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

Abstract

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 BRD4⅐P-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.

Inhibition of bromodomain and extra-terminal proteins (BET) as a potential therapeutic approach in haematological malignancies: emerging preclinical and clinical evidence

“…In addition, p300 sequestration in inactive chromatin domains results in p53 inactivation. BRD4-NUT knockdown restores c-fos expression and p53 activity [Reynoird et al 2010;Yan et al 2011].…”

BRD4 regulates Nanog expression in mouse embryonic stem cells and preimplantation embryos

“…25 The proximal promoter ( À 188) and distal enhancer ( À 4546) regions of Nanog gene have been defined previously. [26][27][28] Primers spanning these regions were used in ChIP-qPCR assays to detect the binding of BRD4 (Figure 5a).…”

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

“…1A; French et al 2008;Reynoird et al 2010;Yan et al 2011; data not shown). The function of these foci is not known; however, they have been observed in all of the BRD-NUT tumor tissue and patient cells examined to date (French et al 2008;Haack et al 2009;Reynoird et al 2010;Yan et al 2011). The critical nature of the bromodomain-acetyl-lysine interaction has been exploited by the development of acetyl lysine mimetic molecules, termed BET inhibitors, that rapidly induce differentiation of NMC cells both in vitro and in vivo (Filippakopoulos et al 2010) with concomitant disappearance of the hyperacetylated nuclear foci.…”

“…This has led to two nonmutually exclusive models for how BRD-NUT drives oncogenesis. In one, BRD-NUT represses the expression of prodifferentiation genes through its sequestration of p300 away from these genes (Reynoird et al 2010;Yan et al 2011;French 2012French , 2014. In the other, BRD-NUT directs p300-dependent transcriptional activity to genes required for proliferation and the blockade of differentiation (French 2014;Wang and You 2015).…”

“…The BET protein tethers NUT to acetylated chromatin via its dual bromodomains, forming ∼80-100 large, hyperacetylated nuclear foci ( Fig. 1A; French et al 2008;Reynoird et al 2010;Yan et al 2011; data not shown). The function of these foci is not known; however, they have been observed in all of the BRD-NUT tumor tissue and patient cells examined to date (French et al 2008;Haack et al 2009;Reynoird et al 2010;Yan et al 2011).…”