Abstract

   Recurrent C11orf95-RELA fusions (RELA^FUS) are the hallmark of
   supratentorial ependymomas. The presence of RELA as the fusion partner
   indicates a close association of aberrant NF-κB activity with
   tumorigenesis. However, the oncogenic role of the C11orf95 has not been
   determined. Here, we performed ChIP-seq analyses to explore genomic
   regions bound by RELA^FUS and H3K27ac proteins in human 293T and mouse
   ependymoma cells. We then utilized published RNA-Seq data from human
   and mouse RELA^FUS tumors and identified target genes that were
   directly regulated by RELA^FUS in these tumors. Subsequent
   transcription factor motif analyses of RELA^FUS target genes detected a
   unique GC-rich motif recognized by the C11orf95 moiety, that is present
   in approximately half of RELA^FUS target genes. Luciferase assays
   confirmed that a promoter carrying this motif is sufficient to drive
   RELA^FUS-dependent gene expression. Further, the RELA^FUS target genes
   were found to be overlapped with Rela target genes primarily via
   non-canonical NF-κB binding sites. Using a series of truncation and
   substitution mutants of RELA^FUS, we also show that the activation
   domain in the RELA^FUS moiety is necessary for the regulation of gene
   expression of these RELA^FUS target genes. Lastly, we performed an
   anti-cancer drug screening with mouse ependymoma cells and identified
   potential anti-ependymoma drugs that are related to the oncogenic
   mechanism of RELA^FUS. These findings suggested that RELA^FUS might
   induce ependymoma formation through oncogenic pathways orchestrated by
   both C11orf95 and RELA target genes. Thus, our study unveils a complex
   gene function of RELA^FUS as an oncogenic transcription factor in
   RELA^FUS positive ependymomas.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s40478-021-01135-4.

   Keywords: Ependymoma, Fusion gene, NF-κB signaling, Transcription
   factor motif

Introduction

   Ependymomas are primary glial tumors that can occur in all ages and
   locations of the central nervous system [[44]39]. Current therapeutic
   options for all tumors largely depend on maximal safe surgical
   resection and radiation therapy, whereas no significant survival
   benefit was observed for standard chemotherapy [[45]34, [46]43,
   [47]68]. Recent large-scale genome sequencing studies of ependymomas
   have identified nine molecular subgroups associated with distinct
   genomic alterations, clinical behavior, age distribution, and
   anatomical location [[48]44]. Supratentorial ependymomas are
   characterized by mutually exclusive recurrent RELA and YAP1-related
   gene fusions, thereby being segregated into two subgroups denoted as
   ST-EPN-RELA and YAP1 [[49]44]. The ST-EPN-RELA subgroup displays a
   dismal prognosis, and incomplete surgical resection of these tumors is
   associated with increased recurrence rates and poor outcome, signifying
   that the development of new therapies is essential for these cases
   [[50]9, [51]34, [52]44, [53]68]. Thus, the identification of clinically
   relevant subtypes and oncogenic drivers could provide the opportunity
   to develop specific targeted therapies for individual tumor types.

   Recurrent C11orf95-RELA fusion (RELA^FUS) genes were identified in a
   large fraction of supratentorial ependymomas [[54]45]. RELA^FUS1 (Type
   1) and RELA^FUS2 (Type 2), the two most frequent fusion variants are
   potent driver oncogenes capable of inducing human ependymoma-like
   tumors in mice, and therefore likely represent the tumor-initiating
   events in human patients [[55]41, [56]45, [57]60]. RELA is a well-known
   master transcription factor in the NF-κB pathway, which is intimately
   involved in various pathophysiological processes such as inflammation
   and cancer [[58]6, [59]69]. Upon external stimuli, RELA is released
   from IκBα-mediated cytoplasmic sequestration and translocated into the
   nucleus, thereby transcriptionally regulating the expression of the
   target genes [[60]69]. Given that the fusion protein preferentially
   localizes in the nucleus, persistent activation of the NF-κB pathway is
   thought as the primary mechanism for the RELA^FUS-driven ependymoma
   formation as bolstered by high NF-κB activity in human and mouse
   RELA^FUS tumors [[61]41, [62]45]. However, dysregulation of many
   non-NF-κB pathways was also commonly identified in these tumors.
   Furthermore, expression of wild type RELA or activating RELA mutants
   failed to induce brain tumor formation in mice, strongly suggesting an
   important role of non-NF-κB pathways in the RELA^FUS-driven ependymoma
   formation [[63]41, [64]45].

   The recent identification of active super-enhancers (SE) specific to
   human RELA^FUS ependymomas gave significant insights into the activated
   oncogenic pathways and potential therapeutic targets in these tumors
   [[65]31]. Although a subset of these super-enhancers highlighted
   oncogenic driver genes and pathways associated with tumorigenesis, not
   all direct targets of RELA^FUS could be identified due to the technical
   limitations in accurately determining enhancer target genes [[66]46].
   Further, transcription factors generally function in a
   context-dependent manner [[67]24]. Therefore, a different approach
   would be useful to further scrutinize genes directly regulated by
   RELA^FUS. Here, to dissect the oncogenic program underlying ependymoma
   formation, we explored transcriptional target genes directly regulated
   by the RELA^FUS1-HA protein using HA tag and H3K27ac-ChIP-seq analyses
   in human 293T and mouse ependymoma cells, and uncovered the complex
   epigenetic regulation by RELA^FUS1. In addition, we performed an
   anti-cancer drug screening to validate the potential therapeutic
   relevance of downstream effectors driven by RELA^FUS1 targets. Our
   study further deepens our understanding of the molecular functions of
   RELA^FUS in driving tumorigenesis, thus providing significant clues to
   identify therapeutic targets for RELA^FUS positive ependymomas
   [[68]31].

Materials and methods

Generation of murine RELA^FUS1 tumors

   All animal experiments were done in accordance with protocols approved
   by the Institutional Animal Care and Use Committees of Fred Hutchinson
   Cancer Research Center (FHCRC) and followed NIH guidelines for animal
   welfare. The RCAS/tv-a system used in this work has been described
   previously [[69]16–[70]18, [71]41, [72]42]. Mouse RELA^FUS1 tumors were
   generated with the injection of RCAS-RELA^FUS1 or RELA^FUS1-HA virus
   into newborn pups brains in N/tv-a;Ink4a-Arf^−/−;Pten^fl/fl mice. The
   mice were sacrificed when they developed symptoms of the disease and
   the brain tumors were used for the generation of tumor cell lines.

Generation of mouse neurosphere and ependymoma cell lines

   Neurosphere lines were generated by mechanical dissociation from
   forebrains of newborn pups in N/tv-a;Ink4a-Arf^−/−;Pten^fl/fl or B/tv-a
   mice and then maintained in serum-free neurosphere medium (Stem Cell
   Technologies) [[73]4, [74]41]. Murine ependymoma cell lines were
   generated by mechanical dissociation from brain tumors driven by
   RCAS-RELA^FUS1 (H41, H57 and H59) or RCAS-RELA^FUS1-HA (H1203) and then
   maintained as adherent cells in serum-free neurosphere medium (Stem
   Cell Technologies) [[75]41]

Cell culture, transfections and infections

   293T cells (ATCC: CRL-3216) and DF-1 cells (ATCC: CRL-12203) were
   maintained according to the manufacture’s protocol. RCAS virus was
   produced in DF-1 packaging cells as previously described
   [[76]16–[77]18]. Transient transfection of luciferase reporter and/or
   RCAS plasmids into 293T or 293T/tv-a cells was performed with
   X-tremeGENE 9 DNA Transfection Reagent according to the manufacture’s
   protocol (Merck by Roche). 293T/tv-a cell lines lentivirally expressing
   the tv-a-myc/6xHis were generated with standard protocol and maintained
   in a medium containing 1 μg/ml puromycin as previously described
   [[78]42]. For the generation of 293T/tv-a cells expressing the relevant
   RCAS virus, cells were subjected to retroviral infection using the RCAS
   viral supernatant in the DF-1 cells.

Chromatin immunoprecipitation and sequencing (ChIP-seq)

   293T/tv-a cells infecting RCAS-RELA^FUS1-HA, RELA^FUS1−S486E-HA and
   mEPN cells (H41 and H1203) were fixed with 1% formaldehyde, stopped the
   fixation with 0.125 M glycine, and then collected ice-cold 1 × PBS(−)
   containing 1 mM PMSF according to the standard protocol. Nuclei
   preparation and chromatin digestion was performed according to
   manufacturer's instructions (Cell Signaling Technology, #9003) with
   modification [[79]20]. Nuclei pellets were resuspended with ChIP buffer
   (50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 0.5% IGEPAL
   CA-630, 5 mM EDTA [pH 8.0], 1 mM PMSF and Protease Inhibitor Cocktail).
   Samples were sonicated by using Bioruptor II (BM Equipment, BR2006A) to
   generate DNA fragments of ~ 200 base pairs. Antibodies (2 µg) for
   H3K27ac (Abcam, #4729, Lot GR3252404) or HA (Abcam, #9110, Lot
   GR235874-5) were added into the sheared chromatin (10 ~ 30 µg), and
   incubated in an ultrasonic water bath for 30 min at 4 °C. After
   centrifugation, supernatants were incubated with FG Beads HM Protein G
   (Tamagawa Seiki, TAB8848N3173) for 30 min at 4 °C. Beads were washed
   twice with ChIP buffer and washed with Wash buffer (50 mM Tris–HCl [pH
   8.0], 300 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate and
   5 mM EDTA pH 8.0) and LiCl buffer (50 mM Tris–HCl [pH 8.0], 250 mM
   LiCl, 1% Triton X-100, 0.5% Na-deoxycholate and 5 mM EDTA [pH 8.0]).
   Immunoprecipitated chromatin was eluted and reverse-crosslinked
   according to manufacturer's instructions (Cell Signaling Technology,
   #9003). Immunoprecipitated DNA was purified by using QIAquick PCR
   Purification Kit (QIAGEN, #28106). DNA libraries were prepared by using
   QIAseq Ultralow Input Library Kit (QIAGEN, #180492). The size
   determination and quantification of DNA libraries was done by qPCR (New
   England Biolabs, E7630) and by using Agilent 2100 Bioanalyzer. DNA
   libraries were sequenced on Illumina sequencers (Illumina HiSeq 3000 or
   NovaSeq 6000). Detailed antibody information, library preparations and
   Illumina sequencers in this study were described in Additional file
   [80]9: Table S1A.

