Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene, which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs, the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming, modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds, sodium butyrate (NaB) and Parnate, led to an increase in FXN expression and correction of repressive marks at the FXN locus, which persisted for several passages. However, prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore, we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together, these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications, thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA.
Get full access to this article
View all access options for this article.
References
1.
CampuzanoV, MonterminiL, MoltoMD, PianeseL, CosseeM, CavalcantiF, MonrosE, RodiusF, DuclosF, et al. (1996). Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science, 5254:1423–1427.
2.
MarmolinoD. (2011). Friedreich's ataxia: past, present and future. Brain Res Rev, 1–2:311–330.
3.
PandolfoM. (2009). Friedreich ataxia: the clinical picture. J Neurol, 256:3–8.
4.
DurrA, CosseeM, AgidY, CampuzanoV, MignardC, PenetC, MandelJL, BriceA and KoenigM. (1996). Clinical and genetic abnormalities in patients with Friedreich's ataxia. N Engl J Med, 16:1169–1175.
5.
FillaA, De MicheleG, CavalcantiF, PianeseL, MonticelliA, CampanellaG and CocozzaS. (1996). The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia. Am J Hum Genet, 3:554–560.
6.
EpplenC, EpplenJT, FrankG, MiterskiB, SantosEJ and ScholsL. (1997). Differential stability of the (GAA)n tract in the Friedreich ataxia (STM7) gene. Hum Genet, 6:834–836.
7.
BurnettR, MelanderC, PuckettJW, SonLS, WellsRD, DervanPB and GottesfeldJM. (2006). DNA sequence-specific polyamides alleviate transcription inhibition associated with long GAA.TTC repeats in Friedreich's ataxia. Proc Natl Acad Sci U S A, 31:11497–11502.
8.
SoragniE, HermanD, DentSY, GottesfeldJM, WellsRD and NapieralaM. (2008). Long intronic GAA*TTC repeats induce epigenetic changes and reporter gene silencing in a molecular model of Friedreich ataxia. Nucleic Acids Res, 19:6056–6065.
9.
KimE, NapieralaM and DentSY. (2011). Hyperexpansion of GAA repeats affects post-initiation steps of FXN transcription in Friedreich's ataxia. Nucleic Acids Res, 19:8366–8377.
10.
SandiC, PintoRM, Al-MahdawiS, EzzatizadehV, BarnesG, JonesS, RuscheJR, GottesfeldJM and PookMA. (2011). Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model. Neurobiol Dis, 3:496–505.
11.
SavelievA, EverettC, SharpeT, WebsterZ and FestensteinR. (2003). DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing. Nature, 6934:909–913.
12.
GrohM, LufinoMM, Wade-MartinsR and GromakN. (2014). R-loops associated with triplet repeat expansions promote gene silencing in Friedreich ataxia and fragile X syndrome. PLoS Genet, 5:e1004318.
13.
ButlerJS and NapieralaM. (2015). Friedreich's ataxia—a case of aberrant transcription termination?. Transcription, 2:33–36.
14.
ChutakeYK, LamC, CostelloWN, AndersonM and BidichandaniSI. (2014). Epigenetic promoter silencing in Friedreich ataxia is dependent on repeat length. Ann Neurol, 4:522–528.
15.
LiL, MatsuiM and CoreyDR. (2016). Activating frataxin expression by repeat-targeted nucleic acids. Nat Commun, 7:10606.
De BiaseI, ChutakeYK, RindlerPM and BidichandaniSI. (2009). Epigenetic silencing in Friedreich ataxia is associated with depletion of CTCF (CCCTC-binding factor) and antisense transcription. PLoS One, 11:e7914.
18.
Al-MahdawiS, PintoRM, IsmailO, VarshneyD, LymperiS, SandiC, TrabzuniD and PookM. (2008). The Friedreich ataxia GAA repeat expansion mutation induces comparable epigenetic changes in human and transgenic mouse brain and heart tissues. Hum Mol Genet, 5:735–746.
19.
KumariD, BiacsiRE and UsdinK. (2010). Repeat expansion affects both transcription initiation and elongation in Friedreich ataxia cells. J Biol Chem, 6:4209–4215.
20.
PungaT and BuhlerM. (2010). Long intronic GAA repeats causing Friedreich ataxia impede transcription elongation. EMBO Mol Med, 4:120–129.
21.
GreeneE, MahishiL, EntezamA, KumariD and UsdinK. (2007). Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia. Nucleic Acids Res, 10:3383–3390.
