The Tumor Suppressor CIC Directly Regulates MAPK Pathway Genes Via Histone Deacetylation Simon Weissmann1,2, Paul A

Published OnlineFirst May 29, 2018; DOI: 10.1158/0008-5472.CAN-18-0342

Cancer Genome and Epigenome Research

The Tumor Suppressor CIC Directly Regulates MAPK Pathway Genes via Histone Deacetylation Simon Weissmann1,2, Paul A. Cloos1,2, Simone Sidoli2,3, Ole N. Jensen2,3, Steven Pollard4, and Kristian Helin1,2,5

Abstract

Oligodendrogliomas are brain tumors accounting for approximately 10% of all central nervous system can- Low MAPK signaling High MAPK signaling cers. CIC is a transcription factor that is mutated in most RTKs patients with oligodendrogliomas; these mutations are believed to be a key oncogenic event in such cancers. MEK Analysis of the Drosophila melanogaster ortholog of ERK SIN3 CIC, Capicua, indicates that CIC loss phenocopies CIC HDAC activation of the EGFR/RAS/MAPK pathway, and HMG C-term studies in mammalian cells have demonstrated a role SIN3 CIC HDAC for CIC in repressing the transcription of the PEA3 HMG C-term Ac Ac subfamily of ETS transcription factors. Here, we address Ac Ac the mechanism by which CIC represses transcription and assess the functional consequences of CIC inactivation. Genome-wide binding patterns of CIC in several cell types revealed that CIC target genes were enriched ETV, DUSP, CCND1/2, SHC3,... for MAPK effector genes involved in cell-cycle regulation Normal Astrocytoma and proliferation. CIC binding to target genes was Glioblastoma multiforme abolished by high MAPK activity, which led to their 1p/19q deletion CIC-Mutants transcriptional activation. CIC interacted with the SIN3 SIN3 deacetylation complex and, based on our results, we SIN3 CIC suggest that CIC functions as a transcriptional repressor HDAC HDAC HMG C-term through the recruitment of histone deacetylases. Inde- CIC R215W SIN3 pendent single amino acid substitutions found in oli- HMG C-term CIC HDAC godendrogliomas prevented CIC from binding its target HMG C-term genes. Taken together, our results show that CIC is a R1515C transcriptional repressor of genes regulated by MAPK

signaling, and that ablation of CIC function leads Ac Ac Ac Ac Ac Ac Ac Ac to increased histone acetylation levels and transcription at these genes, ultimately fueling mitogen- independent tumor growth. Signiﬁcance: Inactivation of CIC inhibits its direct ETV, DUSP, CCND1/2, SHC3,... ETV, DUSP, CCND1/2, SHC3,... repression of MAPK pathway genes, leading to their increased expression and mitogen-independent growth. Oligodendroglioma Graphical Abstract: http://cancerres.aacrjournals. The tumor suppressor CIC represses its target genes via histone deacetylation. High MAPK signaling or loss of CIC function leads to hyperacetylation and gene activation. org/content/canres/78/15/4114/F1.large.jpg. Cancer Res; © 2018 American Association for Cancer Research 78(15); 4114–25. 2018 AACR.

1Biotech Research and Innovation Centre (BRIC), University of Note: Supplementary data for this article are available at Cancer Research Copenhagen, Copenhagen, Denmark. 2Centre for Epigenetics, University Online (http://cancerres.aacrjournals.org/). of Copenhagen, Copenhagen, Denmark. 3Department of Biochemistry S. Weissmann and P.A. Cloos contributed equally to this article. and Molecular Biology, VILLUM Centre for Bioanalytical Sciences, University of Southern Denmark, Odense, Denmark. 4MRC Centre for Corresponding Authors: Kristian Helin and Paul A. Cloos, University of Copen- Regenerative Medicine and Edinburgh Cancer Research UK Centre, The hagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark. Phone: 453-532-5666; University of Edinburgh, Edinburgh, United Kingdom. 5The Novo Nordisk Fax: 453-532-5669; E-mail: [email protected]; [email protected] Foundation Center for Stem Cell Biology (Danstem), University of doi: 10.1158/0008-5472.CAN-18-0342 Copenhagen, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. 2018 American Association for Cancer Research.

