Interrogation of SCAANT1 expression levels revealed an opposite pattern, as SCAANT1 was much higher in the lung and kidney than in cortex, cerebellum, striatum, or liver (Figure 4B). As the presence of the CAG repeat expansion

decreased SCAANT1 promoter activity in our luciferase reporter assays (Figure 2), we tested if the diametrically opposed expression of ataxin-7 sense transcript and SCAANT1 might occur in the selleck chemical context of SCA7 disease. To test this hypothesis, we performed RT-PCR analysis of a SCA7 patient fibroblast cell line, and while we could amplify both the normal and expanded repeat alleles for the ataxin-7 sense transcript, we could not detect antisense SCAANT1 transcript expression from the expanded 55Q allele (Figure 4C).

We then performed quantitative RT-PCR analysis of ataxin-7 sense expression on fibroblasts obtained from two SCA7 patients, one with a moderately sized disease repeat (55Q), and one with a severely expanded repeat (150Q). With increasing expansion size, we observed significantly increased ataxin-7 sense transcript levels (Figure 4D), indicating that expansion of the CAG repeat at the ataxin-7 locus yields increased levels of ataxin-7 transcript in association with reduced expression of SCAANT1. We also obtained a set of peripheral blood samples from three additional SCA7 patients, isolated RNA from their lymphocytes, and performed RT-PCR analysis. Antisense SCAANT1 transcript expression could not Autophagy activator be detected from the expanded allele

of the SCA7 samples, and all three SCA7 patients exhibited significantly increased ataxin-7 sense transcript levels (Figure 4E). As mutation of the 3′ CTCF binding site reduced the activity of the SCAANT1 promoter while derepressing ataxin-7 Urease sense expression from promoter P2A, we hypothesized that CTCF modulates ataxin-7 sense expression from this promoter by driving the expression of SCAANT1. To test this hypothesis, we validated two different CTCF shRNAs and derived a dual CTCF knockdown vector. After subcloning the CTCF dual shRNA knockdown fragment into a lentiviral construct with a linked eGFP expression cassette, we infected human Y-79 retinoblastoma cells and isolated RNA from flow-sorted GFP-positive Y-79 cells. Real-time RT-PCR analysis confirmed CTCF knockdown, and revealed a significant reduction in the expression level of SCAANT1 (Figure 5A). Significant reduction in SCAANT1 expression was accompanied by a marked increase in the ataxin-7 sense transcript from the P2A promoter, but not from the previously defined “standard” ataxin-7 sense promoter, located >40 kb 5′ to the repeat region (Figure 5A). Although direct, physiological comparison of the standard P1 promoter and P2A promoter is complicated by the coexistence of SCAANT1 transcription, analysis of ataxin-7 alternative sense and standard sense expression at baseline in Y-79 cells revealed only modestly (i.e., ∼2.