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

Significance Genetic variants in genes involved in maintenance of genomic stability are modifiers of Huntington’s disease (HD) age of onset. This study shows a connection between the E3 SUMO ligase PIAS1, DNA damage repair protein PNKP, and HD-associated transcriptional dysregulation. Reduction of Pias1 in a knockin HD mouse striatum normalizes disease-associated aberrant transcription, rescues perturbed enzymatic activity of Pnkp, and increases genomic integrity, while also having transcriptional effects in WT animals. PIAS1 reduction in human iPSC neurons alters transcription of synaptic signaling and DNA damage repair, rescues PNKP activity, and increases genomic integrity in HD iPSC-derived neurons. Finally, PIAS1 can modulate SUMO modification of PNKP, which is the first identification of an enzyme that regulates this modification.


Supplementary Results
We evaluated open field, accelerating rotarod and body weight in zQ175 treated mice as described (1). In addition, a running wheel task (2) was used to assess motor deficits in presymptomatic Pias1 KD animals.

Symptomatic
Behavior: For open field task at 12.5 Mo, no significant differences were detected between groups for distance traveled or velocity (SI Appendix, Figure S2A). This data is consistent with the lack of a genotype effect in the presymptomatic cohort assessed with this task at 12.5 Mo and as observed previously for these animals (1). No differences were detected for males or females for percent of time in center separately but combined, animals showed a significant difference, suggesting that this cohort of zQ175 mice may have had increased anxiety (F2, 44 = 3.380, p<0.05, SI Appendix, Figure S2A). However, post-hoc analysis failed to reveal the source of significance.
To assess additional deficits in motor movements, accelerating Rotarod task was performed at 9.5 and 12.5 Mo (SI Appendix, Figure S2B). At 9.5 Mo, zQ175 animals had a significant decrease in latency to fall that was primarily observed in the females only as males alone did not show any significant difference in latency to fall between the groups (SI Appendix, Dataset S1). Pias1 KD had no effect on latency to fall at 9.5 Mo. At 12.5 Mo, no differences in latency to fall were detected for males and females separately, but combined showed a significant difference suggesting a slight decrease in latency to fall for zQ175 mice (Combined: F2, 43 = 3.576, p<0.05). However, post-hoc analysis failed to show significance between groups with combined animals further suggesting that miPias1.3 treatment did not affect behavioral readouts in zQ175 mice (SI Appendix, Figure S2B).
Body weight was also recorded for these animals and assessed separately due to the previously observed differences based on sex in these animals (1). Males did not show any differences in body weight at 12.5 Mo even though decreased body weight is typical in zQ175 mice at this age (1). For females, 1-way ANOVA suggested a significant genotype effect indicating decreased body weight (F2, 24 = 4.226, p<0.05) but with post-hoc analysis again failing to reveal significance (SI Appendix, Figure S2C). Symptomatic Pias1 KD did not affect body weights. Taken together, behavioral data suggest that symptomatic miPias1.3 treatment does not affect behavioral readouts in zQ175 mice.
A decline in mHTT protein levels was previously associated with disease progression in knockin HD mice (5). To determine if Pias1 KD affected this genotype-associated decline, soluble, fulllength HTT (FL HTT) protein was quantified using D7F7 antibody. While significantly lower levels of soluble FL HTT were observed in zQ175 mice, miPias1.3 treatment had no effect in either females (F2, 11 = 19.830, p<0.001) or males (F2, 9 = 12.060, p<0.01, SI Appendix, Figure S4C, F), suggesting that Pias1 is not modulating age-associated decline of mHTT protein (5). Taken together, targeting Pias1 symptomatically in the zQ175 mice did not affect behavioral deficits or accumulation of insoluble mHTT species.