ChIP-seq data analysis

   The RelA ChIP-seq datasets in murine embryonic fibroblasts after three
   hours of TNF stimulation were obtained from [81]GSE132540 [[82]37].
   Sequencing reads from ChIP-seq experiments were mapped to the hg19
   version of the human genome or mm9 version of the mouse genome with
   Bowtie (v2.2.9) and parameters–local, respectively [[83]25]. Gene
   annotations were obtained from Ensemble (v.75). Duplicate reads were
   removed by Samtools (v1.3.1). Heatmaps to visualize enriched regions
   (ERs) around TSS ± 10 kb were generated with NGSplot (v2.63) [[84]56].
   The normalized ERs were visualized with the Integrative Genomics Viewer
   (IGV; v2.3.91) [[85]50]. ChIP-seq ERs were called using MACS (v1.4.2)
   with the default parameters (p value cutoff; 1e-5) with the relevant
   input as control [[86]70]. ChIP-seq peak statistics were summarized in
   Additional file [87]9: Table S1A. A correlation heatmap to evaluate the
   relationship between samples was generated by DiffBind (v2.4.8)
   [[88]52]. For the differential binding analyses, we consolidated the
   peaks into a consensus set using a “minOverlap” of 2. The count reads
   were then TMM normalized implemented in DiffBind. Batch annotation of
   ERs was performed using ChIPpeakAnno (v3.10.2) as a Bioconductor
   package [[89]73] within the statistical programming environment R
   (v3.4.1). Motif analyses of C11orf95-RELA fusion and Rela protein
   binding sites were performed using MEME-ChIP (v5.0.1) with sequences of
   ERs [[90]30]. Overlapping peaks of ERs between different ChIP-seq
   experiments were obtained by using “findOverlapsOfPeaks” function under
   the default setting in ChIPpeakAnno. This setting counts the peaks as
   the minimal involved peaks in any group of connected/overlapped peaks.
   Super-enhancers were defined by H3K27ac peak rank order using ROSE
   algorithm [[91]29, [92]65]. Enhancer profiles specific for human
   ST-EPN-RELA ependymomas and RELA-EnhancerAssociatedGene (Subgroup
   specific enhancers and super enhancers detected in ST-EPN-RELA
   ependymoma) were obtained in the previous study [[93]31]. For the
   purpose of pathway enrichment analysis, gene ontology networks were
   generated using ClueGO (v2.5.1) [[94]3] through Cytoscape (v3.6.1)
   [[95]55]. We used the following ontologies: KEGG_20.11.2017 and
   REACTOME_Pathways_20.11.2017. To calculate enrichment/depletion tests,
   two-sided tests based on the hypergeometric distribution were
   performed. To correct the p values for multiple testing, Bonferroni
   step down method was used. We used min:3 max:8 GO tree interval, a
   minimum of 3 genes per GO term, kappa score of 0.4.

   Lists of the target gene were generated by removing duplicate gene
   annotations from the peak list obtained at TSS ± 10 kb (Additional file
   [96]9: Table S1E). A list of ‘previously reported-NFkB target genes’
   was previously described [[97]12, [98]41]. For comparison of our mouse
   gene list to the human, all mouse gene symbols were converted to humans
   using the MGI homology map with the BioMart browser in the Ensembl
   database. The normalized ChIP-seq tracks were visualized on the IGV
   genome browser (v2.3.91). RELA^FUS1-HA, RELA^FUS1−S486E-HA, Rela,
   H3K27ac and input peaks are shown with the same scale in each figure
   (Fig. [99]5b, c, i, j, Additional file [100]5: S4B, E, F and Additional
   file [101]7: S6D). 5′-BGKGGCCCCBG-3′ (B = C or G or T, K = G or T) and
   5′-GGGRNWYYCC-3′ (R = A or G, N = any base, W = A or T, and Y = C or T)
   sequences were used to find a genomic position of the
   293T-RELA^FUS1-MEME-2 and canonical NF-κB consensus motif,
   respectively. The position of the 293T-RELA^FUS1-MEME-2 and κB site is
   shown as a blue vertical bar on positive (+) and negative (−) DNA
   strands. Transcriptional start sites (TSSs) were analyzed using the
   DBTSS; Data Base of Transcriptional Start Sites online tool [[102]59].
   Representative images in two technical replicates were shown in the
   figures.

Fig. 5.

   [103]Fig. 5
   [104]Open in a new tab

   RELA^FUS1 transcriptionally regulates the target gene expression
   through DNA binding on the RELA^FUS1-MEME-2 sequence. a Venn diagram
   showing the number of the RELA^FUS1 target genes overlapped between
   293T and mEPN cells. 41 common targets were shown in the right table.
   SE-located genes were highlighted in yellow. b, c, i, j RELA^FUS1-HA,
   RELA^FUS1−S486E-HA and H3K27ac binding profiles surrounding the human
   C11orf95 and LMX1B in 293T/tv-a, and mouse 2700081O15Rik and Lmx1b loci
   in mEPN (H1203 and H41) cells. Rela binding profile in MEFs was also
   shown in mouse 2700081O15Rik locus. The position of the MEME-2 and κB
   sites are shown as a blue vertical bar on positive (+) and negative (−)
   DNA strands. d Boxplots of C11orf95 and LMX1B mRNA expression in human
   RELA^FUS positive (n = 14) and negative (n = 54) ependymomas. e, f
   Boxplots of 2700081O15Rik and Lmx1b mRNA expression in mouse normal
   brain (NB) and RCAS-RELA^FUS1-driven ependymoma or PDGFA-driven glioma
   tissues in the N/tv-a (e) or N/tv-a;Ink4a-Arf^−/−;Pten^fl/fl (KO) (f)
   mice (n = 4 in each group). All box plots showing mRNA expression
   extend from the 25th to 75th percentiles. Whiskers of the box plots
   extend to the most extreme data point (d–f). Gene expression analysis
   was done using unpaired two-tailed t-test (d, e) or Ordinary one-way
   ANOVA (f). **p < 0.01; ****p < 0.0001. g Relative C11orf95 and LMX1B
   mRNA expression in 293T cells. Data are displayed as the relative ratio
   to GFP cells. qPCR data (mean ± SD) for C11orf95 and LMX1B expression
   are displayed as the relative ratio to GFP cells (n = 3, in technical
   quadruplicate). Analysis was done using paired two-tailed t-test.
   *p < 0.05; **p < 0.01 (See Methods). h Relative Nanoluc reporter
   activity to the upstream sequence of C11orf95 gene normalized to the
   Firefly luciferase activity and GFP cells in the 293T cells (n = 6, in
   technical triplicate). Analysis was done using paired two-tailed
   t-test. ****p < 0.0001 k Relative Lmx1b mRNA expression in H1203 cells
   introduced with the indicated dCas-sgRNA. Data are displayed as the
   relative ratio to sgGFP vector-infected cells. Data from two
   independent experiments with two technical replicates are shown in the
   figures. Each qPCR was performed in technical quadruplicate. Analysis
   was done using paired two-tailed t-test. **p < 0.01; ***p < 0.001

RNA-seq datasets and gene expression analysis

   For gene expression analyses, RNA-seq datasets of human ependymomas and
   mouse brain tissues were obtained from the Pediatric Cancer Genome
   Project (PCGP, EGAS00001000254) and the Gene Expression Omnibus
   ([105]GSE93765), respectively [[106]7, [107]41, [108]45]. The human
   ependymoma samples were analyzed between RELA^FUS positive ST-EPNs and
   all negative ependymomas including RELA^FUS negative ST-EPNs and
   PF-EPNs. The aligned reads were counted for gene associations against
   the Ensemble genes database with featureCounts (v1.5.0) [[109]27].
   Transcriptomic signature genes of ST-EPN in single-cell RNA-seq
   analysis were obtained in the previous publication and used for an
   overlapping analysis with RELA^FUS1 target genes [[110]13]. Microarray
   gene expression data of the human ST-EPN-RELA and YAP1 subgroup was
   obtained from publicly available data ([111]GSE64415) and used for
   pathway enrichment analysis [[112]44]. Differential expression analyses
   were performed using the R/Bioconductor package edgeR (v3.18.1)
   [[113]51]. Statistical analyses were performed using GraphPad Prism
   (v7.0.0).

Vector constructs

   All vectors and sgRNA target sequences used in this study are listed in
   Additional file [114]14: Table S6A. The DNA fragments for the RELA^FUS1
   and C11orf95 mutant vector construction were PCR-amplified on the
   relevant DNA template to obtain appropriate restriction sites and then
   inserted in RCAS vector.

