Purchase this article with an account.
O. Nichini, D. F. Schorderet; Identification of the Minimal Promoter Region of Tris1, a Transcription Factor Implicated in Eye Development. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2912.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Early ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signaling molecules. Among these regulatory molecules are Pax6, Pax2, Six3, Six6, Mitf, Chx10, Rx, Otx1, Otx2, cyclin D1, BMP4, and BMP7. Together, these molecules regulate cell proliferation and apoptosis, and specify retinal fate. Tris1 is a homeobox transcription factor also implicated in eye development. As part of a global investigation of this gene, we present here the minimal promoter region.
To functionally characterize Tris1 promoter activity, serial deletions were constructed in pGL-3 basic vector (Promega), which contains the Firefly Luciferase reporter gene. Luciferase assays were performed in HeLa cells by co-transfection of these vectors with the reporter Renilla Luciferase vector, pRL-SV40.
The analysis of the 5’-flanking region of the mouse Tris1 gene by PROSCAN (http://thr.cit.nih.gov/molbio/proscan/), revealed a predicted promoter sequence between -416 and -166 with no TATA box. Promoter activity of several 5’- and 3’-deleted constructions made between -1884 and -164 were compared with that of pGL3-basic. The maximum activity was found for the vector -1884/-1, then for the vectors -502/-1 and -350/-1. Next, the activity decreased with the length of the cloned promoter region. Truncation analysis identified the region from -350 to -296 as having high promoter activity. Within this region, several transcription factor-binding sites cluster, particularly several Sp1-binding sites. A deletion of the 20 bp Sp1 region and 10 point mutations were introduced in pGL3-350/-1 by site directed mutagenesis. The activity of these constructs dramatically decreased to levels of the -296/-1 plasmid.
Our results confirm that the minimal promoter predicted by PROSCAN was correct. We demonstrated that the immediate upstream region of Tris1 gene possesses a strong intrinsic promoter activity in vitro, suggesting its potential role as the promoter of Tris1 in vivo. Although no obvious TATA box or CAAT motif was found, we identified several consensus recognition sites for the ubiquitously expressed Sp1 transcription factor. Sp1 sites are especially interesting since they are often present in TATA-less promoters. An examination of basal promoter activity in Hela cells supported the involvment of Sp1 in the expression of Tris1. Binding of Sp1 to this region of the Tris1 promoter was confimed by targeted mutation of the Sp1-binding sites.
This PDF is available to Subscribers Only