Genome‐wide cooperation of EMT transcription factor ZEB 1 with YAP and AP ‐1 in breast cancer
2020; Springer Nature; Volume: 39; Issue: 17 Linguagem: Inglês
10.15252/embj.2019103209
ISSN1460-2075
AutoresNora Feldker, Fulvia Ferrazzi, Harald Schuhwerk, Sebastian A. Widholz, Kerstin Guenther, Isabell Frisch, Kathrin Jakob, Julia Kleemann, Dania Riegel, Ulrike Bönisch, Soeren Lukassen, Rebecca L. Eccles, Christian Schmidl, Marc P. Stemmler, Thomas Brabletz, Simone Brabletz,
Tópico(s)Cancer-related gene regulation
ResumoArticle21 July 2020Open Access Source DataTransparent process Genome-wide cooperation of EMT transcription factor ZEB1 with YAP and AP-1 in breast cancer Nora Feldker Nora Feldker Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Fulvia Ferrazzi Fulvia Ferrazzi orcid.org/0000-0003-4011-4638 Institute of Human Genetics, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Department of Nephropathology, Institute of Pathology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Institute of Pathology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Harald Schuhwerk Harald Schuhwerk Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Sebastian A Widholz Sebastian A Widholz orcid.org/0000-0002-4566-2986 Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Kerstin Guenther Kerstin Guenther Department of Visceral Surgery, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Isabell Frisch Isabell Frisch Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Kathrin Jakob Kathrin Jakob Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Julia Kleemann Julia Kleemann Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Dania Riegel Dania Riegel Regensburg Center for Interventional Immunology (RCI), University Regensburg and University Medical Center, Regensburg, Germany Search for more papers by this author Ulrike Bönisch Ulrike Bönisch Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Sören Lukassen Sören Lukassen orcid.org/0000-0001-7045-6327 Institute of Human Genetics, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Rebecca L Eccles Rebecca L Eccles Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Christian Schmidl Christian Schmidl Regensburg Center for Interventional Immunology (RCI), University Regensburg and University Medical Center, Regensburg, Germany Search for more papers by this author Marc P Stemmler Marc P Stemmler orcid.org/0000-0002-7866-3686 Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Thomas Brabletz Corresponding Author Thomas Brabletz [email protected] orcid.org/0000-0003-2983-9048 Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Comprehensive Cancer Center Erlangen-EMN, Erlangen University Hospital, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, GermanyThese authors jointly supervised this work Search for more papers by this author Simone Brabletz Corresponding Author Simone Brabletz [email protected] orcid.org/0000-0003-0936-1526 Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, GermanyThese authors jointly supervised this work Search for more papers by this author Nora Feldker Nora Feldker Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Fulvia Ferrazzi Fulvia Ferrazzi orcid.org/0000-0003-4011-4638 Institute of Human Genetics, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Department of Nephropathology, Institute of Pathology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Institute of Pathology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Harald Schuhwerk Harald Schuhwerk Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Sebastian A Widholz Sebastian A Widholz orcid.org/0000-0002-4566-2986 Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Kerstin Guenther Kerstin Guenther Department of Visceral Surgery, Faculty of Biology, University of Freiburg, Freiburg, Germany Search for more papers by this author Isabell Frisch Isabell Frisch Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Kathrin Jakob Kathrin Jakob Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Julia Kleemann Julia Kleemann Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Dania Riegel Dania Riegel Regensburg Center for Interventional Immunology (RCI), University Regensburg and University Medical Center, Regensburg, Germany Search for more papers by this author Ulrike Bönisch Ulrike Bönisch Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Search for more papers by this author Sören Lukassen Sören Lukassen