ARTICLE
DOI: 10.1038/s41467-018-05728-x OPEN Polycomb complexes associate with enhancers and promote oncogenic transcriptional programs in cancer through multiple mechanisms
Ho Lam Chan 1,2, Felipe Beckedorff 1,2, Yusheng Zhang1,2, Jenaro Garcia-Huidobro1,2,5, Hua Jiang3, Antonio Colaprico1,2, Daniel Bilbao1, Maria E. Figueroa1,2, John LaCava3,4, Ramin Shiekhattar1,2 & Lluis Morey 1,2 1234567890():,; Polycomb repressive complex 1 (PRC1) plays essential roles in cell fate decisions and development. However, its role in cancer is less well understood. Here, we show that RNF2, encoding RING1B, and canonical PRC1 (cPRC1) genes are overexpressed in breast cancer. We find that cPRC1 complexes functionally associate with ERα and its pioneer factor FOXA1 in ER+ breast cancer cells, and with BRD4 in triple-negative breast cancer cells (TNBC). While cPRC1 still exerts its repressive function, it is also recruited to oncogenic active enhancers. RING1B regulates enhancer activity and gene transcription not only by promoting the expression of oncogenes but also by regulating chromatin accessibility. Functionally, RING1B plays a divergent role in ER+ and TNBC metastasis. Finally, we show that concomitant recruitment of RING1B to active enhancers occurs across multiple cancers, highlighting an under-explored function of cPRC1 in regulating oncogenic transcriptional programs in cancer.
1 Sylvester Comprehensive Cancer Center, Biomedical Research Building, 1501 NW 10th Avenue, Miami, FL 33136, USA. 2 Department of Human Genetics, University of Miami Miller School of Medicine, Miami, FL 33136, USA. 3 Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY 10065, USA. 4 Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA. 5 Centro de Investigaciones Médicas (CIM), Núcleo Científico Multidisciplinario, Escuela de Medicina, Universidad de Talca, Avenida Lircay S/N, Talca 3460000, Chile. Correspondence and requests for materials should be addressed to L.M. (email: [email protected])
NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x
olycomb group genes (PcG) are evolutionarily conserved overrepresented (Supplementary Fig. 1b). Since the breast cancer Pepigenetic regulators that can be divided into two main data sets contain the largest number of patient samples and thus complexes, Polycomb repressive complex 1 and 2 (PRC1 provide the most robust data, we further analyzed PRC1 genes in and PRC2)1. In mammals, the core PRC2 complex contains these patient samples. We found that RNF2 was amplified in up SUZ12, EED, and the histone methyltransferase enzymes EZH1/ to 22% of breast cancers and cPRC1 genes were amplified in a 2, which catalyze di- and trimethylation on lysine 27 of histone large number of samples (Supplementary Fig. 1c–d). Compared H3 (H3K27me2/3)2. The two main PRC1 sub-complexes are the to RING1 which is not amplified, RNF2 amplification correlated canonical and non-canonical PRC1 complexes (cPRC1 and to its significant overexpression in breast cancer compared to ncPRC1). cPRC1 comprises PCGF2/4, Polyhomeiotic (PHC1/2/ normal breast tissues, regardless of breast cancer subtype (Sup- 3), the CBX proteins (CBX2/4/6/7/8), and the E3-ligase subunits plementary Fig. 1e–f). We also noticed that other amplified RING1A/B, which monoubiquitinate histone H2A at lysine 119 cPRC1 genes, including CBX2/4/8 and PCGF2, exhibited distinct (H2AK119ub1). In contrast, ncPRC1 complexes include RYBP/ expression patterns when categorized by breast cancer subtype YAF2, PCGF1/3/5, and RING1A/B, as well as additional co- (Supplementary Fig. 1g). Furthermore, RNF2 expression was factors3. We and others have shown that cPRC1, ncPRC1, and highest in tumors with amplification of the gene (Supplementary PRC2 complexes regulate stem cell pluripotency, cell fate deci- Fig. 2a). However, RNF2, PCGF2, and CBX2/4/8 expression was sions, and development4,5. Historically, Polycomb complexes higher in all four breast cancer stages compared to normal breast have been mostly associated with maintaining gene repression. tissue, suggesting that their overexpression was not predictive of However, increasing evidence indicates that specific PRC1 var- breast cancer aggressiveness (Supplementary Fig. 2b). iants can be recruited to actively transcribed genes in multiple biological processes6–10. While PRC1 genes are not typically mutated, they are dysre- RING1B binding is redistributed in breast cancer cells.We gulated in many cancer types. BMI1, encoding for PCGF4, is the next focused on understanding the specific role of RING1B in best studied PRC1 gene in cancer to date. It is often overexpressed breast cancer (Fig. 1a). To our knowledge, no genome-wide study in cancer and is important for tumor initiation and of RING1B binding to chromatin in breast cancer cells had yet progression11,12. In contrast, PCGF2 is downregulated in prostate been conducted. We performed RING1B chromatin immuno- and colorectal cancers13, suggesting that PCGF paralogs have precipitation followed by massive parallel sequencing (ChIP-seq) distinct functions in cancer. Recent studies suggested that PRC1 of two breast cancer cell lines—estrogen receptor positive (ER+) genes that play important roles in cancer carry out their functions luminal A cell line, T47D, and triple-negative breast cancer independently of their association with PRC114,15. Nonetheless, (TNBC) cell line, MDA-MB-231—and a non-tumorigenic despite great efforts to understand the epigenetic mechanisms transformed mammary epithelial cell line, MCF10A. As a con- that contribute to human maladies, a comprehensive analysis of trol, we also performed RING1B ChIP-seq in human induced genomic alterations of PRC1 genes, and the architecture, func- pluripotent stem cells (iPSCs) since the target genes of tion, and activity of PRC1 complexes in cancer, have yet to be PRC1 subunits have been extensively mapped in stem cells16,17. fully addressed. Additionally, the RING1B antibody used is validated by mass Here, we show that PRC1 genes are genetically amplified in spectrometry. To further confirm the specificity of this antibody, breast cancer. In contrast to its canonical function, RING1B we performed RING1B western blotting and immunoprecipita- (encoded by RNF2) is predominantly recruited to enhancers and tion from control and RING1B-depleted MDA-MB-231 cells specifically regulates oncogenic transcriptional programs in dif- (Supplementary Fig. 3a–b). As additional controls, we performed ferent breast cancer subtypes. Mechanistically, RING1B associates ChIP-qPCR of known RING1B target genes in iPSCs17 using a with specific cPRC1 components that are recruited to enhancers different RING1B antibody as well as H3K27me3, H3K4me3 and containing estrogen receptor alpha (ERα)inER+ cells, and to H3K27ac antibodies (Supplementary Fig. 3c–d) and the enrich- BRD4-containing enhancers in triple-negative breast cancer ment values are in agreement with ChIP-seq binding. (TNBC) cells. We also show a functional crosstalk between We identified 702 RING1B target genes in iPSCs, 2869 in RING1B, FOXA1, and ERα in ER+ cells, resulting in an atte- MCF10A, 2202 in T47D, and 2137 in MDA-MB-231 (Fig. 1b and nuated response to estrogen with RING1B depletion. We provide Supplementary Data 1). Gene ontology (GO) analyses revealed evidence that RING1B directly regulates chromatin accessibility RING1B targets as developmental genes in iPSCs (Fig. 1c), in at enhancers bound by transcription factors involved in breast agreement with published data17. In contrast, GO analysis of cancer. In agreement with survival prognoses of patients with RING1B targets in MCF10A showed enrichment of genes different breast cancer subtypes and RNF2 expression levels, involved in axon guidance and focal adhesion, while in T47D RING1B differentially regulates the metastatic potential of TNBC and MDA-MB-231, genes involved in focal adhesion, cell-to-cell and ER+ breast cancer cells. Finally, we show that RING1B is junctions, and signaling pathways in cancer were enriched recruited to enhancer regions in other cancer types, suggesting (Fig. 1c). As expected based on the GO analyses, the overlap of that this RING1B-mediated mechanism of controlling oncogenic RING1B targets was relatively low between iPSCs, MCF10A, pathways occurs in multiple cancers. T47D, and MDA-MB-231 (Fig. 1d), but higher between MCF10A, T47D, and MDA-MB-231 (Supplementary Fig. 3e–f). To determine the functional significance RING1B genomic Results distribution, we categorized RING1B ChIP-seq peaks into three cPRC1 genes are amplified and overexpressed in breast cancer. main regions: intergenic, intragenic, and promoter regions To initially assess whether PRC1 components are altered in (Methods section). Most RING1B peaks in iPSCs were located cancer, we examined the mutational frequencies of the histone at promoters or inside genes. However, in MCF10A, T47D, and H2A mono-ubiquitin ligases RNF2 (encoding RING1B) and MDA-MB-231, RING1B was distributed to intergenic regions RING1, the cPRC1 genes, and the core PRC1-encoding genes (Fig. 1e). We also found that each of the cell lines had a set of (Supplementary Fig. 1a) in large-scale genomic data sets from distinct RING1B peaks corresponding to cancer-related and cancer patients. We found that PRC1 genes were amplified in epithelial genes in the breast cells but not in iPSCs (Fig. 1f, g and multiple cancer types. Intriguingly, many hormone-related can- Supplementary Fig. 3g). Importantly, RNA-seq analysis indicated cers (e.g., ovarian, uterine, prostate, and breast cancer) were that RING1B target genes in MCF10A, T47D, and MDA-MB-231
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abRING1B c Non-canonical PRC1 Canonical PRC1 target genes RING1B targets in iPSCs RING1B targets in T47D Homeobox Pathways in cancer RYBP/YAF CBX2-8 Pluripotent cells 702 Development Focal adhesion PCGF1/3/5/6 RING1A/B PCGF2/4 iPSCs Neurogenesis Tight junction co-factors PHC1-3 Cell differentiation Rap1 signaling Non-tumorigenic 2869 –150 –120 –90 –60 –30 0 –8 –7 –6 –5 –4 –3 –2 –1 0 MCF10A log2 (p value) log2 (p value) Genetic alterations and RING1B targets in MCF10A RING1B targets in MB-231 overexpression in breast cancer Luminal breast cancer 2202 Axon guidance Central nervous T47D (ER+) Focal adhesion Cadherin signaling Pathways in cancer Wnt signaling Rap1 signaling Ionotropic Basal-like breast cancer 2137 –12 –10 –8 –6 –4 –2 0 –15 –12 –9 –6 –3 0 glutamate receptor MDA-MB-231 (TNBC) log2 (p value) log2 (p value)
d e f RING1B target genes iPSCs MCF10A RING1B peaks RING1B peaks iPSCs MCF10A MDA-MB-231T47D
MCF10A MDA-MB-231 Intergenic Intergenic 26% 46% 978 genes –2.5kb + TSS Cadherin signaling iPSCs iPSCs Intragenic –2.5kb + TSS 39% 8% (1.8e–10 p value) 2725 1814 23% Intragenic 145 119 Other 43% Other 557 583 12% 3% 1052 genes Cell-cell junction MDA-MB-231 T47D (0.0011 p value) T47D RING1B peaks RING1B peaks iPSCs 2082 Intergenic Intergenic 1865 genes 120 56% 50% Cadherin signaling 582 –2.5kb + TSS (3.8e–8 p value) –2.5kb + TSS Intragenic Intragenic 5% 3% 39% 43% Other Cell-type-specific RING1B peaks 439 genes Other 2% Development 2% –2.5Kb +2.5Kb (3.42e–7 p value)
