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DNA Methylation Leads to DNA Repair Gene Down-Regulation and Trinucleotide Repeat Expansion in Patient-Derived Huntington Disease Cells
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DNA Methylation Leads to DNA Repair Gene Down-Regulation and Trinucleotide Repeat Expansion in Patient-Derived Huntington Disease Cells Q22
Peter A. Mollica,*y John A. Reid,z Roy C. Ogle,y Patrick C. Sachs,y and Robert D. Brunoy From the Departments of Biological Sciences,* Engineering and Technology,z and Medical Diagnostics and Translational Sciences,y Old Dominion University, Norfolk, Virginia Accepted for publication March 16, 2016.
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Address correspondence to Robert D. Bruno or Patrick C. Sachs. E-mail: rbruno@odu. edu or [email protected].
Huntington disease (HD) is an autosomal dominantly inherited disease that exhibits genetic anticipation of affected progeny due to expansions of a trinucleotide repeat (TNR) region within the HTT gene. DNA repair machinery is a known effector of TNR instability; however, the specific defects in HD cells that lead to TNR expansion are unknown. We hypothesized that HD cells would be deficient in DNA repair gene expression. To test this hypothesis, we analyzed expression of select DNA repair genes involved in mismatch/loop-out repair (APEX1, BRCA1, RPA1, and RPA3) in patient-derived HD cells and found each was consistently down-regulated relative to wild-type samples taken from unaffected individuals in the same family. Rescue of DNA repair gene expression by 5-azacytidine treatment identified DNA methylation as a mediator of DNA repair gene expression deficiency. Bisulfite sequencing confirmed hypermethylation of the APEX1 promoter region in HD cells relative to control, as well as 5-azacytidineeinduced hypomethylation. 5-Azacytidine treatments also resulted in stabilization of TNR expansion within the mutant HTT allele during long-term culture of HD cells. Our findings indicate that DNA methylation leads to DNA repair down-regulation and TNR instability in mitotically active HD cells and offer a proof of principle that epigenetic interventions can curb TNR expansions. (Am J Pathol 2016, -: 1e10; http://dx.doi.org/10.1016/j.ajpath.2016.03.014)
Huntington disease (HD) is a neurodegenerative disease that affects medium spiny neurons in the basal ganglia, resulting in involuntary muscle movements and ultimately leading to death. It is caused by an expansion of a cytosine-adenineguanine (CAG) trinucleotide repeat (TNR) region in exon 1 of the HTT gene.1 HD is a dynamic mutation disease in which the number of TNRs increases the probability of expression of the mutant phenotype. The disease does not affect individuals with <35 TNRs, but individuals with 35 to 40 repeats are at an increased risk of developing HD. Expansions >40 repeats inevitably lead to HD progression.2 Although the mechanism is not fully understood, HDaffected individuals exhibit genetic anticipation caused by subsequent generations of HD-affected individuals developing expanded versions of the mutated allele.3e5 Increased expansions in successive generations correlate with more severe symptomatic expression and earlier age at onset in 70% of individuals.6,7 Interestingly, postmortem analysis of
affected neuronal cells and other somatic tissues in HD mouse models reveals disease-causing gene CAG mosaicism, in which CAG repeats of affected alleles in different tissues have different sized expansions.8e11 In addition, a myriad of TNR instability effectors have been identified, such as environmental stresses, chemical inducers, and DNA repair gene expression.12e15 This evidence infers that TNR instability is present not only in germline cells but also throughout development and maturation and thus dependent on efficacy of DNA repair and its ability to appropriately respond to endogenous and exogenous DNA-damaging agents and modifiers. Furthermore, emerging data have recently implicated TNR instability in replicating neuronal Supported by institutional start-up support from the College of Health Q1 Sciences at Old Dominion University. Q2 P.C.S. and R.D.B. contributed equally as senior authors. Disclosures: None declared.
Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2016.03.014
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Mollica et al support cells as possible mediators of disease severity and progression.16e18 Thus, deciphering how TNR expansions occur during development and in proliferating cell populations during development and adulthood is vital to our understanding of the pathogenesis of HD. These data would lead to therapeutic strategies aimed at preventing TNR expansions and delaying the onset and severity of HD symptoms in patients inheriting a mutated HTT allele. Unfortunately, the comprehensive mechanism of TNR instability eludes researchers. Of the proposed mechanisms, heteroduplex formation and the inability for successful resolution during replication are the common factors behind TNR instability.19 Through intrastand complementary base pairing, single-stranded DNA generates secondary structures, such as hairpins or loop-outs.20 Although this secondary structure meets the criteria for DNA damage, the lesion is ignored in HD cells, and the expanded allele generally results in the dissemination of a larger CAG repeat region to daughter cells. The inability of DNA repair machinery to stabilize CAG repeats in HD cells led us to hypothesize that HD somatic tissues have abnormal expression of DNA repair genes, sensitizing them to CAG TNR expansions. To test this hypothesis, we analyzed DNA repair gene expression in fibroblasts from HD patients and wild-type controls. Because TNR instability is seen in all tissue types, we chose to look at fibroblasts because they are mitotically active and thus would better represent TNR expansions occurring during development. We determined that key DNA repair genes were down-regulated in HD fibroblasts compared with wild-type controls and that the promoter region of the down-regulated repair gene, APEX1, was hypermethylated compared with controls. Treatment with the DNA methyltransferase inhibitor 5-azacytidine resulted in increased DNA repair expression and marked stabilization of CAG repeats during long-term culture. This is the first study to identify endogenously deficient DNA repair genes in human HD fibroblasts and is proof of principle that pharmacologic intervention can lead to increased TNR stability in patients at risk for the disease. Table 1
Materials and Methods Cell Lines and Cell Culture Three previously confirmed HD (GM03621, GM02079, Q5 and GM03868), two wild-type (GM02187 and GM02153), and two unknown (GM02191 and GM04022) fibroblast cell lines were obtained from Coriell Cell Repositories (Camden, NJ). Unknown fibroblast samples were obtained from donors of whom one parent was confirmed to be affected by HD but remained undiagnosed. Genetic characteristics of exon 1 of the HTT gene of each cell line used in our experiments are detailed in Table 1. Cell lines were ½T1 cultured and maintained at 37 C in a humidified incubator with 5% CO2. Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium with glutamax (Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (Life Technologies) as recommended by Coriell Cell Repositories. Cell media also contained 1.0% antibioticantimycotic (100) (Life Technologies). Cells were seeded in T75-cm2 flasks at 5.0 104 cells, and medium was replenished between 72 and 96 hours of seeding. Cells were passaged every 5 to 6 days using TrypLE Express (Life Technologies) following manufacturer’s instructions.
RNA and Genomic DNA Isolation Total RNA was isolated from cells on growth to 70% to 80% confluency (approximately 1 million cells) with Trizol (Thermo Fisher, Waltham, MA) following the manufacturer’s protocol. Approximately 1.0 106 cells were harvested, and genomic DNA (gDNA) was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Samples were concentrated by ethanol precipitation when needed. RNA and DNA quantity and purity were assessed by the absorbance at 260/280 nm method using a NanoDrop 2000 (Thermo Fisher), with 260/280 ratios of approximately 2.0 and 260/230 ratios slightly higher than 1.8.
Genetic Characteristics of Exon 1 of the HTT Gene of Each Cell Line Used in This Study
Cell line
Experiment designation
Age, years
Reported status
Reported CAGn
Confirmed status
Confirmed CAGn
GM02187 GM02153 GM02079 GM03621 GM03868 GM04022 GM02191
Wt-1 Wt-2 HD-1 HD-2 HD-3 pHD-1 pHD-2
60 40 48 29 45 28 32
Wild type Wild type HD HD HD Unknown Unknown
17e23 16e32 21e44 18e60 18e44 e e
Wild type Wild type HD HD HD HD HD
17e23 16e32 21e45 18e60 18e48 18e44 17e42
HD-affected and wild-type fibroblast samples are all female. All cell lines were purchased from the Coriell Cell Repositories. The status of fibroblast lines GM02191 and GM04022 were designated unknown on ordering and initial experiments because they had not yet been characterized and were subsequently confirmed to be HD-affected through capillary electrophoresis fragment analysis. Experimental designation is the abbreviation used throughout the article. The CAG expansions shown above represent the characterizations previously published by Coriell Cell Repositories, with the exception of our unknown samples (GM04022 and GM02191) in which the CAG is the first characterization by fragment analysis completed. CAG, cytosine-adenine-guanine; HD, Huntington disease; pHD, potential Huntington disease; Wt, wild type.
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DNA Repair and TNR Stability in HD
5-Azacytidine and Trichostatin-A Treatment For gene and protein expression assays, cell medium was supplemented with 5-azacytidine diluted in phosphate buffer solution to achieve 10-mmol/L concentrations for 120 hours. The 10-mmol/L dosage was the predetermined 50% lethal concentration of 5-azacytidine (data not shown). For TNR stability assays, cells were treated intermittently with 10 mmol/L 5-azacytidine during 35 days to reach four population doublings. Treatment schedule was as follows: pass in normal medium for 24 hours, treat with 10 mmol/L 5-azacytidine for 5 days, allow cells to recover for 3 to 5 days in normal medium, repeat with passaging at a 1:2 split ratio. During 5-azacytidne treatment days, media was replaced daily with freshly prepared 5-azacytidine to optimize passive demethylation during DNA synthesis and to facilitate the removal of toxic products. gDNA samples were collected at the beginning of each treatment schedule and at the end of the designated time frame. For histone-deacetylase inhibition treatment, complete fibroblast medium was supplemented with 0.5 mmol/L and 5.0 mmol/L trichostatin-A (TSA) (Sigma-Aldrich, St. Louis, MO) reconstituted in dimethyl sulfoxide to achieve 0.01% v/v. TSA that contained media was applied 16 hours after passaging cells and allowed to remain for 24 hours. On removal of TSA, total RNA was immediately extracted.
Quantitative RT-PCR One microgram of each RNA sample was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. We used TaqMan Gene Expression Assays (Applied Biosystems) to assess mRNA levels. TaqMan Gene Expression Assays provided optimized primers and probes specific for DNA repair genes APEX1 (Hs00959050_g1), BRCA1 (Hs01556193_m1), RPA1 (Hs00161419_m1), and RPA3 (Hs01047933_g1) and the endogenous housekeeping gene ACTB (Hs99999903_m1). Quantitative RT-PCR (RTqPCR) experiments were conducted with a StepOnePlus Real-Time PCR System (Applied Biosystems) using TaqMan Fast Advanced Master Mix (Life Technologies) using 50 ng of cDNA from each sample per the manufacturer’s protocol. All experiment reactions were independently completed in triplicate. Negative template controls were run on each experiment containing nuclease-free water. All cell lines were tested three independent times, with at least two population doubling events occurring between sample experiments. Each experimental sample was also subsampled three times during RT-qPCR reactions. Relative fold-changes were calculated using the 2DDCt method. Significance was determined by comparing the 2DCt value using a one-way analysis of variance with a Dunnett’s Post Hoc Test.
