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. 2020 Dec 25;295(52):17973-17985.
doi: 10.1074/jbc.RA120.015533. Epub 2020 Oct 7.

Inhibition of the SUV4-20 H1 histone methyltransferase increases frataxin expression in Friedreich's ataxia patient cells

Gabriela Vilema-Enríquez  1 , Robert Quinlan  2 , Peter Kilfeather  1 , Roberta Mazzone  2 , Saba Saqlain  1 , Irene Del Molino Del Barrio  1 , Annalidia Donato  1 , Gabriele Corda  1 , Fengling Li  3 , Masoud Vedadi  4 , Andrea H Németh  5 , Paul E Brennan  2 , Richard Wade-Martins  6
Affiliations

Inhibition of the SUV4-20 H1 histone methyltransferase increases frataxin expression in Friedreich's ataxia patient cells

Gabriela Vilema-Enríquez et al. J Biol Chem. .

Abstract

The molecular mechanisms of reduced frataxin (FXN) expression in Friedreich's ataxia (FRDA) are linked to epigenetic modification of the FXN locus caused by the disease-associated GAA expansion. Here, we identify that SUV4-20 histone methyltransferases, specifically SUV4-20 H1, play an important role in the regulation of FXN expression and represent a novel therapeutic _target. Using a human FXN-GAA-Luciferase repeat expansion genomic DNA reporter model of FRDA, we screened the Structural Genomics Consortium epigenetic probe collection. We found that pharmacological inhibition of the SUV4-20 methyltransferases by the tool compound A-196 increased the expression of FXN by ∼1.5-fold in the reporter cell line. In several FRDA cell lines and patient-derived primary peripheral blood mononuclear cells, A-196 increased FXN expression by up to 2-fold, an effect not seen in WT cells. SUV4-20 inhibition was accompanied by a reduction in H4K20me2 and H4K20me3 and an increase in H4K20me1, but only modest (1.4-7.8%) perturbation in genome-wide expression was observed. Finally, based on the structural activity relationship and crystal structure of A-196, novel small molecule A-196 analogs were synthesized and shown to give a 20-fold increase in potency for increasing FXN expression. Overall, our results suggest that histone methylation is important in the regulation of FXN expression and highlight SUV4-20 H1 as a potential novel therapeutic _target for FRDA.

