Ssue samples from mice of seven different age groups were assessed in this study: post-natal

Ssue samples from mice of seven different age groups were assessed in this study: post-natal week 0 (n = 5), 2 (n = 4), 7 (n = 5), 17 (n = 4), 30 (n = 9), 36 (n = 4) and 93 (n = 9). The activity and epigenetic status of the human C9ORF72 transgene was assessed by quantifying levels of mRNAs and PD173074MedChemExpress PD173074 histone methylation within the promoter of the human gene. First, we measured human C9ORF72 mRNA levels in the neocortex of C9-BAC mice across all age groups using quantitative real-time (qPCR). Three primer sets were used to amplify different human C9ORF72 mRNA transcript variants (V1, V2, and V3) as well as three endogenous controls (betaactin, GAPDH and 18S). Due to the observed variability in beta-actin expression levels across age groups (Additional file 1: Figure S1A), we used the average values of GAPDH and 18S for normalization. We found that levels of all three C9ORF72 transcript variants are significantly reduced, starting within the first post-natal weeks of age (Fig. 1). To confirm our findings, we performed digital droplet quantitative PCR (ddPCR) for absolute quantification of the human C9ORF72 levelsEsanov et al. Molecular Neurodegeneration (2017) 12:Page 5 ofFig. 1 C9ORF72 transcription decreases while a repressive histone methylation mark increases in the brain of C9-BAC mice during the first post-natal weeks. Values of human C9ORF72 in the BAC mouse cortex, normalized to the average of GAPDH and 18S, are shown for primers PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28045099 amplifying transcript variants V1, V2, V3 (a); V1, V3 (b) and V2 (c). Age groups are indicated on the x-axis in weeks (wks). Mean and standard error of the mean (SEM) are indicated by long and short bars respectively. For each primer set, a one-way analysis of variance was performed (p < 0.001). Bonferroni's multiple comparison test was performed between neonatal (0wks) and the rest of the age groups, significance is indicated by p < 0.05 * and p < 0.01 **. H3K9me3 levels were assessed by chromatin immunoprecipitation in brain tissues from 0 to 7 week-old C9-BAC mice (n = 5). Two different primer sets were used: C9.A and C9.B amplifying regions within the human C9ORF72 promoter. Relative enrichment of H3K9me3 was calculated by real-time PCR amplification of immunoprecipitants (IP) relative to inputs and an IgG negative control IP, p < 0.01 ** (d)and observed a similar trend (Additional file 1: Figure S1B). This observation suggests that partial epigenetic repression of the C9ORF72 transgene in the brain of post-natal C9-BAC mice is agedependent. Next, we utilized chromatin immunoprecipitation (ChIP) to isolate H3K9me3-bound DNA fragments from brain samples of 0 and 7 weeks-old C9-BAC mice. H3K9me3 is a repressive epigenetic mark that negatively correlates with transcription rates and is enriched within the promoter region of expanded C9ORF72 alleles as compared to unexpended alleles [12, 26, 29]. Using two different primer sets (C9.A and C9.B), we found that H3K9me3 levels within the promoter of the human C9ORF72 transgene were significantly increased in the brain of C9-BAC mice by week 7 (Fig. 1d). Increased H3K9me3 indicates partial epigenetic repression of the mutant gene locus occurs in the first post-natal weeks of life.DNA hypermethylation within the promoter of the human C9ORF72 transgene occurs in a subset of C9-BAC mice, similar to the human C9-ALS patient populationIn about 30 of all C9-ALS cases, methylated CpG dinucleotides within the C9ORF72 promoter occur more frequently [13, 15]. This promoter h.