g HOXA cluster 100 kb hg19 Scale 100 kb hg19 Scale 100 kb hg19 Scale 100 kb hg19 Scale chr3: 18,850,000 18,900,000 18,950,000 19,000,000 19,050,000 19,100,000 19,150,000 19,200,000 19,250,000 27,100,000 27,150,000 27,200,000 27,250,000 27,300,000 chr11: 114,050,000 114,100,000 114,150,000 114,200,000 chr12: 66,050,000 66,100,000 66,150,000 66,200,000 i - - - - 19 - iPSCs 12 - 18 - 15 - iPSCs MCF10A RING1B RING1B peaks RING1B peaks 0 _ 0_ 0 _ 0 _ 19 - MCF10A 12 - 18 - 15 - RING1B 0 _ 0 _ 0 _ 0 _ - - 19 18 - 15 - 19 - MDA-MB-231 12 - 35%
RING1B 0 _ 0 _ 0 _ 0 _ - - - 70% - 15 - 19 - 12 - 18 - T47D
0 _ 0 _ 0 _ RING1B 0 _ U6 HOXA1 BC035889 HOXA HOXA11 EVX1 HOTAIRM1 HOXA HOXA9 HOXA13 HOXA2 HOXA-AS3 LOC402470 RPSAP52 KCNH8 HOXA3 HOXA5 4HOXA-AS HOTTIP ZBTB16 NNMT HOXA6 MIR196 HMGA2 MDA-MB-231 T47D RING1B peaks RING1B peaks h iPSCs MCF10A MDA-MB-231 T47D Scale 100 kb hg19 Scale 50 kb hg19 Scale 500 kb hg19 Scale 1 Mb hg19 chr7: 27,150,000 27,200,000 27,250,000 27,300,000 chr11: 114,050,000 114,100,000 114,150,000 chr8: 29,200,000 29,300,000 29,400,000 29,500,000 29,600,000 29,700,000 29,800,000 29,900,000 30,000,000 chr2: 214,500,000 215,000,000 215,500,000 216,000,000 216,500,000 217,000,000 217,500,000 - - - - 10 - 19 - 33 - 22 - 25% 20% RING1B
0 _ 0 _ 0 _ 0 _ 35 - 19 - 33 - 22 - H3K27me3
0 _ 0 _ 0 _ 0 _ 15 - 19 - 33 - 22 - H2AK119ub1 b 0 _ 0 _ 0 _ 0 _ H3K27me3+ 31 - 33 - 73 - 39 - H3K4me3 H2AK119ub1+
0 _ 0 _ 0 _ 0 HOXA1 BC035889 HOXA HOXA11 EVX1 HOTAIRM1 HOXA HOXA9 HOXA13 HOXA2 HOXA-AS3 LOC402470 HOXA3 HOXA5 HOXA-AS HOTTIP DUSP4 SPAG16 Mir_548 HOXA6 MIR196 ZBTB16 NNMT LINC00589 HOXA10 j
siCTRLsiRING1AsiRING1B siCTRLsiRING1AsiRING1B siCTRLsiRING1AsiRING1B RING1A 55 kDa RING1A 55 kDa RING1A 55 kDa RING1B 43 kDa RING1B 43 kDa RING1B 43 kDa
H2AK119ub1 26 kDa H2AK119ub1 26 kDa H2AK119ub1 26 kDa H3 17 kDa H3 17 kDa H3 17 kDa MCF10A MDA-MB-231 T47D
Fig. 1 Genome-wide occupancy and activity of RING1B in breast cancer cells. a Model depicting RING1B and cPRC1 subunits that are genetically amplified and overexpressed in breast cancer. b Number of RING1B target genes. Representative phase-contrast images of each cell line are shown at ×10 magnification. Scale bar represents 100 µm. c GO analysis of RING1B target genes. d Venn diagrams of overlapping RING1B target genes. e Distribution of RING1B ChIP-seq peaks. f ChIP-seq heat maps of specific RING1B peaks in each of the cell lines. GO analysis performed on target genes identified in each peak cluster. g Genome browser screenshots of unique RING1B-binding sites in each of the cell lines. RING1B peaks are highlighted in green. h Pie chart showing percentage of RING1B peaks overlapping with H2AK119ub1 and H3K27me3. i Genome browser screenshots of RING1B, H3K27me3, H2AK119ub1, and H3K4me3 in each of the cell lines. RING1B peaks are highlighted in green. j Representative western blots of RING1A, RING1B, and H2AK119ub1 of control and RING1B-depleted cells. Histone H3 was used as a loading control (n = 3) are transcriptionally more active and more highly expressed than the degree of overlap between RING1B and the PRC2-associated the RING1B target genes in iPSCs (Supplementary Fig. 3h–i). and PRC1-associated histone modifications, H3K27me3 and H2AK119ub1, respectively. As expected in iPSCs, the majority of sites containing RING1B were also decorated with both histone Most RING1B-bound sites are devoid of H3K27me3/ modifications (Fig. 1h, i and Supplementary Fig. 3j)18.In H2AK119ub1. Since the classical model of PRC1 binding to MCF10A, 35% of RING1B sites were co-occupied by H3K27me3 chromatin is following PRC2 recruitment, we next determined and H2AK119ub1 and this overlap decreased to 25 and 20% in
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MDA-MB-231 and T47D, respectively (Fig. 1h, i and Supple- SEs in MCF10A, MDA-MB-231, and T47D cells, respectively mentary Fig. 3k–l). This observation was confirmed by over- (Fig. 2h and Supplementary Data 2). Interrogation of published lapping RING1B target genes and H3K27me3-marked genes ChIP-seq data sets in ENCODE using EnrichR revealed a further (Supplementary Fig. 3m). These results indicate that in breast potential functional association of RING1B with ERα in T47D. epithelial cells: (1) RING1B function is not exclusively associated Interestingly, the bromodomain-containing protein, BRD4, was to its mono-ubiquitination ligase activity and (2) RING1B is recruited to genes potentially controlled by RING1B-containing recruited to chromatin independently of PRC2. In agreement SEs in MDA-MB-231, while RACK7 (receptor for activated C- with the low overlap between RING1B and H2AK119ub1, kinase 7) bound the RING1B-containing SEs in MCF10A RING1B depletion had no major effect on bulk levels of (Fig. 2h). Overall, these results indicate that RING1B is recruited H2AK119ub1. However, H2AK119ub1 levels were reduced after to SEs and, importantly, that there is a specific functional RING1A depletion in MDA-MB-231 and T47D (Fig. 1j), indi- crosstalk between RING1B and key signaling pathways involved cating that RING1A enzymatic activity at histone H2A is greater in breast cancer. than RING1B in these cells. This line of evidence suggests that RING1A is the main histone H2A mono-ubiquitin ligase in these breast cancer cell lines. RING1B assembles into discrete cPRC1 complexes. Dozens of cPRC1 and ncPRC1 variants can be potentially assembled, and have distinct biological functions in regulating stem cell plur- RING1B binds active enhancers. Since a large number of ipotency, differentiation, and tissue homeostasis3,6,27–30. To assay RING1B sites were not marked with H3K27me3 or H2AK119ub1 the RING1B protein interactome in MDA-MB-231 and T47D, we and RING1B peaks re-localized to intergenic regions, we next performed co-immunoprecipitations of endogenous RING1B- tested whether RING1B is recruited to enhancer regions. associated protein complexes using the anti-RING1B antibody Enhancers are regulatory sites that can be bound by transcription used for ChIP-seq, followed by label-free quantitative liquid factors to increase the transcription of a particular gene19,20 and chromatography-tandem mass spectrometry (LC-MS/MS). can be divided into typical enhancers and super-enhancers (SEs): Unexpectedly, because both cPRC1 and ncPRC1 genes are in cancer, typical enhancers promote transcription at active genes expressed in these cells (Supplementary Fig. 5a), RING1B mainly and SEs regulate the expression of oncogenes and genes asso- co-immunoprecipitated with cPRC1 subunits (Fig. 3a, b and ciated to oncogenic transcriptional programs21. Active typical Supplementary Data 3). Specifically, when captured from T47D enhancers and SEs are also epigenetically distinct: although both cells, RING1B demonstrated interactions with the are marked with H3K4me1, SEs contain increased levels of cPRC1 subunits CBX4/8, PCGF2, and PHC2/3 (Fig. 3a, left), with H3K27ac21,22. We found that both H3K27ac and H3K4me1 CBX8 and PHC3 displaying the highest levels of interaction with ChIP-seq signals were enriched at RING1B-bound sites (Fig. 2a) RING1B (Fig. 3b, left). RING1B co-immunoprecipitated a larger that were simultaneously devoid of H3K27me3 (Supplementary number of proteins in MDA-MB-231 cells than in T47D cells Fig. 4a–b). Only 4%, 8%, and 13% of typical enhancers contained (Fig. 3a, right), but of the proteins observed, cPRC1 subunits, RING1B in MCF10A, MDA-MB-231, and T47D cells, respec- including CBX8, PCGF2, and PHC2 were amongst the most tively, while in contrast, over 45% of SEs in these cells abundant (Fig. 3b, right). We next addressed whether the were decorated with RING1B (Fig. 2b–d and Supplementary RING1B recruited to chromatin in T47D and MDA-MB-231 is a Fig. 4c–d). Virtually none of the SEs in iPSCs contained RING1B part of a cPRC1 complex. We performed ChIP-seq of (Fig. 2c). PCGF2 since it is the predominant RING1B-associated PCGF We next asked whether RING1B was recruited to SEs near subunit in both cell lines and identified 2408 and 4813 PCGF2 genes with established functions in breast cancer. Indeed, we target genes in T47D and MDA-MB-231 cells, respectively observed RING1B recruitment at SE regions near BCL2L1 in (Fig. 3d, g). Almost 60% of PCGF2 targets in T47D cells, and MDA-MB-231 and ESR1 in T47D23,24 (Fig. 2e and Supplemen- about 80% in MDA-MB-231 cells, were also co-occupied by tary Data 1). To confirm that the SEs were unique to each cell RING1B (Supplementary Fig. 5b–c). line, and that RING1B was recruited specifically to these unique To further interrogate the potential functional relationship sites, we determined the RING1B signal at these SEs. We found between RING1B and ERα, we addressed whether a cPRC1 that RING1B signal at MDA-MB-231 specific SEs was stronger in complex (defined by co-occupancy of RING1B and PCGF2) is MDA-MB-231 cells than at the same SE regions in MCF10A and associated with genomic sites bound by ERα. We found that T47D cells; the same was true for MCF10A- and T47D-specific cPRC1 was indeed co-recruited with ERα to a large number of SEs (Fig. 2f and Supplementary Fig. 4e). These results indicate genomic sites (Fig. 3c). Overlapping RING1B, PCGF2, and ERα that RING1B is recruited to cell-type-specific SEs in breast targets indicated that 890 target genes were decorated with cPRC1 epithelial and cancer cells. and ERα (Fig. 3d). Importantly, these genes are involved in In contrast to the broad RING1B ChIP-seq signals in pathways important in carcinogenesis (Fig. 3e and Supplementary pluripotent cells, RING1B peaks in the breast cell lines were Data 4). Furthermore, since we observed that potential genes narrow (Figs. 1g, h and 2e), resembling ChIP-seq signals of regulated by RING1B-containing SEs in MDA-MB-231 cells were transcription factors. Therefore, we assessed whether RING1B is BRD4 targets (Fig. 2h), we also performed ChIP-seq of BRD4. recruited to specific transcription factor-binding sites at SEs20.In Indeed, cPRC1 largely associated with BRD4 targets and 840 the ER + cell line, T47D, analysis of known transcription factor genes were decorated with cPRC1/BRD4 (Fig. 3f, g and motifs revealed an enrichment of the ERα and FOXA1/2 Supplementary Data 4). These cPRC1/BRD4 co-targets are consensus binding sequences25,26 (Fig. 2g), suggesting a func- involved in cancer and focal adhesion pathways (Fig. 3h). tional connection between RING1B and the ER pathway. We next determined the co-recruitment of cPRC1 with either Similarly, motifs for important breast cancer oncogenic tran- ERα or BRD4 to enhancers in T47D and MDA-MB-231 cells, scription factors were overrepresented at RING1B-containing SEs respectively (Fig. 3i–k and Supplementary Fig. 5d). A total of 81% in MDA-MB-231 and MCF10A cells that are ER− (Fig. 2g). and more than 90% of SEs with cPRC1 were also bound by ERα Finally, we associated potential target genes to the SEs in T47D and BRD4 in MDA-MB-231, respectively (Fig. 3l). containing RING1B based on proximity and retrieved 561, 252, Association of BRD4 to RING1B-containing enhancers in MDA- and 398 genes that were potentially functionally associated with MB-231 was further validated by ChIP-qPCR (Supplementary
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a MDA-MB-231 T47D d MCF10A MDA-MB-231 T47D 0.26 0.26 0.6 RING1B RING1B RING1B RING1B RING1B 0.30 0.24 0.24 H3K27ac 0.8 H3K27ac 0.5 0.22 H3K4me1 H3K4me1 0.22