   The luciferase reporter vectors for the RELA^FUS1-motifs were generated
   by insertion of the synthetic oligonucleotide for the relevant motif
   sequence as between Kpn I and EcoRV sites in the pNL3.2 vector
   containing the minimal promoter (Promega). The C11orf95 5′-upstream
   luciferase reporter construct was generated by PCR-amplifying the human
   C11orf95 gene sequence from genomic DNA of 293T cells. The PCR fragment
   was then subcloned into pGEM-T easy vector (Promega) to obtain
   appropriate restriction sites and subsequently inserted into the pNL3.2
   (minimal promoter) vector (Promega). For RCAS-C11orf95-NLS-VP64-HA
   (CNVP-HA) and lentiCRISPRv2-dCas9-sgGFP1 vector construction,
   SV40-NLS-VP64-HA and sgGFP target sequences as previously described,
   were synthesized and inserted in the relevant vector, respectively
   [[115]47, [116]54].

   pLJM1-EGFP was a gift from David Sabatini (Addgene plasmid #19319;
   [117]http://n2t.net/addgene:19319; RRID: Addgene_19319) [[118]53].
   psPAX2 was a gift from Didier Trono (Addgene plasmid #12260;
   [119]http://n2t.net/addgene:12260; RRID: Addgene_12260). pMD2.G was a
   gift from Didier Trono (Addgene plasmid #12259;
   [120]http://n2t.net/addgene:12259; RRID: Addgene_12259). lentiCRISPR
   v2-dCas9 was a gift from Thomas Gilmore (Addgene plasmid #112233;
   [121]http://n2t.net/addgene:112233; RRID: Addgene 112233).

Western blot analysis

   Cells were cultured, lysed, and processed for western blotting as
   previously described [[122]41]. Antibodies were listed in Additional
   file [123]14: Table S6C. All western blot analyses were performed at
   least twice and successfully repeated in the experiments. The
   representative images were shown in the figures.

   For immunoblot and qPCR analyses in 293T cells as shown in Fig.
   [124]3j–l, [125]5g and Additional file [126]5: Fig. S4C, RCAS vectors
   were transiently transfected with the indicated plasmid concentration
   to adjust the protein expression level between samples. After 48 h of
   the transfection, cells were collected and split for RNA and protein
   extractions. Then, the cell lysates were subjected to immunoblot
   analysis with the indicated antibodies. RNAs were used for subsequent
   qPCR analysis.

Fig. 3.

   [127]Fig. 3
   [128]Open in a new tab

   RELA^FUS1 binds on specific DNA regions through the unique DNA-binding
   motif. a Top three transcription factor (TF) binding motifs enriched
   within the RELA^FUS1-HA peaks identified by the Multiple Em for Motif
   Elicitation (MEME) tool in 293T/tv-a cells. E-value, enrichment p
   value. b RCAS-GFP, RELA-HA, RELA^FUS1-HA (FUS1-HA) and
   RELA^FUS1−S486E-HA (4E-HA) vector expression in 293T/tv-a cells. c–e
   Relative Nanoluc reporter activity to the RELA^FUS1-responsive element
   (RE) normalized to the Firefly luciferase activity and GFP cells in the
   293T/tv-a cells (n = 6 or 8, in technical triplicate). MEME-1, 2 and 3
   denote RELA^FUS1-responsive reporters for top three (3×) or five (5×)
   tandem RELA^FUS1-motif as shown in Additional file [129]4: Fig. S3D–F.
   f Top TF binding motif enriched within the RELA^FUS1-HA peaks in mEPN
   cells. g, h Relative Nanoluc reporter activity to the top three tandem
   (3×) or top five single RELA^FUS1-MEME-2 motifs normalized to the
   Firefly luciferase activity and GFP cells in 293T cells (n = 4 or 5, in
   technical triplicate). i Relative Nanoluc reporter activity to the
   NF-κB-RE normalized to the Firefly luciferase activity and GFP cells in
   293T/tv-a cells (n = 8, in technical triplicate). Analysis was done
   using RM one-way ANOVA (c-e) or paired two-tailed t-test (g–i).
   *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. j RCAS vector
   expression in 293T cells. C11-HA, RCAS-C11orf95-HA k Relative NFKBIA
   mRNA expression in 293T cells. Data (mean ± SD) are displayed as the
   relative ratio to GFP cells (n = 3, in technical quadruplicate).
   Analysis was done using paired two-tailed t-test. *p < 0.05;
   **p < 0.01. l IκBα protein expression in 293T cells. See Methods for
   j–l

Quantitative PCR (qPCR) analysis

   Total RNAs were extracted from mEPN cells or 293T cells using the
   miRNeasy or RNeasy Mini kit (QIAGEN) and were used to synthesize cDNA
   by using the SuperScript IV VILO Master Mix (Thermo Fisher Scientific)
   according to the manufacturer’s protocol. SYBR Green real-time PCR was
   performed using the relevant gene-specific primer sets, PowerUp SYBR
   Green Master Mix (2X) (ThermoFisher SCIENTIFIC), and Fast run protocol
   from Applied Biosystems in a QuantStudio 6 Flex Real-Time PCR System.
   The ΔΔCt method was used to calculate the relative gene expression
   normalized to the reference gene (RPS18 or Rps18). Each biological
   replicate in the PCR reaction was assayed in four technical replicates.
   Data (mean ± SD) are displayed as the relative ratio to the relevant
   control sample (e.g. GFP cells). Circles in the figure indicate
   relative mean values of each biological replicate. Analysis was done
   using paired two-tailed t-test. *p < 0.05; **p < 0.01; ***p < 0.001;
   ****p < 0.0001. All primers used in this study are listed in Additional
   file [130]14: Table S6B [[131]64].

Luciferase reporter assay

   293T/tv-a cells retrovirally infecting the relevant RCAS virus were
   seeded at a density of 7.5 × 10^4 cells in a 24 well format in
   triplicates the day prior to transfection. Cells were then
   co-transfected pGL4.53[luc2/PGK] (control vector) and pNL3.2 (test
   vector) with 1:9 ratio (total 0.2 μg/well) using X-treamGENE9
   transfection reagent (Roche) in 500 μL/well of culture medium. After
   24 h of the transfection, cells were lysed with the Passive Lysis
   Buffer (Promega E1941) (100 μL/well) and the lysates of 80 μL/well were
   transferred in white 96 well plates, followed by analyzed for
   luciferase activity using the Nano-Glo Dual-Luciferase Reporter Assay
   System (Promega N1630) on a GloMax Explorer luminometer (Promega)
   according to manufacturer’s protocol.

   For analysis in transient expression of RCAS vectors as shown in Fig.
   [132]3g, h, [133]5h, 293T cells were seeded at a density of 5 × 10^5
   cells in a 6 well format the day prior to transfection. Cells were then
   co-transfected pGL4.53[luc2/PGK] (control vector), pNL3.2 (test vector)
   and RCAS vector with 1:9:10 ratio (total 2 μg) using X-treamGENE9
   transfection reagent (Roche) in 2 mL/well of culture medium. After 24 h
   of the transfection, cells were lysed with the Passive Lysis Buffer of
   500 μL/well and the lysates of 80 μL/well were transferred in white 96
   well plates in triplicate, followed by analyzed for luciferase activity
   as well.

   Relative luciferase activity was calculated as the ratio of NanoLuc
   normalized to Firefly luciferase and GFP control cells. The box plots
   in all luciferase reporter assays extend from the 25th to 75th
   percentiles. Whiskers of all box plots extend to the most extreme data
   point. Circles in the box plots indicate relative mean values of each
   biological replicate. Analysis was done using repeated measures (RM)
   one-way ANOVA or paired two-tailed t-test using Graph-Pad Prism 8
   software, and a value of p < 0.05 was considered significant.
   *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Immunofluorescence

   For analysis of subcellular localization of C11orf95-HA, RELA-HA and
   RELA^FUS1-HA proteins, DF-1 cells infecting the relevant RCAS viruses
   grown on Lab-Tek Chamber Slide (Nalge Nunc International, Rochester,
   NY) were fixed with 4% paraformaldehyde and were then permeabilized
   with 0.2% Triton X-100 in PBS. Subsequently, cells were immunostained
   with an anti-HA tag (Roche, 11867423001) antibody, followed by Alexa
   Fluor 488 rabbit anti-rat IgG secondary antibody (Invitrogen #A21210).
   The analysis was performed by immunofluorescent microscopy (Leica
   DMI6000 microscope, FW4000 software). GFP fusion proteins were observed
   using fluorescent microscopy (OLYMPUS CKX53 microscope and cellSens
   Standard software).

Anti-cancer drug screening

   H41- and H1203-mEPN cells (5,000/well) were seeded on a 384-well
   culture plate in mouse neurosphere medium in duplicate (Stem Cell
   Technologies) and cultured overnight at 37 °C with 5% CO[2] (day 0).
   Ten micromolar of 164 FDA-approved anti-cancer drugs and 15 selected
   potential NF-κB inhibitors (final 0.1% dimethyl sulfoxide, DMSO) was
   then added to the cells using the Bravo Automated liquid handling
   platform (Agilent technologies) (day 1), and cell viability was
   assessed with a CCK-8 kit (Cell Counting Kit-8, Dojindo, Kumamoto,
   Japan) after 72 h of incubation (day1 to day 4) (Additional file
   [134]13: Table S5A, B). The mean cell viability (% of control) was
   calculated (n = 2) as follows: (A sample-A blank)/(A 0.1% DMSO-treated
   control-A blank) (where, A = Absorbance at 450 nm) and experiments were
   repeated twice. Two Sorafenib Tosylate (SPL30 and SPL180) and
   Vorinostat (SPL70 and SPL179) from the different suppliers were tested
   in this screening because of redundancy between two drug libraries.

