Abstract
The fragile X syndrome is caused by a >200 CGG repeat expansion within the FMR1 gene promoter, with consequent DNA hypermethylation and inactivation of its expression. To further clarify the mechanisms that suppress the activity of the mutant gene and the conditions that may permit its reactivation, we investigated the acetylation and methylation status of three different regions of the FMR1 gene (promoter, exon 1 and exon 16) of three fragile X cell lines, using a chromatin immunoprecipitation (ChIP) assay with antibodies against acetylated-H3/H4 histones and against dimethylated lysine residues K4 and K9 of histone H3 (H3-K4 and H3-K9). We then coupled the ChIP assay with real-time PCR, obtaining absolute quantification of immunoprecipitated chromatin. Basal levels of histone acetylation and H3-K4 methylation were much higher in transcriptionally active wild-type controls than in inactive fragile X cell lines. Treatment of fragile X cell lines with the DNA demethylating drug 5-aza-2-deoxycytidine (5-azadC), known to reactivate the FMR1 gene, induced a decrease of H3-K9 methylation, an increase of H3 and H4 acetylation and an increase of H3-K4 methylation. Treatment with acetyl-L-carnitine (ALC), a compound that reduces the in vitro expression of the FRAXA fragile site without affecting DNA methylation, caused an increase of H3 and H4 acetylation. However, H3-K4 methylation remained extremely low, in accordance with the observation that ALC alone does not reactivate the FMR1 gene. Our experiments indicate that H3-K4 methylation and DNA demethylation are the main epigenetic switches activating the expression of the FMR1 gene, with histone acetylation playing an ancillary role.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Fu YH, Kuhl DP, Pizzuti A et al: Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 1991; 67: 1047–1058.
Verkerk AJMH, Pieretti M, Sutcliffe JS et al: Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting lenght variation in fragile X syndrome. Cell 1991; 65: 905–914.
Feng Y, Zhang F, Lokey LK et al: Translational suppression by trinucleotide repeat expansion at FMR1. Science 1995; 268: 731–734.
Pieretti M, Zhang F, Fu YH et al: Absence of expression of the FMR-1 gene in fragile X syndrome. Cell 1991; 66: 817–822.
Brown V, Jin P, Ceman S et al: Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 2001; 107: 477–487.
Zalfa F, Bagni C : Molecular insights into mental retardation: multiple functions for the fragile X mental retardation protein? Curr Issues Mol Biol 2004; 6: 73–88.
Hagerman RJ, Hull CE, Safanda JF et al: High functioning fragile X males: demonstration of an unmethylated fully expanded FMR1 mutation associated with protein expression. Am J Med Genet 1994; 51: 298–308.
Smeets HJ, Smits AP, Verheij CE et al: Normal phenotype in two brothers with a full FMR1 mutation. Hum Mol Genet 1995; 4: 2103–2108.
Chiurazzi P, Pomponi MG, Willemsen R et al: In vitro reactivation of the FMR1 gene involved in fragile X syndrome. Hum Mol Genet 1998; 7: 109–113.
Pietrobono R, Pomponi MG, Tabolacci E et al: Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine. Nucleic Acids Res 2002; 30: 3278–3285.
Jones PL, Veenstra GJC, Wade PA et al: Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat Genet 1998; 19: 187–191.
Nan X, Ng H-H, Johnson CA et al: Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 1998; 393: 386–389.
Razin A : CpG methylation, chromatin structure and gene silencing – a three-way connection. EMBO J 1998; 17: 4905–4908.
Coffee B, Zhang F, Warren ST et al: Acetylated histones are associated with FMR1 in normal but not fragile X-syndrome cells. Nat Genet 1999; 22: 98–101.
Chiurazzi P, Pomponi MG, Pietrobono R et al: Synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of the FMR1 gene. Hum Mol Genet 1999; 8: 2317–2323.
Turner BM : Cellular memory and the histone code. Cell 2002; 111: 285–291.
Grewal SIS, Moazed D : Heterochromatin and epigenetic control of gene expression. Science 2003; 301: 798–802.
Lachner M, O’Sullivan RJ, Jenuwein T : An epigenetic road map for histone lysine methylation. J Cell Sci 2003; 116: 2117–2124.
Coffee B, Zhang F, Ceman S et al: Histone modifications depict an aberrantly heterochromatinized FMR1 gene in fragile X syndrome. Am J Hum Genet 2002; 71: 923–932.
Pomponi MG, Neri G : Butyrate and acetyl-carnitine inhibit the cytogenetic expression of the fragile X in vitro. Am J Med Genet 1994; 51: 447–450.
Tassone F, Hagerman RJ, Taylor AK et al: Elevated levels of FMR1 mRNA in carrier males: a new mechanism of involvement in the fragile-X syndrome. Am J Hum Genet 2000; 66: 6–15.
Litt MD, Simpson M, Gaszner M et al: Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science 2001; 293: 2453–2455.
Torrioli MG, Vernacotola S, Mariotti P et al: Double-blind, placebo-controlled study of L-acetylcarnitine for the treatment of hyperactive behavior in fragile X syndrome. Am J Med Genet 1999; 87: 366–368.
Pascale E, Battiloro E, Reale GC et al: Modulation of methylation in the FMR1 promoter region after long term treatment with L-carnitine and acetyl-L-carnitine. J Med Genet 2003; 40: e76.
Cheutin T, McNairn AJ, Jenuwein T et al: Maintenance of stable heterochromatin domains by dynamic HP1 binding. Science 2003; 299: 721–725.
Kouzarides T : Histone methylation in transcriptional control. Curr Opin Genet Dev 2002; 12: 198–209.
Pietrobono R, Tabolacci E, Zalfa F et al: Molecular dissection of the events leading to inactivation of the FMR1 gene. Hum Mol Genet 2005; 14: 267–277.
Acknowledgements
We gratefully acknowledge Steve Warren for providing the fragile X lymphoblastoid cell line S1. This work was supported by the FRAXA Foundation, Conquer Fragile X, Sigma-Tau, Fondazione Telethon, MIUR, Fondazione Cariplo and Associazione Anni Verdi.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Tabolacci, E., Pietrobono, R., Moscato, U. et al. Differential epigenetic modifications in the FMR1 gene of the fragile X syndrome after reactivating pharmacological treatments. Eur J Hum Genet 13, 641–648 (2005). https://doi.org/10.1038/sj.ejhg.5201393
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/sj.ejhg.5201393
Keywords
This article is cited by
-
Mitochondrial dysfunction in Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome: prospect use of antioxidants and mitochondrial nutrients
Molecular Biology Reports (2024)
-
Incomplete silencing of full mutation alleles in males with fragile X syndrome is associated with autistic features
Molecular Autism (2019)
-
Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome
Clinical Epigenetics (2016)
-
Genome-wide methylation analysis demonstrates that 5-aza-2-deoxycytidine treatment does not cause random DNA demethylation in fragile X syndrome cells
Epigenetics & Chromatin (2016)
-
Defining the role of the CGGBP1 protein in FMR1 gene expression
European Journal of Human Genetics (2016)
