UC Riverside

Nuclear receptors (NRs) are ligand-sensitive transcription factors that regulate a wide array of biological processes including development, metabolism, and circadian rhythms. All NRs share a common protein structure, including highly conserved DNA binding domains and a highly variable N-terminal A/B domain, and are very popular drug targets. To better understand the role of alternative A/B domains between NR isoforms and the impact of genetic variation on gene expression in the liver, we employed two experimental approaches. The NR hepatocyte nuclear factor 4α (HNF4α), a master regulator of liver-specific gene expression, is regulated by two promoters (P1 and P2) in the liver resulting in proteins with different A/B domains. P1-HNF4α is expressed in fetal and normal adult liver while P2-HNF4α is expressed only in the fetal liver and in liver cancer. We compared wildtype mice, which express only HNF4α1 (P1) in the adult liver, to exon-swap mice that express only HNF4α7 (P2) for global changes in gene expression (RNA-seq), chromatin binding (ChIP-seq), and unique protein interactions (RIME). The results show that P1- and P2-HNF4α isoforms differentially regulate hundreds of transcripts in the adult liver, including the NR CAR (Nr1i3), and may be recruited differentially to non-HNF4α binding sites by unique protein interactions. They also exhibit altered metabolic pathways, especially cytochrome P450 (Cyp) genes. All told, the results show that changes in just 16-30 amino acids in the AF-1 region of an NR can have profound effects on gene expression. Utilizing protein binding microarrays (PBM), we can measure the DNA binding affinity of a given NR against both alleles of 125,000 genetic variants in a single experiment to probe for affinity altering SNPs (aaSNPs). By mining SNPs from ChIP-seq peaks and eQTLs from the GTEx project, we have identified thousands of aaSNPs, hundreds of which show significant correlation to changes in gene expression within their regulatory network. Analysis of aaSNPs from GWAS studies associated with Alzheimer’s disease identified a large number of genetic variants that can alter the DNA binding affinity of PPARɣ in the APOE locus. Additionally, we show the power of the PBMs to validate many aaSNPs derived from in vivo analysis and suggest a role for the PBM technology in characterizing how genetic diversity may play a role in personalized medicine.