Characterization of functionally deficient SIM2 variants found in patients with neurological phenotypes

Abstract

Single-minded 2 (SIM2) is a neuron-enriched basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) transcription factor essential for mammalian survival. SIM2 is located within the Down syndrome critical region (DSCR) of chromosome 21, and manipulation in mouse models suggests Sim2 may play a role in brain development and function. During the screening of a clinical exome sequencing database, nine SIM2 non-synonymous mutations were found which were subsequently investigated for impaired function using cell-based reporter gene assays. Many of these human variants attenuated abilities to activate transcription and were further characterized to determine the mechanisms underpinning their deficiencies. These included impaired partner protein dimerization, reduced DNA binding, and reduced expression and nuclear localization. This study highlighted several SIM2 variants found in patients with disabilities and validated a candidate set as potentially contributing to pathology.

Keywords: aryl hydrocarbon nuclear receptor translocator; mutation; single-minded 2; single-nucleotide polymorphisms; transcription factors.

Figure 1.. SIM2 variants have reduced activity on a CME reporter.

(A) Schematic of SIM2s protein with domains and variants tested shown. N-terminal bHLH domain functions in DNA binding and primary dimerization. PAS domains are important for secondary dimerization and partner protein specificity. NLS directs SIM2 protein import into the nucleus. The C-terminal half of SIM2 contains transcriptional regulatory regions. Variants selected for functional testing are displayed above. (B) Dual-luciferase assays with a 6xCME reporter gene. Expression of SIM2s-HF and variants was induced with dox. Variants highlighted in red showed a significant reduction in reporter activity compared with WT SIM2s. Graph represents mean of n = 3 independent experiments, normalized to WT SIM2s control. Error bars represent SEM. Statistical significance determined by one-way ANOVA. *** P ≤ 0.001, **** P ≤ 0.0001, ns, not significant. (C) Western blot from whole-cell extracts. SIM2s-HF and variants were detected with FLAG antibody. Arrow indicates SIM2s variants, with the truncated R163X variant only detectable on long exposure.

Figure 2.. Select SIM2s variants show impaired dimerization with ARNT2.

Co-immunoprecipitation experiments performed to determine the ability of SIM2s-HF and variants to dimerize with ARNT2. FLAG immunoprecipitation followed by western blots for ARNT2, HA (SIM2s-HF) and Tubulin were performed. Blots are representative of 3 independent experiments. (A) Expression of SIM2s-HF and the E19K, E224K, W306R and V326M variants induced by dox. (B) SIM2s-HF and R163X variants expressed at a 1 : 5 ratio.

Figure 3.. Dimerization-deficient variants lose the ability to repress HIF1α transcriptional activity.

Dual-luciferase reporter assay with a HIF1α responsive 4xHRE reporter. HIF1α expression was induced with the hypoxia mimetic DMOG; expression of SIM2s-HF and variants were induced with dox. Variants highlighted in red show no repression of HIF1α-mediated activation of the reporter. Graph represents mean of n = 4 independent experiments, normalized to parent control. Error bars represent SEM. Statistical significance determined by one-way ANOVA. ** P ≤ 0.01, *** P ≤ 0.001, ns not significant.

Figure 4.. Cellular localization of SIM2 variants.

Immunofluorescence of fixed T-REx293 cells dox induced to express WT SIM2s-HF or mutant variants. FLAG antibody used to detect SIM2 and variants shows all variants are localized to the nucleus except the R163X variant, which is localized throughout the entire cell. 2s; 2 second exposure, 4s; 4 second exposure.

Figure 5.. DNA binding of the E19K variant.

SIM2 Chromatin immunoprecipitation assays from cells with dox-induced expression of SIM2s-HF or E19K variant. Enrichment of the 6xCME response element was assessed by qPCR. Graph represents mean of n = 3 independent experiments presented as percent enrichment compared with input. Error bars represent SD. Statistical significance determined by one-way ANOVA. * P ≤ 0.05 *** P ≤ 0.001.

Figure 6.. SIM2 : ARNT2 : DNA structural models.

(A) Homology model of the WT SIM2 : ARNT2 : DNA structure based on the mouse HIF2α : ARNT : DNA co-crystal structure (PDB:4ZPK). SIM2 is depicted in blue, ARNT2 in purple with transparent surface shown to highlight protein–protein interfaces. SIM2 missense variant residues are shown as red spheres. (B) PAS-A/PAS-B interface of the mouse HIF2α : ARNT : DNA co-crystal structure (PDB:4ZPK). Interface residues are shown as sticks and clearly labelled. Surface electrostatics of the HIF2α are shown. (C) PAS-A/PAS-B interface of the WT SIM2 : ARNT2 model and (D) mutant W306R SIM2 : ARNT2 model. Surface electrostatics are shown to demonstrate charge disruption introduced by W306R mutation. (E–G) Mutations E19K, E224K and V326M, respectively. ARNT1/2 residue labels are represented in italics for clear differentiation, and mutant residues are shown in red.