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Ectopic Protein Interactions Within BRD4–Chromatin Complexes Drive Oncogenic Megadomain Formation in NUT Midline Carcinoma

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Ectopic protein interactions within BRD4–chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma Artyom A. Alekseyenkoa,b,1, Erica M. Walshc,1, Barry M. Zeea,b, Tibor Pakozdid, Peter Hsic, Madeleine E. Lemieuxe, Paola Dal Cinc, Tan A. Incef,g,h,i, Peter V. Kharchenkod,j, Mitzi I. Kurodaa,b,2, and Christopher A. Frenchc,2 aDivision of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; bDepartment of Genetics, Harvard Medical School, Boston, MA 02115; cDepartment of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; dDepartment of Biomedical Informatics, Harvard Medical School, Boston, MA 02115; eBioinfo, Plantagenet, ON, Canada K0B 1L0; fDepartment of Pathology, University of Miami Miller School of Medicine, Miami, FL 33136; gBraman Family Breast Cancer Institute, University of Miami Miller School of Medicine, Miami, FL 33136; hInterdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Miami, FL 33136; iSylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136; and jHarvard Stem Cell Institute, Cambridge, MA 02138 Contributed by Mitzi I. Kuroda, April 6, 2017 (sent for review February 7, 2017; reviewed by Sharon Y. R. Dent and Jerry L. Workman) To investigate the mechanism that drives dramatic mistargeting of and, in the case of MYC, leads to differentiation in culture (2, 3). active chromatin in NUT midline carcinoma (NMC), we have Similarly, small-molecule BET inhibitors such as JQ1, which identified protein interactions unique to the BRD4–NUT fusion disengage BRD4–NUT from chromatin, diminish megadomain- oncoprotein compared with wild-type BRD4. Using cross-linking, associated transcription, including at MYC enhancers and TP63, affinity purification, and mass spectrometry, we identified the and also result in differentiation and growth arrest of NMC cells EP300 acetyltransferase as uniquely associated with BRD4 through (3, 8). Thus, it appears that BRD4–NUT directly misregulates the NUT fusion in both NMC and non-NMC cell types. We also dis- these two key genes, and potentially many others, to drive one of covered ZNF532 associated with BRD4–NUT in NMC patient cells but the most aggressive tumors known in cancer biology. BET inhibi- not detectable in 293T cells. EP300 and ZNF532 are both implicated in feed-forward regulatory loops leading to propagation of the on- tors, specifically targeting the BRD4 bromodomains, exhibit on-target cogenic chromatin complex in BRD4–NUT patient cells. Adding key activity in NMC patients (9), albeit with significant dose-limiting functional significance to our biochemical findings, we indepen- toxicity. Multiple BET inhibitor clinical trials are currently enrolling dently discovered a ZNF532–NUT translocation fusion in a newly NMC patients (NCT01587703, NCT01987362, NCT02431260, and diagnosed NMC patient. ChIP sequencing of the major players NCT02259114). NUT, ZNF532, BRD4, EP300, and H3K27ac revealed the formation of ZNF532–NUT–associated hyperacetylated megadomains, dis- Significance tinctly localized but otherwise analogous to those found in BRD4– NUT patient cells. Our results support a model in which NMC is de- Chromatin factors generally act within large, multisubunit pendent on ectopic NUT-mediated interactions between EP300 and complexes; thus, identifying both their normal and aberrant components of BRD4 regulatory complexes, leading to a cascade interactors in cancer should provide important information of misregulation. regarding potential targets for therapeutic intervention. Here, we apply this principle to analysis of BRD4–NUT, a fusion – BioTAP-XL | hyperacetylation | ZNF532 NUT | topological domains | BRD4 oncoprotein that drives an aggressive subtype of squamous cell cancer. We identify ZNF532 as a prominent BRD4–NUT– utations in the subunits of chromatin regulatory complexes interacting protein in an established NUT midline carcinoma Mare found at high frequencies in cancer cells. Thus, the patient cell line, and independently discover ZNF532 fused di- comprehensive identification of the components of chromatin rectly to NUT in a newly analyzed patient. Like BRD4–NUT, – complexes implicated in disease via their protein protein inter- ZNF532–NUT