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Introduction 1 mmol/L sodium pyruvate, penicillin/streptomycin, leukemia inhibitory factor, 1 mmol/L MEK inhibitor (PD0325901), and Oligodendrogliomas represent a subgroup of low-grade glioma 3 mmol/L GSK inhibitor (CT-99021). with a distinct mutational spectrum. The vast majority of oligo- Mouse neural progenitor cells derived from mESCs were dendroglioma cases contain chromosomal deletions of 1p and cultured on PDL/laminin in complete NSC medium: 50:50 19q and 70% harbor mutations in CIC, located on chromosome DMEM/F12:Neurobasal (Invitrogen) supplemented with 19q13.2 (1). CIC encodes a transcription factor containing a N2/B27 (Invitrogen), 0.05 mmol/L b-mercaptoethanol, SOX-like high mobility group (HMG) domain. At least two iso- 2 mmol/L GlutaMAX, 0.1 mmol/L nonessential amino acids, forms of CIC have been identified in humans: a short form CIC-S penicillin/streptomycin, 1 mmol/L sodium pyruvate, 5 mmol/L and a longer isoform CIC-L that shares the HMG domain but HEPES, 2 mg/mL heparin, 50 mg/mL BSA, 10 ng/mL bFGF/EGF. contains an extended N-terminal part (2). Mutations in CIC Human GNS line G144 (19) and human fetal neural stem cell frequently lead to truncations or amino acid substitutions in lines 18.5 and 21.5 (a kind gift of Dr. Steven Goldman, University residues that are thought to be essential for DNA binding of Rochester Medical Center) were cultured on PDL/Laminin in (Supplementary Fig. S1A and S1B). Together with 1p/19q chro- complete NSC medium. mosomal deletions, these recurrent CIC mutations are thought to Flp-In-T-REX-293 cells were cultured in DMEM (Invitrogen) represent an ablation of CIC function (3), being causally involved with 10% FBS (HyClone) and penicillin/streptomycin. Cells in oligodendroglioma formation. Of further interest, CIC muta- were authenticated by measuring the expression of specific tions are mutually exclusive with alterations of EGFR, TP53, ATRX, cellular markers and by observing their morphology and func- and CDKN2A (4, 5). tionality in specific assays. All the cell lines used were routinely Most of our knowledge about CIC stems from studies in (every 4 weeks) tested negative for mycoplasma by PCR. Cell Drosophila melanogaster, where it was genetically defined as a culture time between thawing and experiment did not exceed transcriptional repressor acting downstream of receptor tyrosine five passages. kinase (RTK) signaling regulating developmental patterning (6, 7). MAPK-mediated phosphorylation of Cic leads to its nuclear CRISPR KO export and/or degradation of Cic (8) and expression of Cic target sgRNAs were cloned into pSpCas9(BB)-2A-GFP (Addgene genes. In mammals, RTK signaling has similar effects on 48138), using the protocol described in ref. 20 and transfected CIC-mediated gene repression, possibly involving MAPK and into mESCs (Lipofectamine 2000, Invitrogen), or G144 cells p90RSK (9). How CIC is acting as a transcriptional repressor is (Amaxa Nucleofector II, Program A033). GFP-positive cells were currently not well understood. In Drosophila, Cic-mediated repres- single-cell sorted 48 hours posttransfection, expanded, and sion is dependent on the corepressor protein Groucho (6, 10). screened by immunoblotting for Cic deletion. Gene targeting was Mouse studies have suggested that CIC-mediated repression is in confirmed by PCR amplification and Sanger sequencing. Mouse part dependent on the binding to Ataxin1 (ATXN1) or a related Cic KO clones (KO1, KO2, and KO3) all harbored out-of-frame factor ATXN1L (11–13). mutations causing complete protein depletion. Human CIC KO Several mouse knockout studies have shown that germline clones (KO8 and KO9) harbored a mixture of out-of-frame and deletion of murine Cic leads to perinatal lethality (13–15) and large in-frame mutations, resulting in expression of an HMG deletion of Cic after birth has been linked to T-cell lymphoblastic domain-deleted truncated protein. Primers and sgRNAs are listed lymphoma formation (14, 16). In Ewing sarcomas, a genetic in Supplementary Table S1. fusion of CIC to the exons coding for the transcriptional activator domain of DUX4 causes derepression of PEA3 (polyoma Plasmids and cDNA enhancer activator 3)-family genes (17). Furthermore, CIC dele- The open-reading frame of human CIC-L was amplified from a tion and mutation was shown to influence metastasis in an EGFR cDNA library derived from human fetal brain and cloned into inhibitor-resistant model of lung adenocarcinoma, through TOPO/pCR8, yielding a flawless 7554 bp clone encoding a 2513 the deregulation of the PEA3-family gene Etv4 (18). CIC may amino acid protein (splice variant ENST00000572681, ProteinID therefore act as an important tumor suppressor through the ENSP00000459719). transcriptional repression of its target genes, including the PEA3 The CIC-R215W and R1515C mutants were generated using subfamily, whose deregulation was linked to cancer develop- the Quickchange II Site Directed Mutagenesis Kit (Agilent ment (17, 18). However, the mechanistic consequences of Technologies) and expressed from pPB-CAG-Dest-pA-pgk-bsd inactivation or mutation of CIC and their contribution to (Addgene 74918) by cotransfection with transposase pCyL43 tumorigenesis of oligodendrogliomas are not well understood. (PBase; Sanger Institute's plasmid repository) into mESCs and Furthermore, the mechanism through which CIC achieves gene selected with 5 mg/mL blasticidin. Primers are listed in Supple- repression in mammals is not known. mentary Table S1. In this study, we aimed at elucidating how CIC contributes to normal development, the consequences of CIC loss and how Differentiation this could lead to tumor formation. mESCs were differentiated into NSCs as described in (21). Briefly, 104 cells/cm2 mESCs were seeded onto gelatine into Materials and Methods complete NSC medium without bFGF/EGF. Culture medium was Cell lines and culture changed every day. Four days after initiation of differentiation, mESCs (E14TG2a.4, ATCC) were cultured on gelatin in 2i cells were plated onto PDL/Laminin–coated dishes in complete medium: 50:50 DMEM/F12:Neurobasal (Invitrogen) supple- NSC medium containing bFGF/EGF (10 ng/mL). Cells were mented with N2/B27 (Invitrogen), 0.05 mmol/L b-mercaptoetha- subcultured additional 10 days until homogeneous NSC popula- nol, 0.1 mmol/L nonessential amino acids, 2 mmol/L GlutaMAX, tions were established.

