Published OnlineFirst May 3, 2018; DOI: 10.1158/0008-5472.CAN-17-3460
Cancer Genome and Epigenome Research
SETD2 Haploinsufficiency for Microtubule Methylation Is an Early Driver of Genomic Instability in Renal Cell Carcinoma Yun-Chen Chiang1, In-Young Park2, Esteban A. Terzo3, Durga Nand Tripathi2, Frank M. Mason3, Catherine C. Fahey1, Menuka Karki2,4, Charles B. Shuster4, Bo-Hwa Sohn2, Pratim Chowdhury2, Reid T. Powell5, Ryoma Ohi6, Yihsuan S. Tsai1, Aguirre A. de Cubas3, Abid Khan1,7, Ian J. Davis1, Brian D. Strahl1,7, Joel S. Parker1, Ruhee Dere2, Cheryl L. Walker2, and W. Kimryn Rathmell3
Abstract
Loss of the short arm of chromosome 3 (3p) occurs early in human kidney cells, rescue with a pathogenic SETD2 mutant >95% of clear cell renal cell carcinoma (ccRCC). Nearly ubiqui- deficient for microtubule (aTubK40me3), but not histone tous 3p loss in ccRCC suggests haploinsufficiency for 3p tumor (H3K36me3) methylation, replicated this phenotype. Genomic suppressors as early drivers of tumorigenesis. We previously instability (micronuclei) was also a hallmark of patient-derived reported methyltransferase SETD2, which trimethylates H3 his- cells from ccRCC. These data show that the SETD2 tumor sup- tones on lysine 36 (H3K36me3) and is located in the 3p deletion, pressor displays a haploinsufficiency phenotype disproportion- to also trimethylate microtubules on lysine 40 (aTubK40me3) ately impacting microtubule methylation and serves as an early during mitosis, with aTubK40me3 required for genomic sta- driver of genomic instability. bility. We now show that monoallelic, Setd2-deficient cells retain- Significance: Loss of a single allele of a chromatin modifier ing H3K36me3, but not aTubK40me3, exhibit a dramatic plays a role in promoting oncogenesis, underscoring the grow- increase in mitotic defects and micronuclei count, with increased ing relevance of tumor suppressor haploinsufficiency in tumor- viability compared with biallelic loss. In SETD2-inactivated igenesis. Cancer Res; 78(12); 3135–46. 2018 AACR.
Introduction vided by a large deletion of chromosome 3p (2). Recent large- scale multiplatform sequencing techniques identified that, in Many cancer types are characterized by large-scale, character- addition to VHL, other tumor suppressor genes encoding epige- istic alterations of chromosomal copy number. Clear cell renal cell netic regulators such as Polybromo 1 (PBRM1/BAF180), BAP1, and carcinoma (ccRCC) is characterized by frequent inactivating Set domain containing 2 (SETD2) are implicated in ccRCC mutations of the Von Hippel–Lindau (VHL) tumor suppressor tumorigenesis (3–5). Importantly, all of these commonly mutat- gene (1), with accompanying loss of heterozygosity (LOH) pro- ed genes reside in the same portion of affected chromosome 3p. Loss of 3p results in LOH for all four of these genes, which individually acquire "second-hit" mutations and loss of function 1Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel of the remaining allele in subsets of ccRCC. Of these, SETD2 is of 2 Hill, North Carolina. Center for Precision Environmental Health, Department of particular interest, as this tumor suppressor has been increasingly Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas. identified as a common mutation across cancer types, including 3Vanderbilt-Ingram Cancer Center, Division of Hematology and Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, lung (6), bladder (7), glioma (8), and leukemia (9). Tennessee. 4Department of Biology, New Mexico State University, Las Cruces, SETD2 encodes a methyltransferase known to be the sole New Mexico. 5Texas A&M Health Sciences Center, Institute of Biosciences and enzyme responsible for the trimethylation of lysine 36 on Technology, Houston, Texas. 