orcid.org/0000-0001-7045-6327 Institute of Human Genetics, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Rebecca L Eccles Rebecca L Eccles Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Christian Schmidl Christian Schmidl Regensburg Center for Interventional Immunology (RCI), University Regensburg and University Medical Center, Regensburg, Germany Search for more papers by this author Marc P Stemmler Marc P Stemmler orcid.org/0000-0002-7866-3686 Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Thomas Brabletz Corresponding Author Thomas Brabletz [email protected] orcid.org/0000-0003-2983-9048 Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany Comprehensive Cancer Center Erlangen-EMN, Erlangen University Hospital, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, GermanyThese authors jointly supervised this work Search for more papers by this author Simone Brabletz Corresponding Author Simone Brabletz [email protected] orcid.org/0000-0003-0936-1526 Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, GermanyThese authors jointly supervised this work Search for more papers by this author Author Information Nora Feldker1,‡, Fulvia Ferrazzi2,3,4,‡, Harald Schuhwerk1, Sebastian A Widholz1,9, Kerstin Guenther5, Isabell Frisch1, Kathrin Jakob1, Julia Kleemann1, Dania Riegel6, Ulrike Bönisch7, Sören Lukassen2,10, Rebecca L Eccles1, Christian Schmidl6, Marc P Stemmler1, Thomas Brabletz *,1,8 and Simone Brabletz *,1 1Department of Experimental Medicine 1, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany 2Institute of Human Genetics, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany 3Department of Nephropathology, Institute of Pathology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany 4Institute of Pathology, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany 5Department of Visceral Surgery, Faculty of Biology, University of Freiburg, Freiburg, Germany 6Regensburg Center for Interventional Immunology (RCI), University Regensburg and University Medical Center, Regensburg, Germany 7Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany 8Comprehensive Cancer Center Erlangen-EMN, Erlangen University Hospital, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany 9Present address: Institute of Molecular Oncology and Functional Genomics, TUM School of Medicine, Technical University, Munich, Germany 10Present address: Charité – Universitätsmedizin Berlin, Digital Health Center, Berlin, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 9131 8529104; E-mail: [email protected] *Corresponding author. Tel: +49 9131 8529101; E-mail: [email protected] The EMBO Journal (2020)39:e103209https://doi.org/10.15252/embj.2019103209 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Invasion, metastasis and therapy resistance are the major cause of cancer-associated deaths, and the EMT-inducing transcription factor ZEB1 is a crucial stimulator of these processes. While work on ZEB1 has mainly focused on its role as a transcriptional repressor, it can also act as a transcriptional activator. To further understand these two modes of action, we performed a genome-wide ZEB1 binding study in triple-negative breast cancer cells. We identified ZEB1 as a novel interactor of the AP-1 factors FOSL1 and JUN and show that, together with the Hippo pathway effector YAP, they form a transactivation complex, predominantly activating tumour-promoting genes, thereby synergising with its function as a repressor of epithelial genes. High expression of ZEB1, YAP, FOSL1 and JUN marks the aggressive claudin-low subtype of breast cancer, indicating the translational relevance of our findings. Thus, our results link critical tumour-promoting transcription factors: ZEB1, AP-1 and Hippo pathway factors. Disturbing their molecular interaction may provide a promising treatment option for aggressive cancer types. Synopsis How the EMT-transcription factor ZEB1 stimulates cancer cell plasticity in context-dependent manner is unclear. Here, genome-wide analyses highlight AP-1 and Hippo effector YAP as novel binding partners of ZEB1, cooperating in driving tumour-promoting gene expression in mammary malignancies. ZEB1 and AP-1 factor JUN co-occupy DNA-targets in human TNBC cells. ZEB1 directly interacts with AP-1 factors JUN and FOSL1. ZEB1 mediates dual transcriptional function, being repressive on its own, but trans-activating together with AP-1 and YAP. ZEB1, AP-1 and YAP