forms unusually large (100-kb to 1-Mb) domains actions is an important avenue toward finding potential targets of hyperactive chromatin, including at the MYC locus, and for therapeutic intervention. In NUT midline carcinoma (NMC), drives self-reinforcing regulatory loops that are likely to be a a subtype of squamous cell cancer, the transcriptional machinery powerful strategy for the growth advantage of cancer cells. is hijacked to drive expression of progrowth, antidifferentiation genes (1–3). NMC is defined by chromosomal rearrangement of Author contributions: A.A.A., E.M.W., M.I.K., and C.A.F. designed research; A.A.A., the NUT (NUTM1) gene, which is most commonly fused to the E.M.W., B.M.Z., P.H., and P.D.C. performed research; T.A.I. contributed new reagents/ BRD4 gene (4, 5). The resulting BRD4–NUT chimeric onco- analytic tools; B.M.Z., T.P., M.E.L., and P.V.K. analyzed data; and A.A.A., B.M.Z., P.V.K., protein forms large nuclear foci (6), proposed to form through M.I.K., and C.A.F. wrote the paper. tethering of the BRD4 bromodomains to acetylated chromatin Reviewers: S.Y.R.D., University of Texas MD Anderson Cancer Center; and J.L.W., Stowers Institute for Medical Research. and the acetylation of neighboring histones by EP300 via its in- Conflict of interest statement: T.A.I. has a pending patent application relating to the teraction with NUT (7). Our recent genomic analysis of NMC OCMI-E medium. patient cell lines provides strong evidence that the dual prop- Data deposition: The ChIP-seq and nascent RNA-seq data reported in this paper have been erties of acetyl-histone binding and EP300 recruitment result in a deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih. feed-forward expansion of acetylated chromatin and BRD4– gov/geo (accession no. GSE96775). The RNA-seq data detecting the ZNF532–NUT fusion NUT over massive genomic domains, often filling entire topolog- transcript reported in this paper have been deposited in the Sequence Read Archive ically associating domains (TADs) (3). The number and magnitude (accession no. PRJNA361284). The mass spectrometric proteomics data reported in this “ ” paper have been deposited in the ProteomeXchange Consortium via the PRIDE partner of these megadomains correlate with the characteristic nuclear repository (accession no. PXD005786). foci seen in diagnostic patient tumor samples or in cultured NMC 1A.A.A. and E.M.W. contributed equally to this work. cells stained with a NUT-specific antibody (3, 6, 7). 2To whom correspondence may be addressed. Email: [email protected]. Megadomains encompassing the MYC and TP63 regulatory edu or [email protected]. regions are common to all NMCs examined to date, and RNAi This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. knockdown of either of these genes in patient cells blocks growth 1073/pnas.1702086114/-/DCSupplemental. E4184–E4192 | PNAS | Published online May 8, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1702086114 Downloaded by guest on October 6, 2021 Given the potency of aberrant chromatin regulation by BRD4– A PNAS PLUS NUT, and reasoning that identifying additional therapeutic targets will be necessary to defeat this disease, we have undertaken a BRD4* comprehensive analysis of BRD4–NUT protein interactions on chromatin in TC-797 NMC patient cells and when ectopically 0.4 NUTM1 expressed in 293TRex cells. Rather than taking a candidate ap- BRD3 proach, we have used comprehensive proteomics to probe how CBX4 ATAD5 fusion to NUT alters normal BRD4 function. Our cross-linking CDK9 EP300 WHSC1L1 approach revealed that EP300 is indeed the acetyltransferase 0.3 CCNT1 uniquely attracted by the NUT portion of the fusion oncopro- CREBBP TAF6 WHSC1 CHD8 ZMYND8 tein. We further identified ZNF532 and a short list of additional PWWP2B GLTSCR1 NSD1 TAF3 factors as candidates associated with BRD4–NUT complexes spe- TAF1 BRD2 MED1 cifically in patient cells. Remarkably, our identification of ZNF532 TAF2 – through BRD4 NUT proteomics coincided with our independent XPO1 discovery of a translocation of ZNF532 to NUT in a newly identified ZNF687 NMC patient. We found that ZNF532–NUT fusion protein forms megadomains of hyperacetylated chromatin, similar to those formed – by BRD4 NUT, suggesting a common feed-forward mechanism for 0.1 0.2 megadomain formation. BRD4NUT-NBioTAP(293TRex cells) Results EP300 Acetyltransferase Is