Carreira S, Dexter T J, Yavuzer U, Easty D J, Goding C R
Eukaryotic Transcription Laboratory, Marie Curie Research Institute, Oxted, Surrey RH8 0TL, United Kingdom.
Mol Cell Biol. 1998 Sep;18(9):5099-108. doi: 10.1128/MCB.18.9.5099.
Previous work has demonstrated that two key melanocyte-specific elements termed the MSEu and MSEi play critical roles in the expression of the melanocyte-specific tyrosinase-related protein 1 (TRP-1) promoter. Both the MSEu and MSEi, located at position -237 and at the initiator, respectively, bind a melanocyte-specific factor termed MSF but are also recognized by a previously uncharacterized repressor, since mutations affecting either of these elements result in strong up-regulation of TRP-1 promoter activity in melanoma cells. Here we demonstrate that repression mediated by the MSEu and MSEi also operates in melanocytes. We also report that both the MSEu and MSEi are recognized by the brachyury-related transcription factor Tbx2, a member of the recently described T-box family, and that Tbx2 is expressed in melanocyte and melanoblast cell lines but not in melanoblast precursor cells. Although Tbx2 and MSF each recognize the TRP-1 MSEu and MSEi motifs, it is binding by Tbx-2, not binding by MSF, that correlates with repression. Several lines of evidence tend to point to the brachyury-related transcription factor Tbx2 as being the repressor of TRP-1 expression: both the MSEu and MSEi bind Tbx2, and mutations in either element that result in derepression of the TRP-1 promoter diminish binding by Tbx2; the TRP-1 promoter, but not the tyrosinase, microphthalmia, or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) promoter, is repressed by Tbx2 in cotransfection assays; a high-affinity consensus brachyury/Tbx2-binding site is able to constitutively repress expression of the heterologous IE110 promoter; and a low-affinity brachyury/Tbx2 binding site is able to mediate Tbx2-dependent repression of the G3PDH promoter. Although we cannot rule out the presence of an additional, as yet unidentified factor playing a role in the negative regulation of TRP-1 in vivo, the evidence presented here suggests that Tbx2 most likely is the previously unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box family member.
先前的研究表明，两个关键的黑素细胞特异性元件，即MSEu和MSEi，在黑素细胞特异性酪氨酸酶相关蛋白1（TRP-1）启动子的表达中起关键作用。MSEu和MSEi分别位于-237位和起始子处，它们都能结合一种称为MSF的黑素细胞特异性因子，但也能被一种以前未鉴定的阻遏物识别，因为影响这两个元件中任何一个的突变都会导致黑色素瘤细胞中TRP-1启动子活性的强烈上调。在这里，我们证明了由MSEu和MSEi介导的抑制作用在黑素细胞中也起作用。我们还报告说，MSEu和MSEi都能被与短尾相关的转录因子Tbx2识别，Tbx2是最近描述的T-box家族的成员，并且Tbx2在黑素细胞和成黑素细胞系中表达，但在成黑素细胞前体细胞中不表达。尽管Tbx2和MSF都能识别TRP-1的MSEu和MSEi基序，但与抑制作用相关的是Tbx2的结合，而不是MSF的结合。几条证据倾向于表明与短尾相关的转录因子Tbx2是TRP-1表达的阻遏物：MSEu和MSEi都能结合Tbx2，并且导致TRP-1启动子去抑制的任一元件中的突变都会减少Tbx2的结合；在共转染实验中，TRP-1启动子，而不是酪氨酸酶、小眼畸形或甘油醛-3-磷酸脱氢酶（G3PDH）启动子，受到Tbx2的抑制；一个高亲和力的共有短尾/Tbx2结合位点能够组成性地抑制异源IE110启动子的表达；一个低亲和力的短尾/Tbx2结合位点能够介导Tbx2依赖的G3PDH启动子的抑制。尽管我们不能排除存在另一种尚未鉴定的因子在体内对TRP-1的负调控中起作用的可能性，但这里提供的证据表明Tbx2很可能是以前未鉴定的TRP-1表达的阻遏物，因此很可能代表T-box家族成员转录抑制的第一个例子。