correlate with worse survival in claudin-low aggressive breast cancer. Introduction The transcription factor ZEB1 (zinc finger E-box binding homeobox 1) is an activator of the embryonic epithelial to mesenchymal transition (EMT) programme, which, when hijacked by cancer cells, is considered a major driver of tumour progression (De Craene & Berx, 2013; Stemmler et al, 2019). Importantly, EMT is not a one-way road but a highly dynamic and reversible process, providing cancer cells with the plasticity needed to cope with the different challenges on their way to distant metastasis formation (Chaffer et al, 2016). In addition to ZEB1, EMT can also be induced by other core EMT-transcription factors (EMT-TFs), namely ZEB2, TWIST1 and the two members of the Snail family, which most likely all have non-redundant subfunctions (Stemmler et al, 2019; Yang et al, 2020). Notably, these core EMT-TFs not only exert "classical" EMT properties, like loss of epithelial integrity and increased motility, but also confer other properties important for cancer progression, including stemness, survival and therapy resistance (Nieto et al, 2016). The core EMT-TF ZEB1 was shown to be particularly important for tumorigenicity and metastasis, by triggering the combined activation of cell motility and stemness properties (Vandewalle et al, 2009; Sanchez-Tillo et al, 2011; Krebs et al, 2017). Its expression mediates aggressiveness, metastasis and therapy resistance in many different cancer types (Zhang et al, 2015; Stemmler et al, 2019). This includes epithelial cancers like breast and pancreatic cancer, but also non-epithelial tumours, like glioblastoma (Karihtala et al, 2013; Siebzehnrubl et al, 2013; Bronsert et al, 2014; Kahlert et al, 2015; Lehmann et al, 2016; Krebs et al, 2017). In breast cancer, ZEB1 is highly expressed specifically in a fraction of the aggressive triple-negative subtype (Karihtala et al, 2013; Lehmann et al, 2016). ZEB1 can control target gene transcription by different modes of action. It binds to DNA via the two zinc finger clusters at its N- and C-terminal ends, with each cluster binding to an individual E-box motif (Remacle et al, 1999; Balestrieri et al, 2018). At least one E-box needs to be a high-affinity motif with the consensus sequence CAGGTG/A (Remacle et al, 1999). By recruiting additional cofactors, such as CtBP, ZEB1 represses the expression of epithelial genes (Postigo & Dean, 1999; Vandewalle et al, 2009). In addition to its role as a transcriptional repressor, ZEB1 has also been reported to act as a transcriptional activator (Gheldof et al, 2012). One important mechanism enabling its dual function as a transcriptional repressor and as an activator appears to be cooperativity with different transcription factor complexes (Gubelmann et al, 2014; Sanchez-Tillo et al, 2015; Lehmann et al, 2016; Rosmaninho et al, 2018). We recently described that a selected set of tumour-promoting target genes is activated by ZEB1 together with the Hippo pathway effectors YAP/TEAD (Lehmann et al, 2016). In a genome-wide study using glioblastoma cell lines, ZEB1 was shown to cooperate with factors of the LEF/TCF family to activate expression of tumour-promoting genes in brain cancer (Rosmaninho et al, 2018). We here investigated transcriptional control by ZEB1 on a genome-wide level in aggressive breast cancer cells to further dissect ZEB1 modes of action in a cancer of epithelial origin. In this context, we could not see any cooperation with LEF/TCF factors in this tumour type, but identified AP-1 factors as novel partners of ZEB1 in activating cancer-promoting genes together with YAP/TEAD. Results ZEB1 exhibits overlapping DNA binding with the AP-1 factor JUN To gain genome-wide insights into ZEB1 modes of action, we performed ChIP-seq to analyse its binding pattern using the triple-negative breast cancer cell line MDA-MB-231 as a model. We detected ZEB1 binding at 12,617 genomic sites (Fig EV1A), including promoter regions of many established ZEB1-repressed target genes conferring an epithelial phenotype such as CDH1 or CRB3 (Fig EV1B; Aigner et al, 2007; Eger et al, 2005), as well as known ZEB1 activated genes such as the protumorigenic factors CTGF and CYR61 (Fig EV1C) (Lazarova et al, 2001; Lai et al, 2011; Lehmann et al, 2016). Furthermore, gene annotation of binding peaks in promoter regions and integration with gene expression data of ZEB1 knockdown compared to