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311 312 313 Cell samples were seeded onto Lab-Tek microscope 3 314 chamber slides at a density of 1.5 10 cells/mL and fixed 315 with 10% formalin for 30 minutes at room temperature. 316 Samples were blocked with 10% goat serum for 60 minutes 317 at room temperature and then incubated with primary anti318 body overnight at 4 C in a humidified chamber. Primary 319 antibodies were obtained from ABCAM (Cambridge, MA): 320 APEX1 antibody (ab105081; 1:700), BRCA1 antibody 321 (ab16781; 1:250), RPA1 antibody (ab79398; 1:250), and 322 323 RPA3 antibody (ab6432; 1:50). Samples were then incu324 bated with Alexa Fluor 488 conjugated secondary anti325 rabbit or anti-mouse antibodies (Thermo Fisher, 1:1000) 326 for 60 minutes. Slides were counterstained with DAPI. 327 Fluorescent images were acquired with a Zeiss Axio 328 Observer.Z1 microscope (Carl Zeiss AG, Jena, Germany) at Q6 329 10 objective. Images were collected in 16-bit black and 330 white to maximize resolution. False coloring was added by 331 ImageJ software version x.xxx (NIH, Bethesda, MD; http:// Q7 332 imagej.nih.gov/ij). At least three images of each gene and 333 cell line were collected to ensure global capture of the 334 335 population of cells. Relative quantitation was conducted using ImageJ software version x.xxx (NIH, Bethesda, MD; Q8 336 337 http://imagej.nih.gov/ij). Cell fluorescence was determined 338 by multiplying the total area of the image by mean back339 ground of the image, then subtracted from integrated den340 sity. The cell fluorescence of each image was then divided 341 by the number of complete nuclei in the image to give the 342 corrected total cell fluorescence (CTCF). Mean CTCF 343 is represented graphically as normalized values to the 344 mean CTCF of wild-type fibroblast sample. For statistical 345 analysis, relative fold-changes were compared using the 346 347 Wilcoxon rank sum test. 348 349 Triplet Repeat Primed PCR 350 351 Primers were synthesized by Integrated DNA Technologies 352 (Coralville, IA). The forward primer sequence was 50 -ATG353 AAGGCCTTCGAGTCCCTCAAGTC-30 , with a carboxy354 fluorescein at its 50 end, and the reverse was 50 -CGGTGG355 CGGCTGTTGCTGCTGCTGCTGCTG-30 .21 Amplification 356 357 reactions were run in 20mL volumes and contained the 358 following components; 1 FailSafe Premix J (Epicenter, 359 Madison, WI), 1 U of Platinum Taq DNA Polymerase High 360 Fidelity (Life Technologies), 1.0 mL of 20 to 25 ng/mL of 361 gDNA sample, and 0.5 mmol/L each primer. PCR conditions 362 were as follows: 95.0 C for 1 minute hot start/initial dena363 turation, followed by 35 cycles of 94.0 C for 1 minute, 364 64.0 C for 1 minute, 72.0 C for 2 minutes, and a final 365 extension of 15 minutes at 72.0 C. One microliter of each 366 sample was combined with 1.0 mL of Mapmarker 1000 367 internal size standard (BioVentures Inc., Murfreesboro, 368 369 TN) and 9.0 mL of Hi-Di Formamide (Life Technologies). 370 Just before sample loading of 10 mL onto capillary elec371 trophoresis plates, each sample was heated to 95.0 C for 372
Immunofluorescence of DNA Repair Genes
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2 minutes for denaturation and then immediately placed on ice for 5 minutes. Capillary electrophoresis fragment analysis was conducted on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems) with 36-cm arrays and POP-7 Polymer-CG (Applied Biosystems). Electrokinetic injection of samples was initiated with 12 kV for 16 seconds and then electrophoresed at 15 kV for 1200 seconds at 60 C. Raw data were analyzed using GeneMarker software version 2.6.4 (SoftGenetics LLC, State College, PA).21 We conducted amplified fragment length polymorphism analysis using optimized macros for TNR expansion calling and labeling. Macros for bin calling were customized using gDNA samples obtained from Coriell Cell Repositories and were graciously created by Mohamed Jama (University of Utah). As positive controls, we obtained 6-g DNA samples from various HD-affected and wild-type characterizations. These chosen positive control gDNA samples had previously been tested in multiple laboratories and were sequenced to confirm CAG repeats (data not shown).
Bisulfite Conversion and meCpG Analysis
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The gDNA samples were treated to bisulfite conversion using the EpiTect Fast Bisulfite Kit (Qiagen) according to the manufacturer’s protocol. Primers were designed to evaluate methylation patterns approximately 500 bp upstream of APEX1 using MethPrimer software version x.xx.22 The region was amplified in two segments: forward: 50 -ATAATGTGTTGTGTATTTGGTATAA-30 , reverse: 50 -ATAACAACCTAACTCCTCCTAACCC-30 ; forward: 50 -TGTTAGGAGTTGTGGAGGTTTTTAT-30 , reverse: 50 -TTCTCTAAAACACTTAAATCCCCAA-30 . PCR amplification of 500 ng of bisulfite-treated gDNA was conducted using 1 U of Platinum Taq DNA Polymerase High Fidelity (Life Technologies) as previously described. We used a touchup PCR cycling protocol where the annealing temperature was gradually increased after the first and second set of five cycles, with annealing temperatures increasing from 53 C to 56 C and then finally 60 C for the remaining 30 cycles. PCR cycling conditions were as follows: 95 C for 1-minute hot start, followed by a touch-up protocol with increasing annealing temperatures for 1 minute, and 72 C for 1 minute for 40 total cycles. A final extension of 10 minutes at 72 C was applied at the end. Correct amplicons were assessed using gel electrophoresis on a 1.5% agarose gel. Appropriate bands were extracted using QIAquick Gel Extraction Kit (Qiagen). Each extracted amplicon was then inserted into the pCR4-TOPO TA vector with the TOPO TA cloning kit for sequencing (Thermo Fisher) per the manufacturer’s protocol. Cycle sequencing of fragments was conducted using the M13 forward and M13 reverse primer sets provided in the TOPO TA kit. Methylation analysis and representation were conducted using Methylation Plotter software version x.xx online.23
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Results APEX1, BRCA1, RPA1, and RPA3 Gene Expression Is Deficient in Both HD and Unknown Fibroblasts To determine whether DNA repair gene expression was perturbed in proliferating HD cells, we performed RT-qPCR to detect relative mRNA expression level of four DNA repair genes (APEX1, BRCA1, RPA1, and RPA3) in HD samples. We compared fibroblasts isolated from three confirmed HD patients (GM02079, GM03621, GM03868), two wild-type patients (GM02187 and GM02153), and two unknown patients (GM02079 and GM04022) who had a 50% chance of inheriting the mutant HTT allele. Heretofore, confirmed HD cell lines are designated as HD-n, unknowns are designated as potential HD pHD-n, and wild-type samples designated Wt-n (Table 1). We chose to examine APEX1, BRCA1, RPA1, and RPA3 because they are effectors of TNR instabilityeinduced DNA damage and because we had previously observed their down-regulation in HD cells (unpublished data).24e29 Our analysis revealed statistically significant down-regulation of all four genes in all HD-affected and unknown fibroblast lines (Figure 1). The ½F1 mRNA levels presented are in relation to control sample Wt-1 because this sample had lower overall expression of the four DNA repair genes when compared with the Wt-2 sample and thus represents a more stringent comparison.
Unknown Samples Are Affected by mHTT Expansion Because both of our unknown samples (pHD-1 and pHD-2) had significant down-regulation of all four DNA repair genes, we sought to identify if they did, in fact, have an expanded HTT allele. To determine HD status of unknown fibroblast lines, we conducted capillary electrophoresis fragment analysis using triplet repeat primed PCR on all samples (Table 1 and Supplemental Figure S1). Both unknown fibroblast lines pHD-1 and pHD-2 were confirmed HD, with alleles that contained CAG repeats of 18/44 and 17/42, respectively (Supplemental Figure S1, A and B). Combined with data identifying APEX1, BRCA1, RPA1, and RPA3 down-regulation, this confirmation of unknown samples being affected by HD identifies therapeutic biomarkers, as well as a possible alternate means for HD diagnosis. Interestingly, we witnessed TNR instability in previously characterized HD samples HD-1 and HD-3, whereas HD-2 Q13 remained static (Supplemental Figure S1C, Supplemental Figure S2, A and B, and Table 1). Previously, Coriell Cell Repositories reported HTT gene characterization of HD-1 as containing 21/44 CAG repeats and HD-3 as containing 18/44 CAG repeats. Our initial characterization was con- Q14 ducted after 10 population doublings and revealed HTT expansions in the mutated allele to 45 and 48, respectively. Interestingly, HD-3 had a contraction in the unaffected allele from previously reported 18 to 17. No instability was
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DNA Repair and TNR Stability in HD
detected in either wild-types samples (Wt-1 or Wt-2) after identical population doublings (Supplemental Figure S2C, Supplemental Figure S3, and Table 1).
APEX1, BRCA1, RPA1, and RPA3 Protein Levels Are Down-Regulated in HD Fibroblasts To determine whether protein levels of DNA repair genes matched our findings from our mRNA analysis, we conducted immunocytochemistry of all fibroblast lines ½F2 (Figure 2A). CTCF revealed that protein levels in pHD-1 and pHD-2 samples were approximately 23% of APEX1, 22% of BRCA1, 8% of RPA1, and 32% of RPA3 (Figure 2B) and 54% of APEX1, 35% of BRCA1, 46% of RPA1, and 36% of RPA3 (Figure 2C), respectively. Protein levels are relative to the lowest-expressing wild-type fibroblast sample (Wt-1). These data indicate that DNA repair components are significantly decreased in HD fibroblast samples, possibly having consequences on DNA repair pathways to efficiently address lesions and adducts generated during DNA synthesis.
APEX1, BRCA1, and RPA1 Gene Expression Is Affected by 5-Azacytidine Treatments We next wanted to identify the potential mechanism of the observed DNA repair gene down-regulation. Because no previous DNA repair gene mutational events have been associated with HD, we hypothesized that epigenetic regulators could be a primary cause of the observed downregulations. To test for the involvement of histone acetylation and DNA condensation as a primary regulator, we treated pHD-1 and pHD-2 samples with 5 mmol/L TSA, a potent global deacetylase inhibitor, for 24 hours. RT-qPCR revealed no significant changes in DNA repair gene expres½F3 sion in either pHD-1 (Figure 3A) or pHD-2 (Figure 3B) in response to TSA treatments, suggesting hyperacetylation of histones was not primarily responsible for the suppression of DNA repair genes.
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559 560 561 Figure 1 DNA repair deficiencies in multiple Huntington disease (HD) fibroblast cell samples. APEX1, BRCA1, RPA1, and 562 RPA3 expression in wild-type (Wt-1), potential HD-affected 563 (pHD), and HD-affected (HD) fibroblasts. Using TaqMan Gene 564 Expression Assays, we identified HD DNA repair mRNA down565 regulation of APEX1, BRCA1, RPA1, and RPA3. Gene expres566 DDCt sion is determined by the (2 ) of the mean of each 567 independent triplicate. Error bars represent the SD of the 568 mean between replicate samples. Each tested gene shows 569 significant decreases in expression for all HD and pHD cell 570 lines, relative to the Wt sample. Gene expression is normalized 571 to the Wt-1 fibroblast line GM02187 gene expression. Statistical significance was determined using a two-tailed, homo572 scedastic t-test. ***P < 0.001. 573 574 575 576 577 Next, we chose to investigate the effects of 5-azacytidine 578 to determine possible involvement of DNA methylation 579 in DNA repair gene down-regulation. pHD-1 and pHD-2 580 samples were seeded and cultured in the presence of culture 581 media supplemented with 10 mmol/L 5-azacytidine for 582 120 hours. After treatment, pHD-1 and pHD-2 revealed 583 significant up-regulation of APEX1, BRCA1, and RPA1 584 mRNA as confirmed by RT-qPCR (Figure 3, C and D) and 585 protein expression as measured by immunocytochemistry 586 (Supplemental Figure S4 and Figure 3, E and F). Overall, 587 588 these data suggest that the DNA repair gene expression in 589 HD fibroblasts was at least partially restored by treatment 590 with 5-azacytidine. 591 592 593 Differential Methylation Patterning in APEX1 Promoter 594 in HD Fibroblasts 595 596 To determine whether DNA methylation was increased near 597 promoter regions of DNA repair genes in HD cells and if 598 this was reversed by 5-azacytadine treatment, we conducted 599 bisulfite sequencing analysis of approximately 500 bp up600 stream to approximately 75 bases downstream of the APEX1 601 transcriptional start site. We compared both untreated and 602 5-azacytadine treated Wt-1, pHD-1, and pHD-2 samples. 603 Bisulfite sequencing revealed that no methylated CpG sites 604 605 were present in treated Wt-1 and untreated Wt-1 samples in the analyzed region (Figure 4). In contrast, untreated HD ½F4 606 607 samples had methylated CpG sites in the APEX1 promoter 608 region. Untreated pHD-1 was methylated at 437 and 28 609 bp, whereas untreated pHD-2 was methylated at 435, 610 427, 417, 412, 138, 68, 28, and 7 bp. Impor611 tantly, no methylated CpG sites were seen in pHD-1 or 612 pHD-2 when treated with 5-azacytadine. Not only do these 613 data confirm that 5-azacytidine treatments caused direct 614 hypomethylation through passive cell replication, but this 615 also brings to light the differential methylation patterns seen 616 617 in HD fibroblasts. This differential methylation in the pro618 moter region of APEX1 elucidates a possible cause of gene 619 down-regulation seen in our gene expression data. 620
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Figure 2 Down-regulation of DNA repair proteins in confirmed Huntington disease (HD) fibroblasts. A: Immunocytochemistry staining of APEX1, BRCA1, RPA1, and RPA3 in wild-type 1 (Wt-1), potential HD 1 (pHD-1), and pHD-2 cells. Differences in expression levels of each protein were determined by total fluorescence of cell area of pHD-1 (B) and pHD-2 (C) compared with Wt-1. Primary antibodies were counterstained with secondary conjugated Alexa Fluor 488. Nuclei staining was conducted with DAPI. Images were acquired using a Zeiss Azio microscope (Carl Zeiss AG, Jena, Germany) with a 10 objective. Error bars represent the SD of the mean corrected total cell fluorescence. Statistical significance was determined using a two-tailed, homoscedastic t-test. **P < 0.01, ***P < 0.001.
