As NotI digests were typically used during the former era of EST discovery, the 5′ end of this transcript was likely created during its cloning. To identify the extent of the antisense transcript, we performed strand-specific RT-PCR and 5′ RACE, and mapped the TSS to intron 4 (Figure 2A). We named this antisense transcript SCAANT1 for “spinocerebellar ataxia-7 antisense noncoding transcript 1.” To delineate the regulatory region responsible for transcription of SCAANT1, we cloned a series of human ataxin-7 CAG10 genomic fragments into a luciferase reporter construct in antisense orientation Selleckchem Alpelisib ( Figure 2A) and transfected the different ataxin-7 antisense genomic fragment—luciferase

constructs into primary cerebellar astrocytes. We noted that a short stretch of DNA 5′ to the SCAANT1 TSS was required for transactivation, while a sizable sequence 3′ to the SCAANT1 TSS was needed to achieve robust

Screening Library order transactivation ( Figure 2B). As the two CTCF binding sites lie within the regulatory domain mapped by the luciferase reporter assays, we derived another set of luciferase reporter constructs, based upon our most potent construct 2R, in which we mutated either of the CTCF binding sites ( Figure 2A). When we measured the transactivation competence of the 2R-m2 and 2R-m1 constructs, we observed marked reductions in luciferase activity ( Figure 2B), suggesting that CTCF binding site integrity is required for maximal SCAANT1 expression. We also derived an ataxin-7 antisense construct carrying a CAG92 repeat expansion (2R-exp), and when we measured its transactivation competence, we documented a significant reduction in luciferase activity ( Figure 2B). The existence of an ∼1.4 kb antisense noncoding transcript overlapping Thalidomide a potentially strong sense promoter at the human ataxin-7 locus suggested that their transcription regulation might be linked. As CTCF binding site integrity was required for SCAANT1

transcription, we derived two ataxin-7 minigene constructs that contain the sense P2A promoter and SCAANT1, flanked by ∼5 kb of DNA 5′ to this region and ∼8 kb of DNA 3′ to this region (Figure S2). Within this 13.5 kb human ataxin-7 genomic fragment reside two CTCF binding sites, known as CTCF-I and CTCF-II. To understand the regulatory relationship between SCAANT1 and ataxin-7 transcription from promoter P2A, we introduced an 11 nucleotide substitution mutation at the 3′ CTCF-I binding site (Figure S2). The location of the mutation was based upon DNA footprinting analysis, and validation of abrogated CTCF binding was achieved by electrophoretic mobility shift assays, as we have shown (Libby et al., 2008). In this way, we derived two distinct ataxin-7 genomic fragment constructs with an expanded CAG repeat tract: SCA7-CTCF-I-wt and SCA7-CTCF-I-mut (Figure S2).