In addition, in a transgenic mouse model of achondroplasia, Recifercept restores reduced body weight and long bone growth in these mice. These data suggest that Recifercept treatment could lead to clinical benefits in children treated with this molecule.Achondroplasia is a rare genetic disorder caused by mutations in the Fibroblast Growth Factor receptor 3 (FGFR3). These mutations lead to aberrant increase of inhibitory signaling in proliferating chondrocytes at the growth plate. Recifercept is a potential treatment for this disease using a decoy approach to sequester FGFR3 ligands subsequently normalizing activation of the mutated FGFR3 receptor. Recifercept binds to FGF isoforms  Mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3) are responsible for the phenotypes of several skeletal chondrodysplasias, including achondroplasia, the most common form of short limb and short stature. Achondroplasia is a rare genetic autosomal dominant disorder and has an incidence of 1:20’000 children. Children affected by achondroplasia suffer from abnormal long bone development, resulting in growth deficits [The FGFR3 gene which codes for a tyrosine kinase coupled receptor, is located on the distal short arm of chromosome 4. In the early 90’s, different mutant alleles in FGFR3 were discovered that are causal for achondroplasia . Most ofFGFR3 is one of the four transmembrane FGF receptors (FGFR1-FGFR4) that contains an extracellular ligand binding domain, a transmembrane domain and two intracellular tyrosine kinase domains. The function of FGFR3 during skeletal development and postnatal growth was identified by analyzing the consequences of its mutation in mice. The global knockout of FGFR3 produces large mice with longer than normal limb bones . In contBecause activation of FGFR3 G380R requires ligand binding , 14, a pin vitro and cellular systems and that Recifercept is able to restore normal signaling in these cells. Moreover, treating a transgenic mouse model for achondroplasia [Recifercept is currently in clinical development for treating achondroplasia in children. It is comprised of the extracellular domain of the FGFR3 and it is believed to act as a decoy receptor normalizing the aberrant signaling of the mutated human FGFR3 in achondroplasia. The proposed mechanism is sequestering extracellular FGF, inhibiting its binding to the mutated FGFR3 on chondrocytes and thereby reducing the increased signaling of the receptor in the growth plate promoting normal bone growth. Here, we show that Recifercept is binding with high affinity to FGF isoforms relevant for bone growth roplasia  with RecRecifercept–FGF interactions were characterized using a Biacore T200 instrument . Recifercept was immobilized on a CM5 sensor chip according to standard amine coupling protocol . Briefly, CM5 carboxymethyl groups were first activated using an injection pulse of 50 μl  of an equimolar mix of N-ethyl-N-(dimethyaminopropyl)carbodiimide and N-hydroxysuccinimide . Following activation, Recifercept was diluted to 5 μg/ml in 10 mM sodium acetate (pH 4) buffer and injected for 420sec with an aim ligand level of 400 RU. Excess unreacted sites were deactivated with a 40 μl injection of 1 M ethanolamine. The first flow cell (Fc) was used as a reference with a blank immobilization and Recifercept was immobilized on the second Fc to approximately 400 response units (RU). For kinetic, different concentrations of hFGF in HBS-EP buffer , pH 7.4) were injected over the Recifercept CM5 chips at a flow rate of 50 μl/min. At the end of each sample injection (180 s), a 500 sec dissociation phase was performed with HBS-EP and the sensor surface was fully regenerated by injection of 50 μl of 100 mM sodium acetate, 2M NaCl . Human FGF isoforms were analyzed by subfamily in order to run each subfamily on a new biosensor surface Each single cycle kinetic run was performed as follows, hFGF9 start run control + hFGFa, hFGFb etc, hFGF9 end run control. The FGF8 subfamily was analyzed with 16nM of Heparin in each tested concentration .2 < 2 and U-value < 25. Detailed data are shown in Reference responses from the control Fc (blank immobilization), were subtracted from Recifercept Fc for each analyte injection using BiaEvaluation software . The resulting sensorgrams were used for kinetic parameter determination by globally fitting the entire association and dissociation phases to a 1: 1 interaction. Five different analyte concentrations were used to determine the kinetic parameters for each interaction. Following curve fitting, each sensorgram was manually examined for data quality according the following acceptance criteria green quality control, reliable Rmax (not more than 10-fold the observed RU level response), ChiMurine pro-B lymphocyte BaF3 cells  were transduced with retroviral particles to stably overexpress either human FGFR3 IIIc or FGFR3 IIIc G380R. Briefly, GP-2 HEK packaging cell line (Clontech) was co-transfected with a pantropic VSVg vector (Clontech) and a pBABE-puro-hFGFR3 IIIc or hFGFR3 IIIc G380R  using a standard CaCl2 procedure. After 48h, supernatant containing retroviral particles were harvested and viral RNA copy was determined by RT-qPCR .BaF3 were then transduced by spinocculation  and puromycin (Gibco) was added 3 days later to start the selection. 2μg/mL final puromycin concentration was chosen based on a puromycin kill curve made with non-transfected BaF3.BaF3 mutant cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco), 1% Penicillin/Streptomycin/Glutamine (Gibco), 10ng/mL recombinant murine IL-3 (R&D Systems) and 2μg/mL puromycin.5000 cells were plated in white 96-well plate with optical bottom (Nunc). Prior to plating, cells were centrifuged twice at 800g, 5 minutes at room temperature and washed with assay medium  to remove of IL-3 and puromycin.Cells were either stimulated for 72h with increasing concentrations of human FGF1 (Peprotech) or simultaneously with 10ng/mL human FGF1 and increasing concentrations of Recifercept, both supplemented with 10μg/mL heparin sodium salt (Sigma) acting as a co-factor. Cell proliferation was assessed by measuring intracellular ATP concentration with CellTiter Glo assay (Promega). Luminescence readout was measured with a Varioskan Lux plate reader (Thermo Fisher Scientific).Rat ChondroSarcoma cells (RCS) were kindly provided by Dr Claudio Basilico from New York University School of Medicine. Cells were maintained in DMEM high glucose (Gibco) supplemented with 10% hiFBS (Gibco) and 1% Penicillin/Streptomycin/Glutamine (Gibco). For proliferation assay, 2500 cells were plated in black 96-well plate with optical bottom (Nunc). 24h after plating, medium was removed and replaced by assay medium . Cells were either stimulated for 48h with increasing concentrations of human FGF2 (Peprotech) or 0,3ng/mL human FGF2 and increasing concentrations of Recifercept, both supplemented with 1μg/mL heparin sodium salt. Cell proliferation was assessed by measuring cell nuclei number with CyQuant Direct assay (Thermofisher Scientific). Fluorescence was measured with a Varioskan Lux plate reader.RCS were plated at 100,000 cells in a 6-well plate in full medium. After 24h, medium was removed and replaced by assay medium. Cells were stimulated for 30 minutes with either 10ng/mL human FGF2 alone or in combination with 2μM Recifercept. Heparin sodium salt was added as a co-factor at 1μg/mL. Cells were washed twice in cold DPBS (Gibco) and lysed in RIPA buffer (Millipore) containing proteases and phosphatases cocktail inhibitors . Cell lysates were centrifuged at 16,000g, 10 minutes at +4°C, and supernatants were harvested, and total protein concentration was measured by BCA assay (Thermofisher Scientific).30μg protein under reducing conditions  were loaded on a Bis-Tris 4%-12% precast gel (Novex). After dry transfer, PVDF membrane (Novex) were blocked for 30 minutes and then incubated overnight at +4°C with the following rabbit antibodies : phospho-PLCg1, PLCg1, phospho-MEK, MEK, phospho-ERK, ERK. All primary antibodies were used at 1:1000. Secondary anti-rabbit IgG-HRP  was used at 1:5000 and WesternSure PREMIUM chemiluminescent substrate added. Images were taken with a C-Digit imager (Li-Cor). Phospho/Total protein ratios were calculated using Image 3.1. Software (Li-Cor)ESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPPPGGGPMGPTVWVKDGTGLVPSERVLVGPQRLQVLNASHEDSGAYSCRQRLTQRVLCHFSVRVTDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLLAVPAANTVRFRCPAAGNPTPSISWLKNGREFRGEHRIGGIKLRHQQWSLVMESVVPSDRGNYTCVVENKFGSIRQTYTLDVLERSPHRPILQAGLPANQTAVLGSDVEFHCKVYSDAQPHIQWLKHVEVNGSKVGPDGTPYVTVLKTAGANTTDKELEVLSLHNVTFEDAGEYTCLAGNSIGFSHHSAWLVVLPVSLESNASMSSNT.Recifercept is composed of the extracellular domain of the FGFR3 receptor (accession number P22607) from the amino