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Intermittent 5-Azacytidine Treatments Induces TRN Stability in the HTT Gene Finally, we sought to determine whether the up-regulation of DNA repair genes by 5-azacytidine would stabilize TNR instability during long-term culture of HD cells. Previous studies have reported contradictory findings regarding global hypomethylation and its effect on TNR stability in various transgenic CAG repeat models13,30 We reasoned that the beneficial effect of 5-azacytidine treatment on DNA
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repair gene expression might also result in stabilization of the TNR region in HD-affected human cells. Cell lines pHD-1 and pHD-2 were plated in T-75 flasks and treated intermittently with 10 mmol/L 5-azacytidine during 35 days to reach four population doublings, which in our experience is ample time to witness small expansions of TNR regions of the mutant HTT gene. Genomic DNA samples were taken of pHD-1 and pHD-2 samples at the initial split before the treatment began and at the end of the 35-day time frame. In parallel to the treated samples, we passaged a subpopulation
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DNA Repair and TNR Stability in HD Figure 3 5-Azacytodine (5-Aza) but not trichostatin-A (TSA) increases DNA repair gene expression in Huntington disease (HD) fibroblasts. Potential HD 1 (pHD-1) (A) and 2 (pHD-2) (B) fibroblasts were treated with 5 mmol/L TSA for 24 hours, and gene expression levels of APEX1, BRCA1, RPA1, and RPA3 were analyzed and compared with cells treated with vehicle alone. Significant decreases were seen for all genes analyzed. pHD-1 (C) and pHD-2 (D) fibroblasts were treated with 10 mmol/L 5-Aza for 120 hours, and gene expression was measured relative to cells treated with vehicle alone. Each tested gene has significant increases in expression for the pHD-1 etreated fibroblast line. APEX1, BRCA1, and RPA1 have statistically significant increases in pHD-2 treated cells. E and F: Protein quantity was determined using corrected total cell fluorescence of the collected cell area using immunocytochemistry. E: Analysis reveals statistically significant up-regulation of APEX1 for 5-Azaetreated pHD-1 fibroblasts. F: pHD-2 fibroblasts treated with 5-Aza have statistically significant upregulation of APEX1 and BRCA1. Bars represent the mean corrected total cell fluorescence (CTCF) of at least three images in random fields of view. Error bars represent the SD of the mean CTCF of each image. CTCF is relative to the untreated sample of each fibroblast line. *P < 0.05, **P < 0.01. DMSO, dimethyl sulfoxide.
of each sample as a nontreated control. The pHD-1 sample began the treatment regimen with the HTT geneeaffected allele containing 43 CAG repeats, with a small population of cells showing expansions in the affected allele up to 44 ½F5 and 45 CAG repeats (Figure 5). At the end of the treatment schedule, the untreated control pHD-1 sample indicated the expected TNR expansion with the predominant population containing an allele with 44 CAG repeats and a small population of 45 CAG repeats. Conversely, the pHD-1 sample treated intermittently with 5-azacytidine revealed TNR stability because it maintained a predominant population that contained an affected allele with 43 CAG repeats, with a small population of 44 CAG repeats. Similar results were witnessed in sample pHD-2, where initial expansion of the affected allele was characterized as having predominantly 41 CAG repeats (Figure 5). At the end of the treatment cycles, the untreated pHD-2 sample had
predominantly 42 CAG repeats with instability within the population up to 45. Again, conversely, the treated pHD-2 sample revealed that most cells had maintained 41 CAG repeats, with only a small subset of the cell population having 42 CAGs. Interestingly, these data indicate that the normal expansion of CAG repeats during culture of HD cells is mitigated by 5-azacytidine treatment and is a proof of principle that pharmacologic intervention can be used to prevent TNR instability in HD.
Discussion To the best of our knowledge, this is the first report demonstrating down-regulation of DNA repair genes in multiple proliferating HD cell lines. Down-regulation of APEX1, BRCA1, RPA1, and RPA3 mRNA levels were so Figure 4 Differential methylation of APEX1 promoter region of Huntington disease (HD) and wild-type (Wt) cells. Genomic DNA was isolated from untreated Wt-1 (U-Wt-1), potential HD-1 (U-pHD-1), and potential HD-2 (U-pHD2) cells. Bisulfite sequencing was used to analyze the promoter region of APEX1. Open and closed circles represent unmethylated and methylated CpGs, respectively. U-Wt-1 has no methylated CpG sites in the promoter region, whereas U-pHD-1 (437 and 28 bp) and U-pHD-2 (435, 427, 417, 138, 68, 28, and 7 bp) contain methylated CpGs. Treatment of each cell line with 10 mmol/L 5-azacytadine for 120 hours (T-Wt-1, T-pHD-1, an pHD-2, respectively) results in loss of all methylated CpG sites in T-pHD-1 and T-pHD-2, whereas T-Wt-1 remains unmethlyated at all sites.
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Figure 5
5-Azacytidine treatment stabilizes trinucleotide repeats (TNRs) of mitotically active Huntington disease (HD) fibroblasts. Two HD fibroblast cell lines were treated with 10 mmol/L 5-azacytidine intermittently in 35 days. The top panels represents the cytosine-adenine-guanine (CAG) expansions at treatment day 0. The middle panels represents the CAG expansion of the untreated control at day 35. The bottom panels show the CAG expansion of the 5-azacytidine treated samples after 35 days. Potential HD 1 (pHD-1) fibroblasts have beginning and treated samples with CAG repeats primarily of 43, with a small population of cells having expansions up to 44 to 45. Untreated pHD-1 has a CAG expansion of predominantly 44, with a small population of 45. pHD-2 fibroblasts have beginning and treated samples with CAG repeats of predominantly 41. Untreated pHD-2 have expansions of predominantly 42 but with small subpopulations expanding up to 45.
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consistent, we were able to use them to predict the HD status of two unknown samples. We pursued these genes in particular because they are implicated in TNR contractions/ expansions and were found to be down-regulated in a preliminary screen of DNA repair gene expression in HD cells (unpublished data).24e29 We found that this downregulation is likely mediated, at least in part, by DNA methylation because the DNA methyltransferase inhibitor 5-azatacytadine can partially restore DNA repair expression. Perhaps most strikingly, 5-azacytadine treatment stabilized TNRs during long-term culture of proliferating HD cells. These results offer a novel insight into HD pathogenesis and identify new potential biomarkers and therapeutic strategies. Although there is overwhelming evidence indicating that HD and DNA repair are associated, no comprehensive model has been determined to clearly explain TNR instability in HD. Importantly, the identification of APEX1, BRCA1, RPA1, and, RPA3 down-regulation in HD can potentially provide therapeutic targets and biomarkers for disease progression or treatment success. These genes obviously represent a small subset of all DNA repair genes likely involved in TNR instability, and thus future studies will expand on these findings to determine whether other genes are similarly affected in HD. The mHtt protein produced by the expanded TNR region of exon 1 in the HTT gene interacts nonspecifically with many unintended targets. The resulting interaction leads to cellular dysfunction on multiple levels, including transcriptional dysregulation, mitochondrial dysfunction, altered vesicular transport, reactive oxygen species defense, abnormal endocytosis and secretion, and increased apoptosis. The elongated gene encodes for repetitive glutamine amino acids
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(polyglutamine [poly-Q]), in which these poly-Q chains contain hydrophilic properties. The mHtt protein exhibits a toxic gain of function in which poly-Q chains bind unintended targets, sequestering them from normal function.31 Consequently, expanded poly-Q regions of mHtt disrupt normal cell functions through interaction with multiple basal transcription factors, causing gene expression dysregulation, including those encoding CREB, CREBBP, Sp1, TFII30, NRSF/REST, PFC1a, BCL11b, TBP, and others.32e37 Furthermore, extensive changes in DNA methylation are associated with the expression of mHtt. Interestingly, these changes in methylation patterns seem to be sequence specific to DNA-binding motifs.38 Therefore, it seems likely that mHtt might disrupt normal expression of APEX1, BRCA1, RPA1, and RPA3 through both disruption of relevant transcription factors and methylation patterning. Our observations revealing intermittent treatment of 5-azacytidne and its up-regulation of DNA repair genes further support the finding that mHtt might be interfering with DNA methylation epigenetics. Any dysfunction of methylation pattern maintenance caused by mHtt would clearly alter physiologic epigenetics and would have repercussions long before HD neuronal dysfunction presents, especially when considering the extensive involvement that methylation plays on neurogenesis.39e41 Although the passive demethylation by cell division does not address mitotically inactive disease-associated cells (medium spiny neurons), the stabilization of the CAG expansion in exon 1 of the HTT gene over four population doublings indicates the possibility that controlling TNR instability could prevent HD-associated genetic anticipation. Furthermore, our finding that the DNA repair expression of APEX1 did not return to wild-type levels despite complete promoter hypomethylation suggests
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DNA Repair and TNR Stability in HD additional mechanisms (ie, direct transcriptional disruption) factor into the DNA repair gene down-regulation. Whatever the mechanism, it is clear that mitotically active HD cells have reduced DNA repair gene expression. Future studies should seek to answer key questions raised in this study. First, we are attempting to identify whether this down-regulation persists in mitotically inactive neurons and whether DNA repair deficiencies lead to increased transcription-mediated TNR instability in neurons as well. Furthermore, because RT-qPCR is a relatively quick assay compared with current sequencing and triplet repeat primed PCR technologies, the utility of gene expression as a biomarker for HD disease should be explored. Finally, the stabilization of CAG repeats within the HD gene by 5azacytadineeinduced up-regulation of DNA repair genes is intriguing. Unfortunately, 5-azacytadine is likely not a viable preventive therapeutic option, but the data here are a proof of principle that pharmacologic interventions aimed at up-regulated DNA repair mechanisms during development could delay onset and severity of the disease by preventing CAG expansions in mutant HTT. As such, future work should look to identify other molecules that have a similar effect on DNA repair gene expression and TNR stability and can be administered for long periods in young patients with a mutant HTT allele. DNA repair genes APEX1, BRCA1, RPA1, and RPA3 are significantly down-regulated in mitotically active HD fibroblasts. Gene expression levels of these four genes accurately predicted the HD status of two unknown samples. Furthermore, the suppression of DNA repair gene expression is mediated, in part, by DNA methylation and can be partially restored by treatment with 5-azacytadine. Finally, 5-azacytadine treatment prevented TNR expansion during long-term culture of HD cells. We conclude that downregulation of DNA repair genes leads to increased TNR instability in HD cells, and restoration of normal DNA repair expression can reduce TNR instability in the HTT gene.
