Extended Data Fig. 4: ZFP specificity assessment in vitro by microarray and RT–qPCR.
From: Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington’s disease
a, Microarray assessment of GM02151 (CAG18/45) HD fibroblasts transfected with ZFP-A, -B or -C. Each dot represents the mean fold change compared to control-treated cells for a given gene (x value) and the associated P value (y value) determined by an eBayes ANOVA analysis. Genes with ≥ 2 FC change and P < 0.01 are colored red (downregulated) or green (upregulated). ZFP-A, -B and -C: n = 6 biologically independent samples; control: n = 5 biologically independent samples. b,c, RT–qPCR analysis of normal (blue), mutant (red) and total HTT for transfected (b) GM02151 fibroblast and (c) GENEA020 neuron replicates evaluted for microarray analysis. n = 6 biologically independent samples; mean ± s.d.; one-way ANOVA with Tukey’s multiple comparison test; **** P < 0.0001. Allele-specific RT–qPCR was performed as in Fig. 2b (GENEA020 NSCs) and Fig. 1i (GM02151 fibroblasts). d, Twelve genes were selected for RT–qPCR validation of the microarray results. Three genes were chosen for each ZFP based on two criteria: (1) at least one ZFP treatment resulted in ≥ 2 FC change with P < 0.01, and (2) the gene contained ≥6× CAG repeats (allowing up to three mismatches per 6× CAG) within 1 kb of a TSS (see Methods and Supplementary Table 7). An additional three genes were chosen that had large CAG arrays and which were not regulated by any ZFP in the microarray study (‘No ZFP’ group). The distances of the CAG repeat from the TSS, total CAG count and contiguous CAG count are indicated (see Supplementary Table 7). Normal and mutant HTT CAG alleles for GM02151 fibroblasts are shown for reference. e, Regulation of the CAG repeat genes in d was confirmed by RT–qPCR. Normalized expression levels were scaled to the control treatment. n = 6 biologically independent samples; mean ± s.d. f, Venn diagram of regulated genes in a. No genes were commonly regulated (≥ 2 FC change; P < 0.01) by ZFP-A (n = 10) and ZFP-B (n = 5); there was partial overlap with genes regulated by ZFP-C (n = 53).
