Histone deacetylase inhibitors reverse gene silencing in Friedreich’s ataxia
Friedreich’s ataxia is an inherited disease that causes progressive damage to the nervous system resulting in symptoms ranging from gait disturbance and speech problems to heart disease.
Expansion of GAATTC triplets within an intron in FXN (the gene encoding frataxin) leads to transcription silencing, forming the molecular basis for the neurodegenerative disease Friedreich’s ataxia. Gene silencing at expanded FXN alleles is accompanied by hypoacetylation of histones H3 and H4 and trimethylation of histone H3 at Lys9, observations that are consistent with a heterochromatin-mediated repression mechanism. We describe the synthesis and characterization of a class of histone deacetylase (HDAC) inhibitors that reverse FXN silencing in primary lymphocytes from individuals with Friedreich’s ataxia. We show that these molecules directly affect the histones associated with FXN, increasing acetylation at particular lysine residues on histones H3 and H4 (H3K14, H4K5 and H4K12). This class of HDAC inhibitors may yield therapeutics for Friedreich’s ataxia.
Friedreich’s ataxia (FRDA) is caused by a defect in transcription resulting from hyperexpansion of GAATTC triplet repeats in the first intron of a nuclear gene that encodes the essential mitochondrial protein frataxin1, 2. Frataxin insufficiency leads to progressive spinocerebellar neurodegeneration, resulting in symptoms of gait and hand incoordination, slurred speech, muscle weakness and sensory loss, with extraneural scoliosis, cardiomyopathy and diabetes. At present there is no effective treatment for FRDA, and generally within 15–20 years after the first appearance of symptoms, affected individuals are confined to a wheelchair; in later stages, they become completely incapacitated. Many people with Friedreich’s ataxia die in early adulthood from the associated heart disease, the most common cause of death in FRDA. Normal FXN alleles have 6–34 GAATTC repeats, whereas FRDA-associated alleles have 66–1,700 repeats. Individuals with FRDA have a marked deficiency of FXN mRNA1, 3. Although individuals who are heterozygous for this expansion have 50% of normal FXN mRNA and protein concentrations, they do not show symptoms. Unlike in many triplet-repeat diseases (for example, the polyglutamine-expansion diseases such as Huntington disease and the spinocerebellar ataxias), expanded GAATTC triplets do not alter the coding potential of the FXN gene; thus, gene activation would be of therapeutic benefit.
Numerous biochemical studies have documented that GAATTC repeats adopt non–B-DNA structures4, 5. Long GAATTC repeats form triplexes containing two purine GAA strands and one pyrimidine TTC strand, which flank a single-stranded pyrimidine region. Other structures such as ‘sticky’ DNA have been associated with expanded GAATTC repeats4. Using cloned repeat sequences from individuals with FRDA, investigators have shown that GAATTC repeats interfere with in vitro transcription in a length-dependent manner5, 6. This interference is most pronounced in the physiological orientation of transcription (that is, the synthesis of the GAA-rich transcript). These results are consistent with the observed correlation between GAATTC repeat length and the age at onset and severity of disease.
In contrast, a study using artificial transgenes for a lymphoid cell-surface marker protein (hCD2) has shown that expanded GAATTC repeats induce repressive heterochromatin in vivo in a manner reminiscent of position-effect variegated gene silencing (PEV)7. PEV occurs when a gene is located within or near regions of heterochromatin, and silent heterochromatin is characterized by the presence of particular types of histone modifications (for example, H3K9 methylation), the absence of acetylated histones, and the presence of HDACs, DNA methyltransferases, chromodomain proteins (such as members of the HP-1 family of repressors) and polycomb group proteins8. It has been suggested that molecules that reverse triplex and/or heterochromatin formation in FXN could increase elongation through expanded GAATTC repeats, thereby relieving the deficiency in FXN mRNA and protein in affected individuals7, 9, 10.
Acetylation and deacetylation of histone proteins, and of other proteins involved in transcriptional regulation (such as DNA-binding transcription factors, nuclear hormone receptors and signal-transduction proteins), have critical roles in regulating gene expression. Aberrant protein acetylation stemming from misregulation of either histone acetyltransferases (HATs) or HDACs has been linked to cancer11 and various neurological diseases, including the polyglutamine-expansion diseases and also fragile-X mental retardation and myotonic dystrophy, in which expanded repeats lead to gene silencing12, 13. HDAC inhibitors may revert silent heterochromatin to an active chromatin conformation and restore the normal function of genes that are silenced in these diseases12. Chemically, the HDAC inhibitors can be classified into six structural groups: the small carboxylates, the hydroxamic acids, the benzamides, the epoxyketones, the cyclic peptides, and hybrid molecules containing cyclic peptide motifs and hydroxamic acid moieties11. The human genome encodes at least 17 different HDACs having a wide range of substrate specificities. The various HDAC inhibitors target the various HDAC enzymes and modulate the levels of acetylation both of histone and nonhistone chromosomal proteins and of other cellular targets (for example, microtubule proteins and components of the cell cycle regulatory apparatus). HDAC inhibitors have both positive and negative effects on gene expression11; however, microarray experiments have shown that the expression of a limited number of genes is affected by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA)14. With regard to Friedreich’s ataxia, one study reported a 16% increase in expression of a frataxin reporter construct in cells on treatment with sodium butyrate15. However, HDAC inhibitors that alleviate gene silencing at the endogenous FXN gene in FRDA cells have not been described. We describe herein the identification and optimization of HDAC inhibitors that reverse FXN gene silencing.
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Histone compositions of active and repressed FXN alleles
To assess whether histone modifications have a role in gene silencing in FRDA, we monitored the histone acetylation state of the FXN gene in an Epstein Barr virus–transformed lymphoid cell line derived from an individual with FRDA (line GM15850, alleles with 650 and 1,030 GAATTC repeats in FXN) by chromatin immunoprecipitation (ChIP) with antibodies to the acetylated forms of histones H3 and H4. For comparison, we used a similar cell line from a normal sibling of this person (line GM15851, normal range of repeats). As expected, the cell line from the individual with FRDA had a markedly lower concentration (13% 6%, range of 20 determinations16) of FXN mRNA than did the cell line from the unaffected sibling, as determined by quantitative real time/reverse transcriptase PCR (qRT-PCR). Primers that interrogate the chromatin regions upstream or downstream of the GAATTC repeats in the first intron of FXN and in the promoter element were used in the ChIP experiments, and the levels of immunoprecipitated DNA were quantified by real-time PCR (Fig. 1a). There was no difference in the expression of glyceraldehyde-3-phosphodehydrogenase (GAPDH) mRNA between the two cell lines, and we used GAPDH as a recovery standard in the ChIP experiments. The coding region of active FXN alleles in the GM15851 cell line is enriched in histones acetylated at H3K9, H3K14, H4K5, H4K8, H4K12 and H4K16 compared to the inactive alleles in the GM15850 FRDA cell line, which are clearly depleted of these histone modifications. We found no significant differences in the levels of histone acetylation on the FXN promoter in the two cell lines. Additionally, we examined the methylation status of H3K9 with antibodies to mono-, di- and trimethylated H3K9, and found that H3K9 is highly trimethylated in the FRDA cell line compared to the normal cell line (Fig. 1b). Along with hypoacetylation, trimethylation of H3K9 is a hallmark of heterochromatin8. Thus, the histone postsynthetic modification states within the coding region of inactive FXN alleles are consistent with a chromatin-mediated mechanism as the cause of gene silencing in FRDA7.