acid 22 up to amino acid 358. There is an additional stretch of 13 amino acids added to C-terminus composed of the cytoplasmic domain of the FGFR3 from amino acid 423 up to amino acid 435. The full sequence of the molecule is: For recombinant protein production the sequence was cloned in a eukaryotic expression vector.in vitro assays and the PK study, Recifercept recombinant drug material was generated from a 50L single use bioreactor (SUB) culturing from a GPEX CHO cell line. The media was G12.1, and the bioreactor was harvested on Day 14 at 81% viability. Media was filtered and drug substance was purified using chromatography.For in vivo applications was generated using a 2L bioreactor culturing GPEX CHO cell line. The media was G12.1 and the bioreactor was harvested on Day 13 at 88% viability. Media was filtered and drug substance was purified using chromatography.The drug substance for the http://grants1.nih.gov/grants/olaw/references/phspol.htm) and the European commission guidelines for the protection of animals used for scientific purposes  were followed at all times. All experiments have been approved by the national ethic committee (Ministère de l’Enseignement et de la Rercherche) under protocols APAFIS#l 5330–2018051717046781 v2 and APAFIS#20115–2019032211376608 v2 (PI: E Gouze).The Principles of Laboratory Animal Care , all newborn mice from a single litter received the same dose subcutaneously (sc) with either vehicle control (PBS), Recifercept 3 mg/kg or 10 mg/kg twice weekly. Thereafter, sc injections were done at day 3, 5, 9, 11, 16, and 18. At PND3 77, 31 and 21 Fgfr3er group . At day In 6 weeks old FVB mice . Recifercept was administered once at 3 mg/kg or 10 mg/kg by intravenous (IV) injection. Total volume was supplemented with PBS 1X to inject 100μL per mouse. Blood was harvested at 10 min, 2h, 4h, 6h, 8h and 24h post drug administration. Recifercept levels in serum were measured by ELISA with MAB7662 (R&D Systems) as a coating antibody and a 7D7 custom detection antibody raised against a synthetic peptide of the D1 domain (KDGTGLVPSER).Statistical analyses were performed with GraphPad Prism 7.0 software . To determine the statistical tests to be used, necessary assumptions were verified. To verify normality and equal variance, an Agostino and Pearson omnibus normality test (a = 0.05) and a Brown-Forsythe test (P < 0.05) were performed, respectively.Because all skeletal measurement data sets , fulfilled normality and equal variance requirements, two-tailed Student’s t test for comparisons of two independent groups was used in the different statistical analyses.Different recombinant human FGF isoforms have been used to analyze their interaction with Recifercept using Surface Plasmon Resonance (SPR). Recifercept contains the full extracellular domain of the FGFR3 and the ligands FGF18, 9 and 16 are binding with high affinity to Recifercept . MoreoveRecifercept is thought to act in part as a decoy to trap FGFs . TrappedIn order to functionally test Recifercept, we worked with BaF3 overexpressing FGFR3. Pre-B murine lymphocyte BaF3 don’t express any of the four FGFR’s and can be easily transfected with either one or several receptors. These cells are very useful tools for drug screening purposes. Since we have shown that Recifercept binds to FGF1 with high affinity , we stimNext, to test if Recifercept was able to inhibit this proliferative signal, we tested FGF1 concentration of 10ng/ml (0.4nM) which gave a robust signal and added increasing concentrations of Recifercept . The datRat ChondroSarcoma cells (RCS) express different FGF receptors and in contrast to BAF3 reduce proliferation as in chondrocytes upon stimulation with FGF , Fig 2AFig 2A. IWe then analyzed the effect of Recifercept on signaling pathways downstream of FGFR3. The western blot in in vivo pharmacological properties of Recifercept, we dosed 6-week-old WT mice with a single intra-venous dose at 3mg/kg and 10mg/kg and analyzed the Recifercept exposure in serum after different timepoints post dose. in vivo can be reached.To better understand the ach/+ mice [ach/+ mice show pronounced deficits in body weight, axial length and length of the long bones  at the age of 21 days  lack signal sequences and are thought to remain intracellular . ParacriGrowth of long bones is regulated at the growth plate located at the ends of growing endochondral bones . The groin vitro with high affinity to several FGF isoforms using Surface Plasmon Resonance. Recifercept effectively binds human FGF2, human FGF9 and human FGF18 with nanomolar affinity. This high affinity interaction would prevent FGF from binding to its receptors and suggests, that Recifercept acts as a decoy to the FRGR3 receptor. This is further substantiated using several cellular assays.The data presented here show that Recifercept is binding in vivo activity of Recifercept.In our cellular models, we have shown that Recifercept is influencing directly the pathways involved in regulation of chondrocyte proliferation. However, the exact mechanism of action cannot be deciphered from these experiments. As mentioned above, FGFR3 shows a ligand-dependent activation which involves receptor dimerization. In the model systems, we cannot clearly distinguish between a direct effect on the FRGR3 receptor by a possible formation of heterodimers inhibiting the activation and the decoy effects sequestering exogenous FGF2. Further studies will be needed to better understand the contribution of the different mechanisms to the in vitro activity shown in SPR and different cellular models translates well into in vivo activity on bone growth in transgenic Fgfr3ach/+ mice. Recifercept corrects some of the key deficits of the Fgfr3ach/+ transgenic mice such as body weight and body length and growth of long skeletal bones.The Other molecules targeting bone growth signaling pathways have been reported to be successful in showing treatment effects in transgenic mice and also recently in clinical trials on a pediatric population. Vosoritide is a CNP analog acting on the natriuretic peptide receptor 2 (NRP2). This receptor is strongly expressed on chondrocytes and its downstream signaling also activates MAP-kinase pathway. However, the activation of the NRP2 pathway does not fully rescue the activation of FGFR3; specifically, STAT1 signaling is not activated by NRP2 . NeverthTogether with our data presented here, this supports the approach to modulate cellular signaling on chondrocyte pathways to clinically benefit patients, and that efficacy on transgenic mice can translate into efficacy in patients.in vitro and reverts the FGF dependent activation of FGFR3 in cellular models system. Moreover, in a transgenic Fgfr3ach/+ mice, dose-dependent treatment with Recifercept rescues the disease phenotype and restores skeletal growth. Taken together, these data strongly suggest that Recifercept, currently in clinical development, would be able to reverse the molecular mechanisms responsible for the observed growth deficits in children with achondroplasia.The data presented demonstrate that Recifercept is binding to FGF 1, 9 and 8 subfamilies S1 Checklist(PDF)Click here for additional data file.S1 Table2 < 2 and U-value < 25. hFGF9 was used as an internal run control and was loaded before and after the tested subfamilies. * FGF8 subfamily was analyzed with 16 nM of heparin in order to reach acceptance criteria.Binding between Recifercept and human FGFs isoforms was determined by surface plasmon resonance spectroscopy (SPR). Recifercept was immobilized on a CM5 chip and each FGF subfamily was analyzed on a new immobilized chip. hFGF9 was used as an internal run control and was loaded before and after the tested subfamilies. For each FGF, a single cycle kinetic was performed with 5 concentrations from 0 to 16 nM. Following curve fitting, each sensorgram was manually examined for data quality according the following acceptance criteria green quality control, reliable Rmax (not more than 10-fold the observed RU level response), Chi(DOCX)Click here for additional data file.S1 FigBinding between Recifercept and human FGFs isoforms was determined by surface plasmon resonance spectroscopy (SPR). Reference responses from the control Fc (blank immobilization), were subtracted from Recifercept Fc for each analyte injection. The resulting sensorgrams were used for kinetic parameter determination by globally fitting the entire association and dissociation phases to a 1: 1 interaction. hFGF9 was used as an internal run control and was loaded before and after the tested subfamilies. For each FGF, a single cycle kinetic was performed with 5 concentrations from 0 to 16 nM.(TIF)Click here for additional data file.S2 FigWestern blots from (TIF)Click here for additional data file.