   To determine the cell sensitivity to these drugs, anti-cancer drugs
   serially diluted in mouse neurosphere medium were added to the mEPN
   cells in duplicate and cultured for 72 h. Cell viability was then
   assessed by a CCK-8 kit as well. IC50 values were calculated by drawing
   four-parameter curve fitting using GraphPad Prism (version 7, GraphPad
   Software).

Protein domain illustrations

   Illustrations of protein domain as shown in Fig. [135]4a, Additional
   file [136]2: Fig. S1D, Additional file [137]6: Fig. S5B, G and
   Additional file 8: Fig. S6F were generated using the IBS software
   (illustrator of biological sequences) [[138]28]. In Fig. [139]4a,
   Additional file [140]2: Fig. S1D and Additional file [141]8: Fig. S6F,
   Blue and red boxes represent portions of C11orf95
   (UniProtKB—[142]C9JLR9) and RELA (UniProtKB—[143]Q04206) coding
   sequences, respectively. ZF, zinc finger (SPIN-DOC-like, zinc-finger);
   RHD, Rel homology domain; TAD, transactivation domain; black boxes in
   the C-terminus, HA-tag; NLS, nuclear localization signal. NLS
   prediction in C11orf95 protein was done using the cNLS mapper online
   tool
   ([144]http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi)
   [[145]22]. The amino acid position of three predicted bipartite NLSs
   (210–239, 254–286 and 456–487) in C11orf95 are shown as black bars and
   the yellow box (cut-off score = 4.0) in Fig. [146]4a.

Fig. 4.

   [147]Fig. 4
   [148]Open in a new tab

   C11orf95 determines the RELA^FUS1-binding region through the unique
   GC-rich motif. a Schematic of RCAS vector constructs (See Methods) b
   Subcellular localization of RELA-HA, C11orf95-HA and RELA^FUS1-HA in
   DF-1 cells. c Subcellular localization of GFP and GFP-fusion proteins.
   DF-1 cells expressing the relevant RCAS virus were observed with
   fluorescent microscopy. Representative images were shown in b and c. d,
   f RCAS vectors expression in 293T/tv-a cells. e, g Relative Nanoluc
   reporter activity to the RELA^FUS1-RE-luciferase (MEME-2) normalized to
   the Firefly luciferase activity and GFP cells in the 293T/tv-a cells
   expressing the indicated RCAS virus as shown in the d or f. (e, n = 6;
   g, n = 5 or 9 in technical triplicate) Analysis was done using paired
   two-tailed t-test (e) or RM one-way ANOVA (g). *p < 0.05

Statistical analysis

   Statistical analyses in this study were performed using GraphPad Prism
   7, 8, or R software and described with the significance values and
   sample size in the respective figure legends, corresponding results
   sections, or methods section in detail.

Materials availability

   All cell lines, plasmids and other reagents generated in this study are
   available from the corresponding authors with a completed Materials
   Transfer Agreement if there is potential for commercial application.

Results

HA tag ChIP-seq analyses identified unique genomic binding sites of RELA^FUS1

   Accumulating evidence suggests that—in addition to known NF-κB
   targets—also non-NF-κB target genes contribute to the tumorigenesis of
   RELA^FUS [[149]31, [150]35, [151]41, [152]45]. Therefore, to identify
   direct transcriptional target genes of RELA^FUS and to clarify the
   mechanisms of how RELA^FUS causes tumor formation, we initially
   performed an HA-tag protein immunoprecipitation and sequencing (HA
   ChIP-seq) analysis on 293T/tv-a cells retrovirally infected with either
   RCAS-RELA^FUS1-HA or RELA^FUS1−S486E-HA (A serine-to-glutamine
   substitution at Ser-486 of RELA^FUS1 corresponding to Ser-276 in the
   Rel homology domain of RELA, which has been previously shown to
   severely impair the tumor-forming capacity of RELA^FUS1) (Additional
   file [153]2: Fig. S1A–D; Additional file [154]9: Table S1A) [[155]41].
   HA ChIP-seq analyses identified a large number of significant RELA^FUS1
   DNA-binding sites throughout the genome (Fig. [156]1a, b, Additional
   file [157]2: Fig. S1E; Additional file [158]9: Table S1B, C).
   Interestingly, RELA^FUS1 and RELA^FUS1−S486E presented an overall
   similar DNA-binding pattern (Fig. [159]1a, b). However, RELA^FUS1 peaks
   showed somewhat higher enrichment in intronic and intergenic regions
   but a lower enrichment in proximal promoter regions compared to
   RELA^FUS1−S486E (Fig. [160]1c, d), signifying an existence of DNA
   regulatory elements specific for RELA^FUS1 as previously described
   [[161]31].

Fig. 1.

   [162]Fig. 1
   [163]Open in a new tab

   HA tag ChIP-seq analyses identified unique genomic binding sites of
   RELA^FUS1. a, b Genome-wide RELA^FUS1-HA (a) and RELA^FUS1−S486E-HA (b)
   binding demonstrated by HA ChIP-seq analyses in 293T/tv-a cells.
   Heatmaps represent HA ChIP enrichments at the regions (TSS ± 10 kb)
   ranked by the sum of the enrichment values. Heatmaps for two biological
   replicates are shown individually. Input controls are also shown. c, d
   Bar graphs display the genomic distribution of the RELA^FUS1-HA (c) and
   RELA^FUS1−S486E-HA (d) peaks in 293T/tv-a cells. e Volcano plot
   illustrating differences in gene expression between human RELA^FUS
   positive and all negative ependymomas (DEGs: false discovery rate
   (FDR) < 0.05, n = 5507). Differences in Log2 fold change in gene
   expression values are plotted on the x-axis. Adjusted p values
   calculated using the Benjamin-Hochberg method are plotted on the
   y-axis. RELA^FUS1 target genes annotated within the TSS ± 10 kb are
   shown as large circles. LMX1B, C11orf95, RELA, CCND1 and L1CAM genes
   are indicated by the arrows. f Scatter plots showing the mRNA
   expression of direct RELA^FUS1 target genes in the differentially
   expressed genes (DEGs) (logFC) between human RELA^FUS positive and all
   negative ependymomas. Genes are grouped based on the distance of the
   RELA^FUS1 binding to location to the TSS of the gene
   (FDR < 0.05, ± 50 kb- ± 1 kb; n = 870, 769, 645, 501, 350, 261, 214 and
   179). The expression level of DEGs bound by RELA^FUS1 within the
   TSS ± 10 kb were compared to those bound by RELA^FUS1 at other
   distances as indicated in the x-axis. g, h Venn diagram showing the
   number of the overlapping between the RELA^FUS1 and RELA^FUS1−S486E
   peaks (g) and target genes (h) identified within the TSS ± 10 kb in
   293T/tv-a cells. i Scatter plots showing the mRNA expression of target
   genes differentially bound by RELA^FUS1 and/or RELA^FUS1−S486E in the
   DEGs (logFC) between human RELA^FUS positive and all negative
   ependymomas. The target genes of FUS1 (DBPs for RELA^FUS1:
   logFC^DBPs > 1, FDR < 0.05, n = 64), S486E (DBPs for RELA^FUS1−S486E:
   logFC^DBPs > 1, FDR < 0.05, n = 42) and common (No diffenrential peaks
   between RELA^FUS1 and RELA^FUS1−S486E: |logFC|^DBPs < 1, n = 404) are
   shown in the x-axis. DBPs: Differential binding peaks. Statistical
   differences were assessed with a Mann–Whitney U-test. *p < 0.05;
   **p < 0.01; ***p < 0.001; ****p < 0.0001. n.s.: not significant

   We then determined which transcriptional target genes of RELA^FUS1
   might be involved in RELA^FUS1-driven ependymoma formation. We used
   publicly available expression data of human ependymomas [[164]45] and
   detected significantly higher expression of RELA^FUS1 target genes
   (identified by our ChIP-seq analysis) in human RELA^FUS positive
   ependymomas compared to RELA^FUS negative ependymomas (Fig. [165]1e).
   Further, up-regulation of many RELA^FUS1 target genes was also observed
   in a second ependymoma dataset (Additional file [166]2: Fig. S1F)
   [[167]44]. We focused our subsequent analysis on genes bound by
   RELA^FUS1 at ± 10 kb of the transcription start sites (TSSs), since
   these genes were significantly up-regulated compared to genes bound
   at ±30, 40, or 50 kb of the TSSs (Fig. [168]1f). We observed
   significant peaks of both RELA^FUS1 and RELA^FUS1−S486E in these loci
   and identified 619 (in 887 peaks) and 446 (in 592 peaks) direct target
   genes of RELA^FUS1 and RELA^FUS1−S486E, respectively (Fig. [169]1g, h;
   Additional file [170]9: Table S1B–E). Interestingly, more than half of
   the RELA^FUS1−S486E target genes (64%; 287 out of 446 RELA^FUS1−S486E
   target genes) still overlapped with the RELA^FUS1 target genes (46%;
   287 out of 619 RELA^FUS1 target genes) (Fig. [171]1h). Notably, when
   comparing the expression of genes that were occupied by either only
   RELA^FUS1 or only RELA^FUS1−S486E (at TSS ± 10 kb) in human
   RELA^FUS positive versus negative ependymomas, we observed a
   significantly lower up-regulation of RELA^FUS1−S486E target genes
   (Fig. [172]1i), signifying that the transcriptional activity of the
   mutant form might be somewhat impaired, thus likely explaining the lack
   of the tumor-forming potential [[173]41]. These results suggest that
   RELA^FUS1 might function as a transcription factor and induce aberrant
   gene expression for ependymoma formation.

