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Structural organization of the human vitamin D receptor chromosomal gene and its promoter
K Miyamoto1, R A Kesterson, H Yamamoto
1Department of Clinical Nutrition, Tokushima University, Japan.
Abstract:
The vitamin D receptor (VDR) is known to mediate the pleiotropic biological actions of 1,25-dihydroxyvitamin D3 through its ability to modulate the expression of target genes. The regulation of this ligand-activated cellular transcription factor is reported to occur at both transcriptional and posttranslational levels. To begin to address the molecular basis by which the VDR gene is regulated transcriptionally, we report here an initial characterization of the human VDR gene and its promoter. We isolated several overlapping A-phage and cosmid clones that cover more than 100 kb of human DNA and contained the entire VDR gene. The gene is comprised of 11 exons that, together with intervening introns, span approximately 75 kb. The noncoding 5'-end of the gene includes exons 1A, 1B, and 1C. Eight additional exons (exons 2-9) encode the structural portion of the VDR gene product. While primer extension and S1 nuclease-mapping studies reveal several common transcriptional start sites, three unique mRNA species are produced as a result of the differential splicing of exons 1B and 1C. The DNA sequence lying upstream of exon 1A is GC rich and does not contain an apparent TATA box. Several potential binding sites for the transcription factor SP1 and other activators are evident. Fusion of DNA fragments containing putative promoter sequences upstream of the luciferase structural gene followed by transient transfection of these plasmids into several mammalian cell lines resulted in significant reporter activity. Due to the size and complexity of the 5'-end of the VDR gene, we examined the activity of a DNA fragment surrounding exon 1C. An intron fragment 3' of exon 1C conferred retinoic acid responsivity when fused to a reporter gene plasmid, suggesting a molecular mechanism for the previously observed ability of retinoic acid to induce the VDR. The recovery of the gene for the human VDR will enable further studies on the transcriptional regulation of this gene.
Insights
Researchers characterized the human vitamin D receptor (VDR) gene and its promoter, identifying its structure and regulatory elements. This work provides a foundation for understanding VDR gene transcriptional regulation.
Area of Science:
- Molecular Biology
- Genetics
- Endocrinology
Background:
- The vitamin D receptor (VDR) is a ligand-activated transcription factor crucial for mediating vitamin D's biological effects.
- VDR regulation occurs at both transcriptional and posttranslational levels, but the molecular basis of its transcriptional regulation is not fully understood.
Purpose of the Study:
- To initiate the molecular characterization of the human VDR gene and its promoter.
- To elucidate the transcriptional regulation mechanisms of the VDR gene.
Main Methods:
- Isolation and characterization of overlapping A-phage and cosmid clones containing the human VDR gene.
- Analysis of gene structure, including exons, introns, and transcriptional start sites.
- Reporter gene assays (luciferase) using VDR promoter fragments in mammalian cell lines.
- S1 nuclease mapping and primer extension studies.
Main Results:
- The human VDR gene spans approximately 75 kb and consists of 11 exons, with complex noncoding 5'-end exons (1A, 1B, 1C).
- Differential splicing of exons 1B and 1C generates three unique mRNA species.
- The promoter region upstream of exon 1A is GC-rich, lacks a TATA box, and contains potential SP1 binding sites.
- Reporter gene assays confirmed promoter activity, and an intron fragment 3' of exon 1C showed retinoic acid responsiveness.
Conclusions:
- The characterized human VDR gene structure and promoter provide insights into its transcriptional regulation.
- The findings suggest a molecular mechanism for retinoic acid's induction of VDR.
- This foundational work enables further detailed studies on VDR gene transcriptional control.