Rationale: Macrophages play critical roles in the pathogenesis of type 1 diabetes mellitus (T1DM). Circular RNAs (circRNAs) are a novel class of endogenous RNAs with covalently closed loop structures, implicated in various disease processes. However, their impact on macrophage activation and T1DM pathogenesis remains elusive.Methods: circRNA expression profiles of peripheral blood mononuclear cells (PBMCs) from T1DM children were determined by whole transcriptome microarray. Bioinformatics, quantitative real-time PCR, Western blot, RNA immunoprecipitation (RIP), cell co-culture, cell proliferation, and cell apoptosis assays were performed to investigate the expression, function, and regulatory mechanisms of circPPM1F in vitro. The regulatory role of circPPM1F in vivo was evaluated in the streptozocin-induced diabetic mouse model.Results: We identified 27 upregulated and 31 downregulated differentially expressed circRNAs in T1DM patients. circPPM1F, a circRNA with unknown function, was dominantly expressed in monocytes and significantly upregulated in T1DM patients. Functionally, circPPM1F promoted lipopolysaccharide (LPS)-induced M1 macrophage activation via enhancement of the NF-κB signaling pathway. Mechanistically, circPPM1F competitively interacted with HuR to impair the translation of protein phosphatase, Mg2+/Mn2+ dependent 1F (PPM1F), thus alleviating the inhibitory effect of PPM1F on the NF-κB pathway. Moreover, eukaryotic initiation factor 4A-III (EIF4A3) and fused in sarcoma (FUS) coordinately regulated circPPM1F expression during M1 macrophage activation. In addition, circPPM1F could exacerbate pancreas injury in the streptozocin-induced diabetic mice by activation of M1 macrophages in vivo.Conclusions:circPPM1F is a novel positive regulator of M1 macrophage activation through the circPPM1F-HuR-PPM1F-NF-κB axis. Overexpression of circPPM1F could promote pancreatic islet injury by enhancing M1 macrophage activation and circPPM1F may serve as a novel potential therapeutic target for T1DM in children. Type 1 diabetes mellitus  is a chronic autoimmune disease, driven by the interplay between individual genetics and environmental triggers Emerging evidence has suggested that, throughout the immune system, macrophages play a critical role in insulitis, and support autoimmune T cells to aggravate the infiltration of inflammatory cells during T1DM circ-RasGEF1B promotes the antigen presentation process by positively regulating the stability of intercellular adhesion molecule 1 (ICAM-1) mRNA in macrophages exposed to LPS circZKSCAN1 negatively regulates cancer stem cells by physically binding fragile X mental retardation protein (FMRP) against cell cycle and apoptosis regulator 1 (CCAR1) complex in hepatocellular carcinoma Circular RNA (circRNA) is a covalently closed loop molecule ligated by a 3′-5′ phosphodiester bond at the junction site, which is generated through back-splicing and often considered to be a byproduct of aberrant splicing circPPM1F, which was significantly upregulated in the PBMCs of T1DM patients. Functionally, circPPM1F could promote LPS-induced M1 macrophage activation via enhancement of NF-κB signaling. Mechanistically, circPPM1F competitively interacted with HuR to impair the translation of PPM1F, thus alleviating the inhibitory effect of PPM1F on the NF-κB pathway. Moreover, we found that EIF4A3 and FUS participated in the maintenance of high levels of circPPM1F expression. In addition, circPPM1F could exacerbate pancreas injury in STZ-induced diabetic mice by activating M1 macrophages. Overall, our study indicates that circPPM1F plays an important role in the development of T1DM and suggests a potential therapeutic target for T1DM.In this study, we conducted a genome-wide analysis of circRNA expression profiles in peripheral blood mononuclear cells (PBMCs) from T1DM patients and healthy controls and identified a novel circRNA, T1DM patients were diagnosed according to the criteria of American Diabetes Association (ADA) +, CD3+, and CD19+ cells were sorted from PBMCs with a magnetic cell sorting system . Sorted cells were subjected to RNA extraction and qRT-PCR.PBMCs were isolated using Ficoll-Hypaque . Briefly, peripheral blood was mixed with phosphate-buffered saline (PBS) and overlaid on the Ficoll-Hypaque solution (density: 1.077 g/mL). Following centrifugation at 400 ×g for 30 min, PBMCs were aspirated from the interface. The cell pellet was washed twice with PBS and resuspended in TRIzol for subsequent RNA extraction and quantitative real-time PCR (qRT-PCR). In addition, CD14® Premix Ex Taq™ II  using the Roche 480 Real Time PCR System. β-actin served as internal control for mRNAs and circRNAs. Relative quantification (2-ΔΔCT) was used for results analysis. The primers sequences in qRT-PCR are listed in The nuclear and cytoplasmic fractions or total RNA were extracted using TRIzol  followed by reverse transcription of mRNAs and circRNAs using PrimeScript II 1st Strand cDNA Synthesis Kit  according to the standard manufacturer′s instructions. A qRT-PCR assay was performed to measure mRNAs and circRNAs expression with SYBRThe circRNA microarray analysis was performed at the Shanghai Biotechnology Corporation. In brief, Total RNA of PBMCs from T1DM patients (n = 4) and age-matched healthy controls (n = 4) were extracted and purified followed by amplification and labeling with a Low Input Quick Amp WT Labeling Kit. Labeled cRNAs were purified using the RNeasy mini kit. Each slide was hybridized with 1.65 μg Cy3-labeled cRNA for 17 h using a gene expression hybridization kit, and then scanned with an Agilent microarray scanner using default settings. Data were extracted with the Feature Extraction software 10.7 . Raw data were normalized by the Quantile algorithm and limma package in R. Overall, 88750 circRNAs were tested.2 at 37 °C.The human THP1 cell line was obtained from the American Type Culture Collection , and MIN6 and Raw264.7 cells were obtained from the Fudan IBS cell resource center . THP1 and MIN6 cells were maintained in RPMI-1640 medium  with 10% fetal bovine serum  and 1% penicillin/streptomycin (Gibco), and the MIN6 cell culture medium was supplemented with 50 μM 2-mercaptoethanol. Raw264.7 cells were maintained in Dulbecco′s modified Eagle′s medium with high glucose  supplemented with 10% FBS. All cells were kept in a humidified cell incubator with 5% COcircPPM1F, siPPM1F, siHuR, siEIF4A3, or siFUS  according to standard protocols. Cells were transiently transfected with 500 ng/mL ectopic expression vector of circPPM1F with Lipofectamine 2000 reagent , according to the manufacturer's instructions. qRT-PCR assay was used to assess RNA expression levels 48 h after transfection. Relative sequences of siRNA are listed in Cells were transiently transfected with Lipofectamine RNAiMAX reagent  and chemically synthesized si-For the activation of THP1-derived macrophage, THP1 cells were treated as previously described ® . All results were normalized to those of β-tubulin, which was used as a loading control. Detailed characteristics of the antibodies are presented in Total protein was extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer . Lysates were resolved by electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes, and probed with antibodies directed against PPM1F (Abcam); HuR, phosphorylated NF-κB p65 (Ser536) (p-p65), p65, phosphorylated p38 (T180/Y182) (p-p38), p38, phosphorylated JNK (T183/Y185) (p-JNK), JNK, phosphorylated ERK1/2 (T202/Y204) (p-ERK1/2), ERK1/2, phosphorylated m-TOR (Ser2448) (p-mTOR), mTOR, phosphorylated Stat3 (Y705) (p-Stat3), Stat3, Bcl2 ; Bax , and β-tubulin . The bands were detected by developing with chemiluminescent HRP substrate , and intensity of bands was determined by imaging with a Molecular ImagerFour micrograms total RNA from THP1 cells was either untreated (control) or treated with 20 units of RNase R  in the presence of 1 × Reaction Buffer, and incubated for 30 min at 37 °C. The digested RNA was isolated using acid phenol-chloroform (5:1). Then reverse transcription and qRT-PCR were performed, as described in the RNA extraction and qRT-PCR section.5) were placed in 24-well plates and treated with 250 ng/mL actinomycin D  added to the cell culture medium. The levels of circPPM1F and PPM1F were detected at 0, 3, 6, 12, and 24 h.THP1 cells  and nuclear pellets were used for RNA extraction.Nuclear and cytosolic fractions were separated using the Nuclear and Cytoplasmic Extraction Kit . A total of 1 × 107/dish) were harvested, centrifuged and re-suspended using 100 μL RIP lysis buffer supplemented with protease and RNase inhibitors, and 5 μg IgG or HuR antibody-coated beads were incubated with the cell lysates under rotary agitation at 4 °C overnight. Following proteinase K treatment, the immunoprecipitated RNAs were extracted and reversely transcribed as described in the RNA extraction and qRT-PCR section. Levels of circPPM1F and PPM1F were detected by qRT-PCR assay.The RNA immunoprecipitation (RIP) assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit . Two 10-cm culture dishes of THP1 macrophages (1 × 10POLR2A gene and were inserted upstream of the restriction site Xho I and downstream of Pac I. Primer 1 or primer 2 (circPPM1F upstream (~340 bp) or downstream (~3800 bp) of cDNA or gDNA. Two PCR segments were inserted into the 3D5 vector following digestion with restriction enzymes Xho I and Pac I using a seamless cloning assay  were collected. The conditional media were used for treatment of MIN6 cells, at a ratio of 3:1 with complete medium.Raw264.7 cells were seeded in six-well plates, and transfected with 3D5- of IL-6 and TNF-α in the conditional media were measured with the mouse IL-6 and TNF-α DuoSet ELISA kit (eBioscience) according to the manufacturer's instructions. A microplate reader  was used to read the absorbance at 450/570 nm.The protein levels3) were placed in 96-well plates and cultured in conditional medium. Each sample was assayed in triplicate. Cell viability was determined at 0, 24, 48, and 72 h using 10 μL CCK8 solution treatment for 2 h. The optical density of each well was assessed using a Microplate reader  at 450 nm.The CCK8 assay was performed to assess the proliferative ability of MIN6 cells according to the manufacturer's instructions . Briefly, MIN6 cells (1.8 × 105 cells/well) for 48 h using conditional media, followed by H2O2 (500 μΜ in FBS-free medium) treatment for 15 h, and