control MDA-MB-231 cells (Lehmann et al, 2016) corroborated both direct repressive and direct activating functions of ZEB1 (Fig EV1D and E). As expected, known motif discovery analysis retrieved the canonical ZEB1-binding motif as the highest enriched motif in the ZEB1 peaks (Fig 1A). Sixty-eight percent of the binding sites contained a high-affinity ZEB1 motif (Fig 1A, Appendix Table S1), which was specifically enriched within 100 bp around the peak summit and as such located in a good position to mediate DNA binding of ZEB1 (Fig EV1F). In addition to this motif analysis, which relies on already identified DNA binding motifs, a de novo motif analysis was also performed to search for DNA motifs without relying on prior knowledge. This analysis identified a degenerated low affinity ZEB1 motif that was found at even higher frequency than the canonical ZEB1 motif (Appendix Table S2). Click here to expand this figure. Figure EV1. ZEB1 ChIP-seq in MDA-MB-231 cells Heatmap representing the signal enrichment of ZEB1 ChIP-seq and of control input DNA reads in 2 kb regions centred on the ZEB1 peak summits. Reads are clearly enriched in the peak regions in the ZEB1 ChIP-seq but not in the control input DNA. Genome browser tracks of ZEB1 ChIP-seq signal intensity showing specific enrichment at the promoter regions of known ZEB1-repressed targets. Genome browser tracks of ZEB1 ChIP-seq signal intensity showing specific enrichment at the promoter regions of known ZEB1-activated targets. Distribution of ZEB1 peaks with respect to different genomic features. Promoters were defined as 1.5 kb upstream to 0.5 kb downstream of the TSS. Promoter peaks were annotated to their respective genes and integrated with transcriptome data of ZEB1 knockdown compared to control MDA-MB-231 cells to identify peaks associated with activated/repressed genes. Genes that were downregulated in the ZEB1 knockdown compared to control MDA-MB-231 cells were named "activated", meaning activated in the presence of ZEB1. Genes that were upregulated after ZEB1 knockdown were called "repressed", meaning repressed in the presence of ZEB1. Hypergeometric testing showed that ZEB1 promoter peaks are significantly associated with both, genes that are activated and genes that are repressed in the presence of ZEB1. Odds ratio (OR): measure of association between categorical variables with OR > 1 indicating a positive association. P-value from Fisher's exact test. Enrichment profile of the consensus high-affinity ZEB1 DNA binding motif in 600-bp regions centred on the summits of ZEB1 peaks. The two grey dashed lines highlight the 200-bp regions around the ZEB1 peak summits that were used for known and de novo HOMER motif analysis. Overlap between ZEB1 and YAP DNA binding sites in MDA-MB-231 cells. Sites are counted as overlapping when their peak summit positions are not more than 200 bp apart. P-value from permutation test. Enrichment profile of YAP peak summit positions in 1 kb regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the region of ± 200 bp around the ZEB1 peak summit positions where YAP peaks are counted as overlapping with ZEB1 peaks. Heatmap representing the signal enrichment of YAP ChIP-seq reads in 2 kb regions centred on the ZEB1 peak summits. Download figure Download PowerPoint Figure 1. ZEB1 DNA binding sites overlap with YAP- and JUN-binding sites The three top enriched DNA binding motifs identified by HOMER known motif analysis on 200 bp regions centred on the ZEB1 peak summits. An extended list can be found in Appendix Table S1. Enrichment profiles of TEAD4 and AP-1 consensus motifs in 600 bp regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the 200 bp regions around the ZEB1 peak summits which were used for HOMER known motif analysis shown in (A). Overlap between ZEB1 and JUN DNA binding sites. Sites are counted as overlapping when their peak summit positions are not more than 200 bp apart. P-value from permutation test. Enrichment profile of JUN peak summit positions in 1 kb regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the region of ± 200 bp around the ZEB1 peak summit positions where JUN peaks are counted as overlapping with ZEB1 peaks. Heatmap representing the signal enrichment of JUN ChIP-seq reads in 2 kb regions centred on the ZEB1 peak summits. Circle plot displaying the genome-wide distribution of ZEB1 (outer middle circle, blue), JUN (inner middle circle, green) and