Most RELA^FUS1 target genes are actively transcribed

   To further characterize RELA^FUS1 target genes, we also investigated
   the transcriptional profile of RELA^FUS1 in mouse ependymoma (mEPN)
   cells derived from the RCAS-RELA^FUS1-HA-driven ependymoma (H1203
   cells) (Additional file [174]3: Fig. S2A; Additional file [175]9: Table
   S1A) [[176]41]. HA ChIP-seq analysis of the mEPN cells successfully
   identified a large number of the RELA^FUS1 binding sites throughout the
   entire genome (Fig. [177]2a; Additional file [178]10: Table S2A). We
   observed a higher frequency of RELA^FUS1 peaks in intronic and a lower
   frequency in proximal promoter regions in mEPN cells, compared to
   293T-RELA^FUS1 cells (Figs. [179]1c, [180]2b). We then examined the
   expression levels of RELA^FUS1 target genes in RNA-seq data of normal
   mouse brains, PDGFA-driven gliomas, and RELA^FUS1-driven ependymomas
   (Fig. [181]2c–f) [[182]41]. We observed that many RELA^FUS1 target
   genes were significantly up-regulated in RELA^FUS1-driven ependymomas
   compared to both normal brains (Fig. [183]2c, d) and PDGFA-driven
   gliomas (Fig. [184]2e, f). We again observed that RELA^FUS1 target
   genes bound within ± 10 kb of the TSS were significantly up-regulated
   (Fig. [185]2d, f) and thus focused our subsequent analysis on these
   genes. We identified 520 RELA^FUS1 target genes from 649 RELA^FUS1
   peaks in the mEPN cells (Additional file [186]9: Table S1E, Additional
   file [187]10: Table 2A). We observed that a significant portion of
   these genes was commonly up- or down-regulated in RELA^FUS1-driven
   mouse ependymomas compared to normal brains or PDGFA-driven gliomas,
   indicating the creation of a RELA^FUS1-specific transcriptional network
   (Fig. [188]2g, h). Of note, dysregulation of PDGF signaling in human
   and mouse RELA^FUS tumors has been previously shown [[189]41, [190]44,
   [191]45]. However, it is noteworthy that the RELA^FUS-specific
   transcriptional program was observed even when compared to the
   PDGF-driven mouse glioma samples in our analysis, thus implying that
   RELA^FUS likely induces tumor formation by co-activating several
   oncogenic pathways driven by the activation of several RELA^FUS target
   genes in addition to PDGF signaling.

Fig. 2.

   [192]Fig. 2
   [193]Open in a new tab

   Most RELA^FUS1 target genes are actively transcribed. a Genome-wide
   RELA^FUS1-HA binding or H3K27ac marks in mEPN (H1203) cells. The
   enrichment of H3K27ac within the RELA^FUS1-HA binding region is shown
   in heatmaps. b Bar graphs display the genomic distribution of the
   RELA^FUS1 peaks in H1203 cells. c, e Volcano plots illustrating
   differences in gene expression between RCAS-RELA^FUS1-driven
   ependymomas (FUS1) and mouse normal brains (NB) (c) or
   RCAS-PDGFA-driven glioblastomas (PDGFA) (e) (FDR < 0.05, n = 11,574
   (c), n = 8,059 (e)). Differences in Log2 fold change in gene expression
   values are plotted on the x-axis. Adjusted p values calculated using
   the Benjamin-Hochberg method are plotted on the y-axis. RELA^FUS1
   target genes annotated within the TSS ± 10 kb in H1203 cells are shown
   as large circles. 2700081O15Rik, Ccnd1 and Lmx1b genes are indicated by
   the arrows. d, f Scatter plots showing the mRNA expression of direct
   RELA^FUS1 target genes in the DEGs (logFC) between RELA^FUS1-driven
   ependymomas and normal brains (d) or PDGFA-driven glioblastomas (f).
   Genes are grouped based on the distance of the RELA^FUS1 binding to
   location to the TSS of the gene (FDR < 0.05, ± 50 kb- ± 1 kb;
   n = 1,083, 943, 826, 668, 511, 412, 342 and 295). The expression level
   of DEGs bound by RELA^FUS1 within the TSS ± 10 kb were compared to
   those bound by RELA^FUS1 at other distances as indicated in the x-axis.
   g, h Venn diagram showing the number of the overlapping of up- and
   down-regulated RELA^FUS1 target genes (within TSS ± 10 kb) in
   RELA^FUS1-driven ependymomas relative to normal brains or PDGFA-driven
   glioblastomas. i Venn diagram showing the number of the overlapping of
   H3K27ac peaks in H41 plus H1203 cells and RELA^FUS1 peaks in H1203
   cells. j Venn diagram showing the number of the overlapping of
   RELA^FUS1 peaks and super-enhancers (SEs) identified in H1203 cells. k
   Inflection plots representing SEs in H41 and H1203 cells.
   Representative genes are shown along with their rankings. l, m Venn
   diagrams showing the number of the overlapping of Rela peaks in MEFs
   after 3 h of TNF stimulation (see Methods) and RELA^FUS1 peaks in H1203
   cells (l) or H3K27ac peaks within the entire genome (m) in H41 and
   H1203 cells. n, o Venn diagrams showing the number of the overlapping
   of Rela target genes in MEFs and RELA^FUS1 target genes in H1203 (n) or
   293T (o) cells. Statistical tests are as described in Fig. [194]1

   Subsequently, to examine whether these RELA^FUS1 target genes were
   actively transcribed, we performed H3K27ac ChIP-seq (a histone mark of
   active chromatin) with two mEPN (H41 and H1203) cells and investigated
   actively transcribed regions, including promoters and enhancers
   (Fig. [195]2a, Additional file [196]3: Fig. S2A, B; Additional file
   [197]9: Table S1A, Additional file [198]10: Table S2B, C). We
   identified 36,859 peaks that were present in both mEPN cells
   (Fig. [199]2i, Additional file [200]3: Fig. S2C). Interestingly, most
   of the RELA^FUS1 peaks in the TSS ± 10 kb region overlapped with
   H3K27ac peaks (94%; 812 out of 867 RELA^FUS1 peaks, p = 9.4 × 10^–271)
   (Fig. [201]2i). Furthermore, 41% of RELA^FUS1 peaks overlapped with
   super-enhancers (SEs) identified by an exceptionally high degree of
   enrichment of H3K27ac peak (Fig. [202]2j, k, Additional file [203]3:
   Fig. S2D; Additional file [204]10: Table S2D–F) [[205]48, [206]65]. We
   noticed that some RELA^FUS1 peaks (TSS ± 10 kb) overlapped with SE
   regions that were annotated to well-known cancer-associated genes such
   as CCND1 (a representative NF-κB target gene) [[207]14, [208]15],
   PIK3R2 (proto-oncogene), DOT1L (histone modifier gene) in addition to
   RELA and 2700081O15Rik (mouse homolog of C11orf95) (Fig. [209]2k;
   Additional file [210]10: Table S2F). Further, we also found that
   enhancer- and SE-annotated genes identified in mouse ependymoma cells
   and human RELA^FUS tumors were significantly overlapping (Additional
   file [211]3: Fig. S2E, F), thus supporting a close association of our
   analysis with human ependymomas [[212]31].

   To further dissect the molecular mechanism of RELA^FUS-driven
   ependymoma formation, we examined the implication of RELA target genes
   in the RELA^FUS1 transcription network using publicly available Rela
   ChIP-seq data in murine embryonic fibroblasts (MEFs) after TNF
   stimulation [[213]37]. We found that approximately 22% of RELA^FUS1
   peaks in mEPN cells overlapped with Rela peaks in MEFs (Fig. [214]2l)
   as also supported by a significant overlap between the H3K27ac peaks in
   mEPN cells and Rela peaks in MEFs (Fig. [215]2m). More specifically,
   approximately 27 and 10% of RELA^FUS1 target genes in mEPN and
   293T-RELA^FUS1 cells overlapped with the Rela target genes in MEFs,
   respectively, indicating a critical implication of RELA target genes in
   RELA^FUS1-driven ependymoma formation (Fig. [216]2n, o). Interestingly,
   27% of the previously reported-NF-κB target genes (n = 366) were
   present in the Rela-target genes (Additional file [217]3: Fig. S2G)
   [[218]12, [219]41]. By contrast, only 1.1 and 4.4% of these NF-κB
   target genes were identified in the RELA^FUS1 target genes in
   293T-RELA^FUS1 and H1203 cells, respectively, highlighting the
   importance of yet unknown- or non-NF-κB target genes in the
   RELA^FUS-driven ependymoma formation (Additional file [220]3: Fig. S2H,
   I).

