TP53 deletion occurs in various types of human cancers for which no therapeutic strategies have yet been reported. TCGA database analysis shows that the TP53 homozygous deletion locus mostly exhibits co-deletion of the neighboring gene FXR2, which belongs to the Fragile X gene family. Here, we demonstrate that inhibition of the remaining family member FXR1 selectively blocks cell proliferation in human cancer cells containing homozygous deletion of both TP53 and FXR2 in a collateral lethality manner. Mechanistically, in addition to its RNA-binding function, FXR1 recruits transcription factor STAT1 or STAT3 to gene promoters at the chromatin interface and regulates transcription thus, at least partially, mediating cell proliferation. Our study anticipates that inhibition of FXR1 is a potential therapeutic approach to targeting human cancers harboring TP53 homozygous deletion.Tumor suppressor p53 prevents cell transformation by inducing apoptosis and other responses. Homozygous  TP53 produces one of the most important tumor suppressor proteins, and this gene is inactive or missing in many types of human cancer.Healthy human cells employ many tricks to avoid becoming cancerous. For example, they produce proteins known as tumor suppressors, which sense if a cell shows early signs of cancer and instruct the cell to die. A gene known as TP53 gene is particularly difficult. One way to develop new treatments for these conditions would be to target other proteins that these cancers need to survive; but these proteins first need to be identified.Treating cancers that have completely lost the TP53. Searching databases of DNA sequences from human cancer cells revealed that those without the TP53 gene often also lose a neighboring gene called FXR2. Cancer cells survive without FXR2 because a similar gene, called FXR1, can compensate. Fan et al. therefore decided to experimentally lower the activity of the FXR1 gene and, as expected, cancer cells without TP53 and FXR2 stopped growing. Normal cells, on the other hand, were unaffected by the deletion of the FXR1 gene since FXR2 is still there. This phenomenon, in which cancer cells become vulnerable after the loss of certain genes but only because they have already lost important tumor suppressors, is called “collateral lethality”. Further experiments showed that the protein encoded by FXR1 coordinates with other proteins to activate genes that contribute to cell growth.Fan et al. have now identified one such protein in human cancer cells lacking TP53. For example, if scientists can find small molecules that inhibit the protein encoded by FXR1 and show that these molecules can block the growth of tumors lacking TP53 and FXR2, this could eventually lead to a new anticancer drug. However, like any new drug, these small molecule inhibitors would also need to be extensively tested before they could be taken into human clinical trials.These findings suggest new ways to treat human cancers that have lost  TP53 is a common feature in a majority of human cancers, resulting in the escape from tumor-suppressor activities. Numerous strategies have been explored to reverse dysregulated p53 suppressor function, including stabilizing p53 expression by antagonizing the p53–MDM2 interaction in cancers harboring normal TP53 copy number, and restoring p53's tumor suppressor activity in TP53-mutated cancer , located 100 kb downstream of TP53, is also a neighboring gene of TP53 at chromosome 17p13.1. It belongs to the fragile X gene family that has essential functions in binding and regulating mRNA stability, transportation and translation .p53, a critical tumor suppressor, primarily serves as a transcription factor to control various cellular stress-response signaling pathways, including cell cycle arrest, DNA repair, senescence and apoptosis . Loss ofd cancer . In cancd cancer . This codeletion . Homozygnslation . In thisThe fragile X gene family contains three mammalian members, including fragile X mental retardation protein FMR1  and its structural homologs FXR1 and FXR2 , 1995. TFXR2 undergoes concomitant homozygous deletion in combination with TP53 homozygous deletion in a significant proportion of human cancers. Our data show that FXR1 inhibition blocks cell growth in TP53 and FXR2 co-deletion cell lines, but not in copy number normal or single deletion cell lines. The results were further confirmed in CRISPR-Cas9-generated TP53 and FXR2 knockout cell clones. Through a comprehensive analysis of chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) and ChIP coupling with high-throughput sequencing (ChIP-seq), we uncovered the molecular mechanism through which FXR1 regulates cell proliferation. FXR1 is located in the gene promoter region together with histone H3 lysine 4 trimethylation (H3K4me3), and recruits transcription factor STAT1 or STAT3 at gene promoters to facilitate transcription regulation. In an agreement with a role in the FXR1-assocaited mechanism, inhibition of STAT1 or STAT3 target genes can also reduce cell proliferation. Taken together, our findings not only demonstrate the possibility of inhibiting FXR1 in TP53 and FXR2 homozygous co-deletion cancers as a potential treatment option, but also reveal the novel role of FXR1 in gene transcription.From an analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases, we observed that http://www.cbioportal.org; accessed 18 Aug 2016) , respectively. Our data showed that cancer cells with homozygous co-deletion of TP53 and FXR2 exhibit either absent or significantly lower mRNA and protein levels of p53 and FXR2. By contrast, the copy-number-normal and co-deleted cell lines have comparable FXR1 and FMR1 levels   show tha% to 15% . One of ceptions . Consistceptions , the majdeletion . Howeverand FMR1  is largedeletion . The rec1 levels . It is wTP53/FXR2 co-deleted cancer cell line KATOIII, but not in the copy-number-normal cell line MKN45  but not in the copy-number-normal cells , as indicated in TP53/FXR2 copy-number-normal and co-deleted cancer cells . Five doxycycline (Dox)-inducible FXR1 shRNAs were tested and most of them resulted in robust downregulation of FXR1 protein level and exhibited anti-proliferative activity in the ne MKN45 . We seleer cells .TP53 and FXR2 exhibited sensitivity to FXR1 inhibition suggested a collateral lethality correlation between FXR1 and TP53 deletion. To investigate whether TP53/FXR2 co-deletion is essential in determining cells’ sensitivity to FXR1 inhibition, we monitored cell growth rate upon FXR1 downregulation in cell lines carrying homozygous deletion of either TP53  or FXR2 (Hep3B) alone. The levels of p53, FXR2 and FXR1 were confirmed by q-RT-PCR and WB  cell clone, a TP53 single KO (TP53 KO) cell clone, and a FXR2 single KO (FXR2 KO) cell clone. The knockout strategy is illustrated in TP53 and FXR2 double knockout clones /Cas9 to generate the isogenic homozygous t clones  and had ingle KO  or FXR2 ingle KO  clones. el assay . Resistat clones  and the ne Hep3B  suggesteed cells . InteresKO cells . Moreove or FMR1 . To confferation , furtherTP53 and FXR2 co-deleted cancer cells. Therefore, our data validate the role of FXR1 inhibition in blocking cell proliferation in TP53-deleted cancers in a collateral lethality manner upon passenger deletion of FXR2.FXR1 contains multiple functional domains including the newly discovered N-terminal tandem Tudor domain, KH domains and the RGG box, as illustrated in It has recently been reported that the tandem Tudor domain of FMR1 can recognize methylated lysine at histone H3 and recruit DNA repair protein, thus playing a role in DNA damage repair . We usedTP53/FXR2 co-deleted cancer cells. In order to obtain a comprehensive assessment of FXR1’s function, we conducted chromatin immunoprecipitation using a validated FXR1-specific antibody, followed by mass spectrometry (ChIP-MS), aiming to capture both the chromatin-associated and chromatin-free protein complexes in the cells. Proteins identified in the FXR1 pull-down complex from both H358 and KATOIII cells were analyzed. They are listed in We then investigated how FXR1 regulates proliferation in TP53/FXR2 co-deleted H358 cells using FXR1-, STAT1-, STAT3-, H3K4me3-, H3K9me3-, or H3K27me3-specific antibodies. We identified unfiltered 882 peaks of FXR1  in the  of FXR1 , primari of FXR1 . In agre of FXR1 . Interes of FXR1 . The per of FXR1 . The FXR of FXR1 . At TSS, of FXR1 . Among t of FXR1 . FXR1 pe of FXR1 , indicatARID1A, CASC4, TRAPPC9, GLI1, CITED1, NPAS3, and SSBP2 were consistently downregulated in both H358 and KATOIII cells. Data from H358 cells are shown in We selected FXR1-peak-associated putative target genes  overlap with FXR1 