then pooled together after trypsin without EDTA digestion. Cell apoptosis was analyzed using the Annexin V-FITC/Propidium Iodide (PI) Apoptosis Detection Kit  according to the manufacturer's instructions. MIN6 cells were stained with FITC and PI and then analyzed by fluorescence-activated cell sorting using FACS Canto II . The cell apoptosis data were analyzed by FlowJo V10 software . Cells of quadrant 4 that were considered viable were FITC Annexin V and PI negative; cells of quadrant 3 that were in early apoptosis were FITC Annexin V positive and PI negative; cells of quadrant 2 that were in late apoptosis were both FITC Annexin V and PI positive; and cells of quadrant 1 that were necrotic were FITC Annexin V negative and PI positive. The percentages of cell apoptosis were the sum of that from early apoptosis and late apoptosis.MIN6 cells were cultured in six-well plates (1 × 10i.p.) injected with circPPM1F plasmid  and an equal amount of pZW1 using the Entranster™-in vivo Transfection Reagent , respectively; control and STZ group mice were injected with PBS. From day one, STZ, STZ+pZW1, and STZ+circPPM1F group mice were i.p. injected with repeated low doses of STZ ; control group mice were injected with sodium citrate buffer . On day seven, mice were injected circPPM1F, control plasmids, or PBS again, and then received two injections per week. The animal experiments were repeated independently 3 times.C57BL/6 mice  were purchased from Shanghai Slac Laboratory Animal Co. Ltd. All mice were housed at the Experiment Animal Center of Children′s Hospital of Fudan University at room temperature 22 °C under a 12:12 h light/dark cycle, and were provided with rodent chow and tap water. The mice were randomly divided into four experimental groups: Control, STZ, STZ+pZW1, and STZ+circPPM1F group. On day 0, STZ+circPPM1F and STZ+pZW1 group mice were intraperitoneally . All mice were fasted for 12 h before glucose detection. On day 29, mice were sacrificed and pancreas tissues dissected and analyzed for pathology.This project complied with the institutional guidelines and laws for the care and use of laboratory animals. The study was approved and overseen by the Animal Studies Committee of the Children′s Hospital of Fudan University [No. (2016) 96].Pancreas tissues were fixed with 4% paraformaldehyde and then embedded in paraffin and cut into slices. For immunohistochemical analysis, a portion of paraffin sections were routinely stained with hematoxylin and eosin (H&E), while others were incubated with antibodies against Ki-67 , Insulin , and F4/80 . Islets from consecutive tissue cross-sections were photographed at identical exposure conditions and magnification (400×) using a microscope (Leica). For immunofluorescence analysis, paraffin slides of 3-4 μm were prepared, and then incubated with rabbit anti-mouse F4/80 and rabbit anti-mouse iNOS . FITC (green) and Cy3 (red)-conjugated goat anti-rabbit IgG (Servicebio) were used to visualize F4/80 and iNOS, respectively. DAPI  was used to stain the cell nuclei (blue). Images were captured with a fluorescence microscope (Leica). Detailed characteristics of the antibodies used are presented in Pancreas tissues were collected into the gentleMACS C Tubes  containing the enzyme mix of the Multi Tissue Dissociation Kit 1  in serum-free RPM-1640 and cut into 2 × 4-mm pieces. Tissues were subjected to the run program Multi_37C_m (30 min) on the gentleMACS Octo Dissociator with Heaters . The cell suspensions were passed through a 70-μm MACS SmartStrainer , followed by centrifuging at 300 ×g for 7 min at 4 °C. All isolated cells were suspended in 100 μL iced PBS supplemented with 2% FBS. Cells were then counted with trypan blue and processed for flow cytometry in as indicated below.For surface marker analysis, cells were stained with anti-mouse F4/80  for 30 min at 4 °C. For intracellular cytokine staining, cells were fixed and permeabilized and labeled with anti-mouse iNOS  after anti-F4/80 staining. The concentration of each antibody was used according to the recommended product protocol. Cells were examined by flow cytometry using a BD FACSCanto II instrument  and analyzed with FlowJo V10 software . Detailed characteristics of the antibodies used are presented in t-tests were used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for multifactorial comparisons. A p-value of < 0.05 was considered statistically significant. The relationship between circPPM1F and IL-6, IL-1β, TNF-α, EIF4A3 or FUS was tested using Pearson's correlation and linear regression. Statistical analyses were performed with SPSS v.19.0 software or GraphPad Prism 7.0 software.Results from three independent experiments are expressed as mean ± standard error of the mean (SEM). The two-tailed Student's p < 0.05 as the threshold to define up- or down-regulated circRNAs, we identified 27 upregulated and 31 downregulated differentially expressed circRNAs in T1DM patients compared with healthy controls  analysis, which yielded an area under the curve of 0.839  and age-matched healthy controls (n = 4) by circRNA microarray. Using a two-fold change and d Figure A. Furthehsa_circ_0062444 was 4291 nt long and was the back-spliced circular product of the last three exons of the PPM1F transcript; thus, as an exonic circRNA, it was named as circPPM1F  was observed  digestion while linear mRNAs were severely degraded  targeting the circPPM1F junction site; the knockdown efficiency was confirmed by qRT-PCR and si-circPPM1F-2 was selected for the later study  was downregulated in the cells with circPPM1F knockdown after LPS stimulation . Notably, the analysis revealed that the RBP containing the most potential binding sites with the circPPM1F was HuR  induced “head-to-tail” splicing by bringing the 5′- and 3′-termini of an exon or of consecutive exons into spatial proximity circPPM1F. Therefore, RBPs might be responsible for expression of circPPM1F. Using a bioinformatics method (https://circinteractome.nia.nih.gov/index.html), we queried all RBPs that were reported to be involved in circRNA generation. The result revealed that a binding site for EIF4A3 is present in the downstream region of the circPPM1F transcript, and FUS-binding sites were identified in the mature circPPM1F  injection of STZ  and M1 macrophages (F4/80+/iNOS+) in comparison to the other groups  were present in circPPM1F. Interestingly, FMRP participated in RNA trafficking from the nucleus to the cytoplasm, displaying different subcellular distribution due to alternative splicing circPPM1F. It would be helpful to investigate the interaction of FMRP with circPPM1F to better understand the nuclear location of circPPM1F.Exonic circRNAs are usually abundant in the cytoplasm and function mainly through the microRNA “sponge” mechanism PPM1F gene. Recently, HuR is a well-studied RBP that positively augments stability of a number of linear mRNAs and ncRNAs, but also binds to introns of pre-mRNAs to modulate splicing PABPN1 protein circPPM1F and PPM1F interactions. Instead of impacting on circPPM1F transcription, the interaction with HuR and circPPM1F prevented HuR binding to PPM1F mRNA, resulting in a reduction of PPM1F translation. Such a relationship between circRNA and host mRNA is conceptually intriguing, and it might be generalizable to other RBPs. Additionally, our study was the first to reveal the role of PPM1F in M1 macrophage activation, implying that the circPPM1F-HuR-PPM1F axis may represent a novel potential therapeutic target in T1DM.Recently, circRNAs have attracted increasing attention for their potential roles in regulating parental gene expression circPPM1F expression in response to external stimuli. EIF4A3, a member of the DEAD box protein family, has been implicated in nuclear and mitochondrial splicing, ribosome and spliceosome assembly, and translation initiation. Recent studies have revealed that EIF4A3 promoted circMMP9 and circSEPT9 expression via binding to parental pre-mRNAs circPPM1F expression. Unexpectedly, we identified a binding site for EIF4A3 in the downstream region  of circPPM1F. Therefore, we assume that this unusual binding site might be responsible for the suppression of EIF4A3 on circPPM1F biogenesis. In addition, Errichelli et al. found FUS either increased or repressed circRNA biogenesis by binding to the introns flanking the back-splicing junctions, and the interaction machinery could control a complex interplay between linear and back-splicing circPPM1F at the post-transcriptional level and positively mediate its expression upon stimulation. Overall, these findings demonstrated that diverse RBPs participate in circRNA biogenesis, and that some may contribute to diverse regulatory mechanisms, even antagonistically. Hopefully, our study will prompt future work exploring RBP-based diverse regulation of circRNA biogenesis.RBPs are required for regulation of the biogenesis specific circRNAs in a positive or negative way. In this study, our data showed that EIF4A3 and FUS oppositely regulated circPPM1F on development of STZ-induced diabetes mellitus. Consistent with in vitro studies of increased pancreatic β-cell apoptosis, we found that circPPM1F-mediated M1 macrophage activation could also facilitate pancreas injury in diabetic mice. However, it remains to be clarified whether macrophage-specific expression of circPPM1F facilitates the development of T1DM. Treatment of circPPM1F with a macrophage-specific promoter in the STZ-treated NOD mouse model, or generating a chimeric, macrophage-specific circPPM1F knocked-in T1DM mouse model would be helpful to address the issue.STZ is a broad-spectrum antibiotic possessing antitumor, oncogenic, and diabetogenic properties, and multiple small dose injections of STZ in mice produce pancreatic insulitis, with progression to nearly complete β-cell destruction and diabetes mellitus circPPM1F in LPS-induced M1 macrophage activation through the circPPM1F-HuR-PPM1F-NF-κB axis. In vivo, circPPM1F facilitated pancreas injury in STZ-induced diabetic mice by promoting M1 macrophage activation. Additionally, EIF4A3 and FUS might be required for the maintenance of circPPM1F expression during the progression of T1DM. Taken together, our work provides new insights into the pathogenesis of T1DM and suggests a potential novel biomarker or therapeutic target for T1DM.In summary, our studies demonstrate the positive role of Supplementary figures and tables.Click here for additional data file.