   Recent single-cell RNA sequencing analyses of ependymomas identified
   diverse neoplastic subpopulations characterized by specific
   transcriptomic signatures [[221]11, [222]13]. We also examined an
   association between the RELA^FUS1 target genes and the single-cell
   transcriptomic signature genes of ST-ependymomas [[223]13] and observed
   a significant overlap between “ST-RELA-variable” signature genes and
   RELA^FUS1 target genes in both 293T-RELA^FUS1 and mEPN cells, likely
   implying an important role of this subpopulation on tumorigenesis
   (Additional file [224]10: Table S2G). RELA^FUS1 target genes from
   293T-RELA^FUS1 and mEPN cells were also found in “ST-Radial-Glia-like”
   and ‘ST-Metabolic’ signature genes, respectively. Taken together, these
   results suggest that most RELA^FUS1 target genes were actively
   transcribed, thereby driving specific oncogenic pathways necessary for
   ependymoma formation in significant collaboration with the RELA/NF-κB
   pathway.

RELA^FUS1 binds on specific DNA regions through the unique DNA-binding motif

   High NF-κB activity mediated by the transcriptional activity of
   RELA^FUS1 is thought to play a critical role in the RELA^FUS1-driven
   ependymoma formation [[225]41, [226]45]. Although some overlap between
   RELA^FUS1 and Rela target genes were observed, non-Rela target genes
   were more predominantly identified among the RELA^FUS1 target genes
   (Fig. [227]2n, o), implying the creation of a unique DNA-binding motif
   of RELA^FUS1, which is independent of RELA/NF-κB regulation. Of note,
   the RELA/NF-κB dimer can be associated with many non-NF-κB consensus
   sequences [[228]32, [229]67]. We thus used the Multiple Em for Motif
   Elicitation (MEME) Suite to explore what transcription factor (TF)
   binding motifs are enriched in RELA^FUS1 and RELA^FUS1−S486E ChIP-seq
   peaks in 293T/tv-a cells (Fig. [230]1a–d) [[231]1], and identified
   unique DNA-binding motifs, some of which were shared between them
   (Fig. [232]3a and Additional file [233]4: Fig. S3A). Interestingly, the
   canonical NF-κB consensus motif, termed as κB site (5′-GGGRNWYYCC-3′,
   R = A or G, N = any base, W = A or T, and Y = C or T) was not present
   among the top 10 motifs in either RELA^FUS1 or RELA^FUS1−S486E peaks
   (Additional file [234]11: Table S3A) [[235]5, [236]24, [237]36]. In
   turn, when applying the TF motif analysis to Rela Peaks in MEFs
   [[238]37], the NF-κB-like motif (5′-KGGAAADYCCM-3′, K = G or T, D = A
   or G or T, M = A or C) was identified in only 17.4% of the Rela target
   genes as the most enriched motif, thus confirming the presence of Rela
   binding on non-NF-κB consensus sequence (Additional file [239]4: Fig.
   S3B). The RELA^FUS1 motifs or any related motifs were not present among
   the top 5 motifs in Rela peaks (Additional file [240]11: Table S3B).

   To systematically examine whether RELA^FUS1 activates the gene
   expression via DNA-binding motifs identified by ChIP-seq, we generated
   luciferase reporter constructs for three RELA^FUS1 motifs ranked at the
   top 3 and tested them in 293T/tv-a cells (Fig. [241]3b–e, Additional
   file [242]4: Fig. S3C-F; Additional file [243]11: Table S3A). We found
   that RELA^FUS1 responded to the RELA^FUS1-MEME-2 motif
   (5′-BGKGGCCCCBG-3′, B = C or G or T) but not to MEME-1 and 3
   (Fig. [244]3c–e; Additional file [245]11: Table S3C). Further,
   RELA^FUS1 also responded to both the triple tandem of the MEME-2
   sequence ranked at 1st to 3rd in the de novo motif discovery analysis
   and the single MEME-2 sequence ranked within the top 4 when transiently
   introduced the RELA^FUS1 in 293T cells (Fig. 3g, h, Additional file
   [246]4: Fig. S3G; Additional file [247]11: Table S3C). Interestingly,
   RELA^FUS1−S486E also evidently reacted to the MEME-2 motif to a
   somewhat lower degree compared to the RELA^FUS1, whereas wild-type RELA
   barely responded to the MEME-2 motif (Fig. [248]3d). Of note, RELA^FUS1
   and RELA^FUS1−S486E proteins were less expressed than RELA in the
   293T/tv-a cells (Fig. [249]3b), thus emphasizing their actual
   activities to the MEME-2 motif. Further, when applying the TF motif
   analysis to mEPN cells (Fig. [250]2a, b), a very similar GC-rich motif
   (5′-CNGGGGCCACR-3′) to the 293T-RELA^FUS1-MEME-2 motif was identified
   as the top-ranked motif (Fig. [251]3f; Additional file [252]11: Table
   S3D, E). These GC-rich motifs were present in 44.2 (123 out of 278
   peaks) and 43.5 (194 out of 446 peaks) percent of all RELA^FUS1 peaks
   in 293T-RELA^FUS1 and mEPN cells, respectively. Again, no enrichment
   for the canonical NF-κB consensus motif was detected in the top 10
   motifs in mEPN cells (Additional file [253]11: Table S3D).

   To subsequently test if RELA^FUS1 is able to directly activate the
   expression of NF-κB target genes via binding to the NF-κB consensus
   motif, we used a reporter construct containing the NF-κB responsive
   element (5 × κB sequence). As expected, expression of wild-type RELA
   strongly activated the reporter system and induced mRNA and protein
   expression of NFKBIA, a representative NF-κB target gene
   (Fig. [254]3i–l), whereas expression of RELA^FUS1 only minimally
   activated the system (Fig. [255]3i). Nevertheless, forced-expression of
   RELA^FUS1 steadily induced mRNA and protein expression of NFKBIA in a
   dose-dependent manner, signifying that RELA^FUS1 is competent to
   regulate the NF-κB pathway via the consensus sequence (Fig. [256]3j–l,
   Additional file [257]4: Fig. S3H) [[258]58]. In turn, RELA^FUS1−S486E
   did not activate the reporter assay (Fig. [259]3i). These results
   suggested that RELA^FUS1 might form its unique transcription network
   through the GC-rich MEME-2 motif in collaboration primarily with a yet
   unknown-NF-κB motif, rather than the consensus NF-κB motif.

C11orf95 determines the RELA^FUS1-binding region through the unique GC-rich
motif

   The minimal response of RELA to the RELA^FUS1-MEME-2 motif leads to the
   suggestion that the C11orf95 domain rather than RELA might be a
   critical determinant for the DNA binding of RELA^FUS1 proteins to the
   MEME-2 motif (Fig. [260]3d). RELA^FUS1-HA proteins preferentially
   localized in the nucleus compared to RELA-HA proteins (Fig. [261]4a, b)
   [[262]45]. So far, C11orf95 protein function has not been described
   well. However, as predicted by multiple C2H2 type zinc finger domains
   and the putative nuclear localization signal (NLS), the C11orf95
   proteins predominantly accumulate in the nucleus (Fig. [263]4a, b)
   [[264]22, [265]45]. Interestingly, the C11orf95 portion (C11ΔC) of the
   RELA^FUS1 was sufficient for the nuclear localization despite the fact
   that the putative NLS of C11orf95 is lost, implying the presence of an
   additional NLS (Fig. [266]4a, c). We thus hypothesized that the C11ΔC
   portion preserving one zinc finger domain might contribute to nuclear
   localization and DNA-binding through the unique binding motif of
   RELA^FUS1 proteins, thereby regulating the transcriptional activity of
   the target genes by the RELA’s activation domain in the RELA^FUS1
   protein. To reveal the molecular mechanism by which RELA^FUS1 regulates
   the expression of specific target genes with the MEME-2 motif, we
   generated several C11orf95 mutants and analyzed their ability to
   activate the 5xMEME-2 luciferase reporter assay (Fig. [267]4a). The
   C11ΔC-NLS (CN-HA) and C11ΔC-NLS-GFP fusion (CNG-HA) were unable to
   activate the MEME-2 reporter, likely due to the absence of a functional
   activation domain (Fig. [268]4d, e). In turn, a construct that replaced
   the RELA portion of RELA^FUS1 with SV40NLS-VP64, in which VP64 is a
   tetrameric repeat of herpes simplex VP16 minimal activation domain
   (C11ΔC-NLS-VP64; CNVP-HA), evidently activated the MEME-2 reporter
   (Fig. [269]4a, d, e) [[270]2, [271]47]. In addition, we tested the
   ability of RELA^FUS8 (Type 8)—a naturally occurring variant of
   RELA^FUS, lacking most of the Rel homology domain (RHD)—as well as
   CRHD-HA, a RELA^FUS1 mutant lacking the activation domains in the RELA
   portion (Fig. [272]4a, f) [[273]9, [274]36]. RELA^FUS8 strongly
   activated the MEME-2 reporter at levels comparable to RELA^FUS1
   (Fig. [275]4g). In turn, deletion of the RELA activation domain
   (CRHD-HA) resulted in the inability to activate the MEME-2 reporter
   (Fig. [276]4g). Of note, the RELA^FUS8 failed to induce brain tumor
   formation in mice [[277]60], thus signifying that the RHD was
   indispensable for the tumor-forming potential of RELA^FUS1, whereas
   might be dispensable for the transcriptional activity via the MEME-2
   motif. Taken together, these observations suggested that the C11ΔC
   portion primarily determined the DNA binding loci of RELA^FUS1 on the
   MEME-2 motif. However, the cooperation of both the C11ΔC and RELA
   portion are essential for the regulation of the transcriptional target
   genes of RELA^FUS1.