peaks, indicating that FXR1 cooperates with STAT1/3 in a small number of STAT target genes’ transcription in H358 cells . By cont2 >0.6 as the cutoff  FXR1deletion , and 3) g member . AlthougTP53 deletion is required for cancer cells to respond to FXR1 inhibition. This was confirmed in our engineered FXR2 single knockout cell clones which were only sensitive to FXR1 knockdown when p53 was downregulated. However, the mechanism of action remains elusive. There have been reports suggesting that p53 deficiency promotes the activation of JAK/STAT signaling, which may lead to cellular dependency on FXR1 because it regulates the localization of STAT1/3 at gene promoters  as previously described (http://www.broadinstitute.org/ccle) and analyzed using an R package ComplexHeatmap (https://bioconductor.org/packages/release/bioc/html/ComplexHeatmap.html).Copy-number analysis in human tumor tissues was performed using data from The Cancer Genome Atlas (TCGA) (escribed . Gene cohttp://www.atcc.org). The KMS-11 cell line was obtained from Horizon Discovery Ltd. . The L540 cell line was purchased from Biovector Science Lab, Inc . Although L540 is listed as a misidentified cell line by ICLAC , we used it as a TP53 single deletion cell model after comfirming the abscence of p53 mRNA and protein expression. The cell lines were cultured in RMPI 1640 medium (GIBCO) or DMEM medium (GIBCO) supplemented with 10% fetal bovine serum  and 100 U/mL penicillin-streptomycin (GIBCO) at 37°C with 5% CO2. All cell lines were authenticated by Short Tandem Repeat (STR) profiling and routinely monitored for mycoplasma contamination. The source and mycoplasma status as well as the Research Resource Identifiers (RRIDs) of each cell line are listed in KATOIII, HL-60, H358, MKN45, AGS, HepG2, A549, H1299, MG-63, SKOV3, Hep3B, and HEK 293 T cell lines were purchased from American Type Culture Collection . The detailed procedure and information are in the Supplementary methods.ChIP sample preparation follows the manual of SimpleChIP Enzymatic Chromatin IP Kit . The experimental procedure and reagent information for ChIP-WB, ChIP-MS, ChIP-seq, or ChIP-PCR are in the Supplementary methods.In cell assays, three independent replicates were included in each experiment. Student T Test was performed to compare the differences between two groups with normal distribution. All data were analyzed using GraphPad Prism 6.http://www.sigmaaldrich.com/), and the hairpin sequences were cloned into the doxycycline (Dox)-inducible lentiviral vector pLKO-Tet-On with Puromycinas as a selection marker. Five shRNAs targeting each gene  were screened.Gene-specific shRNA sequences were obtained from the Sigma website  and co-transfected into HEK 293 T cells at 70–80% confluency in a 10 cm Petri dish. At 72 hr post transfection, the supernatant containing virus particles was collected and filtered through a 0.45 µm PVDF Millex syringe filter.Each cell line was subjected to a puromycin tolerance test to determine the optimal concentration for establishing stable cell lines. To generate a shRNA-expressing stable cell line, cells were infected with lentiviral particles with 8 µg/ml polybrene. After 48 hr, the cells were selected by 0.5–2 µg/ml puromycin  for 7 days. shRNA expression was induced by 0.1–1 µg/ml Dox , and the knockdown efficiency was tested by WB assay or q-RT-PCR. Dox concentration was optimized for each stable cell line to achieve the most optimal knockdown efficiency at the minimum dose.FXR1, FXR2 or FMR1 gene open reading frame and selected using 2–6 µg/ml blasticidin  for stable cell line generation. The inserted open reading frames are: FXR1_a, NM_005087.3; FXR1_b, NM_001013438.2; FXR1_c, NM_001013439.2; FXR2, – NM_004860.3; and FMR1, NM_002024.5. The sequence of the FXR1 shRNA resistant mutation was CGCCAGGTTCCATTTAATGAA mutated to CGTCAAGTACCGTTCAACGAG and TTGCGAAGTATTCGTACGAAGTTG mutated to TTACGGAGCATCCGAACAAAATTA.For ectopic expression of proteins of interest, cells were infected with the lentiviral pLenti6.3-MCS construct encoding the FXR1 shRNAs were inserted in adenoviral vector pAd/CMV/IRES/GFP. The adenoviruses were produced in HEK 293 T cells by transient transfection of the plasmids. Virus titer was determined by counting GFP-positive cells after adenoviral infection. In the proliferation assay, adenoviruses were added to the cell culture medium and replenished 4 days afterwards to achieve efficient FXR1 knockdown.The shRNA target sequence is listed in Cells were lysed using RIPA lysis buffer  with complete protease inhibitor cocktail  and phosphatase inhibitor cocktail . The protein concentration was detected by BCA kit . The protein lysate was denatured by adding NuPAGE sample reducing agent  and NuPAGE LDS Sample buffer  followed by heating at 95°C for 5 min. Equal amounts of protein lysate was loaded to the NuPAGE Electrophoresis System (Life Technologies) according to the manufacturer's protocol. Proteins in the gel were transferred to the nitrocellulose membrane in iBlot 2 Transfer Stacks (IB23001 and IB23002) using the iBlot 2 Dry Blotting System . The membrane was blocked in SuperBlock Blocking Buffer in TBS , incubated with the specific primary antibodies for the protein of followed by reaction with the secondary antibodies. The protein signals were detected using SuperSignal West Femto Maximum Sensitivity Substrate , and were scanned and quantified using ChemiDoc MP System (BIO-RAD).The antibodies and their Research Resource Identifiers (RRIDs) used in this study are listed in Cell proliferation rate was detected by MTS assay. Briefly, the cells were seeded in 96-well plates at optimal density in 100 µl medium per well followed by treatment. Cells were added with CellTiter 96 AQueous One Solution Reagent  at the indicated time by following the manual instructions and subjected to absorbance recording at 490 nm using a PowerWave HT Microplate Spectrophotometer (BioTek). The cell proliferation rate in MTS was determined by measuring absorbance at 490 nm. In the duplicated experiment, cell images were scanned after fixing the cells with cold methanol and staining with 0.1% crystal violet. In some cases, the cells were seeded on the solidified Matrigel (BD Bioscience) layer in 96-well plate (50 µl per well) to measure the organoid growth.Cell apoptosis was determined by analyzing Caspase3/7 activity using the Caspase-Glo3/7 assay kit  according to the kit instructions or by detecting the protein level of cleaved PARP  in Western blot. The luminescence signal in the Caspase3/7 assay was recorded by an Envision 2104 Multilabel reader (Perkin Elmer).6 cells in 0.2 ml PBS:Matrigel (1:1 mixture) for tumor development. When tumor size reached approximately 180 mm3 on day 14, mice were randomly grouped (n = 6) and Doxycycline (2 mg/ml) was added to drinking water. Doxycycline was replenished twice per week. Tumor volume and body weight were measured twice a week. Tumor size was measured using a calliper, and tumor volume was calculated according to the following equation: tumor volume (mm3) = length (mm) × width2 (mm2) × 0.5. The experiment was terminated when the tumor volume reached 2,000 mm3 according to animal welfare guidelines. All animal studies were carried out under IACUC number R20150728-Mouse and Rat approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec. Summary statistics, including mean and the standard error of the mean (s.e.m), were provided for the tumor volume of each group at each time point. The A549-derived xenograft model was studied using a similar method.The HL-60-derived xenograft model in female BALB/c nude mice was used to determine FXR1’s function in tumor growth. Briefly, the female BALB/c nude mice (age 6–8 weeks) housed under pathogen-free conditions were inoculated subcutaneously in the right flank with HL-60 cells stably expressing control shRNA (shCtrl) or Doxycycline-inducible FXR1-sh3. Each mouse received 5 × 10TP53-to-FXR2, TP53 alone, FXR1 alone, or FXR2 alone)were designed. The synthesized double-strand DNA of the gRNA was inserted into the pU6-gRNA_SPycas9-2Acherry vector expressing Cas9 protein (Guide RNA (gRNA) was designed according to the formula of GN20GG or CCN20C in the forward or reverse strand of genomic DNA, respectively . Two set protein . To testThe donor plasmid contains the recombination sequences compatible with the 5’ and 3’ arms of the genomic region of interest and the GFP-coding sequence in vector pcDNA3.1. The mixture of gRNA-Cas9 plasmids targeting the 5’ or 3’ sequence of the genomic region and donor plasmid at ratio 1:1:1 was transfected into the candidate cells in a 10 cm Petri dish using XtremeGene. One week post-transfection, the