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Acknowledgments We thank Mohammed Jamma of ARUP Laboratories for his assistance with the design of the macroinstructions for triplet repeat primed PCR analysis and Dr. Tabetha Sundin of Sentara Norfolk General Hospital for her troubleshooting expertise and donation of supplies.
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Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2016.03.014.
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References
19.
1. MacDonald ME, Ambrose CM, Duyao MP, Myers RH, Lin C, Srinidhi L, Barnes G, Taylor SA, James M, Groot N, MacFarlane H,
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20.
Jenkins B, Anderson MA, Wexler NS, Gusella JF; Huntington’s Disease Collaborative Research Group: A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 1993, 72:971e983 Bates G: Huntingtin aggregation and toxicity in Huntington’s disease. Lancet 2003, 361:1642e1644 Ranen NG, Stine OC, Abbott MH, Sherr M, Codori AM, Franz ML, Chao NI, Chung AS, Pleasant N, Callahan C, Kasch LM, Ghaffari M, Chase GA, Kazazian HH, Brandt J, Folstein SE, Ross CA: Anticipation and instability of IT-15 (CAG)n repeats in parent-offspring pairs with Huntington disease. Am J Hum Genet 1995, 57:593e602 Langbehn DR, Brinkman RR, Falush D, Paulsen JS, Hayden MR; International Huntington’s Disease Collaborative Group: A new model for prediction of the age of onset and penetrance for Huntington’s disease based on CAG length. Clin Genet 2004, 65:267e277 Langbehn DR, Hayden MR, Paulsen JS; PREDICT-HD Investigators of the Huntington Study Group: CAG-repeat length and the age of onset in Huntington disease (HD): a review and validation study of statistical approaches. Am J Med Genet B Neuropsychiatr Genet 2010, 153B:397e408 Lee JM, Ramos EM, Lee JH, Gillis T, Mysore JS, Hayden MR, et al: CAG repeat expansion in Huntington disease determines age at onset in a fully dominant fashion. Neurology 2012, 78:690e695 Walker FO: Huntington’s Disease. Semin Neurol 2007, 27:143e150 Gomes-Pereira M, Fortune MT, Monckton DG: Mouse tissue culture models of unstable triplet repeats: in vitro selection for larger alleles, mutational expansion bias and tissue specificity, but no association with cell division rates. Hum Mol Genet 2001, 10:845e854 Fortune MT, Vassilopoulos C, Coolbaugh MI, Siciliano MJ, Monckton DG: Dramatic, expansion-biased, age-dependent, tissuespecific somatic mosaicism in a transgenic mouse model of triplet repeat instability. Hum Mol Genet 2000, 9:439e445 Telenius H, Kremer B, Goldberg YP, Theilmann J, Andrew SE, Zeisler J, Adam S, Greenberg C, Ives EJ, Clarke LA, Hayden MR: Somatic and gonadal mosaicism of the Huntington disease gene CAG repeat in brain and sperm. Nat Genet 1994, 6:409e414 Kennedy L, Shelbourne PF: Dramatic mutation instability in HD mouse striatum: does polyglutamine load contribute to cell-specific vulnerability in Huntington’s disease? Hum Mol Genet 2000, 9: 2539e2544 Kovalenko M, Dragileva E, St Claire J, Gillis T, Guide JR, New J, Dong H, Kucherlapati R, Kucherlapati MH, Ehrlich ME, Lee JM, Wheeler VC: Msh2 acts in medium-spiny striatal neurons as an enhancer of CAG instability and mutant huntingtin phenotypes in Huntington’s disease knock-in mice. PLoS One 2012, 7:e44273 Gomes-Pereira M, Monckton DG: Chemically induced increases and decreases in the rate of expansion of a CAG*CTG triplet repeat. Nucleic Acids Res 2004, 32:2865e2872 Chatterjee N, Lin Y, Santillan BA, Yotnda P, Wilson JH: Environmental stress induces trinucleotide repeat mutagenesis in human cells. Proc Natl Acad Sci U S A 2015, 112:3764e3769 Wheeler VC, Persichetti F, McNeil SM, Mysore JS, Mysore SS, MacDonald ME, Myers RH, Gusella JF, Wexler NS; Group US-VCR: Factors associated with HD CAG repeat instability in Huntington disease. J Med Genet 2007, 44:695e701 Huang B, Wei W, Wang G, Gaertig MA, Feng Y, Wang W, Li XJ, Li S: Mutant huntingtin downregulates myelin regulatory factormediated myelin gene expression and affects mature oligodendrocytes. Neuron 2015, 85:1212e1226 Khakh BS, Sofroniew MV: Astrocytes and Huntington’s disease. ACS Chem Neurosci 2014, 5:494e496 Chan CS, Surmeier DJ: Astrocytes go awry in Huntington’s disease. Nat Neurosci 2014, 17:641e642 Lee DY, McMurray CT: Trinucleotide expansion in disease: why is there a length threshold? Curr Opin Genet Dev 2014, 26:131e140 Sinden RR: Neurodegenerative diseases: origins of instability. Nature 2001, 411:757e758
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Mollica et al 21. Jama M, Millson A, Miller CE, Lyon E: Triplet repeat primed PCR simplifies testing for Huntington disease. J Mol Diagn 2013, 15: 255e262 22. Li LC, Dahiya R: MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002, 18:1427e1431 23. Mallona I, Diez-Villanueva A, Peinado MA: Methylation plotter: a web tool for dynamic visualization of DNA methylation data. Source Code Biol Med 2014, 9:11 24. Beaver JM, Lai Y, Xu M, Casin AH, Laverde EE, Liu Y: AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair. Nucleic Acids Res 2015, 43: 5948e5960 25. Li M, Volker J, Breslauer KJ, Wilson DM 3rd: APE1 incision activity at abasic sites in tandem repeat sequences. J Mol Biol 2014, 426: 2183e2198 26. Mason AG, Tome S, Simard JP, Libby RT, Bammler TK, Beyer RP, Morton AJ, Pearson CE, La Spada AR: Expression levels of DNA replication and repair genes predict regional somatic repeat instability in the brain but are not altered by polyglutamine disease protein expression or age. Hum Mol Genet 2014, 23:1606e1618 27. Andreoni F, Darmon E, Poon WC, Leach DR: Overexpression of the single-stranded DNA-binding protein (SSB) stabilises CAG*CTG triplet repeats in an orientation dependent manner. FEBS Lett 2010, 584:153e158 28. Broderick S, Rehmet K, Concannon C, Nasheuer HP: Eukaryotic single-stranded DNA binding proteins: central factors in genome stability. Subcell Biochem 2010, 50:143e163 29. Spiro C, McMurray CT: Nuclease-deficient FEN-1 blocks Rad51/BRCA1-mediated repair and causes trinucleotide repeat instability. Mol Cell Biol 2003, 23:6063e6074 30. Gorbunova V, Seluanov A, Mittelman D, Wilson JH: Genome-wide demethylation destabilizes CTG.CAG trinucleotide repeats in mammalian cells. Hum Mol Genet 2004, 13:2979e2989 31. Nucifora FC Jr, Sasaki M, Peters MF, Huang H, Cooper JK, Yamada M, Takahashi H, Tsuji S, Troncoso J, Dawson VL, Dawson TM, Ross CA: Interference by huntingtin and atrophin-1 with
10
32.
33.
34.
35.
36.
37.
38.
39. 40.
41.
cbp-mediated transcription leading to cellular toxicity. Science 2001, 291:2423e2428 Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, Hersch SM, Mouradian MM, Young AB, Tanese N, Krainc D: Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s disease. Science 2002, 296:2238e2243 Zhai W, Jeong H, Cui L, Krainc D, Tjian R: In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets. Cell 2005, 123:1241e1253 Kazantsev A, Preisinger E, Dranovsky A, Goldgaber D, Housman D: Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells. Proc Natl Acad Sci U S A 1999, 96:11404e11409 Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, Gohler H, Wanker EE, Bates GP, Housman DE, Thompson LM: The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc Natl Acad Sci U S A 2000, 97:6763e6768 Li SH, Cheng AL, Zhou H, Lam S, Rao M, Li H, Li XJ: Interaction of Huntington disease protein with transcriptional activator Sp1. Mol Cell Biol 2002, 22:1277e1287 Seredenina T, Luthi-Carter R: What have we learned from gene expression profiles in Huntington’s disease? Neurobiol Dis 2012, 45: 83e98 Ng CW, Yildirim F, Yap YS, Dalin S, Matthews BJ, Velez PJ, Labadorf A, Housman DE, Fraenkel E: Extensive changes in DNA methylation are associated with expression of mutant huntingtin. Proc Natl Acad Sci U S A 2013, 110:2354e2359 Covic M, Karaca E, Lie DC: Epigenetic regulation of neurogenesis in the adult hippocampus. Heredity 2010, 105:122e134 Sun J, Sun J, Ming GL, Song H: Epigenetic regulation of neurogenesis in the adult mammalian brain. Eur J Neurosci 2011, 33: 1087e1093 Szulwach KE, Li X, Smrt RD, Li Y, Luo Y, Lin L, Santistevan NJ, Li W, Zhao X, Jin P: Cross talk between microRNA and epigenetic regulation in adult neurogenesis. J Cell Biol 2010, 189:127e141
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Supplemental Figure S1 Fragment analysis characterization of fibroblast lines. Using triplet repeat primed PCR in combination with capillary electrophoresis, unknown fibroblast samples of potential Huntington disease (HD) 1 (pHD-1) (A) and 2 (pHD-2) (B) were determined to be affected by HD, with expansions of 18/44 and 17/42 cytosine-adenine-guanine (CAG) repeats, respectively. HD-confirmed cell lines HD-1 (C) contain 21/45. The analysis reveals trinucleotide repeat instability with a subpopulation of multiple HD samples, relative to their previously published expansions by Coriell Cell Repositories (Table 1). ROX1000 MapMarker dye serves as an allelic ladder and is indicated by the red peaks. Fragment analysis characterizing of the fibroblast lines. The cell lines Huntingdon disease (HD) 2 (HD-2) (A) contains 18/60, HD 3 (HD-3) (B) contains 17/48, and wild-type 1 (Wt-1) (C) contains 17/23 cytosine-adenine-guanine (CAG) repeats. Our analysis reveals trinucleotide repeat instability with a subpopulation of multiple HD samples, relative to their previously published expansions by Coriell Cell Repositories (Table 1). Alleles containing >35 CAG repeats are diagnosed as HD. ROX1000 MapMarker dye serves as an allelic ladder and is indicated by the red peaks.
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Supplemental Figure S3 Fragment analysis characterizing fibroblast lines. Wild-type cell line 2 (Wt-2) shows 16/32 cytosine-adenine-guanine (CAG) repeats. ROX1000 MapMarker dye serves as an allelic ladder and is indicated by the red peaks. Supplemental Figure S4 Immunocytochemistry of DNA repair genes in the potential Huntington disease (HD) 1 (pHD-1) and (pHD-2) cells treated with 5-azacytidine. The pHD-1 (A) and pHD-2 (B) fibroblast lines were exposed to 10 mmol/L 5-azacytidine for 120 hours, alongside untreated controls. Immunocytochemistry images show slight increases in fluorescence of DNA repair genes. Nuclei are counterstained with DAPI. Images were acquired using a Zeiss Q17 Q18 Azio microscope (Carl Zeiss AG, Jena, Germany) with a short-working distance 20 objective.