0.6 0.20 0.25 0.4 0.20 0.18 0.18 0.3 0.4 0.16 0.20 0.16 0.2 0.2 0.14 0.14 Read count per million mapped reads Read count per million mapped reads Read count per million mapped reads Read count per million mapped reads Read count per million mapped reads −2500 −1250 Center 1250 2500 −2500 −1250 Center 1250 2500 −2500 5′End 33% 66% 3′End 2500 −2500 5′End33%66%3′End 2500 −2500 5′End 33% 66% 3′End 2500
Scale 200 kb hg19 Scale 50 kb hg19 chr6: 151,750,000 151,800,000 151,850,000 151,900,000 151,950,000 152,000,000 152,050,000 152,100,000 152,150,000 152,200,000 152,250,000 152,300,000 152,350,000 152,400,000 152,450,000 chr20: 30,220,000 30,230,000 30,240,000 30,250,000 30,260,000 30,270,000 30,280,000 30,290,000 30,300,000 30,310,000 30,320,000 30,330,000 30,340,000 30,350,000 30,360,000 30,370,000 b e 12 - 5 - RING1B iPSCs MCF10A 1 _ 1 _ 31 - 48 - H3K4me3 1 _ 1 _ 10,000 1e+05 12 - 13 - H3K27ac 1 _ 1 _ 8000 8e+04 12 - 8 - H3K36me3 1 _ 1 _ 6000 6e+04 12 - 5 - H3K27me3 1 _ 1 _ 4000 4e+04 12 - 5 -
Super-enhancers (633) H2AK119ub 1 _ 1 _ Super-enhancers (1106) 2000 2e+04 12 - 5 - H3K27ac signal at enhancers H3K27ac signal
H3K27ac signal at enhancers RING1B 1 _ 1 _ 62 - 54 - 0 0e+00 H3K4me3 0 2000 4000 6000 8000 0 5000 10,000 15,000 20,000 1 _ 1 _ 15 - 24 - Enhancers ranked by H3K27ac Enhancers ranked by H3K27ac H3K27ac 1 _ 1 _ 12 - 9 - H3K36me3 1 _ 1 _ 12 - 5 - MDA-MB-231 MDA-MB-231 T47D H3K27me3 1 _ 1 _ 120,000 12 - 5 - 250,000 H2AK119ub 1 _ 1 _ 100,000 12 - 5 - 200,000 RING1B 1 _ 1 _ 80,000 68 - 56 - 150,000 H3K4me3 1 _ 1 _ 60,000 24 - 26 - 100,000 H3K27ac 40,000 1 _ 1 _ 12 - 10 - Super-enhancers (1092) Super-enhancers (710) 50,000 T47DH3K36me3 MCF10A 20,000 1 _ 1 _ 12 - 5 - H3K27ac signal at enhancers H3K27ac signal H3K27ac signal at enhancers H3K27ac signal H3K27me3 0 0 1 _ 1 _ 12 - 5 - 0 5000 10,000 15,00020,000 25,000 0 5000 10,000 15,000 H2AK119ub Enhancers ranked by H3K27ac Enhancers ranked by H3K27ac 1 _ 1 _ RMND1 CCDC170 ESR1 COX4I2 BCL2L1 TPX2 c f g h RING1B signal at Known motif enrichment RING1B ±200 Kb iPSCs-SEs MCF10A-SEs MDA-MB-231-specific SEs SEs with RING1B Super-enhancer Closest expressed 0.26 1% MCF10A MDA-MB-231 T47D gene 0.24 FRA1 (1e–166)
0.22 FOSL2 (1e–160) 57% 0.20 ATF3 (1e–159) MCF10A
0.18 MCF10A RACK7 targets (6.78e–21) (561 genes) ELK3 targets (1.56e–19) 0.16
0.14 ***
Read count per million mapped reads FRA1 (1e–163) MDA-MB-231-SEs T47D-SEs 0.12 −2500 5′End 33% 66% 3′End 2500 BATF (1e–152) MDA-MB-231 cJUN targets (4.94e–18) (252 genes) BRD4 targets (3.67e–17) ATF3 (1e–152)
45% 57% RING1B signal at T47D-specific SEs MDA-MB-231
MCF10A MDA-MB-231 T47D T47D ESR1 targets (4.15e–10) 0.30 (398 genes) ESR2 targets (2.71e–11) GRHL1 (1e–115) 0.25 α RING1B+ ER (1e–56) FOXA1 (1e–40) 0.20 T47D FOXM1 (1e–39) FOXA2 (1e–38) 0.15 *** Read count per million mapped reads
−2500 5′End 33% 66% 3′End 2500
Fig. 2 RING1B is recruited to super-enhancers. a H3K27ac and H3K4me1 ChIP-seq signals relative to RING1B peak summit. b Super-enhancers (SEs) identified in each cell line. c Pie charts showing percentage of SEs containing RING1B. d RING1B ChIP-seq signal at SEs. e Genome browser screenshots of RING1B and histone modifications. SE regions near ESR1 and BCL2L1 are highlighted in yellow. f RING1B ChIP-seq signal at T47D-specific SEs (top) or MDA- MB-231–specific SEs (bottom). RING1B ChIP-seq signal in RING1B-T47D SEs compared to RING1B ChIP-seq signal in the same genomic region in MDA- MB-231 (p-value = 3.07e − 24) and MCF10A (p-value = 2.39e − 29). RING1B ChIP-seq signal in RING1B-MDA-MB-231 SEs compared to RING1B ChIP-seq signal in the same genomic region in T47D (p-value = 1.15e − 16) and MCF10A (p-value = 1.36e − 09). Significance was determined by the Kolmogorov–Smirnov test. ***p-value < 0.001. g Transcription factor motif analysis of SEs containing RING1B. h Enrichr analysis of ENCODE ChIP-seq data using the nearest genes from SEs containing RING1B
Fig. 5e). We conclude that cPRC1 complexes are co-recruited to facilitates gene activation (Fig. 4a, left, and Supplementary Fig. genes and enhancers targeted by key factors that regulate 6a). In contrast, in MDA-MB-231, RING1B depletion had a transcriptional networks in breast cancer. more modest effect on gene regulation as only about 90 genes were significantly deregulated (Fig. 4a, right). Deregulated genes in both cell lines included key genes involved in breast RING1B regulates oncogenic pathways and enhancer RNAs. cancer progression and metastasis (Fig. 4b and Supplementary Next, we determined the effects of RING1B depletion on gene Fig. 6b). Additionally, these deregulated genes were sig- expression in T47D and MDA-MB-231 cells. We found that in nificantly enriched as ERα targets in T47D cells (Supplementary T47D, more genes were downregulated than upregulated (62% Fig. 6c). CD36, which regulates fatty acid metabolism and versus 38%) after RING1B depletion, suggesting that RING1B metastasis31, was the second most upregulated gene in
NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x
Main PRC1 in T47D Main PRC1 in MDA-MB-231 a Endogenous RING1B IP in T47D (n =3) b 1.0 Endogenous RING1B IP in MDA-MB-231 (n =3) 1.0 3.5 5
RING1B 4.5 RING1B 0.8 3 0.8 PCGF2 CBX8 PCGF2 PHC2 PCGF2 CBX8 CBX8 PHC3 4 CBX4 CBX2 CBX8 PHC2 RING1B 2.5 PHC2 CBX4 3.5 PHC3 RING1B 0.6 KDM2B PHC1 PCGF4 0.6 PCGF2 3 BCORL value RING1B/IgG PHC3 value RING1B/IgG 2 PCGF1 p p MGAP RYBP 0.4 2.5 TREF1 FBRS 0.4 UBP2L BCORPCM1 test
test RING1 t t 1.5 DCAF7
s 2 s ′ ′
1.5 Relative abundance 0.2 0.2
1 Relative abundance >0.04 1 0.5 0.5 0.0 0.0 –Log student –Log student FDR=0.01 FDR=0.01 0 0 CBX8 PHC3 CBX4 PHC2 CBX8PHC2 CBX2CBX4PHC3RL22PHC1PCM1 –8 –6 –4 –2 0 2 4 6 8 10 12 –10 –8 –6 –4 –2 0 2 4 6 8 10 RING1B PCGF2 RING1B PCGF2PCGF4 Student′s t test difference RING1B/IgG Student′s t test difference RING1B/IgG
c d f g MDA-MB-231 α T47D RING1B PCGF2 BRD4 RING1B PCGF2 ER RING1B 377 RING1B 2137 2202 PCGF2 585 786 4813 134 840 392 PCGF2 335 2408 890 1041 BRD4 ERα 940 663 2955 2146 2961 1273 463
ChIP-seq peaks T47D h e PRC1+ BRD4 targets PRC1+ ERα targets ChIP-seq peaks MDA-MB-231 Pathways in cancer CCKR signaling RAS signaling WNT signaling Focal adhesion EGFR signaling PI3K-AKT pathway Endothelin signaling –2.5 Kb +2.5 Kb –2.5 Kb +2.5 Kb –10 –8 –6 –4 –2 0 –4 –3 –2 –1 0 log2 (p value) log2 (p value)
Scale 100 kb hg19 chr9: 117,450,000 117,500,000 117,550,000 117,600,000 117,650,000 117,700,000 117,750,000 ijk23 -
α RING1B 1 _ RING1B PCGF2 ER H3K27ac H3K4me1 H3K27me3 RING1B PCGF2 BRD4 H3K27ac H3K4me1 H3K27me3 7 - PCGF2
1 _ 15 -
BRD4
1 _ 24 -
H3K27ac
MDA-MB-231 1 _ 26 - H3K4me1
1 _ 23 - enhancer marks in T47D H3K27me3 +
α 1 _ C9orf91 TNFSF15 TNFSF8 LOC100505478 Mir_633
Scale 100 kb hg19 chr11: 101,050,000 101,100,000 101,150,000 101,200,000 101,250,000 101,300,000 28 -
PRC1/ER RING1B
1 _ 14 - PRC1/BRD4 + enhancer marks in MDA-MB-231 PCGF2 –2.5 Kb +2.5 Kb –2.5 Kb +2.5 Kb 1 _ 32 -
ERα cPRC1 SEs cPRC1 SEs 1 _ l 20 - – BRD4 7.5% T47D H3K27ac α – ER 1 _ 19% 24 - + ERα + BRD4 H3K4me1 81% 92.5% 1 _ 28 - H3K27me3
1 _ PGR TRPC6 LOC101054525
Fig. 3 The RING1B interactome and its genome-wide association with ERα and BRD4 in ER+ and TNBC cells. a Endogenous RING1B immunoprecipitation with whole-cell extracts. Proteins bound to RING1B were identified by LC-MS/MS, and enrichment was calculated based on LFQ intensities. IgG was used as a negative control. Experiments were performed in three biological replicates. b Relative abundance of RING1B interactors. c ChIP-seq heat maps of RING1B, PCGF2, and ERα in T47D. d Overlapping of RING1B, PCGF2, and ERα target genes in T47D. e GO analysis of RING1B/PCGF2/ERα co-target genes. f ChIP-seq heat maps of RING1B, PCGF2, and BRD4 in MDA-MB-231. g Overlapping of RING1B, PCGF2, and BRD4 target genes in MDA-MB-231. h GO analysis of RING1B/PCGF2/BRD4 co-target genes. i–j ChIP-seq heat maps of RING1B, PCGF2, ERα, and histone modifications associated with active enhancers and SEs in T47D, and PCGF2, BRD4 in MDA-MB-231. k Genome browser screenshots of SEs. SE regions are highlighted in yellow. l Pie charts of cPRC1-SEs with ERα in T47D and BRD4 in MDA-MB-231 shRING1B T47D (Fig. 4a, left and Supplementary Fig. 6b) and in both shRING1B T47D and MDA-MB-231 cells confirmed the fatty acid metabolism pathway was upregulated after the RNA-seq results, and further suggested that fatty acid RING1B depletion (Fig. 4b, left, Supplementary Fig. 6b, and metabolism (represented by CD36 and HMGCS2)mayplaya Supplementary Data 5). In RING1B-depleted MDA-MB-231 major role in the tumorigenesis of ER+ breast cancer (Fig. 4c). cells, several well-known oncogenic signaling pathways were Although RNF2 amplification did not correlate with over- also deregulated after RING1B depletion (Fig. 4b, right and expression in patients with HER2+ tumors, RNF2 expression Supplementary Fig. 6b). RT-qPCR of select cancer-related genes was significantly elevated compared to normal breast tissues
6 NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x ARTICLE
a b c shCTR T47D MDA-MB-231 T47D shRING1B shCTR shRING1B shCTR shRING1B GSEA analysis shRING1B GSEA analysis shRING1B 20 1.2 T47D EGR3 MAP3K3 MDA-MB-231 15 0.9 ADAMTS15 HSD17B10 COL6A1 Protein secretion EMT TGFBR3 10 0.6 NFKB signaling ADAMTS14 Fatty acid metabolism KRAS signaling up 5 0.3 BCL2 mTOR signaling Relative to RPO FGFR2 WNT signaling 0 0.0 TGFB1 CDKN1A EMT NOTCH signaling SNAI1 CD36 GATA5 ID1 hypoxia SOX2 EGR3 RING1B FGFR2 GATA4 Estrogen resp. early HMGCS2 NOTCH1 TGF beta signaling MDA-MB-231 MAP3K3 Estrogen resp. late MYC targets TCF3 6 1.2 E2F targets CDH1 Hypoxia ID3 0.9 CD9 mTOR signaling 4 CD44 KRAS signaling down FGFR4 Protein secretion 0.6 ID1 DUSP1 DOT1L NOTCH signaling Fatty acid metabolism 2 0.3 EREG MMP1 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 Relative to RPO FZD8 –3.0–2.5–2.0–1.5–1.0–0.5 –3.0–2.5–2.0–1.5–1.0–0.5 SOX2 0 0.0 COL7A1 NES NES CDH10 IL6 (NOM p value<0.05) (NOM p value<0.05) CD36 MMP9 ID1 q value<0.05 HMGCS2 q value<0.05 DOT1L MMP1 MMP9 RING1B MAP3K3
±200 Kb d e T47D f RING1B g T47D T47D eRNA upregulated at RING1B-SEs Super-enhancer ALL expressed p = 0.003502 0.16 SE eRNA upregulated SE eRNA downregulated shCTR genes shRING1B 6 *** 4 5 0.12 T47D T47D 10 4 3 (404 SEs) (2484 genes) 3 2 *** RPKM RPKM 2 5 1 1 0.08 0 0 shCTR shRING1B Log2(FPKM) Read count per 0 shCTRL shRING1B shCTRL shRING1B million mapped reads 0.04 −450 −225 Center 225 450
MDA-MB-231 MDA-MB-231 T47D shCTR shCTR SE eRNA upregulated SE eRNA downregulated eRNA downregulated at RING1B-SEs shRING1B shRING1B 4 3 Cluster 1 Cluster 2 *** shCTR Cluster 1 3 2 0.30 shRING1B 2 ***
RPKM 1 RPKM 1 0.25 0 0 0.20 shCTRL shRING1B shCTRL shRING1B
0.15 107 genes differentially expressed Read count per Cluster 2
million mapped reads ENCODE ChIP-seq: 0.10 ESR1_T47D_hg19 (8.953e–7) −700 −350 Center 350 700 h i TCGA ER+ breast cancer GFP-Luc GFP-Luc shCTR shRING1B Luminescence T47D metastasis in lungs ++++++++ 12,000 +++++++++++++++++++++++++++++++ 07 1.00 +++++++++++++ +++++++++++++++++++++++ 4.0×10 +++++ +++ ++++++++ +++ + +++ ++++ +++ +++++++++ +++ +++++++++++++ ++++ 10,000 * 0.75 ++++ +++++++ High (n = 231) 07 + +++ ++++++++++ ++++ 3.0×10 ++ ++ ++++ 8000 0.50 ++ +++ 2.0×1007 6000 0.25 p = 0.004 + Low (n = 223) 07 + 1.0×10
4000 (photons/sec) Luminescence Survival probability 0.00 0 2000 4000 2000 0 GFP-Luc GFP-Luc Counts shCTR shRING1B Time since diagnosis (days) Color scale T47D Min = 1300 Max = 12,000
j shCTRGFP-Luc shRING1BGFP-Luc TCGA basal breast cancer Luminescence MDA-MB-231 metastasis 10 ++++++++++++++++++ +++++++++++++++ 1.5×10 1.00 +++ ++++ NoAmp (n = 23) ++++++++ 60,000 ++ * +++ ++ +++++++++ 0.75 ++ + ++++ Amp (n = 75) 1.0×1010 0.50 40,000
