RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis

Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 1 of 16 CANCER RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis Sara Sánchez-Molina1*, Elisabet Figuerola-Bou1, Enrique Blanco2,3, María Sánchez-Jiménez1, Pablo Táboas1, Soledad Gómez1, Cecilia Ballaré2,3, Daniel J. García-Domínguez4, Estela Prada1, Lourdes Hontecillas-Prieto4, Ángel M. Carcaboso1, Óscar M. Tirado5, Inmaculada Hernández-Muñoz1,6, Enrique de Álava4,7, Cinzia Lavarino1,8, Luciano Di Croce2,3,9*†, Jaume Mora1,8*† Ewing sarcoma (EwS) is an aggressive tumor that affects adolescents and young adults. EwS is defined by a chromosomal translocation, EWSR1-FLI1 being the most common, that causes genome reprogramming through remodeling of enhancers. Here, we describe an unexpected function of RING1B, which is highly expressed in EwS. While retaining its repressive activity at Polycomb developmental regulated genes, RING1B colocalizes with EWSR1-FLI1 at active enhancers. We demonstrate that RING1B is necessary for the expression of key EWSR1-FLI1 targets by facilitating oncogene recruitment to their enhancers. Knockdown of RING1B impairs growth of tumor xenografts and expression of genes regulated by EWSR1-FLI1 bound enhancers. Pharmacological inhibition of AURKB with AZD1152 increases H2Aub levels causing down-regulation of RING1B/EWSR1-FLI1 common targets. Our findings demonstrate that RING1B is a critical modulator of EWSR1- FLI1–induced chromatin remodeling, and its inhibition is a potential therapeutic strategy for the treatment of these tumors. INTRODUCTION Ewing sarcoma (EwS) is an aggressive, poorly differentiated, human tumor characterized by a chromosomal translocation involving a member of the FET family of genes (FUS, EWSR1 and TAF15) and a member of the ETS family of transcription factors, with the EWSR1-FLI1 gene fusion the most common one (1). EwS genomes present low mutation rates with FET-ETS rearrangements as the dominant genetic aberration in the majority of tumors (2). Notably, the cell of origin of EwS is still a controversial field, although human mesenchymal stem cells (hMSCs) and human neural crest stem cells are the most accepted (3–5). The EWSR1-FLI1 fusion protein, which contains the transcriptional activation and RNA binding domains of EWSR1 and the DNA binding domain of FLI1, is the main driver of tumorigenesis (3,6). The resulting fusion oncoprotein has the ability to act as an aberrant transcription factor, leading to gene activation and repression for a well-described set of genes (3,7). A decade ago, EWSR1-FLI1 was found to bind preferentially to DNA sites containing GGAA microsatellite repeats (8,9). Recent studies have reported that binding of EWSR1-FLI1 multimers to GGAA repeats acts as a pioneer factor and induces the formation of de novo active enhancers by recruiting the acetyl transferases CBP/p300, E2F3, and the BRG1/ BRM-associated factor chromatin remodeling complex (10–12). On the other hand, it was hypothesized that monomeric EWSR1-FLI1 inhibits transcription at enhancers by displacing endogenous ETS transcription factors from GGAA motifs (10). Therefore, the mechanisms by which EWSR1-FLI1 acts as either a gene activator or repressor depend on both DNA sequence and cofactors. Several proteins from the Polycomb group (PcG) have previously been implicated in EwS tumorigenesis. PcG was first described in Drosophila melanogaster as a key regulator of Hox genes expression. PcG proteins not only prevent differentiation by repressing lineagespecific genes but also mark bivalent chromatin regions for subsequent activation. EZH2 (the enzymatic subunit of PRC2) methylates histone H3 at lysine 27 (H3K27me3), while RING1B (the enzymatic subunit of PRC1) ubiquitinates H2A at lysine 119 (H2Aub), both considered repressive histone marks (13). The canonical PRC1 complex (defined by the presence of four subunits, comprising one variant each of PCGF, PHC, CBX, and RING1) has mostly been associated with maintaining gene repression. However, increasing evidence indicates that PRC1 complexes containing RING1B have the potential for transcription activation, via their catalytic-independent association with UTX, an H3K27me3 demethylase, and p300 acetyltransferase (14,15). With respect to EwS, it was recently shown that EZH2 blocks endothelial and neuroectodermal differentiation (16), BMI1 promotes tumorigenicity (17), and RING1B represses the nuclear factor B pathway (18). The molecular mechanisms behind the contribution of PcG to EwS have not been addressed. Notably, the GGAA repeats are significantly decorated with H3K27me3in H1 human embryonic cell lines and human umbilical 1Developmental Tumor Biology Laboratory, Institut de Recerca Sant Joan de Déu, Esplugues de Llobregat, 08950 Barcelona, Spain. 2Center for Genomic Regulation (CRG), Barcelona Institute of Science and Technology, 08003 Barcelona, Spain. 3Universitat Pompeu Fabra (UPF), 08002 Barcelona, Spain. 4Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocio/CSIC/ Universidad de Sevilla-CIBERONC, Department of Pathology, 41013 Seville, Spain. 5Sarcoma Research Group, Laboratori d’Oncologia Molecular, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL)-CIBERONC, L’Hospitalet de Llobregat, 08908 Barcelona, Spain. 6Fundació Institut Hospital del Mar d'Investigacions Mèdiques (FIMIM), 08003 Barcelona, Spain. 7Department of Normal and Pathological Cytology and Histology, School of Medicine, University of Seville, 41009 Seville, Spain. 8Pediatric Cancer Center Barcelona (PCCB), Hospital Sant Joan de Déu, Esplugues de Llobregat, 08950 Barcelona, Spain. 9Institucio Catalana de Recerca i Estudis Avançats (ICREA), Pg Lluis Companys 23, 08010 Barcelona, Spain. *Corresponding author. Email: [email protected] (S.S.-M.); [email protected] (L.D.C.); [email protected] (J.M.) †These authors contributed equally to this work as co-senior authors. Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 2 of 16 vein endothelial cells (HUVECs) (19). This is in stark contrast with the lack of H3K27me3 mark at EWSR1-FLI1 binding sites in EwS cells (10,11), thus suggesting a different role of PcG in EwS. Last, comparison between malignant and nonmalignant tissues revealed a misregulation of PcG target genes in EwS (20). Together, these findings suggest a potential role of the PcG during the early steps of EwS pathogenesis. Here, we report that RING1B and EWSR1-FLI1 interact and colocalize at the same genomic loci. Notably, we find that RING1B is present at promoters and enhancers of actively transcribed EWSR1-FLI1 target genes. Furthermore, we demonstrate that modulation of RING1B interferes with EWSR1-FLI1 recruitment and with the expression of EWSR1- FLI1 targets, thus unveiling an interdependent cooperation between both proteins. RESULTS EWSR1-FLI1 targets PcG-repressed regions in hMSC Human pediatric MSCs (hpMSCs) have been proposed as a plausible cell of origin for EwS (21). Nevertheless primary human endothelial HUVECs share high similarity in gene expression profiles with EwS cells (22). Thus, to investigate the potential contribution of epigenetic alteration in the initiation of EwS, we analyzed the role of epigenetic marks in these models and compared to established EwS cell lines. We first analyzed the levels of H3K27me3 and H3K4me3in the human EwS-derived cell line A673 at several bona fide direct targets of EWSR1-FLI1 (table S1) by chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR). Promoter of genes that are transcriptionally activated by EWSR1-FLI1, such as FCGRT, NR0B1, CACNB2, EZH2, IGF1, NKX2-2, and HOXD11, was enriched for the H3K4me3 mark, and lacked the H3K27me3 mark, in agreement with previous data (8,20,23,24) (fig. S1A). On the other hand, transcriptionally repressed genes, such as KCNA5 (25), were enriched for H3K27me3. We next compared the levels of H3K27me3 and H3K4me3 at the same loci in HUVECs and in hpMSCs. In an apparently reversed situation to the A673 EwS cell line, analysis of those promoters presented strong enrichment for H3K27me3 but not for H3K4me3 (fig. S1B). Accordingly, infection of HUVECs with the EWSR1-FLI1 oncogene (Fig.1A) not only led to the activation of these targets (FCGRT, NR0B1, CACNB2, EZH2, IGF1, NKX2-2, and HOXD11) (Fig.1B) but also decreased the levels of H3K27me3 (Fig.1C). This demonstrates that, although H3K27me3 is not present at oncogene binding regions in EwS cell lines such as A673, these regions are repressed by PcG before oncogene expression. To explore the chromatin and transcriptional states of EWSR1- FLI1 binding sites (10), we measured the frequency of each chromatin state at these regions (26) and compared to the corresponding value obtained for the whole genome in several cell lines [HUVECs, H1, and H9 human embryonic cells, H1-derived MSCs, bone marrow (BM)–derived MSCs, and adipose-derived MSCs]. This analysis indicated that EWSR1-FLI1 binding sites are overrepresented in chromatin states associated with zinc finger genes and repeats (ZNF/repeats) and active promoters (Fig. 1D and table S1). In cells with MSC origin (such as H1, adipose, and BM-derived cell lines), EWSR1-FLI1 binding sites are overrepresented in PcG weak repressed state, which represents flanking regions of H3K27me3 peaks summit (Fig.1D and fig. S1, C and D). Similar results were obtained when we grouped chromatin states of similar categories (Fig.1E). This suggests that EWSR1- FLI1 occupies flanking regions of H3K27me3 summit peaks in hMSC, which are considered to be the potential cell of origin for EwS. Data from our group have revealed that the PRC1 subunit RING1B, is highly overexpressed in EwS primary tumors (18). We thus assessed whether RING1B modulates the growth rate of EwS cells as has been reported for other PcG subunits, such as EZH2 and BMI1 (16,17). RING1B depletion caused a reduction in cell viability in the A673, SK-ES1, and, with a lesser extent, in A4573 EwS cell lines but not in the control cell line 293T (Fig.1F and fig. S1E), suggesting that RING1B represents an epigenetic vulnerability for EwS cells. RING1B colocalizes genome-wide with EWSR1-FLI1 at active enhancers Chan etal. (27) recently proposed that RING1B might play a role in modulating enhancer activity. Together with its role in promoter regulation, EWSR1-FLI1 has been recently reported to generate de novo enhancers (10). This led us to postulate whether EWSR1-FLI1 and RING1B might cooperate during EwS tumorigenesis. We first aimed to define the genome-wide localization of RING1B and its repressive histone mark H2Aub in the A673 cell line by chromatin immunoprecipitation sequencing (ChIP-seq). In two independent experiments, we identified 2573 and 3945 peaks of RING1B, and 26424 and 10269 peaks of H2Aub. Using differential binding analysis (DiffBind), which allows for the identification of statistically common peaks (28), we found 2459 RING1B and 5392 H2Aub significant peaks between duplicates (P < 0.05, fig. S2A), corresponding to 1264 target genes and 3013 target genes, respectively (table S2). Genomic distribution of peaks showed that RING1B is more abundant in intergenic regions, whereas H2Aub is mainly located in promoters (Fig.2A). Moreover, 38% of RING1B peaks were found at intergenic regions with respect to 21.5% of H2Aub peaks, and 29.2% of RING1B peaks were in promoters with respect to 40.5% of H2Aub peaks, further supporting the potential role of RING1B at enhancers. We then categorized peaks for RING1B, H2Aub, and EWSR1-FLI1 in active or poised enhancers, and in active or poised promoters, based on H3K27me3, H3K4me3, H3K27ac, and H3K4me1 (29). To complement the above data, we performed a ChIP-seq analysis using a different antibody directed against FLI1 (fig. S2B and table S2). We found that an important fraction of RING1B peaks (35%) and EWSR1-FLI1 (46%) are located at transcriptionally active enhancers and promoters