Comprehensive Study of Nuclear Receptor DNA Binding Provides a Revised Framework for Understanding Receptor Specificity

The type II nuclear receptors (NRs) function as heterodimeric transcription factors with the retinoid X receptor (RXR) to regulate diverse biological processes in response to endogenous ligands and therapeutic drugs. DNA-binding specificity has been proposed as a primary mechanism for NR gene regulatory specificity. We use protein-binding microarrays (PBMs) to comprehensively analyze the DNA binding of 12 NR:RXRα heterodimers. We find more promiscuous NR-DNA binding than has been reported, challenging the view that NR binding specificity is defined by half-site spacing. We show that NRs bind DNA using two distinct modes, explaining widespread NR binding to half-sites in vivo. Finally, we show that the current models of NR specificity better reflect binding-site activity rather than binding-site affinity. Our rich dataset and revised NR binding models provide a framework for understanding NR regulatory specificity and will facilitate more accurate analyses of genomic datasets. Protein-binding microarray experiments were performed for type II Nuclear Receptors (NR) dimers. Purified or in-vitro transcribed translated (IVT) NR proteins were combined generate heterodimer and homodimers. NRs can bind half-sites sequences organized as direct repeats with a variable length spacer of 0-5 base pairs (bp) (DR0-DR5). To characterize both DNA-base preferences and the DR-spacing preferences, we measured NR binding to more than 1500 unique sequences at six DR spacer lengths (DR0-DR5). Specifically, for each DR spacer length, we measured NR binding to 24 sequences, which we refer to as seed sequences. These seed sequences exhibit a range of degeneracy from the consensus 5’-RGKTCA-3’ half-sites. To assay DNA base preferences for each seed sequence we also measured binding to all possible single-nucleotide variants (SNVs), with each SNV included as a separate probe on the PBM. To capture any binding preferences for flank and DR spacer sequences, we have included SNVs for the five nucleotides upstream and downstream of the DR and across the spacer sequence.