DOI: 10.5061/dryad.0k6djhb98
title: Data from: Suppression of Huntington’s disease somatic instability by transcriptional repression and direct CAG repeat binding

description: Huntington’s disease arises from a CAG expansion in the huntingtin gene
 beyond a critical threshold. Current therapeutics primarily aim to reduce
 toxicity by lowering levels of mutant HTT mRNA and protein. Genetic data
 support a role for somatic instability in HTT’s CAG repeat as a driver of
 age of motor dysfunction onset, but currently, the relationship between
 instability and HTT lowering remains unexplored. Here, we investigate
 various HTT-lowering modalities to establish the relationship between HTT
 lowering and instability in Huntington’s disease knock-in mice. We find
 that repressing transcription of mutant Htt reduces instability, using
 genetic and pharmacological approaches. Remarkably, zinc finger proteins
 that target CAG repeats, but lack a repressive domain, protect from
 somatic instability despite not reducing HTT mRNA or protein levels. These
 results suggest that DNA-targeted HTT-lowering treatments may have
 advantages compared to other HTT-lowering approaches, and that steric
 blockage of CAG repeats may reduce instability while sparing HTT
 expression.
description: All data was either collected directly by the Carroll lab at
 Western Washington University and University of Washington, or sent to us
 by collaborating labs where data was collected: Evotec SE, Hamburg,
 Germany; RNA Therapeutics Institute, University of Massachusetts Chan
 Medical School; Ionis Pharmaceuticals; CHDI Management, Inc.; Center for
 Genomic Medicine, Massachusetts General Hospital.  The
 data is in its raw form, with the exception of somatic instability data,
 which has been processed through an R script (available on Zenodo) to
 generate expansion indices for each mouse.
description: # Data from: Suppression of Huntington’s disease somatic instability by
 transcriptional repression and direct CAG repeat binding
 [https://doi.org/10.5061/dryad.0k6djhb98](https://doi.org/10.5061/dryad.0k6djhb98) ## Description of the data and file structure This data collections contains all data associated with the attached manuscript.  ### Files and variables #### File: Source_Data.xlsx **Description:** The main spreadsheet contains all the raw data from the associated manuscript. It is divided into pages, one for each figure. The page names start with the associated figure number.  ##### Variables 1b - PAF protein: Western blot data from our peripheral ASO follow-up study (PAF) * Blot: Denotes which gel the samples were run on * Genotype: The genotype of the associated mouse * Sample: The internal mouse ID, used to differentiate individual mice * Treatment: Denotes which experimental treatment the associated mouse received * TPS: Raw value of the total protein stain lane corresponding to the sample * Load_norm: Total protein stain value, normalized to a 0-1 scale * wtHTT_raw: Raw value of the experimental sample wildtype HTT band * mHTT_raw: Raw value of the experimental sample mutant HTT band * wtHTT_norm: Wildtype band raw value multiplied by the Load_norm value * mHTT_norm: Mutant band raw value multiplied by the Load_norm value 1c - PAF mRNA: qPCR data from our peripheral ASO follow-up study (PAF) * Sample: The internal mouse ID, used to differentiate individual mice * Genotype: The genotype of the associated mouse * Group: Used to differentiate different experimental groups that are plotted together * Age_at_sac_m: The mouse's age at sacrifice, in months * Treatment_duration_m: Length of treatment, in months * Treatment: Denotes which experimental treatment the associated mouse received * Reference: For qPCR data, denotes which housekeeping gene was used as the reference value * Target: For qPCR data, denotes which gene was used as the experimental target * Reference_CT: Average CT value of the reference gene * Target_CT: Average CT value of the target gene * dCT: delta CT, as calculated by the delta delta CT method * ddCT: delta delta CT, as calculated by the delta delta CT method * Fold_Increase: Fold increase compared to the control treatment, as calculated by the delta delta CT method 1e - PAF SI: Somatic instability data from our PAF study * Mouse: The internal mouse ID, used to differentiate individual mice * Threshold: Percentage of the tallest peak to exclude all values that fall below (noise reduction) * Expansion.index: Expansion index calculated by the attached script * Contraction.index: Contraction index calculated by the attached script * Genotype: The genotype of the associated mouse * Tissue: Tissue the data was gathered from * Group: Used to differentiate different experimental groups that are plotted together * Treatment: Denotes which experimental treatment the associated mouse received * Sex: Sex of the mouse * HTT silencing window: Ages at which mice mice were treated with HTT ASO * treatment_age_start_months: Mouse age at beginning of HTT ASO treatment * treatment_age_end_months: Mouse age at end of HTT ASO treatment * sac_age_months: Mouse age at sacrifice, in months * HTT_silencing_duration_mo: Length of ASO treatment, in months * exclude: Whether or not the data point was excluded - notes: Explanation for excluded data points 1f - PAF premRNA: Pre-mRNA qPCR data from our PAF study * Mouse: The internal mouse ID, used to differentiate individual mice * Genotype: The genotype of the associated mouse * Group: Used to differentiate different experimental groups that are plotted together * Age_at_sac_m: Mouse age at sacrifice, in months * Treatment_duration_m: Length of ASO treatment, in months * Treatment: Denotes which experimental treatment the associated mouse received * Exon: Denotes the exon/intron boundary targeted with qPCR probes * Average Tfrc: Average CT value of the reference gene * Average Target: Average CT value of the target gene * ddCT: Delta delta CT, as calculated by the delta delta CT method * Fold Increase: Fold increase compared to the control treatment, as calculated by the delta delta CT method * Normalized: Fold increase value normalized on a 0-1 scale for comparison with other groups 2c - LacO protein: Meso scale discovery (MSD) data from our *Lac* operon study * Sample: The sample number on the MSD plate * Mouse: The internal mouse ID, used to differentiate individual mice * IPTG: Length of isopropyl β-D-1-thiogalactopyranoside (IPTG) treatment * Well: The well each sample was run in on the MSD plate * Signal: Raw MSD signal for each well * Mean: Average MSD signal between technical replicates * CV: Coefficient of variation (CV) of MSD signal between technical replicates * Calc.conc.pg.ml: Calculated concentration in pg/mL, based on standard curve * pg.ml.mean: Average concentration between technical replicates * Calc.conc.CV: CV of the calculated concentration values between technical replicates * Detection Range: Denotes whether or not the signal is within the MSD detection range * Assay: MSD assay used, here either wildtype HTT (wtHTT) or mutant HTT (mHTT) 2d - LacO mRNA: Taqman qPCR data from our *Lac* operon study * Sample: The internal mouse ID, used to differentiate individual mice * Exon: Denotes which qPCR probeset was used * Genotype: The genotype of the associated mouse * IPTG: Length of IPTG treatment * Sex: Sex of the associated mouse * Fold.Increase: Fold increase compared to the control treatment, as calculated by the delta delta CT method 2e - LacO SI: Somatic instability data from our *Lac* operon study * Sample: The internal mouse ID, used to differentiate individual mice * Threshold: Percentage of the tallest peak to exclude all values that fall below (noise reduction) * Expansion.index: Expansion index calculated by the attached script * Contraction.index: Contraction index calculated by the attached script * Genotype: The genotype of the associated mouse * Tissue: The tissue used in the experiment * Experiment: Used to differentiate different experimental groups that are plotted together * Treatment type: Route of IPTG administration * IPTG: Length of IPTG treatment * Sex: Sex of the associated mouse * Sac.age.m: Age of the mouse at sacrifice, in months 3b - QPro2 MSD: MSD data from our CRISPR HTT knockout study * Mouse: The internal mouse ID, used to differentiate individual mice * Treatment: Denotes which experimental treatment the associated mouse received * Sample: The internal mouse ID, used to differentiate individual mice * Well: The well each sample was run in on the MSD plate * Signal: Raw MSD signal for each well * Mean: Average MSD signal between technical replicates * CV: Coefficient of variation (CV) of MSD signal between technical replicates * pg.ml.mean: Average concentration between technical replicates * calc.conc.CV: CV of the calculated concentration values between technical replicates * Detection Range: Denotes whether or not the signal is within the MSD detection range 3c - QPro2 SI: Somatic instability data from our CRISPR HTT knockout study * Mouse: The internal mouse ID, used to differentiate individual mice * Threshold: Percentage of the tallest peak to exclude all values that fall below (noise reduction) * Expansion.index: Expansion index calculated by the attached script * Contraction.index: Contraction index calculated by the attached script * Instability.index: Instability index calculated by the attached script * MinBP: Minimum basepair value used in calculation of the instability indices * MaxBP: Maximum basepair value used in calculation of the instability indices * BPrange: Range of basepair values used in the calculations, MaxBP - MinBP * CAGrange: Range of basepair values converted into number of CAG repeats (divided by 3 and rounded to the nearest integer) * Treatment: Denotes which experimental treatment the associated mouse received 4b - GalNAc MSD: MSD data from our