Analysis of transcription complexes and effects of ligands by microelectrospray ionization mass spectrometry

The human vitamin D receptor (VDR) and retinoid X receptor-alpha (RXR alpha) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in the presence of their known endogenous ligands 1 alpha,25-dihydroxyvitamin D-3 (1,25-[OH](2)D-3) and 9-cis-retinoic acid (9-c-RA). We used rapid buffer exchange gel filtration in conjunction with microelectrospray ionization mass spectrometry (mu ESI-MS) to study the binding of these receptors to the osteopontin vitamin D response element (OP VDRE). In the absence of DNA, both VDR and RXR alpha existed primarily as monomers, but in the presence of OP VDRE, homodimeric RXR alpha and heterodimeric RXR alpha-VDR complexes were shown to bind OP VDRE. Addition of 9-c-RA increased RXR alpha homodimer-OP VDRE complexes, and addition of 1,25-(OH)2D3 resulted in formation of 1,25-(OH)(2)D-3-VDR-RXR alpha-OP VDRE complexes. Addition of low-affinity binding ligands had no detectable effect on the VDR-RXR alpha-OP VDRE transcription complex. These results demonstrate the utility of mu ESI-MS in analyzing multimeric, high-molecular-weight protein-protein and protein-DNA complexes, and the effects of ligands on these transcriptional complexes.