of A673 cells (Fig.2B, left). On the other hand, as expected, 35% of RING1B peaks and 37% of H2Aub peaks showed a preference for transcriptionally repressed regulatory regions (Fig.2B, left). We then intersected the list of genes associated to RING1B and H2Aub peaks with published data of EWSR1-FLI1 target genes in A673 cells, producing a common set of 162 genes (fig. S2C and table S3). Comparing this set with 386 genes containing only RING1B and H2Aub or the group of 324 EWSR1-FLI1/RING1B genes without H2Aub confirmed that the presence of EWSR1-FLI1 correlated with higher level of transcription (P < 10−16; fig. S2D, left). Functional analysis of the common gene set of 324 EWSR1-FLI1/RING1B genes (table S3) returned Gene Ontology (GO) categories related to chondrocyte and neuronal differentiation (fig. S2D, right). EWSR1-FLI1/RING1B/H2Aub genes were also enriched in neuronal differentiation category, while the RING1B/H2Aub genes were related to general transcription. These data suggest that RING1B is a positive regulator of a specific set of genes implicated in EwS and that this activity is independent of its canonical repressive mark. Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 3 of 16 0 2 4 6 8 10 12 024487296 Cell number × 106 293T siCTRL siRING1B#1 siRING1B#2 0 0.5 1 1.5 024487296 Cell number × 106 A673 0 1 2 3 4 5 024487296 Cell number × 106 SK-ES1 0 1 2 3 4 5 024487296 Cell number × 106 Hours A4573 AB C F FLI1 Tubulin pLIV EWSR1 -FLI1pLIV D E Heterochromatin Bivalent/poised TSS Flanking bivalent TSS/Enh Bivalent enhancer Repressed Polycomb Weak repressed Polycomb 913101514188914 17 16 17 30 28 37 36 25 14 34 36 28 21 29 21 28 30 28 24 26 31 28 20 30 30 28 26 211311 07231717 10 411613 10 10 11 7274 623121102110 431021312110 11 987191914 19 18 21 18 24 BG FLI1 BG FLI1 BG FLI1 BG FLI1 BG FLI1 BG FLI1 Min Max % Active TSS Enhancers Transcription Heterochromatin Repressed PcG Bivalent Enh Bivalent TSS ZNF/repeats H1 H9 HUVEC H1-MSC Ad-MSC BM-MSC Active TSS Flanking active TSS Transcr. at gene 5' and 3' Strong transcription Weak transcription Genic enhancers Enhancers BG Adiposed-derived MSC BM-derived MSCH1-derived MSC ** * * *** *** * *** *** *** *** Quiescent ZNF genes + repeats 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 0.6 0.8 1.0 FLI1 BG FLI1 BG FLI1 0 5 10 15 20 25 30 Rel. mRNA HUVEC pLIV EWSR1- FLI1pLIV 0 2 4 6 8 10 12 % Input H3K27me3 0 5 10 15 20 pLIV EWSR1- FLI1pLIV Fig. 1. EWSR1-FLI1 targets PcG-repressed regions in hMSC. (A) Western blot showing ectopic expression of EWSR1-FLI1 upon infection of HUVECs with an empty pLIV vector or EWSR1-FLI1pLIV. (B) RT-qPCR determination of relative mRNA expression of EWSR1-FLI1 target genes upon infection of HUVECs with an empty pLIV vector or EWSR1-FLI1pLIV. Values are normalized to TBP. (C) H3K27me3 ChIP-qPCR at EWSR1-FLI1 target gene promoters in HUVECs infected with an empty pLIV vector or EWSR1-FLI1pLIV. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input with subtracted immunoglobulin G (IgG). (D) Bar plots of chromatin state relative frequencies in the whole genome [background (BG)] and in published EWSR1-FLI1 binding sites (FLI1) for three selected cell lines. Genome segmentations were extracted from the Epigenome Roadmap Consortium. (E) Heatmap with percentages of each chromatin state in the whole genome (BG) as compared to the frequency within published EWSR1-FLI1 binding regions for indicated cell lines by grouping in 8 similar chromatin states the initial classification containing 15 (quiescent segments were excluded). Bold format indicates enrichments greater than 10%. Enrichment scores were calculated as the difference between the value in EWSR1-FLI1 and the value at the whole genome, normalized by the value at the whole genome. (F) Cell proliferation expressed as cell number in 293T, A673, SK-ES1, and A4573 cells transiently transfected with small interfering RNA (siRNA) against a control (siCTRL) or two different RING1B sequences (siRING1B#1 and #2). Error bars in (B), (C), and (F) indicate SD of three biological independent experiments. Statistical significance in (D) and (F) is as follows: ***P < 0.001 and *P < 0.05. Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 4 of 16 EWSR1-FLI1 RING1B RING1B-FLI1 H2Aub H3K27ac H3K4me3 H3K27me3 4 0 1 0 15 0 30 0 2 0 4 0 SEnh 10 kb AD F B G RING1B only 1504 RING1B RING1B FLI1 H3K27ac H2Aub H3K27me3 Common 955 EWSR1- FLI1 only 4673 –5000 +5000 –5000 +5000–5000 +5000–5000 +5000 –5000 +5000 H2Aub 40.5% 21.5% 38% Intergenic TSS (±2.5 kb) Gene body CE 29.2% 38% 32.8% H3K27ac H2Aub H3K27me3 Common FLI1 only RING1B only Common FLI1 only RING1B only Common FLI1 only RING1B only FLI1 RING1B Input Active enhancer Active promoter Poised enhancer Poised promoter RING1B FLI1 RING1B Common H2Aub FLI1 RING1B EWSR1-FLI1 0 10 20 30 40 50 60 70 0 20 40 60 80 0 0.5 1 1.5 2 2.5 –4000 –2000 0 2000 4000 0.1 0.2 0.3 0.4 0.5 –4000 –2000 0 2000 4000 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 –4000 –2000 0 2000 4000 Normalized count of reads Normalized count of reads Normalized count of reads 0.2 0.4 0.6 0.8 –4000 –2000 0 2000 4000 Normalized count of reads 1.5 1.0 0.5 0 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 1.0 1.20 0.010 0.020 0.0300 0.2 0.4 0.6 0.8 RING1B H2Aub H3K27ac SEnh CCND1 CAV2 10 0 1 0 15 0 25 0 5 0 CAV1 10 0 EWSR1-FLI1 RING1B-FLI1 H3K4me3 H3K27me3 50 kb 4 0 1 0 15 0 30 0 2 0 4 0 VRK1 200 kb 2 0 1 0 10 0 20 0 2 0 3 0 EWSR1-FLI1 RING1B RING1B-FLI1 H2Aub H3K27ac H3K4me3 H3K27me3 NKX2-2 SEnh 20 kb EWSR1-FLI1 RING1B RING1B-FLI1 H2Aub H3K27ac H3K4me3 H3K27me3 SEnh Fig. 2. RING1B colocalizes genome-wide with EWSR1-FLI1 at active enhancers. (A) Pie chart showing genomic distribution of RING1B and H2Aub peaks relative to functional categories including promoter (±2.5 kb from TSS), gene body (intragenic region not overlapping with promoter), and intergenic (rest of the genome). (B) Boxplot depicting percentage of regulatory elements (active/bivalent enhancers and promoters) in each described group. (C) Venn diagram depicting the overlap between RING1B and EWSR1-FLI1 in A673 cells at the peak level. (D) Aggregated plot showing the average ChIP-seq signal of RING1B and EWSR1-FLI1 at EWSR1-FLI1 binding sites. (E) Aggregated plots showing the average ChIP-seq signal of H3K27ac, H2Aub, and H3K27me3 in the three sets of RING1B and EWSR1-FLI1 peaks. (F) Heatmap showing RING1B, EWSR1-FLI1, H3K27ac, H2Aub, and H3K27me3 ChIP-seq signals segregating in the three sets of RING1B and EWSR1-FLI1 peaks. Top MEME motif for every group is shown. (G) University of California Santa Cruz (UCSC) genome browser ChIP-seq signal tracks for RING1B, EWSR1-FLI1, H2Aub, H3K27ac, H3K4me3, and H3K27me3 at NKX2-2, CCND1, VRK1, and CAV1 gene promoters and intergenic enhancer regions. Gray boxes represent EWSR1-FLI1 and RING1B colocalization and ES super-enhancers (SEnh; as shown at VRK1 and CAV1/2). Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 5 of 16 To fully understand the association of RING1B with transcriptional activation in EwS, we intersected EWSR1-FLI1 peaks with those of RING1B and obtained 955 common regions (Fig.2C). Notably, intersection between H2Aub and RING1B peaks returned only 589 common peaks. Among the 955 overlapping EWSR1-FLI1/RING1B peaks, we inspected for genes containing an enhancer within 100 kb and obtained 1276 genes, of which 235 (18%) were reported to be regulated by EwS super-enhancers (table S4) (11). The common targets of RING1B and EWSR1-FLI1 sites were found within active enhancers, while the majority of RING1B peaks not overlapping with EWSR1-FLI1 were located in transcriptionally repressed regulatory elements (Fig.2B, right). The distribution of RING1B peaks was centered on EWSR1-FLI1 binding sites (Fig.2D), suggesting that their binding occurs at the same loci. We next assessed the distribution of H3K27ac, H2Aub, and H3K27me3in genomic regions occupied by EWSR1-FLI1, RING1B, or shared (Fig.2E). Common peaks were decorated with H3K27ac, lacking H2Aub (Fig.2,EandF), and presented narrow RING1B peaks located in intergenic or intronic regions (fig. S2E, right). These data suggest that common sites likely represent enhancers. Known EWSR1-FLI1 target genes such as NKX2-2, CCND1, VRK1, or CAV1 presented an intergenic peak of RING1B, which overlaps with defined super-enhancers in the case of VRK1 and CAV1 (Fig.2G). Intronic enhancers such as JARID2 or MYOM2 (fig. S2G) constitute the majority of the 162 common RING1B, EWSR1-FLI1, and H2Aub genes (53% of sites, fig. S2C). On the other hand, RING1B-specific peaks were associated with H3K27me3 and H2Aub (Fig.2,EandF) and presented a broader distribution [e.g., HNF1B and TAL1 (fig. S2H)] mainly located within promoter or gene body regions (fig. S2E, left). The bivalent marks H3K4me3 and H3K27me3 decorated 63% of the 932 downstream genes associated to RING1B-specific peaks (P < 10−300, table S4) (29). RING1B– transcription start sites (TSS) do not overlap with EWSR1-FLI1 and are decorated with H2K27me3 and H2Aub, while RING1B-distal sites overlap with EWSR1-FLI1 and with H3K27ac (fig. S2F). Last, de novo motif analysis revealed that EWSR1-FLI1–specific sites contained predominantly (P < 10−282) one single occurrence of the canonical ETS motif GGAA (Fig.2F). When EWSR1-FLI1 was associated with RING1B, we observed a significant enrichment for multimeric GGAA repeats (P < 10−1072) (10). Furthermore, RING1B- sepecific sites were enriched for CG sequence, as previously reported (P < 10−176) (30). Together, we identified two major types of RING1B peaks in EwS: a prominent group with narrow peaks that colocalizes with EWSR1-FLI1 at enhancers of actively transcribed genes and a second group with broader peaks located at promoters, where RING1B is associated with H2Aub. RING1B regulates the expression of EWSR1-FLI1 activated and repressed targets To further characterize RING1B binding regions (table S4), we analyzed several EWSR1-FLI1 active promoters (CAV1, FCGRT, NR0B1, CACNB2, FEZF1, and KIAA1797) and enhancers (CCND1, IGF1, CAV2, JARID2, VRK1, and NKX2-2) by ChIP-qPCR. Both groups showed enrichment for RING1B, with stronger signals at enhancers (Fig.3A). Known repressed targets of the oncogene (e.g., IGFBP3, TGFBR2, and LOX) also showed binding of RING1B. At these repressed promoters, RING1B was accompanied by its canonical repressive mark H2Aub (fig. S3A). We also validated the occupancy of RING1B in EWSR1-FLI1–activated promoters (CAV1, FCGRT, NR0B1, and FEZF1) and enhancers (CCND1, CAV2, JARID2, and VRK1) in SK-ES1 cells (fig. S3B). Similar to A673 cells, H2Aub correlated with RING1B at promoters of repressed genes (IGFBP3, TGFBR2, and LOX) (fig. S3C). Last, we observed that the PRC1 and PRC2 subunits, BMI1 and EZH2, respectively, were present at repressed promoters but not in active enhancers (fig. S3, D and E), as well as in promoters with broad peaks of RING1B concomitant with H3K27me3 and H2Aub but no EWSR1-FLI1 (e.g., TAL1, IGF1R, and HNF1B) (fig. S3F). Furthermore, genome-wide analysis demonstrated that BMI1 and CBX7 (31) subunits of the PRC1 canonical complex colocalize with RING1B only at repressed regions (TAL1) as shown in Fig.3B, while no detectable peaks are present at active enhancers where EWSR1-FLI1 is present (VRK1). Thus, while RING1B decorates EWSR1-FLI1–activated promoters and enhancers, it also maintains its canonical role at several oncogene repressed regions, as well as in a subgroup of genes with no EWSR1-FLI1. To understand whether RING1B behaves as a canonical repressor and/or activator in EwS, we analyzed the expression changes after knocking down RING1B using two different sets of short hairpin RNA (shRNA, seq#1 and seq#2; fig. S4A). The data obtained showed that 71.94 and 63.85% of genes were down-regulated in the A673 and SK-ES1 cell lines, respectively (FC < -1.5, Fig.3C). This confirms our finding that RING1B acts predominantly