GFP-expressing cells were sorted and seeded into a 96-well plate at a density of one cell per well using FACSArisII (BD). The single cell clones were cultured for two weeks to identify and filter out the cells with transient GFP signal. The clones with strong GFP signal, which would have potential insertion and replacement of the genomic region of interest by the donor sequence, were subjected to knockout validation by genomic PCR-sequencing, q-RT-PCR, and Western blot.The sequence of genomic PCR primers is listed in ChIP sample preparation: ChIP samples were prepared using SimpleChIP Enzymatic Chromatin IP Kit  according to the manufacturer’s protocol. Briefly, cells cultured in 15 cm Petri dishes were crosslinked with 1% formaldehyde when reaching 70–80% confluence. Crosslinking was terminated by adding 10 × glycine followed by rinsing the cells twice with cold PBS, and 4 × 107 cells were collected into one 15 ml conical tube. The cell pellet was resuspended and incubated in Buffer A on ice for 10 mins for cell lysis. The nuclei were collected by centrifugation at 3,000 rpm for 5 mins at 4°C and resuspended in Buffer B followed by centrifugation and supernatant removal. The nuclei pellets were resuspended in 1.0 ml Buffer B, and transferred to a 1.5 ml microcentrifuge tube. 7.5 µl of Micrococcal Nuclease was added and incubated for 20 mins at 37°C to digest the DNA to lengths of approximately 150–300 bp. After the termination of digestion by adding 100 µl of 0.5 M EDTA, the nuclei pellets were collected by centrifuging at 13,000 rpm for 1 min at 4°C. The nuclear pellets were resuspended in 1 ml ChIP buffer and separated into two tubes, and incubated on ice for 10 mins followed by subsequent sonication of lysates at 9W, 10 s ON/30 s OFF, nine cycles. Lysates were collected by centrifugation at 10,000 rpm for 10 mins at 4°C. The supernatant (single nucleosome lysate) was transferred to a new tube, and stored at −80°C for the following experiments. 50 µl of lysate was used for the analysis of chromatin digestion.Pulldown: The single nucleosome lysate was diluted with ChIP buffer with protease inhibitor cocktail . 10 µl of sample was saved as input. To reduce nonspecific binding, the lysate was pre-cleared with 10% BSA and 30 μl ChIP Grade Protein G Magnetic Beads  at 4°C with rotation for 1 hr. The supernatant was collected by placing the tube on a magnetic separation rack. Antibodies of interest or IgG control were added and incubated on rotation at 4°C overnight followed by incubation with 30 µl of magnetic beads for 2 hr at 4°C. Beads were washed with low salt wash buffer (100 µl 10 × ChIP Buffer in 900 ml water) three times and with high salt wash buffer (100 µl 10 × ChIP Buffer and 70 µl 5 M NaCl in 830 µl water) once on rotation.AB_1849204); anti-FXR2 ; anti-STAT1 ; anti-STAT3 ; rabbit IgG ; anti-H3K4me3 ; anti-H3K9me3 ; and anti-H3K27me3 .The antibodies used in ChIP pulldown were: anti-FXR1  for 5 min. Eluted proteins were separated in SDS-PAGE gel and analyzed by WB.For ChIP-MS, eluted proteins were separated in SDS-PAGE gel followed by gel fixing in buffer containing methanol, acetic acid, and water at 5:1:4 ratio. The gel was stained in EZBlue Gel Staining Reagent  and subjected to mass spectrometry (MS) analysis.For ChIP-seq or ChIP-PCR, beads were eluted with 150 µl of ChIP elution buffer for 30 mins at 65°C with gentle vortexing  in thermomixer. Eluted supernatant was incubated with 6 µl 5 M NaCl and 2 µl Proteinase K at 65°C for 2 hr in thermomixer to digest the protein. DNA was purified using spin columns, and sent for ChIP-seq or ChIP-PCR analysis.−5. The distribution of FXR1 ChIP-seq peaks were investigated by the annotations on the human genome. The peaks were binned into eight groups according to the positions relative to annotated genes: 5’ Distal (up to 100 kb upstream TSS), 5’ Proximal (up to 10 kb upstream TSS), 5’ TSS (5 kb upstream TSS to 1 kb downstream TSS), Intragenic (1 kb downstream TSS to 1 kb upstream TTS), 3’ TTS (1 kb upstream TTS to 5 kb downstream TTS), 3’ Proximal (up to 10 kb downstream TTS), 3’ Distal (up to 100 kb downstream TTS), and the remaining sequence defined as Intergenic region. The aggregation signals of FXR1, H3K4me3, STAT1, and STAT3 at genebody (TSS to TTS) plus upstream and downstream 5 kb region were extracted and plotted using in-house perl and MATLAB code. A hub for the UCSC genome browser was constructed in order to investigate the detailed information of the peaks. The summit positions of the FXR1 ChIP-seq peaks were extended to 100 bp both upstream and downstream as input region for HOMER (http://bioinfogp.cnb.csic.es/tools/venny/index.html). Protein function enrichment was analyzed using DAVID (http://www.broadinstitute.org/igv/).DNA from ChIP pull down complexes was subjected to library preparation according to Illumina's instructions (Part # 11257047). Sequencing was performed on Illumina HiSeq 2000. ChIP-seq data quality was analyzed using FastQC . Reads ior HOMER  to call ng DAVID , 2009. TThe sequence of ChIP-PCR primers is listed in 4HCO3 (Sigma Aldrich) and Acetonitrile (ACN) (Fisher Scientific) at 1:1 ratio, dried with SpeedVac, rehydrated in 300 μl of 10 mM DTT (Sigma Aldrich) solution at 56°C for 1 hr, and reacted in 25 mM IAA (Sigma Aldrich) at 37°C for 30 mins. After washing with 25 mM NH4HCO3 (Sigma Aldrich) for 10 mins, the gel pieces were digested with trypsin solution (0.01 g/L trypsin in 25 mM NH4HCO3) (Promega) at 37°C for 12 hr. The digested peptides were extracted using 300 μl of 50% ACN-0.1% FA. The extracted solution was collected and concentrated with SpeedVac. The samples were resolved with 0.1% FA and analyzed by nanoLC-MS/MS (Thermofisher). MS/MS was performed in a data-dependent mode in which the top 10 most abundant ions (S/N > 30) for each MS scan were selected for MS/MS analysis by HCD. Parameters used during searching included enzyme, trypsin; missed cleavages, One; dynamic modifications, oxidation (M); static modifications, Carbamidomethyl (C); precursor mass tolerance, 10 ppm; fragment mass tolerance, 0.1 Da. Parameters in data filtration with Prohits included kinds of exclusion proteins: Heat Shock, Ribosomal, Cytoskeleton, Keratin, Artifact Protein, Rib, Nucleoprotein and Albumin; Max SAINT < 0.7 . The stained gel was cut into multiple parts based on protein molecular weight. Gel pieces were destained in solution containing 50 mM NHNT < 0.7 . All MS NT < 0.7 , 2012.FXR1 interacting proteins were classified according to the GO (gene ontology) database. Protein function clustering was analyzed with DAVID. Visualization and network map of FXR1-interacting proteins were delineated using BioGRID in Cytoscape. Protein–protein interaction was analyzed using the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database.Total RNA was extracted using RNeasy Mini Kit  and Qiagen QIAcube 230 V w.starter Pack-2 machine, and reverse-transcribed using Oligo (dT) primer and the SuperScript III First-Strand synthesis system . q-RT-PCR using cDNA product as the template was performed using Power SYBR Green PCR Master Mix (Applied Biosystems (AB), 4367659) and an ABI 7900HT Fast Real-Time PCR-3 Machine with specific primers. Gene expression was quantified using the comparative Ct (cycle threshold) method and the results were normalized to GAPDH. Sequences of q-RT-PCR primers are listed in 2FC cutoff 0.6 and adjusted p value cutoff 0.01. KEGG pathway enrichment was investigated using GSEA  (save 10 µl as input), followed by rotation at 4°C for 30 mins. The reaction solution was spun down  to collect the supernatant in a new tube. 15 μl of Ni-NTA beads (Qiagen) was added to the collected supernatant followed by rotation at 4°C for 1 hr. Beads were collected by spinning at 1,000 g for 2 min, washed with 1 ml of Histone MLA Binding Buffer five times, suspended in 30 μl of 2 × SDS sample buffer and boiled to denature the proteins. The FXR1-bound histone MLA proteins were detected in a WB assay.Histone H3 MLA proteins with methylation at various residues were purchased from Active Motif (31208–31222). His-tagged-FXR1-Tudor (2-132) protein were expressed and purified from an escribed . In briehttp://www.selleck.cn/). Owing to the instability of the compound, the medium with inhibitor was replaced every 5 days.JAK inhibitor S-Ruxolitinib  was purchased from selleck . The siRNA oligonucleotides were produced by Invitrogen, and the MISSION siRNA Universal Negative Control #2 (Sigma) was used as a negative control. Five siRNAs for each gene were tested. siRNAs were introduced into cells at a final concentration of 5–20 nM using Lipofectamine RNAiMAX Transfection Reagent  and Opti-MEM  according to the manufacturer’s protocol. Knockdown efficiency was assessed 48 hr post-transfection by q-RT-PCR or WB. Sequences of siRNAs are listed in Gene-specific siRNA sequence was designed using the software in Hannon Lab  with complete protease inhibitor cocktail , phosphatase inhibitor cocktail , and PMSF . Lysate was incubated, at 4°C overnight, with 100 μl Flag-M2 magnetic beads  to purify Flag-tagged-FXR1 protein or with 100 μl Influenza Hemagglutinin (HA) magnetic beads  to purify HA-tagged-STAT protein. Beads were washed with 1 ml lysis buffer three times and with 600 μl elution buffer  once. Target protein was eluted with 30 μl of Flag peptide solution  to obtain Flag-tagged-FXR1 protein or with HA peptide  to harvest HA-tagged-STAT protein.For in vitro pulldown, 25 µl purified Flag-tagged-FXR1 protein was incubated with 75 µl purified HA-tagged-STAT1 or -STAT3 protein in 500 µl binding buffer  at 4°C overnight. 