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Starvation and inhibitor treatment filtration column using an AKTA€ purification platform (GE Eighty percent confluent NSC cultures were washed twice with Healthcare). PBS and cultured in NSC medium without bFGF/EGF for 16 m hours. Inhibitor treatment with 1 mol/L MEK inhibitor Chromatin immunoprecipitation fl (PD0325901) was performed in 80% con uent NSC or GNS Cells were cross-linked (1% formaldehyde, 10 minutes), cultures grown in complete NSC medium for 4 and 24 hours, quenched with 125 mmol/L glycine, washed twice with PBS and respectively. harvested in SDS buffer (50 mmol/L Tris at pH 8.1, 0.5 % SDS, 100 mmol/L NaCl, 5 mmol/L EDTA). Cells were pelleted, resus- Monoclonal antibody generation pended in Triton-X IP buffer (100 mmol/L Tris at pH 8.6, 0.3% The C-terminal 195 amino acids of CIC (cloning primers in SDS, 1.7% Triton X-100, and 5 mmol/L EDTA) and chromatin Supplementary Table S1) were expressed by generating a bacu- was sonicated (fragment size 200–500bp). The 250 mg chromatin lovirus through the cotransfection of pVL-Flag-His-CIC-C- (measured by Bradford) was precleared with protein-A Sepharose terminus and Bsu36I-linearized Bakpak6 baculovirus DNA. beads (GE healthcare) for 1 hour and incubated with primary Flag-His-tagged proteins were expressed in Trichoplusia ni,High antibody overnight at 4C. Protein-A Sepharose beads were fi – fi Five, cells and puri ed by Flag Sepharose af nity chromatog- blocked with 10 mg/mL BSA overnight at 4 C. Next day, beads raphy as described previously (22) and used to immunize mice. and antibody/chromatin-mixture were incubated for 3 hours at Spleens were isolated, homogenized, and fused to the myeloma 4C. Beads were washed three times with low salt buffer (1% cell line SP2/0-Ag14 by addition of PEG1500. Supernatants Triton X-100, 0.1% SDS, 150 mmol/L NaCl, 2 mmol/L EDTA, from the resulting hybridomas were screened by ELISA, pH 8.0, 20 mmol/L Tris-HCl, pH 8.0) and twice with high salt followed by further subcloning. Selected clones were tested for buffer (1% Triton X-100, 0.1% SDS, 500 mmol/L NaCl, 2 mmol/L fi fi speci city and quali ed for immunoblotting and immunopre- EDTA, pH 8.0, 20 mmol/L Tris-HCl, pH 8.0). DNA was eluted cipitation using both human and mouse samples. All mouse with elution-buffer (1% SDS, 0.1 mol/L sodium bicarbonate) work was approved by the Danish Animal Ethical Committee at 65C overnight and purified using QIAquick PCR Purification ("Dyreforsøgstilsynet"). Kit (Qiagen). DNA was analyzed using the LightCycler 480 SYBR Green I system (Roche). qPCR was performed in technical tripli- Immunoblotting cates. Primers and antibodies are listed in Supplementary To prepare whole-cell extracts, cells were lysed in high salt Table S1. buffer (300 mmol/L NaCl, 50 mmol/L Tris-HCl pH7.5, 0.5% For chromatin immunoprecipitation (ChIP)-seq, 1–2ngof Triton X-100, 0.1% SDS, 1 mmol/L EDTA, 1 mmol/L DTT, ChIP DNA was used for library preparation, using the NEBNext aprotinin, leupeptin, 0.1 mmol/L PMSF). Equal amounts of Ultra II DNA Library prep Kit (E7370; NEB). Libraries were whole-cell extracts were analyzed by standard SDS-PAGE/immu- sequenced using Illumina NextSeq 500 sequencer 75 bp Single noblotting. Primary antibodies and dilutions are listed in Sup- End. plementary Table S1. Bioinformatics and motif finding RT-qPCR Sequenced reads were aligned using Bowtie2 (mouse-mm10 Cells were washed once with PBS, lysed directly in RLT and human-hg38; ref. 25). Duplicates were removed using buffer (Qiagen), and total RNA was extracted using the Samtools RmDUP. Peaks were called in EaSeq (26) using stan- RNAeasy Kit (Qiagen). Two-hundred nanograms of total dard settings and corresponding IgG control (FDR < 10 5). For RNA was reverse transcribed using TaqMan Reverse Transcrip- motif analysis, 50 bp surrounding the apex of each peak was fi tion Reagents (Applied Biosystems) and quanti ed using used by MEME-ChIP (27) using standard settings in discrimi- the LightCycler 480 SYBR Green I system. qPCR was performed native mode. Control peak set matching size and distance-to- in technical triplicates. Primers are listed in Supplementary transcriptional start site (TSS) was generated in EaSeq (26). Table S1. Data availability RNA sequencing RNA-seq and ChIP-seq data have been deposited in the Five-hundred nanograms of total RNA was used for library ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) preparation using TruSeq RNA Library Prep Kit v2 (Illumina) under accession numbers E-MTAB-6681 and E-MTAB-6682, according to the manufacturer's recommendations. Libraries were respectively. sequenced using Illumina NextSeq 500 sequencer 75 bp Single End. RNA sequencing in G144 cells was done in technical tripli- Tandem affinity purification cates for parental cells and technical duplicates for one WT and Nuclear extracts (500 mg, 3 109 cells) from Flp-In-T-REX-293 two CIC KO clones (KO8, KO9). Sequencing data were aligned via cells expressing Flag–HA-tagged human CIC-L were precleared Tophat2 (23) and differential expression was called using DESeq2 and incubated with a 700 mL packed volume of anti-Flag beads (24). Genes were called as differential expressed for 2-fold (anti-Flag-M2-agarose, Sigma) overnight at 4C with rotation. The change and false discovery rate (FDR) 0.01. Data from parental beads were collected by centrifugation (700 g, 5 minutes) and cells and WT clone were treated as replicates. Similarly, data from washed five times with buffer A (20 mmol/L Tris-HCl, pH 8.0, the two KO clones were treated as replicates. 300 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 10% glycerol, 0.2 mmol/L PMSF, 1 mmol/L DTT, 1 mg/mL aprotinin, Size-exclusion chromatography and 1 mg /mL leupeptin) and once with buffer A containing 293FT cell nuclear extracts were prepared as described pre- Benzonase at 4 C for 1 hours. The complexes were eluted with viously (22) and fractionated on a Superose 6 PC 3.2/30 gel 1 bed volume of buffer A supplemented with 0.5 mg/mL Flag

