PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington's disease

Abstract

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.

Keywords: DNA damage repair; Huntington's disease; PIAS; PNKP; SUMO.

Fig. 1.

Presymptomatic Pias1 KD regulates transcription in WT and HD mice at 13.5 mo of age. (A) PCA shows separation by both treatment and genotype. (B) Barplot showing number of DEGs per contrast at 8 and 13.5 mo with Pias1 KD. (C) Fold-change heatmap of 22 shared DEGs between age groups; of these, only Pde4a is a likely off-target DEG as determined by siSPOTR (70, 71) (SI Appendix, Table S1). (D) Fold-change heatmap of Pias1 KD modulated DEGs in WT and zQ175. (E) Heatmap (column min-max transformed log2 adjusted P values) and hierarchical clustering of top significantly enriched GO biological processes for WT Pias1 effect (WT miPias1.3 vs. miSafe), Het Pias1 effect (Het miPias1.3 vs. miSafe), overall Pias1 effect (279 genes shared between WT Pias1 effect and Het Pias1 effect), and disease DEG Pias1 effect (521 disease-associated DEGs modulated by miPias1.3 treatment). (F) Representative fold-change heatmap of GO process transsynaptic signaling shows inverse fold change of DEGs associated with treatment. (G) Bar chart from combined mRNAseq datasets showing the number and significance of rescued and exacerbated genes within disease-associated transcriptional modules originally identified in the allelic series transcriptional data (18). n = 3 animals per age per group.

Fig. 2.

Pias1 is part of the TCR complex and KD affects DNA damage repair in zQ175 mice. (A) PLA in SY5Y cells with PNKP, HTT, and PIAS1 antibodies. (B) Coimmunoprecipitation of the nuclear extract from SY5Y cells with HTT antibody. (C) mHTT-perturbed enzymatic activity of repair enzyme PNKP in the striatum is rescued with Pias1 KD in zQ175 mice (n = 3). (D) LA-qPCR of normalized transcriptional targets in males at 8 mo (n = 4 to 5/group) and (E) quantification of PCR products. Neurod1: treatment, F_{1, 15} = 25.110, P < 0.001, F_{1, 15} = 1.011, P > 0.05, Neurod2: treatment, F_{1, 15} = 29.000, P < 0.0001, genotype, F_{1, 15} = 35.460, P < 0.0001, Bdnf: treatment, F_{1, 14} = 19.970, P < 0.001, genotype, F_{1, 14} = 1.152, P > 0.05, Arc: treatment, F_{1, 15} = 17.89, P < 0.001, genotype, F_{1, 15} = 1.897, P > 0.05, Bcl2l2: treatment, F_{1, 15} = 7.636, P < 0.05, genotype, F_{1, 15} = 2.694, P < 0.05. Long amplicon (L) normalized to short amplicon (S). *P < 0.05, **P < 0.01, ****P < 0.0001, ns = not significant, values represent means ± SEM and individual values.

Fig. 3.

SUMO2 PNKP modification is mediated by PIAS1 in vitro. (A) In-cell, HeLa SUMOylation assay shows PNKP is SUMOylated by SUMO2. Black arrowheads indicate corresponding molecular weight shift of SUMOylated substrate by addition of SUMO moieties (orange boxes). SUMOylated PIAS1 serves as a positive control. (B) Significant KD (P < 0.0001) of PIAS1 with siRNA (siPIAS1) shows a reduction in SUMOylated PNKP by His-SUMO2 (blue box). Asterisk represents SUMOylated PIAS1 used for quantification (n = 4). (C) Under SUMO-limiting conditions (1/3 normal input, blue box), PIAS1 overexpression significantly increases PNKP SUMOylation by His-SUMO2 (n = 3, orange box). *P < 0.05, **P < 0.01, ****P < 0.0001, ns = not significant, values represent means ± SEM and individual values.

Fig. 4.

Neuronal KD of PIAS1 in iPSC derived neurons mRNAseq. (A) Barplot showing number of DEGs per contrast. (B) Venn diagram showing the DEGs with and without PIAS1 KD shows a majority overlap between control and HD as a result of PIAS1 KD. (C) Fold-change heatmap of the 2,228 shared DEGs between control and HD. The top 10 significantly enriched GO biological processes for (D) control samples generated from the PIAS1 KD DEGs and (E) for HD samples generated from the PIAS1 KD DEGs. (F) Hypergeometric analysis of the siPIAS1 vs. siLuciferase DEGs in control iPSC-derived neurons shows a significant overlap of genes in the M2 and M39 modules, but not M20 module. (G) Hypergeometric analysis of the siPIAS1 vs. siLuciferase DEGs in HD iPSC-derived neurons shows a significant overlap of DEGs with the M2 and M39 modules, but not M20 module. Modules are from ref. .

Fig. 5.

PIAS1 modulates endogenous PNKP enzymatic activity in iPSC-derived neurons and genomic DNA integrity of key genes is increased. (A) Nuclear PNKP activity assay from differentiated neurons (n = 3). (B) Genomic DNA integrity of key genes is increased with PIAS1 KD (n = 3). NEUROD1: treatment, F_{1, 8} = 78.640, P < 0.0001; genotype, F_{1, 8} = 8.246, P < 0.05; interaction, F_{1, 8} = 13.780, P < 0.01, BDNF: treatment, F_{1, 8} = 0.958, P > 0.05; genotype, F_{1, 8} = 3.854, P > 0.05; interaction, F_{1, 8} = 9.992, P < 0.05, BCL2L2: treatment, F_{1, 8} = 0.895, P > 0.05; genotype, F_{1, 8} = 23.700, P < 0.01; interaction, F_{1, 8} = 23.730, P < 0.01. (C) Integrity of mitochondrial DNA is increased with PIAS1 KD (n = 3): treatment, F_{1, 8} = 45.490, P < 0.001; genotype, F_{1, 8} = 2.963, P > 0.05; interaction, F_{1, 8} = 7.919, P < 0.05. (D) SUMO 2/3 coimmunoprecipitation shows endogenous unmodified PNKP and higher molecular weight PNKP immunoreactivity suggesting endogenous PNKP SUMOylation in iPSC-derived neurons (n = 2). KD of PIAS1 suggests reduction in high molecular weight PNKP signal. Endogenous SUMOylated PIAS1 served as a control for enrichment of SUMOylated proteins. γH2AX immunostaining suggests no increase in DNA damage levels with H₂O₂ treatment in these cells. Long amplicon (L) normalized to short amplicon (S). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant, values represent means ± SEM and individual values. β-Actin served as a loading control for whole cell lysate input samples.