6Department of Cell and Molecular Biology, histone H3 (H3K36me3; refs. 7, 8, 10–12). Biallelic deficiency 7 Vanderbilt University, Nashville, Tennessee. Department of Biochemistry and of SETD2 via deletions and inactivating mutations occur in up Biophysics, University of North Carolina, Chapel Hill, North Carolina. to 20% of primary human RCC tumors and it is associated with Note: Supplementary data for this article are available at Cancer Research more advanced disease and the metastatic phenotype, typically Online (http://cancerres.aacrjournals.org/). lethal within 1–5 years (13). Biallelic loss of SETD2 has been Y.C. Chiang, I.Y. Park, and E.A. Terzo are co-first authors and contributed shown to result in loss of H3K36me3 in ccRCC-derived cells equally to this article. and tumors (9, 14, 15). Examination of H3K36me3 status in Corresponding Author: W. Kimryn Rathmell, Vanderbilt-Ingram Cancer Center, ccRCC cells of metastatic tumor specimens suggest that SETD2 2220 Pierce Avenue, Preston Research Building, 798C, Nashville, TN 37232. mutations may occur in over 50% of metastatic lesions (16). Phone: 615-343-0250; Fax: 615-343-0250; E-mail: Furthermore, a study of ccRCC intratumoral heterogeneity [email protected] identified distinct SETD2 mutations across subsections of an doi: 10.1158/0008-5472.CAN-17-3460 individual tumor, suggesting a selection bias for SETD2 muta- 2018 American Association for Cancer Research. tion in the course of ccRCC development (7).
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SETD2 is a multidomain-containing protein with distinct ing a previously unrecognized effect of SETD2 dosage. Comparing functions for each domain. The methyltransferase activity is different SETD2 mutations, we also found that the SRI domain mediated by a centrally located SET domain. Mutations in this mutation, which rescues H3K36me3, but not aTubK40me3, fails domain are common in ccRCC (10, 14), suggesting loss of to rescue micronuclei formation and mitotic defects in SETD2- catalytic activity is a critical event in tumor development. We deleted cells compared with WT SETD2. Importantly, micronuclei previously characterized a pathogenic SET domain mutation were also a common feature of 3p-deleted ccRCC cell lines and found in ccRCC, an arginine-to-cysteine mutation at residue were seen in a significant fraction of tumor cells from all patients 1625 of SETD2 (R1625C; ref. 15), which abolishes methylation with ccRCC. These data indicate that SETD2 is haploinsufficient activity. At its C-terminus, SETD2 also contains the Set2–Rpb1 for aTubK40 trimethylating activity, with loss of microtubule interaction (SRI) domain (17). This domain mediates the methylation driving early genomic instability in tumors with 3p interaction between SETD2 and the phosphorylated C-terminal loss, preceding the loss of histone H3K36 trimethylation, and domain of RNA polymerase II (RNAPII). We also identified a contributing to the progression of ccRCC. Moreover, our results recurrent mutation in the SRI domain, an arginine-to-histidine shed light on a new mechanism by which monoallelic inacti- mutation at residue 2510 (R2510H; ref. 15). This mutation vation of SETD2 or 3p loss can drive tumorigenesis in other preserves the H3 trimethylation catalytic activity of SETD2, cancers, and points to microtubule methylation as a key tumor suggesting SETD2 may have other key functions in addition suppressive activity of SETD2. to its well-characterized role as a histone methyltransferase. We recently discovered that SETD2 also functions as a micro- tubule methyltransferase, in addition to the well-characterized Materials and Methods role of SETD2 in histone methylation (18). SETD2 trimethy- Cell cultures lates a-tubulin on lysine 40 (aTubK40me3) of microtubules Human renal cell carcinoma ACHN, UMRC2, Caki-1, and A498 and loss of this marker results in genomic instability. SETD2- cells were acquired from the ATCC. RCC4 cells were generously null cells exhibit mitotic spindle and cytokinesis defects, poly- provided by Dr. William Kim (University of North Carolina, ploidy, and formation of micronuclei.Importantly,pathogenic Chapel Hill, NC). The SV-40–transformed human renal tubule SETD2 mutationsintheSETdomainaswellastheSRIdomain epithelial cell line (HKC) was obtained from Dr. Lorraine were unable to methylate microtubules, and caused an increase Racusen, (Johns Hopkins Hospital, Baltimore, MD). All cells in