Presymptomatic KD (13.5-month old sacrifice)
Open field task: To assess motor deficits and anxiety, the open field task was performed at 7.5 and 12.5 Mo and analyzed by Ethovision™ video tracking software. Deficits in total distance traveled were previously reported at 7.5 Mo for zQ175 heterozygous mice (1). At 7.5 Mo, both zQ175 males and females had significantly less distance traveled with slower velocity. This was retained analyzing both sexes combined with no effect of Pias1 KD treatment observed (SI Appendix, Dataset S1, Figure S3A). These data support a clear zQ175 motor deficit but miPias1.3 treatment had no effect on motor behavior in these animals at 7.5 Mo. No effects were observed for percent of time spent in center zone for any group indicating a lack of anxiety phenotype in these animals that was not affected by miPias1.3 treatment. At 12.5 Mo, zQ175 motor deficits were no longer observed and there was no miPias1.3 treatment effect (SI Appendix, Figure S3). No anxiety phenotype was observed for percent time spent in center zone. Loss of genotype effect is common in these animals at later ages (1) and overall Pias1 KD did not affect these behavioral readouts.
Rotarod: Rotarod detects genotype differences starting at 7.5 Mo in zQ175 mice (1). No overt miPias1.3 treatment effects were observed for either males or females in latency to fall in this study (SI Appendix, Figure S3C, Dataset S1). Specifically assessing latency to fall at 9.5 Mo both males and females showed a significant genotype effect that was retained when combined, indicating that zQ175 mice had a decreased latency to fall (SI Appendix, Figure S3C). No treatment effect was observed for either sex when analyzed separately or for males and females combined. Interestingly, at 12.5 Mo, male heterozygote animals with miPias1.3 treatment may have had a slight rescue in latency to fall as observed by a significant interaction detected by 2way ANOVA (Interaction, F1, 26 = 4.473, p<0.05). This effect was not observed in females and only the genotype effect was observed analyzing the combined group (Genotype: F1, 53 = 9.958, p<0.01, SI Appendix, Figure S3C), further suggesting that miPias1.3 treatment does not affect motor phenotypes in these animals.
Body weight: zQ175 heterozygous animals reliably show decreased body weight (1). Male zQ175 animals had significantly lower body weights than their WT counterparts with Pias1 KD not altering this genotype effect (SI Appendix, Dataset S1, Figure S3D). In female animals, only a decrease in weight was observed for miPias1.3 treated zQ175 animals, but with no significant treatment effect (Treatment: F1, 23 = 2.592, p>0.05). Overall, males showed a significant genotype effect while female zQ175 animals did not show a genotype effect but may have decreased body weight with Pias1 KD.
Running Wheel: The running wheel task allows for uninterrupted, voluntary assessment of motor function and motor learning (2). Therefore, males were assessed at 9 Mo for two weeks. Females were not analyzed due to the influence of estrus cycle on running wheel activity (2). Running wheel data trends suggest a promotion of motor deficits in mice treated with miPias1.3 in both WT and zQ175 mice compared to miSafe-treated controls, but with no significant treatment effect over time (Treatment: F3, 15 = 3.257, p>0.05, SI Appendix, Figure S3E). Overall, miPias1.3 treated zQ175 mice performed worse throughout the task and WT animals with miPias1.3 treatment showed a significant reduction in wheel-usage at days 3 and 5, suggesting a negative impact on this task with miPias1.3 treatment on WT and HD mice at this age (SI Appendix, Figure S3E).
The running wheel task can also be used to assess motor learning based on the slope of mean running between days 2 and 10 (2). Using this analysis, no differences in motor learning were observed between our groups (Genotype, F1, 15 = 0.510, p>0.05; Treatment, F1, 15 = 0.741, p>0.05, SI Appendix, Figure S3E). Overall, running wheel data may suggest an exacerbation in motor deficits in zQ175 animals and a decreased usage effect in WT animals with miPias1.3 presymptomatic treatment when assessed at 9 Mo, with no effect on motor learning.  Figure S5B, C), suggesting that presymptomatic KD of Pias1 did not impact age-associated decline of mHTT. In female animals, Pias1 protein was also not significantly reduced in either WT or zQ175 mice for either the Insoluble (F1, 10 = 4.464, p>0.05) or Soluble fractions (F1, 10 = 2.512, p>0.05, SI Appendix, Figure S5E, F), however, insoluble HMW mHTT showed a higher intensity in females with miPias1.3 treatment (p<0.05). No treatment effect was observed on levels of soluble, FL HTT (F1, 10 = 1.879, p>0.05) which was also reduced in zQ175 female mice (F1, 10 = 43.570, p<0.0001). Therefore, Pias1 KD appears to have a selective influence on the accumulation of insoluble, HMW mHTT in females only by 13.5 Mo.

