1. ¹Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
2. ²Departments of Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA
3. ³Boston University School of Medicine, Boston, MA, USA
4. ⁴Department of Oncology, Lombardi Comprehensive Cancer Center and Pediatrics, Georgetown University, Washington, DC, USA
5. ⁵Allina Hospitals and Clinics, Minneapolis, MN, USA
6. ⁶Telethon Institute for Child Health Research, and Centre for Child Health Research, The University of Western Australia, Perth, Australia

Correspondence: Dr CA French, Department of Pathology, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115, USA. E-mail: Cfrench@partners.org

Received 28 March 2007; Revised 10 August 2007; Accepted 17 September 2007; Published online 15 October 2007.

Abstract

An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3–NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4–NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3–NUT and BRD4–NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4–NUT and BRD3–NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD–NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3–NUT and BRD4–NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD–NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.