chromosome bridges and lagging chromosomes relative to undergo annual short tandem repeat (STR) confirmation. Human wild-type SETD2, indicating that in addition to the catalytic cell lines were cultured in DMEM (Gibco/Life Technologies) domain, a functional SRI domain was also required for supplemented with 10% FBS (Gemini Bio-Products), nonessen- aTubK40me3 (18). tial amino acids, L-glutamine, penicillin, and streptomycin. For w/f f/f These mitotic alterations caused by loss of aTubK40me3 Setd2 and Setd2 MEFs, cells were maintained in phenol can lead to chromosomal abnormalities and genomic instabil- red–free media (DMEM, high glucose, HEPES, no phenol red) ity, hallmarks of tumorigenesis, and are thought to be an supplemented with sodium pyruvate and GlutaMAX (Thermo important source of genetic diversity and development of cell Fisher Scientific), to prevent any spontaneous activation of clones during tumor progression (19). In the case of the type of the Cre recombinase by estrogenic compounds found in phenol defects observed with SETD2 mutants deficient in microtubule red–containing media. All cultures were maintained at 37 C methylation (lagging and bridging chromosomes), this genomic in 5% CO2. The identify of all human cell lines used in this study instability results in the formation of micronuclei. Micronuclei has been authenticated using the STR profiling test offered by contain acentric chromosome fragments, acentric chromatid ATCC as a service. All cell lines have been tested for Mycoplasma fragments, or whole chromosomes that failed to migrate during contamination using the PCR-based ATCC's Universal mitosis, which are enclosed by nuclear membrane (20). The Mycoplasma Detection test (catalog number: 30-10102K). presence of micronuclei is a reliable cytologic indicator of w/f f/f chromosome instability (21), and micronuclei are a common Generation of Setd2 and Setd2 MEFs feature of many solid tumors and preneoplastic lesions (19, 20), The detailed information for generating Setd2w/f and Setd2f/f but have not been studied in any detail in ccRCC to date. MEFs was described in a previous publication (18). In brief, Here, we report that SETD2's ability to trimethylate microtu- Setd2w/f or Setd2f/f MEFs were generated from 13.5 days postcoi- bules and preserve genomic stability is dose dependent, and tum (d.p.c.) embryos of Setd2w/f or Setd2f/f mice, respectively. The SETD2 haploinsufficiency or reduced dosage is sufficient to cells were spontaneously immortalized via serial passaging. The impair genomic stability and induce micronuclei formation. immortalized cells were transfected with an ER-Cre vector expres- Using micronuclei as a readout of genomic instability in wild- sing Cre recombinase fused with a mutated ligand-binding type (WT) SETD2 and SETD2 disrupted human kidney proximal domain for the human estrogen receptor (ER-Cre). Stable cell tubule epithelial cells (HKC), and we confirmed that SETD2 loss lines expressing ER-Cre were generated by selection using 5 mg/mL causes a significant increase in micronuclei. To directly demon- blasticidin (Thermo Fisher Scientific). strate that SETD2 haploinsufficiency was sufficient to induce Setd2w/f or Setd2f/f MEFs expressing ER-Cre were treated with genomic instability (micronuclei), we induced loss of a single 3 mmol/L 4-hydroxytamoxifen (4-HT, the active metabolite of copy of Setd2 using Setd2w/f mouse embryonic fibroblasts (MEF). tamoxifen; Sigma-Aldrich) or vehicle (0.01% ethanol) for 2 days Monoallelic loss of Setd2 was sufficient to increase lagging and (or as indicated) for efficient Setd2 knockout. The phenotype bridging chromosomes, and caused a significant increase in of Setd2w/f MEFs is similar to that observed with biallelic loss micronuclei formation. Importantly, while promoting micronu- (Setd2f/f; ref. 18), for which micronuclei were counted after two clei accumulation, monoallelic SETD2 loss had no measurable days of tamoxifen treatment, while the Setd2w/f MEFs remain effect on H3K36me3, but did cause loss of aTubK40me3, reveal- viable as a culture for several passages.