Presymptomatic KD (8-month old sacrifice)
Behavior: For this group, only a significant genotype effect in percent time spent in the center was observed for the Open Field task at 7.5 Mo (Genotype: F1, 34 = 13.09, p<0.001, SI Appendix, Figure S6A). Bodyweight was recorded and assessed as in previous cohort until 8 Mo. No differences in weight gain were detected for either genotype or treatment by 8 Mo (SI Appendix, Figure S6B). However, at 8 Mo, zQ175 mice only exhibit minor deficits in body weight (1). It is possible that this cohort of animals had not begun showing detectable genotype effects with this analysis by time of sacrifice.
The running wheel apparatus has a finite number of animals that can be tested at a time due to single housing. Therefore, groups of male mice were of comparable size to the cohort assessed at 9 Mo and data was similarly processed (SI Appendix, Figure S6C). In contrast to mice tested at 9 Mo, which showed some additional impairment in this task with Pias1KD, at this earlier time point, KD increased running wheel usage. Specifically, a significant treatment effect (Treatment: F3, 12 = 3.526, p<0.05) was observed with WT animals treated with miPias1. 3 showing an apparent increase in usage of the running wheel over time compared to WT control treated animals. A similar trend was observed between zQ175 miSafe and miPias1.3 treated animals over time. No differences were detected in motor learning (Genotype: F1, 12 = 1.339, p>0.05; Treatment, F1, 12 = 4.663, p>0.05). Overall, data suggests that miPias1.3 treatment led to increased usage of the running wheel and increased motor capabilities in at least WT animals and potentially zQ175 animals at this time point (7 months, SI Appendix, Figure S6C). Therefore, miPias1.3 presymptomatic treatment may have differential effects on behavior during different stages of disease pathogenesis.
Levels of insoluble Pias1 were not elevated in untreated zQ175 animals at this age, similar to 13.5month old animals, supporting a different baseline from R6/2 mice in these animals. However, this reduction of Pias1 still did not affect formation of insoluble HMW mHTT accumulated species (p>0.05). A modest treatment effect on the reduced FL HTT in zQ175 mice (Genotype: F1, 10 = 31.990, p<0.0001) was observed with miPias1.3 treatment (Treatment: F1, 10 = 7.564, p<0.05).
Striata from female mice were also assessed (SI Appendix, Figure S7B). In the Soluble fraction, a significant treatment effect was observed (F1, 10 = 14.72, p<0.01) but post-hoc analysis failed to reveal source of significance due to a large amount of variability in zQ175 miPias1.3 treated samples. This variability inversely corresponded to levels of detected GFP, suggesting that lack of significant reduction was due to insufficient viral transduction (SI Appendix, Figure S7B).
Excluding zQ175 KD sample with the least amount of GFP, a significant reduction in Pias1 is observed in both WT and zQ175 treated animals (F1, 9 = 26.90, p<0.001). Insoluble Pias1 levels also showed a significant treatment effect with (F1, 10 = 9.124, p<0.05) and without (F1, 9  showed separation between genotype but not by treatment (SI Appendix, Figure S10A).
DEseq2 analysis to determine DEGs (6) revealed only 157 disease-associated DEGs between WT and zQ175 miSafe treated animals. 194 DEGs were observed in WT animals with miPias1.3 treatment, and 57 DEGs observed in zQ175 with miPias1.3 treatment (SI Appendix, Dataset S2, Figure S10B). Compared to male mice at this age, an overlap of only 3 shared DEGs was observed, with Pias1 being down-regulated in both (SI Appendix, Figure S10C). Further, only 4 DEGs were in common between genotypes in females at this age (SI Appendix, Figure S10D) suggesting differential impact of Pias1 KD between genotypes in female mice. Supporting this, GO enrichment revealed differential biological processes for WT compared to zQ175 animals with Pias1 KD (SI Appendix, Dataset S3, Figure S10E, F) as well as a significant negative concordance between males and females with miPias1.3 treatment from this cohort as seen by a scatter plot of Z scores calculated using the log2FC from each dataset (SI Appendix, Figure  S10G). However, comparison with previously published allelic series showed significant concordance, suggesting a consistent molecular genotype effect (SI Appendix, Figure S10H).