0.25 5.0×1009 p = 0.05 20,000 (photons/sec) Luminescence Survival probability 0.00
0 2000 4000 Counts 0 GFP-Luc GFP-Luc Color scale shCTR shRING1B Time since diagnosis (days) MDA-MB-231 Min = 2500 Max = 75,000
Fig. 4 RING1B regulates specific oncogenic pathways and metastasis in breast cancer subtypes. a RNA-seq heat maps of upregulated and downregulated genes in RING1B-depleted (shRING1B) T47D and MDA-MB-231 cells. RNA-seq experiments were performed in two biological replicates. b GSEA analyses after RING1B depletion. c Real-time qPCR of selected genes in control and RING1B-depleted T47D and MDA-MB-231. Expression was normalized to the housekeeping gene RPO. Data represent the average of two independent experiments. d Box plots of deregulated eRNAs in SEs after RING1B depletion. e eRNA signal at RING1B-occupied SE regions. f Heatmap of deregulated genes near SEs containing RING1B in shRING1B T47D. g Genes in f that are downregulated (cluster 1) or upregulated (cluster 2) in shRING1B T47D. h Kaplan–Meier survival analysis of patients from TCGA with ER+ tumors (top) or Basal (TNBC) breast cancer tumors segregated by RNF2 expression. i–j Representative images of metastatic signal detected by IVIS in NSG mice 65 days after injection of control and shRING1B T47DGFP-luc and MDA-MB-231GFP-luc cells in the mammary fat pad (n = 5/group). Quantification of luciferase signal by IVIS in control and shRING1B T47DGFP-luc and MDA-MB-231GFP-luc cells. Error bars represent SD. *p-value < 0.05; ***p-value < 0.001, two-tailed t-test. Center line of box plots represent the median and upper and lower bounds of whiskers represent the maximum and minimum values, respectively
(Supplementary Fig. 2a). To assess whether RING1B depletion fold change > 2) upon RING1B KD, with 255 and 419 genes also affected oncogenic pathways in HER2+ cells, we stably upregulated and downregulated, respectively (Supplementary depleted RING1B in the commonly used HER2+ cell line, Fig. 6e), suggesting that RING1B may also positively regulate SKBR3, and performed RNA-seq experiments (Supplementary gene expression in HER2+ cells. GSEA analysis revealed a Fig. 6d). A total of 674 genes were deregulated (q-value < 0.05, strong deregulation of cancer-related pathways, including cell
NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x cycle, TGF-β and PPAR signaling, and fatty acid metabolism signaling pathway through an ERα/FOXA1 transcriptional reg- (Supplementary Fig. 6e–f). ulatory axis. Interestingly, in MDA-MB-231 cells that do not Since RING1B was bound to enhancers, we next asked whether express FOXA1, RING1B was recruited to the FOXA1 promoter RING1B depletion affected the expression of enhancer RNAs and had a canonical repressive function, co-localizing with (eRNAs)32. RING1B depletion significantly dysregulated eRNAs H2AK119ub1 and H3K27me3 histone marks (Fig. 5a). In con- transcribed from active typical enhancers and SEs (Fig. 4d and trast, in T47D cells, RING1B bound to a putative SE downstream Supplementary Fig. 6g). Importantly, RING1B was recruited to 64 of FOXA1, suggesting that it plays an activating role in regulating and 53% of SE eRNAs that were differentially expressed after FOXA1 expression (Fig. 5a). RING1B ChIP-qPCR of several RING1B depletion in both cell lines (Fig. 4e, Supplementary RING1B-SEs in control and RING1B-depleted T47D cells con- Fig. 6h). firmed the binding of RING1B to enhancer regions identified by Finally, we assessed whether RING1B depletion affected ChIP-seq, including the FOXA1 putative enhancer (Fig. 5b and expression of genes potentially regulated by RING1B-containing data not shown). We then assessed whether RING1B directly SEs (as identified in Fig. 2h). In T47D, of the 2484 genes regulates FOXA1 expression in both T47D and MDA-MB-231 identified that are potentially regulated by 404 RING1B-SEs, 107 cells. While RING1B depletion in MDA-MB-231 was not suffi- were deregulated upon RING1B depletion. Although most were cient to activate FOXA1 expression (data not shown), acute downregulated (cluster 1) and included important genes for depletion of RING1B by siRNA reduced FOXA1 protein levels breast epithelial homeostasis (e.g., LRIG1, CYP27B1, HES1, ~50% in T47D cells (Fig. 5c, left panel). Although FOXA1 levels THBS1), a set of genes were upregulated (cluster 2) (Fig. 4f). remained unaffected upon stable RING1B depletion by shRNA These results suggest that at enhancer regions, RING1B (Fig. 5c, right panel), cellular fractionation assays showed that potentially plays a dual function in gene expression (cluster 1) FOXA1 was displaced from chromatin and relocated to the and gene repression (cluster 2) (Fig. 4g). soluble nuclei fraction (Fig. 5d). Since FOXA1 is a transcription factor important for ERα recruitment to chromatin26, displace- ment of FOXA1 from chromatin also impaired chromatin loca- Role of RING1B in breast cancer tumorigenesis and metastasis. lization of ERα (Fig. 5d). This set of data suggests that RING1B We next sought to determine the function of RING1B in breast mediates the estrogen response by affecting FOXA1 and ERα cancer tumorigenesis and metastasis in vivo. We hypothesized recruitment to chromatin. that RING1B depletion increases the aggressiveness of T47D cells We then asked whether FOXA1 depletion affected RING1B due to the strong upregulation of CD36, a marker for metastasis- levels. While acute FOXA1 depletion affected the RING1B initiating cells (Fig. 4a, c and Supplementary Fig. 7a). However, protein levels moderately (Fig. 5e, left panel), stable FOXA1 RING1B depletion in MDA-MB-231 cells resulted in both posi- depletion strongly reduced RING1B global levels (Fig. 5e, right tive and negative deregulation of genes involved in breast cancer, panel). Importantly, RING1B binding to chromatin was also thus we could not anticipate the role of RING1B in TNBC in vivo. severely reduced (Fig. 5f). Analysis of FOXA1 ChIP-seq in T47D Our initial analysis of the TCGA breast cancer data set (Fig. 4h) cells did not reveal binding of FOXA1 to the RNF2 promoter indicated that patients with ER+ breast cancer and high levels of (data not shown). RNF2 survive longer than patients with lower RNF2 levels. In Finally, since we observed reduced levels of both FOXA1 and contrast, patients with basal breast cancer and high levels of RNF2 ERα at chromatin upon RING1B depletion, we asked whether have a lower survival probability. This data suggested that RING1B-depleted cells can respond to estrogen stimulation. To RING1B might exert divergent functions in tumor formation or this end, we cultured control and RING1B KD cells for 72 h in metastasis in specific breast cancer subtypes. To assess whether hormone-deprived (HD) media prior to induction of ERα T47D and MDA-MB-231 cells recapitulate the results obtained signaling with 10 nM of E2 (estradiol) for 12h34. In agreement with the TCGA data set, we injected control and shRING1B cells with the global gene expression profiles of RING1B-depleted into the mammary fat pad of NSG mice . Cells were engineered to T47D cells (Fig. 4b), there was reduced expression of prominent express a GFP-luciferase transgene to monitor tumor formation E2-responsive genes in shRING1B T47D compared to control and metastasis by IVIS (Supplementary Fig. 7b). Although we did cells (Fig. 5g). Altogether, these results demonstrated that not detect significant changes in primary tumor development RING1B is a novel epigenetic factor that directly and indirectly between control and RING1B-depleted T47D and MDA-MB-231 regulates the FOXA1–ERα axis by multiple mechanisms (Fig. 5h). cells (Supplementary Fig. 7c–d), mice with tumors derived from T47D-shRING1B cells lost more weight than control animals. In contrast, mice with tumors derived from shRING1B-MDA-MB- RING1B regulates chromatin accessibility at enhancers. Since 231 were heavier than control animals (Supplementary Fig. 7e). RING1B was recruited to regions targeted by transcription factors T47D cells are not highly metastatic33, yet shRING1B T47D but and its depletion deregulated breast cancer signaling pathways as not control cells metastasized to the lungs (Fig. 4i). In the highly well as FOXA1 and ERα localization to chromatin, we next metastatic MDA-MB-231 tumors33, depletion of RING1B hypothesized that RING1B regulates transcriptional programs in reduced the metastatic potential of these cells (Fig. 4j). Impor- breast cancer by orchestrating chromatin accessibility. To test tantly, these results are in agreement with our TCGA survival this, we performed transposase-accessible chromatin sequencing analysis (Fig. 4h), and further support the concept of RING1B (ATAC-seq)35 in RING1B-depleted cells (Fig. 6a). As expected, being a pro-metastatic gene in basal breast cancer and a sup- ATAC-seq peaks in control cells were at promoter, intronic, and pressor of metastasis in ER+ tumors. intergenic regions (Supplementary Fig. 8a). Importantly, ATAC- seq peaks co-localized with a large number of RING1B peaks in A novel RING1B-FOXA1-ERα transcriptional axis in ER+ control cells, and the majority of this co-localization occurred at cells. In T47D cells, RING1B was recruited to SEs containing introns and intergenic regions, but not at promoters (Fig. 6b). FOXA1 and ERα-binding sites (Figs. 2g and 3c, i, l). Among These results indicate that RING1B depletion affects chromatin those, RING1B bound to the SE that regulates ESR1 (encoding accessibility at enhancer regions. ERα) (Fig. 2e). Moreover, RING1B depletion strongly affected the We next asked whether RING1B depletion-induced de novo “Estrogen Response” gene signature (Fig. 4b). These results sug- generation and/or loss of accessibility sites. RING1B depletion gested that RING1B is functionally involved in the estrogen generally affected chromatin accessibility, suggesting that
8 NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x ARTICLE
Scale 20 kb hg 19 abchr14: 38,000,000 38,010,000 38,020,000 38,030,000 38,040,000 38,050,000 38,060,000 38,070,000 RING1B binding at enhancers in T47D H3K27ac at RING1B-enhancers 83 RING1B 0.20 80 1 56 * H3K4me3 * 1 0.15 10 60 H3K27ac 1 10 0.10 H3K4me1 40 1
9 % Input % Input H3K36me3 0.05 20 1 13 MDA-MB-231 H3K27me3 1 10 0.00 0 H2AK119ub shCTR shRING1B shCTR shRING1B H3K27me3 H3K27ac 1 49 RING1B IgG RING1B 1 15 (antibody #2) ERα 1 56 H3K4me3 c 1 25 H3K27ac 1 20 siCTR siRING1B shCTR shRING1B
T47D H3K4me1 1 8 43 kDa 43 kDa H3K36me3 RING1B RING1B 1 8 H3K27me3 FOXA1 55 kDa FOXA1 55 kDa 1 8 H2AK119ub 130 kDa 130 kDa 1 VINCULIN VINCULIN MIPOL1 FOXA1
d Soluble Soluble Insoluble e shFOXA1 Cytoplasm nuclei chromatin chromatin siCTR siFOXA1 shCTR #1 #2 #3 #4 #5 130 kDa VINCULIN FOXA1 55 kDa FOXA1 55 kDa ERα 72 kDa High exposure 43 kDa RING1B 43 kDa RING1B ERα 72 kDa Low exposure 130 kDa 130 kDa FOXA1 55 kDa VINCULIN VINCULIN High exposure FOXA1 55 kDa Low exposure 43 kDa RING1B g FM HD HD + E2 h Promoter H3 17 kDa 72 h 12 h MDA-MB-231 RING1B CXCL12 0.008 0.06 (TNBC) RING1B FOXA1 shCTR shCTR shCTR shCTR shCTR shRING1B shRING1B shRING1B shRING1B 0.006 shRING1B Enhancer 0.04 ERα
f Soluble Soluble Insoluble 0.004 T47D RING1B Cytoplasm nuclei chromatin chromatin (ER+) 0.02 FOXA1
130 kDa Relative to RPO 0.002 VINCULIN Enhancer ?