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SETD2 Haploinsufficiency Promotes Genomic Instability
Generation of SETD2-null or mutation human kidney pH 7.5, 150 mmol/L NaCl, and 0.1% Tween 20) for 1 hour epithelial cells and immunoblotted using primary antibodies for overnight To establish HKC SETD2 null cells, HKC cells were transfected and econdary goat anti-mouse IgG-HRP (sc-2005; Santa Cruz as described previously (15) with a pair transcription activator– Biotechnology) or goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz like effector nucleases (TALEN) constructs targeting exon 3 (50- Biotechnology) for 2 hours. TCATGTAACATCCAGGCC-30 and 50-ACAGCAGTAGCATCTC- For SETD2 knockdown HKC cells, lysates were separated on a CA-30) using Amaxa cell Line Nucleofector Kit V (Lonza) and 4%–15% SDS-PAGE gel and the proteins were subsequently single cell sorted to isolate single clones. Individual clones were transferred to a PVDF membrane. Membranes were blocked in screened for loss of H3K36me3 and sequenced at the SETD2 5% nonfat milk in TBST and incubated O/N with primary anti- target site to confirm the presence of inactivating mutations. bodies in milk. Anti-b-tubulin (05-661; EMD Millipore) antibody The allelic sequencing confirmed the SETD2 inactivation was used as loading control. through frameshift mutations at the TALEN target site in both SETD2 alleles. Immunofluorescence staining for micronuclei HKC cells were cultured on chamber slides (Corning) for Generation of SETD2 knockdown human kidney epithelial 16–18 hours and fixed using a 4% paraformaldehyde for 15 min- cells utes. After three washes with cold PBS for 5 minutes each, the HKC cells were infected with shRNA virus expressing either a samples were permeabilized using a 0.25% Triton X-100 in PBS Scramble (SCR) or shRNA against SETD2 (TRCN0000237839; for 10 minutes and followed by 10-minute incubation with 1% Sigma). shSETD2 sequence obtained from the Broad Institute hydrogen peroxide. After blocking in 5% BSA, cells were incubat- shRNA Library. Cells were passaged 24 hours postinfection ed at 4 C overnight with mouse anti LAP2 (1:500; Abcam) or in media containing puromycin (1 mg/mL). Following drug human anti-CREST (1:6,000; Antibody Inc.) primary antibody. selection, cells were passaged once more at day 3 post shRNA After four rounds of vigorous washing for 10 minutes each using virus infection. Subsequently, cells were fixed with either PBST, cells were incubated for 1 hour at room temperature with paraformaldehyde or methanol for immunostaining at 6 days goat anti-mouse IgG Alexa Fluor 488 (Life Technologies, 1:1,000) postinfection. and goat anti-human IgG HþL 568 (Life Technologies, 1:1,000) secondary antibody. After washing, cells were counterstained SNP array and 3p structural variation analysis using DAPI for subsequent visualization of nuclei. Coverslips Genomic DNA was isolated from confluent 10-cm plates were mounted into chamber slides with ProLong Gold Antifade using the Qiagen DNeasy Blood & Tissue Kit (Qiagen 69504) Mountant (Life Technologies). Fluorescence signals were visual- per manufacturer's standard protocols. SNP array was conducted ized using confocal microscopy (LSM 880, Zeiss). The number on the Infinium Core-24 Kit BeadChip (Illumina) by UNC of micronuclei per 300 cells was counted and repeated for two Mammalian Genotyping Core according to manufacturer's times per coverslip. Data from three different coverslips in three standard protocols. The SNP data were preprocessed with independent experiments were manually quantitated. Genomestudio v2 (Illumina) and the R package, genoCNV was For shRNA SETD2 knockdown of HKC