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

The human vitamin D receptor (VDR) and retinoid X receptor-α (RXRα) 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 1α,25-dihydroxyvitamin D3 (1,25-[OH]2D3) and 9-cis-retinoic acid (9-c-RA). We used rapid buffer exchange gel filtration in conjunction with microelectrospray ionization mass spectrometry (μESI-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 RXRα existed primarily as monomers, but in the presence of OP VDRE, homodimeric RXRα and heterodimeric RXRα–VDR complexes were shown to bind OP VDRE. Addition of 9-c-RA increased RXRα homodimer–OP VDRE complexes, and addition of 1,25-(OH)2D3 resulted in formation of 1,25-(OH)2D3–VDR–RXRα–OP VDRE complexes. Addition of low-affinity binding ligands had no detectable effect on the VDR–RXRα–OP VDRE transcription complex. These results demonstrate the utility of μESI-MS in analyzing multimeric, high-molecular-weight protein–protein and protein–DNA complexes, and the effects of ligands on these transcriptional complexes.

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