as an activator, despite its presence at several EWSR1-FLI1–repressed targets. Furthermore, H2Aub levels remained unchanged after RING1B knockdown (Fig.3D), while RING1A knockdown produces a notable decrease in H2Aub levels (fig. S4B). These data suggest that RING1B main function in EwS is uncoupled from its ubiquitin ligase activity toward H2A and that RING1A is the main histone H2A mono-ubiquitin ligase. To further elucidate to what extent RING1B cooperates with EWSR1-FLI1in transcription regulation, we intersected differentially expressed genes in RING1B knockdown cells (absolute FC>1.25) with those affected by EWSR1-FLI1 knockdown (absolute FC > 1.5) (10), obtaining an overlap of 1078 genes. After segregating these data into down- and up-regulated genes, we found that RING1B and EWSR1- FLI1–activated 229 genes and repressed 162 genes (Fig.3E and table S5). Among the 229 activated genes, we found several developmental genes, including SOX2, SIX3, LYAR, and KIT. GO analysis showed regulation of the potassium channel and mechanisms that control actin monomers and filaments as the main categories (fig. S4C), in agreement with previous publications (25,32). Among the activated genes, SOX2 and KIT harbored RING1B and EWSR1-FLI1 peaks in intergenic and intronic enhancer regions, respectively (fig. S4E). TGFBR2, a gene repressed by both EWSR1-FLI1 and RING1B, also contained an intronic enhancer where both proteins colocalized. Notably, the expression of known targets of EWSR1-FLI1, such as NKX2-2 or IGF1 (fig. S4D), was just below our logFC cutoff value. Nonetheless, we confirmed by reverse transcription (RT)–qPCR the changes in expression levels of selected repressed and activated genes cobound by EWSR1-FLI1 and RING1B. We noticed that RING1B knockdown causes a significant reduction in the expression levels of those genes where both EWSR1-FLI1 and RING1B were co-occupying enhancer regions (Fig.3,FandG). The expression of CAV1, NKX2-2, SOX2, IGF1, JARID2, and VRK1 was affected in stronger manner upon EWSR1-FLI1 knockdown, indicating that some cofactors could remain when RING1B is depleted (fig. S4F). The effect of RING1B knockdown was less pronounced when both proteins were enriched at promoter regions of active genes (fig. S4, G and H, left). As expected, at those genes where EWSR1-FLI1 acts as a repressor, RING1B knockdown induces a promoter reactivation (fig. S4, G and H, right). Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 6 of 16 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.25 1.5 % DEG A673 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.25 1.5 SK-ES1 RING1B repressed RING1B activated 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Rel. mRNA A673 shCTRL shRING1B#1 shRING1B#2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Rel. mRNA SK-ES1 shCTRL shRING1B#1 shRING1B#2 D EActivated Repressed B EWSR1-FLI1 RING1B EWSR1-FLI1 RING1B C A FG H2Aub H4 SK-ES1 shCTRL shRING1B shCTRL shRING1B A673 shCTRL shRING1B shCTRL shRING1B RING1B Lamin B SK-ES1A673 RING1A H3K27me3 FC FC ** * *** ** *** *** * ** ** * * * * *** * H2Aub CBX7 BMI1 RING1B H3K27me3 5 0 2 0 7 0 5 0 TAL1 1 0 3 0 EWSR1-FLI1 10 kb H2Aub CBX7 BMI1 5 0 2 0 7 0 5 0 1 0 3 0 RING1B H3K27me3 EWSR1-FLI1 100 kb VRK1 0.0 0.5 1.0 1.5 2.0 2.5 % Input Active promoters IGG RI NG1B 0 0.5 1 1.5 2 2.5 3 % Input Active enhancers 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Control regions IgG RING1B 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Repressed promoters IgG RING1B Fig. 3. RING1B regulates the expression of EWSR1-FLI1–activated and EWSR1-FLI1–repressed targets. (A) RING1B ChIP-qPCR of EWSR1-FLI1 bound active promoters, repressed promoters, and active enhancers. Control regions indicate the absence of RING1B and EWSR1-FLI1 binding at these sites. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. (B) UCSC genome browser ChIP-seq signal tracks for EWSR1-FLI1, RING1B, CBX7, BMI1, H2Aub, and H3K27me3 at TAL1 promoter and VRK1 enhancer. (C) Histogram depicting percentages of activated and repressed genes in A673 and SK-ES1 cells with stable RING1B knockdown seq#2 (shRING1B#2) versus control seq#2 (shCTRL#2), with P < 0.05 and an absolute fold change (FC) > 1.25 or 1.5. (D) Western blot showing RING1B, RING1A, H2Aub, and H3K27me3 in A673 and SK-ES1 cells with either shCTRL#2 or shRING1B#2. Lamin B and histone H4 are used as loading controls. (E) Venn diagram showing intersection between differentially activated or repressed genes for EWSR1-FLI1 and RING1B in A673 cells; P < 0.05. (F) RT-qPCR determination of mRNA expression of EWSR1-FLI1 target genes with active enhancers in shCTRL and shRING1B A673 cells (#1 and #2). Values are normalized to GAPDH. (G) Same analysis as in (F) for SK-ES1 cells. Error bars in (A), (F), and (G) indicate SD of four independent biological experiments and ***P < 0.001, **P < 0.01, and *P < 0.05. Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 7 of 16 Overall, these data indicate that RING1B and EWSR1-FLI1 cooperate in gene activation, at both the promoter and enhancer levels, while RING1B retains its canonical role at those targets repressed by the oncogene. Since a large number of EWSR1-FLI1 and RING1B cotargets were not altered by RING1B knockdown, we postulate compensatory mechanism(s) or additional cofactors involved in their regulation. RING1B interacts with EWSR1-FLI1 and affects its recruitment to chromatin Wild-type EWSR1 interacts with RING1B in the VCaP prostate cancer cell line (33). We also confirmed this interaction in SK-ES1 cells (Fig.4A). Since RING1B and EWSR1-FLI1 are enriched at transcriptionally active regions, we next aimed to investigate whether both proteins interact. Coimmunoprecipitation experiments in HeLa cells where EWSR1-FLI13xFlag was overexpressed (34) confirmed that indeed oncogene interacts with RING1B (Fig.4,BandC). Analysis of published mass spectrometry data demonstrated that several SWI/SNF subunits interact