Htt-targeting GalNAc-conjugated siRNA study * Sample: The sample number on the MSD plate * Mouse: The internal mouse ID, used to differentiate individual mice * Treatment: Denotes which experimental treatment the associated mouse received * Well: The well each sample was run in on the MSD plate * Signal: Raw MSD signal for each well * Mean: Average MSD signal between technical replicates * CV: Coefficient of variation (CV) of MSD signal between technical replicates * Calc.conc.pg.ml: Calculated concentration in pg/mL, based on standard curve * pg.ml.mean: Average concentration between technical replicates * Calc.conc.CV: CV of the calculated concentration values between technical replicates * Detection Range: Denotes whether or not the signal is within the MSD detection range * Replicate: Labels technical replicates * Assay: MSD assay used, here either wildtype HTT (wtHTT) or mutant HTT (mHTT) 4c - GalNAc steady-state: Taqman qPCR data from our Htt-targeting GalNAc-conjugated siRNA study * Mouse: The internal mouse ID, used to differentiate individual mice * Treatment: Denotes which experimental treatment the associated mouse received * Average Tfrc: Average CT value of the reference gene (Tfrc) * Average Htt: Average CT value of the target gene (Htt) * dCT: Delta CT, as calculated by the delta delta CT method * ddCT: Delta delta CT, as calculated by the delta delta CT method * Fold.Increase: Fold increase compared to the control treatment, as calculated by the delta delta CT method 4d - GalNAc pre-mRNA: Pre-mRNA qPCR data from our Htt-targeting GalNAc-conjugated siRNA study * Exon: Denotes the exon/intron boundary targeted with qPCR probes * Mouse: The internal mouse ID, used to differentiate individual mice * Treatment: Denotes which experimental treatment the associated mouse received * Average Tfrc: Average CT value of the reference gene (Tfrc) * Average Htt: Average CT value of the target gene (Htt) * dCT: Delta CT, as calculated by the delta delta CT method * ddCT: Delta delta CT, as calculated by the delta delta CT method * Fold.Increase: Fold increase compared to the control treatment, as calculated by the delta delta CT method 4e - GalNAc SI: Somatic instability data from our Htt-targeting GalNAc-conjugated siRNA study * Sample.Name: The internal mouse ID, used to differentiate individual mice * Threshold: Percentage of the tallest peak to exclude all values that fall below (noise reduction) * Expansion.index: Expansion index calculated by the attached script * Contraction.index: Contraction index calculated by the attached script * Instability.index: Instability index calculated by the attached script * MinBP: Minimum basepair value used in calculation of the instability indices * MaxBP: Maximum basepair value used in calculation of the instability indices * BPrange: Range of basepair values used in the calculations, MaxBP - MinBP * CAGrange: Range of basepair values converted into number of CAG repeats (divided by 3 and rounded to the nearest integer) * Treatment: Denotes which experimental treatment the associated mouse received 5b - Hemi null protein: Western blot data from our hemizygous HTT knockout study * Image Name: Denotes which gel the samples were run on * Name: Internal sample name, used to distinguish individual mice and mutant vs wildtype HTT bands * Signal: Real signal measured by ImageStudio, calculated by normalizing total signal to band area and background signal * Total: Total signal measured by ImageStudio * Area: Area of each band measured by ImageStudio * Bkgnd.: User-defined background signal measured by ImageStudio * Genotype: Genotype of the associated mouse * Sample: The internal mouse ID, used to differentiate individual mice * Allele: Denotes which HTT allele is measured, can be either wildtype (wtHTT) or mutant (mHTT) * Load_norm: The signal of the total protein stain, converted to a 0-1 scale * HTT_norm: The HTT signal multiplied by Load_norm to normalize for total protein amount * Age: Age of the associated mouse at sacrifice, in months * Tissue: The tissue used in the experiment 5c - Hemi null SI: Somatic instability data from our hemizygous HTT knockout study * Mouse ID: The internal mouse ID, used to differentiate individual mice * Sex: Sex of the associated mouse * Age: Age of the associated mouse at sacrifice, in months * Genotype: Genotype of the associated mouse * Modal CAG Ear at Weaning: Number of CAG repeats of the associated mouse, measured from weaning ear punch * Tissue: Tissue used in the experiment * 10% Expansion Index: Expansion index calculated by the attached script, with a 10% threshold applied (percentage of the tallest peak to exclude all values that fall below, for noise reduction) 6cd - ZFP HTT bDNA: QuantiGene Branched DNA (bDNA) data from our CAG-targeting ZFP study * zfp_treatment: Denotes which ZFP treatment was given to the associated mouse * htt_levels_bdna: Signal from the QuantiGene bDNA (branched