The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains

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.

Ectopic protein interactions within BRD4–chromatin complexes drive oncogenic megadomain formation in NUT midline carcinoma

“…To this end, we induced expression of BioTAP-tagged BRD4-NUT (BRD4-NUT-BioTAP) or the short isoform of BRD4 (BRD4short-BioTAP), encoding only the portion of BRD4 included in the BRD4-NUT fusion oncoprotein (10). We expressed the epitopetagged proteins from single-copy transgenes integrated in a non-NMC cell line, 293T, the derivative of which we term 293TRex (3,11). 293TRex cells serve as a useful model, as they do not normally harbor the oncogenic fusion but, when induced to express BRD4-NUT, form de novo nuclear foci and hyperacetylated megadomains (3).…”

“…Our recent genomic analysis of NMC patient cell lines provides strong evidence that the dual properties of acetyl-histone binding and EP300 recruitment result in a feed-forward expansion of acetylated chromatin and BRD4-NUT over massive genomic domains, often filling entire topologically associating domains (TADs) (3). The number and magnitude of these "megadomains" correlate with the characteristic nuclear foci seen in diagnostic patient tumor samples or in cultured NMC cells stained with a NUT-specific antibody (3,6,7).…”

“…Similarly, small-molecule BET inhibitors such as JQ1, which disengage BRD4-NUT from chromatin, diminish megadomainassociated transcription, including at MYC enhancers and TP63, and also result in differentiation and growth arrest of NMC cells (3,8). Thus, it appears that BRD4-NUT directly misregulates these two key genes, and potentially many others, to drive one of the most aggressive tumors known in cancer biology.…”

“…Megadomains encompassing the MYC and TP63 regulatory regions are common to all NMCs examined to date, and RNAi knockdown of either of these genes in patient cells blocks growth and, in the case of MYC, leads to differentiation in culture (2,3). Similarly, small-molecule BET inhibitors such as JQ1, which disengage BRD4-NUT from chromatin, diminish megadomainassociated transcription, including at MYC enhancers and TP63, and also result in differentiation and growth arrest of NMC cells (3,8).…”

“…Thus, the comprehensive identification of the components of chromatin complexes implicated in disease via their protein-protein interactions is an important avenue toward finding potential targets for therapeutic intervention. In NUT midline carcinoma (NMC), a subtype of squamous cell cancer, the transcriptional machinery is hijacked to drive expression of progrowth, antidifferentiation genes (1)(2)(3). NMC is defined by chromosomal rearrangement of the NUT (NUTM1) gene, which is most commonly fused to the BRD4 gene (4,5).…”