD, and Myc/ΔPOZ tumorspheres with antibodies to cleaved caspase-3 (cell death), Ki67 (proliferation), and DAPI (nuclei). Scale bar represents 50 μm. (D) Bromodeoxyuridine (BrdU) pulse and FACS analysis to measure tumorsphere proliferation. (E) Apoptosis measured by Annexin V staining and FACS analysis. p Values (shown at the top) are calculated by an unpaired two-tailed t test from three independent experiments. Data are represented as the mean ± SD. See also Figure S2. Cancer Cell 2016 29, 5-16DOI: (10.1016/j.ccell.2015.12.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Promoter Binding of Myc and Miz1 Determines the Transcriptional Output (A) Venn diagram displaying the number of binding sites for Myc and Miz1 in promoter regions (±5 kb) of RNA polymerase II transcribed genes in G3 MB. (B) Genome browser picture documenting binding of Miz1 and Myc to a large genomic region (chr3:48,99,758–56,129,010) in G3 MB and neural progenitor cells (NPCs) (Wolf et al., 2013). The arrow indicates the position of the Ccrn4l promoter, which contains a Miz1 binding motif. (C) Heatmap documenting binding of Myc and Miz1 to genes transcribed by RNA-Pol-II in G3 MB. All 31,194 refseq genes are shown and Myc/Miz1 peaks are centered to the adjacent transcriptional start site (TSS, ±5 kb). Genes are sorted according to the occupancy by Miz1. (D) Schematic representation of DNA binding motifs and transcriptional function of Miz1 alone, Miz1 bound to the Myc/Max complex, and Myc bound to Max. nc, non-consensus. (E) The diagram shows the sum of Myc and Miz1 binding tags versus the ratio of Myc/Miz1 tags for promoters that are bound by Myc and Miz1 in G3 MB. Color dots designate regulated genes after 12 hr of activation of Myc-ER with 4OHT in GNPs: gray dot, Myc/Miz1 bound; red dot, Myc activated; green dot, Myc repressed. Microarray analysis identified 475 downregulated (log2FC < −0.322, green dots) and 696 upregulated (log2FC > 0.322, red dots) genes upon activation of Myc. FC, fold change. (F) Quantification of the distribution of repressed (green bars) and induced (red bars) genes with respect to the Myc/Miz1 ratio. p Value (shown at the top) was calculated by a chi-squared test. (G) Gene ontology (GO) terms (DAVID) of the analysis of the 475 genes that are repressed (log2FC < −0.322) 12 hr after the activation of Myc with 4OHT. See also Figure S3. Cancer Cell 2016 29, 5-16DOI: (10.1016/j.ccell.2015.12.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 MycVD and Myc/ΔPOZ Tumors and Tumorspheres Express Primary Cilia the Loss of Which Did Not Affect Tumor Onset or Penetrance (A and B) Detection of primary cilia in tumor sections (A) and tumorspheres (B) from indicated tumors by immunofluorescence staining for Arl13b (green), to detect primary cilia, and γ-tubulin (purple), to identify basal bodies. DAPI (blue) was used to detect nuclei. Scale bar represents 50 μm. (C and D) The number of basal bodies (C) or nuclei (D) were used as a denominator to calculate the percentage of ciliated cells. p Values were calculated by an unpaired two-tailed t test. Data are represented as the mean ± SD. (E) ChIP-Seq analysis for Myc and Miz1 binding to the Atoh1 locus (black bar). A canonical E-box sequence is shown as a blue bar below the binding trace. (F) Immunofluorescence of RFP marked Myc, MycVD, and Myc/ΔPOZ tumorspheres with antibodies against Atoh1. Scale bar represents 50 μm. (G) Kaplan-Meier survival curves of mice transplanted with three individual G3 tumorsphere lines (19,251, 19,554, and 19,568) infected with retroviruses empty vector control (blue) and encoding Atoh1 (red). Top panel: line 19,251 median survival (ms) = 33 days for control (n = 5) and ms = 26 days for Atoh1 (n = 5). ∗∗p = 0.0018 control versus Atoh1. Middle panel: line 19,554, ms = 43 days for control (n = 5) and ms = 38 days for Atoh1 (n = 5). ∗p = 0.0373 control versus Atoh1. Bottom panel: line 19,568, ms = 13.5 days for control (n = 12) and ms = 8 days for Atoh1 (n = 7). ∗∗∗p = 0.0001 control versus Atoh1. See also Figure S4 and Table S1. Cancer Cell 2016 29, 5-16DOI: (10.1016/j.ccell.2015.12.