YAP (inner circle, red) peaks across all chromosomes (outer circle). Peak density is visualised as a heatmap with more intense colours indicating a higher number of peaks in a specific location. ZEB1, JUN and YAP DNA binding overlaps at 1,993 genomic sites in MDA-MB-231 cells. Triple overlap is assumed when both JUN and YAP peak summit positions are within 200 bp of a ZEB1 peak summit position. P-value from permutation test. Download figure Download PowerPoint Strikingly, the second top enriched motif within the ZEB1 peaks was the consensus binding site for AP-1 transcription factors. This motif was still present in more than 1/5 of all peaks (Fig 1A, Appendix Table S1), and similarly to the classical ZEB1-binding motif, it was overrepresented within 100 bp around the peak summit (Fig 1B). This enrichment of AP-1 motifs at the ZEB1 peak summit indicates joint binding of AP-1 factors together with ZEB1 at the same genomic sites. To investigate this hypothesis, we compared the ZEB1-binding sites to a published ChIP-seq data set of the AP-1 factor JUN (also known as c-JUN), also generated in MDA-MB-231 cells (Zanconato et al, 2015). More than 33% of the ZEB1 peaks overlap with sites of JUN-binding (4,201 out of 12,617) (Fig 1C). Furthermore, JUN peak summits coincide with ZEB1 peak summits (Fig 1D) and JUN ChIP-seq signal intensity increases around sites of ZEB1 binding (Fig 1E). These data suggest a cooperation between ZEB1 and the AP-1 factor JUN. The third top enriched motif was the TEAD-binding motif, the main platform via which YAP binds to DNA (Zhao et al, 2008; Fig 1A, Appendix Table S1). The similar confined enrichment around the ZEB1 peak summit (Fig 1B) is consistent with the concept that ZEB1 can form a transcriptional activator complex together with the Hippo pathway effector YAP on genome-wide level. Notably, in contrast to glioblastoma (Rosmaninho et al, 2018), we did not detect a significant enrichment of the LEF/TCF motif within the ZEB1 peaks in the breast cancer cells (Appendix Table S1). To investigate whether ZEB1, AP-1 and YAP bind all together or ZEB1 with either YAP or AP-1 individually, we compared the DNA binding patterns of all three factors. To this end, we first verified the genome-wide distribution of all three factors including a published data set of YAP binding peaks also generated by ChIP-seq in MDA-MB-231 cells (Zanconato et al, 2015; Fig 1F). When we assessed the overlap of the ZEB1 and YAP peaks, we found that approximately 19% of the ZEB1-binding sites overlap with YAP-binding (2,372 out of 12,617) (Fig EV1G) and that YAP peak summits coincide with ZEB1 peak summits (Fig EV1H and I). Comparison among all three ChIP-seq data sets showed that the largest fraction of common ZEB1-YAP sites is additionally bound by JUN (approx. 84%; 1,993 out of 2,372) (Fig 1G). Moreover, approximately half of the common ZEB1-JUN sites also showed YAP-binding (1,993 out of 4,201). These findings suggest a genome-wide cooperation of the EMT inducer ZEB1 with the Hippo effector YAP and the AP-1 factor JUN in breast cancer. A cooperation of ZEB1 with LEF/TCF factors, as shown in glioblastoma, was not detected. ZEB1 interacts with the AP-1 factors JUN and FOSL1 To identify whether ZEB1 and AP-1 factors physically interact, we first tested if both ZEB1 and JUN are co-expressed in MDA-MB-231 cells. We included the JUN co-factor FOSL1 (also known as FRA-1) in our analysis due to its known role in cancer progression and metastasis (Bakiri et al, 2015). Immunofluorescence labelling showed nuclear co-localisation of ZEB1 with JUN and FOSL1 in MDA-MB-231 cells (Fig EV2A). An in situ proximity ligation assay for ZEB1 and the two AP-1 factors further demonstrated that ZEB1 proteins are found in close proximity to JUN and FOSL1 proteins in the nuclei of MDA-MB-231 cells (Fig 2A), as well as in two additional basal type breast cancer cell lines, BT549 and Hs578T (Fig EV2B). As control, we used the Hippo pathway effector TAZ, for which we did not detect an interaction with ZEB1 in any of the three cell lines, although TAZ co-localises with ZEB1 in the nucleus, as shown for MDA-MB-231 (Fig EV2A). We further tested whether ZEB1 interacts with JUN and FOSL1 by performing co-immunoprecipitation (co-IP) for all three factors and identified a physical interaction of endogenous ZEB1 with both JUN and FOSL1 (Fig 2B). Antibody and RNAi controls are shown in Appendix Fig S1A and