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peptide for 4 hours. The eluate was collected by passing (Fig. 1E). We also identified three members of the dual through a 0.45-mm, 1.5 mL centrifugal unit (Merck Millipore). specificity protein phosphatase subfamily Dusp4, Dusp5,and The Flag-IP elute was incubated with 120 mL of 50 % slurry HA- Dusp6 as direct CIC targets. Other significant targets include beads overnight. The beads were washed six times with buffer the genes Fos,Fosb,Fosl1,Id1,Ccnd1,Rictor,Vgf,Ptpn9and A and eluted with 100 mL buffer A supplemented with 1 mg/mL Spred1, Spred2, and Spred3 (Fig. 1E; Supplementary Table S2). HA peptide for 2 hours. The samples were boiled in SDS Interestingly CIC also binds to its own promoter, suggesting loading buffer and run shortly into an SDS-PAGE gel. A gel an auto-regulatory loop. Many of the CIC-bound genes are slice containing the purified proteins was isolated for mass implicated in the regulation of RAS/MAPK signaling, either spectrometry analysis. For comparative purposes, nuclear as effectors or as members of feedback mechanisms. Indeed, extracts from an equivalent number of Flp-In-T-REX-293 cells we found CIC target genes significantly enriched for RAS expressing Flag-HA–tagged human FUBP1 were processed in signaling and other signaling pathways that are frequently parallel. perturbed in cancer using pathway analysis (Supplementary Fig. S1D). Mass spectrometry In agreement with the reported function of CIC to repress Proteins were in-gel digested using trypsin. Peptides were ETV4 and ETV5 (9), deletion of Cic ledtoupregulationofEtv4 loaded on a nanoHPLC RSLC Ultimate 3000 (Thermo Scien- and Etv5 expression. Moreover, ectopic expression of human tific) coupled online with an Orbitrap Fusion Tribrid (Thermo). wild-type (WT) CIC, but not the HMG mutant R215W or the Peptides were separated using C18 nanochromatography with a C-terminal mutant R1515C, reverted the increased levels of 60-minute gradient, and acquired using Data-Dependent Etv4 and Etv5, suggesting that both mutants are nonfunctional Acquisition with MS/MS fragmentation using Higher energy (Supplementary Fig. S1E and S1F). The inability of CIC-R215W Collision Dissociation (HCD). Spectra identification was per- to reduce target gene expression is likely due to its reduced formed with Proteome Discoverer (v1.4, Thermo) using as ability to bind DNA (3). Indeed, ChIP-qPCR at the promoter reference the UniProt human database. Results were filtered region of the two target genes showed binding of exogenously for 1% FDR. expressed WT CIC, similar to enogenous levels, but no binding of CIC-R215W (Supplementary Fig. S1G). Interestingly, binding of CIC-R1515C–mutant protein was also undetectable, Results which is in agreement with a recent publication showing CIC binds directly to the promoter region of its target that the C-terminal portion of CIC is critical for DNA binding genes and is required for repressing Pea3 group genes in (30) and mutations within this domain result in activation mouse ESCs of Etv4 (18). To investigate the mechanism by which CIC regulates transcription and the functional consequences of its inactivation, MAPK signaling prevents CIC from binding to its target genes we initiated our studies in mouse embryonic stem cells and associated cis-regulatory elements (mESC). mESCs can be selectively differentiated into neural Previous studies have linked the MAPK pathway signaling stem cells (NSC) and further to oligodendrocyte precursor cells to CIC regulation (7, 9, 31). To investigate the influence of (21, 28). CIC deletions most likely occur in one of these MAPK signaling on CIC target gene regulation, we established immature cell types during oligodendroglioma formation. NSC cultures from WT and Cic KO mESCs. In mitogen (bFGF/ mESCs therefore appeared to be appropriate to test the con- EGF) containing NSC medium, we were unable to detect sequences of Cic loss. In addition the 2i growth conditions used any binding of CIC at the promoter of target genes. In contrast, to expand mESCs enables investigation of CIC in the absence of removal of mitogens for 16 hours led to a strong recruitment MEK/ERK signaling (29). Thus, we generated Cic knockout of CIC to Etv4 and Etv5 (Supplementary Fig. S2A). This was (KO) mESCs using CRISPR-Cas9 (Supplementary Fig. S1C). accompanied by a significant reduction of Etv4 and Etv5 Targeted knockout clones showed no detectable levels of CIC expression in WT NSCs, whereas only insignificant changes (Fig. 1A). in gene expression were detected in Cic KO NSCs (Supple- To obtain a comprehensive view of the genes regulated by CIC, mentary Fig. S2B). we mapped the genome-wide location of CIC by ChIP, followed To extend this observation to human cells and in an effort to by high-throughput sequencing (ChIP-seq) in mESCs using Cic- eliminate pleiotropic effects of growth factor withdrawal, we KO cells as a negative control (Fig. 1B). We identified a strong set performed ChIP analysis for CIC in primary human fetal NSCs of 159 specific, CIC-bound regions, of which approximately two (hNSC18.5 and hNSCs21.5) and the human glioma stem cell thirds were located in close proximity to a gene (Fig. 1C), marking (GNS) line G144, after treatment with a small-molecule 112 unique genes. inhibitor against MEK (PD0325901). Cells cultured in mitogen Next, we performed motif enrichment analysis by compar- (bFGF/EGF) containing medium showed minimal CIC recruiting motif enrichments 50 bp surrounding the apex of each ment to the promoter region of ETV4. However, CIC binding was CIC peak to a control dataset of matching size and distance to strongly increased in response to MEK inhibition (Fig. 2A). closest TSS. This comprehensive analysis revealed the highly Similarly, we observed a strong global recruitment of CIC at its enriched sequence TSAATGR (Fig. 1D) representing the target sites after MEK inhibition, using ChIP-seq (Fig. 2B and C; reported CIC consensus sequence (7, 17). Supplementary Table S2) with a significant overlap in the three The expression of the PEA3 family ETS transcription factors studied cell lines (Fig. 2D). Conversely, almost no peaks were ETV1, ETV4,andETV5 is reported to be directly regulated by detected in mock control samples of hNSC18.5 (n ¼ 16), mammalian CIC (9, 13). Indeed, among the most prominent hNSC21.5 (n ¼ 9), and G144 (n ¼ 8). By intersecting the set of targets in our ChIP-seq experiments were Etv1, Etv4,andEtv5 human and mouse CIC target genes we identified 58 genes that

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A B IP:IgG IP:CIC IP:CIC kDa WT2 WT3 KO1 KO2 KO3 WT1 Short exposure WT WT KO CIC-L 250

CIC-S 148

CIC-L Long exposure 250

CIC-S 148 CIC Peaks ( n = 159) Sorted according to Q-value Vinculin 70 10,000 bp 50 Peaks 60 C D Sequence motif 40 2 E Value 3.6e-398

