Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of melanocyte differentiation, essential for pigmentation and melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate melanocyte differentiation through interactions with MITF. Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented melanocytes and results in de-pigmentation of differentiated melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF. These findings indicate a requirement for BET proteins in the regulation of pigmentation and melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, melanocytes, and melanoma cells.

中文翻译：

---

溴结构域和末端外结构域（BET）蛋白调节黑素细胞的分化

溴结构域和末端外（BET）蛋白的药理抑制作用目前正在探索，作为癌症的一种新治疗方法。一些研究还暗示BET蛋白通过它们与谱系特异性因子的相互作用来作为细胞身份和分化的调节剂。但是，尚未研究BET蛋白在黑素细胞分化中的作用。黑色素细胞诱导转录因子（MITF）是黑色素细胞分化的主要调节剂，对色素沉着和黑色素细胞存活至关重要。在这项研究中，我们测试了BET蛋白通过与MITF相互作用调节黑素细胞分化的假设。在这里，我们表明对BET蛋白的化学抑制作用可防止未着色的黑素母细胞分化为有色的黑素细胞，并导致分化的黑素细胞脱色素。BET抑制作用还可以减缓细胞生长，而不会引起细胞死亡，从而增加了G1细胞的数量。转录谱分析表明，BET抑制导致色素特异性基因（包括许多MITF靶标）的表达下降。色素特异性基因的表达在黑色素瘤细胞中也下调，但程度较小。我们发现，BET家族成员，含溴结构域的蛋白质4（BRD4）和含溴结构域的蛋白质2（BRD2）的RNAi耗竭抑制了两种黑色素合成酶TYR和TYRP1的表达。在MITF结合位点周围的黑素细胞启动子上检测到BRD4和BRD2，与开放的染色质结构相关，并促进MITF结合到这些位点。此外，BRD4和BRD2在物理上与MITF相互作用。这些发现表明在调节色素沉着和黑素细胞分化中需要BET蛋白。我们确定了黑素母细胞，黑色素细胞和黑色素瘤细胞中BET抑制后发生的色素沉着特异性基因表达的变化。