RELA^FUS1 transcriptionally regulates the target gene expression through DNA
binding on the RELA^FUS1-MEME-2 sequence

   In general, transcription factors regulate the expression of target
   genes in a context-dependent manner [[278]24]. In fact, L1 cell
   adhesion molecule (L1CAM), a well-known downstream marker of RELA^FUS
   was identified as a RELA^FUS1 target gene in 293T-RELA^FUS1 but not in
   mEPN cells (Additional file [279]5: Fig. S4A–D) [[280]45]. Therefore,
   we examined the overlap between RELA^FUS1 ChIP-seq peaks in
   293T-RELA^FUS1 and mEPN cells (Fig. [281]1a, [282]2a). Unexpectedly, we
   found that only 41 genes were shared between these cells, implying a
   flexible DNA-binding capacity of RELA^FUS as a TF function, depending
   on the cellular context (Fig. [283]5a; Additional file [284]9: Table
   S1E). However, it is noteworthy that CCND1, H-Ras, PIK3R2, and DOT1L in
   addition to C11orf95 were identified in the common target genes. 24 out
   of the 41 shared genes were located within SE regions, presumably
   serving as core target genes responsible for RELA^FUS1-driven
   ependymoma formation (Fig. [285]2k, [286]5a and Additional file [287]5:
   Fig. S4E–G).

   The fact that C11orf95 was a RELA^FUS1 target in both cell types
   provides significant insights into the understanding of the oncogenic
   mechanism of RELA^FUS1. Prominent peaks of RELA^FUS1 binding were
   detected around the TSS within both the C11orf95 and 2700081O15Rik loci
   in the HA ChIP-seq analyses (Fig. [288]5b, c). Interestingly, multiple
   MEME-2 sites were concomitantly found in the RELA^FUS1 peaks, thus
   strongly suggesting a direct transcriptional regulation of C11orf95
   gene expression by RELA^FUS1 (Fig. [289]5b, c). Indeed, human RELA^FUS
   positive ependymomas exhibited significantly higher C11orf95 mRNA
   expression than negative ones (Fig. [290]1e, [291]5d and Additional
   file [292]2: Fig. S1F). Further, as bolstered by the overlapping of
   H3K27ac peaks with the RELA^FUS1 peaks in the 2700081O15Rik gene
   (Fig. [293]5c), 2700081O15Rik was remarkably up-regulated in
   RELA^FUS1-driven ependymomas relative to normal brains and PDGFA-driven
   gliomas (Fig. [294]5e, f). Forced-expression of RELA^FUS1 in 293T cells
   was able to induce C11orf95 mRNA expression in a dose-dependent manner
   (Fig. [295]3j, [296]5g). Further, to confirm if RELA^FUS1 had a direct
   impact on the C11orf95 expression, we cloned an immediate upstream
   sequence of the C11orf95 gene, including three MEME-2 motifs into a
   luciferase reporter vector (Additional file [297]4: Fig. S3C,
   Additional file [298]6: Fig. S5A). We found that the expression of
   RELA^FUS1 strongly activated this reporter, suggesting that RELA^FUS1
   is able to up-regulate its own expression directly (Fig. [299]5h).
   Since RELA^FUS is caused by the genomic rearrangement involving
   C11orf95 and RELA loci [[300]45], the expression of the RELA^FUS gene
   is thought to be controlled by the C11orf95 promoter, thus possibly
   resulting in the formation of an autoregulatory feedback loop in the
   tumors (Additional file [301]6: Fig. S5B). Of note, 2700081O15Rik was
   found to be a direct Rela target gene, as shown by a Rela peak with
   multiple κB sites in the gene locus, which are different from MEME-2
   sites (Fig. [302]5c; Additional file [303]9: Table S1E). However, the
   lack of RELA^FUS1 binding to the κB sites suggests a specific
   2700081O15Rik gene regulation by RELA^FUS independent of Rela.

   The distribution of RELA^FUS1 binding loci in our ChIP-seq analyses
   suggests that RELA^FUS1 might also control the gene expression via an
   intronic enhancer regulatory region. Thus, to further investigate the
   TF function of RELA^FUS1, we focused on the LMX1B gene, a
   brain-developmental transcription factor, which was one of the common
   RELA^FUS1 target genes in 293T-RELA^FUS1 and mouse ependymoma cells and
   was also selected as one of the enhancer-associated genes specific for
   human RELA^FUS tumors and mouse ependymoma cells (Fig. [304]2k,
   [305]5a) [[306]31]. RELA^FUS1 bound similar positions on the LMX1B gene
   locus in 293T and mouse ependymoma cells. Two RELA^FUS1 peaks
   containing the MEME-2 motif were found in the putative promoter region
   (upstream of the TSS) and second long intron of both human and mouse
   genes (Fig. [307]5i, j). The H3K27ac peaks were also concomitantly
   identified in these loci in mEPN cells, suggesting the direct gene
   regulation by RELA^FUS1 (Fig. [308]5j). As expected, a significant
   upregulation of LMX1B was observed in both human and mouse RELA^FUS
   tumors (Fig. [309]1e, [310]5d–f). Further, forced-expression of
   RELA^FUS1 in 293T cells was able to induce LMX1B expression in a
   dose-dependent manner (Fig. [311]3j, [312]5g). Interestingly,
   GeneHancer profiling indicated an association between the promoter and
   enhancer regulatory elements in the second intron of the human LMX1B
   gene (Additional file [313]6: Fig. S5C) [[314]8]. Therefore, to dissect
   a mechanism of LMX1B gene regulation by RELA^FUS1, we examined if
   physical perturbation of RELA^FUS1 binding on these loci affected the
   gene expression using the CRISPR-dCas9 system [[315]49]. We designed
   multiple sgRNAs to target the CRISPR-dCAS9-sgRNA complexes in two
   regions around the MEME-2 motif denoted as Region-1 and -2 (R1 and R2),
   and then lentivirally introduced them in mEPN cells (Fig. [316]5j,
   Additional file [317]6: Fig. S5D-F). Targeting R1 (intronic region) but
   not R2 (promoter region) resulted in significant downregulation of
   Lmx1b gene expression (Fig. [318]5k, Additional file [319]6: Fig. S5G).
   Collectively, these findings appear to represent prototypic examples
   for epigenetic gene regulation of RELA^FUS1, thus confirming the
   oncogenic TF function of RELA^FUS1 on the MEME-2 sequence.

Anti-cancer drug screening highlights oncogenic signaling driven by RELA^FUS1
target genes

   We finally used Gene Ontology (GO) analysis based on our ChIP-seq
   experiments to explore the signaling network directly regulated by
   RELA^FUS1 target genes. As expected by the small number of RELA^FUS1
   target genes shared between 293T-RELA^FUS1 and mEPN cells, diverse
   signaling networks were enriched in these cells with a little overlap
   between both cell types (Figs. [320]5a, [321]6a, b) [[322]3]. Target
   genes of the 293T-RELA^FUS1 were notably involved in the MAPK signaling
   pathway, signaling pathways regulating pluripotency of stem cells, VEGF
   signaling, and Regulation of PTEN gene transcription (Fig. [323]6a;
   Additional file [324]12: Table S4A). On the other hand, GO terms
   enriched in mEPN cells converged to Glioma, PI3K-Akt signaling pathway,
   Signaling by PDGF, VEGF signaling pathway, RNA Polymerase II
   Transcription, Protein processing in the endoplasmic reticulum, and
   non-integrin membrane-ECM interactions (Fig. [325]6b; Additional file
   [326]12: Table S4B). Target genes of 293T-RELA^FUS1−S483E presented
   somewhat different signaling pathways from those of 293T-RELA^FUS1 as
   suggested by the number of target genes shared between these cells,
   presumably due to an impairment of DNA and/or protein binding due to
   the mutation in the RHD (Figs. [327]1g, h, [328]6a, Additional file
   [329]7: Fig. S6A, Additional file [330]12: Table S4A, C).
   Interestingly, when focusing on common pathways between the
   293T-RELA^FUS1 and mEPN cells, we identified an enrichment of
   ‘Signaling by Receptor Tyrosine Kinases (RTKs)’, particularly VEGF
   signaling (Fig. [331]6c). Association of aberrant RTK activity, such as
   EFN, PDGF, and FGFR signaling, with ependymomagenesis was previously
   reported [[332]19, [333]31, [334]41, [335]44] and thereby implicated as
   potential therapeutic targets for this tumor type (Additional file
   [336]7: Fig. S6B, C, Additional file [337]12: Table S4D-G). Further,
   PDGF signaling was found to be a direct transcriptional target of
   RELA^FUS1 in mEPN cells (Additional file [338]7: Fig. S6D), consistent
   with previous observations that PDGF signaling was up-regulated in
   human and mouse RELA^FUS1 tumors [[339]41, [340]44, [341]45].
   Therefore, to examine if blockade of these signaling pathways had a
   significant inhibitory effect on the tumor growth, we performed an
   anti-cancer drug screening using the FDA-approved drug library with
   additional selected-NF-κB inhibitors in two mEPN cells (Additional file
   [342]3: Fig. S2A; Additional file [343]13: Table S5A) [[344]40]. We
   treated the cells with these drugs and focused on drugs presented over
   85% growth inhibition (Fig. [345]6d, e, Additional file [346]8: Fig.
   S6E; Additional file [347]13: Table S5B). As expected, multi-tyrosine
   kinase inhibitors such as Sorafenib (targeting VEGFR, PDGFR and RAF)
   and Ponatinib (targeting BCR-ABL, Src, VEGFR, FGFR, and PDGFR) were
   able to effectively inhibit the growth of these cells (Fig. [348]6e;
   Additional file [349]13: Table S5B) [[350]38, [351]66]. Interestingly,
   in addition to an IκB kinase inhibitor (IKK-16), HDAC inhibitors
   (Belinostat, Romidepsin, Vorinostat) and a Proteasome inhibitor
   (Bortezomib), both of which were known to block NF-κB signaling were
   able to effectively inhibit the growth of mEPN cells, likely supporting
   the contribution of NF-κB activity in RELA^FUS1 ependymomas [[352]26,
   [353]33, [354]62]. We then determined the half-maximal inhibitory
   concentration values (IC50) of these drugs and selected six
   representatives from these drug categories. The IC50 values of these
   drugs were very similar between both mEPN cells, indicating the high
   specificity of these inhibitors to RELA^FUS1 (Fig. [355]6f, g). These
   results suggest that inhibitors against NF-κB and RTK signaling (most
   notably drugs targeting VEGFR and PDGFR) could be promising therapeutic
   agents for RELA^FUS tumors.