30 µl Flag or HA magnetic beads were added and incubated at 4°C for another 3 hr. Beads were washed with wash buffer  three times. The eluted products in 30 µl elution buffer were subjected to WB analysis.2, 30 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol [DTT], protease inhibitor cocktail, 100 U/ml RNasin, 0.1% Nonidet P40 [NP40]) and divided into two parts. 2% of lysate was saved as input. The lysate was pre-cleared by 10% BSA and 30 µl ChIP Grade Protein G Magnetic Beads  on rotation for 1 hr at 4°C. The supernatant was collected by a magnetic separation rack, incubated with FXR1 antibody  or IgG control  for 1 hr at 4°C, and incubated with 30 µl of magnetic beads for 1 hr at 4°C on rotation. Beads were washed with NT2 buffer  five times. Endogenous RNA protein complexes (RNP) were eluted from antibody/Protein G beads by incubation with 150 µl of ChIP elution buffer for 30 mins at 65°C with gentle vortexing (1200 rpm) in thermomixer. Proteins in the elution were digested by Proteinase K. RNA was purified using spin columns from RNeasy Mini Kit  and reverse-transcribed using Oligo (dT) primer and the SuperScript III First-Strand synthesis system . The cDNA was subjected to RT-PCR assay using AccuPrime Pfx SuperMix   . The PCRChIP-seq data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE79707. RNA-seq data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE101754. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Kevin Struhl (Senior Editor) and three reviewers, one of whom, Irwin Davidson, is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Pierre Massion (Reviewer #2).Thank you for submitting your article "FXR1 regulates transcription and is required for tumor growth in TP53 homozygous deletion cancers" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This study reports that FXR1 plays an essential role in the proliferation of tumour cells bearing a homozygous double TRP53-FXR2 deletion. The paper shows that knockdown of FXR1 in cells with the double deletion arrests their proliferation. The authors go onto show that FRX1 binds to a small number of promoters and facilitates the recruitment of STAT1 that activates their expression and that some of these target genes are essential for the proliferation of the TRP53-FXR2 deleted cells. The reviewers found that the data are novel and move the field forward, strengthening the evidence for targeting FXR1 in cancers where TRP53 and FXR2 are deleted.Essential revisions:The additional experiments that should be provided concern two main points. The relationship between FXR1 and FXR2 function and secondly the crosstalk between FXR1 and STAT1/3 that is not properly established. The reviewers are aware that these may take more than two months to complete, but they will be required.Cells with a double TRP53/FXR2 double deletion are dependent on FXR1. The authors should use CRISPR/Cas9 to make a TRP53/FXR1 double deletion and show that the resulting cells are now FXR2 dependent. This would strengthen the idea that FXR1 and FXR2 fulfill at least partially redundant and essential functions. For example, the authors show that depletion of FXR2 reduces expression of the FXR1 target gene CASC4 in cells where FXR1 is normally expressed . This reAlong the same lines: the authors performed FXR1 ChIP-seq and they performed FXR2 ChIP-qPCR. As they have a ChIP-grade FXR2 antibody, they should perform FXR2 ChIP-seq to determine the overlap in genomic binding and function of FXR1 and FXR2.According to the ChIP-seq experiments FXR1 has only a limited number of binding sites around half of which were also marked by H3K4me3. What about the sites that showed no H3K4Me3, what do these sites correspond to, promoters of repressed genes? Also, as only 37-49% of FXR1 sites correspond to STAT-binding sites, what do the others correspond to? The authors should perform motif enrichment analyses (like for example MEME ChIP) to identify other transcription factors that may be enriched at these sites. Also without performing STAT1/3 ChIP-seq how can the authors be sure that there is no STAT protein present at these sites in these cells? More importantly, the authors state that only a small subset of STAT sites is co-occupied by FXR1. The relationship between FXR1 and the STATs is therefore not clear and the factors that dictate FXR1 genomic binding remain unknown. There are perhaps other factors that recruit FXR1, that may or may not have been detected by mass-spectrometry. A more thorough bioinformatic analyses of the FXR1 binding sites is required. Otherwise one could argue that FXR1 acts as a cofactor for a transcription factor, other than STAT1/3, present at almost all target genes.In relation to this, there is some inconsistency between the results shown in Some of these issues could be clarified by performing RNA-seq to get an idea of the overall effects of FXR1 silencing on gene expression. Are there a small number of affected genes perhaps in line with the small number of occupied sites? Or alternatively, FXR1 silencing may have widespread effects on gene expression that go well beyond the small number of affected genes investigated. If so how could this be explained?According to IARC database (http://p53.iarc.fr/CellLines.aspx), and the literature (https://www.ncbi.nlm.nih.gov/pubmed/2155427), Hep3B should be a p53-null cell line. Can the authors comment on this and have they verified the identity of the Hep3B cells they used? Essential revisions:The additional experiments that should be provided concern two main points. The relationship between FXR1 and FXR2 function and secondly the crosstalk between FXR1 and STAT1/3 that is not properly established. The reviewers are aware that these may take more than two months to complete, but they will be required.Cells with a double TRP53/FXR2 double deletion are dependent on FXR1. The authors should use CRISPR/Cas9 to make a TRP53/FXR1 double deletion and show that the resulting cells are now FXR2 dependent. This would strengthen the idea that FXR1 and FXR2 fulfill at least partially redundant and essential functions. For example, the authors show that depletion of FXR2 reduces expression of the FXR1 target gene CASC4 in cells where FXR1 is normally expressed . This reTP53/FXR1 double knockout (DKO) cells using CRISPR-Cas9, and stably expressed FXR2 shRNA-2 or FXR2 shRNA-3 in these DKO cells. We observed that knockdown of FXR2 upon Dox induction suppressed proliferation in these cells  both FXR1 and FXR2 can rescue FXR1 shRNA-inhibited cell proliferation , 2) TheyAccording to the ChIP-seq experiments FXR1 has only a limited number of binding sites around half of which were also marked by H3K4me3. What about the sites that showed no H3K4Me3, what do these sites correspond to, promoters of repressed genes? Also, as only 37-49% of FXR1 sites correspond to STAT-binding sites, what do the others correspond to? The authors should perform motif enrichment analyses (like for example MEME ChIP) to identify other transcription factors that may be enriched at these sites. Also without performing STAT1/3 ChIP-seq how can the authors be sure that there is no STAT protein present at these sites in these cells? More importantly, the authors state that only a small subset of STAT sites is co-occupied by FXR1. The relationship between FXR1 and the STATs is therefore not clear and the factors that dictate FXR1 genomic binding remain unknown. There are perhaps other factors that recruit FXR1, that may or may not have been detected by mass-spectrometry. A more thorough bioinformatic analyses of the FXR1 binding sites is required. Otherwise one could argue that FXR1 acts as a cofactor for a transcription factor, other than STAT1/3, present at almost all target genes.We thank the