Isolation of genomic DNA sequences that bind vitamin D receptor complexes.
Vitamin D signaling is believed to be transduced by a heterodimeric receptor complex that binds to specific sequences of DNA termed vitamin D response elements (VDREs) in the promoter regions of target genes. However, recent studies have suggested that considerable flexibility exists in the types of binding sites the vitamin D receptor (VDR) is capable of recognizing, including some that bind VDR homodimers. In this report, a screening method involving immunoselection and PCR amplification was utilized to examine genomic binding sites for the receptor. Four individual fragments ranging in size from ca. 250-320 bp were nominally isolated from the amplified pool of captured fragments for further analysis. Each of the four sequences was capable of forming specific, unique VDR complexes using recombinant human VDR (rhVDR) alone or rhVDR heteromers formed in conjunction with the addition of recombinant human retinoid X receptor alpha (rhRXRalpha). Two of these fragments exhibited significant hormone-dependent repression of luciferase activity when linked to a thymidine kinase driven reporter vector. DNaseI footprinting revealed specific binding over DR+3 or related half-site sequences found within both of these DNA fragments. The results from this study demonstrate that specific, functional binding sites for the VDR can be successfully isolated from genomic DNA and should aid in the discovery of genes regulated by the steroid hormone.
Koszewski
N J
NJ
Division of Nephrology, Bone and Mineral Metabolism, University of Kentucky Medical Center, Lexington 40536-0298, USA. njhosz0@pop.uky.edu
Kiessling
S
S
Malluche
H H
HH
D000818:Animals; D001483:Base Sequence / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D018983:DNA Footprinting; D019281:Dimerization; D005240:Feasibility Studies; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D015698:Genomic Library; D006801:Humans; D007158:Immunologic Techniques; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D046911:Macromolecular Substances; D009893:Opossums; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D018167:Receptors, Calcitriol / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D047488:Retinoid X Receptors; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D046911:Macromolecular Substances; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D004247:DNA