Generation of PIAS1 heterozygous null iPSC lines using CRISPR/CAS9 genome editing
Clones were generated by targeting exon 2 of the PIAS1 locus (SI Appendix, Figure S16A).
Several clones were validated for in-dels by genomic DNA sequencing and the specific mutation shown to be a deletion in the 33Q and an insertion in the 66Q iPSC clones generating two heterozygote PIAS1 KD lines (SI Appendix, Figure S16B, C); both produced shortened transcripts that resulted in significantly reduced levels of PIAS1 protein product as assessed by western blot in iPSCs (p<0.05, SI Appendix, Figure S16D, E). iPSC clones were assessed by immunofluorescence for pluripotency (SI Appendix, Figure S16F, G) and lines had normal karyotype (46XX-CgH array at Cell Linge Genetics, WI, USA). Next, the capacity to differentiate into neurons was assessed using a 37-day protocol to derive MSNs (SI Appendix, Figure S17A (7)). Cell lines showed comparable DARPP32 and BCL-11B co-staining, and MAP-2 staining at the end of differentiation indicating successful differentiation (SI Appendix, Figure S17B, C), and revealing that PIAS1 reduction does not impact differentiation into neurons.

Supplementary Discussion
Unexpectedly, Pias1 KD did not show significant or consistent effects or alter behavior in the zQ175 mice, unlike what we previously observed in the R6/2 model (4). This may be due to the subtle behavioral phenotype observed for zQ175 mice that can be challenging to measure and in fact even lost over time (8,9). Supporting this, studies have reported subtle or no behavioral alterations while still having robust molecular changes utilizing zQ175 mice as a model for HD (10,11).
We also do not observe robust changes in accumulation of HMW mHTT species in the presence of Pias1 KD as in R6/2 mice. However, there was a significant accumulation of insoluble Pias1 in R6/2 striatum, that we do not observe in zQ175 striatum (4). In R6/2 mice, miPias1.3 treatment specifically reduced insoluble Pias1 protein levels, in parallel with reduction of insoluble HMW mHTT (4), whereas in zQ175 mice, reduction of Pias1 is confined to the Soluble fraction.
Therefore, it is possible that zQ175 mice display a differential baseline of Pias1 activity and homeostasis reflected in its localization compared to the R6/2 mouse model. This difference may explain why insoluble Pias1 remained unchanged in zQ175 mice with KD and in part, may account for the differences in effects on behavior and HMW mHTT accumulation in this study. Future studies will investigate the functional contributions of insoluble versus soluble Pias1 on molecular pathways and whether the transition between fractions is a reflection of the pathogenic process that impacts early versus later neuropathology We observed differences based on sex in genes affected by Pias1 KD, and opposite effects of Pias1 KD on HMW mHTT accumulation in female mice at 13.5 Mo compared to males; these differences remain to be explored. A direct influence of estrogen on gene expression is possible for BDNF, ARC, and BCL2L2 (12)(13)(14) and BDNF contains a putative estrogen response element within its promoter (15). However, there is a limited amount of evidence for hormone transcription factor binding in human and mammalian brains (16) and while sex-specific fluctuations in gene expression may exist, recent transcriptomic profiling in multiple human brain regions including the caudate and putamen did not identify sex-specific expression bias for BDNF, ARC, NEUROD1, NEUROD2, or BCL2L2 (17). It is noteworthy that the vast majority of transcriptomic and behavioral studies in R6/2 mice have been carried out in males, and previous transcriptional profiling in zQ175 mice removed sex-associated variation (18). Of interest, transcriptional analysis of LNP-mediated PIAS1 KD predicted activation of β-estradiol and estrogen receptor 1 (ESR1) in control and HD iPSC-derived neurons ( Figure 4F), suggesting that PIAS1 may have a sexdependent function. This hypothesis is the basis for future work.