APOBEC3B  as a source of mutations in breast, bladder, cervical, lung, head, and neck cancers. However, the role of APOBEC3B in adrenocortical carcinoma (ACC) and the mechanisms through which its expression is regulated in cancer are not fully understood. Here, we report that APOBEC3B is overexpressed in ACC and it regulates cell proliferation by inducing S phase arrest. We show high APOBEC3B expression is associated with a higher copy number gain/loss at chromosome 4 and 8 and TP53 mutation rate in ACC. GATA3 was identified as a positive regulator of APOBEC3B expression and directly binds the APOBEC3B promoter region. Both GATA3 and APOBEC3B expression levels were associated with patient survival. Our study provides novel insights into the function and regulation of APOBEC3B expression in addition to its known mutagenic ability.Recent evidence has implicated  Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine malignancy , 2. TherMutagenesis occurs from exogenous sources present in the environment and endogenous sources that are present intracellularly. Exogenous sources include ultraviolet radiation, that results in the transitions of cytosine to thymine (C-to-T) due to oxidative damage which eventually forms pyrimidine dimers, aristolochic acid, a dietary supplement derived from plants which is responsible for A-to-T transversion mutations in the liver and tobacco, an established carcinogen . EndogenThe distinct pattern of DNA base alterations has been characterized in the cancer genome using high throughput deep sequencing technologies, that reflect the underlying mutational process. The kataegis pattern of hypermutation clusters, largely dominated by C-to-T transitions, had been identified as a hallmark of various cancers. Whole genome and exome mutation analysis of The Cancer Genome Atlas (TCGA) data on multiple cancers have revealed that this pattern is consistent with the deaminase activity of the AID/APOBEC  family of enzymes, therefore, implying its significance as an endogenous mutator and a crucial contributor to somatic mutations and genomic instability , 8.In humans, the AID/APOBEC family has eleven members including activation-induced cytidine deaminase  and apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBECs): APOBEC1 (A1), APOBEC2 (A2), APOBEC3 (A3), APOBEC3A (A3A), APOBEC3B (A3B), APOBEC3C (A3C), APOBEC3DE (A3DE), APOBEC3F (A3F), APOBEC3G (A3G), APOBEC3H (A3H), and APOBEC4 (A4) . The enzin vitro and in vivo.Given there are no well-established exogenous factors associated with ACC, we postulated whether APOBEC3B could be an endogenous mechanism of genomic instability/mutations in ACC and investigated its function APOBEC3B gene expression in 21 normal adrenal cortices, 69 benign adrenocortical tumors and 38 primary ACC samples and found that APOBEC3B mRNA is significantly overexpressed in ACC (p < 0.001)  and EMBL-EBI  for the expression of APOBEC3B and observed that in both cohorts, there was a 5–6 fold higher expression in ACC compared to normal and benign adrenocortical tissue samples (p < 0.01) with no difference in expression between benign tumors and normal adrenocortical tissue samples  . We also samples  and 1C.  samples . We eval samples  and 1F. p < 0.001)   and 2D a< 0.001)  and 2F. APOBEC3B is known to induce DNA damage, a critical molecular event in multiple cancers, we investigated the levels of phospho-Histone H2AX (Ser139), an indicator of DNA double-strand breaks. We observed high levels of γ-H2AX in ACC as compared to normal adrenal cortex and benign adrenocortical tumor samples  array in the ACC cohort and analyzed the association of APOBEC3B gene expression with gene copy number. We found that ACC with higher APOBEC3B gene expression had higher rates of chromosomal gain/loss particularly in chromosomes 4 and 8 as compared to samples with lower APOBEC3B expression . Next, wAPOBEC3B and no differential CpG methylation in the APOBEC3B promoter or enhancer regions. Therefore, we performed a functional knockdown screen of 92 cancer associated transcription factors and identified GATA3, SPDEF, IRF9, and YY1 as candidate transcriptional activators/repressors for APOBEC3B gene expression (APOBEC3B gene. We carried out a luciferase reporter assay under the APOBEC3B gene promoter after transient knockdown of GATA3 transcription factor with two different siRNAs in BD140 and H295R cells. We found a significant decrease in the APOBEC3B promoter activity when compared to the vector control upon knockdown of GATA3 in both cell lines (p = 0.00000) when compared to the patients with a higher expression of GATA3.  in the 5′-promoter region  of the in vivo, we performed xenograft studies using an immunodeficient mouse model: NOD Scid gamma (NSG) mice. First, we established H295R cell lines that were stably overexpressing empty vector or four different shRNAs (sh1–4) targeting APOBEC3B transcripts. We selected two H295R cell lines (A3B_sh2 and A3B_sh4) that demonstrated a higher knockdown efficiency when compared to the other two, shRNA1 and shRNA3 (p = 0.018) and A3Bsh4 (p = 0.0009) as compared to the empty vector control  compared to corresponding normal tissues , 17, 18.The members of the APOBEC3 family possess the ability to induce detectable DNA damage and activation of the DNA repair machinery as associated with strong pan-nuclear γ-H2AX signal , 23–25. TP53 gene is frequently mutated in many cancers and tumors with TP53 mutations have also been reported to have higher levels of APOBEC3B expression in breast cancer [TP53 mutations in ACC [TP53 mutations in the intron of the gene which have been found to alter TP53 protein expression through several mechanisms such as aberrant pre-messenger mRNA splicing and disruption of DNA-transcription factor interactions in a variety of cancer types, as well as, single nucleotide polymorphism in introns associated with higher cancer risk [The t cancer  and ovart cancer . Our ress in ACC , 18. We cer risk –29. Thiset al. reported gains in the DNA copy number in chromosome 17 or 17q to represent the earliest genomic alterations which are seen in benign adrenocortical tumors [Several studies looking at ACC have identified molecular events that may be responsible for disease progression . Zhao etl tumors . A studyl tumors . In our Several reports showed that higher levels of APOBEC3B expression was associated with decreased survival rates among estrogen receptor-positive (ER+) breast cancer patients  and a hiAPOBEC3B gene expression. In breast cancer, a low expression of GATA3 has been reported to be strongly associated with a higher histologic grade, poor differentiation, positive lymph nodes, Estrogen Receptor (ER) and progesterone receptor (PR) negative and overexpressed HER2/neu, all of which are indicators of poor prognosis [Very little is known about the mechanism by which APOBEC3B gene expression is regulated. Several genomic studies have revealed that there was no evidence of copy number alterations, presence of miRNA binding sites and methylation CpG sites in the genomic region of APOBEC3B suggesting that it is not regulated by any of the globally known mechanisms . We alsorognosis . GATA3 lrognosis . In ACC,rognosis  and throTP53 mutations in ACC. For the first time, we demonstrated that GATA3 directly regulates the expression of APOBEC3B and that both are prognostic markers in ACC.In summary, APOBEC3B overexpressed in ACC, and is associated with DNA damage, S phase arrest, higher copy number alterations and Human adrenocortical tissue samples were collected according to an institutional review board-approved clinical protocol after written informed consent was obtained (NCT01005654 and NCT01348698).Human ACC cell lines H295R and SW13 were purchased from ATTC  and were grown in DMEM supplemented with 1% insulin transferrin selenium and 2.5% NuSerum I . The ACC BD140A cell line , 37 was The microarray data from the GEO database, GSE10927 was analyzed through an online GEO2R expression analysis software using the default parameters and the APOBEC3B gene expression values for each patient samples were collected and plotted in Box and whiskers graph. The microarray gene expression data from EMBL-EBI, E-TABM-311 dataset was directly extracted from the author submitted normalized expression data and all the data points for APOBEC3B gene probe were shown in Box-whiskers plots.5 cells in 6-well plates, using the transfection reagent RNAiMax  according to the manufacturer’s protocol. Four lentiviral psi-LVRU6P-01, psi-LVRU6P-02, psi-LVRU6P-03 and psi-LVRU6P-04 vectors  with U6 promoter expressing shRNAs targeting APOBEC3B and one shRNA scrambled control clone for psi-LVRU6P  were obtained from GeneCopoeia . H295R cells were transfected with four shRNAs and vector control using Lipofectamine 2000 and selected with 1 μg/ml puromycin for one month. Stable H295R cells expressing shRNAs 2 and 4 were used for xenograft studies as they have shown high knockdown efficiency. For luciferase reporter experiments, the cells were seeded into a 96-well plate . After 24 hours, the cells were co-transfected with a GoClone reporter vector containing the promoter region of APOBEC3B  or mutant vectors and the siRNAs targeting GATA3 , using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Luciferase activity was measured 24 hours after transfection, using the LightSwitch Luciferase Assay Reagent (SwitchGear Genomics) according to the manufacturer’s protocol.For transient transfection, two siRNAs targeting APOBEC3B , two siRNAs targeting GATA3  and a negative control (Scrambled); Life Technologies, Thermo Fisher Scientific), were transiently transfected into 2 × 10Chromatin immunoprecipitation (ChIP) assays were performed using the Chromatin Immunoprecipitation Assay Kit  according to the manufacturer’s instructions. Briefly, the cells were cross-linked by 1% formaldehyde for 10 minutes. The formaldehyde was quenched using 2 ml of 10× glycine for 5 minutes at room temperature before harvesting. Cells were collected by centrifugation in phosphate-buffered saline (PBS) containing protease inhibitors and were lysed in SDS lysis buffer. Soluble chromatin was prepared after sonication to a DNA length of 200 to 500 base pairs. Fragmented chromatin was immunoprecipitated using antibodies against GATA3 (Abcam) or IgG control overnight at 4°C on a rotating platform. The magnetic beads were washed, the chromatin was extracted and protein-DNA complexes were reverse cross-linked. DNA was purified and analyzed by PCR using the specific primers for the APOBEC3B promoter. The total input was the supernatant from the no-antibody control. The following ChiP primers were used for site A (for: CTCCCAGGCTCAGGCTGC and rev: CTGGTTTTCCCCGGACCCT), site B (for: AGGAAGTGAAACCACAGA and rev: GACCACCAGGCAGGAAGG), Exon 5 as negative control (for: CTGGGAGGACAGGCCAG and rev: TTTTCATCGAATTTGTAC), GCMB gene as a positive control from literature .  according to the manufacturer’s protocol. RNA yield was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).