FOXA1 55 kDa 0.000 0.00 High exposure – E2 + E2 – E2 + E2 FOXA1 55 kDa Low exposure Positive feedback loop RING1B/FOXA1 α 43 kDa GREB1 TFF1 Does RING1B regulate FOXA1/ER chromatin accesibility? RING1B 0.05 0.05 High exposure RING1B 43 kDa shCTR 0.04 0.04 Low exposure shRING1B H3 17 kDa 0.03 0.03
0.02 0.02 shCTR shCTR shCTR shCTR Relative to RPO shFOXA1 #5 shFOXA1 #5 shFOXA1 #5 shFOXA1 #5 0.01 0.01
0.00 0.00 – E2 + E2 – E2 + E2
Fig. 5 RING1B regulates FOXA1 and ERα through multiple mechanisms. a Genome browser screenshots of the profiles of RING1B and histone modifications in T47D and MDA-MB-231 cells at the FOXA1 locus. b RING1B, H3K27me3 and H3K27ac ChIP-qPCR of RIN1GB-containing enhancers in control and RING1B-depleted T47D cells. IgG antibody was used as a negative control. As additional control, RING1B ChIP-qPCR were performed using a different RING1B antibody from the one used for ChIP-seq. Error bars represent the SD of two independent experiments. *p-value < 0.05, two-tailed t-test. c Western blot of RING1B and FOXA1 from control and RING1B-depleted cells 72 h after siRNA transfection (left panel) or after puromycin selection of shRING1B T47D cells (right panel). VINCULIN was used a loading control. d Western blot of RING1B, FOXA1 and ERα after cellular fractionation of control and RING1B-depleted T47D cells. VINCULIN and histone H3 were used as a cytoplasmic and chromatin fraction controls, respectively. e Western blot of RING1B and FOXA1 from control and FOXA1-depleted cells 72 h after siRNA transfection (left panel) or after puromycin selection of shFOXA1 T47D cells (right panel). VINCULIN was used a loading control. f Western blot of RING1B and FOXA1 after cellular fractionation of control and FOXA1-depleted T47D cells. VINCULIN and histone H3 were used a cytoplasmic fraction and chromatin fraction control, respectively. All the cellular fractionation experiments and total protein extracts shown in the figure were performed at least three times. g RT-qPCR of E2-responsive genes in control and RING1B-depleted T47D after administration of E2 (10 mM) for 12 h in cells cultured in hormone-deprived (HD) media for 72 h. FM full media. Error bars represent SD of two independent experiments. h Model of RING1B action in MDA-MB-231 and T47D cells
RING1B is involved in both opening and closing chromatin (Fig. 6e, f, bottom). These results suggest that RING1B plays a (Fig. 6c, d). Upon RING1B depletion, the ATAC-seq peaks either dual role in regulating chromatin accessibility. lost or gained de novo were located at introns and intergenic We next analyzed the impact of RING1B depletion on regions (Supplementary Fig. 8b–c). Notably, RING1B was chromatin accessibility at enhancers. In T47D cells, RING1B recruited to genomic regions not accessible to transposase in depletion resulted in the loss of about 500 peaks and gain of more control cells but became accessible in RING1B-depleted cells than 600 de novo peaks at enhancers (Fig. 6g). RING1B binds to (Fig. 6e, f, top). Further, RING1B was recruited to open 55% of SEs and 23% of typical enhancers (Fig. 6g). Transcription chromatin sites and its depletion-induced chromatin compaction factor motif analysis revealed that ATAC-seq peaks lost at
NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x
a b 1200 ATAC-seq peaks c d containing RING1B siCTR vs siRING1B siCTR vs siRING1B MDA-MB-231 T47D 1000 in control cells T47D T47D MDA-MB-231 siCTRL siRING1B siCTRL siRING1B MB-231 siRING1B specific peaks siRING1B specific peaks 43 kDa 800 RING1B 10.6% 15.3% 600 TUBULIN 55 kDa siCTR-specific peaks 400 siCTR-specific peaks 6.6% ATAC-seq peaks ATAC-seq 24.7% 200 siRNA CTR/RING1B Common peaks Common peaks 0 64.6% 78.1% ATAC-seq after 72 h er (2 biological replicates) Exon Intron Intergenic Promot e f g h New ATAC-seq peaks siRING1B-T47D New ATAC-seq peaks siRING1B MB-231 ATAC-seq peaks lost at ATAC-seq peaks lost at enhancers in siRING1B-T47D siCTRL 0.16 siCTRL enhancers in siRING1B-T47D 600 0.20 siRING1B siRING1B ChIP_RING1B 0.14 ChIP_RING1B 500 RING1B+ SCL (1e–17) 0.12 85 (54.8%) FOXA2(1e–13) 0.15 400 0.10 NF1 (1e–11) 300 0.10 0.08 AR (1e–11) 200 82 (22.8%) 0.06 ATAC-seq peaks ATAC-seq CTCF (1e–11) 0.05 Super enhancer Read count per million mapped reads Read count per million mapped reads 100 −2500 −1250 Center 1250 2500 −2500 −1250 Center 1250 2500 Typical enhancer FOXA1 (1e–11) 0
ATAC-seq peaks lost in siRING1B-T47D ATAC-seq peaks lost in siRING1B MB-231 New ATAC-seq peaks New ATAC-seq peaks 0.16 siCTRL 0.18 siCTRL at enhancers in siRING1B-T47D siRING1B siRING1B 700 at enhancers in siRING1B-T47D 0.14 ChIP_RING1B 0.16 ChIP_RING1B RING1B+ 600 CTCF (1e–260) 0.14 93 (63%) 0.12 500 GRHL2 (1e–13) 0.12 0.10 400 0.10 IRF2 (1e–11) 0.08 300 113 (22.5%) 0.08 NRFS (1e–11) 200 0.06 0.06 ATAC-seq peaks ATAC-seq AP-2gamma (1e–11) Read count per million mapped reads Read count per million mapped reads 100 Super enhancer −2500 −1250 Center 1250 2500 −2500 −1250 Center 1250 2500 Typical enhancer FOSL2 (1e–11) 0
T47D
Scale 1 kb hg19 Scale 5 kb hg19 ,chr20:, 49,343,000 49,343,000 49,343,00049,344,000 49,344,000 49,345,000 49,345,000 chr14: 94,796,000 94,797,000 94,798,000 94,799,000 94,800,000 94,801,000 94,802,000 94,803,000 94,804,000 94,805,000 94,806,000 94,807,000 94,808,000 94,809,000 94,810,000 i ATAC-seq peaks lost at jk11 _ 25 _ ATAC-seq peaks lost at ATAC siCTRL enhancers in siRING1B MDA-MB-231 1 1 enhancers in siRING1B MDA-MB-231 11 _ 25 _ 400 ATAC siRING1B 1 1 13 _ 26 _ FRA1 (1e–119) RING1B RING1B+ 1 1 20 _ 24 _ 300 ERα 1 1 34 (44.1%) CTCF (1e–98) 13 _ 8 _ PCGF2 1 1 JUN-AP1(1e–95) 23 _ 26 _ 200 H3K27ac 1 1 7 _ 18 _ 39 (16%) FOSL2 (1e–94) H3K4me1 1 1 7 _ 8 _ RUNX1(1e–21) H3K4me3 100 1 1 ATAC-seq peaks ATAC-seq Typical enhancer 7 _ 8 _ H3K36me3
1 1 Super enhancer GATA3 (1e–6) 7 _ 8 _ H3K27me3
0 1 1 7 _ 8 _ H2AK119ub New ATAC-seq peaks New ATAC-seq peaks 1 1 at enhancers in siRING1B MDA-MB-231 at enhancers in siRING1B MDA-MB-231 MDA-MB-231
Scale 1 kb hg19 Scale 2 kb hg19 chr8: 23,617,500 23,618,000 23,618,500 23,619,000 23,619,000 23,620,000 23,620,500 23,621,000 23,621,500 chr13: 106,802,500 106,803,000 106,803,500 106,804,000 106,804,500 106,805,000 106,805,500 106,806,000 106,806,500 106,807,000 106,807,500 106,808,000 106,808,500 106,809,000 106,809,500 106,810,000 106,810,500 800 44 _ 100 _ RING1B+ ATAC siCTRL 1 1 CTCF (1e–234) 44 _ 100 _ 95 (53.7%) ATAC siRING1B
1 1 FRA1 (1e–219) 19 _ 10 _ 600 RING1B
1 1 14 _ 16 _ FOSL2 (1e–187) BRD4
1 1 400 6 _ 9 _ 77 (14.2%) PCGF2 NF-E2 (1e–26) 1 1 6 _ 9 _ H3K36me3
1 1 200 AR (1e–9) 18 _ 23 _ ATAC-seq peaks ATAC-seq Typical enhancer H3K27ac 1 1 SMAD3(1e–6) 17 _ 22 _ Super enhancer H3K4me1
1 1 8 _ 9 _ 0 H3K4me3
1 1 6 _ 9 _ H3K27me3
1 1 6 _ 9 _ H2AK119ub
1 1 5S rRNA Fig. 6 RING1B regulates chromatin accessibility at enhancers. a Western blot of RING1B from control and RING1B-depleted cells 72 h after siRNA transfection. TUBULIN was used a loading control. ATAC-seq experiments were performed in two biological replicates after siRNAs transfections. b ATAC- seq peak distribution in genomic sites bound by RING1B in RING1B-depleted cells. c, d Pie charts showing percentage of ATAC-seq peaks not affected by RING1B depletion (common peaks), lost after RING1B depletion (siCTR-specific peaks), or gained after RING1B depletion (siRING1B-specific peaks. Two ATAC-seq experiments were performed after two independent siRING1B transfections. e, f RING1B ChIP-seq signals and ATAC-seq signals at acquired and lost ATAC-seq peaks. g ATAC-seq peaks at enhancers after RING1B depletion, number of enhancers containing RING1B ChIP-seq signals in T47D. h Transcription factor-binding motif analysis of peaks acquired or lost at enhancers in T47D. i Acquired and lost ATAC-seq peaks at enhancers after RING1B depletion, number of enhancers containing RING1B ChIP-seq signals in MDA-MB-231. j Transcription factor-binding motif analysis in peaks acquired or lost at enhancers in MDA-MB-231. k Genome browser screenshots of ChIP-seq and ATAC-seq profiles at selected enhancers enhancer regions contained FOXA1/2-binding sites (Fig. 6h, top), line with the modest gene expression changes in shRING1B further confirming a functional association between RING1B and MDA-MB-231 cells. However, about 300 and 700 ATAC-seq ERα. In contrast, de novo ATAC-seq peaks in T47D-contained peaks were lost and gained at enhancers, respectively, after CTCF-binding sites, suggesting that RING1B might be involved RING1B depletion (Fig. 6i). Interestingly, in addition to CTCF in maintaining topological-associated domains (TADs)36 (Fig. 6h, sites, accessibility was altered for breast cancer-specific transcrip- bottom). tion factors (Fig. 6j). Furthermore, altered chromatin accessibility The influence of RING1B on chromatin accessibility in MDA- at enhancers were co-bound by cPRC1/ERα and cPRC1/BRD4 in MB-231 was less profound than in T47D (Fig. 6d), which is in T47D and MDA-MB-231 cells, respectively (Fig. 6k). Overall,
10 NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x ARTICLE
a b HepG2-SEs c HepG2 K562 Known motif enrichment super-enhancers with RING1B in HepG2 200,000 – RING1B + RING1B CLOCK-Liver ChIP-seq (1e–249) 3e+05 34% 66% 150,000 BMAL-Liver ChIP-seq (1e–236) 3e+05 NPAS2--Liver ChIP-seq (1e–182) 100,000 K562-SEs 1e+05 – RING1B bHLHE40-HepG2 ChIP-seq (1e–156) 50,000 5% Super-enhancers (1246) Super-enhancers (852) HNF4a-HepG2 ChIP-seq (1e–114) H3K27ac signal at enhancers H3K27ac signal at enhancers 0 0e+00 + RING1B 95% Erra-HepG2 ChIP-seq (1e–28) 0 5000 10,000 15,000 0 5000 10,000 15,000 20,000 Enhancers ranked by H3K27ac Enhancers ranked by H3K27ac
d e HepG2 g
20 _ 50 kb A1 RING1B AT RING1B H3K27ac H3K4me1 H3K27me3 G RING1B H3K27ac H3K4me1 H3K27me3bHLHE40 1 81 _ bHLHE40
1 39 _ H3K27ac
1 70 _ H3K4me1
1 20 _ H3K27me3
1 INSR
f Known motif enrichment super-enhancers with RING1B in K562
GATA1-K562 ChIP-seq (1e–1601)
GATA2-K562 ChIP-seq (1e–1583) RING1B/enhancers/TF peaks in K562 RING1B/enhancers/TF
RING1B/enhancers/TF peaks in HepG2 RING1B/enhancers/TF cJun-AP1/K562 ChIP-seq (1e–596)
NFE2-K562 ChIP-seq (1e–312)
Bach1-K562 ChIP-seq (1e–278)
–2.5 kb +2.5 kb –2.5 kb +2.5 kb
h K562 i 50 kb 50 kb
10 kb 243 _ 73 _ 154 _ RING1B RING1B
1 1 1 23 _ 71 _ 73 _ GATA1 bHLHE40 1 60 _ 1 1 151 _ 194 _ H3K27ac
1 H3K27ac 30 _
1 1
HepG2 89 _ 58 _ H3K4me1
1 154 _ H3K4me1
1 1 H3K27me3 89 _ 42 _ 1 H3K27me3
1 1 GAB2 243 _ 73 _ RING1B
1 1 j 71 _ 80 _ FOXA1 ERα ER+ breast cancer GATA1 1 1 151 _ 194 _
cPRC1 H3K27ac 1 1 BRD4 TNBC breast cancer 89 _ 58 _ K562 RING1B H3K4me1
1 1 GATA1 Leukemia 89 _ 42 _ H3K27me3
1 1 bHLHE40 Hepatocellular carcinoma NFE2L2 AGPS CDCP1 H3K27me3 H3K27ac PRC2 Canonical Oncogenic enhancers RING1B function H3K4me1 H3K4me3 H2AK119ub1
Fig. 7 RING1B is recruited to super-enhancers in other cancer types. a SEs identified in HepG2 and K562 cells. b Percentage of SEs containing RING1B in HepG2 and K562. c Transcription factor-binding motif enrichment of SEs containing RING1B in HepG2 cells. d ChIP-seq heat maps of RING1B and histone modifications associated with active enhancers and SEs. e Genome browser screenshots of co-occupancy of RING1B and bHLHE40 at enhancers in HepG2. f Transcription factor-binding motif enrichment of SEs containing RING1B in K562 cells. g ChIP-seq heat maps of RING1B and histone modifications associated with active typical enhancers and SEs, and GATA1 in K562 cell lines. h Genome browser screenshots of co-occupancy of RING1B and GATA1 at enhancers in K562. i Genome browser screenshots of RING1B and GATA1 or RING1B and bHLHE40 co-occupancy at specific SEs in K562 and HepG2 cells, respectively. j Model: In cancer, cPRC1 complexes have a dual function. cPRC1 is recruited to gene promoters to repress gene expression and to active cancer-specific enhancers in different cancer subtypes to modulate their expression and chromatin accessibility to oncogenic transcription factors these results confirm that RING1B has dual function in regulating this end, we used public RING1B ChIP-seq data sets from transcriptional programs in breast cancer cells and does so by ENCODE in a leukemia cell line, K562, and in a hepatocellular altering chromatin accessibility for key transcription factors and carcinoma cell line, HepG2. Notably, in both cell lines, RING1B chromatin organization proteins. co-localized with the enhancer-associated histone modifications (Supplementary Fig. 9a). We identified 1246 SEs in HepG2 and RING1B is recruited to enhancers in other cancer types.We 852 SEs in K562 cells, of which 66 and 95% contain RING1B, finally sought to determine whether RING1B recruitment to SEs respectively (Fig. 7a, b). Moreover, RING1B peaks that co- only occurs in breast cancer cells or if, in contrast, RING1B localized with H3K4me1 and H3K27ac were devoid of acquired the ability to bind to enhancers in other cancer types. To H3K27me3 (Supplementary Fig. 9b) and about 40% of typical
NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x enhancers were occupied by RING1B in both HepG2 and K562 Although the classical PRC1-mediated gene regulation is to cells (Supplementary Fig. 9c). compact chromatin, PRC1 complexes are also involved in facil- RING1B-containing SEs in breast cancer cells included binding itating gene transcription6,7,28,44,45. The exact function of PRC1 sites for oncogenic transcription factors (Fig. 2g). In HepG2 cells, complexes in gene activation, and the molecular mechanisms that RING1B was recruited to SEs bound by key circadian rhythm permit Polycomb complexes to activate genes, are under intense transcription factors, including CLOCK, BMAL, and NPAS2, that investigation. Recently, it has been shown that the PRC1 complex directly regulate the expression of BHLHE40, another core clock is redistributed genome-wide during oncogenesis in Drosophila component identified in our analysis (Fig. 7c)37. Importantly, to sites decorated with H3K27ac7. In melanoma, RING1B is disruption of the circadian clock has been implicated in liver recruited to chromatin to repress gene activity, but it is also cancer and the abnormal expression of clock genes correlates with recruited to transcriptionally active genes devoid of H3K27me3 increased tumor size and cell proliferation38,39. Moreover, using and H2AK119ub144. More recently, it has been shown than in the TCGA liver hepatocellular carcinoma (LIHC) data set, we adult epidermal stem cells, PRC1 is recruited to gene promoters found that 9% of patients with LIHC have amplification of RNF2 containing histone modifications typically found in active (Supplementary Fig. 9d), and its expression is significantly higher enhancers45. Here, we have provided the first evidence that in liver tumors compared to normal liver (Supplementary Fig. 9e). RING1B and cPRC1 complexes are recruited to enhancer regions Finally, we confirmed that RING1B-containing enhancers in in cancer cells and that RING1B depletion has a major impact on HepG2 cells were also decorated with BHLHE40 (Fig. 7d, e), and chromatin accessibility at enhancers. Mechanistically, we show a 72% of SEs contained both RING1B and BHLHE40 (Supplemen- functional crosstalk between RING1B and the FOXA1/ERα axis, tary Fig. 9f, left). which ultimately resulted in an attenuated response to estrogen. In K562 cells, RING1B was recruited to SEs containing binding FOXA1, as a nuclear receptor regulatory factor, is not limited to motifs for GATA1 and GATA2 factors (Fig. 7f). GATA2 is often ERα as it also interacts with the androgen receptor (AR) to reg- mutated in myeloid malignancies while GATA1 is overexpressed ulate its deposition to chromatin in prostate cancer cells46. Thus, in acute myeloid leukemia (AML), highlighting the role of GATA it remains to be determined whether RING1B functionally factors in leukemia40. Importantly, we also confirmed that associates with FOXA1 and AR in other cancer types or during GATA1 is recruited to RING1B-containing enhancers and to embryonic development. 65% of the SEs in K562 cells (Fig. 7g, h and Supplementary None of the cPRC1 components have the ability to directly Fig. 9f, right). As expected, RING1B binding to SEs in K562 and bind DNA. Therefore, we anticipate that non-genomic factors are HepG2 cells was cell type specific (Fig. 7i). These observations involved in their recruitment to chromatin. One possibility could lead us to conclude that RING1B is a novel epigenetic factor that be that cPRC1 components are post-transcriptionally modified at promotes important transcriptional regulatory networks at SEs. Since CBX proteins can bind RNA47, another possible enhancers to promote oncogenic pathways in multiple cancer mechanism could involve CBX8 interacting with eRNAs to types (Fig. 7j). recruit RING1B and the cPRC1 complex to specific enhancers. Interestingly, physical interactions between PRC1 and Fs(1)h and Br140, the Drosophila orthologs of BRD4 and BRD1, have been Discussion recently described48. Although we did not detect a physical During the last decade, extensive sequencing of cancer genomes interaction of RING1B with BRD4 in the MDA-MB-231 cell line has revealed mutations of transcription factors, epigenetic under our experimental conditions, we observed a strong co- machineries, and signaling pathway factors, and led to the occupancy of BRD4 and cPRC1 at active genes, in agreement with development of novel therapeutic targets. Nonetheless, it remains the observation of Kuroda and colleagues. Our studies also crucial to investigate the molecular mechanisms, enzymes, and indicate that both of these epigenetic machineries are co-recruited epigenetic machineries that are dysregulated and altered in can- to enhancer regions in breast cancer cells. cer. The importance of this complementary approach is exem- Recruitment of RING1B to active enhancers would appear to plified by the PRC1-mediated mechanisms we propose. While suggest that RING1B is solely involved in positively regulating PRC1-encoding genes are not typically mutated in cancer, we their expression. Surprisingly, we discovered a much more found that several canonical PRC1 genes are amplified and dys- complicated scenario, in which RING1B can exert canonical and regulated in many hormone-related cancers, including breast non-canonical functions at enhancers, both at the levels of their cancer. Hormone-related cancers share a unique mechanism of transcriptional activity and transcription factor accessibility. We action, as hormones drive proliferation which induces the accu- theorize that in a set of highly active enhancers, RING1B is mulation of mutations41. Whether hormones contribute to required for their activity, while in another set of enhancers with chromosome instability and genomic rearrangements of genomic diminished activity, RING1B is required to prevent a hyper- sites of PRC1 genes in breast cancer remain to be addressed. activation of the enhancer. This model is in agreement with a We propose that in normal breast epithelial and breast cancer recent report showing that RACK7 and KDM5C are recruited to cells, RING1B function is uncoupled from its classical role as a enhancers, where they act to hamper full enhancer activation in repressor of lineage genes2. In pluripotent cells, RING1B is the cancer cells49. Overall, we suggest that intricate epigenetic main E3-ligase that mono-ubiquitinates histone H2A42. In con- mechanisms mediate enhancer activity and disruption of this trast, in the breast cancer cells used in this study, RING1A is regulation may contribute to tumorigenesis. more enzymatically active towards histone H2A than RING1B. The contribution of Polycomb complexes in breast cancer TRIM37 was recently proposed as a novel histone H2A ubiquitin tumorigenesis and metastasis is largely unknown. Here we show ligase in breast cancer cells, with a chromosomal copy-number that high levels of RNF2 in patients with ER+ breast cancer amplification at 17q2343. T47D and MDA-MB-231 cells do not tumors correlate with good survival outcome, while high RNF2 have amplification of this chromosome arm, which is consistent levels in patients with basal breast cancer correlate with lower with our results suggesting that RING1A is the main histone H2A survival probability. These surprising results are consistent with mono-ubiquitin ligase in these cells. Further analyses at the bio- our xenografts experiments and with other reports showing that chemical level are required to determine the exact mechanisms RING1B is required for migration of TNBC cells50. It has been underlying RING1A and TRIM37 deposition of H2AK119ub1 in also shown that high levels of RING1B correlated with metastatic the context of breast cancer. squamous cell carcinoma50,51. The molecular mechanisms by
12 NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x ARTICLE which RING1B either prevents or enhances metastasis in specific scraper and washed 1× with PBS, then centrifuged for 5 min at 400×g. Cell pellet breast cancer subtypes remain to be fully understood. Future was resuspended 1:5 (w:v) in Buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, work coupling genomics and genome architecture with functional 10 mM KCl, 0.05% NP-40) supplemented with protease inhibitors and 0.5 mM DTT. After 10 min on ice, cells were centrifuged for 5 min at 400×g. The super- assays may help reveal which of the RING1B-mediated molecular natant, representing the cytosolic fraction, was collected and stored at 4 °C. The mechanisms contribute to breast cancer metastasis. Finally, we remaining nuclear pellet was resuspended in ¾ of the initial volume with Buffer B propose that development of small molecules to impair RING1B (5 mM HEPES [pH 7.9], 1.5 mM MgCl2, 0.2 mM EDTA, 26% glycerol) supple- recruitment to specific genomic sites in TNBC tumors may have mented with protease inhibitors and 0.5 mM DTT, and homogenized with 20 strokes in a dounce homogenizer fitted with pestle A. After 20 min on ice, important therapeutic implications. extracts were centrifuged for 20 min at 16,000×g. The supernatant, representing the soluble nuclei fraction, was collected and stored at 4 °C. The remaining pellet was Methods resuspended in ½ of the original volume with high-salt buffer (300 mM NaCl, 50 mM Tris-HCl [pH 8], 10% glycerol, and 0.2% NP-40) supplemented with Cell lines. Human iPSCs (ATCC #ACS-1021) were maintained in complete feeder- protease inhibitors and sonicated with a Bioruptor for 5 min in 30” ON-OFF cycles. free mTESR1 culture medium (STEMCELL Technologies #85850) on matrigel- After centrifugation at 16,000×g for 20 min, the supernatant, representing the coated plates (Corning #354277) at 37 °C with 5% CO . The culture medium was 2 soluble chromatin fraction, was stored at 4 °C. The remaining pellet, representing changed daily and iPSCs colonies were enzymatically passaged with StemPro the insoluble chromatin fraction, was resuspended in volume equal to original Accutase Cell Dissociation Reagent (Thermo Fisher Scientific #A1110501) at a volume with 2×Laemmli buffer and sonicated with a Bioruptor for 10 min in 30” 1:4–1:6 split ratio every 4–7 days. DMEM/F-12 media (STEMCELL Technologies ON-OFF cycles. Protein concentration of cytosolic, soluble nuclei and soluble #36254) was used to detach colonies. ROCK inhibitor Y-27632 (STEMCELL chromatin fractions were determined by Bradford assay, and 20 μg of protein and Technologies #72302) was used in every split and when cells were thawed from 1/5 of the insoluble chromatin fraction material were loaded onto SDS-PAGE gels liquid nitrogen. If identified, spontaneously differentiated cells were mechanically followed by western blotting. removed prior to passaging. MCF10A, MDA-MB-231, T47D, and SKRB3 (ATCC #CRL-10317, HTB-26, HTB-133, and HTB-30) were maintained at 37 °C with 5% – CO2 and split every 2 3 days according to ATCC recommendations. Culture media Transfection of siRNAs. The day before siRNA transfection, 2 × 105 cells were fi was supplemented with 1× penicillin/streptomycin (Thermo Fisher Scienti c seeded in 6-well plates and maintained in antibiotic-free culture medium. 