Survival motor neuron gene 2 silencing by DNA methylation correlates with spinal muscular atrophy disease severity and can be bypassed by histone deacetylase inhibition

Abstract

Spinal muscular atrophy (SMA), a common neuromuscular disorder, is caused by homozygous absence of the survival motor neuron gene 1 (SMN1), while the disease severity is mainly influenced by the number of SMN2 gene copies. This correlation is not absolute, suggesting the existence of yet unknown factors modulating disease progression. We demonstrate that the SMN2 gene is subject to gene silencing by DNA methylation. SMN2 contains four CpG islands which present highly conserved methylation patterns and little interindividual variations in SMN1-deleted SMA patients. The comprehensive analysis of SMN2 methylation in patients suffering from severe versus mild SMA carrying identical SMN2 copy numbers revealed a correlation of CpG methylation at the positions -290 and -296 with the disease severity and the activity of the first transcriptional start site of SMN2 at position -296. These results provide first evidence that SMN2 alleles are functionally not equivalent due to differences in DNA methylation. We demonstrate that the methyl-CpG-binding protein 2, a transcriptional repressor, binds to the critical SMN2 promoter region in a methylation-dependent manner. However, inhibition of SMN2 gene silencing conferred by DNA methylation might represent a promising strategy for pharmacologic SMA therapy. We identified histone deacetylase (HDAC) inhibitors including vorinostat and romidepsin which are able to bypass SMN2 gene silencing by DNA methylation, while others such as valproic acid and phenylbutyrate do not, due to HDAC isoenzyme specificities. These findings indicate that DNA methylation is functionally important regarding SMA disease progression and pharmacological SMN2 gene activation which might have implications for future SMA therapy regimens.

Figure 1.

Transcriptional survival motor neuron gene 2 (SMN2) activation by the DNA-demethylating drug 5-aza-2′-deoxycytidine (Aza) in SMN1-deleted SMA (spinal muscular atrophy) fibroblasts. Bar graphs show SMN2 transcript (A) and SMN2 protein levels (B) in ML5 and ML16 fibroblast cell lines treated with various doses of Aza for 72 h (mean ± SEM). Expression levels in solvent- and time-matched controls were set to 100%. Increased SMN2 transcript levels (normalized to ß-actin) were observed following Aza treatment (1, 10, 100 µm) and a dose-dependent SMN2 gene activation was confirmed on protein level (normalized to ß-tubulin). A representative western blot analysis (ML16) is shown. (C) Quantification of SMN2 transcript levels (normalized to ß-actin) in ML16 cells after treatment with different doses of the SMN2-activating HDAC (histone deacetylase) inhibitor M344 either alone or in combination with 10 µm Aza for 48 h. Transcriptional SMN2 gene activation either by M344 alone or in combination with Aza (10 µm) reached levels of significance in all cases (P < 0.05, t-test). A >5-fold induction was observed following treatment with 10 µm M344 (546 ± 53%), while no additive effects were observed after co-treatment with 10 µm Aza (P > 0.05, t-test).

Figure 2.

Survival motor neuron gene 2 (SMN2) is a methylated gene containing four CpG islands (CGIs). Putative CGIs within the genomic region 3000 nucleotides (nt) upstream and downstream the translational SMN2 start site (NCBI36: 5:70254287:70248287) were identified using the CGI finder and plotting tool (www.EBI.ac.uk/emboss). This algorithm identified four putative CGIs within the respective genomic region (^SMN2CGI 1: nt −896 to −645, ^SMN2CGI 2: nt −469 to −247, ^SMN2CGI 3: nt −151 to +295, ^SMN2CGI 4: nt +844 to +1146). ^SMN2CGI 2 contains the first out of three transcriptional start sites (TSS) of SMN2 at nt −296, while the second TSS at nt position −242 is located close to the downstream border of ^SMN2CGI 2 (30,31). (A) Schematically illustrates the localizations of all four ^SMN2CGIs within the analysed genomic region. The localizations of the TSS at nt positions −296, −242 and −163 are indicated (30,31). (B) Frequency plots illustrating the methylation levels of each CpG dinucleotide within each ^SMN2CGI in the respective SMA(Spinal muscular atrophy)-fibroblasts. The mean methylation levels of each ^SMN2CGI, indicated by a horizontal line, are as follows: ML5: ^SMN2CGI 1: 87.1%, ^SMN2CGI 2: 36.6%, ^SMN2CGI 3: 6.9%, ^SMN2CGI 4: 75.9%; ML16: ^SMN2CGI 1: 87.7%, ^SMN2CGI 2: 37.7%, ^SMN2CGI 3: 6.2%, ^SMN2CGI 4: 73.4%. (C) Methylation analysis of ^SMN2CGI 1, ^SMN2CGI 2 and ^SMN2CGI 4 in ML5 and ML16 fibroblasts cell lines by bisulphite treatment, followed by PCR amplification of the respective ^SMN2CGI, cloning of PCR products, and sequencing. The methylation patterns of nine independent clones for each ^SMN2CGI and each cell line are shown. Empty circles represent unmethylated CpGs, whereas full circles correspond to their methylated status.

