Survival motor neuron gene 2 silencing by DNA methylation correlates with spinal muscular atrophy disease severity and can be bypassed by histone deacetylase inhibition - PubMed (original) (raw)

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 m

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), phenylbutyrate (PB) (1 m

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) or SAHA (10 µ

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) for 48 h did not affect _SMN2_CGI 2 methylation levels. Frequency plot illustrating methylation levels of each CpG dinucleotide within the _SMN2_CGI 2 in treated ML17 cells compared with time- and solvent-matched ML17 control fibroblasts. The DNA-demethylating drug zebularine (ZEB, 100 µ

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) was used as positive control. The mean methylation levels of _SMN2_CGI 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 µ

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, 48 h) results in a significant demethylation of nine out of 12 CpG dinucleotides located in _SMN2_CGI 2, including the nt positions −296 and −290. (B) ZEB-induced _SMN2_CGI 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 µ

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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).