# Peaks 20 1 Figure 1. Bits Genome-wide characterization of CIC GA TTTA GGC T targets in mESCs. A, CIC Immunoblot of CAG AA G C C C C 0 CAGT CGT 0 A Cic 1 2 3 5 4 6 9 7 8 G three WT mESC clones and three -KO 14 13 12 T AA10 T11 Exon Other clones. B, Heatmap at CIC peaks showing 5' UTR Intron3' UTR IgG and CIC in WT and Cic KO. C, Distribution TSS (-5 kb) TTS (+1 kb) of peaks according to gene elements. D, E Motif analysis using MEME-ChIP. Top motif 50 chr11 100 chr16 50 chr10 50 chr8 50 chr18 identiﬁed is depicted with corresponding E value. E, ChIP-seq tracks of CIC occupancy

IP:IgG at selected target genes in WT and KO 50 100 50 50 50 mESCs. WT WT

50 100 50 50 50 KO IP:CIC IP:CIC

Etv4 Etv5 Dusp6 Dusp4 Spry4

25 chr4 25 chr4 25 chr5 25 chr7 25 chr15 IP:IgG 25 25 25 25 25 WT WT

25 25 25 25 25 KO IP:CIC IP:CIC

Gpr3 Plk3 Vgf Ccnd1 Maff

3,000 bp

were found in mESCs and in at least one human cell line shown in Fig. 3A and B, CIC was found in regions strongly (Supplementary Fig. S2C). Twenty-one genes were found across enriched for H3K27Ac and H3K4me1, whereas a random all samples (Supplementary Table S2). Taken together, these control dataset generated to match size and distance to closest results demonstrate that MAPK signaling prevents CIC from TSS (generated in EaSeq; ref. 26) was not enriched and conse- binding to its target genes, and that these are largely conserved quently showed little overlap. between mouse and human. Collectively, we have generated high conﬁdence results Interestingly, in human NSCs as well as in GNS cells, the for the genome-wide binding of CIC. These results show that majority of peaks were found >10 kb distal to a TSS (Fig. 2E), CIC binds to a deﬁned set of target regions in a MAPK- indicating that CIC might act at cis-regulatory elements. dependent manner and those regions are largely conserved To investigate whether CIC binding to distal gene regions is between mouse and human. Furthermore, our results show linked to enhancer elements we performed ChIP-seq experi- that CIC is recruited to both TSS and to enhancers, suggest- ments in G144 cells for two histone marks, H3K27ac and ing that CIC can repress transcription via both of these H3K4me1, which are used as markers of enhancers (32). As regulatory regions.

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A D hNSC21.5 hNSC18.5 hNSC 18.5 hNSC 21.5 G144 8130 0.8 0.4 2.0 IgG - DMSO 1 0 2 0.6 0.3 1.5 IgG - MEKi DMSO 5 0.4 0.2 1.0 CIC - DMSO CIC - MEKi

% of Input 0.2 0.1 0.5 G144 0.0 0.0 0.0 RPLP0 ETV4 RPLP0 ETV4 RPLP0 ETV4 hNSC18.5 hNSC21.5 B hNSC 18.5 hNSC 21.5 G144 1 181 329 peaks 68 peaks 672 peaks 21 10 10 10 IgG 46 DMSO CIC 0 102 MEKi CIC

24 h MEKi 524

0 0 0 G144 -3,000 bp| 3,000 bp -3,000 bp| 3,000 bp -3,000 bp| 3,000 bp

C hNSC 18.5 MEKi 50 chr17 30 chr1 25 chr20 50 chr9 30 chr14 E 30 IgG 20

50 30 25 50 30 10 hNSC # of Peaks 18.5 0 0246 50 30 25 50 30 Log10 (distance to tss) hNSC

DMSO 21.5 hNSC 21.5 MEKi 50 30 25 50 30 4 G144 2 50 30 25 50 30 hNSC # of Peaks 0 18.5 1357 Log10 (distance to tss) 50 30 25 50 30 hNSC G144 MEKi 21.5 80

24 h MEKi 50 30 25 50 30 60 40 G144 20 # of Peaks 0 -1 1 3 5 7 ETV4 GPR3 ID1 SHC3 FOS Log10 (distance to tss) 6,000 bp

Figure 2. MAPK signaling prevents CIC from binding to its target genes A, ChIP-qPCR for CIC-binding at the promoter of ETV4 or control gene (RPL30)intwo human neural stem cell lines (18.5 and 21.5) and one glioma neural stem cell (GNS) line (G144) after 24 hours mock (DMSO) or MEK inhibitor (MEKi) treatment. Data are represented as mean SD, n ¼ 3 B, Average intensity plot of all CIC peaks found in each cell line after 24-hour MEK inhibition. C, ChIP-seq tracks of CIC occupancy at selected targets genes in response to DMSO or 24-hour MEKi. D, Overlaps of CIC peaks detected by ChIP-seq in human neural stem cells and GNS cells with and without MEKi. E, Number of peaks found in MEKi-treated cells depicted according to their distance to the closest TSS.

CIC interacts with the SIN3 histone deacetylating complex them by mass spectrometry. Previously identified interaction and loss of CIC leads to increased histone acetylation of CIC partners Ataxin-1 (ATX1) and Ataxin-1–like (ATX1L; ref. 11) as target genes well as 14-3-3 proteins (9) were highly enriched. Among the CIC is believed to act as a transcriptional repressor (17); most strongly enriched proteins were members of the SIN3 however, the molecular mechanism through which CIC regu- transcriptional repressive complex, including core deacetylating lates gene expression is unknown. To obtain insights into CIC- enzymes HDAC1 and HDAC2 and structural components like mediated transcriptional regulation in mammalian cells, we SIN3A, SIN3B, and SAP30 (Fig. 4A and B; Supplementary purified proteins interacting with human CIC-L and identified Table S3). This suggests that CIC could regulate transcription

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AB H3K27ac H3K4me1 H3K27ac H3K4me1

G144 Peaks (n = 672) n = 672) Control peaks (n = 672) 80

20 % of Total peaks

0 Peaks G144 ( n = 672)

H3K27ac H3K4me1 Sorted according to increasing distance tss Sorted according to increasing distance tss Controls for G144 peaks matched to TSS hg38 (

100,000 bp 100,000 bp 100,000 bp 100,000 bp 50 CIC Peaks G144 CIC Peaks G144 Control peaks Control peaks