25 nM fi #15140-122) and 1× glutamax (Thermo Fisher Scienti c #35050-061), and com- siRNAs (Sigma-Aldrich #SIC007 for CTR, EHU230291 for RING1A, EHU109061 — ’ plete culture media for each cell line were as follows: MCF10A DMEM/Ham sF- for RING1B, and EHU155811 for FOXA1) were transfected using Lipofectamine fi 12 (1:1) (Corning #45000-348) with 5% horse serum (Thermo Fisher Scienti c RNAiMAX (Thermo Fisher Scientific #13778150) following the manufacturer’s fi #16050-122), 10 ng/ml EGF (Thermo Fisher Scienti c #PHG0311), 50 ng/ml instructions. cholera toxin (Sigma-Aldrich #C8052), 10 μg/ml insulin (Sigma-Aldrich #91077 C), and 500 ng/ml hydrocortisone (Sigma-Aldrich #H0888); MDA-MB-231—DMEM (Lonza #12001-576) with 10% FBS (Benchmark #100-106); T47D—RPMI-1640 Animal studies. The University of Miami Institutional Animal Care and Use (Lonza #95042-506) with 10% FBS and 10 μg/ml insulin; SKBR3—McCoy’s5a Committee (IACUC) approved all animal procedures. shCTR and shRING1B Medium Modified (Lonza #12001-562) with 10% FBS. For experiments in which T47D and MDA-MB-231 cells were transduced with retroviruses expressing GFP- estrogen (10 nM E2) was added to T47D, cells were maintained in phenol-free luciferase (pMSCV-IRES-Luciferase-GFP), and successful transduction confirmed RPMI-1640 (Thermo Fisher Scientific #32404014) supplemented with 5% charcoal- by imaging cells on an Olympus IX70 fluorescence microscope with a GFP filter. stripped FBS (Benchmark #100-119) for 72 h prior to treatment. In experiments After transduction of cells, GFP-positive cells were collected by FACS. 1 × 106 with BRD4 inhibition, MDA-MB-231 cells were treated either with DMSO or shCTR and shRING1B-MDA-MD-231GFP-luc cells and 5 × 106 shCTR and 500 nM JQ1, obtained from the Bradner lab. Cells were routinely tested to be free shRING1B T47DGFP-luc cells were injected into the mammary fat pad of 8-week- of mycoplasma infection. Cells were imaged using an Olympus IX70 inverted old female NSG mice (Jackson Labs #005557) (n = 5 per group). Sample size was microscope fitted with a phase-contrast filter. chosen to generate enough power for statistical significance and mice were ran- domly allocated to experimental groups, estimating variance is similar for the two groups. Injection of tumor cells were not blinded. Tumor development and Generation of cells stably expressing shRNAs . To produce shRNA lentiviruses, metastasis were monitored weekly using an in vivo imaging system (IVIS, Perkin 2×106 HEK293T cells (ATCC #CRL-3216) were plated into a 10 cm2 plate and Elmer) during the course of 65 and 72 days for MDA-MB-231 cells and TD47, transfected 16 h later with 8 μg of pLKO-shRNAs (Addgene #10879 for CTR; respectively. Specifically, 10 min prior to imaging, mice were injected intraper- Sigma-Aldrich #TRCN0000033696 and TRCN0000033697 for RING1B; and itoneally with D-luciferin (Perkin Elmer #760504) at a dose of 150 mg/kg. Tumor Sigma-Aldrich #TRCN0000014881 for FOXA1), 2 μg of pCMV-VSV-G, and 6 μg size and metastasis were quantified using the Living Image software (Perkin of pCMV-dR8.91 plasmids using calcium phosphate. 72 h after transfection, viral Elmer). Luciferase signal is represented as Luminescence (Photons/s). Mice were supernatant was collected, passed through a 0.45 μM polyethersulfone filter, and sacrificed at the indicated time points and primary tumors were collected and used to transduce MDA-MB-231 and T47D cells. Specifically, 3 × 105 cells were weighed. No animals were excluded from the analysis. plated into a 6-well plate followed by the addition of viral media with 8 μg/ml polybrene (Millipore-Sigma #TR-1003-G). Cells were centrifuged for 1 h at 1000×g at 32 °C then incubated overnight with fresh viral media. Viruses were removed Preparation of ATAC reactions and libraries. ATAC-seq experiments were and complete culture media was added for cell recovery. Cells were selected 24 h performed as previously described35 with modifications. Briefly, 25,000 cells of after recovery with 2 μg/ml of puromycin (Biogems #5855822) and were main- each cell line were used to perform the transposition reaction. Samples were eluted tained in selection. All experiments were performed within 3 weeks post in 13 μl of Buffer EB (Qiagen #28206). To calculate the number of cycles for library transduction. amplification, 2 μl of transposed DNA were amplified by qPCR for a total of 25 cycles. The 10 μl qPCR reaction was set up as follows: 2 μl of transposed DNA, μ μ μ μ Western blotting and immunoprecipitation 0.3 l25 M Ad1_noMX, 0.3 l25 M Ad2.X (custom oligos synthesized by . Cells were lysed in high-salt buffer μ (300 nM NaCl, 50 mM Tris-HCl [pH 8], 10% glycerol, and 0.2% NP-40) supple- Integrated DNA Technologies, see Supplementary Data 7), 5 l NEBNext High- Fidelity 2X PCR Master Mix (New England BioLabs #M0541S), 0.1 μl 100X SYBR mented with protease inhibitors (Sigma-Aldrich #04693132001) and sonicated fi μ 5 min at 4 °C with a Bioruptor in 30” ON-OFF cycles. After centrifugation at Green I (Thermo Fisher Scienti c #S7563) and 2.3 l nuclease-free water with the 16,000×g for 15 min, soluble material was quantified by Bradford assay (Bio-rad following program on a Bio-Rad CFX96 Optics Module Thermal Cycler machine: #5000006), and 1 mg of protein was used for each immunoprecipitation (IP), or (1) 72 °C for 5 min, (2) 98 °C for 30 s, (3) 98 °C for 10 s, 63 °C for 30 s and 72 °C for 30–50 μg of protein was loaded onto SDS-PAGE gels for western blotting. IP 30 s, 25 cycles, (4) 72 °C for 1 min, and (5) hold at 10 °C. The Ct value of each μ sample reflects the number of PCR cycles for optimal amplification in the linear samples were incubated overnight with 5 g of antibody (see Supplementary Data 6 μ μ for a list of antibodies used) followed by 30 μl of protein A/G agarose bead slurry range of the reaction. A 50 l PCR reaction was then set up as follows: 10 l transposed DNA, 1.5 μl25μM Ad1_noMX, 1.5 μl25μM Ad2.X (unique for each (Santa Cruz #sc-2003) for 2 h. IP material was washed 3× with high-salt buffer and μ μ eluted with Laemmli buffer, then loaded for SDS-PAGE. Western blotting was sample), 12 l nuclease-free water, and 25 l NEBNext High-Fidelity 2X PCR performed using standard protocols and imaged on an Odyssey CLx imaging Master Mix with the same program as for the qPCR, but substituting the cycle system (Li-COR), and various exposures within the linear range captured using number with the Ct-value obtained from the qPCR reaction. The PCR was per- formed on a Bio-Rad C1000 Touch Thermal Cycler. After PCR, the 50 μl reactions ImageStudio software (Li-COR). Images were rotated, resized, and cropped using μ Adobe Photoshop CC 2018 and imported into Adobe Illustrator CC 2018 to be were cleaned up and size selected by adding 25 l AMPure XP beads (Beckman fi fi Coulter #A63881) to remove fragments higher than 800 bp. The supernatant was assembled into gures. Unprocessed images for all western blots in main gures are μ provided in Supplementary Fig. 10. transferred to a new tube and 65 l AMPure XP beads were added to remove fragments smaller than 100 bp, then washed twice with freshly prepared 80% ethanol and eluted in 25 μl nuclease-free water. To determine the average fragment Subcellular fractionation. Two 150 cm2 dishes with 80% confluent T47D and size of each library, samples were run through a high sensitivity DNA screentape MDA-MB-231 cells (control, RING1B or FOXA1-depleted cells) were used for (Agilent Technologies #5067–5584) following the manufacturer’s instructions on each experiment. All steps were carried out at 4 °C. Cells were collected with a cell an Agilent Technologies 2200 TapeStation machine. To determine the
NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x concentration of each library, Qubit dsDNA high sensitivity reagents (Thermo gently at RT. The supernatant was aspirated, cells washed 1× with PBS, and har- Fisher Scientific #Q32851) were used following the manufacturer’s instructions on vested on ice using cell scrapers into 15 ml sonication tubes (Diagenode a Qubit 3 fluorometer. Finally, the samples were pooled and sequenced, paired-end, #C01020031). Cells were pelleted at 400×g for 3 min at 4 °C, washed 1× with 10 ml 75 bp on a NextSeq 500. cold PBS, and pelleted once more. Cell pellet was resuspended in 1.3 ml cold ChIP Buffer (two volumes of SDS ChIP Buffer [100 mM NaCl, 50 mM Tris-HCl pH 8.1, 5 mM EDTA pH 8.0, 0.5% SDS] with one volume TXT ChIP Buffer [100 mM Tris- ATAC-seq analysis . FASTQ data were processed with Trimmomatic v0.32 and HCl pH 8.6, 100 mM NaCl, 5 mM EDTA pH 8.0, 5% Triton X-100]). Cells were Skewer v0.2.2 to remove low-quality reads, and paired-end reads were aligned to then sonicated with a Bioruptor Pico at 4 °C for 10 min of 30” ON-OFF cycles for the hg19 genome (UCSC) using Bowtie2 v2.2.6. Duplicate reads were removed iPSCs or 20 min for MCF10A, MDA-MB-231 and T47D. After sonication, samples using Picard tools (version 1.126 -http://broadinstitute.github.io/picard). Read 35 were centrifuged at 16,000×g for 15 min at 4 °C and supernatant transferred to a alignment was offset as previously described . Peaks were called using the MACS2 new 1.5 ml microtube. To check sonication efficiency, 20 µl of sonicated samples v2.1.0.20150731 algorithm with the parameters: -g hs -p 0.01—nomodel—shift − — was transferred to a new microtube with 80 µl PBS and incubated at 65 °C for 3 h 75 extsize 150 and a cutoff of q-value < 0.05. Bedtools v2.26.0 intersect was used on an Eppendorf Thermomixer shaking at 1000 rpm to decrosslink. DNA was to determine peak overlaps. NGS Plot was used to generate heat maps and density purified with the QIAquick PCR Purification Kit (Qiagen #28106) and eluted in plots. Homer annotatePeaks was used for peak annotation. 30 µl H2O. 6 µl of orange DNA dye was added and 12 µl and 24 µl of each sample were run in a 1% agarose gel at 100 V for 15 min and imaged on a Bio-rad Purification of endogenous RING1B complexes. Cell pellets in triplicate from ChemiDoc XRS + . Protein concentration in sonicated samples was measured by MDA-MB-231 or T47D cell lines were snap frozen in liquid nitrogen. Each Bradford assay, and 200 µg of total protein was transferred to a 1.5 ml LoBind tube replicate was processed as follows, modified from established procedures52. Each (Eppendorf #0030108051) and brought up to 500 µl final volume with ChIP buffer. pellet was resuspended 1:4 (w:v) in a solution of 50 mM Tris pH 8.0, 300 mM NaCl, 5 µl was removed as input material (1%) and placed in a separate microtube at 4 °C. 0.2% (v/v) NP-40, supplemented with protease inhibitors (Sigma-Aldrich 2 µg antibody was used for each histone ChIP, except for H2AK119ub in which #11836170001). Samples were lysed by ultrasonication at 4 °C using a QSonica 1.5 µg was used (see Supplementary Data 6). The samples were rotated end-to-end S4000 equipped with an S4717 microtip probe. For each sample, 2-s-long pulses at overnight at 4 °C. Protein A sepharose beads (GE Healthcare #17528001) were 1Amp were applied, with 1 s pauses, until ~20 J of output per 100 mg of cell pellet washed 3× with ChIP buffer and 30 µl bead slurry was added to each sample. was reached. After sonication, samples were centrifuged at 20,000×g at 4 °C for Samples were incubated with beads for 2 h at 4 °C rotating end-to-end. Following 10 min, producing a clarified cell extract. For MDA-MB-231, 400 μl of clarified incubation, samples were centrifuged at 400×g for 3 min at 4 °C, washed 2× with extract from each replicate was used in IP and control experiments, respectively. In ChIP Low Salt Buffer (50 mM HEPES pH7.5, 140 mM NaCl, 1% Triton X-100, 1x IP experiments, extracts were combined with 10 μl of magnetic affinity medium protease inhibitors), and 1× with ChIP High-Salt Buffer (50 mM HEPES pH7.5, (Thermo Fisher Scientific #14301) coupled to anti-RING1B antibodies (MBL, see 500 mM NaCl, 1% Triton X-100, 1× protease inhibitors). Beads were dried after the Supplementary Data 6). In control experiments mouse IgG (Sigma-Aldrich, see last wash with a 28 G needle fitted to a 1 ml syringe. Elution Buffer (1% SDS, 0.1 M Supplementary Data 6) was used. 7.3 μg of anti-RING1B antibody and 10 μgof sodium carbonate) was prepared fresh before use and 110 µl was added to each mouse IgG were used, respectively, per mg of magnetic medium in epoxy-based sample and 95 µl to each input sample that was previously set aside. To elute covalent coupling (as per manufacturer’s instructions). For T47D IP/control immunocomplexes from beads, samples were incubated at 65 °C for 3 h on an experiments, 200 μl of extract were combined with 5 μlofaffinity medium. Clar- Eppendorf Thermomixer shaking at 1000 rpm. Tubes were centrifuged for 3 min at ified cell extracts were incubated with magnetic media for 1 h at 4 °C with gentle 400×g at RT and 100 µl of supernatant was transferred to a new tube, being careful end-over-end mixing. After mixing, the supernatants were removed and the beads not to aspirate beads. DNA purification was performed with the QIAquick PCR fi fi were washed three times with 1 ml of the extraction solution. The bound fraction Puri cation Kit (Qiagen cat# 28106) and eluted in 60 µl H2O and quanti ed by was released from IP/control experiments by the addition of 15 μl of 1× LDS Qubit. For non-histone ChIP targets, samples were processed using the High sample buffer (Thermo Fisher Scientific #NP0007) with incubation at 70 °C for Sensitivity ChIP-IT Kit (Active Motif #53040, see Supplementary Data 6). 