Yavuzer U, Keenan E, Lowings P, Vachtenheim J, Currie G, Goding C R
Eukaryotic Transcription Laboratory, Marie Curie Research Institute, Oxted, Surrey, UK.
Oncogene. 1995 Jan 5;10(1):123-34.
Little is known of the molecular mechanisms underlying the differentiation of the melanocyte from the melanoblast or the progression from the melanocyte to a malignant melanoma. Since the adenovirus E1A products have proved a useful tool for understanding control of differentiation in other systems, we explored the possibility of using E1A as a probe for factors controlling melanocyte-specific gene expression and differentiation. The results obtained show that the adenovirus E1A 13S, but not the 12S, product can transform the highly pigmented and TPA-dependent melanocyte cell line melan-a. Transformation is characterised by a morphological change, loss of TPA-dependence, the ability to grow in soft agar and strikingly, loss of pigmentation which correlates with loss of expression of the melanocyte-specific TRP-1 and tyrosinase genes. Cotransfection assays demonstrated that repression of TRP-1 by E1A correlated with E1A binding to p105Rb and p300, with the target in the TRP-1 promoter being the M-box, and 11 bp basic-Helix-loop-Helix (bHLH) factor-binding motif conserved between melanocyte-specific promoters. Consistent with the M-box acting as a target for E1a-mediated transcription repression, we also show that the basic-helix-loop-helix-leucine zipper (bHLH-LZ) protein (Mi) encoded by the microphthalmia gene (mi), which is required for pigment cell differentiation, is a positive acting transcription factor which can interact with the retinoblastoma product in vitro and activate the TRP-1 promoter. Moreover, expression of the mi gene was reduced around 50-fold in the non-pigmented E1a-transformed melan-a cells compared to the nontransformed melan-a cell line, with ectopic expression of Mi able to prevent repression of the tyrosinase and TRP-1 promoters in the presence of E1A. Mi therefore appears to play a crucial role in melanocyte-specific gene expression. The parallels between repression of myogenesis and muscle cell bHLH factors, and Mi and melanocyte differentiation are discussed.
黑素母细胞分化为黑素细胞以及黑素细胞发展为恶性黑色素瘤背后的分子机制鲜为人知。由于腺病毒E1A产物已被证明是理解其他系统中分化控制的有用工具，我们探索了使用E1A作为控制黑素细胞特异性基因表达和分化因子的探针的可能性。所得结果表明，腺病毒E1A 13S产物而非12S产物能够转化高度色素沉着且依赖佛波酯（TPA）的黑素细胞系黑素-a。转化的特征在于形态变化、对TPA依赖性的丧失、在软琼脂中生长的能力，以及显著的色素沉着丧失，这与黑素细胞特异性TRP-1和酪氨酸酶基因表达的丧失相关。共转染实验表明，E1A对TRP-1的抑制与E1A与p105Rb和p300的结合相关，TRP-1启动子中的靶标是M盒，以及在黑素细胞特异性启动子之间保守的11 bp碱性螺旋-环-螺旋（bHLH）因子结合基序。与M盒作为E1a介导的转录抑制靶标一致，我们还表明，小眼畸形基因（mi）编码的碱性螺旋-环-螺旋-亮氨酸拉链（bHLH-LZ）蛋白（Mi）是色素细胞分化所必需的，它是一种正向作用的转录因子，能够在体外与视网膜母细胞瘤产物相互作用并激活TRP-1启动子。此外，与未转化的黑素-a细胞系相比，在无色素的E1a转化的黑素-a细胞中，mi基因的表达降低了约50倍，在存在E1A的情况下，Mi的异位表达能够防止酪氨酸酶和TRP-1启动子的抑制。因此，Mi似乎在黑素细胞特异性基因表达中起关键作用。本文讨论了肌生成和肌肉细胞bHLH因子的抑制与Mi和黑素细胞分化之间的相似之处。