Figure 3. CIC targets are enriched for enhancer-associated histone marks. A, Heatmaps showing ChIP-seq signals of H3K27ac and H3K4me1 at CIC peaks in G144 cells. A set of control regions with matched distance to TSS is depicted to the right. B, Percentage of CIC peaks or control regions overlapping with H3K27ac or H3K4me1 peaks in G144 cells.

by recruiting the SIN3 complex. Size-exclusion chromatogra- firmed a significant increase in H3K9ac (6/7 genes) and H3K27ac phy supported the interaction between CIC and SIN3A (Sup- (2/7 genes; Supplementary Fig. S3C). Taken together, these results plementary Fig. S2D). show that the recruitment of CIC to its target genes leads to their The affinity purification also identified the Dual specificity deacetylation, which is in agreement with our demonstration tyrosine phosphorylation–regulated kinases DYRK1A and that CIC associates with the SIN3A complex. DYRK1B as well as their partner DCAF7 (Fig. 4A). Size-exclusion chromatography showed that a fraction of DYRK1A also MEK inhibition in human glioma cells causes repression of comigrated with CIC (Supplementary Fig. S2D). The Drosoph- a subset of CIC target genes and increased proliferation in a ila orthologs Minibrain (mnb) and Wings apart (wap) can act as CIC-dependent manner ERK-independent regulators of CIC (33). Thus, our results Having established that CIC binding to its target genes is suggest that this mechanism could be conserved during ablated by MAPK activity, we investigated global gene expression evolution. changes caused by MEK inhibition and the effect of CIC deletion To investigate a possible link between CIC and the SIN3 (Supplementary Fig. S4A and Supplementary Table S4). To this histone deacetylation complex in regulating the expression of end we defined "CIC target" genes as genes with a CIC peak 5kb CIC target genes, we established a CIC KO G144 line and used around the TSS after 4 and 24 hours MEK inhibition, respectively. ChIP-seq to correlate CIC recruitment and changes in histone For these sets of genes, we compared RNA-seq data of parental acetylation. The human GNS cell line G144 exhibits an oligo- G144 cells and one CIC WT single cell clone to two independent dendrocyte precursor-like phenotype (19) and represents a CIC KO clones. A large proportion of CIC target genes showed a disease relevant models that is experimentally tractable. We downregulation after 4h of MEK inhibition (9/19) and to a lesser detected a strong recruitment of CIC in response to MEK extent after 24 hours of MEK inhibition (14/111). More impor- inhibition, whereas no recruitment was detected in CIC KO tantly, this loss of CIC target gene expression was reduced or cells (Fig. 5A; Supplementary Fig. S3A). completely abolished in CIC KO cells (Fig. 6A; Supplementary To understand the functional consequences of CIC-recruitment Table S4), which was confirmed by qPCR (Fig. 6B). Given that to its target genes, we performed genome-wide localization deacetylation of histones could be one of CIC-mediated mechan- studies of histone acetylation. This revealed a strong depletion isms to downregulate its target genes, we used histone deacetylase of histone H3 acetylation around the promoter of CIC target genes (HDAC) inhibition (Panobiostat) to test if HDACs were involved upon CIC recruitment (Fig. 5B). We observed a strong deacetyla- in the repression of ETV4 and ETV5. Although, ETV4 and ETV5 tion at CIC peaks after 4 hours and somewhat less pronounced expression was still decreased in response to MEKi, HDAC inhi- deacetylation after 24 hours of MEKi treatment. This loss in bition significantly dampened this repression (Supplementary acetylation was not observed in CIC knockout cells (Fig. 5C and Fig. S4B). D; Supplementary Fig. S3B). The effect of CIC on histone acety- Previous studies have indicated that deletion of CIC confers lation levels was further addressed in mESCs by ChIP-qPCR in resistance to the MAPK pathway (34) or EGFR inhibition (35). wild type and Cic KO ESCs. A number of candidate genes con- Indeed, prolonged treatment with MEK inhibitors caused a

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Direct Regulation of MAPK Pathway Genes by CIC

Family Protein Coverage # Unique (%) peptides IP CIC 62.00 106 kDa CICL FUBP1 SIN3A 60.57 101 SIN3B 44.49 50 ARI4A 22.59 24 α-CIC 188 ARI4B 36.97 54 SATB1 38.66 15 98 Figure 4. SATB2 50.48 27 98 CIC interaction partners. A, Table HDAC1 56.22 20 α-HA (FUBP1) of selected CIC-interacting proteins HDAC2 56.97 21 62 identified by mass-spectrometry of SIN3 complex SP130 40.55 33 affinity-purified exogenously SAP30 47.27 15 expressed Flag-HA-CIC in HEK293 SP30L 42.08 10 cells. Interacting proteins are grouped 188 RBBP4 45.41 9 α-SIN3A according to functionality. B, 98 Validation of SIN3A interaction RBBP7 68.47 16 by immunoprecipitation ING2 27.14 6 (IP)-immunoblotting. An BRMS1 34.55 6 98 immunoprecipitation performed in BRM1L 55.42 22 α-PABP1 parallel for HEK293-expressing SDS3 62.50 23 62 Flag-HA-FUBP1 (immunoblotting for SIN3 associated FOXK1 68.49 42 its interaction with PABP1 and HUR) is 38 α-HUR TFs FOXK2 79.55 38 included as a negative control. ATX1L 55.01 29 ATX1 22.70 12 1433Z 40.41 7 Ataxin & 14-3-3 1433T 69.80 15 1433G 46.96 6 1433E 66.27 15 1433B 28.86 1 DCAF7 56.43 17 DYRK DYRK1A 29.23 18 DYRK1B 34.02 14