– (Ct of gene of interest – Ct of GAPDH) × 100%, where Ct is the PCR cycle threshold. All PCR reactions were performed in a final volume of 20 μl with 1 μl of cDNA template on an ABI PRISM®7900 Sequence Detection System (Applied Biosystems). The PCR thermal cycler condition was 95°C for 12 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All experiments were performed in triplicate.Gene expression levels were measured using specific primers and probes. Briefly, 500 to 1,000 ng of total RNA was reverse transcribed using a High Capacity Reverse Transcription cDNA kit  and the resulting cDNA was diluted and amplified according to the manufacturer’s instructions. The taqman probes were purchased from Thermofisher scientific. For human APOBEC3B (ID#Hs00358981_m1), human GATA3 (ID#Hs00231122_m1) and human GAPDH, ID#Hs02786624_g1). GAPDH was used as an endogenous control –41. GeneCell proliferation experiments were performed using CyQUANT cell proliferation assays (Life Technologies) according to the manufacturer’s protocol. For transiently transfected cells, the cells were first transfected in a six-well plate, trypsinized 24 hours later, counted and then seeded in quadruplicate on ninety-six-well black plates to monitor cell growth. Absorbance at 485 nm/538 nm was determined using a ninety-six-well fluorescence microplate reader SpectraMax M5 . H295R cells were seeded at 8,000 cells/well and BD140A cells at 4,000 cells/well.5 cells in a 6 well dish with 2 mL of culture medium. Three days later, cells were trypsinized, washed with PBS and fixed with ice-cold 70% ethanol. Cells were then washed with PBS and resuspended in a solution consisting of PBS, ribonuclease A (0.1 mg/mL) and propidium iodide (0.05 mg/mL). The DNA content of cells was then measured by FACS analysis using Canto II (Becton Dickinson). Cell cycle analysis of the gated propidium iodide (PI) distribution was done using ModFit software .Cells were reverse transfected with control and APOBEC3B specific siRNAs, plated at a density of 2 × 10Tissues were fixed with 10% formalin and embedded in paraffin. Tissue-section slides were deparaffinized in xylene and rehydrated in ethanol, followed by antigen retrieval with 10% citrate buffer (pH 6.0) in a pressure cooker for 10 minutes at 120°C. Slides were incubated with the primary antibody at 4°C overnight. For APOBEC3B and ɣH2AX staining, Anti-APOBEC3B antibody  and Anti-gamma H2AX (phospho S140) antibody (ab22551) was used at 1:200 dilution. The rabbit and mouse secondary antibodies were used from Invitrogen Alexa fluorescently conjugated system.SureFIND transcriptome PCR array cancer associated transcription factor assay was purchased directly from SABiosciences , . Briefly, MCF7, breast cancer cells were transiently knockdown with a pool of siRNAs targeting each of the 92 cancer associated transcription factors independently and the RNA was extracted and subsequent cDNA was generated from these cells and aliquoted onto a 96-well PCR plate. The APOBEC3B gene expression was then assayed and the log2 expression values were calculated with reference to the house keeping gene, GAPDH, according to the manufacturer’s recommendation and their provided template.The mutations in the APOBEC3B promoter constructs were generated by site-directed mutagenesis of a wild-type (WT) human APOBEC3B promoter clone obtained from Switchgear genomics using QuickChange Site-directed mutagenesis kit from Stratagene . The following primers were used for generating the mutant constructs for site A (for: CCTGCCCAGGACACATAAAGACAGAGCAGC and rev: GCTGCTCTGTCTTTATGTGTC CTGGGCAGG) and site B (for: CAGGGACAAGCGTATGTAAGAGGCTGAACATG and rev: CATGTTCAGCCTCTTACATACGCTTGTCCCTG). The numbering of the position was made based on the promoter region of the clone obtained from Switchgear genomics.TP53 were sequenced at the NCI Sequencing Facility .The exon and interon regions of et al. [For the SNP-CGH array experiment, Illumina HumanCytoSNP-12v2.1 BeadChip was used. Data generated were then analyzed by using the ASCAT method described by Van Loo et al. . We compCells were lysed in a buffer containing 10 mM Tris and 1% SDS with 1× Halt Protease and a phosphatase inhibitor cocktail (Thermo Fisher Scientific) was used. The lysates were quantified for protein concentrations using a BCA Protein Assay kit . Cell lysates were analyzed in SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes . The membranes were blocked with 5% BSA in a TBS-Tween buffer and then incubated with primary antibodies overnight at 40°C. The primary antibodies used were: APOBEC3B antibody ; β-actin antibody , phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody  and p44/42 MAPK (Erk1/2) antibody . Secondary antibodies used were goat anti-mouse IgG-horseradish peroxidase (HRP) and goat anti-rabbit IgG-HRP .2 or isoflurane) for a vaporizer followed by a secondary physical method of cervical dislocation to ensure the animal death as per the NIH animal safety guidelines. Mice were sacrificed when the tumor size reached 2 cm in greatest diameter. The animal experiments were approved by the NIH-ARAC.NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ  breeder female mice were purchased from The Jackson Laboratory, they were maintained and bred according to the guidelines of the National Institutes of Health Animal Research Advisory Committee (NIH-ARAC). NSG mice were xenografted when they were 3–4 months old. A total of 24 mice, no more than 5 animals in each cage, weighing 27–33 g, were randomly assigned to three groups, each group receiving a subcutaneous injection on both sides of the flank with 3 × 10^6 H295R cells stably expressing either APOBEC3B shRNA2, APOBECB shRNA4 or the control vector in a 100-μl suspension (cell culture medium: Matrigel = 1:1). A blinded measurement of the tumor size was taken each week. Tumor volumes were calculated using the equation ×2y/2, where x is the smallest and y is the largest diameters. Isoflurane was used as an anesthetic agent as it is safer than the other anesthetics. Mice were euthanized primarily by inhalant anesthetics (COP < .05 was considered statistically significant. All calculations were performed using GraphPad Software 6.0.Overall survival was defined as the time from preoperative blood samples drawn to disease-related mortality or last follow-up, which was censored for those patients who underwent repeated abdominal surgery. The association between APOBEC3B gene expression levels and survival was assessed using the Kaplan–Meier method, with statistical differences determined by log-rank test. For gene expression data in


N. benthamiana plants. All chickens immunized with purified H5 trimers were fully protected against the severe HPAIV H5N1 challenge. We further developed a proof-of-principle approach by using disulfide bonds, homoantiparallel peptides or homodimer proteins to combine H5 trimers leading to production of H5 oligomers. Mice vaccinated with crude leaf extracts containing H5 oligomers induced neutralizing antibodies better than those induced by crude leaf extracts containing trimers. As a major result, eleven out of twelve chickens (92%) immunized with adjuvanted H5 oligomer crude extracts were protected from lethal disease while nine out of twelve chickens (75%) vaccinated with adjuvanted H5 trimer crude extracts survived. The solid protective immune response achieved by immunization with crude extracts and the stability of the oligomers form the basis for the development of inexpensive protective veterinary vaccines.Since 2003, H5N1 highly pathogenic avian influenza viruses (HPAIV) have not only caused outbreaks in poultry but were also transmitted to humans with high mortality rates. Vaccination is an efficient and economical means of increasing immunity against infections to decrease the shedding of infectious agents in immunized animals and to reduce the probability of further infections. Subunit vaccines from plants are the focus of modern vaccine developments. In this study, plant-made hemagglutinin (H5) trimers were purified from transiently transformed  The vaccination of humans and animals to protect against infectious diseases is an efficient preventive measure . VaccineVaccination is an efficient and economical means of increasing immunity against infections to decrease the shedding of infectious agents in immunized animals and to reduce the probability of further infections , thus acN. benthamiana, and synthesized . DNA sequences coding for H5-TG and H5HT trimers were cloned into pRTRA-35S-H5pII [The DNA hemagglutinin sequences of A/duck/Viet Nam/TG24-01/2005 (H5N1) (hereafter referred to as H5-TG), A/duck/Viet Nam/HT2/2014 (H5N1) (hereafter referred to as H5HT) strains and others were codon-optimized for expression in 5S-H5pII  containi5S-H5pII . DNA seq5S-H5pII  N. benthamiana leaves by using the agrobacterium vacuum infiltration method as described by Phan and Conrad [g, 30 min, 4 °C) and resuspended in infiltration buffer (10 mM 2-(N-morpholino) ethanesulfonic acid (MES), 10 mM MgSO4, pH 5.6). Both agrobacterial cultures were combined and diluted in infiltration buffer to a final OD600 of 1.0. N. benthamiana plants (six to eight weeks old) were infiltrated by completely submerging each plant in an agrobacterium-containing cup standing inside a desiccator. Vacuum was applied for 2 min and then quickly released. The plants were then grown in the greenhouse at 21 °C with 16 h light/8 h dark conditions. Five days post vacuum infiltration, the infiltrated leaves were taken and stored at −80 °C.Recombinant hemagglutinins were transiently expressed in d Conrad . Recombig, 30 min, at 4 °C. Protein concentrations of clarified plant extracts were determined by Bradford assay.Twenty grams of the infiltrated leaves were ground in liquid nitrogen and homogenized in phosphate buffered saline (PBS)  with a commercial blender. The extracts were centrifuged at 16,200× H5 contents in crude plant extracts were semi-quantified by Western blotting. Known concentrations of the anti-tumour necrosis factor nanobody-elastin-like polypeptide (aTNF-ELP) standard protein  for the v/v) glycerol; 1% (w/v) SDS; 0.005% (w/v) bromophenol blue; 2.5% (v/v) β-mercaptoethanol), cooked 95 °C for 5 min. Treated proteins were separated on 10% SDS gels at reducing conditions. After electrophoresis, proteins were transferred into nitrocellulose membranes. The blotted membranes were incubated with monoclonal anti-c-Myc antibodies, and subsequently incubated with horseradish peroxidase-linked sheep anti-mouse IgG antibodies [Proteins were prepared in an sodium dodecyl sulfate (SDS) sample buffer  fat-free milk powder dissolved in a PBS buffer for 2 h. Each lane of the membrane was separated by cutting and incubated at room temperature for 90 min with a 1:20 dilution of a mixture of sera from ten chickens of each group after the second immunization. The membranes were incubated with a 1:2000 dilution of an alkaline-conjugated goat anti-chicken IgY secondary antibody for 1 h at room temperature. Specific signals were detected by incubating membranes with 3,3–diaminobenzidine  dissolved in 0.05 M tris-HCl and 0.04% hydrogen peroxide for 10 min in the dark.To detect H5-specific chicken antibodies raised against plant extracts containing H5 oligomer-tailpiece (TP) and H5 trimer, 700 ng of the size exclusion chromatography (SEC)-purified H5 trimer proteins were separated on 6 lanes of a reducing SDS–PAGE gel (10% polyacrylamide) and transferred to a nitrocellulose membrane. The membrane was blocked with a 5% . The clarified extracts were mixed with Ni Sepharose 6 Fast Flow  (washed twice with water before) for 30 min at 4 °C. The mixture was applied to a chromatography column. The column was extensively washed . Recombinant proteins were eluted from the column with elution buffer , added to dialysis bags, concentrated in PEG 6000 and dialyzed against PBS.Infiltrated leaves were ground in liquid nitrogen, and resuspended in 50 mM tris buffer, pH 8.0. The extracts were centrifuged . The column was fed with 48 mL of PBS buffer at 0.5 mL/min flow rate. Fractions containing five hundred microliters were collected for hemagglutination test and Western blot analyses. The high molecular weight standard proteins (44 kDa to 669 kDa) were loaded onto the column to estimate the molecular weight of the proteins of interest.Affinity-purified trimeric hemagglutinin (A/duck/Vietnam/TG24-01/2005) was further purified via the SuperoseTM 6 increase 10/300 GL column with starting concentrations of 1.25 mg in 0.5 mL. The resulting SEC purified H5 trimer was coated to plates for indirect ELISA.