Frederic Jehan

DNA bending is induced by binding of vitamin D receptor-retinoid X receptor heterodimers to vitamin D response elements

The ability of vitamin D receptor‐retinoid X receptor (VDR‐RXR) heterodimers to induce a DNA bend upon binding to various vitamin D response elements (VDRE) has been investigated by circular permutation and phasing analysis. Recombinant rat VDR expressed in the baculovirus system and purified recombinant human RXRβ have been used. The VDREs were from 1,25‐dihydroxyvitamin D3 (1,25‐[OH]2D3) enhanced genes (rat osteocalcin, rOC; mouse osteopontin, mOP, and rat 1,25‐dihydroxyvitamin D3‐24‐hydroxylase, r24‐OHase), and a 1,25‐(OH)2D3 repressed gene (human parathyroid hormone, hPTH). As shown by circular permutation analysis, VDR‐RXR induced a distortion in DNA fragments containing various VDREs. Calculated distortion angles were similar in magnitude (57°, 56°, 61°, and 59°, respectively for rOC, mOP, r24‐Ohase, and hPTH). The distortions took place with or without a 1,25‐(OH)2D3 ligand. The centers of the apparent bend were found in the vicinity of the midpoint of all VDREs, except for rOC VDRE which was found 4 bp upstream. Phasing analysis was performed with DNA fragments containing mOP VDRE and revealed that VDR‐RXR heterodimers induced a directed bend of 26°, not influenced by the presence of hormone. In this study we report that similar to other members of the steroid and thyroid nuclear receptor superfamily, VDR‐RXR heterodimers induce DNA bending. J. Cell. Biochem. 74:220–228, 1999.

Cloning and expression of the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA

Using a cDNA probe from the rat 24-hydrovitamin D3 24-hydroxylase, the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA has been isolated from a chicken kidney lambda gt11 library. The high degree of similarity with the mammalian 24-hydroxylase cDNAs strongly supports the belief that it is the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA. The deduced amino acid sequences are also very well conserved and 325 of them are identical among the four known 25-hydroxyvitamin D3 24-hydroxylases. This cDNA expressed in E. coli produces 24-hydroxylase activity.

Isolation and characterization of the chicken vitamin D receptor gene and its promoter.

The sequences from several independent cDNA clones encoding the chicken vitamin D receptor as well as primer extension assay have clearly delineated the 5′ terminus and the transcriptional start site. Screening a chicken genomic library produced genomic clones containing vitamin D receptor (VDR) gene fragments. Restriction map of clone 8 showed that the 18.6‐kb chicken VDR fragment has exons 1 and 2, intron 1, part of intron 2, and 7‐kb 5′ flanking region. Exons 1, 2 , and 3 found in the chicken VDR gene shares low homology with its mammalian counterparts (i.e., E1A, E1B, and E1C in human). By contrast, the fourth exon and following exons for the coding region of VDR gene are highly conserved between avian and mammalian species. While the fourth exon bears the ATG sites for translation initiation in mammals, the third exon in birds has two extra ATG sites for leaky translation as determined previously. Thus, the avian VDR has more N‐terminal sequence than the mammalian VDR and is found in two distinct forms. The 5′ flanking region from genomic clone 8 shares considerable homology in several regions with the human and mouse VDR promoters. Moreover, the 5′ flanking region of chicken VDR gene possesses promoter activity, as shown by its ability to drive the luciferase reporter gene in cell transfection assays. Like other steroid receptor promoters, the chicken VDR promoter contains no TATA box but possesses several GC boxes or SP1 sites. A series of deletional promoter constructs established that the proximal GC boxes are the major drivers of gene transcription, while the more upstream sequences have repressive elements. J. Cell. Biochem. 77:92–102, 2000.