Figure 3.

Survival motor neuron gene 2 (SMN2) methylation patterns are highly conserved in blood samples derived from spinal muscular atrophy (SMA) patients. Bar charts show the mean methylation levels (±SEM) of each CpG dinucleotide within the respective ^SMN2CGIs in DNA isolated from blood samples drawn from SMN1-deleted SMA patients carrying two SMN2 copies (A, B). Type I (n = 10) and type III SMA patients (n = 7) show highly conserved SMN2 methylation patterns as can be deduced from the comparatively low SEM for the methylation levels of each CpG dinucleotide. The mean methylation levels of each ^SMN2CGI (see frequency plot) in both groups are as follows: type I SMA – ^SMN2CGI 1: 89.1%, ^SMN2CGI 2: 58.0%, ^SMN2CGI 3: 4.6%, ^SMN2CGI 4: 79.9%; type III SMA – ^SMN2CGI 1: 85.5%, ^SMN2CGI 2: 52.6%, ^SMN2CGI 3: 4.8%, ^SMN2CGI 4: 78.1%. SMN2 methylation correlates with the disease severity. Bar chart shows differences in DNA methylation at each CpG dinucleotide in type I versus type III SMA patients (C). Seven CpG dinucleotides (nt −871, −695, −296, −290, +855, +988, +1103) were identified whose methylation frequencies differ by at least 10% in type I versus type III SMA patients. Correlations between CpG methylation frequencies and disease severity reached levels of significance in all seven cases. Three levels of statistical significance were discriminated: *P < 0.05, **P < 0.01, ***P < 0.001 (t-test).

Figure 4.

Low survival motor neuron gene 2 (SMN2) methylation levels at the positions −296 and −290 are associated with mild spinal muscular atrophy (SMA) in SMA fibroblasts. (A) Bar chart showing the mean methylation levels (±SEM) at positions −296 and −290 in fibroblasts derived from SMN1-deleted SMA patients carrying two SMN2 copies. At both sites, type I SMA fibroblasts (ML17) show significantly higher methylation levels than fibroblasts derived from type IIIa (ML22) and type IIIb (ML74) patients. Three levels of statistical significance were discriminated: *P < 0.05, **P < 0.01, ***P < 0.001 (t-test). Methylation levels at positions −296 and −290 correlate with the activity of the transcriptional start site (TSS) at position −296 in SMA fibroblasts. Relative to type I SMA fibroblasts (ML17), the low methylation levels at positions −296 and −290 observed in type IIIa and type IIIb SMA fibroblasts are associated with high SMN2 transcripts levels originated from the TSS at position −296 (LT-SMN2) as shown by semi-quantitative RT–PCR analyses using two different primer combinations. (B) ß-Actin primers were used as internal control to verify equal loading of cDNA. CDK4 primers were used to detect potential contaminations with genomic DNA (amplicon size cDNA: 262 bp, amplicon size gDNA: 420 bp). (C) Differences in LT-SMN2 expression levels were confirmed by real-time PCR (normalized to ß-actin) using the LT-SMN2^{−279,−128} primer pair. Data are given as mean percentage (±SEM) relative to LT-SMN2 transcript levels in type I SMA fibroblasts (ML17) which were set to 100%.

Figure 5.

Histone deacetylase (HDAC) inhibitor treatment bypasses LT-SMN2 gene silencing mediated by DNA methylation. Treatment of type I SMA (spinal muscular atrophy) fibroblasts (ML17) with increasing doses of SAHA (hydroxamic acid) or FK-228 (cyclic tetrapeptide) elevates LT-SMN2 transcript levels in a dose-dependent manner as shown by semi-quantitative RT–PCR analyses. (A) ß-Actin primers were used as internal control to verify equal loading of cDNA. LT-SMN2 and total SMN2 transcript levels in type I SMA fibroblasts (ML17) following treatment with HDAC inhibitors for 48 h were further analysed by real-time PCR. (B) LT-SMN2 and total SMN2 transcript levels (both normalized to ß-actin) following HDAC inhibitor treatment are given as mean percentages (±SEM) relative to LT-SMN2 and total SMN2 transcript levels in solvent- and time-matched controls which were set to 100%.