with RING1B (33), further supporting an active role of RING1B in EwS gene regulation. Together, our results indicate that EWSR1-FLI1 and RING1B not only colocalize at the same genomic regions but also physically interact, mainly through the EWSR1 component of the fusion protein. Next, we analyzed whether RING1B depletion affects the EWSR1- FLI1 recruitment to chromatin. As expected, after knockdown, we observed a notable reduction of RING1B in the chromatin bound fraction (Fig.4D). EWSR1-FLI1 was also evicted from chromatin bound and enriched in the soluble chromatin fraction (Fig.4D). We then monitored the occupancy of EWSR1-FLI1, RING1B, and H3K27ac at several enhancers (e.g., SOX2, NKX2-2, and IGF1). The data in Fig.4E showed that upon RING1B knockdown, enrichments at those enhancers decreased to control values [immunoglobulin G (IgG) or ENC1 region]. To assess the decrease of EWSR1-FLI1 recruitment genome-wide, we performed ChIP-seq analysis of RING1B and FLI1in shCTRL and shRING1B A673 cells. The analysis indicated that upon RING1B depletion, EWSR1-FLI1 binding to chromatin was reduced (Fig.4,FandG). In sum, we conclude that in EwS, RING1B exerts its main role as activator by promoting recruitment of EWSR1-FLI1 to enhancer regions. RING1B knockdown decreases tumor growth in vivo RING1B stimulates tumor growth and metastasis in melanoma, leukemia, and breast cancers (14,27). We observed a reduction in colony number when RING1B is depleted in the SK-ES1 cell line (fig. S5A). To gain functional insight into the cancer pathways potentially modulated by RING1B, we performed gene set enrichment analysis (GSEA) by comparing SK-ES1 shCTRL versus shRING1B cells. The top 10 most significant pathways included interferon-, epithelial-to-mesenchymal transition, hedgehog signaling, and angiogenesis, with a 0.25 Q value cutoff (fig. S5B). In EwS, disruption of angiogenic pathways has been described (4,22). Further inspection of angiogenic gene list revealed that key genes such as PDGFA, FGFR1, SLCO2A1, CXCL6, and S100A4 were downregulated upon RING1B depletion (fig. S5C). To assess the relevance of RING1B invivo, we generated xenografts by injecting SK-ES1 shCTRL or shRING1B cells (seq#1 and seq#2) subcutaneously into athymic nude mice. Cells with reduced RING1B levels showed delayed engraftment and slower tumor growth (Fig.5A). At 21 days after injection, tumors derived from shRING1B cells were significantly smaller than those from control cells (fig. S5D). Notably, the median survival increases from 26 days for shCTRL cells to 30 days for shRING1B seq#1 and from 20 to 27 days for shRING1B seq#2 (Fig.5B). Immunohistochemical analyses of tumors confirmed reduced levels of RING1B, while the ES marker CD99 remained essentially unchanged (Fig.5C and fig. S5E). Furthermore, shCTRL tumors displayed higher proliferation rates than shRING1B, as shown by Ki-67 staining (Fig.5C). To better characterize xenograft derived tumors, we performed RNA sequencing (RNA-seq) of a cohort of tumors (six for each group, Fig.5D). GSEA analysis confirmed the enrichment of angiogenic genes in the shCTRL tumors (fig. S5, F and G). Since RING1B retains its repressive function at several promoters, we hypothesized that the delay in survival and in tumor growth upon RING1B knockdown could be related to up-regulation of tumor suppressor genes (TSG). GSEA applied to 983 genes from TSG database (https://bioinfo.uth.edu/TSGene), indicated that this gene list was enriched in shCTRL phenotype, suggesting that tumor growth and survival differences observed were not due to RING1B repression of TSG (fig. S5H). The NKX2-2, SOX2, and IGF1 genes are necessary for EwS tumor proliferation (21,23,35). In agreement, confirmed RING1B and EWSR1-FLI1 expression reduction (Fig.5E) is associated to down-regulation of these genes in xenograft tumors (Fig.5F, left), as we previously shown in EwS cells (Fig.3,FandG). Furthermore, after RING1B knockdown, we also validated down-regulation of S100A4, SLCO2A1, and VEGFA, which are main activators of angiogenic signaling pathways (Fig.5F, right). All these data highlight the role of RING1B as an activator in EwS tumorigenesis. EWSR1-FLI1/RING1B targets are regulated by AURKB inhibition Several kinases (including AURKB, MEK1, and CK2) have been reported to modulate the activating transcriptional function of RING1B (14,15,36). To investigate which pathway(s) regulates RING1B at active enhancers in EwS, we analyzed the expression levels of these three kinases in a publicly available database (4) comprising a cohort of 27 tumor samples and BM-MSCs. While MEK1 and CK2 were not expressed in primary tumors with respect to BM-MSCs (control), 11 of 27 EwS tumors (40%) showed higher levels of AURKB compared to control (fig. S6A). EWSR1-FLI1 directly regulates the expression of AURKB (37), as also demonstrated by AURKB down-regulation in EwS cell lines upon oncogene knockdown (fig. S6A). AZD1152 is a specific AURKB inhibitor, with a median inhibitory concentration (IC50) of 19 nM in EwS cell lines (38). Accordingly, we observed IC50 values of 5 and 6 nM in SK-ES1 and A4573 cells, respectively; in contrast, the IC50 for A673 was 5 M, and AZD1152 had no effect on the control cell line 293T (fig. S6B). EwS cells that survived to the treatment showed an atypical phenotype, suggesting enhanced differentiation (fig. S6C). Furthermore, viability of EwS cell lines was not affected by the inhibition of RING1B E3 ubiquitin ligase activity with PRT4165 (fig. S6B). To further elucidate the effect of AZD1152in EwS, cell death was analyzed by Annexin V staining. A 72-hour AZD1152 treatment of A673, SK-ES1, and A4573 cells led to an increase in the early and late apoptosis populations as compared to 293T cells (Fig.6A). Analysis of cleaved PARP levels further demonstrated that AZD1152 stimulated apoptotic pathways in EwS cell lines, with SK-ES1 being the most sensitive (Fig.6B). It is worth noting that the levels