Identification of nuclear localization signal that governs nuclear import of BRD7 and its essential roles in inhibiting cell cycle progression

Abstract

BRD7, a novel bromodomain gene, is identified to be associated with nasopharyngeal carcinoma (NPC). Decreased or loss of expression of BRD7 was detected in NPC biopsies and cell lines. Overexpression of BRD7 could inhibit NPC cell growth and arrest cells in cell cycle by transcriptionally regulating some important molecules involved in ras/MEK/ERK and Rb/E2F pathway, and downregulate the promoter activity of E2F3. In the present study, the subcellular localization of BRD7 was investigated. It was found that BRD7 was mainly localized in nucleus without distinct cell-specific difference between COS7 and HNE1. Furthermore, a functional nuclear localization signal (NLS) sequence ranging from amino acid 65 to 96 was identified and characterized. The NLS is composed of a cluster of four bipartite nuclear targeting sequences, which are tightly linked and extremely overlapped. We found that whether the entire NLS or the four bipartite nuclear targeting sequences could respectively determine the nuclear import of green fluorescent protein (GFP). The most important is that NLS-deleted BRD7 shifted the nuclear localization to be mostly in cytoplasm, and failed or reduced to negatively regulate the expression of cell cycle related molecules, cyclin D1 and E2F3, and cell cycle progression from G1 to S phase. In conclusion, NLS is an essential motif affecting BRD7 nuclear distribution, and the nuclear localization of BRD7 is critical for the expression of cell cycle related molecules and cell biological function.