Keywords: Friedreich's ataxia; drug screening; epigenetics; frataxin; histone methylation.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Screening the SGC epigenetic probe set identifies histone methyltransferases as regulators of FXN repression.A, schematic representation of the FXN–Luc and FXN–GAA–Luc reporter cell lines. B, luciferase assay of the FXN–GAA–Luc cell line treated with the SGC epigenetic probes collection. The FXN–Luc cell line was used as a reference for FXN levels. C, concentration-response curves assessed by luciferase assay of the FXN–GAA–Luc cell line treated for 6 days with A-196 EC50 5.2 μm (left panel), GSK343 EC50 596 μm (middle panel), and SGC0946 EC50 6.8 μm (right panel). The data are relative to the vehicle and are presented as means ± S.E.M. (n = 3 performed in duplicate, one-way ANOVA followed by Bonferroni test). *, p < 0.05; **, p < 0.01. Conc, concentration, MBS, MS2 protein-binding sites, luc, luciferase.
Figure 2
Figure 2
siRNA knockdown of the SUV4-20 family of methyltransferases identifies SUV4-20 H1 as a critical protein for the repression of FXN.A, relative SUV4-20 H1 mRNA expression after SUV4-20 H1 siRNA-mediated knockdown assessed by qRT-PCR. B, FXN-Luc protein expression after SUV4-20 H1 siRNA-mediated knockdown assessed by luciferase assay. C, relative SUV4-20 H2 mRNA expression after SUV4-20 H2 siRNA-mediated knockdown assessed by qRT-PCR. D, FXN-Luc protein expression after SUV4-20 H2 siRNA-mediated knockdown assessed by luciferase assay. E, relative SUV4-20 H1 mRNA expression after SUV4-20 H1 knockdown assessed in the fibroblast line GM04078. F, relative FXN mRNA expression after SUV4-20H1 down-regulation in fibroblasts GM04078. Experiments performed in the line GM04078 were carried out using two different control siRNA. The data are relative to control siRNA, and control siRNA 1, correspond to 6 days treatments, and are presented as means ± S.E.M. (n = 3 performed in triplicate, one-way ANOVA followed by Bonferroni test). *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
Figure 3
Figure 3
A-196 increases frataxin expression in patient-derived cells.A, representative Western blotting of mature frataxin protein expression in the primary fibroblast line GM04078 after A-196 treatment (n = 3 in duplicate). B, quantification of the experiment shown in A. C and D, relative frataxin protein expression in the lymphoblastoid cell lines GM16220 and GM15850 assessed by AlphaLISA (n = 4 in triplicate). E, frataxin mRNA expression after A-196 treatment in PBMCs extracted from four FRDA patients. The data are relative to the vehicle, a treatment of 6 days, and are presented as means ± S.E.M. (one-way ANOVA followed by Bonferroni test). *, p < 0.05; **, p < 0.01.
Figure 4
Figure 4
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.
Figure 5
Figure 5
RNA sequencing of primary FRDA fibroblast line GM04078 after A-196 treatment.A, relative abundance of FXN mRNA in A-196 and SGC2043-treated samples, demonstrating an increase in FXN expression. B, PCA bi-plot of sequenced samples, illustrating a dose-dependent separation of untreated and A-196–treated samples along PC1. C, kernel density plot of significantly differentially expressed genes in all A-196–treated samples and HDACi 109–treated samples from Lai et al. (33). A-196 induces significantly lower transcriptional perturbation at all concentrations. 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.
Figure 6
Figure 6
Medicinal chemistry synthesis of new A-196 derivatives capable of increasing frataxin protein expression.A, screen of A-196 derivatives using the FXN–GAA–Luc cell line (n = 3 in triplicate). B, concentration-response curve of compound A12 (EC50 = 2.7 μm) in the line FXN–GAA–Luc (n = 3 in triplicate). C, concentration-response curve of compound A3 (EC50 = 0.21 μm) in the line FXN–GAA–Luc (n = 3 in triplicate). D and E, FXN mRNA expression of primary fibroblast GM04078 and GM03816 after treatment with compound A3 (n = 3 in duplicate). The data are relative to the vehicle, a treatment of 6 days and are presented as means ± S.E.M. (one-way ANOVA followed by Bonferroni test). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
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References

    1. Campuzano V., Montermini L., Moltò M.D., Pianese L., Cossée M., Cavalcanti F., Monros E., Rodius F., Duclos F., Monticelli A., Zara F., Cañizares J., Koutnikova H., Sanjay I., Gellera C. Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science. 1996;271:1423–1427. doi: 10.1126/science.271.5254.1423. 8596916. - DOI - PubMed
    1. Cossée M., Schmitt M., Campuzano V., Reutenauer L., Moutou C., Mandel J.-L., Koenig M. Evolution of the Friedreich's ataxia trinucleotide repeat expansion: founder effect and premutations. Proc. Natl. Acad. Sci. U.S.A. 1997;94:7452–7457. doi: 10.1073/pnas.94.14.7452. 9207112. - DOI - PMC - PubMed
    1. Chamberlain S., Shaw J., Rowland A., Wallis J., South S., Nakamura Y., Von Gabain A., Farrall M., Williamson R. Mapping of mutation causing Friedreich's ataxia to human chromosome 9. Nature. 1988;334:248–250. doi: 10.1038/334248a0. 2899844. - DOI - PubMed
    1. Pastore A., Puccio H. Frataxin: a protein in search for a function. J. Neurochem. 2013;126:43–52. doi: 10.1111/jnc.12220. 23859340. - DOI - PubMed
    1. Pandolfo M. Friedreich ataxia: the clinical picture. J. Neurol. 2009;256:3–8. doi: 10.1007/s00415-009-1002-3. 19283344. - DOI - PubMed

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