of EWSR1-FLI1 were decreased after AZD1152 treatment in SK-ES1 Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 8 of 16 0.00 0.05 0.10 0.15 0.20 0.25 IgGFLI1RING1B % Input NKX2-2 0 5 10 15 20 25 IgGK27ac 0.00 0.05 0.10 0.15 0.20 0.25 IgGFLI1RING1B % Input IGF1 0 2 4 6 8 10 12 14 IgGK27ac 0.00 0.05 0.10 0.15 0.20 0.25 IgGFLI1RING1B % Input SOX2 0 5 10 15 20 25 IgGK27ac 0.00 0.05 0.10 0.15 0.20 0.25 IgGFLI1RING1B % Input ENC1 0 5 10 15 20 25 IgGK27ac shCTRL shRING1B 0 5 10 15 20 Rel. prot. level RING1B EWSR1-FLI1 AB C EWSR1 Input RING1B IgG EWSR1 RING1B SK-ES1: D 0 RING1B Flag Calnexin Dox (µg/ml): 0.01 0.05 0.1 0.5 1 5 RING1B Flag Input RING1B IgG Flag HeLa EWSR1-FLI1 3XFlag: FLI1 GAPDH H4 shCTRL shRING1B shCTRL shRING1B shCTRL RING1B Cit. Sol. Bound Chrom. Chrom. shRING1B FG RING1B peaks FLI1 peaks EWSR1-FLI1 shRING1B EWSR1-FLI1 RING1B shRING1BshCTRL shCTRL RING1B E * SOX2 VRK1 Normalized count of reads 0.1 0.2 0.3 0.4 0.5 0.6 Normalized count of reads 0 0.1 0.2 0.3 0.4 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 0.5 1.0 1.5 2.0 2.5 3.0 –4000 –2000 0 2000 4000 –4000 –2000 0 2000 4000 shCTRL shRING1B shCTRL input shRING1B input shCTRL shRING1B shCTRL shRING1B shCTRL shRING1 B ** *** shCTRL input shRING1B input 100 kb 100 kb EWSR1-FLI1 EWSR1-FLI1 RING1B RING1B Fig. 4. RING1B interacts with EWSR1-FLI1 and affects its recruitment to chromatin. (A) Western blot showing endogenous coimmunoprecipitation of RING1B with EWSR1 in the SK-ES1 cell line. (B) Western blot showing overexpression of EWSR1-FLI1-3xFlag and RING1B levels in HeLa stably transfected cells upon induction with indicated doxycycline concentrations for 24 hours. Calnexin is used as loading control. (C) Coimmunoprecipitation of RING1B with EWSR1-FLI1-3xFlag under induction conditions (0.5 g/ml). Inputs in (A) and (C) contain 10% of immunoprecipitated material and IgG is used as control. (D) Western blot showing RING1B and EWSR1-FLI1 in cytoplasm, soluble, and bound chromatin fractions in shCTRL#1 or shRING1B#1 SK-ES1 cells. Histone H4 is used as a control of bound chromatin, and GAPDH as a control of cytoplasmic fraction. Blot quantification of the same ordered samples is depicted below. (E) ChIP-qPCR analysis of FLI1, RING1B, and H3K27ac at EWSR1-FLI1–activated enhancers of NKX2-2, SOX2, or IGF1 genes in shCTRL#2 and shRING1B#2 A673 cells. ENC1 is used as negative control region. The values of the Y axis represent the enrichment ratio of immunoprecipitated samples relative to input. Error bars indicate SD of three independent biological experiments. Statistical significance is as follows ***P < 0.001, **P < 0.01, and *P < 0.05. (F) Aggregated plot and boxplot showing the average ChIP-seq signal of RING1B and FLI1 peaks at RING1B and EWSR1-FLI1 binding sites, respectively, in shCTRL#2 and shRING1B#2 A673 cells. (G) UCSC genome browser ChIP-seq signal tracks for EWSR1-FLI1 and RING1B in shCTRL#2 and shRING1B#2 A673 cells at SOX2 and VRK1 enhancer regions. Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 9 of 16 and A4573, yet RING1B levels were unaffected (Fig.6B and fig. S6, D and E, right). To understand how AURKB modulates RING1B in EwS, we analyzed H2Aub levels after AZD1152 treatment. We observed increased levels of H2Aub repressive mark after AURKB inhibition, suggesting that this kinase indeed inhibits the ubiquitin ligase activity of RING1B in EwS (Fig.6C). Furthermore, in SK-ES1 and A4573 cells, the increase in ubiquitin ligase activity correlated with decreased expression of EWSR1-FLI1 targets co-occupied by AC D E shCTRL H&E CD99 Ki67 RING1B F shRING1B 2 0 –2 shCTRL shRING1B B Z score Fig. 5. RING1B knockdown decreases tumor growth in vivo. (A) Tumor volume curve in xenografts established by subcutaneous injection of shCTRL and shRING1B#1 (n = 9 and n = 10, respectively, above) or shRING1B#2 (n = 12 both groups, below) SK-ES1 cells in athymic nude mice. (B) Kaplan-Meier xenograft survival curves in shCTRL and shRING1B SK-ES1 cells (#1 and #2). (C) Immunohistochemistry staining of EWSR1-FLI1, CD99, and RING1B on sections of tumors excised from shCTRL#1 and shRING1B#1 SK-ES1 xenografts. Proliferation was analyzed by Ki67 immunohistochemistry; hematoxylin and eosin (H&E) was used as control. (D) Heatmap depicting fold changes in gene expression in six tumors excised from shCTRL#1 and shRING1B#1 SK-ES1 groups. (E) RT-qPCR levels of mRNA expression for RING1B and EWSR1-FLI1 in shCTRL#1 and shRING1B#1 SK-ES1–derived tumors; ***P < 0.001. (F) RT-qPCR levels of mRNA expression for genes regulated by EWSR1-FLI1/RING1B enhancers (left) and angiogenic genes (right) in shCTRL#1 and shRING1B#1 SK-ES1 derived tumors; *P < 0.05. Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Sánchez-Molina et al., Sci. Adv. 2020; 6 : eaba3058 23 October 2020 SCIENCE ADVANCES | RESEARCH ARTICLE 16 of 16 39. G. Boulay, A. Volorio, S. Iyer, L. C. Broye, I. Stamenkovic, N. Riggi, M. N. Rivera, Epigenome editing of microsatellite repeats defines tumor-specific enhancer functions and dependencies. Genes Dev. 32, 1008–1019 (2018). 40. S. L. Kloet, M. M. Makowski, H. I. Baymaz, L. van Voorthuijsen, I. D. Karemaker, A. Santanach, P. W. T. C. Jansen, L. Di Croce, M. Vermeulen, The dynamic interactome and genomic targets of Polycomb complexes during stem-cell differentiation. Nat. Struct. Mol. Biol. 23, 682–690 (2016). 41. A. Banito, X. Li, A. N. Laporte, J.-S. Roe, F. Sanchez-Vega, C.-H. Huang, A. R. Dancsok, K. Hatzi, C.-C. Chen, D. F. Tschaharganeh, R. Chandwani, N. Tasdemir, K. B. Jones, M. R. Capecchi, C. R. Vakoc, N. Schultz, M. Ladanyi, T. O. Nielsen, S. W. Lowe, The SS18-SSX oncoprotein hijacks KDM2B-PRC1.1 to drive synovial sarcoma. Cancer Cell 34, 346–348 (2018). 42. V. van den Boom, H. Maat, M. Geugien, A. Rodríguez López, A. M. Sotoca, J. Jaques, A. Z. Brouwers-Vos, F. Fusetti, R. W. Groen, H. Yuan, A. C. Martens, H. G. Stunnenberg, E. Vellenga, J. H. Martens, J. J. Schuringa, Non-canonical PRC1.1 targets active genes independent of H3K27me3 and is essential for leukemogenesis. Cell Rep. 14, 332–346 (2016). 