Supplemental Experimental Procedures zQ175 knockin mice
CAG repeat sizing of genomic DNA harvested from tails was performed by Laragen. Male and female littermates were subjected to bilateral intrastriatal injections to deliver treatment at either 2.5 (presymptomatic) or 7.5 (symptomatic) Mo. Littermates of the same sex were randomly assigned to experimental groups and were group housed, mixed genotype, for majority of experiments with the exception of male mice subjected to running wheel task which were singlehoused during and after completion of running wheel assessment. Experiments were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the NIH and an approved animal research protocol by the Institutional Animal Care and Use Committee (IACUC) at the University of California, Irvine. Animals were humanely euthanized by an over-dose injection of Euthasol followed by whole body perfusion with 1x-PBS and decapitation.

iPSC derivation and maintenance
HD and non-disease repeat iPSCs were generated and characterized as described (7,19 CO2, then passaged using Versene (Gibco) at a ratio of 1:3 to 1:6, depending on confluency.

HeLa and SH-SY5Y cell culture
HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with High glucose supplemented to 10% fetal bovine serum (FBS) at 37°C, 5% CO2 and passaged at 95% confluency. as previous (4). 5 µl of AAV2/1 virus containing miPias1.3 CMVeGFP or miSafe CMVeGFP at ~ 3e12 vg/ml was injected using a Hamilton syringe at a rate of 0.5 µl/min. Syringe was left in place for 5 minutes after each injection to ensure complete ejection of viral solution into brain region. Incisions were sutured and animals were allowed to fully recover on a heating pad then monitored daily for 1 week prior to beginning behavioral analysis.

Behavioral Paradigms
For zQ175 Pias1 presymptomatic KD cohorts, animal body weights were measured weekly over the course of the studies. Motor deficits were assessed by a running wheel (0297-0521-D60; Columbus Instruments) at 7 or 9 Mo depending on date of sacrifice. Mice had free access to running wheel usage that was constantly monitored over two weeks and collected in three-minute bins using Multi-Device System software 24-Channel Version 1.55 (160640; Columbus Instruments). Data from dark phase only was analyzed for significance over the two-week period.
Motor learning was calculated as previously described (2). Briefly, slopes were calculated using formula (Y2-Y1)/(X2-X1) where Y2 is wheel usage per bin on night 10 and X2 is 10 (night 10), and Y1 is wheel usage per bin on night 2 with X1 as 2.
The rotarod apparatus was used to measure fore and hind limb motor coordination and balance and mice were tested using an accelerating assay (Dual Species Economex Rota-Rod; 0207-003M; Columbus Instruments) at 9.5 and 12.5 Mo under blinded conditions. Animals were tested over two days with four trial runs each day consisting of 5-minute trial periods. Animals were acclimated to behavior room for 1 hour prior to testing. The first trial for each test day was used as a training trial and not used in final calculations. The latency to fall for the three final trials on day 2 of testing were averaged and analyzed for significance.
To assess anxiety and measure an additional motor task, animals were subjected to open field test at 7.5 Mo and 12.5 Mo when applicable. Animals were acclimated to behavior room for 1 hour prior to testing. Mice were allowed to explore an empty open field box for 5 minutes. Activity during these five minutes was recorded and analyzed using Ethovision™ Software that determines time spent in the center or on the periphery, average velocity and distance traveled over the 5 minute test period. Velocity and distance traveled during the open field trials were used as measures of motor performance while percent time spent in center was used as a measure of anxiety.

