ZNF91 is an endogenous repressor of the molecular phenotype associated with X-linked dystonia-parkinsonism (XDP)

Background X-linked dystonia-parkinsonism (XDP) is a severe neurodegenerative disorder resulting from the insertion of an intronic SINE-Alu-VNTR (SVA) retrotransposon in the TAF1 gene. Recent research has revealed that the pathogenic XDP-SVA insertion leads to dysregulation of TAF1 transcription, including increased intron retention and decreased expression of exons surrounding the insertion. The Krüppel-associated box (KRAB) zinc finger protein, ZNF91, is a critical repressor of SVA retrotransposons. However, it remains unclear whether ZNF91 is able to repress the XDP-SVA insertion and how this influences the XDP-associated molecular phenotype. In this study, we investigate the role of ZNF91 in repressing the XDP-SVA insertion and its impact on the molecular phenotype associated with XDP. Methods Here, we used CRISPR/Cas9 to genetically delete ZNF91 in induced pluripotent stem cell (iPSC) lines derived from XDP patients, as well as isogenic control iPSC lines that lack the XDP-SVA insertion. Total RNA sequencing and capture RNA-sequencing were used to confirm ZNF91 deletion and to assess TAF1 transcriptional changes between conditions. Furthermore, publicly available transcriptomic data from whole blood and different brain regions were used to assess ZNF91 expression levels across ages. Results We found that genetic deletion of ZNF91 exacerbates the molecular phenotype associated with the XDP-SVA insertion in patient cells, while no difference was observed when ZNF91 was deleted from isogenic control cells. Additionally, we observed a significant age-related reduction in ZNF91 expression in whole blood and brain, indicating a potential role of ZNF91 in the age-dependent onset of XDP. Conclusions These findings indicate that ZNF91 plays a crucial role in controlling the molecular phenotype associated with XDP. Since ZNF91 is a critical epigenetic repressor of SVAs, this suggests that epigenetic silencing of the XDP-SVA minimizes the severity of the molecular phenotype. Our results showing that ZNF91 expression levels significantly decrease with age provide a potential explanation for the age-related progressive neurodegenerative character of XDP. Collectively, our study provides important insights into the protective role of ZNF91 in XDP pathogenesis and suggests that modulating ZNF91 levels or targeted repression of the XDP-SVA could be novel therapeutic strategies worth exploring. ### Competing Interest Statement The authors have declared no competing interest. * ARH : Autosomal recessive hypercholesterolemia Cap-seq : Capture RNA-sequencing Cas9 : CRISPR-associated protein 9 cDNA : Complementary DNA ChIP-seq : Chromatin Immunoprecipitation Sequencing CRISPR : Clustered regularly interspaced short palindromic repeats DE : Differential expression dSVA : XDP-SVA excised DYT3 : X-linked dystonia-parkinsonism (Alternative name) ESC : Embryonic Stem Cell FACS : Fluorescence-activated cell sorting FCMD : Fukuyama-type congenital muscular dystrophy FGF : Fibroblast Growth Factor G4 : G-quadruplexes gDNA : Genomic DNA gRNA : Guide RNA hESCs : Human Embryonic Stem Cells iPSC : Induced pluripotent stem cell KAP1 : KRAB-associated protein 1 KO : Knockout KRAB : Krüppel-associated box KZNF : KRAB zinc finger L2FC : Log2 fold change NF1 : Neurofibromatosis type I OLS : Ordinary least squares padj : Adjusted P-value PBS : Phosphate-Buffered Saline PEI : Polyethylenimine Poly-A : Poly-adenosine RNA-seq : RNA-sequencing SINE : Short Interspersed Nuclear Element SVA : SINE-VNTR-Alu TAF1 : TATA-binding protein-associated factor-1 TE : Transposable Element TFIID : Transcription Factor II D VCM : Virus Containing Media VNTR : Variable Number Tandem Repeat WT : Wild type XDP : X-linked dystonia-parkinsonism XDP-SVA : XDP-specific SVA XLA : X-linked agammaglobulinemia ZNF91 : Zinc Finger Protein 91