43. Z. Wang, M. D. Gearhart, Y. W. Lee, I. Kumar, B. Ramazanov, Y. Zhang, C. Hernandez, A. Y. Lu, N. Neuenkirchen, J. Deng, J. Jin, Y. Kluger, T. A. Neubert, V. J. Bardwell, N. B. Ivanova, A non-canonical BCOR-PRC1.1 complex represses differentiation programs in human ESCs. Cell Stem Cell 22, 235–251.e9 (2018). 44. B. E. Gryder, M. E. Yohe, H.-C. Chou, X. Zhang, J. Marques, M. Wachtel, B. Schaefer, N. Sen, Y. Song, A. Gualtieri, S. Pomella, R. Rota, A. Cleveland, X. Wen, S. Sindiri, J. S. Wei, F. G. Barr, S. Das, T. Andresson, R. Guha, M. Lal-Nag, M. Ferrer, J. F. Shern, K. Zhao, C. J. Thomas, J. Khan, PAX3–FOXO1 establishes myogenic super enhancers and confers BET bromodomain vulnerability. Cancer Discov. 7, 884–899 (2017). 45. A. Puissant, S. M. Frumm, G. Alexe, C. F. Bassil, J. Qi, Y. H. Chanthery, E. A. Nekritz, R. Zeid, W. C. Gustafson, P. Greninger, M. J. Garnett, U. McDermott, C. H. Benes, A. L. Kung, W. A. Weiss, J. E. Bradner, K. Stegmaier, Targeting MYCN in neuroblastoma by BET bromodomain inhibition. Cancer Discov. 3, 308–323 (2013). 46. P. N. Gollavilli, A. Pawar, K. Wilder-Romans, R. Natesan, C. G. Engelke, V. L. Dommeti, P. M. Krishnamurthy, A. Nallasivam, I. J. Apel, T. Xu, Z. S. Qin, F. Y. Feng, I. A. Asangani, EWS/ETS-driven Ewing sarcoma requires BET bromodomain proteins. Cancer Res. 78, 4760–4773 (2018). 47. G. E. Winter, U. Rix, A. Lissat, A. Stukalov, M. K. Mullner, K. L. Bennett, J. Colinge, S. M. Nijman, S. Kubicek, H. Kovar, U. Kontny, G. Superti-Furga, An integrated chemical biology approach identifies specific vulnerability of Ewing’s sarcoma to combined inhibition of Aurora kinases A and B. Mol. Cancer Ther. 10, 1846–1856 (2011). 48. S. Wang, E. E. Hwang, R. Guha, A. F. O’Neill, N. Melong, C. J. Veinotte, A. C. Saur, K. Wuerthele, M. Shen, C. McKnight, G. Alexe, M. E. Lemieux, A. Wang, E. Hughes, X. Xu, M. B. Boxer, M. D. Hall, A. Kung, J. N. Berman, M. I. Davis, K. Stegmaier, B. D. Crompton, High-throughput chemical screening identifies focal adhesion kinase and Aurora kinase B inhibition as a synergistic treatment combination in Ewing sarcoma. Clin. Cancer Res. 25, 4552–4566 (2019). 49. A. Dobin, C. A. Davis, F. Schlesinger, J. Drenkow, C. Zaleski, S. Jha, P. Batut, M. Chaisson, T. R. Gingeras, STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013). 50. M. Zhao, J. Sun, Z. Zhao, TSGene: A web resource for tumor suppressor genes. Nucleic Acids Res. 41, D970–D976 (2013). 51. Y. Zhang, T. Liu, C. A. Meyer, J. Eeckhoute, D. S. Johnson, B. E. Bernstein, C. Nusbaum, R. M. Myers, M. Brown, W. Li, X. S. Liu, Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008). 52. W. J. Kent, C. W. Sugnet, T. S. Furey, K. M. Roskin, T. H. Pringle, A. M. Zahler, D. Haussler, The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002). Acknowledgments: We thank N. Riggi for reagents and technical advice and M. Martínez-Balbás for technical advice and critical reading of the manuscript. We also thank G. Pascual-Pasto, S. Mateo, and M. Suñol for technical advice, S. Perez-Jaume for statistical advice, and L. Nonell from the Microarray Analysis Service, Hospital del Mar Medical Research Institute (IMIM, Barcelona) for technical advice. Last, we are grateful to the Band of Parents at Hospital Sant Joan de Déu for supporting the overall research activities of the developmental tumor laboratory, PCCB. Funding: S.S.-M. and the project were supported by the Spanish Association Against Cancer (AECC) consolidated groups grant (GCB13131578) consortium. The project also had the support from the Asociacion Pablo Ugarte (APU). E.F.-B. was supported by the Spanish government grant, Instituto de Salud Carlos III (PI16/00245) to J.M. The work in the Di Croce laboratory was supported by grants from the Spanish of Economy, Industry and Competitiveness (MEIC) (BFU2016-75008-P), and Fundacion Vencer El Cancer (VEC). Author contributions: S.S.-M., L.D.C., and J.M. designed the study, conducted experiments, and wrote the manuscript. J.M. supervised all the work. S.S.-M., E.F.-B., M.S.-J., P.T., C.B., E.P., L.H.-P., and D.J.G.-D. performed the experiments. E.B. and S.G. performed all the bioinformatic analysis. I.H.-M., O.M.T., A.M.C., C.L., and E.Á. provided expertise and feedback. All authors reviewed the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. Submitted 3 December 2019 Accepted 9 September 2020 Published 23 October 2020 10.1126/sciadv.aba3058 Citation: S. Sánchez-Molina, E. Figuerola-Bou, E. Blanco, M. Sánchez-Jiménez, P. Táboas, S. Gómez, C. Ballaré, D. J. García-Domínguez, E. Prada, L. Hontecillas-Prieto, Á. M. Carcaboso, Ó. M. Tirado, I. Hernández-Muñoz, E. de Álava, C. Lavarino, L. Di Croce, J. Mora, RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis. Sci. Adv. 6, eaba3058 (2020). Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023 Use of this article is subject to the Terms of service Science Advances (ISSN 2375-2548) is published by the American Association for the Advancement of Science. 1200 New York Avenue NW, Washington, DC 20005. The title Science Advances is a registered trademark of AAAS. Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC). RING1B recruits EWSR1-FLI1 and cooperates in the remodeling of chromatin necessary for Ewing sarcoma tumorigenesis Sara Snchez-Molina, Elisabet Figuerola-Bou, Enrique Blanco, Mara Snchez-Jimnez, Pablo Tboas, Soledad Gmez, Cecilia Ballar, Daniel J. Garca-Domnguez, Estela Prada, Lourdes Hontecillas-Prieto, ngel M. Carcaboso, scar M. Tirado, Inmaculada Hernndez-Muoz, Enrique de lava, Cinzia Lavarino, Luciano Di Croce, and Jaume Mora Sci. Adv., 6 (43), eaba3058. DOI: 10.1126/sciadv.aba3058 View the article online https://www.science.org/doi/10.1126/sciadv.aba3058 Permissions https://www.science.org/help/reprints-and-permissions Downloaded from https://www.science.org at Universidad de Sevilla on May 17, 2023