Soluble/Insoluble Fractionation tissue lysis for protein quantification and Western Blot analysis
Flash frozen brain tissue was prepared as previous for Soluble/Insoluble fractionation (3,4).

RNA purification, mRNAseq and qPCR Analysis
For zQ175 mice, GFP+ micro-dissected flash frozen brain regions were homogenized in TRIzol reagent (Invitrogen) and RNA was extracted according to manufacturer's protocol. RNA was then purified using RNEasy Mini kit (QIAGEN). Residual DNA was removed by DNase treatment incorporated into the RNEasy protocol as per manufacturer's suggestion prior to qPCR or submitting for mRNAseq as previously described (21). Potential off-target genes for miPias1.3 were determined using siSPOTR as previously described (22,23) with TPOTS score above 0.1 representing the potential for being real (SI Appendix, Table S1). For iPSC-derived neurons, RNA was extracted from day 37 cell pellets using Qiagen RNeasy kit and QIAshredders, 1 µg of RNA with Rin values >8 for each sample were utilized for library preparation and RNAseq as previously described (7). To confirm Pias1 knock-down in zQ175 mice, reverse transcription was performed using SuperScript 3 First-strand synthesis system according to manufacturer's protocol (Invitrogen, 18080085) from harvested and purified RNA. Both oligo (dT) and random hexamer primers were used in a 1:1 ratio. Final synthesized cDNA was diluted to 5ng/ul in DEPC treated water and stored at -20°C until use. For qPCR, technical triplicates for each individual animal were used to determine mean CT values. DeltaCT was calculated by subtracting mean CT of Eif4a2, a house-keeping gene which does not have a genotype effect in zQ175 mice (9), from mean CT of target gene per animal. Primers used for qPCR are listed in SI Appendix Table S2.

Differential expression analysis of combined data
mRNAseq datasets from this study were compared to previously published allelic series transcriptional profiles (18). Genes were first filtered for at least 1cpm in at least ¼ of the total number of samples. To make DE analysis more robust against potential outlier measurements (counts) that may remain even after outlier sample removal, we defined individual observation weights designed to downweigh potential outliers as described in (24) and (25). DE analysis was carried out using DESeq2 (6)
The radioactive bands were visualized in PhosphorImager (GE Healthcare, USA). The data were represented as % of the phosphate release (% product) with the total radiolabeled substrate as 100.

Genomic DNA purification and Long-Amplification qPCR
Integrity of target genes was assessed using Long Amp Taq DNA polymerase (NEB, M0323L) to amplify products of >6kB. To ensure reaction remained within the linear range of amplification, cycle number and DNA concentration were standardized before final quantification to ensure the product remained within the linear amplification range (31).
For mouse genomic material, optimized conditions for each target gene were as follows: Neurod1 Short PCR used 50 ng of the template from the same DNA aliquot for nuclear genes and 5 ng DNA for mitochondrial genes. Products were then analyzed using agarose gel electrophoresis followed by densitometry analysis on ImageJ software. Primers used for LA-qPCR can be found in SI Appendix Table S3.

Construction of plasmid expressing Myc-tagged PNKP
Plasmid DNA clone carrying human PNKP cDNA was purchased from the Origene Technologies, USA (RC207551L1). The PNKP cDNA was PCR amplified and sub-cloned into plasmid pcDNA3.1-Hygro (+) (Invitrogen, USA). Synthetic DNA linkers encoding MYC-tag sequences were introduced at the N-terminal of PNKP to construct the final plasmid pcDNA-Myc-PNKP.
The sequence integrity of the final recombinant plasmid was verified by sequencing, and appropriate expression of PNKP was verified by western blot and measuring PNKP activity.

