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1.
Figure 4

Figure 4. Mild overexpression of Atxn1L restores expression levels of some genes altered in Atxn1−/− cerebella.. From: Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis.

Real-time quantitative RT-PCR on RNA extracted from wild-type (n = 8), Atxn1−/− (n = 10) and Atxn1−/−; Atxn1Ldp/+ cerebella (n = 6) for (A) Ccnd1, (B) Igfbp5, (C) Apba2bp, (D) Robo1, and (E) Grid2 reveals that these transcripts were restored close to wild-type levels in Atxn1−/−; Atxn1Ldp/+ cerebellum. Real-time qRT-PCR experiments were performed in triplicate for each sample. Error bars represent +/−SEM, *p<0.05.

Juan Crespo-Barreto, et al. PLoS Genet. 2010 July;6(7):e1001021.
2.
Figure 1

Figure 1. Comparison of transcriptional profiles of Atxn1154Q/+ and Atxn1−/− cerebella.. From: Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis.

(A) Venn diagram reporting the number of significant gene expression changes in Atxn1−/− and Atxn1154Q/+cerebella. A total of 197 transcripts were significantly altered in both Atxn1−/− and Atxn1154Q/+mice, using a p-value of less than 0.01, and a minimal fold change of |±0.1| log2. (B) The majority of shared changes (68% of genes or 135 out of 197) went in the same direction.

Juan Crespo-Barreto, et al. PLoS Genet. 2010 July;6(7):e1001021.
3.
Figure 6

Figure 6. Model of Atxn1L rescue through Cic stabilization in Atxn1−/− mice.. From: Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis.

(A) In wild-type mice, Atxn1-Cic and Atxn1L-Cic complexes bind the promoters of target genes and repress them effectively. (B) In mice expressing polyglutamine-expanded Atxn1, mutant Atxn1 associates preferentially with Rbm17, while the decrease in Atxn1-Cic complexes destabilizes Cic and reduces its levels at the promoters, thus leading to de-repression of its target genes. (C) A similar Cic destabilization occurs in the absence of wild-type Atxn1 in Atxn1−/− mice, also resulting in increased expression of target genes. (D) When Atxn1L is moderately overexpressed in Atxn1−/− mice, it stabilizes Cic levels by forming functional Atxn1L-Cic complexes that can substitute for Atxn1-Cic at the promoters, thus rescuing target gene repression. We propose that this mechanism might also act to rescue loss-of-function of Atxn1 in Atxn1154Q/+; Atxn1Ldp/+ mice.

Juan Crespo-Barreto, et al. PLoS Genet. 2010 July;6(7):e1001021.
4.
Figure 3

Figure 3. Atxn1Ldp stabilizes Cic protein levels in Atxn1−/− mice by enhancing Atxn1L-Cic complex formation.. From: Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis.

(A) Western blot analysis shows that overexpression of Atxn1L rescues the reduced Cic levels in Atxn1−/− mice (* p<0.05). (B) Co-immunoprecipitation of Atxn1L using Cic antibodies show that, despite the reduced levels of Cic protein in Atxn1−/− cerebella, the relative fraction of Atxn1L co-immunoprecipitated with Cic in Atxn1−/− protein extracts was greater than in wild-type cerebella. Atxn1L overexpression further increased the levels of Atxn1L-Cic co-immunoprecipitation in Atxn1−/−; Atxn1Ldp/+ mice. Images show representative blots and the quantification of three independent experiments. Error bars in graphs represent +/−SEM. * p<0.05. (C) To determine if Atxn1L-Cic complexes are functional, we measured the transcriptional effect of Cic, Atxn1 and Atxn1L on the expression of a luciferase reporter construct containing a tandem array of Cic binding sites (CBS). Luciferase activity is expressed as fraction of the activity when the reporter is expressed alone (100%). Co-transfection of Cic and Atxn1[2Q]) results in synergistic co-repression of this luciferase reporter (Cic+Atxn1[2Q]). Interestingly, co-transfection of constructs expressing Cic and Atxn1L (Cic+Atxn1[2Q]) results in synergistic repression of the reporter similar to co-transfection of Cic and wild-type Atxn1. These results suggest that Atxn1L-Cic complexes are functional in Capicua-dependent repression. Assays were performed in duplicate in 5 independent experiments. Error bars in graph represent +/− SEM, *p<0.05,***p<0.005.

Juan Crespo-Barreto, et al. PLoS Genet. 2010 July;6(7):e1001021.
5.
Figure 5

Figure 5. Atxn1Ldp suppresses the behavioral deficits in Atxn1−/− mice.. From: Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis.