cells, samples were used to estimate the copy number states and genotype calls for fixed with cold methanol for 5 minutes at 20 C, followed by all interested cell lines. The allele-specific copy number analysis three 5 minutes washes in PBS. Cells were then blocked in 2.5% of tumors (ASCAT) algorithm (version 2.5; ref. 22) was used BSA, 1% normal goat serum (NGS) in PBS for 1 hour followed to calculate somatic whole-genome ASCAT-profiles, as well as by incubation with anti-LaminB1 (ab16048; Abcam; 1:250) estimates of cell ploidy. antibody in PBS-BSA-NGS for 1 hour at room temperature (RT). Cells were washed with PBS-0.1% Tween 20-NGS Western blot analysis (PBST-NGS), followed by secondary antibody incubation for Cells were lysed by gentle pipetting several times and incu- 30 minutes at room temperature. Cells were counterstained bated on ice for 10 minutes in a cell fractionation buffer using DAPI (1:1,000) for 5 minutes and then mounted on [320 mmol/L sucrose, 10 mmol/L Tris-HCl (pH 8.0), slides using Prolong Gold Antifade mounting media. Images 2 mmol/L magnesium acetate, 3 mmol/L calcium chloride, were acquired on a Zeiss 800 Upright confocal microscope 0.1 mmol/L EDTA, 0.5% NP-40, fresh 1 mmol/L dithiothreitol, usinga63 /1.4 NA under default filter settings and pinhole protease inhibitor cocktail, 1 mmol/L PMSF, and 1 mmol/L of 1 airy unit. For every image, approximately 10 Z-sections sodium orthovanadate]. Cytoplasmic fractions were collected were acquired with an average of 0.4-mm thickness per slice. after centrifugation at 1,000 g for 5 minutes and were used Z-stacks were processed in FIJI (Version 2.0.0) and images for Western blots using antibodies against a-TubK40me3 presented are maximum intensity projections of Z-stacks. The (18, 23) or a-tubulin (MA1-19162; Thermo Fisher Scientific). number of micronuclei per 150 cells per sample (shScramble The nuclear proteins were extracted in cell lysis buffer [20 mmol/L and shSETD2) was counted and data from three independent Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L experiments were manually quantitated. EGTA, 1% Triton X-100, and protease inhibitor cocktail] by Setd2 knockout was induced in MEFs, then fixed in 3.7% sonication for three cycles of 30 seconds on/off using Bioruptor formaldehyde solution for 15 minutes, permeabilized in Tri- 300 (Diagenode) and used for Western blots using antibodies ton-X 100 for 20 minutes, and incubated for an hour in blocking against SETD2 (HPA042451; Sigma-Aldrich), H3K36me3 buffer containing 3% BSA in 1 PBS at room temperature. MEFs (61101, Active Motif), or histone H3 (ab1791; Abcam). The were then probed with human anti-CREST antibody (Antibody samples were separated on a 4%–15% SDS-PAGE gel and trans- Inc.) and anti-rabbit CENPB (Abcam, ab25734) for an hour at ferred onto a polyvinylidene difluoride (PVDF) membrane. Mem- room temperature. Alexafluor 568- and 488-conjugated second- branes were blocked in 5% nonfat milk in TBST (50 mmol/L Tris ary antibodies (Invitrogen) were used to detect anti-CREST and
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anti-CENPB, respectively, whereas DNA was stained using (Version 2.0.0) and images presented are maximum intensity Hoechst 33342 (Invitrogen). Immunofluorescence images were projections of individual cells. taken using Zeiss Axiovert 200M-inverted Microscope equipped with epifluorescence optics and an Apotome structured illumi- IHC nation module (Carl Zeiss) or a 60 1.4 NA objective (Olympus) For H3K36me3 IHC, patient deidentified, formalin-fixed on a DeltaVision Elite Imaging System (GE Healthcare) equip- and paraffin-embedded (FFPE) ccRCC samples were