Denaturing SUMOylation Assay
Denaturing SUMOylation assay was carried out as described with modifications (20

Quantification and Statistical Analysis
Animal tissue data analyzed by Li-Cor Odyssey CLx Imager on western blot was quantified using ImageStudio software to determine IR-fluorescent intensity values. Relative target signal was normalized to total protein revert intensity signal. ECL analyzed western blots were quantified using ImageJ densitometry analysis and normalized to detected levels of α-Tubulin on the same blot. SUMO-modified species of PNKP were quantified using ImageStudio software and normalizing to abundance of 5% input from the same western and multiplying by 100. All data represented as mean ± SEM with a p value of p<0.05 considered statistically significant. Analyses were completed in GraphPad Prism™ software. Statistical analysis was completed of the delta CT (dCT) values obtained from qPCR reactions normalized to Eif4a2. For animal studies, "n" represents individual animals used for each assessment. For cell culture studies, "n" represents experimental replicates. All 2-way ANOVAs were followed by Tukey's multiple comparisons test.
All 1-way ANOVAs were followed by Tukey's multiple comparisons test.

Data and Code Availability
The datasets generated during this study are available at GEO, accession number GSE162349.
Original/source data for allelic series mRNA-seq comparison in the paper is available online at 10.1038/nn.4256 and available through HDinHD (https://www.hdinhd.org).

Supplemental Figures and Legends Supplemental Figure S1
Supplemental Figure S1: Efficiency of viral transduction and HWM mHTT formation in zQ175 mouse striatum. A) Time-lines for presymptomatic and symptomatic injections of AAV2/1 miRNA, behavior, and sacrifice dates. B). Western blot indicating HMW Insoluble mHTT is detectable at 6 Mo, and increases at 10 Mo. C Insoluble HMW mHTT in both male and female zQ175 mice at 12 Mo is detectable by 5492 antibody, which was used for all subsequent assessments of this species and D) N-terminal recognizing antibodies only (n=3/sex). E) Mouse brain section showing GFP viral reporter is present 11 months post-injection, indicating continued presence of successful transduction (LV=Lateral Ventricle, STR=Striatum, CTX=Cortex) F) HeLa cell lysates with siRNA against PIAS1 and control siLuciferase shows specificity of Cell Signaling #3550 antibody against PIAS1 which was used for subsequent analysis (n=3). Protein normalized to total protein stain. HTT antibodies: 5526 (Abcam, ab109115), em38 (Millipore, MAB5374), 2166 (Millipore, MAB2166), D7F7 (Cell Signaling, 5656S), 5492 (Millipore MAB5492). Scale bar 500µm.

Supplemental Figure S2
Supplemental Figure S2: Symptomatic Pias1 KD in zQ175 mice has no effect on behavior. A) Open field task at 12.5 Mo for combined group shows no effect for distance traveled or velocity but with a significant difference in % center time by 1-Way ANOVA, with no post-hoc reaching significance (males n=6-7, females n=7-13). B) Rotarod of combined males and females at 9.5 Mo (males n=6-7, females n=7-12) and 12.5 Mo (males n=6-7, females n=7-12) shows significant genotype effects but no treatment effects. C) No differences were detected in body weight at 12.5 Mo (males n=6-7, females n=7-12). Open symbols represent female mice. All samples were analyzed by 1-way ANOVA followed by Tukey's multiple comparison test. Statistical outputs are detailed in Supplemental Table 1. ns=not significant, *p<0.05, **p<0.01, ***p<0.001, values represent means ± SEM and individual values. Shaded bars represent miPias1.3 treated animals.
Supplemental Figure S3: Presymptomatic Pias1 KD in zQ715 mice may exacerbate motor deficits. A) Open field task at 7.5 Mo for combined group shows significant genotype effect for distance traveled and velocity but not % center time (males n=7-8, females n=5-9). B) No differences were detected in open field task at 12.5 Mo (males n=7-8, females n=5-9). C) Rotarod of combined males and females at 9.5 Mo (males n=7-8/group, females n=5-9/group) and 12.5 Mo (males n=7-8/group, females n=5-9/group) shows significant genotype effects. D) Male mice show genotype effect in body weight, only observed for females treated with miPias1.3 (males n=7-8/group, females n=5-9/group). E) Running wheel task at 9 Mo in males (n=4-5/group) suggests a negative impact on motor tasks with miPias1.3 treatment but with no effect on motor learning. Genotype effects were observed for day 1 (F1, 15  Supplemental Figure S4: Symptomatic Pias1 KD does not affect mHTT accumulation at 13.5 months of age. A) qPCR analysis of mRNA harvested from male GFP+ microdissected striata shows significant reduction in levels of Pias1 transcript with miPias1.3 treatment (F2, 9 = 13.410, p<0.01). B) Western blot analysis of Soluble/Insoluble fractionation of striatal tissue from male mice and C) quantification shows no significant reduction of Pias1 protein in both fractions when detected with antibody validated against Pias1 (SI Appendix, Figure S1F). HMW insoluble mHTT detected with 5492 antibody remains unchanged with treatment. Soluble Full length (FL) HTT detected with D7F7 antibody shows a significant genotype effect with no treatment effect. D) qPCR analysis of mRNA harvested from female GFP+ microdissected striata shows significant reduction in levels of Pias1 transcript with miPias1.3 treatment as well as a significant genotype effect compared to WT vehicle control mice (F2, 8

