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.
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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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