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Characterization of human and mouse angiopoietin-like factor CDT6 promoters
1Department of Ophthalmology, University of Washington School of Medicine, Seattle 98195-6485, USA.
Purpose:
Angiogenesis refers to the latter stage of vascular development. It has been reported that angiopoietin-like factor cornea-derived transcript 6 (CDT6) encodes a protein homologous to angiopoietins that could play a critical role in blocking a receptor of angiopoietin (Tie2) and therefore contribute to the avascularity and transparency of the cornea in the developing embryo and the adult. This study was focused on isolation and characterization of the CDT6 promoter.
Methods:
Rapid amplification of cDNA ends (5'-RACE) was used to isolate the CDT6 promoter from an adaptor-ligated genomic DNA fragment library and to identify the transcription initiation site of the CDT6 gene. The RNase protection assay was performed to confirm the initiation site. The sequence similarity, binding sites for putative transcription factors, and transcriptional activity of human and mouse CDT6 promoters were compared. Corneal and noncorneal cells from humans and other animals were transiently transfected with CDT6 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs to analyze the transcriptional activity of the promoter.
Results:
A 2956-bp human CDT6 promoter fragment and a 3142-bp mouse CDT6 promoter fragment were isolated. The major transcription initiation sites of the human and mouse CDT6 genes were located at 224 and 168 bp, respectively, upstream of the translation initiation site. Human and mouse CDT6 promoter sequences were very similar. Both promoters were minus TATA and CAAT boxes close to the transcription initiation site. Transfection into human corneal and noncorneal cells and into nonhuman cells revealed that the human CDT6 promoter probably contains positive and negative cis-regulatory elements that modulate cell, tissue, and species specificity. The human CDT6 promoter contains four interferon (IFN)-stimulated response elements (ISREs). No ISREs could be identified in the mouse promoter. IFN-alpha stimulated transcriptional activity of the human promoter.
Conclusions:
The human and mouse CDT6 promoters have similar sequences and share many cis-regulatory elements. IFN-alpha appears to have an important role in regulating transcription of the human, but not the mouse, CDT6 promoter.