Specific to BRD4–NUT Affinity Purification. −0.2 −0.1 0.0 0.1 0.2 0.3 To identify factors that may contribute to BRD4–NUT–driven BRD4short-NBioTAP (293TRex cells) oncogenesis, we sought to affinity purify the fusion oncoprotein and compare a comprehensive list of its interacting proteins with B Joint enriched factors copurified with BRD4 lacking NUT. To this end, we Name Mwt BRD4 short BRD4NUT BRD4NUT induced expression of BioTAP-tagged BRD4–NUT (BRD4– – – N-BioTAP N-BioTAP C-BioTAP NUT BioTAP) or the short isoform of BRD4 (BRD4short BRD4* 152.12 167 83 108 BioTAP), encoding only the portion of BRD4 included in the BRD2 91.98 35 23 22 BRD4–NUT fusion oncoprotein (10). We expressed the epitope- BRD3 79.49 39 20 12 tagged proteins from single-copy transgenes integrated in a non- ATAD5 207.44 51 24 52 NMC cell line, 293T, the derivative of which we term 293TRex WHSC1L1 161.51 40 21 16 (3, 11). 293TRex cells serve as a useful model, as they do not WHSC1 152.16 38 12 25 normally harbor the oncogenic fusion but, when induced to ex- CREBBP 265.18 31 28 8 press BRD4–NUT, form de novo nuclear foci and hyperacetylated CBX4 61.33 30 15 9 megadomains (3). Chromatin cross-linking, affinity purification, CHD8 290.34 26 10 31 and mass spectrometry (BioTAP-XL) (3, 12) allowed stringent CCNT1 80.63 22 14 4 purification of N- and C-terminally BioTAP-tagged BRD4–NUT CDK9 53.33 19 9 4 and N-BioTAP–BRD4–associated proteins from 293TRex cells.
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    ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en EPIGENETIC REGULATORS IN NEUROBLASTOMA: BRG1, A FUTURE THERAPEUTIC TARGET PhD thesis presented by Luz Jubierre Zapater To obtain the degree of PhD for the Universitat Autónoma de Barcelona (UAB) PhD thesis carried out at the Translational Research in Child and Adolescent Cancer Laboratory, at Vall d’Hebron Research Institute (VHIR), under the supervision of Dr. Miguel F. Segura and Dr. Soledad Gallego Thesis affiliated to the Department of Biochemistry and Molecular Biology from the UAB, in the PhD program of Biochemistry, Molecular Biology and Biomedicine, under the tutoring of Dr. José Miguel Lizcano Universidad Autónoma de Barcelona, Septiembre 5th 2017 Dr. Miguel F. Segura Dr. Soledad Gallego (Director) (Director) Luz Jubierre Zapater (Student) 2017 Is this the real life? Is this just fantasy? Caught in a landslide No escape from reality; Open your eyes Look up to the skies and see Bohemian Rhapsody, Queen To my Mom To my Dad To Adri ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Acknowledgement Desde que escribí la primera palabra en el libro de mi vida mucha gente se ha cruzado en mi camino para dejar su huella en él.
  • Polyclonal Antibody to ZNF687

    Polyclonal Antibody to ZNF687

    TA319985 OriGene Technologies Inc. OriGene EU Acris Antibodies GmbH 9620 Medical Center Drive, Ste 200 Schillerstr. 5 Rockville, MD 20850 32052 Herford UNITED STATES GERMANY Phone: +1-888-267-4436 Phone: +49-5221-34606-0 Fax: +1-301-340-8606 Fax: +49-5221-34606-11 [email protected] [email protected] Polyclonal Antibody to ZNF687 (C-term) - Aff - Purified Alternate names: KIAA1441, Zinc finger protein 687 Catalog No.: TA319985 Quantity: 0.1 mg Concentration: 1.0 mg/ml Background: The zinc finger protein 687 (ZNF687) was initially identified as a translocation partner gene with RUNX1 in patients with acute myeloid leukemia (AML). Little is known of the function of the ZNF687 protein, but it has been shown to weakly interact with the Ring1/Rnf2 RING finger protein member of the Polycomb group of proteins, suggesting it may be involved in the chromatin-modifying complexes essential for embryonic development and stem cell renewal. Other evidence suggests that ZNF687 may be part of a transcriptional network that also includes ZNF592 and ZMYMD8. Uniprot ID: Q8N1G0 NCBI: NP_065883 GeneID: 57592 Host / Isotype: Rabbit / IgG Immunogen: 17 amino acid synthetic peptide near the carboxy terminus of Human ZNF687 (AP55449CP- N) Format: State: Liquid purified Ig fraction Purification: Affinity chromatography purified via peptide column Buffer System: PBS containing 0.02% Sodium Azide as preservative Applications: Western blot: 0.5-1 µg/ml. Positive Control: Jurkat cell lysate. Immunofluorescence: Start at 20 μg/ml. Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user. Specificity: This antibody is Human specific.