sectioned ped with a Cool SnapHQ2 CCD camera (Roper), and processed and hematoxylin and eosin (H&E) stained by Vanderbilt using ratio deconvolution in SoftWorx (GE Healthcare). Total University Medical Center Translational Pathology Shared CREST (þ) micronuclei were counted per 100 micronuclei per Resource. To perform H3K36me3 IHC, FFPE sections were coverslip from three independent experiments. deparaffinized, then incubated for 45 minutes in heated citric acid buffer (10 mmol/L citric acid, 0.05% Tween-20, pH 6) for Immunofluorescence staining for mitotic defects antigen retrieval. Slides were then stained with H3K36me3 Cells were cultured on coverslips and fixed using ice-cold antibody (Abcam, ab9050, 1:500), using the Cell and Tissue methanol for 10 minutes and followed by permeabilization using Staining Kit (Rabbit, HRP-DAB System, R&D Systems), accord- a 0.2% Triton X-100 solution in PBS buffer for 5 minutes. After ing to the manufacturer's protocol. three washes using PBS buffer, the cells were blocked in 2% BSA blocking buffer for 30 minutes at room temperature. Cells were Reverse transcriptase-PCR incubated in blocking buffer using primary antibodies overnight The total RNAs were isolated from Setd2w/w,Setd2f/w,and at 4 C. Primary antibodies were diluted as follows; rabbit anti- Setd2f/f MEFs treated with 4-HT for 0, 3, and 6 days using H3K36me3 (1:1,000; Abcam), mouse anti-a-tubulin (1:400, RNeasy Mini Kit (Qiagen), and their complementary DNAs clone DMIA; Sigma), and human anti-centromere (1:6,000; Anti- were synthesized using Superscript III reverse transcriptase body Inc.). After five rounds of vigorous washing for 10 minutes (Invitrogen). The quantities of Setd2 mRNAs were assessed by each using PBST, cells were incubated for 1 hour in blocking buffer real-time quantitative PCR using specificTaqManprobes with corresponding secondary antibodies [donkey anti-rabbit (Thermo Fisher Scientific, Mm01250234_m1) amplifying exon (clone 488; Life Technologies), goat anti-mouse IgG H&L 6, which is the region to be deleted by Cre recombination, and a Cy5 (Abcam), and goat anti-human IgG HþL (clone 568; Life TaqMan Fast Universal master mix on QuantStudio Flex Real- Technologies)] at a dilution of 1:1,000. Following more than Time PCR system (Thermo Fisher Scientific). The quantities of three washes, for 10 minutes each using PBST, cells were counter- Setd2 mRNAs were normalized to those of glyceraldehyde stained using DAPI for subsequent visualization of nuclei. Cover- 3-phosphate dehydrogenase, which was used as an endogenous slips were mounted in ProLong Gold Antifade Mountant (Life control. The following set of conditions were used for each Technologies) and cells visualized using confocal microscopy real-time reaction: 95 C for 20 minutes followed by 40 cycles of (LSM 880; Zeiss). 1secondat 95 C and 20 seconds at 60 C. The real-time PCR reactions were performed in triplicate twice and quantified fl Immuno uorescence staining for micronuclei in human using the DD cycle threshold (Ct)method. ccRCC Human primary RCCs were obtained under an approved Statistical analysis Institutional Review Board protocol with patients providing writ- Densitometry analyses using ImageStudio Ver2.0 were per- ten informed consent to deidentified use of tissue. The studies formed on immunoblots of H3K36me3 and aTubK40me3 nor- were conducted in accordance with recognized ethical guidelines malized to H3 and aTubulin, respectively. Significance was (e.g., Belmont Report). inferred with Tukey multiple comparison test. To determine Approval was granted by the Vanderbilt Biomedical Insti- significance of micronuclei count or percentage and