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DNA Methylation Leads to DNA Repair Gene Down-Regulation and Trinucleotide Repeat Expansion in Patient-Derived Huntington Disease Cells Q22
Peter A. Mollica,*y John A. Reid,z Roy C. Ogle,y Patrick C. Sachs,y and Robert D. Brunoy From the Departments of Biological Sciences,* Engineering and Technology,z and Medical Diagnostics and Translational Sciences,y Old Dominion University, Norfolk, Virginia Accepted for publication March 16, 2016.
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Address correspondence to Robert D. Bruno or Patrick C. Sachs. E-mail: rbruno@odu. edu or [email protected].
Huntington disease (HD) is an autosomal dominantly inherited disease that exhibits genetic anticipation of affected progeny due to expansions of a trinucleotide repeat (TNR) region within the HTT gene. DNA repair machinery is a known effector of TNR instability; however, the specific defects in HD cells that lead to TNR expansion are unknown. We hypothesized that HD cells would be deficient in DNA repair gene expression. To test this hypothesis, we analyzed expression of select DNA repair genes involved in mismatch/loop-out repair (APEX1, BRCA1, RPA1, and RPA3) in patient-derived HD cells and found each was consistently down-regulated relative to wild-type samples taken from unaffected individuals in the same family. Rescue of DNA repair gene expression by 5-azacytidine treatment identified DNA methylation as a mediator of DNA repair gene expression deficiency. Bisulfite sequencing confirmed hypermethylation of the APEX1 promoter region in HD cells relative to control, as well as 5-azacytidineeinduced hypomethylation. 5-Azacytidine treatments also resulted in stabilization of TNR expansion within the mutant HTT allele during long-term culture of HD cells. Our findings indicate that DNA methylation leads to DNA repair down-regulation and TNR instability in mitotically active HD cells and offer a proof of principle that epigenetic interventions can curb TNR expansions. (Am J Pathol 2016, -: 1e10; http://dx.doi.org/10.1016/j.ajpath.2016.03.014)
Huntington disease (HD) is a neurodegenerative disease that affects medium spiny neurons in the basal ganglia, resulting in involuntary muscle movements and ultimately leading to death. It is caused by an expansion of a cytosine-adenineguanine (CAG) trinucleotide repeat (TNR) region in exon 1 of the HTT gene.1 HD is a dynamic mutation disease in which the number of TNRs increases the probability of expression of the mutant phenotype. The disease does not affect individuals with <35 TNRs, but individuals with 35 to 40 repeats are at an increased risk of developing HD. Expansions >40 repeats inevitably lead to HD progression.2 Although the mechanism is not fully understood, HDaffected individuals exhibit genetic anticipation caused by subsequent generations of HD-affected individuals developing expanded versions of the mutated allele.3e5 Increased expansions in successive generations correlate with more severe symptomatic expression and earlier age at onset in 70% of individuals.6,7 Interestingly, postmortem analysis of
affected neuronal cells and other somatic tissues in HD mouse models reveals disease-causing gene CAG mosaicism, in which CAG repeats of affected alleles in different tissues have different sized expansions.8e11 In addition, a myriad of TNR instability effectors have been identified, such as environmental stresses, chemical inducers, and DNA repair gene expression.12e15 This evidence infers that TNR instability is present not only in germline cells but also throughout development and maturation and thus dependent on efficacy of DNA repair and its ability to appropriately respond to endogenous and exogenous DNA-damaging agents and modifiers. Furthermore, emerging data have recently implicated TNR instability in replicating neuronal Supported by institutional start-up support from the College of Health Q1 Sciences at Old Dominion University. Q2 P.C.S. and R.D.B. contributed equally as senior authors. Disclosures: None declared.
Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2016.03.014
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Mollica et al support cells as possible mediators of disease severity and progression.16e18 Thus, deciphering how TNR expansions occur during development and in proliferating cell populations during development and adulthood is vital to our understanding of the pathogenesis of HD. These data would lead to therapeutic strategies aimed at preventing TNR expansions and delaying the onset and severity of HD symptoms in patients inheriting a mutated HTT allele. Unfortunately, the comprehensive mechanism of TNR instability eludes researchers. Of the proposed mechanisms, heteroduplex formation and the inability for successful resolution during replication are the common factors behind TNR instability.19 Through intrastand complementary base pairing, single-stranded DNA generates secondary structures, such as hairpins or loop-outs.20 Although this secondary structure meets the criteria for DNA damage, the lesion is ignored in HD cells, and the expanded allele generally results in the dissemination of a larger CAG repeat region to daughter cells. The inability of DNA repair machinery to stabilize CAG repeats in HD cells led us to hypothesize that HD somatic tissues have abnormal expression of DNA repair genes, sensitizing them to CAG TNR expansions. To test this hypothesis, we analyzed DNA repair gene expression in fibroblasts from HD patients and wild-type controls. Because TNR instability is seen in all tissue types, we chose to look at fibroblasts because they are mitotically active and thus would better represent TNR expansions occurring during development. We determined that key DNA repair genes were down-regulated in HD fibroblasts compared with wild-type controls and that the promoter region of the down-regulated repair gene, APEX1, was hypermethylated compared with controls. Treatment with the DNA methyltransferase inhibitor 5-azacytidine resulted in increased DNA repair expression and marked stabilization of CAG repeats during long-term culture. This is the first study to identify endogenously deficient DNA repair genes in human HD fibroblasts and is proof of principle that pharmacologic intervention can lead to increased TNR stability in patients at risk for the disease. Table 1
Materials and Methods Cell Lines and Cell Culture Three previously confirmed HD (GM03621, GM02079, Q5 and GM03868), two wild-type (GM02187 and GM02153), and two unknown (GM02191 and GM04022) fibroblast cell lines were obtained from Coriell Cell Repositories (Camden, NJ). Unknown fibroblast samples were obtained from donors of whom one parent was confirmed to be affected by HD but remained undiagnosed. Genetic characteristics of exon 1 of the HTT gene of each cell line used in our experiments are detailed in Table 1. Cell lines were ½T1 cultured and maintained at 37 C in a humidified incubator with 5% CO2. Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium with glutamax (Life Technologies, Carlsbad, CA) with 10% fetal bovine serum (Life Technologies) as recommended by Coriell Cell Repositories. Cell media also contained 1.0% antibioticantimycotic (100) (Life Technologies). Cells were seeded in T75-cm2 flasks at 5.0 104 cells, and medium was replenished between 72 and 96 hours of seeding. Cells were passaged every 5 to 6 days using TrypLE Express (Life Technologies) following manufacturer’s instructions.
RNA and Genomic DNA Isolation Total RNA was isolated from cells on growth to 70% to 80% confluency (approximately 1 million cells) with Trizol (Thermo Fisher, Waltham, MA) following the manufacturer’s protocol. Approximately 1.0 106 cells were harvested, and genomic DNA (gDNA) was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Samples were concentrated by ethanol precipitation when needed. RNA and DNA quantity and purity were assessed by the absorbance at 260/280 nm method using a NanoDrop 2000 (Thermo Fisher), with 260/280 ratios of approximately 2.0 and 260/230 ratios slightly higher than 1.8.
Genetic Characteristics of Exon 1 of the HTT Gene of Each Cell Line Used in This Study
Cell line
Experiment designation
Age, years
Reported status
Reported CAGn
Confirmed status
Confirmed CAGn
GM02187 GM02153 GM02079 GM03621 GM03868 GM04022 GM02191
Wt-1 Wt-2 HD-1 HD-2 HD-3 pHD-1 pHD-2
60 40 48 29 45 28 32
Wild type Wild type HD HD HD Unknown Unknown
17e23 16e32 21e44 18e60 18e44 e e
Wild type Wild type HD HD HD HD HD
17e23 16e32 21e45 18e60 18e48 18e44 17e42
HD-affected and wild-type fibroblast samples are all female. All cell lines were purchased from the Coriell Cell Repositories. The status of fibroblast lines GM02191 and GM04022 were designated unknown on ordering and initial experiments because they had not yet been characterized and were subsequently confirmed to be HD-affected through capillary electrophoresis fragment analysis. Experimental designation is the abbreviation used throughout the article. The CAG expansions shown above represent the characterizations previously published by Coriell Cell Repositories, with the exception of our unknown samples (GM04022 and GM02191) in which the CAG is the first characterization by fragment analysis completed. CAG, cytosine-adenine-guanine; HD, Huntington disease; pHD, potential Huntington disease; Wt, wild type.
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5-Azacytidine and Trichostatin-A Treatment For gene and protein expression assays, cell medium was supplemented with 5-azacytidine diluted in phosphate buffer solution to achieve 10-mmol/L concentrations for 120 hours. The 10-mmol/L dosage was the predetermined 50% lethal concentration of 5-azacytidine (data not shown). For TNR stability assays, cells were treated intermittently with 10 mmol/L 5-azacytidine during 35 days to reach four population doublings. Treatment schedule was as follows: pass in normal medium for 24 hours, treat with 10 mmol/L 5-azacytidine for 5 days, allow cells to recover for 3 to 5 days in normal medium, repeat with passaging at a 1:2 split ratio. During 5-azacytidne treatment days, media was replaced daily with freshly prepared 5-azacytidine to optimize passive demethylation during DNA synthesis and to facilitate the removal of toxic products. gDNA samples were collected at the beginning of each treatment schedule and at the end of the designated time frame. For histone-deacetylase inhibition treatment, complete fibroblast medium was supplemented with 0.5 mmol/L and 5.0 mmol/L trichostatin-A (TSA) (Sigma-Aldrich, St. Louis, MO) reconstituted in dimethyl sulfoxide to achieve 0.01% v/v. TSA that contained media was applied 16 hours after passaging cells and allowed to remain for 24 hours. On removal of TSA, total RNA was immediately extracted.
Quantitative RT-PCR One microgram of each RNA sample was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. We used TaqMan Gene Expression Assays (Applied Biosystems) to assess mRNA levels. TaqMan Gene Expression Assays provided optimized primers and probes specific for DNA repair genes APEX1 (Hs00959050_g1), BRCA1 (Hs01556193_m1), RPA1 (Hs00161419_m1), and RPA3 (Hs01047933_g1) and the endogenous housekeeping gene ACTB (Hs99999903_m1). Quantitative RT-PCR (RTqPCR) experiments were conducted with a StepOnePlus Real-Time PCR System (Applied Biosystems) using TaqMan Fast Advanced Master Mix (Life Technologies) using 50 ng of cDNA from each sample per the manufacturer’s protocol. All experiment reactions were independently completed in triplicate. Negative template controls were run on each experiment containing nuclease-free water. All cell lines were tested three independent times, with at least two population doubling events occurring between sample experiments. Each experimental sample was also subsampled three times during RT-qPCR reactions. Relative fold-changes were calculated using the 2DDCt method. Significance was determined by comparing the 2DCt value using a one-way analysis of variance with a Dunnett’s Post Hoc Test.
