Variation in TAF1 expression in female carrier induced pluripotent stem cells and human brain ontogeny has implications for adult neostriatum vulnerability in X-linked Dystonia Parkinsonism

Abstract

X-linked Dystonia-Parkinsonism (XDP) is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. The mechanisms underlying regional differences in degeneration and adult onset are unknown. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due, in part, to a partial loss of TAF1 function. A disease-specific SINE-VNTR-Alu (SVA) retrotransposon insertion occurs within intron 32 of TAF1, a subunit of TFIID involved in transcription initiation. While all XDP males are usually clinically affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight iPSC lines from three XDP female carrier individuals for X chromosome inactivation status and identified clonal lines that express either the wild-type X or XDP haplotype. Furthermore, we characterized XDP-relevant transcript expression in neurotypical humans, and found that SVA-F expression decreases after 30 years of age in the brain and that TAF1 is decreased in most female samples. Uniquely in the caudate nucleus, TAF1 expression is not sexually dimorphic and decreased after adolescence. These findings indicate that regional-, age- and sex-specific mechanisms regulate TAF1, highlighting the importance of disease-relevant models and postmortem tissue. We propose that the decreased TAF1 expression in the adult caudate may synergize with the XDP-specific partial loss of TAF1 function in patients, thereby passing a minimum threshold of TAF1 function, and triggering degeneration in the neostriatum.Significance StatementXDP is an inherited, X-linked, adult-onset movement disorder characterized by degeneration in the neostriatum. No therapeutics alter disease progression. Developing therapeutics requires a deeper understanding of how XDP-relevant features vary in health and disease. XDP is possibly due to a partial loss of TAF1 function. While all XDP males are usually affected, females are heterozygous carriers generally not manifesting the full syndrome. As a resource for disease modeling, we characterized eight stem cell lines from XDP female carrier individuals. Furthermore, we found that, uniquely in the caudate nucleus, TAF1 expression decreases after adolescence in healthy humans. We hypothesize that the decrease of TAF1 after adolescence in human caudate, in general, may underlie the vulnerability of the adult neostriatum in XDP.

Keywords: SVA retrotransposon; X-linked Dystonia Parkinsonism; induced pluripotent stem cells; movement disorder; neurodegeneration; repeat expansion.

Figure 1.

Characterization of XDP female carrier-derived iPSCs for XCI status of the XDP haplotype. a, Family trees showing that the iPSC clones were derived by daughters/obligated carriers of XDP probands. b, Long-PCR products obtained from amplification of genomic DNA extracted from one control, one XDP mutant line, and all eight XDP female carrier-derived iPSC clones. Primers flanking the SVA insertion site were used to confirm the presence of the SVA retrotransposon in the genome. The control iPSC line shows only a product of ∼599 bp (lower band), whereas the male XDP-derived iPSC line shows the predicted 3229 bp SVA product (upper band). Both PCR products were detectable in all eight XDP female carrier-derived iPSCs. c, Illustration of XDP female carrier-derived iPSCs. Because of clonal expansion of iPSCs and random XCI, female iPSC clones can inactivate two different X chromosomes. If the X chromosome carrying the healthy wild-type (WT) allele is active, the cell will express the canonical TAF1 transcript; in contrast, if the X chromosome carrying the mutant XDP allele is active, the cell will express the mutant TAF1 transcript, including the XDP haplotype. XDP female carrier-derived iPSCs were subjected to strand-specific RNA sequencing to perform gene expression analysis and allele-specific expression analysis. d, Bar plots of the fraction of allele-specific transcripts derived from the X chromosomes, which are shared between the different iPSC clones. Expression from the same or different alleles was determined by counting the number of shared and unique homozygous SNPs, respectively. The specific number of SNPs analyzed for each pair is shown to the right of the bar. e, Illustration of the mutant TAF1 splice variant characterized by retention of intron 32 (TAF1-32i) close to the SVA insertion. We used a designed custom TaqMan primer/probe to detect the presence of the exon 32/intron 32 splice site (Aneichyk et al., 2018). Yellow arrows show that forward and reverse primers bind to exon 32 and intron 32, respectively, so that the probe spans the splice junction. f, The graph shows the relative expression of TAF1-32i in XDP female carrier-derived iPSCs in comparison to iPSCs derived from control, XDP, and relatively isogenic SVA-deleted lines (ΔSVA-XDP). All values were normalized to the XDP sample. One-way ANOVA was performed on the mean and SEM from three independent experiments, and significance was determined as follows: ***p ≤ 0.001; ns = not significantly different. g, Table summarizing features of the eight XDP female carrier-derived iPSCs used in this study. See also Extended Data Figures 1-1, 1-2, and 1-3. Figure contribution: Ricardo S. Jacomini and Apua C. M. Paquola performed the allele-specific expression analysis. Laura D’Ignazio performed the long-PCR and TAF1-32i Taqman assay and analyzed the data.

