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Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

ABSTRACT: The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GR^dim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GR^D/D) have previously helped to define the functions of GR monomers and dimers. Since GR^D/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GR^D+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GR^L/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and K_d values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

SUBMITTER: Timmermans S

PROVIDER: S-EPMC8808175 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

Timmermans Steven S Verhoog Nicolette J D NJD Van Looveren Kelly K Dewaele Sylviane S Hochepied Tino T Eggermont Melanie M Gilbert Barbara B Boerema-de Munck Anne A Vanderhaeghen Tineke T Vanden Berghe Joke J Garcia Gonzalez Natalia N Vandewalle Jolien J Bloch Yehudi Y Provost Mathias M Savvides Savvas N SN De Bosscher Karolien K Declercq Wim W Rottier Robbert J RJ Louw Ann A Libert Claude C

The Journal of biological chemistry 20220108 2

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D ...[more]

PMID: 35007536

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Project description:The mouse Btaf1 gene, an ortholog of yeast MOT1, encodes a highly conserved general transcription factor. The function of this SNF-2 like ATPase has been studied mainly in yeast and human cells, which has revealed that it binds directly to TBP, forming the B-TFIID complex. This complex binds to core promoters of RNA polymerase II- transcribed genes and, of crucial importance, BTAF1-TBP interactions have been shown to affect the kinetics of TBP-promoter interactions. Here we report the isolation of a mouse line carrying a Btaf1 allele containing an ENU-induced point mutation that causes a substitution mutation in the BTAF1 ATPase domain. Embryos homozygous for this loss-of-function mutation appear to be morphologically normal until early somite stages, but die between embryonic day 9 and 10.5 displaying growth arrest and edema. Analyses in vitro suggest that the altered protein is less stable and, independent from this, functionally impaired in releasing of TBP from chromatin, but not in binding to TBP. A microarray screen for genes with altered expression in the Btaf V1330M mutant as compared to WT embryos was performed with RNA from whole embryos. We compared mRNA transcripts from pools of Btaf1-/- and Btaf1+/+ embryos. Where possible, we used 3-5-somite embryos, a stage we considered to be immediately prior to the emergence of a perceptible phenotype. The experiments were done in duplo, i.e. two sets of comparisons were made: in one case stage matching (i.e. number of somites counted) was the highest priority, in the other case the pools were designed such that a maximum number of litter mates were compared while a difference of up to five somites was allowed. cDNAs were synthesized and Cy-3/Cy-5 labeled cRNAs were generated by using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Santa Clara, CA). The labeled cRNAs were hybridized on 4X44K Agilent Whole Mouse Genome Dual Color Microarrays (G4122F). Two dye-swap experiments were performed, resulting in four individual arrays. Microarray signal and background information were retrieved with Feature Extraction (V9.5.3, Agilent technologies). All data analyses were performed by using ArrayAssist (5.5.1, Stratagene Inc, La Jolla, CA) and Microsoft Excel (Microsoft Corporation, Redmont, WA). Genetic background: C57BL/6J were mutagenized, and crossed against FVB/NJ mice for positional mapping. The background of the mice used in the experiment is hybrid C57BL/6J *FVB/NJ with a percentage FVB between 50 and 90%.

Dataset Information

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

Publications

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

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