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311 312 313 Cell samples were seeded onto Lab-Tek microscope 3 314 chamber slides at a density of 1.5 10 cells/mL and fixed 315 with 10% formalin for 30 minutes at room temperature. 316 Samples were blocked with 10% goat serum for 60 minutes 317 at room temperature and then incubated with primary anti318 body overnight at 4 C in a humidified chamber. Primary 319 antibodies were obtained from ABCAM (Cambridge, MA): 320 APEX1 antibody (ab105081; 1:700), BRCA1 antibody 321 (ab16781; 1:250), RPA1 antibody (ab79398; 1:250), and 322 323 RPA3 antibody (ab6432; 1:50). Samples were then incu324 bated with Alexa Fluor 488 conjugated secondary anti325 rabbit or anti-mouse antibodies (Thermo Fisher, 1:1000) 326 for 60 minutes. Slides were counterstained with DAPI. 327 Fluorescent images were acquired with a Zeiss Axio 328 Observer.Z1 microscope (Carl Zeiss AG, Jena, Germany) at Q6 329 10 objective. Images were collected in 16-bit black and 330 white to maximize resolution. False coloring was added by 331 ImageJ software version x.xxx (NIH, Bethesda, MD; http:// Q7 332 imagej.nih.gov/ij). At least three images of each gene and 333 cell line were collected to ensure global capture of the 334 335 population of cells. Relative quantitation was conducted using ImageJ software version x.xxx (NIH, Bethesda, MD; Q8 336 337 http://imagej.nih.gov/ij). Cell fluorescence was determined 338 by multiplying the total area of the image by mean back339 ground of the image, then subtracted from integrated den340 sity. The cell fluorescence of each image was then divided 341 by the number of complete nuclei in the image to give the 342 corrected total cell fluorescence (CTCF). Mean CTCF 343 is represented graphically as normalized values to the 344 mean CTCF of wild-type fibroblast sample. For statistical 345 analysis, relative fold-changes were compared using the 346 347 Wilcoxon rank sum test. 348 349 Triplet Repeat Primed PCR 350 351 Primers were synthesized by Integrated DNA Technologies 352 (Coralville, IA). The forward primer sequence was 50 -ATG353 AAGGCCTTCGAGTCCCTCAAGTC-30 , with a carboxy354 fluorescein at its 50 end, and the reverse was 50 -CGGTGG355 CGGCTGTTGCTGCTGCTGCTGCTG-30 .21 Amplification 356 357 reactions were run in 20mL volumes and contained the 358 following components; 1 FailSafe Premix J (Epicenter, 359 Madison, WI), 1 U of Platinum Taq DNA Polymerase High 360 Fidelity (Life Technologies), 1.0 mL of 20 to 25 ng/mL of 361 gDNA sample, and 0.5 mmol/L each primer. PCR conditions 362 were as follows: 95.0 C for 1 minute hot start/initial dena363 turation, followed by 35 cycles of 94.0 C for 1 minute, 364 64.0 C for 1 minute, 72.0 C for 2 minutes, and a final 365 extension of 15 minutes at 72.0 C. One microliter of each 366 sample was combined with 1.0 mL of Mapmarker 1000 367 internal size standard (BioVentures Inc., Murfreesboro, 368 369 TN) and 9.0 mL of Hi-Di Formamide (Life Technologies). 370 Just before sample loading of 10 mL onto capillary elec371 trophoresis plates, each sample was heated to 95.0 C for 372
Immunofluorescence of DNA Repair Genes
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2 minutes for denaturation and then immediately placed on ice for 5 minutes. Capillary electrophoresis fragment analysis was conducted on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems) with 36-cm arrays and POP-7 Polymer-CG (Applied Biosystems). Electrokinetic injection of samples was initiated with 12 kV for 16 seconds and then electrophoresed at 15 kV for 1200 seconds at 60 C. Raw data were analyzed using GeneMarker software version 2.6.4 (SoftGenetics LLC, State College, PA).21 We conducted amplified fragment length polymorphism analysis using optimized macros for TNR expansion calling and labeling. Macros for bin calling were customized using gDNA samples obtained from Coriell Cell Repositories and were graciously created by Mohamed Jama (University of Utah). As positive controls, we obtained 6-g DNA samples from various HD-affected and wild-type characterizations. These chosen positive control gDNA samples had previously been tested in multiple laboratories and were sequenced to confirm CAG repeats (data not shown).
Bisulfite Conversion and meCpG Analysis
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The gDNA samples were treated to bisulfite conversion using the EpiTect Fast Bisulfite Kit (Qiagen) according to the manufacturer’s protocol. Primers were designed to evaluate methylation patterns approximately 500 bp upstream of APEX1 using MethPrimer software version x.xx.22 The region was amplified in two segments: forward: 50 -ATAATGTGTTGTGTATTTGGTATAA-30 , reverse: 50 -ATAACAACCTAACTCCTCCTAACCC-30 ; forward: 50 -TGTTAGGAGTTGTGGAGGTTTTTAT-30 , reverse: 50 -TTCTCTAAAACACTTAAATCCCCAA-30 . PCR amplification of 500 ng of bisulfite-treated gDNA was conducted using 1 U of Platinum Taq DNA Polymerase High Fidelity (Life Technologies) as previously described. We used a touchup PCR cycling protocol where the annealing temperature was gradually increased after the first and second set of five cycles, with annealing temperatures increasing from 53 C to 56 C and then finally 60 C for the remaining 30 cycles. PCR cycling conditions were as follows: 95 C for 1-minute hot start, followed by a touch-up protocol with increasing annealing temperatures for 1 minute, and 72 C for 1 minute for 40 total cycles. A final extension of 10 minutes at 72 C was applied at the end. Correct amplicons were assessed using gel electrophoresis on a 1.5% agarose gel. Appropriate bands were extracted using QIAquick Gel Extraction Kit (Qiagen). Each extracted amplicon was then inserted into the pCR4-TOPO TA vector with the TOPO TA cloning kit for sequencing (Thermo Fisher) per the manufacturer’s protocol. Cycle sequencing of fragments was conducted using the M13 forward and M13 reverse primer sets provided in the TOPO TA kit. Methylation analysis and representation were conducted using Methylation Plotter software version x.xx online.23
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Results APEX1, BRCA1, RPA1, and RPA3 Gene Expression Is Deficient in Both HD and Unknown Fibroblasts To determine whether DNA repair gene expression was perturbed in proliferating HD cells, we performed RT-qPCR to detect relative mRNA expression level of four DNA repair genes (APEX1, BRCA1, RPA1, and RPA3) in HD samples. We compared fibroblasts isolated from three confirmed HD patients (GM02079, GM03621, GM03868), two wild-type patients (GM02187 and GM02153), and two unknown patients (GM02079 and GM04022) who had a 50% chance of inheriting the mutant HTT allele. Heretofore, confirmed HD cell lines are designated as HD-n, unknowns are designated as potential HD pHD-n, and wild-type samples designated Wt-n (Table 1). We chose to examine APEX1, BRCA1, RPA1, and RPA3 because they are effectors of TNR instabilityeinduced DNA damage and because we had previously observed their down-regulation in HD cells (unpublished data).24e29 Our analysis revealed statistically significant down-regulation of all four genes in all HD-affected and unknown fibroblast lines (Figure 1). The ½F1 mRNA levels presented are in relation to control sample Wt-1 because this sample had lower overall expression of the four DNA repair genes when compared with the Wt-2 sample and thus represents a more stringent comparison.
Unknown Samples Are Affected by mHTT Expansion Because both of our unknown samples (pHD-1 and pHD-2) had significant down-regulation of all four DNA repair genes, we sought to identify if they did, in fact, have an expanded HTT allele. To determine HD status of unknown fibroblast lines, we conducted capillary electrophoresis fragment analysis using triplet repeat primed PCR on all samples (Table 1 and Supplemental Figure S1). Both unknown fibroblast lines pHD-1 and pHD-2 were confirmed HD, with alleles that contained CAG repeats of 18/44 and 17/42, respectively (Supplemental Figure S1, A and B). Combined with data identifying APEX1, BRCA1, RPA1, and RPA3 down-regulation, this confirmation of unknown samples being affected by HD identifies therapeutic biomarkers, as well as a possible alternate means for HD diagnosis. Interestingly, we witnessed TNR instability in previously characterized HD samples HD-1 and HD-3, whereas HD-2 Q13 remained static (Supplemental Figure S1C, Supplemental Figure S2, A and B, and Table 1). Previously, Coriell Cell Repositories reported HTT gene characterization of HD-1 as containing 21/44 CAG repeats and HD-3 as containing 18/44 CAG repeats. Our initial characterization was con- Q14 ducted after 10 population doublings and revealed HTT expansions in the mutated allele to 45 and 48, respectively. Interestingly, HD-3 had a contraction in the unaffected allele from previously reported 18 to 17. No instability was
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DNA Repair and TNR Stability in HD
detected in either wild-types samples (Wt-1 or Wt-2) after identical population doublings (Supplemental Figure S2C, Supplemental Figure S3, and Table 1).
APEX1, BRCA1, RPA1, and RPA3 Protein Levels Are Down-Regulated in HD Fibroblasts To determine whether protein levels of DNA repair genes matched our findings from our mRNA analysis, we conducted immunocytochemistry of all fibroblast lines ½F2 (Figure 2A). CTCF revealed that protein levels in pHD-1 and pHD-2 samples were approximately 23% of APEX1, 22% of BRCA1, 8% of RPA1, and 32% of RPA3 (Figure 2B) and 54% of APEX1, 35% of BRCA1, 46% of RPA1, and 36% of RPA3 (Figure 2C), respectively. Protein levels are relative to the lowest-expressing wild-type fibroblast sample (Wt-1). These data indicate that DNA repair components are significantly decreased in HD fibroblast samples, possibly having consequences on DNA repair pathways to efficiently address lesions and adducts generated during DNA synthesis.
APEX1, BRCA1, and RPA1 Gene Expression Is Affected by 5-Azacytidine Treatments We next wanted to identify the potential mechanism of the observed DNA repair gene down-regulation. Because no previous DNA repair gene mutational events have been associated with HD, we hypothesized that epigenetic regulators could be a primary cause of the observed downregulations. To test for the involvement of histone acetylation and DNA condensation as a primary regulator, we treated pHD-1 and pHD-2 samples with 5 mmol/L TSA, a potent global deacetylase inhibitor, for 24 hours. RT-qPCR revealed no significant changes in DNA repair gene expres½F3 sion in either pHD-1 (Figure 3A) or pHD-2 (Figure 3B) in response to TSA treatments, suggesting hyperacetylation of histones was not primarily responsible for the suppression of DNA repair genes.
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559 560 561 Figure 1 DNA repair deficiencies in multiple Huntington disease (HD) fibroblast cell samples. APEX1, BRCA1, RPA1, and 562 RPA3 expression in wild-type (Wt-1), potential HD-affected 563 (pHD), and HD-affected (HD) fibroblasts. Using TaqMan Gene 564 Expression Assays, we identified HD DNA repair mRNA down565 regulation of APEX1, BRCA1, RPA1, and RPA3. Gene expres566 DDCt sion is determined by the (2 ) of the mean of each 567 independent triplicate. Error bars represent the SD of the 568 mean between replicate samples. Each tested gene shows 569 significant decreases in expression for all HD and pHD cell 570 lines, relative to the Wt sample. Gene expression is normalized 571 to the Wt-1 fibroblast line GM02187 gene expression. Statistical significance was determined using a two-tailed, homo572 scedastic t-test. ***P < 0.001. 573 574 575 576 577 Next, we chose to investigate the effects of 5-azacytidine 578 to determine possible involvement of DNA methylation 579 in DNA repair gene down-regulation. pHD-1 and pHD-2 580 samples were seeded and cultured in the presence of culture 581 media supplemented with 10 mmol/L 5-azacytidine for 582 120 hours. After treatment, pHD-1 and pHD-2 revealed 583 significant up-regulation of APEX1, BRCA1, and RPA1 584 mRNA as confirmed by RT-qPCR (Figure 3, C and D) and 585 protein expression as measured by immunocytochemistry 586 (Supplemental Figure S4 and Figure 3, E and F). Overall, 587 588 these data suggest that the DNA repair gene expression in 589 HD fibroblasts was at least partially restored by treatment 590 with 5-azacytidine. 591 592 593 Differential Methylation Patterning in APEX1 Promoter 594 in HD Fibroblasts 595 596 To determine whether DNA methylation was increased near 597 promoter regions of DNA repair genes in HD cells and if 598 this was reversed by 5-azacytadine treatment, we conducted 599 bisulfite sequencing analysis of approximately 500 bp up600 stream to approximately 75 bases downstream of the APEX1 601 transcriptional start site. We compared both untreated and 602 5-azacytadine treated Wt-1, pHD-1, and pHD-2 samples. 603 Bisulfite sequencing revealed that no methylated CpG sites 604 605 were present in treated Wt-1 and untreated Wt-1 samples in the analyzed region (Figure 4). In contrast, untreated HD ½F4 606 607 samples had methylated CpG sites in the APEX1 promoter 608 region. Untreated pHD-1 was methylated at 437 and 28 609 bp, whereas untreated pHD-2 was methylated at 435, 610 427, 417, 412, 138, 68, 28, and 7 bp. Impor611 tantly, no methylated CpG sites were seen in pHD-1 or 612 pHD-2 when treated with 5-azacytadine. Not only do these 613 data confirm that 5-azacytidine treatments caused direct 614 hypomethylation through passive cell replication, but this 615 also brings to light the differential methylation patterns seen 616 617 in HD fibroblasts. This differential methylation in the pro618 moter region of APEX1 elucidates a possible cause of gene 619 down-regulation seen in our gene expression data. 620
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Figure 2 Down-regulation of DNA repair proteins in confirmed Huntington disease (HD) fibroblasts. A: Immunocytochemistry staining of APEX1, BRCA1, RPA1, and RPA3 in wild-type 1 (Wt-1), potential HD 1 (pHD-1), and pHD-2 cells. Differences in expression levels of each protein were determined by total fluorescence of cell area of pHD-1 (B) and pHD-2 (C) compared with Wt-1. Primary antibodies were counterstained with secondary conjugated Alexa Fluor 488. Nuclei staining was conducted with DAPI. Images were acquired using a Zeiss Azio microscope (Carl Zeiss AG, Jena, Germany) with a 10 objective. Error bars represent the SD of the mean corrected total cell fluorescence. Statistical significance was determined using a two-tailed, homoscedastic t-test. **P < 0.01, ***P < 0.001.