reviewer for raising this point that only about half of FXR1 binding sites overlapped with H3K4me3. We found that the majority of the overlapped peaks located at TSS region added in . We analUpon the reviewer’s comment on our conclusion regarding FXR1 and STAT relationship, we conducted STAT1 and STAT3 ChIP-seq in the same H358 cells to re-evaluate FXR1 and STAT genomic binding instead of using STAT ChIP-seq database as in the original manuscript. Our ChIP-seq results are in an agreement with our previous results obtained from Encode database. STAT1 and STAT3 showed sporadic binding in the genome of H358 cells. In the same cells, about 24.38% (215 in 882) and 25.74% (227 in 882) of FXR1 binding sites overlap with STAT1 and STAT3 binding, respectively . EspeciaFurther, the majority of the FXR1-STATs overlapped peaks located at TSS region . We also-2-associated transcription factor 1) was revealed in FXR1 protein complex  showed moderate downregulation , and the literature (https://www.ncbi.nlm.nih.gov/pubmed/2155427), Hep3B should be a p53-null cell line. Can the authors comment on this and have they verified the identity of the Hep3B cells they used?Based on the CCLE database, Hep3B carries 1.3 copies of TP53 suggesting a partial deletion. Our WB and Q-PCR assay also indicated the expression of p53 mRNA and protein in Hep3B, while FXR2 level is undetectable. Therefore, Hep3B served as a cell model of FXR2 single deletion in this study .


We present a purification and biochemical characterization of recombinant B. burgdorferi RecA protein. We find that B. burgdorferi RecA displays the expected properties of being a DNA-dependent ATPase, of having an intrinsic binding preference for ssDNA over dsDNA enhanced by ATP binding, of promoting DNA pairing and strand exchange reactions and of having a detectable coprotease activity with E. coli LexA repressor. DNA pairing and strand exchange reactions promoted by B. burgdorferi RecA show an unusually strong dependence upon the presence of the cognate ssDNA binding protein (SSB) but are very sensitive to inhibition by SSB when the ssDNA was prebound by SSB. This indicates B. burgdorferi RecA may have an enhanced requirement for recombinational mediators to promote RecA-SSB exchange, despite the absence of homologues of the RecF pathway proteins that normally play this role in eubacteria. Finally, we do not find any unusual, intrinsic properties of B. burgdorferi’s RecA protein to explain the unusual phenotype of recA mutation and suggest that there may be alternative recombinase functions that could explain the ‘missing’ phenotypes.RecA plays key roles in DNA recombination, replication and repair. Mutation of  E. coli and other eubacteria reveal that RecA is a DNA-dependent ATPase that possesses DNA pairing and strand exchange activities. Additionally, the activated form of RecA (the ATP-bound presynaptic filament formed on ssDNA) senses excessive ssDNA production to signal the presence of DNA damage or invading DNA through activation of a specialized DNA damage response referred to as the SOS response. Activated RecA triggers the SOS response by virtue of its coprotease activity, wherein it chaperones the transcriptional repressor, LexA, to cleave itself, thereby derepressing a suite of ~40 genes involved in the DNA damage response ATP in a total volume of 60 μL. After incubation on ice for 5 min the 1.5 mL reaction tubes were placed on a transilluminator and subjected to UV irradiation (312 nm) for the indicated times. Where indicated, ϕX174 virion DNA was present at 10 μg/ml. The samples were prepared for gel loading by addition of 5X SDS-load dye to a 1X concentration, followed by denaturation of the samples at 95°C for 6 min. After the gel run the gel was washed for 30 min in several changes of water then Coomassie stained to visualize the position of the bands. The gel was then wrapped in plastic and exposed to a phosphorimaging screen to detect protein that had become crosslinked to [γ32P]ATP.The ability to bind ATP was assayed by incubating 2 μM RecA in reaction buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl2, 1 mM DTT, 100 μg/ml bovine serum, 50 mM NaCl, 100 μM ATP and 66 nM [γ32P]ATP in a total volume of 30 μL. After incubation at 37°C for the times indicated in the figure legend, an aliquot of the reaction was spotted onto a polyethyleneimine thin-layer chromatography plate that was developed with 1M formic acid and 0.5M LiCl. The plate was air dried and then exposed to a phosphorimaging screen for determination of the free phosphate to total ATP ratio. When present, the indicated DNA effectors were present at 10 μg/mL.ATPase activity was determined by incubating 1 μM RecA in buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl2, 1 mM DTT, 100 μg/ml BSA, 50 mM NaCl and 2 mM ATP or AMPPNP. The reactions were performed in 120 μL reaction volumes by incubating 2 μM RecA with 20 nM of 5’-32P endlabeled 95 nt OGCB396 oligonucleotide (1.9 μM nt) at 30°C for 5 minutes followed by addition of pUC19 (77 μM nt) and incubation at 37°C for the indicated times. Where indicated, B. burgdorferi SSB was added to the indicated concentration. When added first, SSB was pre-incubated with the OGCB396 donor at 30°C for 5 min prior to RecA and pUC19 addition. When added second, SSB was added immediately before pUC19 addition. Reaction timepoints were examined by withdrawing 28 μL aliquots into 5X reaction stop dye to a final concentration of 1X. The 5X stop dye contains 100 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 3% SDS, 30% glycerol, 0.024% bromophenol blue. Samples were deproteinated by addition of pronase to a final concentration of 50 μg/mL and by continued incubation at 37°C for 10 min. D-loops were visualized by loading the deproteinated samples on a 0.8% agarose 1X TAE gel electrophoresed at 3V/cm. The gel was transferred onto a Hybond N membrane on a 3MM Whatman paper backing and dried at 80°C under vacuum for 45 min prior to exposure to a phosphorimaging screen.The ability to form D-loops was assayed in a buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl2, 1 mM DTT, 100 μg/ml BSA, 50 mM NaCl and 2 mM ATP, AMPPNP or ATP[γS]. The reactions were performed in 60 μL reaction volumes by incubating 2 μM RecA with 20 nM of the 70 nt OGCB748 oligonucleotide (1.4 μM nt) at 30°C for 5 minutes followed by addition of 20 nM of the 5’-endlabeled 35 bp duplex assembled from oligonucleotides OGCB664/665 (1.4 μM nt) and continued incubation at 37°C for the indicated timepoints. Where present, SSB was added either just prior to addition of the duplex DNA substrate (added 2nd) or pre-incubated at 30°C for 5 min with the single-stranded donor DNA followed by RecA and duplex DNA addition and continued incubation at 37°C. Reaction timepoints were examined by withdrawing 18 μL aliquots into 5X reaction stop dye to a final concentration of 1X. The 5X stop dye contains 100 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 3% SDS, 30% glycerol, 0.024% bromophenol blue. Samples were deproteinated by addition of pronase to a final concentration of 50 μg/mL followed by continued incubation at 37°C for 10 min. The strand exchange reaction timecoureses were visualized by loading the deproteinated samples on an 8% PAGE 1X TAE/0.1% SDS gel electrophoresed at 10V/cm. The gel was transferred to 3MM Whatman paper and dried at 80°C under vacuum prior to exposure to a phosphorimaging screen.The ability to perform strand exchange was assayed in a buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl2 indicated in the legends. The reactions were performed in 120 μL reaction volumes by incubating 2 μM RecA with ϕX174 virion (5.1 μM nt) at 30°C for 5 minutes followed by addition of XhoI-linearized, ϕX174 RF1 DNA (5.1 μM nt) followed by continued incubation at 37°C for the indicated timepoints. When added first, SSB was incubated with ϕX174 virion at 30°C for 5 min prior to the addition of RecA and linear ϕX174 duplex DNA followed by continued incubation at 37°C for the times indicated above the gel. When SSB was added second, RecA was incubated with ϕX174 virion at 30°C for 5 min prior to the addition of SSB and linear duplex DNA, followed by continued incubation at 37°C for the times indicated above the gel. Reaction timepoints were examined by withdrawing 18 μL aliquots into 5X reaction stop dye to a final concentration of 1X. The 5X stop dye contains 100 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 3% SDS, 30% glycerol, 0.024% bromophenol blue. Samples were deproteinated by addition of pronase to a final concentration of 50 μg/mL followed by continued incubation at 37°C for 30 min. Strand exchange products