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Myc/Miz1-Mediated Repression Discriminates G3 MB from Other Subgroups (A) ATOH1 expression in WNT, SHH, G3, G4 MBs from St. Jude Children's Research Hospital–Washington University Pediatric Cancer Genome Project (PCGP) database. The black line represents the median value, the boxes reflect the first and third quartiles of the interquartile range, and the red dots represent the jitter outliers. (B) Immunofluorescence of Myc or MycN (green) in GNPs cells from Trp53−/−;Cdkn2c−/− mice expressing a conditional Myc-ER or MycN-ER chimeric construct and RFP, before and after treatment with 4OHT. Co-expression of Myc and RFP (red and yellow). DAPI (blue) was used to detect nuclei. Scale bar represents 50 μm. (C) The median regulation of all genes (n = 713) that are bound by Myc and Miz1 in G3 MB and are repressed in G3 MB and re-expressed in MycVD and Myc/ΔPOZ tumors after 2 hr, 4 hr, 12 hr, and 24 hr activation of Myc- and MycN-ER by 4OHT. p Values (above each time point) depict if target genes are more repressed by Myc than by MycN (non-parametric one-tailed two-sample Wilcoxon signed rank test). (D and E) Co-immunoprecipitation of exogenous HA-tagged versions of Myc (MycN or Myc) and untagged Miz1 expressed in HEK293 cells by precipitation of Myc with HA antibody (D, αHA) or of Miz1 with an Miz1 antibody (E, αMiz1). (F) Binding of Miz1 at Vsp72, Foxg1, and Egr1 loci in G3 and SHH MB tumorspheres. Miz1 binding motifs (orange bars) and canonical E-box sequences (blue bars) are shown below the binding traces. (G) Boxplot comparing Miz1 binding in Myc-driven G3 and SHH tumorspheres (log ratio) of Miz1 target genes in NPCs, Myc target genes in G3 tumors and MycN target genes in SHH tumorspheres. The black line indicates the median value, the bottom and top of the boxes reflect the first and third quartile, whiskers represent the 1.5 interquartile range, and outliers are shown as dots (Tukey boxplot). p Values (shown at the top) are calculated by an unpaired two-tailed t test. (H) Protein expression in GNPs from the cerebella of 7-day-old Trp53−/−;Cdkn2c−/− mice infected or not with retroviruses encoding Myc or MycN and RFP. (I) Survival of mice transplanted with GNPs transduced with MSCV-IRES-GFP vector empty (control, black line, n = 5), encoding MycN (red line, n = 7), or MycNVD (dashed blue line, n = 10). ∗∗∗∗p < 0.0001 control versus MycN. See also Figure S5. Cancer Cell 2016 29, 5-16DOI: (10.1016/j.ccell.2015.12.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Comparison of Global Gene Expression between Murine MB Subgroups (A) Gene expression profiles using publicly available datasets of the mouse WNT subgroup (Ctnnb1+/lox(ex3);Blbp-Cre;Trp53−/−) of murine SHH MBs engineered by enforced expression of MycN in GNPs from Trp53−/−;Cdkn2c−/− mice (MycN) or spontaneously arisen from Trp53−/−;Cdkn2c−/− (Trp53), and Ptch1+/−;Cdkn2c−/− (Ptch), and of G3 MBs (Uziel et al., 2005). The heatmaps contain genes that are repressed by the Myc/Miz1 complex (bound by Myc and Miz1, repressed by Myc in tumors, and re-expressed in MycVD- and Myc/ΔPOZ-genes; n = 713) and are sorted according to fold expression after 2 hr, 4 hr, 12 hr, and 24 hr induction of Myc-ER (left). (B) Expression data from different MB subgroups (GEO: GSE37382) were RMA normalized, median centered, and the expression of each gene was averaged within the subgroups. The black line indicates the median value, the bottom and top of the boxes reflect the first and third quartile, whiskers represent the 1.5 interquartile range, and outliers are not shown (Tukey boxplot). p Values were calculated using a paired two-tailed Wilcoxon signed-rank test. See also Figure S6. Cancer Cell 2016 29, 5-16DOI: (10.1016/j.ccell.2015.12.003) Copyright © 2016 Elsevier Inc. Terms and Conditions