DNA) assay targeting Htt * htt_I1pA1_levels: Signal from the QuantiGene bDNA (branched DNA) assay targeting Htt1a 6f - ZFP SI: Somatic instability data from our CAG-targeting ZFP study * expansion_index: Expansion index calculated by the attached script * zfp_construct: Denotes which ZFP treatment was given to the associated mouse 6g - Click-it: Data from an Invitrogen Click-iT assay on cells treated with our CAG-targeting ZFP * Treatment: Denotes which experimental treatment the associated mouse received * Value: qPCR signal * Assay_full_name: Denotes the exon/intron boundary targeted with qPCR primers * Assay: Shorthand name of the qPCR primer set 6h - ZFP MSD in vitro: Protein data generated by MSD from cells treated with our CAG-targeting ZFP * MOI: Multiplicity of infection: Viral dose that the cells were treated with * Target: The variable being measured * Treatment: ZFP construct given to the cells * Value: MSD data value S2 - ActD pre-mRNA: Pre-mRNA qPCR study from our supplemental Actinomycin D (ActD) study * Sample: The internal mouse ID, used to differentiate individual mice * Exon: Denotes the exon/intron boundary targeted with qPCR probes * Treatment: Denotes which experimental treatment the associated mouse received * Average Tfrc: Average CT value of the reference gene (Tfrc) * Average Gene: Average CT value of the reference gene (Htt) * dCT: Delta CT, as calculated by the delta delta CT method * ddCT: Delta delta CT, as calculated by the delta delta CT method * Fold.Increase: Fold increase compared to the control treatment, as calculated by the delta delta CT method S4c - ZFP off-target: QuantiGene Branched DNA (bDNA) panel for predicted off-target genes from our CAG-targeting ZFP study * Group: Internal sample group number for QuantiGene bDNA runs * Sample Number: The internal mouse ID, used to differentiate individual mice * Side: Denotes which side of the brain the sample came from * Luminex ID Number: Internal ID for the Luminex assay * Stk32c - Pum1: These columns are in wide format; each column denotes a QuantiGene bDNA probeset. The values denote bDNA signal.  * Treatment: Denotes which experimental treatment the associated mouse received Unpublished - ASO timecourse * Exon: Denotes the exon/intron boundary targeted with qPCR probes * Mouse: The internal mouse ID, used to differentiate individual mice * Treatment: Denotes which experimental treatment the associated mouse received * Doses: Number of doses of ASO the mouse received, at intervals of one dose per week * Average Tfrc: Average CT value of the reference gene (Tfrc) * Average Htt: Average CT value of the target gene (Htt) * dCT: Delta CT, as calculated by the delta delta CT method * ddCT: Delta delta CT, as calculated by the delta delta CT method * Fold.Increase: Fold increase compared to the control treatment, as calculated by the delta delta CT method #### File: Inherited_CAG_sizes.xlsx **Description:** This spreadsheet contains CAG sizes from mice included in experiments in the associated manuscript. It is divided into pages, one for each group of mice. ##### Variables * Mouse: Mouse ID * Genotype: Genotype of the mouse * CAGs: The CAG size of the mouse #### File: Statistics.xlsx **Description:** The main spreadsheet contains all the statistical results used in the associated manuscript. This includes one-way and factorial ANOVAs, T-tests, Tukey's HSD tests, and mean and percent change calculations. The type of test and figure it corresponds to is denoted in the page name. The first page contains a clickable table of contents to help with navigation.  ##### Variables AVOVAs: * Df: Degrees of freedom * Sum.Sq: Sum of squares * Mean.Sq: Mean squares * F.value: F-value * Pr..F.: p-value Tukey's HSD tests:  * diff: Mean difference * lwr: Lower confidence interval * upr: Upper confidence interval * p.adj: p-value T-tests:  * Dependent Variable:  * t: t-value * df: Degrees of freedom * p: p-value * d: Cohen's d * CI_lower: lower confidence interval * CI_upper: upper confidence interval #### File: Raw_blot_fig_5a.pdf * This page contains the raw Western blot image and associated information used in figure 5a.  **References, funding sources, and detailed method information can be found in the associated manuscript.**  ## Code/software The only required software is R. Code was written on R version 4.3.2.  The included scripts are labeled with the associated figure names. These scripts were used to generate the figures in the manuscript.  There are also two scripts marked "Exemplar SI Script". They contain the code used to calculate somatic instability expansion indices.  ## Access information Other publicly accessible locations of the data: * N/A Data was derived from the following sources: * N/A ## Version Changes **12-sep-2025**: Added raw Western blot from figure 5a. Added unpublished data used in reviewer communications as requested by the journal. Uploaded raw blot data file as a separate pdf.