We assessed the effects of mild overexpression of Atxn1L on Atxn1−/− phenotypes (A) Mice of the following genotypes: Atxn1+/+ (n = 5), Atxn1+/− (n = 12), Atxn1−/− (n = 9), Atxn1+/+; Atxn1Ldp/+(n = 10), Atxn1+/−; Atxn1Ldp/+(n = 17), and Atxn1−/−; Atxn1Ldp/+(n = 12), were tested at 8 weeks for contextual conditioned fear. Atxn1−/− mice exhibited significantly reduced freezing behavior in the contextual fear-conditioning test compared to wild-type and Atxn1+/− littermates. However, Atxn1−/− mice carrying the Atxn1L duplication performed significantly better than Atxn1−/− littermates in this task (p<0.05). Wild-type and Atxn1+/− mice expressing the Atxn1Ldp allele performed similarly to wild-type and Atxn1+/− mice without the Atxn1L duplication. (B–E) An independent cohort of mice was generated to test the effects of Atxn1L duplication on the motor deficits of Atxn1−/− mice. Atxn1+/− (n = 16), Atxn1+/−; Atxn1Ldp/+(n = 23), Atxn1−/− (n = 14), and Atxn1−/−; Atxn1Ldp/+ mice (n = 17) were tested on the dowel and wire hang paradigms at 8 weeks. The latency of Atxn1−/− mice to reach the side for the first time was increased compared to Atxn1+/− and Atxn1+/−; Atxn1dp/+ littermates on the rod (B); they also walked off the rod fewer times in the 120 s interval (C). In contrast, Atxn1−/−; Atxn1Ldp/+ mice took less time to walk off the dowel (B), and they also crossed the dowel more times in 120 s than Atxn1−/− littermates (C). In the wire hang test, Atxn1−/− mice showed increased latency to reach the sides of the wire compared Atxn1+/− and Atxn1+/−; Atxn1Ldp/+controls (D). Additionally, Atxn1−/− mice reached the sides fewer times than control littermates (E). In contrast, Atxn1−/− mice overexpressing Atxn1L exhibited marked reduction in the time for the first touch (D), and increased number of side touches in the 120 s interval, when compared to Atxn1−/− mice (E). Error bars in graphs represent +/− SEM, *p<0.05.

Juan Crespo-Barreto, et al. PLoS Genet. 2010 July;6(7):e1001021.
6.
Figure 2

Figure 2. Chromatin immunoprecipitation (ChIP) reveals co-occupancy at the promoters of Cic target genes by Atxn1 and Cic.. From: Partial Loss of Ataxin-1 Function Contributes to Transcriptional Dysregulation in Spinocerebellar Ataxia Type 1 Pathogenesis.

(A) ChIP using Cic antisera confirmed Cic binding at the promoter of two direct targets of Capicua, Ccnd1 and Etv5, that were up-regulated in the Atxn1−/− and Atxn1154Q/+ cerebella. (B) ChIP using Atxn1 anti-sera in cerebellar extracts from Atxn1+/−, Atxn1154Q/−, and Atxn1−/− mice reveals a signal in mice expressing only wild-type (Atxn1+/−) but not polyQ-Atxn1 (Atxn1154Q/−) compared to negative controls (pre-immune sera, and Atxn1−/−) (C) ChIP as in (B), this time using Cic antibody. In contrast to (B), Cic is present at comparable levels at the target promoters in Atxn1+/−, Atxn1154Q/−, and Atxn−/− mice. All ChIP assays were repeated three times on independent samples, representative results shown. (D) ChIP followed by quantitative PCR (ChIP-qPCR) on the promoter of Etv5 confirms that the proportion of immunoprecipitated DNA by Cic antibody is comparable in all three genotypes (as seen in C). A region containing two Capicua binding sites (promoter-CBS) of Etv5 is more enriched by Cic antibody than a region lacking CBSs (promoter-no CBS) compared to preimmune sera. (E) ChIP-qPCR on the promoter of Ccnd1 also shows similar enrichment of immunoprecipitated DNA by Cic antibody in all three genotypes (as seen in C). ChIP-qPCR using primers designed for a region within 100 bps of the CBS in the Ccnd1 promoter (which is highly conserved across species) show more Cic binding than primers designed for a poorly conserved region further downstream (∼400 bps) of the CBS at the promoter of Ccnd1, compared to preimmune sera. ChIP-qPCR experiments in (D) and (E) were performed in triplicate on three independent sets of samples (3 cerebella per genotype) using SYBR Green Dye. N.S. = not significant, ** p<0.01, *** p<0.005.

Juan Crespo-Barreto, et al. PLoS Genet. 2010 July;6(7):e1001021.

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