complete stop of cell proliferation in G144 cells, whereas CIC tional activator (38). However, we have not been able to confirm KO cells continued to grow even after 11 days of MEK inhib- this observation, because we only observed robust CIC binding ition (Fig. 6C). after MAPK inhibition. The PEA3 transcription factors have been shown to be MAPK- and CIC-regulated genes and have been linked to cancer progres- Discussion sion (17, 18). Here, we show that these genes are likely to have The high frequency of CIC mutations in oligodendroglioma similar roles in oligodendroglioma. In addition, we have identi- suggests that CIC is a bona fide tumor-suppressor gene. It is fied new target genes of CIC whose deregulation could play an therefore crucial to investigate the effects of CIC (mis)regulation important role in oligodendroglioma development. Some of as this knowledge will give insight into the molecular mechanisms these genes, as for example ID1, CCND1, and SHC3 are strongly leading to oligodendroglioma and help design future treatments bound by CIC and have been implicated in tumor formation. The for the disease. In this study, we have mapped the genome-wide members of the inhibitor of DNA-binding (ID) protein family are binding pattern of mammalian CIC. We find that CIC binds to an master regulators of cancer stem cells and tumor aggressiveness overlapping set of core regulatory regions in mouse and human (39), and ID1 is directly implicated in neural stem cell identity genomes, including promoter regions of established MAPK effec- and gliomagenesis (40). The gene CCND1 encoding cyclin D1, tor genes, as well as distal regions associated with enhancer which is a key regulatory subunit of CDK4 and CDK6, is instru- features. Furthermore, the detection of a conserved CIC-binding mental during G1–S-phase transition. SHC proteins function as motif, similar to the one previously reported for Drosophila Cic adaptors for translating RTK signals into the activation of down- suggests that CIC function is conserved during evolution. Our stream pathways like RAS/MAPK (41). Interestingly, SHC3 is the results identify CIC as a key transcriptional repressor of MAPK predominantly expressed in the brain, where it is involved in effector genes that are controlling cellular proliferation. A number neuronal differentiation and prevents apoptosis in mature neu- of these genes are deregulated in CIC-deleted cell lines (36) and rons (42, 43). Furthermore, it was found to be misregulated in CIC-mutated oligodendrogliomas (37). On the basis of our data, high-grade astrocytoma where it increased cell survival (44). we propose that these genes are directly affected by the loss of CIC. CIC targets also include groups of genes that are negative A recent study suggested that CIC binds a number of genes regulators of the MAPK pathway. The DUSP phosphatases and unrelated to MAPK signaling and could function as a transcrip- Sprouty-Related EVH1 Domain (SPRED) containing family

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ACH3K9ac H3K27ac WT KO 15 DMSO DMSO 4 h MEKi 24 h MEKi DMSO 24 h MEKi MEKi WT - 4 h

15 n = 672) WT - 24 h

15 Peaks 24 h MEKi ( Sorted according to decreasing -log10 (FDR) KO - 24 h 10,000 bp Peaks | | 50 -5,000 bp 5,000 bp -5,000 bp 5,000 bp B D ETV4 CIC H3K9ac H3K27ac 50 IgG CIC DMSO 1.0 4 WT DMSO 50 0.8 WT 4 h WT 4 h MEKi 3 WT 24 h 50 0.6 24 h MEKi KO DMSO 2 0.4 KO 24 h % of Input 50 % of Input DMSO 0.2 1 KO 50 24 h MEKi 0.0 0 chr17 ETV4 ETV5 ETV4 ETV5 ETV4 ETV4 ETV4 GAPDH DUSP4 SPRY4 GAPDH DUSP4 SPRY4 H3K9ac SPRY4 H3K27ac 50 2.0 10 DMSO ns ns ** 8

ut 1.5 50 WT 4 h MEKi 6 *** * f Inp 1.0 f Input

50 o * * o 4

24 h MEKi % ** * % * 0.5 50 2 DMSO KO 0.0 0 50 24 h MEKi ETV4 ETV5 ETV4 ETV5 chr5 GAPDH DUSP4 SPRY4 GAPDH DUSP4 SPRY4 SPRY4 SPRY4 SPRY4

5,000 bp

Figure 5. Loss of CIC leads to increased histone acetylation at CIC target genes. A, Heatmap of all CIC ChIP-seq peaks found in glioma neural stem cells (G144) after 24 hours of MEK inhibition showing CIC in WT and KO cells after 4 or 24 hours of MEK inhibition. B, ChIP-seq tracks showing increase of CIC occupancy at selected target genes and concurrent reduction of histone acetylation following MEK inhibition. C, Average ChIP-seq signal of H3K9ac and H3K27ac at CIC peaks after 4- and 24-hour MEK inhibition. D, ChIP-qPCR for CIC and histone acetylation marks at selected CIC target genes, with GAPDH as a negative control. Data are represented as mean SD, n ¼ 3(, P <0.05; , P <0.01; , P <0.001).

members are ERK-speciﬁc phosphatases (45) and SOS inhibitory simultaneous transcriptional induction of negative RTK regula- binding proteins (46), respectively. Conversely, Sprouty RTK tors, which might protect from acquiring mutations in RTK coding Signaling Antagonist 4 (SPRY4) blocks MAPK activation by bind- genes. Importantly, CIC mutations are not found in other types of ing to RAF1 (47). This suggests that CIC fulﬁlls a central role in a brain tumors such as high-grade astrocytoma and Glioblastoma delicate feedback mechanism that keeps mitogen activated sig- multiforme (GBM) that display an accelerated disease progres- naling in check under physiological conditions. The mutual sion and frequently harbor aberrant RTK signaling (5). This exclusivity of CIC mutations and RTK mutants in oligodendro- suggests that increased RTK signaling can induce malignant cell glioma could, therefore, be explained not only by activation of growth through CIC-dependent and -independent pathways lead- proliferative genes in response to CIC loss, but also by the ing to mutual exclusiveness with CIC mutations and a more

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Direct Regulation of MAPK Pathway Genes by CIC