22.
CastaldoI, PinelliM, MonticelliA, AcquavivaF, GiacchettiM, FillaA, SacchettiS, KellerS, AvvedimentoVE, ChiariottiL and CocozzaS. (2008). DNA methylation in intron 1 of the frataxin gene is related to GAA repeat length and age of onset in Friedreich ataxia patients. J Med Genet, 12:808–812.
23.
LufinoMM, SilvaAM, NemethAH, Alegre-AbarrateguiJ, RussellAJ and Wade-MartinsR. (2013). A GAA repeat expansion reporter model of Friedreich's ataxia recapitulates the genomic context and allows rapid screening of therapeutic compounds. Hum Mol Genet, 25:5173–5187.
24.
XuC, SoragniE, ChouCJ, HermanD, PlastererHL, RuscheJR and GottesfeldJM. (2009). Chemical probes identify a role for histone deacetylase 3 in Friedreich's ataxia gene silencing. Chem Biol, 9:980–989.
25.
SoragniE, ChouCJ, RuscheJR and GottesfeldJM. (2015). Mechanism of action of 2-aminobenzamide HDAC inhibitors in reversing gene silencing in Friedreich's ataxia. Front Neurol, 6:44.
26.
BuganimY, FaddahDA and JaenischR. (2013). Mechanisms and models of somatic cell reprogramming. Nat Rev Genet, 6:427–439.
27.
UrbachA, Bar-NurO, DaleyGQ and BenvenistyN. (2010). Differential modeling of fragile X syndrome by human embryonic stem cells and induced pluripotent stem cells. Cell Stem Cell, 5:407–411.
28.
CoffeeB, ZhangF, WarrenST and ReinesD. (1999). Acetylated histones are associated with FMR1 in normal but not fragile X-syndrome cells. Nat Genet, 1:98–101.
29.
Evans-GaleaMV, HannanAJ, CarrodusN, DelatyckiMB and SafferyR. (2013). Epigenetic modifications in trinucleotide repeat diseases. Trends Mol Med, 11:655–663.
30.
BhattacharyyaA and ZhaoX. (2016). Human pluripotent stem cell models of Fragile X syndrome. Mol Cell Neurosci, 73:43–51.
31.
EigesR, UrbachA, MalcovM, FrumkinT, SchwartzT, AmitA, YaronY, EdenA, YanukaO, BenvenistyN and Ben-YosefD. (2007). Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos. Cell Stem Cell, 5:568–577.
PolakU, HirschC, KuS, GottesfeldJM, DentSYR and NapieralaM. (2012). Selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts. J Vis Exp, DOI: 10.3791/e3416.
34.
LiY, PolakU, BhallaAD, RozwadowskaN, ButlerJS, LynchDR, DentSY and NapieralaM. (2015). Excision of expanded GAA repeats alleviates the molecular phenotype of Friedreich's Ataxia. Mol Ther, 6:1055–1065.
35.
WangG, GuoX, HongW, LiuQ, WeiT, LuC, GaoL, YeD, ZhouY, et al. (2013). Critical regulation of miR-200/ZEB2 pathway in Oct4/Sox2-induced mesenchymal-to-epithelial transition and induced pluripotent stem cell generation. Proc Natl Acad Sci U S A, 8:2858–2863.
36.
LiY, LuY, PolakU, LinK, ShenJ, FarmerJ, SeyerL, BhallaAD, RozwadowskaN, et al. (2015). Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus. Hum Mol Genet, 24:6932–6943.
37.
MirandaCJ, SantosMM, OhshimaK, SmithJ, LiL, BuntingM, CosseeM, KoenigM, SequeirosJ, KaplanJ and PandolfoM. (2002). Frataxin knockin mouse. FEBS Lett, 1–3:291–297.
38.
TakahashiK, OkitaK, NakagawaM and YamanakaS. (2007). Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc, 12:3081–3089.
39.
TakahashiK, TanabeK, OhnukiM, NaritaM, IchisakaT, TomodaK and YamanakaS. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 5:861–872.
40.
HickA, Wattenhofer-DonzeM, ChintawarS, TropelP, SimardJP, VaucampsN, GallD, LambotL, AndreC, et al. (2013). Neurons and cardiomyocytes derived from induced pluripotent stem cells as a model for mitochondrial defects in Friedreich's ataxia. Dis Model Mech, 3:608–621.
41.