Fig. 6.

   [356]Fig. 6
   [357]Open in a new tab

   Anti-cancer drug screening highlights oncogenic signaling driven by
   RELA^FUS1 target genes. a, b Pathway enrichment analysis for RELA^FUS1
   target genes in 293T-RELA^FUS1 (a) and H1203-mEPN (b) cells. Color
   nodes and the size represent the enriched gene set and the number of
   genes in each gene set, respectively. c Venn diagram showing the number
   of the overlapping of pathways involved with the 293T-RELA^FUS1,
   293T-RELA^FUS1−S486E, and H1203-RELA^FUS1 target genes. Common GO term
   of dysregulated pathways driven by RELA^FUS1 target genes within the
   TSS ± 10 kb in 293T-RELA^FUS1 and H1203 cells is shown in the right
   panel. d Correlation of cell viability in two mEPN cells (H41 and
   H1203). Cells were treated with 179 anti-cancer drugs with 10 μM for
   72 h in two technical replicates. The mean values of two independent
   experiments for each cells are shown in x and y axis, respectively.
   Analysis was done using two-tailed Pearson's correlation. e List of
   twenty drugs presented over 85% growth inhibition relative to the 0.1%
   DMSO-treated control. The mean values of cell viability in H41 and
   H1203 cells are shown in the figure. f, g Cell viability of mEPN cells
   (f H41 and g H1203 cells) treated with six selected drugs. The IC50
   values of each anti-cancer drug were shown in the figures

Discussion

   In this study, we performed ChIP-seq experiments to explore target
   genes that are directly regulated by RELA^FUS1 and unveiled the unique
   epigenetic regulation of RELA^FUS in ependymoma formation (Additional
   file [358]8: Fig. S6F). Activation of the NF-κB pathway has been so far
   well-documented in human and mouse RELA^FUS ependymomas [[359]41,
   [360]45]. However, it remained to be determined how this pathway
   contributes to tumorigenesis and if it might serve as an actual
   therapeutic target in patients. Our findings suggest that RELA^FUS1
   might induce brain tumor formation through two main oncogenic pathways
   regulated by C11orf95 and RELA target genes (Additional file [361]8:
   Fig. S6F). The NF-κB pathway driven by the RELA portion was essential
   for tumorigenesis, although unknown-RELA/NF-κB target pathways were
   likely more common than canonical pathways via the κB site. Thus,
   blockade of active NF-κB pathways will likely be one option for
   RELA^FUS positive ependymoma therapy.

   RELA regulates NF-κB target genes by forming homo- or heterodimers, and
   the selectivity of the NF-κB response is variable according to the
   dimerization partner [[362]23, [363]57]. Of note, the RELA/NF-κB dimer
   can interact with both NF-κB consensus motif and many non-consensus
   sequences [[364]32, [365]67]. It is not known whether RELA^FUS forms
   dimers or if the dimerization is necessary for tumorigenesis. However,
   our reporter assays with the MEME-2 and NF-κB motifs and the absence of
   the κB site in the DNA sequences bound by RELA^FUS1 indicate that DNA
   binding of RELA^FUS1 might have deviated from that of RELA, thus
   suggesting unusual dimerization of RELA^FUS1.

   The RELA^FUS1−S486E mutant remarkably responded to the MEME-2 motif but
   completely failed to recognize the κB site. Phosphorylation of S276 in
   the RHD by PKAc induces the conformation change of RELA and subsequent
   recruitment of p300/CBP, thereby resulting in promoting the
   transcriptional activity [[366]71]. Thus, severe impairment of the
   oncogenicity of the mutant might be explained by the inability to exert
   a precise conformation change, consequently losing the capacity to
   activate the NF-κB pathway. Similarly, the RELA^FUS8 variant, which is
   lacking the RHD, was capable of recognizing the MEME-2 motif and
   displayed prominent transcriptional activity but failed to induce brain
   tumors [[367]60]. These observations support that both RELA/NF-κB
   activity and binding to the MEME-2 motif on its own is insufficient but
   essential for the tumor-forming potential of RELA^FUS1.

   We observed RELA^FUS1 binding peaks concomitant with MEME-2 sequences,
   which is recognized by the C11orf95 portion of RELA^FUS1, within the
   regulatory regions of RELA^FUS1 target genes. The RELA subunit
   preferentially binds to a DNA sequence consisting of a series of G
   bases at the 5′ position followed by a central A/T base for fine
   transcriptional regulation [[368]10, [369]36, [370]63]. Therefore, the
   absence of the central A/T bases, characteristic for the consensus
   NF-κB motif sequence, in the MEME-2 motif strongly suggests that
   transcriptional regulation of these C11orf95 target genes by RELA^FUS1
   was independent of gene regulation via the consensus NF-κB motif
   [[371]63]. Interestingly, we have recently shown that RELA^FUS variants
   with one C2H2 type zinc finger domain in the C11orf95 portion
   (RELA^FUS1 and RELA^FUS4) presented a more aggressive phenotype
   compared to those with two zinc finger domains (RELA^FUS2 and
   RELA^FUS3), supporting the importance of the RELA^FUS function via the
   C2H2 type zinc finger domain [[372]60]. Taken together, these
   observations suggested that two independent programs driven by C11orf95
   and RELA target genes are likely central players in the tumorigenic
   functions of RELA^FUS.

   Our anti-cancer drug screening highlighted several compounds targeting
   signaling pathways associated with the oncogenic mechanisms of
   RELA^FUS1, such as RTK, HDAC and NF-κB inhibitors including a
   proteasome inhibitor. Interestingly, Actinomycin D, previously
   identified as a potential drug for RELA^FUS tumors, was also selected
   in the top-ranked drugs, thus supporting a specificity of our screening
   to the RELA^FUS [[373]61]. Further, a Phase II Clinical Trial of
   Marizomib, a second-generation irreversible proteasome inhibitor is
   currently ongoing for “Recurrent Low-Grade and Anaplastic
   Supratentorial, Infratentorial and Spinal Cord Ependymoma”
   ([374]NCT03727841). The therapeutic effect is predicted especially
   against RELA^FUS tumors. However, it is of note that such a screening
   is generally biased toward identifying cytotoxic agents, as also
   demonstrated by our screening. Thus, a more careful selection would be
   essential for precisely evaluating the specificity of compounds.

   Our understanding of the molecular mechanisms underlying the
   RELA^FUS-driven ependymoma formation is increasingly deepened by recent
   key reports [[375]31, [376]72]. In this study, we not only successfully
   reproduced many of the data shown in the previous studies but also
   presented a more detailed functional analysis of RELA^FUS in several
   aspects [[377]31, [378]72]. Our analyses similarly identified L1CAM,
   IGF2, C11orf95, DOT1L, CCND1 and RELA genes as direct RELA^FUS targets
   that contain the specific RELA^FUS binding motif. Further, we
   experimentally demonstrated the transcriptional regulation of target
   genes by RELA^FUS and highlighted a potential autoregulatory feedback
   loop by RELA^FUS of itself in these tumors. Interestingly, several
   RELA^FUS target genes such as L1CAM and IGF2 were not necessarily
   common in 293T and mouse ependymoma cells in our analyses, indicating
   the fact that the transcriptional programs activated by RELA^FUS are
   context-dependent. Thus, our study emphasizes the significance to
   examine TF functions in various experimental settings. In contrast to
   gene fusions involving protein kinases, such as the BCR-ABL fusion in
   chronic myeloid leukemia [[379]21], therapeutic targets for cancers
   harboring gene fusion involving transcription factors are more elusive
   owed to their complex functions, as shown in this study. Therefore, our
   experimental approach integrating with ChIP-seq, RNA-seq, functional
   studies, and drug screenings will be helpful for not only a better
   understanding of ependymoma biology but also the identification of the
   precise therapeutic targets. Ependymomas are still lethal brain tumors,
   and hopefully, the results of this study will greatly contribute to the
   advancement of ependymoma research.

Conclusions

   So far, the contribution of the C11orf95 moiety to RELA^FUS-driven
   ependymoma formation has been considerably underestimated. Our study
   revealed that the C11orf95 moiety was a key determinator for the
   nuclear localization and DNA binding of RELA^FUS, consequently forming
   the complex oncogenic signaling networks in significant collaboration
   with the RELA targets. These findings will provide therapeutic insights
   for patients with RELA^FUS positive ependymoma.

Supplementary Information

   [380]Additional file1.^ (141.6KB, docx)
   [381]Additional file2 (JPG 497 KB)^ (497.2KB, jpg)
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   [391]Additional file12.^ (546KB, xlsx)
   [392]Additional file13.^ (27KB, xlsx)
   [393]Additional file14.^ (15.8KB, xlsx)

Acknowledgements