Supplemental Figure S6
Supplemental Figure S6: Presymptomatic Pias1 KD in zQ175 mice may rescue early motor deficits. A) Open field task at 7.5 Mo for combined group shows no effect for distance traveled and velocity but a significant genotype effect for % center time (males n=4, females =5-7). B) No differences were detected in body weight at 8 Mo (males n=4, females =5-7). C) Running wheel task at 7 Mo in males suggests a significant increase in motor activity with miPias1.3 treatment in at least WT animals but with no effect on motor learning (n=4). Significant treatment effects were observed on day 1 (F1, 12   Supplemental Figure S11: Pias1 is part of the TCR complex but KD does not affect genomic stability in females or levels of Pnkp in vivo. A) Antigen controls for PLA in SH-SY5Y cells supports specific interaction between PNKP and HTT, HTT and PIAS1, and PNKP and PIAS1. B) LA-qPCR of normalized transcriptional targets in females (n=5/group) and C) quantification of PCR product. Neurod1: Treatment, F1, 16

Supplemental Figure S12
Supplemental Figure S12: PNKP may be a substrate for SUMOylation. A) SUMO is covalently linked to target substrates through a four-step enzymatic pathway: 1) SUMO processing by SUMO-specific proteases (SENPs) expose the diglycine motif, 2) activation of SUMO by E1 activating enzymes generates a thioester bond, 3) SUMO transfer onto E2 conjugating enzyme followed by 4) transfer to E3 ligase through transthioesterification reactions to conjugate SUMO moiety onto target substrate. B) Human PNKP is predicted to be SUMOylated at numerous lysine residues by Abcepta SUMOplot™. C) Several predicted PNKP SUMO consensus sites are highly conserved in mammals and lower vertebrates. Human lysines are listed with corresponding alignments from Clustal Omega™ output. Yellow highlights conserved residues (compared to human) if consensus site was predicted by SUMOplot™.

Supplemental Figure S13
Supplemental Figure S13: PNKP is SUMOylated and may be a substrate for STUbL activity in vitro. PNKP is SUMOylated by A) SUMO1 and B) SUMO2 as shown using in-cell SUMOylation assay in HeLa cell-lysates assessed under denaturing conditions. Black arrowheads indicate corresponding molecular weight shift of SUMOylated substrate by numerous SUMO moieties. SUMOylated PIAS1 serves as a positive control. High molecular weight laddering after denaturing pulldown suggests PNKP is modified beyond two SUMO moieties. Asterisk indicated high molecular weight laddering. n=3 per SUMO isoform. Overlay (yellow) shows immunoreactivity of modified species with both anti-PNKP (green) and anti-Myc (red) antibodies.