Fig 2: Pharmacological inhibition of the SUV4-20 methyltransferases with A-196 decreases H4K20me2/3 in the FXN–GAA–Luc cell line and in FRDA patient–derived cells.A, representative Western blotting of the FXN–GAA–Luc cell line after A-196, A-197, and SGC2043 treatment. B–D, quantification of the global level of H4K20 methylation after inhibition of SUV4-20. E and F, representative Western blots of patient-derived cells after treatment with the above-mentioned probes. The data are relative to the vehicle, a treatment of 6 days, and are presented as means ± S.E.M. (n = 3, one-way ANOVA followed by Bonferroni test). *, p < 0.05; **, p < 0.01.

Fig 3: Frataxin expression levels. (A) Dipstick immunoassay of frataxin protein in primary fibroblasts (FRDA, GM03816 and normal, GM07492) following synergistic treatment with BIX01294 (100 nM) and GSK126 (2 µM) for different time points. Values are expressed as percentage of the vehicle treated samples of normal fibroblasts at the corresponding time point. (B) Western blot analysis indicating the relative frataxin protein expression levels in human fibroblasts (FRDA, GM04078 and FA3, and normal GM08399 and GM08333) following synergistic treatment with BIX01294 (100 nM) and GSK126 (2 µM) for 3 days. Values were normalized to actin levels and were expressed as percentage of the vehicle treated samples of normal fibroblasts. Representative Immunoblots are shown. Error bars indicate SEM and values represent mean ± SEM (n = 3). Asterisks indicate significant differences between drug and vehicle treated cell lines, assessed by unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01).

Fig 4: FRDA-DMR hypermethylation and deficiency of FXN-E transcript in tissues from the YG8sR humanized mouse model of FRDA. (A) Cerebellum (CBL), heart, and skeletal muscle (SkM) from the non-FRDA control mouse (Y47R; with 9 GAA triplets) express FXN-M (isoform I) and FXN-E isoforms (IIa, IIb, and IIc). Note: Uncropped gel images are included in Fig. S14. (B) Relative FXN-E transcript levels in CBL, heart, and SkM in Y47R mice (n = 3) and in the FRDA mouse model (YG8sR; with 480 GAA repeats; n = 3). (C) Relative FXN-M transcript levels in CBL, heart, and SkM in Y47R (n = 3) and YG8sR (n = 3) mice. (D) FRDA-DMR methylation levels in CBL, heart, and SkM from the Y47R (GAA-9; n = 3) and YG8sR (GAA-480; n = 3) mice. All graphs show mean ± SD; 2way ANOVA with Tukey’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. = not significant.

Fig 5: Generation of the FXNI151F model. A Alignment of the C-terminal region from human (Hu, Uniprot Q16595) and mouse (Ms, Uniprot O35943) frataxin protein sequences. Amino acid I151 in mice corresponds to I154 in humans (boxed). B ribbon representation of human frataxin (pdb code 3s4m) showing the position of I154 (spacefill, in violet). Ribbons are colored according to sequence, from dark blue (N-terminal) to red (C-terminal). Molecular graphics were performed with the UCSF Chimera package. C DNA sequence analyses of WT, HET, and FXNI151F mice. The top panel shows a schematic diagram of the Fxn Gene. The amplified sequence used in genotyping assays includes exon 4 and is indicated. The region containing the mutations is highlighted and shown below. Dashed boxes indicate the ACC ? ACT (silent) and ATC ? TTC (I151F) mutations introduced by gene editing into the frataxin gene. D Genotyping of litters from HET intercrosses