Gain-of-Function Alleles in Caenorhabditis elegans Nuclear Hormone Receptor nhr-49 Are Functionally Distinct.
Nuclear hormone receptors (NHRs) are transcription factors that regulate numerous physiological and developmental processes and represent important drug targets. NHR-49, an ortholog of Hepatocyte Nuclear Factor 4 (HNF4), has emerged as a key regulator of lipid metabolism and life span in the nematode worm Caenorhabditis elegans. However, many aspects of NHR-49 function remain poorly understood, including whether and how it regulates individual sets of target genes and whether its activity is modulated by a ligand. A recent study identified three gain-of-function (gof) missense mutations in nhr-49 (nhr-49(et7), nhr-49(et8), and nhr-49(et13), respectively). These substitutions all affect the ligand-binding domain (LBD), which is critical for ligand binding and protein interactions. Thus, these alleles provide an opportunity to test how three specific residues contribute to NHR-49 dependent gene regulation. We used computational and molecular methods to delineate how these mutations alter NHR-49 activity. We find that despite originating from a screen favoring the activation of specific NHR-49 targets, all three gof alleles cause broad upregulation of NHR-49 regulated genes. Interestingly, nhr-49(et7) and nhr-49(et8) exclusively affect nhr-49 dependent activation, whereas the nhr-49(et13) surprisingly affects both nhr-49 mediated activation and repression, implicating the affected residue as dually important. We also observed phenotypic non-equivalence of these alleles, as they unexpectedly caused a long, short, and normal life span, respectively. Mechanistically, the gof substitutions altered neither protein interactions with the repressive partner NHR-66 and the coactivator MDT-15 nor the subcellular localization or expression of NHR-49. However, in silico structural modeling revealed that NHR-49 likely interacts with small molecule ligands and that the missense mutations might alter ligand binding, providing a possible explanation for increased NHR-49 activity. In sum, our findings indicate that the three nhr-49 gof alleles are non-equivalent, and highlight the conserved V411 residue affected by et13 as critical for gene activation and repression alike.
Graduate Program in Medical Genetics, University of British Columbia, Vancouver, BC, Canada.|Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada.
Kayoung
K
Lee
Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada.|Graduate Program in Cell and Developmental Biology, University of British Columbia, Vancouver, BC, Canada.
Grace Ying Shyen
GY
Goh
Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada.
Marcus Andrew
MA
Wong
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada.
Tara Leah
TL
Klassen
Graduate Program in Medical Genetics, University of British Columbia, Vancouver, BC, Canada.|Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, University of British Columbia, Vancouver, BC, Canada.|Graduate Program in Cell and Developmental Biology, University of British Columbia, Vancouver, BC, Canada.|Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.
Stefan
http://orcid.org/0000-0002-2432-7257
S
Taubert
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D008969:Molecular Sequence Data; D017354:Point Mutation; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D018160:Receptors, Cytoplasmic and Nuclear