Figure 2.

XDP female carrier-derived iPSCs have proper X chromosome dosage compensation. a, Diagram illustrating differences between “proper” and “eroded” XCI status. Proper XCI occurs when X-linked genes show higher monoallelic expression than do autosomal genes. Erosion of XCI occurs when the fraction of monoallelic transcripts is similar between the X chromosome and autosomes. b, Fraction of monoallelic expression from chromosome X compared with that from all autosomal chromosomes. An increased fraction of monoallelic expression indicates silencing of the X chromosome, and that proper XCI occurs in all XDP female carrier-derived iPSCs. The XCI status (proper or eroded) is reported for each iPSC clone. c, The relative expression of the X chromosome (RXE) is the difference between the log2-transformed mean TPM values of the X chromosome and all autosomes. If the expression of X and autosomes is equal, the RXE value will be 0, suggesting proper X dosage compensation. Positive RXE values indicate that the expression of X-linked genes is greater than that of autosomal genes; thus, dosage compensation is complete. Negative RXE values indicate that the expression of X-linked genes is lower than that of autosomes, and there is incomplete dosage compensation. d, Box plots showing log2-transformed relative expression of the X chromosome (RXE, blue) and all autosome chromosomes (red) for XDP female carrier-derived iPSCs. Red dots indicate the relative expression of each autosome pair (RGE) over all other chromosomes. e, RXE values of XDP female carrier-derived iPSC lines in comparison to female and male iPSC lines from public HipSci collection (Kilpinen et al., 2017) and adult female and male postmortem caudate nucleus samples from the BrainSeq Consortium (Collado-Torres et al., 2019; Benjamin et al., 2020). One-way ANOVA was performed on the mean and SEM calculated for each sample, and significance was determined as follows: *p ≤ 0.05, ***p ≤ 0.001; ns = not significant. Figure contribution: Apua C. M Paquola and Ricardo S. Jacomini performed the allele-specific transcriptomic analysis. Apua C. M Paquola and Kynon J. M. Benjamin performed the dosage compensation analysis.

Figure 3.

Dosage compensation in female iPSCs inversely correlates with XIST expression. a, Simplified map of the XIST, TSIX, HUWE1, ATRX, and XACT loci on the X chromosome, and expression of X-linked gene transcripts in XDP carrier-derived iPSCs. Out of these genes, XIST shows higher variability among clones. b, RT-qPCR analysis of XIST expression in XDP female carrier-derived iPSCs compared with a control male iPSC line. Primers flanking exons 1 and 3 of XIST have been used. One-way ANOVA was performed on the mean and SEM from three independent experiments, and significance was determined as follows: *p ≤ 0.05, **p ≤ 0.01. c, Representative images from iPSC clone 33360.D showing localization of RNA scope-specific probes for XIST (red) and XACT (green) transcripts. DAPI was used to stain nuclei. Scale bar: 20 μm. Based on the expression of those transcripts, different categories of nuclear localization were identified: (i/v) detection of one RNA molecule of XIST/XACT; (ii/vi) detection of two RNA molecules of XIST/XACT; (iii/vii) detection of several dispersed molecules of XIST/XACT transcripts forming a diffuse cloud; (iv/viii) detection of several molecules of XIST/XACT transcripts organized in a compact cloud. Bar charts report quantification of the four categories in the isogenic set of XDP female carrier-derived iPSCs. For each clone, >700 cells have been analyzed. The percentage of cells not expressing XIST or XACT transcripts is also reported. d, Representative images from iPSC clone 33360.A and 33360.B showing localization of RNA scope-specific probes for XIST (red) and HUWE1 (green) transcripts. DAPI was used to stain nuclei. Scale bar: 20 μm. The HUWE1 gene is usually subject to XCI; thus, four different cellular conditions were identified: (1) monoallelic expression of HUWE1 accompanied by XIST expression (in any form, i.e., single RNA molecules, multiple RNA molecules, diffuse clouds, compact cloud); (2) monoallelic expression of HUWE1 in the absence of XIST expression; (3) biallelic expression of HUWE1 accompanied by XIST expression (in any form, i.e., single RNA molecule, multiple RNA molecules, diffuse cloud, compact cloud); (4) biallelic expression of HUWE1 in the absence of XIST expression. Bar graphs report the percentage of HUWE1-positive cells showing each of the four cellular conditions in the isogenic iPSC pair 33360.A and 33360.B. For each clone, >600 cells have been analyzed. See also Extended Data Figure 3-1. Figure contribution: Kynon J. M. Benjamin analyzed the RNA-seq data. Laura D’Ignazio and Bareera Qamar performed and analyzed both the XIST qPCR analysis and the RNA scope.