of mitotic tutional Review Board. Following pathologic assessment, extra defects from triplicates, we performed a two-way ANOVA test, tissue was freshly provided, and the cells were dispersed by which shows significant interactions between samples. We per- mechanical dissociation, followed by enzymatic digestion using formed Student t test using a paired, two-tailed distribution to collagenase and DNase. Single-cell suspension was passed over determine the significance of qRT-PCR and micronuclei count in 70-mm strainer then subjected to red blood cell lysis. Samples SETD2 knockdown HKC datasets. A reported P value of less than were then mounted with Wescor Cytopro cytocentrifuge and fixed 0.05 is considered significant. All analyses were performed using with 4% PFA (Electron Microscopy Sciences) in cytoskeleton Prism 7 (GraphPad) and Excel (Microsoft) software. buffer (10 mmol/L MES pH 6.1, 138 mmol/L KCl, 3 mmol/L MgCl2, 2 mmol/L EGTA, 320 mmol/L sucrose) for 10 minutes. Cells were permeabilized with 0.1% TritonX-100 in PBS (PBSTx), Results blocked with 1% BSA in PBSTx, stained for pan-cytokeratin SETD2 loss is sufficient to induce genomic instability in (AE1/AE3þ5D3, Abcam ab86734, 1:100 in 1% BSA in PBSTx) normal human kidney proximal tubule epithelial cells and DAPI, and then mounted with Prolong Gold Antifade reagent Previous studies have linked SETD2 inactivation to genomic (Thermo Fisher Scientific). For secondary antibody staining, instability in RCC utilizing tumor-derived analysis (24). We first Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (Jackson asked whether loss of this tumor suppressor alone was sufficient ImmunoResearch, 715-545-151, 1:500) was used. Images were to promote genomic instability in nontumor kidney epithelial acquired on a Zeiss LSM880 or LSM710 confocal microscopes cells. To answer this question, and to examine the domain(s) of using 63 /1.40 Plan-Apochromat oil objective and 405 and SETD2 required for maintenance of genomic stability in normal 488 nm lasers, default filter settings and pinhole of 1 airy unit kidney epithelial cells, we utilized a cell line generated from or Deltavision Elite Imaging System. Images were processed in FIJI immortalized normal human kidney proximal epithelial cells
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SETD2 Haploinsufficiency Promotes Genomic Instability
A 100 C Normal Multipolar Lagging Bridging Normal Multipolar spindle spindle 7.92 1.98 0 90.10 50 HKC + GFP Bridging SETD2-/- HKC 11.21 5.60 0 83.18 + GFP Lagging SETD2-/- % Mitotic cells HKC 7.47 2.87 1.15 88.51 Figure 1. 0 tSETD2 SETD2 loss and pathogenic mutations SETD2-/- HKC 11.30 9.57 0 79.13 in SET and SRI domains promote GFP GFP R1625C mitotic defects. A, Mitotic cells were tSETD2 -/- R1625CR2510H SETD2 HKC 11.82 9.09 1.18 77.27 plotted for the entire tSETD2 HKC R2510H panel. Percentage (%) of cells tSETD2 undergoing normal (light gray) mitosis, SETD2-/- or displaying multipolar spindle HKC HKC (orange), chromosome bridging (dark gray), or chromosome lagging defects B SETD2-/- HKC SETD2-/- HKC SETD2-/- HKC are represented. B, Representative GFP tSETD2-R1625C tSETD2-R2510H immunofluorescence images of SETD2 / HKC GFP, SETD2 / HKC tSETD2-R1625C, and SETD2 / HKC tSETD2-R2510H costained with DAPI (chromosomes, blue) and CREST Merge (centromeres, magenta) are shown. Light blue arrowheads, lagging chromosome. Light gray arrowhead, chromosome bridge. Scale bar, 10 mm. C, Quantitation of cells undergoing normal mitosis or displaying lagging DAPI chromosome, bridging, or multipolar spindle defects for all tSETD2 HKC panel. CREST
Lagging Lagging Chromosome chromosome chromosome bridge