Perturbation of BRD4 Protein Function by BRD4-NUT Protein Abrogates Cellular Differentiation in NUT Midline Carcinoma

Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2011; 286(31):27663-75. DOI: 10.1074/jbc.M111.246975
Source: PubMed


NUT midline carcinoma (NMC) belongs to a class of highly lethal and poorly differentiated epithelial cancers arising mainly
in human midline organs. NMC is caused by the chromosome translocation-mediated fusion of the NUT (nuclear protein in testis) gene on chromosome 15 to a few other genes, most frequently the BRD4 gene on chromosome 19. The mechanism by which the BRD4-NUT fusion product blocks NMC cellular differentiation and contributes to oncogenesis remains elusive. In this study, we show
that BRD4-NUT and BRD4 colocalize in discrete nuclear foci that are hyperacetylated but transcriptionally inactive. BRD4-NUT
recruits histone acetyltransferases to induce histone hyperacetylation in these chromatin foci, which provide docking sites
for accumulation of additional BRD4 and associated P-TEFB (positive transcription elongation factor b) complexes in the transcriptionally
inactive BRD4-NUT foci. These molecular events lead to repression of a BRD4·P-TEFB downstream target gene c-fos, a component of activator protein 1 (AP-1), that directly regulates epithelial differentiation. Knockdown of BRD4-NUT in NMC cells disperses the transcriptionally inactive chromatin foci and releases the transcriptional activators to stimulate
c-fos expression, leading to restoration of cellular differentiation. Our study provides a novel mechanism by which the BRD4-NUT oncogene perturbs BRD4 functions to block cellular differentiation and to contribute to the oncogenic progression in the
highly aggressive NMC.

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    • "Despite the lack of overlap between most regions bound in TC-797 and 293T cells, BRD4-NUT megadomains in TC-797 cells are also strongly enriched for H3K27ac (Fig. 1F) and show other chromatin features associated with active transcription, as previously noted for BRD4-NUT nuclear foci (Reynoird et al. 2010; Yan et al. 2011). Specifically , ChIP-seq in TC-797 cells revealed concurrent enrichment of megadomains for H3K9Ac, H3K14Ac, and H3K36Me3 (Fig. 2A). "
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    ABSTRACT: NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we found correlate with ∼100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These "megadomains" appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones that drive transcription of underlying DNA in NMC patient cells and naïve cells induced to express BRD4-NUT. Megadomain locations are typically cell lineage-specific; however, the cMYC and TP63 regions are targeted in all NMCs tested and play functional roles in tumor growth. Megadomains appear to originate from select pre-existing enhancers that progressively broaden but are ultimately delimited by topologically associating domain (TAD) boundaries. Therefore, our findings establish a basis for understanding the powerful role played by large-scale chromatin organization in normal and aberrant lineage-specific gene transcription. © 2015 Alekseyenko et al.; Published by Cold Spring Harbor Laboratory Press.
    Full-text · Article · Jul 2015 · Genes & Development
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    • "Cells cultured on cover slips were fixed with 3% paraformaldehyde in PBS for 20 min. Immunofluorescent staining was performed as previously described [40,43]. The following primary antibodies were used: anti-Xpress (Invitrogen), anti-HA (Santa Cruz), and anti-FLAG (Sigma). "
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    ABSTRACT: The human papillomavirus (HPV) vaccines effectively protect against new infections of up to four HPV subtypes. However, these vaccines are not protective against many other clinically relevant HPV subtypes and are ineffective at treating established HPV infections. There is therefore a significant need for antiviral treatments for persistent HPV infections. A promising anti-HPV drug target is the interaction between the HPV E2 protein and cellular bromodomain-containing protein 4 (Brd4) since this protein complex mediates several processes important for the viral life cycle including viral genome maintenance, replication, and transcription. Using bimolecular fluorescence complementation (BiFC) technology, we demonstrate the E2 and Brd4 interaction on both interphase chromatin and mitotic chromosomes throughout mitosis. The E2-Brd4 BiFC was significantly diminished by mutating the Brd4 binding sites in E2 or by a dominant negative inhibitor of the E2-Brd4 interaction, demonstrating the potential of BiFC for identifying inhibitors of this important virus-host interaction. Importantly, when Brd4 was released from chromatin using the bromodomain inhibitor JQ1(+), the E2-Brd4 interacting complex relocated into foci that no longer associate with mitotic chromosomes, pointing to JQ1(+) as a promising antiviral inhibitor of HPV genome maintenance during HPV persistent infection.
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    ABSTRACT: Chromatin structure organization is crucial for regulating many fundamental cellular processes. However, the molecular mechanism that regulates the assembly of higher-order chromatin structure remains poorly understood. In this study, we demonstrate that Brd4 (bromodomain-containing protein 4) protein participates in the maintenance of the higher-order chromatin structure. Brd4, a member of the BET family of proteins, has been shown to play important roles in cellular growth control, cell cycle progression, and cancer development. We apply in situ single cell chromatin imaging and micrococcal nuclease (MNase) assay to show that Brd4 depletion leads to a large scale chromatin unfolding. A dominant-negative inhibitor encoding the double bromodomains (BDI/II) of Brd4 can competitively dissociate endogenous Brd4 from chromatin to trigger severely fragmented chromatin morphology. Mechanistic studies using Brd4 truncation mutants reveal that the Brd4 C-terminal domain is crucial for maintaining normal chromatin structure. Using bimolecular fluorescence complementation technology, we demonstrate that Brd4 molecules interact intermolecularly on chromatin and that replacing Brd4 molecules by BDI/II causes abnormal nucleosome aggregation and chromatin fragmentation. These studies establish a novel structural role of Brd4 in supporting the higher chromatin architecture.
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