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Intermittent 5-Azacytidine Treatments Induces TRN Stability in the HTT Gene Finally, we sought to determine whether the up-regulation of DNA repair genes by 5-azacytidine would stabilize TNR instability during long-term culture of HD cells. Previous studies have reported contradictory findings regarding global hypomethylation and its effect on TNR stability in various transgenic CAG repeat models13,30 We reasoned that the beneficial effect of 5-azacytidine treatment on DNA
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repair gene expression might also result in stabilization of the TNR region in HD-affected human cells. Cell lines pHD-1 and pHD-2 were plated in T-75 flasks and treated intermittently with 10 mmol/L 5-azacytidine during 35 days to reach four population doublings, which in our experience is ample time to witness small expansions of TNR regions of the mutant HTT gene. Genomic DNA samples were taken of pHD-1 and pHD-2 samples at the initial split before the treatment began and at the end of the 35-day time frame. In parallel to the treated samples, we passaged a subpopulation
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DNA Repair and TNR Stability in HD Figure 3 5-Azacytodine (5-Aza) but not trichostatin-A (TSA) increases DNA repair gene expression in Huntington disease (HD) fibroblasts. Potential HD 1 (pHD-1) (A) and 2 (pHD-2) (B) fibroblasts were treated with 5 mmol/L TSA for 24 hours, and gene expression levels of APEX1, BRCA1, RPA1, and RPA3 were analyzed and compared with cells treated with vehicle alone. Significant decreases were seen for all genes analyzed. pHD-1 (C) and pHD-2 (D) fibroblasts were treated with 10 mmol/L 5-Aza for 120 hours, and gene expression was measured relative to cells treated with vehicle alone. Each tested gene has significant increases in expression for the pHD-1 etreated fibroblast line. APEX1, BRCA1, and RPA1 have statistically significant increases in pHD-2 treated cells. E and F: Protein quantity was determined using corrected total cell fluorescence of the collected cell area using immunocytochemistry. E: Analysis reveals statistically significant up-regulation of APEX1 for 5-Azaetreated pHD-1 fibroblasts. F: pHD-2 fibroblasts treated with 5-Aza have statistically significant upregulation of APEX1 and BRCA1. Bars represent the mean corrected total cell fluorescence (CTCF) of at least three images in random fields of view. Error bars represent the SD of the mean CTCF of each image. CTCF is relative to the untreated sample of each fibroblast line. *P < 0.05, **P < 0.01. DMSO, dimethyl sulfoxide.
of each sample as a nontreated control. The pHD-1 sample began the treatment regimen with the HTT geneeaffected allele containing 43 CAG repeats, with a small population of cells showing expansions in the affected allele up to 44 ½F5 and 45 CAG repeats (Figure 5). At the end of the treatment schedule, the untreated control pHD-1 sample indicated the expected TNR expansion with the predominant population containing an allele with 44 CAG repeats and a small population of 45 CAG repeats. Conversely, the pHD-1 sample treated intermittently with 5-azacytidine revealed TNR stability because it maintained a predominant population that contained an affected allele with 43 CAG repeats, with a small population of 44 CAG repeats. Similar results were witnessed in sample pHD-2, where initial expansion of the affected allele was characterized as having predominantly 41 CAG repeats (Figure 5). At the end of the treatment cycles, the untreated pHD-2 sample had
predominantly 42 CAG repeats with instability within the population up to 45. Again, conversely, the treated pHD-2 sample revealed that most cells had maintained 41 CAG repeats, with only a small subset of the cell population having 42 CAGs. Interestingly, these data indicate that the normal expansion of CAG repeats during culture of HD cells is mitigated by 5-azacytidine treatment and is a proof of principle that pharmacologic intervention can be used to prevent TNR instability in HD.
Discussion To the best of our knowledge, this is the first report demonstrating down-regulation of DNA repair genes in multiple proliferating HD cell lines. Down-regulation of APEX1, BRCA1, RPA1, and RPA3 mRNA levels were so Figure 4 Differential methylation of APEX1 promoter region of Huntington disease (HD) and wild-type (Wt) cells. Genomic DNA was isolated from untreated Wt-1 (U-Wt-1), potential HD-1 (U-pHD-1), and potential HD-2 (U-pHD2) cells. Bisulfite sequencing was used to analyze the promoter region of APEX1. Open and closed circles represent unmethylated and methylated CpGs, respectively. U-Wt-1 has no methylated CpG sites in the promoter region, whereas U-pHD-1 (437 and 28 bp) and U-pHD-2 (435, 427, 417, 138, 68, 28, and 7 bp) contain methylated CpGs. Treatment of each cell line with 10 mmol/L 5-azacytadine for 120 hours (T-Wt-1, T-pHD-1, an pHD-2, respectively) results in loss of all methylated CpG sites in T-pHD-1 and T-pHD-2, whereas T-Wt-1 remains unmethlyated at all sites.
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Figure 5
5-Azacytidine treatment stabilizes trinucleotide repeats (TNRs) of mitotically active Huntington disease (HD) fibroblasts. Two HD fibroblast cell lines were treated with 10 mmol/L 5-azacytidine intermittently in 35 days. The top panels represents the cytosine-adenine-guanine (CAG) expansions at treatment day 0. The middle panels represents the CAG expansion of the untreated control at day 35. The bottom panels show the CAG expansion of the 5-azacytidine treated samples after 35 days. Potential HD 1 (pHD-1) fibroblasts have beginning and treated samples with CAG repeats primarily of 43, with a small population of cells having expansions up to 44 to 45. Untreated pHD-1 has a CAG expansion of predominantly 44, with a small population of 45. pHD-2 fibroblasts have beginning and treated samples with CAG repeats of predominantly 41. Untreated pHD-2 have expansions of predominantly 42 but with small subpopulations expanding up to 45.
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consistent, we were able to use them to predict the HD status of two unknown samples. We pursued these genes in particular because they are implicated in TNR contractions/ expansions and were found to be down-regulated in a preliminary screen of DNA repair gene expression in HD cells (unpublished data).24e29 We found that this downregulation is likely mediated, at least in part, by DNA methylation because the DNA methyltransferase inhibitor 5-azatacytadine can partially restore DNA repair expression. Perhaps most strikingly, 5-azacytadine treatment stabilized TNRs during long-term culture of proliferating HD cells. These results offer a novel insight into HD pathogenesis and identify new potential biomarkers and therapeutic strategies. Although there is overwhelming evidence indicating that HD and DNA repair are associated, no comprehensive model has been determined to clearly explain TNR instability in HD. Importantly, the identification of APEX1, BRCA1, RPA1, and, RPA3 down-regulation in HD can potentially provide therapeutic targets and biomarkers for disease progression or treatment success. These genes obviously represent a small subset of all DNA repair genes likely involved in TNR instability, and thus future studies will expand on these findings to determine whether other genes are similarly affected in HD. The mHtt protein produced by the expanded TNR region of exon 1 in the HTT gene interacts nonspecifically with many unintended targets. The resulting interaction leads to cellular dysfunction on multiple levels, including transcriptional dysregulation, mitochondrial dysfunction, altered vesicular transport, reactive oxygen species defense, abnormal endocytosis and secretion, and increased apoptosis. The elongated gene encodes for repetitive glutamine amino acids
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(polyglutamine [poly-Q]), in which these poly-Q chains contain hydrophilic properties. The mHtt protein exhibits a toxic gain of function in which poly-Q chains bind unintended targets, sequestering them from normal function.31 Consequently, expanded poly-Q regions of mHtt disrupt normal cell functions through interaction with multiple basal transcription factors, causing gene expression dysregulation, including those encoding CREB, CREBBP, Sp1, TFII30, NRSF/REST, PFC1a, BCL11b, TBP, and others.32e37 Furthermore, extensive changes in DNA methylation are associated with the expression of mHtt. Interestingly, these changes in methylation patterns seem to be sequence specific to DNA-binding motifs.38 Therefore, it seems likely that mHtt might disrupt normal expression of APEX1, BRCA1, RPA1, and RPA3 through both disruption of relevant transcription factors and methylation patterning. Our observations revealing intermittent treatment of 5-azacytidne and its up-regulation of DNA repair genes further support the finding that mHtt might be interfering with DNA methylation epigenetics. Any dysfunction of methylation pattern maintenance caused by mHtt would clearly alter physiologic epigenetics and would have repercussions long before HD neuronal dysfunction presents, especially when considering the extensive involvement that methylation plays on neurogenesis.39e41 Although the passive demethylation by cell division does not address mitotically inactive disease-associated cells (medium spiny neurons), the stabilization of the CAG expansion in exon 1 of the HTT gene over four population doublings indicates the possibility that controlling TNR instability could prevent HD-associated genetic anticipation. Furthermore, our finding that the DNA repair expression of APEX1 did not return to wild-type levels despite complete promoter hypomethylation suggests
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DNA Repair and TNR Stability in HD additional mechanisms (ie, direct transcriptional disruption) factor into the DNA repair gene down-regulation. Whatever the mechanism, it is clear that mitotically active HD cells have reduced DNA repair gene expression. Future studies should seek to answer key questions raised in this study. First, we are attempting to identify whether this down-regulation persists in mitotically inactive neurons and whether DNA repair deficiencies lead to increased transcription-mediated TNR instability in neurons as well. Furthermore, because RT-qPCR is a relatively quick assay compared with current sequencing and triplet repeat primed PCR technologies, the utility of gene expression as a biomarker for HD disease should be explored. Finally, the stabilization of CAG repeats within the HD gene by 5azacytadineeinduced up-regulation of DNA repair genes is intriguing. Unfortunately, 5-azacytadine is likely not a viable preventive therapeutic option, but the data here are a proof of principle that pharmacologic interventions aimed at up-regulated DNA repair mechanisms during development could delay onset and severity of the disease by preventing CAG expansions in mutant HTT. As such, future work should look to identify other molecules that have a similar effect on DNA repair gene expression and TNR stability and can be administered for long periods in young patients with a mutant HTT allele. DNA repair genes APEX1, BRCA1, RPA1, and RPA3 are significantly down-regulated in mitotically active HD fibroblasts. Gene expression levels of these four genes accurately predicted the HD status of two unknown samples. Furthermore, the suppression of DNA repair gene expression is mediated, in part, by DNA methylation and can be partially restored by treatment with 5-azacytadine. Finally, 5-azacytadine treatment prevented TNR expansion during long-term culture of HD cells. We conclude that downregulation of DNA repair genes leads to increased TNR instability in HD cells, and restoration of normal DNA repair expression can reduce TNR instability in the HTT gene.