®-D adjuvant  at 20% final concentration. Twelve male C57/Bl6/J mice per group (Charles River Laboratories GmbH) were subcutaneously vaccinated with Emulsigen®-D adjuvant-formulated plant extracts at days 0, and boosted at days 14 and 28. Mouse sera were collected one week after the second and third immunizations for hemagglutination inhibition and ELISA tests.Western blotting was used to semi-quantitatively estimate H5 contents in plant extracts. Plant crude extracts were prepared to contain equal H5 contents (100 ng of either H5 oligomers or H5 trimers). Wild-type (WT) plant extract containing equal total soluble protein amount as H5 oligomer plant extracts was included in mouse experiments as a negative control group. Plant lysates were mixed with the EmulsigenThe mouse animal experiments  were approved by the Landesverwaltungsamt Sachsen-Anhalt, Halle/Saale, Referat Verbraucherschutz, Veterinärangelegenheiten and by the Landkreis Harz, Amt für Veterinärwesen und Lebensmittelüberwachung, Halberstadt. All animals received humane care according to the requirements of the German Animal Welfare Act, §8 Abs. 1.The hemagglutination test was performed with 1% chicken erythrocytes . The higTo analyze HI titers from chicken sera, inactivated A/Chicken/DL/NAVET_0292/2013 (H5N1) viruses were used.w/v) bovine serum albumin (BSA), 0.05% (v/v) Tween20 in PBS (PBST) for 2 h and incubated at 25 °C for 1 h. Plates were washed 5 times with PBST, incubated with alkaline phosphatase-conjugated anti-chicken IgY diluted  in 1% (w/v) BSA and washed again. One hundred microliters of p-nitrophenyl phosphate (pNPP) substrate prepared in 0.1 M diethanolamine-HCl, pH 9.8 was applied. Plates were incubated at 37 °C for 1 h. The absorbance signal was measured at 405 nm. End-point titers were determined as the reciprocal highest serum dilutions that produced mean optical density values four-fold greater than the geometric mean of those from the negative control (injected with N. benthamiana WT plant extracts) sera.Each 100 µL per well of 1 µg/mL of IMAC- and SEC-purified hemagglutinin trimers in phage PBS  were put into flat-bottom microtiter plates  and incubated overnight at room temperature for coating. One hundred microliters of serum samples as serial dilutions starting at 1:100 was applied after blockage with 3% (® Influenza H5 Antibody Competition) was used to detect anti-H5 chicken antibodies raised by the purified H5 trimers. The procedure followed the the manufacturer’s instructions (ID.vet).A commercial competitive ELISA (ID Screen®-D adjuvanted H5-TG trimer (10 µg antigen/chicken) in PBS at the second and fifth weeks of age. Seven chickens received neither antigens, adjuvants nor buffer (non-vaccinated group). At 7 weeks of age (two weeks after the second vaccination), each chicken was inoculated by 107 plaque forming unit of HPAIV A/duck/Viet Nam/TG24-01/2005 (H5N1). All birds were clinically observed for 10 days. Clinical scoring was done as following: 0 for healthy birds, 1 for birds with one clinical sign , 2 for birds that showed more than one clinical sign, and 3 for dead birds. The pathogenicity index (PI) was calculated as the sum of the daily arithmetic mean values divided by 10 (the number of observation days). The PI ranged from 0 (avirulent) to 3 (highly virulent).Seven chickens per group were intramuscularly immunized with EmulsigenThe experiment was conducted in the biosafety level-3 animal facilities of the Friedrich-Löffler-Institut (FLI) following the German Regulations for Animal Welfare and authorized by the ethics committee of the State Office of Agriculture, Food Safety, and Fishery in Mecklenburg-Western Pomerania (permission number: 7221.3-1.1-051/12). The experiment was performed after approval from the authorities and the commissioner for animal welfare at the FLI representing the Institutional Animal Care and Use Committee (IACUC).N. benthamiana WT plant extract that had the same amount of total soluble proteins as plant extracts containing H5HT oligomers were used. Plant extracts containing H5HT oligomer-TP or H5HT trimer, N. benthamiana WT plant extract, and commercial inactivated H5N1 virus vaccine  were formulated with in-house water in oil adjuvant (NAVETCO) at 70% final concentration. To monitor the effect of the in-house water in oil adjuvant, plant extracts containing either H5HT oligomer-TP or H5HT trimer without adjuvant were included in the experiment. Twelve chickens (10 chickens for the negative control group) per group were intramuscularly immunized with antigens at the second and fifth weeks of age. Two weeks after the second vaccination, each chicken was inoculated by 106 egg lethal dose 50 of HPAIV A/chicken/DL/NAVET 0292/2013 (H5N1). One day before viral inoculation, chicken sera were collected individually for hemagglutination inhibition and ELISA tests. All birds were observed for 10 days after the viral inoculation.The hemagglutinin contents in plant extracts were semi-quantified by Western blotting. Plant extracts containing 70 ng of either H5HT oligomer-TP or H5HT trimer were selected for chicken immunization. In the control group, Experiments with chicken were conducted in accordance with the “3Rs” and the relevant rules of both Institute of Biotechnology  and NAVETCO ethics committees under the decision number 07/2015/HĐ-NĐT. p-values less than 0.05 were defined as significant.Statistical analyses of HI data and ELISA results were performed using the Mann–Whitney rank-sum test (Sigma Plot software). N. benthamiana, and cloned it into a plant expression vector  in plants  by fusion vector . After iThe trimeric form of purified H5 proteins was determined by a crosslinking reaction using a BS3 (bissulfosuccinimidyl suberate) cross linker. H5 proteins were exposed to BS3 and crosslinked products were separated on a SDS gel under reducing and denaturing conditions, blotted and immunodetected using an anti-c-Myc monoclonal antibody. Immunoblot results reveal a single band with an apparent molecular weight of approximately 300 kDa corresponding to a trimer  strain can be obtained by the fusion of the trimeric GCN4-pII motif at the C-terminus.®-D adjuvant at 20% final concentration. Seven chickens per group were intramuscularly vaccinated with purified H5 trimers (10 µg antigen/chicken), and PBS at the second and fifth weeks of age. A group of seven non-vaccinated chickens were included. At one day old (before the first immunization) and seven weeks old , chicken sera were collected. Serum samples were examined using a commercial competitive ELISA against anti-H5 avian influenza antibodies . As summarized in Purified H5 trimers were tested for their ability to induce protective immunity against homologous highly pathogenic avian virus  induced stronger antibody responses than trimers and monomers ,13. In aWe hypothesized that stabilized H5 trimers can be linked via connectors that are introduced after the GCN4pII trimeric motif to form H5 oligomers, outlined in N. benthamiana leaves, the expression of all four proteins was confirmed. Expression levels of these proteins  were approximately 0.02, 0.02, 0.003, and 0.003% of totally soluble, respectively, as semi-quantitatively estimated by Western blot (N. benthamiana and PBS buffer (negative controls) did not show hemagglutination activity  were further characterized under native conditions. After affinity purification, all three potential oligomers were separated by SEC, and the fractions were characterized by Western blotting and hemagglutination titer estimation. As demonstrated in As represented in Neutralizing antibodies (inhibiting hemagglutination) raised by oligomer-TP, oligomer-HDP, oligomer-HAP, H5 trimer, and WT crude extracts were then measured. Due to unavailability of the A/duck/Viet Nam/TG24-01/2005 (H5N1) inactivated virus, the heterologous inactivated virus strain rg A/swan/Germany/R65/2006(H5N1) was used for the HI assay. The two strains shared 96% deduced hemagglutinin amino acid sequence similarity. As shown in The humoral immune responses were tested against purified SEC hemagglutinin H5 by an indirect ELISA d,e. No iTaken together, plant crude extracts containing H5 oligomers and trimers induced specific antibodies against H5. Oligomers induced neutralizing antibodies with titers significantly higher compared to trimers. Here, oligomer-TP performed best.During this study, a new H5N1 strain of clade 2.3.2.1 was introduced into Vietnam from Southern China. The H5 sequence (H5HT) of the A/duck/Viet Nam/HT2/2014 (H5N1) strain (clade 2.3.2.1c)  Table S was therTo confirm these observations, H5-specific chicken IgY titers of all chicken groups were estimated by an indirect ELISA. In Neutralizing antibodies were measured by the hemagglutination inhibition test. Inactivated A/Chicken/DL/NAVET_0292/2013 (H5N1) virus was used in the HI test. A/duck/Viet Nam/HT2/20144 (H5N1) and A/Chicken/DL/NAVET_0292/2013 (H5N1) are both grouped in clade 2.3.2.1c and share 98% similarity of the deduced hemagglutinin amino acid sequence . The dat6 egg lethal dose 50 (106 ELD50). All negative control animals died within four days after viral challenge. Animals that were vaccinated with the non-adjuvant H5HT trimer crude extract died within three days. Animals vaccinated with the non-adjuvant H5HT oligomer-TP crude extract also died, but with a delay of 1–3 days. In contrast, animals vaccinated with adjuvant H5HT trimer and the H5HT oligomer-TP crude extracts showed high survival rates. Nine out of twelve animals (75%) immunized with adjuvant H5HT trimer crude extracts and eleven out of twelve animals (92%) immunized with adjuvant H5HT oligomer-TP crude extracts were protected from lethal disease. One animal died with a delay at day six after virus challenge. All chickens in the positive control group (immunized with the standard vaccine) survived. To monitor the stability of H5 antigens in plant extracts, two independent experiments were performed  in clinical vaccine development. Multivalent antigen presentation can be achieved by using nanoparticles for antigen display  or by ateraction , which reraction ,34 that Recombinant H5 expression in eukaryotic expression systems results in superior antigens compared to other expression systems in terms of posttranslational modifications such as disulfide bond formation, glycosylation, protein folding and trimerization. We and others have shown that recombinant influenza H5 was N-glycosylated both in plant cells ,36 and ain planta allow the direct use of plant extracts containing antigens for animal vaccination. However, N. benthamiana plants are known to contain alkaloids, especially a high amount of nicotine , and anabasine (8.4%) [N. benthamiana variants. Although all animals in our study were healthy and alive after vaccination with plant extracts, nicotine contents present in plant extracts should be monitored in the future studies dependent on the extraction method to further improve this vaccination strategy.The results presented in the actual study and data provided by others  showed te (8.4%) . NicotinOur results clearly show the potential of H5 oligomers in plant crude extracts for the development of cheap protective veterinary vaccines meeting the demands of highly effective vaccines, simple and severe production strategies and efficient and applicable methods for distribution and use. Together with the high stability of the H5 oligomers in plant crude extracts, this is a very well-founded concept, especially for developing countries. The developments and experiments in Vietnam strengthen the value of these data in the fight against avian flu as an international goal. Such a vaccine against avian flu has the potential to decrease virus shedding in poultry significantly and thus could help to build herd immunity. As a zoonosis, avian flu is of significant importance for human medicine, especially because of the rather high mortality rate of infected patients. In the light of specific epidemiologic conditions in south-east Asia, such a vaccine could be very useful in reducing the danger of infection in human beings.