Figure 4.

Sex-dependent expression of TAF1 in female iPSCs. a, Normalized expression of the TAF1 gene in XDP carrier-derived iPSCs. b, RT-qPCR analysis of TAF1 expression in XDP female carrier-derived iPSCs compared with iPSCs derived from control, XDP, and a relatively isogenic SVA-deleted line (ΔSVA-XDP). Primers flanking exons 32 and 33 of TAF1 were used in this analysis. One-way ANOVA was performed on the mean and SEM from three independent experiments, and significance was determined as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns = nonsignificant. c, Box plots comparing normalized TAF1 transcript expression levels in female (red) and male (blue) iPSCs from public HipSci collection (Kilpinen et al., 2017); *p ≤ 0.05. d, Percentage of X-linked genes in female iPSCs (Kilpinen et al., 2017) differentially expressed with sex (orange) and expressed independently of sex (gray). e, Number of sex-differentially expressed X-linked genes that are upregulated or downregulated in female iPSCs (Kilpinen et al., 2017). TAF1 is among the eight genes under-expressed in female iPSCs compared with male iPSCs. Figure contribution: Laura D’Ignazio performed the TAF1 qPCR analysis. Laura D’Ignazio and Kynon J.M. Benjamin analyzed the RNA-seq data from XDP female carrier-derived iPSCs and HipSci collection.

Figure 5.

TAF1 and SVA-F expression over the lifespan in the postmortem brain. a–c, Box plots comparing residualized gene expression of TAF1 in (a) the caudate nucleus (n = 394), (b) DLPFC (n = 379), and (c) hippocampus (n = 376) of female (red) and male (blue) individuals, from the BrainSeq Consortium (Collado-Torres et al., 2019; Benjamin et al., 2020); ns = not significant. d–i, Box plots comparing residualized gene expression of individuals from the BrainSeq Consortium (Collado-Torres et al., 2019; Benjamin et al., 2020) for: (d) TAF1 in the caudate nucleus, (e) TAF1 in the DLPFC, (f) TAF1 in the hippocampus, (g) SVA-F in the caudate nucleus, (h) SVA-F in the DLPFC, (i) SVA-F in the hippocampus. Samples were divided into four groups based on the age of the individuals: 0–15 years; 16–30 years; 31–51 years; and older than 51 years; ns = not significant; *FDR ≤ 0.05, **FDR ≤ 0.01, ***FDR ≤ 0.001. j, SVA expression in iPS cells. Plot shows normalized RNA-Seq coverage along the SVA sequence for both sense (+) and antisense (–) strands. Mutant trace (red) indicates female XDP carrier lines expressing the XDP allele: 33110.2A, 33110.2E, 33110.2F, 33360.A, 33360.D. Wild-type trace (light blue) indicates female XDP carrier lines expressing the wild-type X chromosome: 33360.B, 33811.A, 33811.B. The schematic illustration describes the structure of the human SVA retrotransposons. k, Diagram depicting our proposed model to explain the differential degeneration in the neostriatum of individuals with XDP. The decrease in TAF1 expression after 15 years of age may synergize with a XDP-specific partial loss of TAF1 function in the caudate nucleus, a brain region that seems to be selectively sensitive to TAF1 levels. See also Extended Data Figures 5-1, 5-2, 5-3, and 5-4. Figure contribution: Ria Arora performed the sex differential expression of TAF1. Kynon J. M. Benjamin performed the age-differential expression of TAF1 across lifespan. Taylor Evans performed the SVA-F differential expression analysis across lifespan. Apua C. M. Paquola and Jennifer A. Erwin performed the SVA-derived transcript analysis on iPSCs.