were visualized by loading the deproteinated samples to a 0.8% agarose 1X TAE gel electrophoresed at 3V/cm for 4 h. The gel was transferred to DE81 paper and dried at 80°C under vacuum for 45 min prior to exposure to a phosphorimaging screen.The ability to perform strand exchange with long substrates was assayed in a buffer containing 25 mM HEPES (pH 7.6), 1 mM DTT, 100 μg/ml BSA, 50 mM NaCl and 2 mM ATP or ATP[γS] and the concentration of MgClB. burgdorferi RecA was assayed for coprotease activity with E. coli LexA. 60 μL reactions containing 25 mM HEPES (pH 8.2), 1 mM MgCl2, 1 mM DTT, 50 mM NaCl and, where indicated 1 mM ATPγS or ATP. In reactions with ssDNA 2 μM (nt) of OGCB396 was pre-incubated with 1.25 μM RecA at 30°C for 5 min prior to addition of 1.5 μM LexA and continuation of the incubation at 30°C for 16 h. Control reactions without RecA were conducted as above without inclusion of RecA. LexA autocleavage was visualized by denaturation of the samples in SDS/DTT load dye and application to a 5/18% SDS-PAGE gel. The gel was stained with Coomassie Brilliant Blue. Reactions supplemented with B. burgdorferi SSB contained 190 nM SSB that was added just before LexA. This gel was Coomassie stained and then silver stained to improve detection of the low abundance SSB.E. coli RecA has an intrinsic, kinetic preference for binding ssDNA over duplex DNA and this intrinsic, high affinity state for ssDNA is stabilized by ATP binding  formed only joint molecules and and some aggregates rather than the full length recombinant product of nicked circular DNA (st ATP[γS] panel). This is consistent with what is observed for bacterial RecA’s but contrasts with eukaryotic Rad51, which does not have a strong requirement for ATP hydrolysis in this assay [The ability of elevated ,41. Usining time . The bufproteins . As obseis assay ,42.B. burgdorferi RecA was shown in Figs 2+ and, when added, SSB was added after pre-incubation of RecA with ssDNA. The reaction without SSB failed to yield products of any kind, while addition of 0.3 μM SSB supported slow formation of some joint molecules. Addition of 0.6 and 0.9 μM SSB supported progressively faster reactions that started to produce detectable levels of nicked circular strand exchange products in the 2-hour timecourse tested  but to be independent of RecA [Finally, it seems possible that  of RecA ,20,22. B of RecA  and a pu of RecA . Additio of RecA ,51. Sing of RecA .S1 FigE. coli K12 (UniProtKB accession # P0A7G6) and B. burgdorferi B31 (GenBank accession # AAC66507) aligned. Amino acid identities are indicated with coloured boxes. The Zappo colouring scheme that groups amino acids by their physiochemico properties was followed [http://www.bioinformatics.org/sms/; [RecA sequences from followed . Boxed irg/sms/; ).(TIF)Click here for additional data file.S2 FigE. coli K12 (UniProteKB accession # P0AGE0.2) and B. burgdorferi B31 (GenBank accession # AAC66492.1) aligned. Amino acid identities are indicated with coloured boxes. The Zappo colouring scheme that groups amino acids by their physiochemico properties was followed [http://www.bioinformatics.org/sms/; [SSB sequences from followed . The alirg/sms/; ).(TIF)Click here for additional data file.S3 FigA) 12% SDS-PAGE analysis of RecA (K88R) ATP photoaffinity binding assays.B) Summary of ATPase assay results with the indicated concentrations of RecA -/+ 10 μg/mL ϕX174 virion (ssDNA) and ϕX174 RF1 (dsDNA). ATPase assays containing 1 μM RecA were incubated at 37°C for 120 min for ϕX174 virion (ssDNA) containing reactions or at 37°C for 120 min for DNA-free and ϕX174 RF1 (dsDNA) containing reactions. The mean and standard deviation of at least three independent experiments is shown.C) Summary of ATPase assay results with RecA vs. RecA (K88R) -/+ 10 μg/mL ϕX174 virion DNA. ATPase assays containing 1 μM wild type RecA were incubated at 37°C for 30 min for ϕX174 virion (ssDNA) containing reactions or at 37°C for 120 min for DNA-free containing reactions. ATPase assays containing 1 μM RecA (K88R) were incubated at 37°C for 120 min for both DNA-free and ssDNA containing reactions. The mean and standard deviation of at least three independent experiments is shown.(TIF)Click here for additional data file.S4 Fig0.8% agarose 1X TAE gel analysis of D-loop formation conducted -/+ the presence of an ATP regeneration system. RecA was present at 2 μM, donor ssDNA at 1.9 μM (nt) and pUC19 at 77 μM (nt). Where indicated, SSB was present at 300 nM and was added after pre-incubation of RecA with the donor ssDNA. The ATP regeneration system consisted of 3.3 mM phosphoenolpyruvate (PEP) and 10 units/mL pyruvate kinase.(TIF)Click here for additional data file.S5 FigA) Schematic representation of the strand invasion assay indicating the effect of the order of SSB/RecA addition and ATP hydrolysis on D-loop recovery.B) 0.8% agarose 1X TAE gel analysis of strand invasion timecourses performed with 0.3 μM SSB and 2 μM RecA in buffer containing ATP (left panel) or AMPPNP (right panel).2, 1 mM DTT, 100 μg/ml BSA, 50 mM NaCl and 2 mM ATP or AMPPNP in a 120 μL reaction volume. The assay was conducted as a staged reaction with pre-incubation of SSB, RecA or both SSB and RecA simultaneously with the donor ssDNA  followed by addition of SSB or RecA (where required) and supercoiled pUC19 target plasmid and continued incubation at 37°C for the indicated times.D-loop formation was assayed in a buffer containing 25 mM HEPES (pH 7.6), 2 mM MgCl(TIF)Click here for additional data file.S6 Fig32P-endlabels, red/shaded lines represent endlabeled strands.B) 8% PAGE 1X TAE/0.1% SDS gel analysis of strand exchange reactions with RecA (K88R) and SSB. RecA was present at 2 μM, donor ssDNA at 1.4 μM (nt) and target duplex at 1.4 μM (nt). When added, SSB was added after pre-incubation of RecA with the single stranded donor and was present at 300 nM. The proteins and nucleotide co-factor present are indicated in the loading key above the gels.A) Schematic representation of the oligonucleotide substrates used. The ssDNA donor oligonucleotide is 70 nt in length while the 5’-endlabeled duplex target DNA is 35 bp. * denote the (TIF)Click here for additional data file.S7 FigE. coli RecA and SSB. RecA was present at 2 μM, donor ssDNA at 1.4 μM (nt) and target duplex at 1.4 μM (nt). When added, SSB was added after pre-incubation of RecA with the single stranded donor and was present at 300 nM. The proteins and nucleotide co-factor present are indicated in the loading key above the gels.8% PAGE 1X TAE/0.1% SDS gel analysis of strand exchange reactions with (TIF)Click here for additional data file.S8 FigE. coli RecA and SSB and a buffer containing 2 mM ATP. RecA was present at 2 μM, ϕX174 virion (css) at 5.1 μM (nt) and XhoI-linearized ϕX174 duplex DNA (lds) at 5.1 μM (nt). SSB was added after pre-incubation of RecA with the single stranded donor and was present at 0.9 μM. The concentration of added MgCl2 is noted in the loading key above the gel. The migration position of the substrate DNA is noted to the left of the gel and of the products to the right. Under our gel conditions css and lss have identical gel mobilities.0.8% agarose 1X TAE gel analysis of strand exchange reactions performed with (TIF)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.S1 Sequence(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s−1) induced a sustained increase in [Ca2+]i in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca2+. Thrombus formation was studied on collagen-coated surfaces using DiOC6-stained platelets. In addition, [Ca2+]i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear.The role of mechanosensitive (MS) Ca Platelet activation plays a crucial role in the physiological process of hemostasis but is also the key precipitating event leading to arterial thrombosis and thus potentially life threatening pathological events such as myocardial infarction or stroke. In the circulation, shear stress exerted by laminar flow of blood is regarded as a vital environmental factor during platelet activation in both normal and pathological situations. For example, shear stress is required at the early stages of the hemostatic machinery where it unfolds von Willebrand factor to reveal the binding domains to glycoprotein Ib and thus allow attachment to collagen exposed at an injury site .2+]i is a pivotal signaling event that is essential for most major functional responses during platelet activation, including cytoskeletal rearrangements and integrin inside-out signaling : F/F0 increased from 1.0 ± 0.1 at no shear (0.0 s−1) to 1.1 ± 0.2, 1.2 ± 0.3, and 1.4 ± 0.4 at normal arterial (1002.6 s−1), stenotic (low) (2282.7 s−1), and stenotic (high) (3989.3 s−1) shear rates, respectively (C). In the absence of extracellular Ca2+  shear-dependent increases in the Fluo-3 signal were abolished except for a small, residual response (F/F0 = 1.2 ± 0.2) at the highest flow rate . Pretreating the attached Meg-01 cells with 2.5 μm GsMTx-4 abolished all increases in Fluo-3 signal in response to applied shear stress . We also compared the effect of shear flow in parallel-plate flow chambers on [Ca2+]i in human umbilical vein endothelial cells, which are known to express functional Piezo1 channels  389.3 s−1 ress see  and a moress see . In the 2+ entry observed in Meg-01 cells led us to develop a method to examine whether a similar pathway exists in human platelets. Previous measurements of Ca2+ responses under arterial shear in single platelets have used glass coverslips coated with adhesive receptor ligands such as fibrinogen and collagen or synthetic peptides mimicking their binding domains . Exposure of attached unstimulated platelets to normal arterial shear stress (1002.6 s−1) resulted in multiple transient increases in cytosolic Ca2+ after a delay of 1–2 min (B and C). Subsequent arrest of flow led to a reduction but not complete inhibition of this Ca2+ response, although a second application of arterial shear caused a further increase in the number of Ca2+ transients . The Ca2+ responses were quantified using the F/F0 integral for a total of 4 min . Prior to the application of flow, when only occasional Ca2+ transients were observed, this value was 0.4 ± 0.6; during normal arterial shear it increased significantly to 1.9 ± 1.9 . In every platelet sample, a proportion of attached platelets did not show increased Ca2+ transients in response to arterial shear. However, in the platelets that did respond, the F/F0 integrals during a first and second exposure to either arterial or stenotic shear were not significantly different (E and G). This allowed the effects of GsMTx-4 and removal of Ca2+ to be assessed during the second cycle of shear. The shear-induced Ca2+ transients were significantly reduced by either the addition of 2.5 μm GsMTx-4  or removal of extracellular Ca2+  . A higher arterial shear (3989.3 s−1), equivalent to the situation when stenosis or narrowing occurs in the arteries, induced a larger and more significant increase  above prestimulus (no flow) levels when compared with normal arterial shear  and was also inhibited by GsMTx-4 . Together, these results suggest that platelets, like Meg-01 cells, possess a MS Ca2+ influx pathway induced by physiological levels of shear. The major difference between the response in these two cell types was the longer delay between stimulus application and the [Ca2+]i increase in platelets compared with Meg-01 cells. This may result from the greater rigidity of platelet surface membranes, a consequence of the extensive cortical cytoskeleton that will resist deformation and thus activation of MS ion channels . All three aspects of thrombus dimension analyzed  were reduced compared with control conditions; mean thrombus height to 52% of control (1.2 ± 0.5 to 0.6 ± 0.4 μm), mean thrombus volume to 52% of control , and mean surface coverage to 62% of control (26.4 ± 10.2 to 16.3 ± 8.6%) (B). In contrast, GsMTx-4 had no effect on the aggregation response of platelets to 1 μg/ml collagen measured in stirred suspensions, which induce minimal levels of shear (C). This response depends upon activation of the αIIbβ3 integrin (C). Together, these data suggest that the underlying GsMTx-4-sensitive pathway is crucially dependent upon application of shear stress for its activation.Pretreating whole blood with GsMTx-4 for 30 s consistently resulted in a marked reduction in thrombus formation under arterial flow on a collagen surface A. All th ± 8.6%) B. In conof shear C. This rintegrin , as demointegrin C. Togeth2+ entry pathways are unknown. Store-operated Ca2+ entry through Orai1 channels is a major Ca2+ entry pathway evoked by multiple agonists and can be selectively activated by depletion of intracellular Ca2+ stores with the SERCA inhibitor thapsigargin . Furthermore, GsMTx-4 caused no inhibition of Ca2+ entry through transient receptor potential cation channel subfamily C member 6 (TRPC6) ion channels directly activated using the diacylglycerol analogue 1-oleoyl-2-acetyl-glycerol (OAG) (60 μm) . A third Ca2+-permeable pathway in platelets is the ATP-gated P2X1 channel, which can be selectively activated by α,β-meATP (2+ response (to 60% of control) to a supramaximal concentration of α,β-meATP  . Loss of P2X1 receptor activity did not contribute to the inhibition of platelet shear-dependent Ca2+ responses by GsMTx-4 (A). However, P2X1 receptors will be functional in the thrombus formation experiments using whole blood because of the ectonucleotidase activity of plasma and leukocytes , consistent with previous reports . Using NF449 alone, mean thrombus volume was reduced from 99,856 ± 30,184 μm3 to 62,678 ± 28,802 μm3(63% of control), whereas using GsMTx-4 alone and GsMTx-4 and NF449 combined, there were reductions to 31%  and 25%  of control in thrombus volume, respectively . Similarly, mean percentage surface coverage was reduced from 46.9 ± 7.6 to 36.1 ± 11.6% using NF449 and to 24.6 ± 4.5 and 18.4 ± 3.7% using GsMTx-4 and both GsMTx-4 and NF449, respectively . Mean thrombus height decreased from 2.4 ± 0.7 to 1.5 ± 0.7 μm using NF449 and to 0.8 ± 0.2 and 0.6 ± 0.2 μm using GsMTx-4 and both GsMTx-4 and NF449, respectively . Together, these results indicate that a pathway other than P2X1 receptors is the main target for GsMTx-4 during inhibition of thrombus formation.GsMTx-4 is widely used as an inhibitor of MS ion channels; however, its effects on well established platelet Casigargin , 4. GsMT (60 μm)   was detected at trace levels in this recent study; thus we repeated the qPCR assay using a larger sample volume. Parallel qPCR runs with primers for GYPA and CD45 were used as described previously . Meg-01 cells were found to express higher levels of FAM38A transcripts compared with platelets and to also express FAM38B (A). In contrast, TRPC6 mRNA was not detected in Meg-01 cells, although it was present in platelets as reported previously (B), and this further suggested a lower level of expression in platelets compared with Meg-01. Piezo1 protein in humans is known to be an N-linked glycoprotein . Although three alternatively spliced forms of Piezo2 protein were detected in Meg-01 cells, they were all absent in platelets,4 in agreement with the lack of Piezo2 mRNA (A). Piezo1 channels have been previously studied in various tissues including erythrocytes with the aid of the blocker GsMTx-4 . Most or all of this response was lost in Ca2+-free external saline , as expected if the predominant location of Piezo1 channels is on the surface membrane (B and C). The residual response to Yoda1 in platelets in Ca2+-free medium can be explained by the suggested presence of Piezo1 channels on membranes of the intracellular stores (A). Yoda1 also enhanced intracellular Ca2+ transients in platelets attached to slides via PECAM-1 (D and E). Yoda1 increased the occurrence of Ca2+ transients both in the absence of flow and upon application of shear . Under static conditions, the F/F0 integral increased more than 3-fold from a F/F0·4 min value of 0.3 ± 0.3 to 1.2 ± 0.9. Under normal arterial flow, in the absence of Yoda1 the F/F0·4 min value was 1.2 ± 0.6, which showed a 1.7-fold increase to 2.1 ± 1.1 in the presence of the Piezo1 agonist .To further assess the contribution of Piezo1 channels to platelet signaling and functional events, we used the recently characterized Piezo1 activator, Yoda1 . In Furar milieu A. Most omembrane , B and Cr stores . These er stores A. Yoda1  PECAM-1 , D and ESince their identification in 2010 by the Patapoutian group , the MS 2+ influx mechanisms that are activated by flow, and thus shear forces, and that the underlying pathway is Piezo1. First, expression of mRNA transcripts and protein for Piezo1 was detected in both platelets and Meg-01 cells -free conditions used to record shear-induced Ca2+ transients in our study 2+ influx studies . Second,e Piezo2 , 15, dir-workers , 13. The-workers  and henclatelets . Althougur study . Furtherur study –32, the agonists , 43. Ca22+ influx in platelets, the presence of such a pathway has been suggested in earlier studies    was belo2+ responses of Meg-01 cells and platelets was the more immediate nature of the cell line response compared with the delayed increase in platelets (B and 2C). A likely explanation is the very different cytoskeletal arrangement of platelets, which consists of a cortical cytoskeleton