Supercharging BRD4 with NUT in carcinoma

Abstract

NUT carcinoma (NC) is an extremely aggressive squamous cancer with no effective therapy. NC is driven, most commonly, by the BRD4-NUT fusion oncoprotein. BRD4-NUT combines the chromatin-binding bromo- and extraterminal domain-containing (BET) protein, BRD4, with an unstructured, poorly understood protein, NUT, which recruits and activates the histone acetyltransferase p300. Recruitment of p300 to chromatin by BRD4 is believed to lead to the formation of hyperacetylated nuclear foci, as seen by immunofluorescence. BRD4-NUT nuclear foci correspond with massive contiguous regions of chromatin co-enriched with BRD4-NUT, p300, and acetylated histones, termed "megadomains" (MD). Megadomains stretch for as long as 2 MB. Proteomics has defined a BRD4-NUT chromatin complex in which members that associate with BRD4 also exist as rare NUT-fusion partners. This suggests that the common pathogenic denominator is the presence of both BRD4 and NUT, and that the function of BRD4-NUT may mimic that of wild-type BRD4. If so, then MDs may function as massive super-enhancers, activating transcription in a BET-dependent manner. Common targets of MDs across multiple NCs and tissues are three stem cell-related transcription factors frequently implicated in cancer: MYC, SOX2, and TP63. Recently, MDs were found to form a novel nuclear sub-compartment, called subcompartment M (subM), where MD-MD interactions occur both intra- and inter-chromosomally. Included in subM are MYC, SOX2, and TP63. Here we explore the possibility that if MDs are simply large super-enhancers, subM may exist in other cell systems, with broad implications for how 3D organization of the genome may function in gene regulation and maintenance of cell identity. Finally, we discuss how our knowledge of BRD4-NUT function has been leveraged for the therapeutic development of first-in-class BET inhibitors and other targeted strategies.

Fig.1.

Schematic of BRD4-NUT fusion protein. Arrows denote breakpoints. ET, extra-terminal domain; Bromo, bromodomain; TAD, transcriptional activation domain; NES, nuclear export signal; NLS, nuclear localization signal; CTD, C-terminal domain. TAD1 corresponds with “NUTF1C” fragment that binds p300 described in Reynoird N et al., 2010. Numbers denote amino acid numbers for the indicated domains.

Fig. 2.

De-repression of p300 causes p300 foci to form, resembling BRD4-NUT. A. Cartoons of inactive and activated conformations of p300. These cartoons derived and simplified by those presented in Ortega E et al., 2018. In this model, p300 activation requires dimerization (not shown), and removal of the auto-inhibitory CH3/TAZ2 and RING domains to allow for HAT activation and engagement of the bromodomain, BD, with acetylated histones. B. Constitutionally activated p300, through deletion of its ring or CH3 domains, results in the formation of nuclear foci. HA-tagged p300 was stained with anti-HA antibody and the images shown are from Ortega E et al., 2018. C. BRD4-NUT immunofluorescence is seen in the NC cell line, TC-797. Left are interphase nuclei, and right are metaphase chromosomes revealing that megadomains cover discrete, chromosomal regions. Immunofluorescence was performed using an antibody to NUT protein (anti-NUT, clone C52B1). Images taken from Alekseyenko A et al., 2015. D. NUT and p300 co-immunofluorescence in the BRD4-NUT+ NC cell line, HCC2429. Taken from Reynoird N et al., 2010.

Fig. 3.

BRD-NUT forms megadomains over the MYC locus in five NMCs, but not in 293TRex cells. Shown is anti-NUT ChIP-seq and RNA-seq over a time course of JQ1. 293TRex and 797TRex cells were induced to express BRD4-NUT. TC-797, 797TRex, PER-403, and 1015 (rust) express endogenous BRD4-NUT. 10326 NMC cells (pea soup green) express BRD3-NUT. Figure taken from Alekseyenko A et al., 2015.

Fig. 4.

Mechanistic model of how BRD4-NUT drives growth and blocks differentiation in NUT carcinoma. Ac, acetyl-lysine; MED, mediator complex.

Fig. 5.

The nuclear foci of BRD4-NUT seen in this biopsy of a NUT carcinoma correspond with megadomains and subM. Immunohistochemistry was performed using an antibody to NUT (clone C52B1, Cell Signaling Technologies, Danvers, MA). Scale bar is 15μm.