B. Together, our results provide strong evidence that ZEB1 forms complexes with the AP-1 factors JUN and FOSL1. Click here to expand this figure. Figure EV2. Proximity ligation assay Immunofluorescence staining of ZEB1, the AP-1 factors JUN and FOSL1, and the Hippo pathway effector TAZ in MDA-MB-231 cells. All four proteins show a clear nuclear localisation. Scale bar = 20 μm. In situ proximity ligation assay (PLA) of ZEB1 with JUN, FOSL1 and TAZ shows close proximity of ZEB1 with JUN and FOSL1 but not TAZ in the nucleus of BT549 and Hs578T breast cancer cells indicated by red fluorescent dots. In the upper panel, representative microscopic images are shown. Scale bar = 20 μm. The lower panel shows a quantification of the PLA shown above. n = 2–3; shown is one representative experiment with means ± s.e.m. of at least 900 cells per condition; ***P ≤ 0.001; 2-way ANOVA plus Tukey′s post-test. Download figure Download PowerPoint Figure 2. ZEB1 interacts with JUN and FOSL1 In situ proximity ligation assay (PLA) of ZEB1 with JUN, FOSL1 and TAZ shows close proximity of ZEB1 with JUN and FOSL1 but not TAZ in the nucleus of MDA-MB-231 cells indicated by red fluorescent dots. As negative control, ZEB1 was transiently knocked down by siRNA. In the left panel, representative microscopic images are shown. Scale bar = 20 μm. Quantification of the PLA is shown on the right. n = 3; one representative experiment with means ± s.e.m. of at least 900 cells per condition is shown; ***P ≤ 0.001; 2-way ANOVA plus Tukey′s post-test. Co-immunoprecipitation (Co-IP) of endogenous ZEB1 and FOSL1 or JUN in MDA-MB-231 cells shows co-precipitation of ZEB1 with FOSL1 and JUN. Source Data for Figure 2 [embj2019103209-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint ZEB1-mediated transcriptional control has at least two modes of action To further investigate whether cooperative binding of ZEB1 together with YAP and AP-1 factors represents a specific mode of ZEB1-mediated transcriptional control, we individually analysed two subsets of ZEB1-binding sites in our ChIP-seq data, hereafter called the ZEB1-only and the ZEB1/YAP/JUN peaks. Importantly, ZEB1 binding to both subsets occurs in the same cell, here the aggressive, triple-negative MDA-MB-231 cells. The ZEB1-only peaks comprised a set of 5,963 locations where ZEB1 binds without any evidence for YAP- or JUN-binding in close proximity. In contrast, at the 1,993 ZEB1/YAP/JUN sites, binding peaks of all three factors overlap pointing to the formation of putative ZEB1/YAP/AP-1 complexes (Fig 3A). Peaks of both subsets were distributed over the whole genome (Fig 3B). However, examination of the specific genomic localisation of ZEB1-only compared to ZEB1/YAP/JUN peaks revealed binding to different genomic elements. ZEB1-only peaks were mostly located within 1 kb distance of a transcription start site (TSS) (Fig 3C). More specifically, 65.8% of the binding sites lay within promoter regions, defined as 1.5 kb upstream to 0.5 kb downstream of a TSS (Fig 3D). In contrast, ZEB1/YAP/JUN peaks were mostly located more distant to TSSs, with almost 40% of the peaks being 10–50 kb up- or downstream (Fig 3C). They were predominantly found in intronic and intergenic regions, with only 14.8% of peaks locating to promoters (Fig 3D). This analysis suggests that while ZEB1 without YAP and JUN preferentially controls transcription from promoter regions, ZEB1, YAP and JUN together act mainly from distal regulatory regions such as enhancers. Figure 3. Characterisation of a ZEB1-only and a ZEB1/YAP/JUN peak set Definition of two different ZEB1 peak subsets based on the presence or absence of overlapping YAP and JUN peaks. The two illustrative genome browser images show one example of a ZEB1-only peak and one example of a ZEB1/YAP/JUN peak (at the EFEMP1 gene and within the SHROOM3 gene, respectively). Control (ctrl) is the input for ZEB1 and IgG for the YAP and JUN ChIP-seqs. ChIP-seqs and respective controls were scaled accordingly. Circle plot displaying the genome-wide distribution of ZEB1-only (middle circle, blue) and ZEB1/YAP/JUN peaks (inner circle, orange) across all chromosomes (outer circle). Peak density is visualised as a heatmap with more intense colours indicating higher accumulation of peaks at a specific location. Localisation of ZEB1 peak summits relative to the closest TSS. Distribution of
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