FSHD2- and BAMS-associated mutations confer opposing effects on SMCHD1 function

Abstract

Structural maintenance of chromosomes flexible hinge domain-containing 1 (Smchd1) plays important roles in epigenetic silencing and normal mammalian development. Recently, heterozygous mutations in SMCHD1 have been reported in two disparate disorders: facioscapulohumeral muscular dystrophy type 2 (FSHD2) and Bosma arhinia microphthalmia syndrome (BAMS). FSHD2-associated mutations lead to loss of function; however, whether BAMS is associated with loss- or gain-of-function mutations in SMCHD1 is unclear. Here, we have assessed the effect of SMCHD1 missense mutations from FSHD2 and BAMS patients on ATP hydrolysis activity and protein conformation and the effect of BAMS mutations on craniofacial development in a Xenopus model. These data demonstrated that FSHD2 mutations only result in decreased ATP hydrolysis, whereas many BAMS mutations can result in elevated ATPase activity and decreased eye size in Xenopus Interestingly, a mutation reported in both an FSHD2 patient and a BAMS patient results in increased ATPase activity and a smaller Xenopus eye size. Mutations in the extended ATPase domain increased catalytic activity, suggesting critical regulatory intramolecular interactions and the possibility of targeting this region therapeutically to boost SMCHD1's activity to counter FSHD.

Keywords: ATPase; SMC; Xenopus; craniofacial development; epigenetics; hinge domain; muscular dystrophy; small-angle X-ray scattering (SAXS).

Figure 1.

SMCHD1 mutations in FSHD2 and BAMS patients frequently occur within the GHKL-containing N-terminal region, which has structural homology to Hsp90. a, domain architectures of full-length Hsp90 comprising three domains (GHKL ATPase, middle, and C-terminal (CTD) domain) with domain boundaries indicated above as amino acids (top) and full-length SMCHD1 comprising the GHKL-containing N-terminal region (residues 111–702) and an SMC hinge domain (residues 1683–1899) (bottom). b and c, illustration of studied SMCHD1 mutations associated with FSHD2 (blue) and BAMS (yellow) located within SMCHD1's N-terminal region, where the GHKL ATPase is colored in red and the extended region is shown in gray; depicted in b is the gene structure and in c a homology model of Smchd1's N-terminal region based on Hsp90, indicating the catalytic motif I in purple. d, multiple-sequence alignment of Smchd1 (residues 111–702) orthologue sequences from Homo sapiens, Rattus norvegicus, M. musculus, Gallus gallus, and Xenopus tropicalis. Conserved residues are colored in red, whereas nonconserved residues are shown in black; the four GHKL motifs are highlighted in purple, and mutations associated with FSHD2 (blue) and BAMS (yellow) are indicated. The alignment was generated with ClustalW and ESPript version 3.0.

Figure 2.

Protein purification and thermal stability analyses of studied Smchd1 N-terminal region mutants. a and b, SEC profiles of FSHD2-associated (blue) (a) and BAMS-associated (red) (b) recombinant proteins, indicating absorbance at 280 nm (y axis, arbitrary units) and elution volume (x axis, milliliters), obtained via a Superdex 200 10/30 column. WT Smchd1 elution is shown in black as a comparison in both a and b. Elution times of molecular mass markers are indicated above in kDa. c, representative Coomassie-stained 4–12% (w/v) reducing SDS-polyacrylamide gel indicating collected fractions following SEC and the high purity of protein obtained. Molecular mass markers are shown on the left. d and e, TSA results of all studied FSHD2 (d) and BAMS (e) mutants, indicating fluorescence intensity (y axis, 530 nm), temperature (x axis, °C) and the melting temperature (T_m) at which 50% protein has denatured as a horizontal dotted line. The TSA plots are representative of two independent experiments for each mutant. f, table summary. g–i, ab initio bead models of representative FSHD2 and BAMS mutants derived from in-solution SAXS data indicate a conserved structural topology. WT Smchd1 (g) was analyzed previously (20) and used to compare with newly acquired analyses of the G478E FSHD2 (h) and S135C BAMS (i) mutants. Ab initio models (panels i) are represented as a gray surface, superimposed with the predicted structural model of Smchd1's N-terminal region based on the Hsp90 crystal structure (PDB code 2CG9), shown in cyan for WT and FSHD-associated mutants and in yellow for BAMS-associated mutants. Affected residues are depicted as red spheres across all models. Images were obtained via PyMOL. Panels ii depict scattering profiles, with intensity of scattered X-rays, I(q), as a function of momentum transfer, q, in Å⁻¹, showing inset Guinier plots for the corresponding mutants, where linearity indicates monodisperse particles. The R_g and initial scattering intensity (I(0)) were approximated from the Guinier plots via the software PRIMUS. Panels iii indicate the pair-distribution functions, P(r), obtained via Fourier transformations of the scattering intensity data (panels ii), via the software GNOM. R_g and D_max (maximum dimension) values were also calculated from the P(r) analyses. Calculated values are summarized in Table 1 and presented in their entirety in Table S1. SAXS data and analyses for seven FSHD2 and seven BAMS mutants are presented in Fig. S1.

Figure 3.

In vitro ATPase analyses indicate FSHD-associated mutants display a loss of catalytic ability, whereas varied changes are observed across BAMS mutants. a–t, ATPase activity analyses of FSHD2-associated (b–h) and BAMS-associated (i–t) mutants, alongside representative WT-protein activity (a). Each graph indicates ATP concentration (x axis, μm), concentration of ADP produced (y axis, μm), and protein concentration (right panels), where measurements were performed in technical triplicates, and error bars represent S.D., with at least two independent experiments performed for each protein variant. Plots (a–t) depict individual experiments carried out at separate times, where WT protein was assessed within each assay (not shown), and the profile (a) serves as a representative result. ATPase analyses for mutants A134S, S135C, E136G, D420V, Y353C, and T527M were previously reported by Gordon et al. (6), with additional repetitions included in these studies. u, relative -fold change in ATPase activity (y axis) of all studied mutants compared with WT protein, where individual mutations are indicated (x axis), alongside overall changes in catalytic activity depicted by overhead arrows (FSHD2 in blue, BAMS in red) or a horizontal line if no significant change was apparent. G137E is indicated with a number symbol, as the mutation was reported in both a BAMS patient and an FSHD2 patient, yet we included it in the BAMS cohort for analysis purposes. -Fold change values were calculated by direct comparison of 16 points (four ATP and protein concentrations tested per mutant) obtained for each mutant, compared with the corresponding 16 values obtained for the WT protein within each assay performed. Statistical analysis was carried out using the Wilcoxon matched-pairs signed-rank test, additionally applying a correction for multiple testing. p values were obtained, as indicated in the outlined box. See u to comparing ATPase activity of mutants to WT protein.

Figure 4.

BAMS-associated mutant forms of SMCHD1 result in a decreased eye diameter in X. laevis. a, synthesized mRNA encoding full-length human SMCHD1 was injected into the two dorsal animal blastomeres at the 8-cell stage to target the head structures. b–e, representative images of stage 45 tadpoles that were uninjected (b), injected with WT mRNA (c), or injected with R552Q mRNA (d–e). f, Western blotting showing that all injected mRNAs produced full-length SMCHD1 protein. Y353C mRNA has been tested previously (6). g, measurements of eye diameter of tadpoles show that all BAMS-associated mutants cause a significant reduction in eye size. n indicates the number of embryos analyzed; data are shown as means ± S.D. (error bars), and p values were calculated by one-way ANOVA followed by Dunn's post-test. n.s., not significant; ***, p < 0.001.