Reduced C9orf72 gene expression in c9FTD/ALS is caused by histone trimethylation, an epigenetic event detectable in blood

Abstract

Individuals carrying (GGGGCC) expanded repeats in the C9orf72 gene represent a significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Elucidating how these expanded repeats cause "c9FTD/ALS" has since become an important goal of the field. Toward this end, we sought to investigate whether epigenetic changes are responsible for the decrease in C9orf72 expression levels observed in c9FTD/ALS patients. We obtained brain tissue from ten c9FTD/ALS individuals, nine FTD/ALS cases without a C9orf72 repeat expansion, and nine disease control participants, and generated fibroblastoid cell lines from seven C9orf72 expanded repeat carriers and seven participants carrying normal alleles. Chromatin immunoprecipitation using antibodies for histone H3 and H4 trimethylated at lysines 9 (H3K9), 27 (H3K27), 79 (H3K79), and 20 (H4K20) revealed that these trimethylated residues bind strongly to C9orf72 expanded repeats in brain tissue, but not to non-pathogenic repeats. Our finding that C9orf72 mRNA levels are reduced in the frontal cortices and cerebella of c9FTD/ALS patients is consistent with trimethylation of these histone residues, an event known to repress gene expression. Moreover, treating repeat carrier-derived fibroblasts with 5-aza-2-deoxycytidine, a DNA and histone demethylating agent, not only decreased C9orf72 binding to trimethylated histone residues, but also increased C9orf72 mRNA expression. Our results provide compelling evidence that trimethylation of lysine residues within histones H3 and H4 is a novel mechanism involved in reducing C9orf72 mRNA expression in expanded repeat carriers. Of importance, we show that mutant C9orf72 binding to trimethylated H3K9 and H3K27 is detectable in blood of c9FTD/ALS patients. Confirming these exciting results using blood from a larger cohort of patients may establish this novel epigenetic event as a biomarker for c9FTD/ALS.

Fig. 1

Frontal cortex and cerebellum tissue samples from the C9orf72+ group exhibit a significant decrease in C9orf72 mRNA expression levels. a Schematic representation showing the three known transcript variants of C9orf72. The star marks the repeat location. b, c qRT-PCR for C9orf72 transcript variants 1, 2, 3 and C9orf72 transcript variants 2, 3 were performed in frontal cortex (b) and cerebellum (c). Data on graphs were normalized to disease control group (mean value set to 1). Statistical differences were calculated by one-way ANOVA with Tukey post hoc test. *p < 0.05, **p < 0.01, ***p < 0.005. d, e ddPCR was used to calculate absolute levels of the less abundant C9orf72 transcript variant 1 in frontal cortex (d) and cerebellum (e). Transcript expression is expressed as number of copies per microliter. The mean expression and the range of expression across all samples tested are shown for each group. A clinical description of participants is available in Table 2 (online resource)

Fig. 2

Reduced C9orf72 mRNA expression levels in the C9orf72+ group result from aberrant binding to trimethylated histone residues. a Electrophoretic representation of chromatin immunoprecipitated DNA in a subgroup of participants tested. Chromatin immunoprecipitation (ChIP) was performed on two different human tissues, frontal cortex (F) and cerebellum (C), using antibodies specific for total histones H3 and H4 or trimethylated histones H3K9, H3K27, H3K79, and H4K20. Following pull-down, bound DNA was purified and used for PCR amplification of the C9orf72 promoter region. This region was successfully amplified in the C9orf72+ group when ChIP was carried out with antibodies targeting trimethylated histone residues; under the same conditions, this region was not amplified in C9orf72− and disease controls, indicating an absence of binding. The complete figure is provided in the online resource. b, c, d, e Relative quantifications of all brain DNA were performed by measuring band intensity (complete gels in the online resource, Fig. 2) for each immunoprecipitated histone and presented as a ratio to the input. Each graph is normalized to total histone levels in the disease control group (mean value set to 1). Statistical differences were calculated by one-way ANOVA with Tukey post hoc test. *p < 0.05, **p < 0.01, *** p < 0.005. A clinical description of participants is available in Table 2 (online resource)

Fig. 3

C9orf72 mRNA expression is increased and H3K9me3 binding is decreased in C9orf72+ fibroblasts upon 5-AZA treatment. a, b qRT-PCR of RNA obtained from human fibroblasts grown in DMSO or 5-AZA demethylating agent. Both assays targeting transcript variants 1, 2, 3 (a) and 2, 3 (b) show a significant increase in expression after 5-AZA treatment only in C9orf72 repeat expansion carriers. c qRT-PCR of H19, an imprinted gene, showing effectiveness of the 5-AZA treatment in C9orf72+ and C9orf72− fibroblasts. Statistical differences were assessed by unpaired Student t test. *p < 0.05, **p < 0.01. NS no significant difference. d Electrophoretic representation of chromatin immunoprecipitated DNA from a fibroblast subgroup using antibodies specific for total H3 or trimethylated histone H3K9. Fibroblasts were grown in DMSO or 5-AZA. Chromatin immunoprecipitation (ChIP) was performed in fibroblasts from C9orf72+ and C9orf72− participants. Following pull-down, bound DNA was purified and used for PCR amplification of the C9orf72 promoter region. Upon treatment with vehicle (DMSO), the binding to trimethylated histone H3K9 in C9orf72+ cells, as assessed by the level of amplified C9orf72 promoter region, was higher as compared to C9orf72−. Treatment with 5-AZA reduced this binding in C9orf72+ cases. The complete figure of all fibroblast lines is provided in the online resource. e, f Relative quantifications of all fibroblast lines were performed by measuring band intensity (complete gels in the online resource, Fig. 3) for each immunoprecipitated histone and presented as a ratio to the input. Graphs are normalized to total histone levels of disease control group (mean value set to 1). Statistical differences were calculated by one-way ANOVA with Tukey post hoc test. *p < 0.05. A clinical description of participants is available in Table 3 (online resource)

Fig. 4

Strong binding of expanded C9orf72 to H3K9me3 and H3K27me3 is detectable in the blood of c9FTD/ALS patients. Electrophoretic representation of chromatin immunoprecipitated blood DNA from two repeat expansion carriers (C9orf72+) and two participants carrying normal alleles (C9orf72−). Antibodies specific for total H3 or trimethylated histone H3K9 and H3K27 were used to pull-down DNA. Following pull-down, bound DNA was purified and used for PCR amplification of the C9orf72 promoter region. The binding to trimethylated H3K9 and H3K27 in C9orf72+ blood cells, as assessed by the level of amplified C9orf72 promoter region, was higher as compared to C9orf72−

Fig. 5

Schematic representation of the proposed haploinsufficiency mechanism resulting from epigenetic changes in c9FTD/ALS