BRACHYURY-RELATED TRANSCRIPTION FACTOR TBX2 AND REPRESSION OF THE MELANOCYTE-SPECIFIC TRP-1 PROMOTER

Previous work has demonstrated that two key melanocyte-specific elemen ts termed the MSEu and MSEi play critical roles in the expression of t he 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 al so recognized by a previously uncharacterized repressor, since mutatio ns affecting either of these elements result in strong up-regulation o f 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 brac hyury-related transcription factor Tbx2, a member of the recently desc ribed T-box family, and that Tbx2 is expressed in melanocyte and melan oblast cell lines but not in melanoblast precursor cells. Although Tbx 2 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 transcriptio n factor Tbx2 as being the repressor of TRP-1 expression: both the MSE u and MSEi bind Tbx2, and mutations in either element that result in d erepression 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 cotr ansfection assays; a high-affinity consensus brachyury/Tbx2-binding si te is able to constitutively repress expression of the heterologous IE 110 promoter; and a low-affinity brachyury/Tbx2 binding site is able t o mediate Tbx2-dependent repression of the G3PDH promoter, Although we cannot rule out the presence of an additional, as yet unidentified fa ctor playing a role in the negative regulation of TRP-1 in vivo, the e vidence presented here suggests that Tbx2 most likely is the previousl y unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box f amily member.