that is responsible for its discoid resting shape and also results in a less flexible plasma membrane compared with other cell types, including its precursor and related cell lines  was from Alomone Labs , and the agonist of Piezo1, Yoda1 (5586), was from Tocris Bioscience . Fura-2 AM and Fluo-3 AM were from Invitrogen. PECAM-1 antibody (WM59) (MCA1738T) for platelet attachment was purchased from AbD Serotec . Type I collagen  was from Takeda . Unless otherwise stated, all other materials were from Sigma-Aldrich.m Phe-Pro-Arg-chloromethylketone  as anticoagulant. For all other studies, blood was taken into acid-citrate-dextrose (ACD) solution  at a ratio of 6:1 (blood:ACD). For extraction of mRNA, 1.5 ml of platelet inhibitor mixture containing 0.1 μm prostaglandin E1, 2 mm EDTA, and 0.3 mm acetylsalicylic acid in ACD was added per 10 ml of whole blood or Meg-01 cell suspension. Platelet-rich plasma (PRP) was prepared by centrifugation at 150 × g for 20 min for mRNA extraction, 100 × g for 20 min for aggregometry, or 700 × g for 5 min for other experiments. For preparation of washed platelets (WP) for aggregometry, ACD was added to PRP in 1:80 (ACD:PRP) ratio and 1 μm prostaglandin E1 and then centrifuged at 700 × g for 10 min, and the platelets were resuspended in Tyrode's HEPES buffer , after which they were centrifuged at 700 × g for 10 min. WP were resuspended in a volume of Tyrode's HEPES buffer equivalent to that of the original PRP. For intracellular Ca2+ measurements, the PRP was incubated at room temperature for 15 min with 100 μm acetylsalicyclic acid and 0.32 unit/ml apyrase  and then incubated with either 2 μm Fura-2 AM  or 5 μm Fluo-3 AM . The PRP was then centrifuged at 350 × g for 20 min, and the platelets were resuspended in an equal volume of nominally Ca2+-free saline  that also contained 0.32 unit/ml apyrase for experiments when P2X1 receptor responses were being studied. Immediately prior to Fura-2 measurements in cuvettes or before introducing Fluo-3-loaded platelets to biochips for attachment, the platelet suspension was diluted 1:1 with Ca2+-free saline. For platelet lysate preparation for Western blotting, the protocol for Fura-2-loaded platelet preparation was followed without the dye addition step, and the washed platelet suspension as lysed with radioimmunoprecipitation assay buffer  with EDTA-free protease inhibitor mixture  on ice for 1 h.Blood was collected by venepuncture from informed, consenting healthy volunteers, in accordance with the Declaration of Helsinki. This study was approved by the University of Leicester Research Ethics Committee for Human Biology . In experiments where human platelets were used, the data were obtained from three to seven donors. Whole blood for thrombus formation was collected into 40 μd-lysine hydrobromide  for 15 min. The slides were then rinsed with sterile water and dried for 2 h. Meg-01 cells in medium were added to the coated slides and incubated at 37 °C for 15 min to promote attachment. Attached cells were then treated with 2 μm Fluo-3 AM at room temperature for 45 min. After washing in Hanks' balanced salt solution , slides were immediately inserted in a parallel-plate flow chamber for experimentation. Meg-01 cell lysates were prepared by washing with PBS and lysed as described for platelets. The lysates were centrifuged at 20,000 × g at 4 °C for 10 min, and the supernatant was stored at −80 °C. Meg-01 cells used in all experiments were from passage numbers 1–14.Meg-01 cells were obtained from the European Collection of Authenticated Cell Cultures and grown in RPMI 1640 supplemented with 10% FBS and 100 units/ml penicillin-streptomycin. Sterilized glass coverslips were coated with 0.1 mg/ml poly-2+]i recordings from single platelets or Meg-01 cells was carried out on an Olympus IX81 inverted confocal microscope with a FV1000 laser scanning module  using a 60×, 1.35 NA oil immersion lens (UPLSAPO). The confocal aperture was set for optimal optical slicing . Fluo-3 fluorescence images were captured at a rate of 1.74 Hz for platelets and 0.37 Hz for Meg-01 cells  attached to a parallel-plate flow chamber was used to initially introduce HEPES-buffered saline solution  to remove air bubbles. 1 ml of whole blood was then introduced into the system for a period of 5 min at the required shear level before perfusing HEPES-buffered saline to clear the components unbound to the collagen surface. The blood was applied at a flow rate of 0.235 ml/min, which equals to a normal arterial shear rate of 1002.6 s−1, calculated according to Q = flow rate (ml/s), w = microslide lumen width in cm (0.15), h = microslide lumen height in cm (0.0125), t = shear stress (Pa), and μ = viscosity of whole blood (0.001002 Pa/s). To calculate shear rate (s−1), t was divided by μ.The glass coverslips were coated with collagen (100 μg/ml) overnight in a humidified chamber at 4 °C. Whole blood was stained with 1 μZ-stack analyses of stable thrombi were carried out within 15 min of their formation using step changes (ΔZ) of 0.69 μm. The data represent averages of at least four randomly chosen fields per experiment. Analysis and 3D reconstruction of Z-stacks were performed with ImageJ 1.49 Volume Viewer 2.0 plugin . Surface coverage and thrombus volume were calculated according to the Cavalieri principle as previously described  as necessary before inserting the slides into the parallel-plate flow chamber. To apply fluid shear stress, the reservoir was filled with either HBSS, Ca2+-free HBSS containing 1 mm EGTA or HBSS containing 2.5 μm GsMTx-4 as appropriate, and the AL-1000 syringe pump (World Precision Instruments) was set to draw fluid through the system at the shear rates of 1002.6, 2282.67, and 3989.248 s−1, which represent shear conditions in normal arteries, moderately stenotic arteries, and severely stenotic arteries, respectively (F) were normalized against prestimulus fluorescence level (F0) to yield F/F0 values.Fluo-3-loaded Meg-01 cells attached to slides were treated with 2.5 μectively . The flum Fluo-3-AM for 45 min, at room temperature) and attached onto glass-bottomed Vena8 GCS biochips  that were coated with monoclonal mouse anti-human PECAM-1 (CD31) antibody by incubation at 37 °C for 1 h. Excess antibody was removed, and nonspecific sites were blocked with 2% BSA at 37 °C for 1 h to prevent glass-induced platelet activation. Immediately prior to each experiment, Fluo-3-loaded platelets were introduced into the biochip channel and incubated at 37 °C for 10 min with occasional gentle shaking. The biochip was then mounted on the microscope stage, and HBSS was introduced into the channel under gravity. Pharmacological reagents (2.5 μm GsMTx-4 or 25 μm Yoda1) were introduced at a very low shear rate (410.16 s−1) prior to the experimental recording. Captured time-series images were analyzed on ImageJ, version 1.47 . The baseline was set manually, and peaks more than 1 point above baseline were included within the calculation of a Ca2+ increase.Platelets were loaded with Fluo-3 , at 37 °C. Platelets were resuspended in nominally Ca2+ measurements were performed in stirred suspensions using a Cairn spectrofluorimeter system  at 37 °C as described elsewhere  using an extracellular calibration after digitonin permeabilization  represent the increases in [Ca2+]i above the prestimulus concentration. The materials used to study Ca2+ influx were as follows: 0.2 mm EGTA, 1 μm thapsigargin to induce store depletion , to selectively activate P2X1 receptors.Fura-2 ratiometric Calsewhere . After llization . Peak [Cepletion ; 60 μm O agonist , 60; and4 mRNA expression values relative to GAPDH were calculated as previously described , as previously described . The reaescribed .FAM38A (Piezo1) rabbit anti-human polyclonal antibody , 1:2000; P2X1 rabbit anti-human polyclonal antibody , 1:1000; and FAM38B (Piezo2) rabbit anti-human polyclonal antibody , 1:200. The specificity of the antibodies for their intended target in Western blots . One-way analysis of variance followed by Tukey's post hoc multiple comparison analyses or paired two-tailed Student's 2+ transients. A. H. G. contributed to the preparation of the manuscript, and M. P. M.-S. conceived and coordinated the study and wrote the manuscript. All authors approved the final version of the manuscript.Z. I. designed, performed, and analyzed the experiments, prepared the figures and wrote the manuscript. J. R. W. devised the mRNA extraction and quantitative-PCR protocols and generated the initial transcriptome database leading up to this project. J. M. G. and C. I. J. developed the single-platelet attachment approach using the PECAM-1 antibody that allowed the imaging of Ca