A 4 h CIC target genes 24 h CIC target genes C 100 ETV5 SHC3 DUSP6 SPON2 4 ETV4 ETV5 DUSP4 VGF ETV1 GPR3 ZSWIM4 GGN FOS ETV4 SPRED2 1 PER2 PLK3 ID1 SPRED1 CDC14A SHC3 MAFF ZC3HAV1 RBPJ MAFG 10 HCRTR1 CAMK1G OSTC 2 DUSP4 DNAJB11 LOC101929705 CIC MIDN 0 UBALD2 LARS2-AS1 DUSP6 EPHA2 DUSP5 MIR9-1 FGFBP3 ADO RHEBL1 GRIK3 SPON2 SPRY4 ZNF219 PLXNA2 MYEOV PXMP2 CCND1 ANKRD28 PER2 NR2C2AP -1 DPF1 0 1 OLIG2 MFSD2A Million cells FOSB RNF24 SPRED1 EPC2 FZD7 FLJ37453 MAB21L2 URM1 CREB3L1 LOC100507194 LOC101929705 STON1 HOXC13-AS AKIRIN2 EXT1 FLJ13224 -2 HES5 MAFF APBB2 HBP1 CHD3 FOXB1 -2 LINC01351 PGAP1 0.1 CDC42EP2 GGN CPM LSM10 ALKBH3-AS1 RNF44 TSC22D2 024681012 OSGIN1 CDC14A NHS UBASH3B MARCKS -3 WNK3 TBL1X VAX1 ANGPT2 SPRED2 ACOT1 Days LPIN2 LHX4 CRY1 TEX2 -4 FOSL1 NR2C2AP COX8A EP400NL UGP2 DUSP10 PCDH7 ADAMTS1 DMSO - Parental MEKi - Parental GOLGB1 CHD3 -4 ZIC1 IGFBP2 NRIP1 TCF7L2 LOC101927248 ATP6V1E2 MTSS1L PLXNA2 POU3F2 ACLY DMSO - CIC-KO8 MEKi - CIC-KO8 GBX2 CXCR4 DLX1 LINC01111 NAIF1 PCSK4 -6 DMSO - CIC-KO9 WT KO WT KO MEKi - CIC-KO9 B Parental WT-clone KO8 KO9 ETV4 ETV5 DUSP4 0.04 0.20 0.0015

0.03 0.15 0.0010 RPLP0) 0.02 0.10 0.0005

Expression 0.01 0.05 (norm to 0.00 0.00 0.0000 DMSO 4 h 24 h DMSO 4 h 24 h DMSO 4 h 24 h

SHC3 SPRY4 GPR3 0.020 0.006 0.0025

0.0020 0.015

PLP0) 0.004

R 0.0015

o 0.010 t 0.0010 0.002

Expression 0.005 0.0005 norm ( 0.000 0.000 0.0000 DMSO 4 h 24 h DMSO 4 h 24 h DMSO 4 h 24 h

Figure 6. Effect of CIC depletion on gene expression and proliferation. A, Gene expression changes of CIC target genes after 4 and 24 hours of MEK inhibition in WT

and CIC KO G144 cells (colors depict log2-fold changes between DMSO and MEKi). B, Expression changes quantiﬁed by qPCR in parental G144, one WT clone, and two independent CIC KO clones. C, Growth curves measured by cell count of parental and CIC KO G144 cells during continuous MEK inhibition or mock treatment (DMSO).

aggressive phenotype. In addition, CIC mutations could have acetylation levels in response to CIC recruitment. Furthermore, synergistic effects with the cell of origin of oligodendroglioma. we provide evidence that HDAC inhibitors could counteract We also investigated the effect of different single amino acid CIC-mediated repression on selected target genes. Interestingly, substitutions, which are found in human oligodendroglioma we observed that not all CIC targets changed their acetylation patients. As expected, the DNA-binding mutant R215C was levels in response to CIC loss, indicating that additional factors or unable to target and repress genes. More surprisingly was that pathways can cause histone deacetylation and gene repression this was also true for the C-terminal mutant R1515C. This synergistically with CIC. In fact, a recent study investigating Cic in observation is in line with reports that a variety of CIC mutant Drosophila reported that the Hippo pathway and Cic both act as proteins, including R1515H, were unable to suppress metas- brakes of the Ras-responsive transcriptional network and the tasis (18) and that the C-terminal part of CIC aids DNA perturbation of both is needed for full activation of target binding (30). genes (48). On the basis of our ﬁnding that CIC is associated with the In summary, our data together with previous ﬁndings show that SIN3A deacetylation complex, we propose a model in which CIC CIC acts as a transcriptional repressor of genes regulated by the recruitment can lead to histone deacetylation. This model is MAPK signaling pathways. We provide novel insights into the supported by our demonstration that a number of genes decrease spectrum of CIC-regulated genes revealing potential mechanisms

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for how loss of CIC could lead to oligodendroglioma formation. Administrative, technical, or material support (i.e., reporting or organizing Moreover, our demonstration that the SIN3A repressive complex data, constructing databases): P.A. Cloos, K. Helin interacts with CIC, and that CIC recruitment to target genes lead Study supervision: P.A. Cloos, K. Helin to their deacetylation suggest a mechanism by which CIC exerts its function. Acknowledgments We thank the members of the Helin lab for discussion, technical advice, and Disclosure of Potential Conflicts of Interest support. We thank Lucia Simon Carrasco and Anne Laugesen for critical comments on the article. This work was supported by the Danish National No potential conflicts of interest were disclosed. Research Foundation (DNRF 82; to K. Helin), the Brain Tumour Charity (GN-000358; to K. Helin and S. Pollard), the European Research Council Authors' Contributions (294666_DNAMET; to K. Helin), and through a center grant from the Novo Conception and design: S. Weissmann, P.A. Cloos, K. Helin Nordisk Foundation (NNF17CC0027852; to K. Helin) Development of methodology: S. Weissmann, P.A. Cloos, O.N. Jensen, S. Pollard The costs of publication of this article were defrayed in part by the payment Acquisition of data (provided animals, acquired and managed patients, of page charges. This article must therefore be hereby marked advertisement provided facilities, etc.): S. Weissmann, S. Sidoli, O.N. Jensen in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Weissmann, P.A. Cloos, S. Sidoli, K. Helin Writing, review, and/or revision of the manuscript: S. Weissmann, P.A. Cloos, Received January 31, 2018; revised March 25, 2018; accepted May 14, 2018; S. Pollard, K. Helin published first May 29, 2018.

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The Tumor Suppressor CIC Directly Regulates MAPK Pathway Genes via Histone Deacetylation

Simon Weissmann, Paul A. Cloos, Simone Sidoli, et al.

Cancer Res 2018;78:4114-4125. Published OnlineFirst May 29, 2018.

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