Figure 6.

HDAC (histone deacetylase) inhibitor treatment elevates LT-SMN expression in human organotypic hippocampal brain slice cultures (OHSCs). (A–D) Bar charts showing LT-SMN and total survival motor neuron (SMN) transcript levels in human OHSCs derived from four different individuals. OHSCs were treated with the indicated doses of M344, SAHA or VPA (valproic acid) for 48 h. LT-SMN and total SMN transcript levels (both normalized to ß-actin) are given as mean percentages (±SEM) relative to LT-SMN and total SMN transcript levels in solvent- and time-matched controls which were set to 100%. Using OHSCs derived from the first patient (A), a dose-dependent SMN induction was confirmed on protein level (relative to ß-tubulin; 8 µm: 138 ± 2%; 16 µm: 188 ± 30%; 32 µm: 219 ± 41%). A representative human OHSC and a representative western blot analysis are shown. Induction of SMN protein expression by M344 reached levels of significance at all indicated doses (P < 0.05, t-test).

Figure 7.

LT-SMN2 induction by HDAC (histone deacetylase) inhibitors is not mediated by survival motor neuron gene 2 (SMN2) promoter demethylation. (A) Treatment of type I spinal muscular atrophy (SMA) fibroblasts (ML17) with valproic acid (VPA) (10 mm), phenylbutyrate (PB) (1 mm) or SAHA (10 µm) for 48 h did not affect ^SMN2CGI 2 methylation levels. Frequency plot illustrating methylation levels of each CpG dinucleotide within the ^SMN2CGI 2 in treated ML17 cells compared with time- and solvent-matched ML17 control fibroblasts. The DNA-demethylating drug zebularine (ZEB, 100 µm) was used as positive control. The mean methylation levels of ^SMN2CGI 2, indicated by horizontal lines, are as follows – control: 47.0%, VPA: 44.5%, PB 44.1%, SAHA: 45.0%, ZEB: 37.3%. Demethylation of CpG dinucleotides at positions −296 and −290 is associated with increased LT-SMN2 expression. Treatment of type I SMA fibroblasts (ML17) with ZEB (100 µm, 48 h) results in a significant demethylation of nine out of 12 CpG dinucleotides located in ^SMN2CGI 2, including the nt positions −296 and −290. (B) ZEB-induced ^SMN2CGI 2 demethylation is associated with increased LT-SMN2 levels as shown by quantitative real-time PCR. LT-SMN2 transcript levels (normalized to ß-actin) are given as mean percentages (±SEM) relative to LT-SMN2 expression levels in solvent- and time-matched controls set to 100%. (C) siRNA-mediated knockdown of MeCP2 elevates LT-SMN2 transcript levels in ML17 SMA fibroblasts. (D) MeCP2 and LT-SMN2 transcript levels (normalized to ß-actin) are given as mean percentages (±SEM) relative to MeCP2 or LT-SMN2 expression levels in time-matched controls transfected with AllStars negative control siRNA. The transcriptional co-repressor methyl-CpG-binding-protein 2 (MeCP2) is associated with the SMN2 promoter region and binds in a methylation-dependent fashion. (E, F) Using SMN1-deleted SMA fibroblasts cells (ML17), a significant binding of MeCP2 to the SMN2 promoter region was observed by chromatin immunoprecipitation (ChIP) analysis using an anti-MeCP2 antibody and primers amplifying the genomic SMN2 promoter region from nt −372 to −266. An unrelated antibody (negative Ctrl IgG, rabbit, Diagenode) was used as negative control. Treatment of ML17 SMA fibroblast cells with 100 µm ZEB resulted in a significant reduction of MeCP2 binding to the SMN2 promoter region. MeCP2 is enriched at a 286 bp SMN2 promoter region. (G) ChIP analyses using ML17 fibroblasts and six different primer pairs covering the genomic SMN2 promoter region from nt −631 to +59. Enrichment after MeCP2 ChIP was detectable for all primer pairs while two primer pairs showed considerably higher ChIP-PCR signal intensities. Three levels of statistical significance were discriminated: *P < 0.05, **P < 0.01, ***P < 0.001 (t-test).