5 min with agitation. After incubation, the eluate was removed, reduced with DTT, Immunoprecipitated DNA from both methods were used to either perform qPCR alkylated with iodoacetamide, and then run a ~6 mm into a 4–12% Bis-Tris or generate libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina NuPAGE gel (1 mm, 12-well; Thermo Fisher Scientific #NP0322BOX). The gel was (New England Biolabs #E7370) following the manufacturer’s instructions. Libraries stained with Coomassie Brilliant Blue G-250, the samples (gel plugs) were excised, were visualized on a Tapestation 2200 using D1000 DNA screentape (Agilent and cut into ~1 mm cubes for processing, as follows. Samples were destained with Technologies #5067–5582). Libraries were quantified on a Qubit 3 fluorometer several washes of 0.5–1 ml 50% v/v acetonitrile (ACN) in 50 mM ammonium with Qubit dsDNA high sensitivity reagents (Thermo Fisher Scientific #Q32851) bicarbonate at 37 °C with shaking. Destained gel pieces were dehydrated by following the manufacturer’s instructions, then pooled and sequenced, single-end, washing with 500 μl ACN, and placed in a speed-vac for ~10 min at RT. Trypsin 75 bp on a NextSeq 500. Processed data was viewed using the UCSC genome working solution (~40 μl at 12.5 ng/μl in 50 mM ammonium bicarbonate) was browser with a smoothing window of 5 pixels. ChIP-qPCR was performed using added to the gel pieces on ice, which were allowed to swell for 45 min. After primers targeting developmental or enhancer regions identified (see Supplementary swelling, additional 50 mM ammonium bicarbonate was added in order to sub- Data 8 for list of primers) on a Bio-Rad CFX96 Real-Time System with iTaq merge the swollen gel pieces (typically ~15 μl). The samples were incubated at universal SYBR green supermix (Bio-rad #1725124) and analyzed with CFX 37 °C to undergo tryptic proteolysis. Trifluoroacetic acid (TFA) was added to each Manager software (Bio-Rad). tube at 0.5% (w/v) final concentration and incubated 5 min at RT. The supernatant (tryptic digest supernatant) was recovered and transferred to a 0.5 ml low protein- binding microfuge tube (Sorenson Bioscience #11300). An aliquot of 50 μl 0.1% w/ TCGA data preparation and analysis. The legacy level 3 data of breast invasive v TFA was added to the gel pieces, which were extracted a further 45 min at RT, carcinoma (BRCA) and liver hepatocellular carcinoma (LIHC) from the The with agitation. The supernatants were removed and pooled with the appropriate Cancer Genome Atlas (TCGA) cohort were obtained from the Genomic Data tryptic digest supernatant. Pooled extracted peptides were desalted using C18 Commons (GDC) data portal. RNA-seq raw counts of 1211 BRCA and 421 LIHC reversed-phase OMIX tips (Agilent #A57003100) as per the manufacturer’s cases as legacy archive, and using the hg19 human reference genome, were instructions. The peptides were eluted from the tips first with 100 µl of aqueous downloaded, normalized and filtered using the R/Bioconductor package, TCGA- 40% (v/v) ACN, 0.5% (v/v) acetic acid (E1) and then with 100 µl of 80% (v/v) ACN, biolinks version 2.5.9. GDCquery, GDCdownload and GDCprepare were used for 0.5% (v/v) acetic acid (E2). E1 and E2 were combined, frozen in liquid nitrogen, both tumor types (“BRCA” and “LIHC”, level 3, and platform “IlluminaHi- and dried in a centrifugal vacuum concentrator. Seq_RNASeqV2”). Integrative analysis using mutation, clinical classification, and gene expression were performed following our recent TCGA’s workflow53. Among Strand-specific total RNA library preparation. RNA was isolated from fresh or BRCA samples 1097 were Primary Solid Tumor (TP) and 114 were solid Tissue frozen cell pellets using TRIzol reagent (Thermo Fisher Scientific #15596018). Normal (NT). Among LIHC samples 371 were TP and 50 NT. The aggregation of Ribosomal RNA was removed using the NEBNext rRNA Depletion Kit (New the two matrices (tumor and normal) for both tumor types was then normalized England Biolabs #E6310) starting with 1 µg total RNA and following the manu- using within-lane normalization to adjust for GC-content effect on read counts and facturer’s instructions. Ribosomal RNA-depleted samples were then further pro- upper-quantile between-lane normalization for distributional differences between cessed with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina lanes, applying the TCGAanalyze_Normalization function and adopting the (New England Biolabs #E7420) for library preparation following the manu- EDASeq protocol. Molecular subtypes, mutation data, and clinical data were pulled facturer’s instructions. Libraries were pooled and sequenced, single-end, 75 bp on a using TCGAbiolinks and the following functions: TCGAquery_subtype, NextSeq 500. GDCquery_maf retrieving somatic variants that were called by the MuTect2 pipeline, and GDCquery_clinic respectively. BRCA tumors with PAM50 classifi- cation54 were stratified into five molecular subtypes: Basal-like (n = 98), HER2- ChIP and ChIP-seq library preparation. Cells were grown to 80% confluence on enriched (n = 58), Luminal A (n = 231), Luminal B (n = 127), and Normal-like 150 cm2 plates and processed for ChIP of histone modifications or ChIP of non- (n = 8). Normal-like samples were not considered in this analysis due for the histone targets. For histone modifications, cells were fixed with 1% formaldehyde limited number of sample availability. For LIHC, tumors with iCluster classifica- (Sigma-Aldrich #252549) added directly to culture media for 15 min, shaking tion were stratified into three molecular subtypes: iCluster:1 (n = 65), iCluster:2 (n gently at RT. During crosslinking, 1.25 M glycine solution (10X stock) was pre- = 55), and iCluster3 (n = 63). Tumor stage information was retrieved from the pared, then added to plates at a final concentration of 125 mM for 5 min, shaking clinical data grouping to main stages (I, II, III, IV) and each subgroup (Ia, IIb, IIIc
14 NATURE COMMUNICATIONS | (2018) 9:3377 | DOI: 10.1038/s41467-018-05728-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05728-x ARTICLE etc.). Amplification data obtained from the GISTIC 2.0 tool was then used to of ±200 bp. Next, BEDtools intersect was used with the option –v to discard any identify regions of the genome that were significantly amplified or deleted across a peak overlapping any exons from the hg19 reference genome (Gencode version 27), set of samples55. GISTIC2 data was retrieved from cBioPortal for both tumor types with additional 2 kb surrounding every exon. A ±600bp window at the center of the considering samples with high amplification greater than 2 and excluding high H3K27ac peak was used to calculate RPKM across the entire eRNA locus using deletion samples lower than −2. The ggpubr R/CRAN (https://CRAN.R-project. total RNA-seq data, considering regions with eRNA expression to have RPKM > org/package=ggpubr) package was used to draw box plots showing relative 0.3. Next a cutoff of fold change >2 or <−2 (shCTR versus shRING1B) was used to expression for each cancer type, stage, and molecular subtype and to perform detected differential expression. eRNA loci was overlapped with super-enhancer multiple means comparisons using a non-parametric Wilcox test. All analyses and regions or typical enhancer regions using BEDtools intersect. plots were generated using the R environment (see Supplementary Data 9 for list of software). Motif analysis. Motif finding was performed with Homer findMotifs v4.8.3. A window of ±100 bp (option -d 200) relative to peak summits was used to perform ChIP-Seq analysis. ChIP-seq of RING1B, H3K27ac, H3K27me3, and H3K4me1 the analyses. (in K562 and HepG2 cells), GATA1 (in K562 cells) and bHLHE40 (in HepG2 cells) were re-analyzed from ENCODE data sets (GSE95908, GSE91837, GSM733658, Statistical analysis. Significance was determined by either Student’s t-test, non- GSM733754, GSM733656, GSM733743, GSM733692, GSM798321, GSM1003608, parametric Wilcox test, Mann–Whitney test, or Kolmogorov–Smirnov test, as GSM935566, GSM733780, GSM733732). All ChIP-seq data, generated in this study indicated. Error bars in figures represent standard deviation (SD) of at least two or deposited into ENCODE, were analyzed according to the following methodol- independent experiments. ogy: FASTQ data were processed with Trimmomatic v0.32 to remove low-quality reads and then aligned to the human genome hg19 using BWA v0.7.13-r1126 with the following parameters: aln -q 5 -l 32 -k 2. Duplicate reads were removed using Data availability. All of the genome-wide data of this study have been deposited in Picard tools (version 1.126—http://broadinstitute.github.io/picard). Peaks were the NCBI Gene Expression Omnibus (GEO) database, GSE number: GSE107176. called using MACS2.1 with default parameters –shiftsize 160 –nomodel –p 0.01 for The mass spectrometry proteomics data have been deposited to the Proteo- all data except RING1B in human iPSCs. For all H3k27me3 and H2AK119ub1 meXchange Consortium via the PRIDE partner repository with the data set fi peak calling, the option –broad was added to identify broad peaks. Whole-cell identi er PXD009570. extract input from the corresponding cell lines were used as controls. Peaks with fold change > 4 and a q-value < 0.05 were used for downstream analysis. Bigwig file Received: 21 December 2017 Accepted: 25 July 2018 output from MACS v 2.1.0.20150731 was visualized in the UCSC genome browser. Homer annotatePeaks v4.8.3 was used for peak annotation. Intergenic peaks are located greater than −2.5 kb from the TSS of a gene; intragenic peaks are inside genes, including intron, exons, and UTRs; –2.5 kb + TSS peaks are located at the TSS and a maximum of 2.5 kb upstream of the TSS; and other peaks are located at uRNAs, microRNAs, and pseudogenes. Bedtools v2.26.0 intersect was used to References determine peak overlaps. NGS Plot v2.61 was used to generate heat maps and 1. Schuettengruber, B., Bourbon, H.-M., Di Croce, L. & Cavalli, G. Genome density plots (see Supplementary Data 9 for list of software). regulation by Polycomb and Trithorax: 70 years and counting. Cell 171,34–57 (2017). Identification of super-enhancers. Super-enhancers (SE) and typical enhancers 2. Laugesen, A. & Helin, K. Chromatin repressive complexes in stem cells, were defined using the ROSE pipeline (Supplementary Data 9) with default development, and cancer. Cell Stem Cell 14, 735–751 (2014). parameters using H3K27ac ChIP-seq peaks as input. 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Cell Stem Cell 9, 233–246 (2011). sequencing. We also thank the Flow Cytometry Core Facility for assistance with cell 30. O’Loghlen, A. et al. MicroRNA regulation of Cbx7 mediates a switch of sorting, and the Cancer Modeling Shared Resource for assistance with the animal studies. Polycomb orthologs during ESC differentiation. Cell Stem Cell 10,33–46 Dr. Ramiro E. Verdun kindly provided the shRING1B plasmids. Kelly Molloy assisted (2012). with the mass spectrometry. Dr. Derek M. Dykxhoorn assisted with the iPSC culture. 31. Pascual, G. et al. Targeting metastasis-initiating cells through the fatty acid Dr. Aznar-Benitah provided the pMSCV-IRES-Luciferase-GFP vector. This work was receptor CD36. Nature 541,41–45 (2017). supported by Sylvester Comprehensive Cancer Center funds to L.M. 32. Ørom, U. A. et al. Long noncoding RNAs with enhancer-like function in human cells. Cell 143,46–58 (2010). 33. Holliday, D. L. & Speirs, V. 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