, 1995) and observed an ∼47 kDa protein in brain lysates Ibrutinib from each SCA7-CTCF-I-mut transgenic line (Figure 3C). We noted higher expression in the SCA7-CTCF-I-mut-(2) line, consistent with its more severe phenotype. Low-level expression of the ∼47 kDa protein was detected in SCA7-CTCF-I-wt mice (Figure 3C),

and this ∼47 kDa protein product corresponds to an open reading frame starting at the initiator ATG codon in exon 3 and continuing through exon 4 until the first nonsense codon in intron 4. The production of a protein product and disease phenotype in the SCA7-CTCF-I-mut mice is reminiscent of the R6/2 mouse model of HD, in which a small fragment from the htt gene was introduced into mice to model repeat Stem Cell Compound Library cost instability, but also yielded a truncated protein product resulting in a HD-like phenotype—despite the fact that the construct lacked a 3′ polyadenylation site or characterized

promoter (Mangiarini et al., 1996). Our findings indicate that mutation of the 3′ CTCF binding site is responsible for initiation of robust sense transcription in SCA7-CTCF-mut-I mice, as SCA7-CTCF-I-wt mice carrying an ataxin-7 genomic fragment with an intact 3′ CTCF binding site express low levels of ataxin-7 mRNA and protein. To determine if the levels of ataxin-7 sense and antisense transcription within the repeat region domain correlate in SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice, we performed quantitative strand-specific RT-PCR amplification, and detected ataxin-7 sense and antisense transcripts in each line. We found that

ataxin-7 sense transcript levels were elevated ∼370-fold in the brains of SCA7-CTCF-I-mut mice compared to SCA7-CTCF-I-wt mice, and this was accompanied by an ∼140-fold decrease in SCAANT1 expression (Figure 3D). In situ hybridization analysis confirmed robust expression of SCAANT1 in the cerebellum of SCA7-CTCF-I-wt mice but did not detect strong SCAANT1 expression in SCA7-CTCF-I-mut mice (Figure 3E). In situ hybridization analysis indicated moderate Cediranib (AZD2171) to strong expression of SCAANT1 in SCA7-CTCF-I-wt mice throughout the brain (Figure S4A). Correspondingly, in situ hybridization did not yield evidence for much SCAANT1 expression in the brain of SCA7-CTCF-I-mut mice (Figure S4B). Taken together, these findings show that reduced SCAANT1 expression correlates with increased P2A promoter activity, resulting in increased sense expression of the ataxin-7 gene. Our studies of the SCA7-CTCF-I-wt and SCA7-CTCF-I-mut mice suggested that expression of the ataxin-7 sense transcript inversely correlates with expression of SCAANT1. To determine if this reciprocal expression relationship exists in normal human tissues, we performed qRT-PCR analysis on a panel of human tissue RNAs.