Vitamin D receptor interactions with positive and negative DNA response elements: an interference footprint comparison.
Interference footprinting protocols were utilized to examine the interactions of the vitamin D receptor (VDR) with either a positive or a negative vitamin D response element (VDRE). A sequence from the human osteocalcin (hOC) gene was chosen for the prototypical positive DR+3 VDRE, while an analogous sequence linked to the avian parathyroid hormone gene (aPTH) was used as the negative VDRE. Both types of response elements were examined for phosphate backbone contacts, as well as base-specific interactions with guanine and thymine residues. Sources of VDR included partially purified canine intestinal preparations, as well as extracts of recombinant human VDR and retinoid X receptor alpha prepared from baculovirus-infected Sf9 insect cells. Cold competition experiments using variable amounts of these oligonucleotides in the mobility shift assay revealed that the hOC element was a five-fold better competitor for heterodimer complex binding than the negative VDRE. Interference footprints revealed extensive strong contacts to the phosphate backbone and individual guanine and thymine nucleotides of the hOC element. The composite hOC footprint was asymmetric for the number and strength of interactions observed over each of the respective direct repeat half-sites. In contrast, the aPTH VDRE footprints revealed fewer points of DNA contact that were limited to the hexanucleotide repeat regions and were strikingly weaker in nature. The alignment of DNA contact points for both elements produced a 5' stagger that was indicative of successive major groove interactions, and consistent with dimer binding. DNA helical representations indicate that the heterodimer contacts to these response elements are substantially different and provide insight into functional aspects of each complex.
Koszewski
N J
NJ
Division of Nephrology, Bone and Mineral Metabolism, Department of Internal Medicine, University of Kentucky Medical Center, Lexington, KY 40536, USA. njhosz0@pop.uky.edu
Malluche
H H
HH
Russell
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018983:DNA Footprinting; D004285:Dogs; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D015675:Osteocalcin / Q000235:genetics; D010281:Parathyroid Hormone / Q000235:genetics; D018167:Receptors, Calcitriol / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000235:genetics; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000235:genetics
D010281:Parathyroid Hormone; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D015675:Osteocalcin; D004247:DNA