Interaction between RAR and VDR genomic signaling and the role of RXR in the interference of nuclear receptor pathways in macrophages

Abstract

Nuclear receptors (NRs) are ligand-dependent transcription factors that regulate gene expression in response to hormones and other signaling molecules by binding to specific genomic sequences called response elements. The distinct biological functions of NRs are shaped by their ligand specificity, dimerization properties, and DNA-binding preferences. Retinoic acid receptors (RARs) and the vitamin D receptor (VDR) are members of the NR superfamily that mediate the actions of retinoids (vitamin A) and vitamin D, respectively. The abundance and characteristics of regulatory regions, occupied by both RARs and VDR are largely unexplored. RARs, VDR, and many other NRs predominantly function as heterodimers with retinoid X receptors (RXRs), a common dimerization partner that coordinates multiple NR signaling pathways. Because several RXR-containing heterodimers can be activated simultaneously by either RXR agonists or partner-specific ligands, competition for RXR may influence transcriptional responses. In this thesis, we investigated shared RARα and VDR genomic binding regions and their contribution to pathway convergence in macrophage-like cells. We also examined ligand-dependent changes in the RXR genomic binding landscape and evaluated whether RXR availability becomes limiting during simultaneous NR pathway activation. To address these questions, we primarily used integrative genome-wide approaches, including ChIP-seq, RNA-seq, and ATAC-seq, and bioinformatics tools. We employed differentiated THP-1 cells as a macrophage-like cellular model for these investigations. Macrophages sense and integrate diverse metabolic and environmental cues by expressing a wide array of receptors, including NRs. We found that the cistromes of ligand-activated RARα and VDR largely overlapped, and their agonists (AM580 and calcitriol) co-regulated several genes, often cooperatively. Common binding regions were frequently (but not exclusively) annotated with co-regulated genes and exhibited increased MED1 occupancy upon ligand stimulation, suggesting their involvement in gene regulation. Chromatin accessibility was typically higher in the common regions than in regions occupied exclusively by RARα or VDR. DNA response elements for RARα (DR1/2/5) and VDR (DR3) were enriched in the common regions, albeit the co-occurrence of the two types of canonical motifs was low (8.4%), suggesting that “degenerate” DR1/2/5 and DR3 motifs or other sequences could mediate the binding. We next used ChIP-seq to profile RXR occupancy in PMA-differentiated THP-1 cells treated with agonists of RXR or partner receptors, including RARα, VDR, PPARδ, PPARγ, LXRs, and TR, or a “cocktail” containing multiple agonists. The RXR agonist LG268 produced a stronger increase in RXR occupancy than any of the six partner-receptor agonists or their combination. The relevance of ligand-induced RXR peaks was confirmed by the analyses of motif enrichment and RXR occupancy at regulatory elements of target genes. RXR binding was investigated in more detail in cells treated with the VDR agonist, calcitriol. Calcitriol markedly enhanced VDR binding, but the corresponding increase in RXR occupancy was less pronounced. We found that both ligand-induced and unresponsive RXR peaks were involved in gene regulation, and only a small subset (∼3%) of calcitriol-regulated genes exhibited decreases in both RXR binding and mRNA levels in response to combined agonist treatment. In summary, common binding regions of RARα and VDR are at the crossroads of the retinoid and vitamin D pathways, playing important roles in their convergence and cooperation. Moreover, these results support a model in which RXR functions as a nonlimiting module in a macrophage-like cell type, and interference between pathways is minimally attributable to RXR sequestration.