Role of distal upstream sequence in vitamin D-induced expression of human CYP24 gene
Section snippets
Materials and methods
Preparation of deletion constructs of the CYP24 gene’s upstream sequence. Human genomic DNA was isolated from skin-derived fibroblasts TIG-112 (Health Science Research Resources Bank (HSRRB), Osaka, Japan), using DNeasy tissue kit (Qiagen) according to the manufacturer’s instruction. Various regions of the CYP24 gene’s upstream sequence were amplified by polymerase chain reaction (PCR). The reaction was carried out in a 50 μl mixture containing 1 U KOD-Plus DNA polymerase (TOYOBO), 5 μl of (10×)
Deletion analysis of the upstream sequence of the human CYP24 gene
Various regions of the upstream sequence of the human CYP24 gene were inserted into the promoter site of the luciferase gene in the expression vector pGL3-basic. These constructs were transfected into TIG-112 cells, and transient expression of the luciferase gene was monitored 12 h after addition of 1,25(OH)2D3 (Fig. 1A). Although the construct −294/+209 includes both of the two VDREs, it was not enough to fully respond to 1,25(OH)2D3. An extension of the insert to nucleotide −548 increased the
Discussion
We have described deletion analyses of the 5′-flanking sequence as well as 5′-UTR of the human CYP24 gene. Most of our effort was directed to the analyses of the region between nucleotides −548 and −294, which is located immediately upstream of the two VDREs, and was not fully studied previously. We found that this region contains sequence elements that synergistically function with the nearby two VDREs. Neither of the sequences was dispensable for the expression of the CYP24 gene. The sequence
Acknowledgment
This work was supported in part by a grant awarded to M.R. from the Hiroshima Prefectural Government in Japan.
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Present address: Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, 7-6-8, Saito, Asagi, Ibaraki, Osaka 567-0085, Japan.