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Acknowledgments We thank Mohammed Jamma of ARUP Laboratories for his assistance with the design of the macroinstructions for triplet repeat primed PCR analysis and Dr. Tabetha Sundin of Sentara Norfolk General Hospital for her troubleshooting expertise and donation of supplies.
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Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2016.03.014.
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References
19.
1. MacDonald ME, Ambrose CM, Duyao MP, Myers RH, Lin C, Srinidhi L, Barnes G, Taylor SA, James M, Groot N, MacFarlane H,
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-
20.
Jenkins B, Anderson MA, Wexler NS, Gusella JF; Huntington’s Disease Collaborative Research Group: A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 1993, 72:971e983 Bates G: Huntingtin aggregation and toxicity in Huntington’s disease. Lancet 2003, 361:1642e1644 Ranen NG, Stine OC, Abbott MH, Sherr M, Codori AM, Franz ML, Chao NI, Chung AS, Pleasant N, Callahan C, Kasch LM, Ghaffari M, Chase GA, Kazazian HH, Brandt J, Folstein SE, Ross CA: Anticipation and instability of IT-15 (CAG)n repeats in parent-offspring pairs with Huntington disease. Am J Hum Genet 1995, 57:593e602 Langbehn DR, Brinkman RR, Falush D, Paulsen JS, Hayden MR; International Huntington’s Disease Collaborative Group: A new model for prediction of the age of onset and penetrance for Huntington’s disease based on CAG length. Clin Genet 2004, 65:267e277 Langbehn DR, Hayden MR, Paulsen JS; PREDICT-HD Investigators of the Huntington Study Group: CAG-repeat length and the age of onset in Huntington disease (HD): a review and validation study of statistical approaches. Am J Med Genet B Neuropsychiatr Genet 2010, 153B:397e408 Lee JM, Ramos EM, Lee JH, Gillis T, Mysore JS, Hayden MR, et al: CAG repeat expansion in Huntington disease determines age at onset in a fully dominant fashion. Neurology 2012, 78:690e695 Walker FO: Huntington’s Disease. Semin Neurol 2007, 27:143e150 Gomes-Pereira M, Fortune MT, Monckton DG: Mouse tissue culture models of unstable triplet repeats: in vitro selection for larger alleles, mutational expansion bias and tissue specificity, but no association with cell division rates. Hum Mol Genet 2001, 10:845e854 Fortune MT, Vassilopoulos C, Coolbaugh MI, Siciliano MJ, Monckton DG: Dramatic, expansion-biased, age-dependent, tissuespecific somatic mosaicism in a transgenic mouse model of triplet repeat instability. Hum Mol Genet 2000, 9:439e445 Telenius H, Kremer B, Goldberg YP, Theilmann J, Andrew SE, Zeisler J, Adam S, Greenberg C, Ives EJ, Clarke LA, Hayden MR: Somatic and gonadal mosaicism of the Huntington disease gene CAG repeat in brain and sperm. Nat Genet 1994, 6:409e414 Kennedy L, Shelbourne PF: Dramatic mutation instability in HD mouse striatum: does polyglutamine load contribute to cell-specific vulnerability in Huntington’s disease? Hum Mol Genet 2000, 9: 2539e2544 Kovalenko M, Dragileva E, St Claire J, Gillis T, Guide JR, New J, Dong H, Kucherlapati R, Kucherlapati MH, Ehrlich ME, Lee JM, Wheeler VC: Msh2 acts in medium-spiny striatal neurons as an enhancer of CAG instability and mutant huntingtin phenotypes in Huntington’s disease knock-in mice. PLoS One 2012, 7:e44273 Gomes-Pereira M, Monckton DG: Chemically induced increases and decreases in the rate of expansion of a CAG*CTG triplet repeat. Nucleic Acids Res 2004, 32:2865e2872 Chatterjee N, Lin Y, Santillan BA, Yotnda P, Wilson JH: Environmental stress induces trinucleotide repeat mutagenesis in human cells. Proc Natl Acad Sci U S A 2015, 112:3764e3769 Wheeler VC, Persichetti F, McNeil SM, Mysore JS, Mysore SS, MacDonald ME, Myers RH, Gusella JF, Wexler NS; Group US-VCR: Factors associated with HD CAG repeat instability in Huntington disease. J Med Genet 2007, 44:695e701 Huang B, Wei W, Wang G, Gaertig MA, Feng Y, Wang W, Li XJ, Li S: Mutant huntingtin downregulates myelin regulatory factormediated myelin gene expression and affects mature oligodendrocytes. Neuron 2015, 85:1212e1226 Khakh BS, Sofroniew MV: Astrocytes and Huntington’s disease. ACS Chem Neurosci 2014, 5:494e496 Chan CS, Surmeier DJ: Astrocytes go awry in Huntington’s disease. Nat Neurosci 2014, 17:641e642 Lee DY, McMurray CT: Trinucleotide expansion in disease: why is there a length threshold? Curr Opin Genet Dev 2014, 26:131e140 Sinden RR: Neurodegenerative diseases: origins of instability. Nature 2001, 411:757e758
ajp.amjpathol.org
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1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116
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Mollica et al 21. Jama M, Millson A, Miller CE, Lyon E: Triplet repeat primed PCR simplifies testing for Huntington disease. J Mol Diagn 2013, 15: 255e262 22. Li LC, Dahiya R: MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002, 18:1427e1431 23. Mallona I, Diez-Villanueva A, Peinado MA: Methylation plotter: a web tool for dynamic visualization of DNA methylation data. Source Code Biol Med 2014, 9:11 24. Beaver JM, Lai Y, Xu M, Casin AH, Laverde EE, Liu Y: AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair. Nucleic Acids Res 2015, 43: 5948e5960 25. Li M, Volker J, Breslauer KJ, Wilson DM 3rd: APE1 incision activity at abasic sites in tandem repeat sequences. J Mol Biol 2014, 426: 2183e2198 26. Mason AG, Tome S, Simard JP, Libby RT, Bammler TK, Beyer RP, Morton AJ, Pearson CE, La Spada AR: Expression levels of DNA replication and repair genes predict regional somatic repeat instability in the brain but are not altered by polyglutamine disease protein expression or age. Hum Mol Genet 2014, 23:1606e1618 27. Andreoni F, Darmon E, Poon WC, Leach DR: Overexpression of the single-stranded DNA-binding protein (SSB) stabilises CAG*CTG triplet repeats in an orientation dependent manner. FEBS Lett 2010, 584:153e158 28. Broderick S, Rehmet K, Concannon C, Nasheuer HP: Eukaryotic single-stranded DNA binding proteins: central factors in genome stability. Subcell Biochem 2010, 50:143e163 29. Spiro C, McMurray CT: Nuclease-deficient FEN-1 blocks Rad51/BRCA1-mediated repair and causes trinucleotide repeat instability. Mol Cell Biol 2003, 23:6063e6074 30. Gorbunova V, Seluanov A, Mittelman D, Wilson JH: Genome-wide demethylation destabilizes CTG.CAG trinucleotide repeats in mammalian cells. Hum Mol Genet 2004, 13:2979e2989 31. Nucifora FC Jr, Sasaki M, Peters MF, Huang H, Cooper JK, Yamada M, Takahashi H, Tsuji S, Troncoso J, Dawson VL, Dawson TM, Ross CA: Interference by huntingtin and atrophin-1 with
10
32.
33.
34.
35.
36.
37.
38.
39. 40.
41.
cbp-mediated transcription leading to cellular toxicity. Science 2001, 291:2423e2428 Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, Hersch SM, Mouradian MM, Young AB, Tanese N, Krainc D: Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s disease. Science 2002, 296:2238e2243 Zhai W, Jeong H, Cui L, Krainc D, Tjian R: In vitro analysis of huntingtin-mediated transcriptional repression reveals multiple transcription factor targets. Cell 2005, 123:1241e1253 Kazantsev A, Preisinger E, Dranovsky A, Goldgaber D, Housman D: Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells. Proc Natl Acad Sci U S A 1999, 96:11404e11409 Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, Gohler H, Wanker EE, Bates GP, Housman DE, Thompson LM: The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc Natl Acad Sci U S A 2000, 97:6763e6768 Li SH, Cheng AL, Zhou H, Lam S, Rao M, Li H, Li XJ: Interaction of Huntington disease protein with transcriptional activator Sp1. Mol Cell Biol 2002, 22:1277e1287 Seredenina T, Luthi-Carter R: What have we learned from gene expression profiles in Huntington’s disease? Neurobiol Dis 2012, 45: 83e98 Ng CW, Yildirim F, Yap YS, Dalin S, Matthews BJ, Velez PJ, Labadorf A, Housman DE, Fraenkel E: Extensive changes in DNA methylation are associated with expression of mutant huntingtin. Proc Natl Acad Sci U S A 2013, 110:2354e2359 Covic M, Karaca E, Lie DC: Epigenetic regulation of neurogenesis in the adult hippocampus. Heredity 2010, 105:122e134 Sun J, Sun J, Ming GL, Song H: Epigenetic regulation of neurogenesis in the adult mammalian brain. Eur J Neurosci 2011, 33: 1087e1093 Szulwach KE, Li X, Smrt RD, Li Y, Luo Y, Lin L, Santistevan NJ, Li W, Zhao X, Jin P: Cross talk between microRNA and epigenetic regulation in adult neurogenesis. J Cell Biol 2010, 189:127e141
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Supplemental Figure S1 Fragment analysis characterization of fibroblast lines. Using triplet repeat primed PCR in combination with capillary electrophoresis, unknown fibroblast samples of potential Huntington disease (HD) 1 (pHD-1) (A) and 2 (pHD-2) (B) were determined to be affected by HD, with expansions of 18/44 and 17/42 cytosine-adenine-guanine (CAG) repeats, respectively. HD-confirmed cell lines HD-1 (C) contain 21/45. The analysis reveals trinucleotide repeat instability with a subpopulation of multiple HD samples, relative to their previously published expansions by Coriell Cell Repositories (Table 1). ROX1000 MapMarker dye serves as an allelic ladder and is indicated by the red peaks. Fragment analysis characterizing of the fibroblast lines. The cell lines Huntingdon disease (HD) 2 (HD-2) (A) contains 18/60, HD 3 (HD-3) (B) contains 17/48, and wild-type 1 (Wt-1) (C) contains 17/23 cytosine-adenine-guanine (CAG) repeats. Our analysis reveals trinucleotide repeat instability with a subpopulation of multiple HD samples, relative to their previously published expansions by Coriell Cell Repositories (Table 1). Alleles containing >35 CAG repeats are diagnosed as HD. ROX1000 MapMarker dye serves as an allelic ladder and is indicated by the red peaks.
Supplemental Figure S2
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Supplemental Figure S3 Fragment analysis characterizing fibroblast lines. Wild-type cell line 2 (Wt-2) shows 16/32 cytosine-adenine-guanine (CAG) repeats. ROX1000 MapMarker dye serves as an allelic ladder and is indicated by the red peaks. Supplemental Figure S4 Immunocytochemistry of DNA repair genes in the potential Huntington disease (HD) 1 (pHD-1) and (pHD-2) cells treated with 5-azacytidine. The pHD-1 (A) and pHD-2 (B) fibroblast lines were exposed to 10 mmol/L 5-azacytidine for 120 hours, alongside untreated controls. Immunocytochemistry images show slight increases in fluorescence of DNA repair genes. Nuclei are counterstained with DAPI. Images were acquired using a Zeiss Q17 Q18 Azio microscope (Carl Zeiss AG, Jena, Germany) with a short-working distance 20 objective.
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