RaiM, SoragniE, JenssenK, BurnettR, HermanD, CoppolaG, GeschwindDH, GottesfeldJM and PandolfoM. (2008). HDAC inhibitors correct frataxin deficiency in a Friedreich ataxia mouse model. PLoS One, 4:e1958.
42.
GottesfeldJM, RuscheJR and PandolfoM. (2013). Increasing frataxin gene expression with histone deacetylase inhibitors as a therapeutic approach for Friedreich's ataxia. J Neurochem 126 Suppl, 1: 147–154.
43.
SoragniE, MiaoW, IudicelloM, JacobyD, De MercantiS, ClericoM, LongoF, PigaA, KuS, et al. (2014). Epigenetic therapy for Friedreich ataxia. Ann Neurol, 4:489–508.
44.
CandidoEP, ReevesR and DavieJR. (1978). Sodium butyrate inhibits histone deacetylation in cultured cells. Cell, 1:105–113.
45.
LeeMG, WynderC, SchmidtDM, McCaffertyDG and ShiekhattarR. (2006). Histone H3 lysine 4 demethylation is a target of nonselective antidepressive medications. Chem Biol, 6:563–567.
46.
KingstonWR. (1962). A clinical trial of an antidepressant, tranylcy-promine (“Parnate”). Med J Aust, 49:1011–1012.
47.
ShiY, LanF, MatsonC, MulliganP, WhetstineJR, ColePA, CaseroRA and ShiY. (2004). Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell, 7:941–953.
48.
MetzgerE, WissmannM, YinN, MullerJM, SchneiderR, PetersAH, GuntherT, BuettnerR and SchuleR. (2005). LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature, 7057:436–439.
49.
ConsortiumEP. (2012). An integrated encyclopedia of DNA elements in the human genome. Nature, 7414:57–74.
50.
BallasN, BattaglioliE, AtoufF, AndresME, ChenowethJ, AndersonME, BurgerC, MoniwaM, DavieJR, et al. (2001). Regulation of neuronal traits by a novel transcriptional complex. Neuron, 3:353–365.
51.
HakimiMA, BocharDA, ChenowethJ, LaneWS, MandelG and ShiekhattarR. (2002). A core-BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes. Proc Natl Acad Sci U S A, 11:7420–7425.
52.
LeeMG, WynderC, BocharDA, HakimiMA, CoochN and ShiekhattarR. (2006). Functional interplay between histone demethylase and deacetylase enzymes. Mol Cell Biol, 17:6395–6402.
53.
DottoriM and PeraMF. (2008). Neural differentiation of human embryonic stem cells. Methods Mol Biol, 438:19–30.
54.
RaiM, SoragniE, ChouCJ, BarnesG, JonesS, RuscheJR, GottesfeldJM and PandolfoM. (2010). Two new pimelic diphenylamide HDAC inhibitors induce sustained frataxin upregulation in cells from Friedreich's ataxia patients and in a mouse model. PLoS One, 1:e8825.
55.
DingVM, LingL, NatarajanS, YapMG, CoolSM and ChooAB. (2010). FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3-K/GSK3beta signaling. J Cell Physiol, 2:417–428.
56.
DuJ, CampauE, SoragniE, KuS, PuckettJW, DervanPB and GottesfeldJM. (2012). Role of mismatch repair enzymes in GAA.TTC triplet-repeat expansion in Friedreich ataxia induced pluripotent stem cells. J Biol Chem, 35:29861–29872.
57.
GerhardtJ, BhallaAD, ButlerJS, PuckettJW, DervanPB, RosenwaksZ and NapieralaM. (2016). Stalled DNA replication forks at the endogenous GAA repeats drive repeat expansion in Friedreich's ataxia cells. Cell Rep, 16:1218–1227.
58.
ChanPK, TorresR, YandimC, LawPP, KhadayateS, MauriM, GrosanC, Chapman-RotheN, GiuntiP, PookM and FestensteinR. (2013). Heterochromatinization induced by GAA-repeat hyperexpansion in Friedreich's ataxia can be reduced upon HDAC inhibition by vitamin B3. Hum Mol Genet, 13:2662–2675.
59.
GafniO, WeinbergerL, MansourAA, ManorYS, ChomskyE, Ben-YosefD, KalmaY, ViukovS, MazaI, et al. (2013). Derivation of novel human ground state naive pluripotent stem cells. Nature, 7479:282–286.
60.
TheunissenTW, PowellBE, WangH, MitalipovaM, FaddahDA, ReddyJ, FanZP, MaetzelD, GanzK, et al. (2014). Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell, 4:471–487.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
