The promoters of the human and mouse genes were analyzed. Luciferase reporter constructs were designed containing various fragments of the 5’ regulatory region from 200bp to –9.6 kilobases upstream of the ATG and transfected into various cell lines. In both human and mouse promoters we found that 400-500bp fragments containing multiple Sp1 sites confer basal promoter activity. We also studied the effect of low phosphate levels on klotho gene regulation. It was recently shown that a transcription factor TFE3 responsive 6 bp element can mediate increased gene expression in vitro under reduced phosphate conditions. Two such phosphate response elements were found in the human klotho gene, one at –1.3kb upstream of exon 1 and the other just downstream of it. Luciferase constructs containing these elements, alone and in tandem, were transfected into a well-differentiated kidney cell line grown without phosphate in the media. However, no significant effect on luciferase expression was observed. The mouse promoter also contains a large polypurine /polypyrimidine tract 7kb upstream of exon 1. Such repeats have been implicated in strong regulation of downstream genes. Luciferase constructs have been designed containing these CT rich tracts and their effects on gene expression are currently under investigation.
Understanding the transcriptional elements regulating klotho gene expression may also allow us to improve the design of klotho gene transfer vectors and achieve more efficient expression. We have recently developed lentivirus-based vectors for efficient delivery and sustained long-term expression of the secreted form of the klotho gene. Our vectors can be produced at titers of 106-7 transducing units per ml, and also contain a marker gene encoding the green fluorescent protein (GFP), whose transcript is directly linked to that of the klotho gene via an internal ribosome entry site (IRES). Gene delivery and expression were confirmed by flow cytometry to detect the GFP marker, and by Western blot using an anti-klotho antibody. These vectors are currently being tested in vivo to determine whether re-expression of the secreted form of klotho alone can rescue the knockout phenotype, and to investigate the effects of over-expressing secreted klotho in wild type mice.
Key words:
Klotho, Gene expression, Lentiviral vectors
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