H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation is prevalent in bone tumors. In contrast to K27M mutation, it remains elusive how G34 mutations affect the epigenome. Here we performed whole-genome bisulfite sequencing of four G34R-mutated gliomas and the G34V-mutated glioma cell line KNS-42 for comparison with gliomas harboring K27M and no mutations in H3F3A and with G34W-mutated bone tumors. G34R-mutated gliomas exhibited lower global methylation levels, similar CpG island (CGI) methylation levels, and compromised hypermethylation of telomere-proximal CGIs, compared to the other two glioma subgroups. Hypermethylated regions specific to G34R-mutated gliomas were enriched for CGIs, including those of OLIG1, OLIG2, and canonical histone genes in the HIST1 cluster. They were notably hypermethylated in osteosarcomas with, but not without, G34W mutation. Independent component analysis revealed that G34 mutation-specific components shared a significant similarity between glioma and osteosarcoma, suggesting that G34 mutations exert characteristic methylomic effects regardless of the tumor tissue-of-origin. CRISPR/Cas9-mediated disruption of G34V-allele in KNS-42 cells led to demethylation of a subset of CGIs hypermethylated in G34R-mutated gliomas. These findings will provide a basis for elucidating epigenomic roles of G34 oncohistone in tumorigenesis.Two recurrent mutations, K27M and G34R/V, in  Global CpG hypomethylation and focal hypermethylation at CpG islands (CGIs) are well-known hallmarks of cancers7. These alterations include IDH1/2 mutations and 1p/19q co-deletion, which have served as the bases for the new nomenclature of central nervous system tumors8. The association of epigenetic alterations with glioma has also been reported. For instance, IDH1/2-mutated gliomas harbor a characteristic methylation pattern defined as the glioma-CGI methylator phenotype and associate with favorable prognosis. Methylation of the promoter of MGMT, encoding O6-methylguanine-DNA methyltransferase, was demonstrated to be a predictive factor for chemosensitivity to alkylating agents9. A recent study demonstrated the utility of methylation profiling in auxiliary diagnosis of central nervous system tumors10.Genetic alterations involved in gliomagenesis have been extensively investigatedH3F3A gene is one of the two genes encoding histone H3.3, a non-canonical histone variant, and has been established as a major driver gene of malignant gliomas, particularly pediatric and young adult gliomas13. Histone H3.3 is loaded to transcriptionally active regions in a replication-independent manner and is conserved among various species, ranging from yeast to human14. Several cancers bear missense mutations that affect the N-terminal tail of histone H3.3. Two recurrent mutations of H3F3A, K27M and G34R/V, have been identified in glioma, whereas G34W/R/L mutations of H3F3A are prevalent in giant cell tumor of bone (GCTB) and K36M mutation of H3F3B recurs in chondrosarcoma17.The 18. Distinct patterns of gene expression and DNA methylation, which likely affect disease phenotypes, have also been reported for these gliomas primarily by microarray analyses12. Epigenetic mechanisms of tumor development have been well elucidated for the K27M subgroup, which is defined as "diffuse midline glioma, H3 K27M-mutant" in the latest WHO classification10. Histone H3.3 bearing K27M substitution traps the Polycomb Repressive Complex 2 (PRC2) to inhibit its histone methyltransferase activity, thereby leading to a global decrease and a local increase of histone H3 trimethylated at its K27 residue20. Accordingly, EZH2, the catalytic component of PRC2, is a promising target of new molecular targeted therapy22.Previous studies on gliomas revealed that K27M and G34R/V subgroups correlate with distinct clinicopathological features such as disease prevalence, age, tumor location, prognosis, MRI findings, and histopathological diagnosis24 to delineate single base-resolution methylomes of four G34-mutated glioma cases characterized in our previous study25. Comparison of the WGBS data with those of other types of gliomas and bone tumors highlighted a unique methylomic signature shared with bone tumors bearing G34 mutations.On the other hand, the mechanism behind the contribution of G34 mutations to tumorigenesis remains elusive. As an approach to address the epigenetic mechanisms of action for G34 variants, we conducted whole-genome bisulfite sequencing (WGBS) by post-bisulfite adaptor tagging (PBAT)H3F3A gene (WT subgroup), respectively, as well as the G34V-mutated glioma cell line KNS-42 , K27M mutation (K27 subgroup), and no mutation in the 2 Tables  and S2. 2 Tables . This or2 Tables . All theTo further evaluate the methylation pattern among the three subgroups, we divided the genome into non-overlapping 1-Mb sliding windows, calculated mean methylation levels of individual windows, and examined their distribution Figure . While t26. Almost all PMDs common to the four G34R-mutated gliomas overlapped with those identified in at least one case of the other two subgroups , which are large genomic regions with intermediate methylation levels and often overlap with the lamina-associated domains (LADs)s Figure . Thus, t12. However, current WGBS data indicated that mean methylation levels of these regions are comparable to the genome-wide mean in all the three subgroups  as a characteristic of G34-mutated gliomaTo reconcile this apparent discrepancy, we separately analyzed CGIs and non-CGI portions of the genome. Notably, the mean CGI methylation level in telomere-proximal 4-Mb regions was higher than the genome-wide mean in both normal and tumor samples Fig. c. Gene bWe next compared the extent of elevation in telomere-proximal CGI methylation among the three subgroups. Compared to K27 and WT subgroups, G34 subgroup showed a sign of mitigation in the elevation of methylation  between G34 and the other two subgroups . For this purpose, we first identified DMRs between the G34 and K27 subgroups, between the G34 and WT subgroups, and between the G34 subgroup and the normal brain tissue. Next, we determined the intersection of these three DMR sets to define 687 G34 subgroup-specific hyper-DMRs  and 2,064 G34 subgroup-specific hypo-DMRs  Figure . SimilarHIST1 cluster  to such a subgroup-specific IC should serve as good methylation markers for the subgroup.As an alternative approach to identifying G34 subgroup-specific alterations, we applied independent component analysis (ICA) to the WGBS data. ICA is a method for blind source separation to decompose a data matrix into the product of a mixing matrix and independent components (ICs), the latter of which represent hidden source signalsWe separately applied ICA to the methylation data for promoters, gene bodies, and CGIs Figs. a and S4aHIST1 cluster and the GO term “nucleosome assembly” . In ICA, the two disruptants had lower weightings on G34-specific ICs than their parental KNS-42 cells  and that CGIs were enriched for H3p3 and G34V peaks .We also performed ChIP-seq using two monoclonal antibodies, one specific to histone H3.3 and the other specific to histone H3.3-G34V. We hereinafter refer to ChIP-seq peaks obtained by the former and latter antibodies as H3p3 and G34V peaks, respectively. Both H3p3 and G34V peaks were enriched in CGIs and promoters in KNS-42 cells, sharing a largely similar distribution pattern Figures  and S7b.To determine the relationship between histone H3.3 deposition and CGI methylation, we first classified CGIs into four distinct groups based on the presence and absence of overlapping ChIP-seq peaks in KNS-42 cells and then compared methylation levels among the four groups. CGIs with histone H3.3 were less methylated than those without it, irrespective of whether the 34th residue was Gly or Val Fig. c. Among 24 to generate WGBS data on 14 gliomas, including the first four WGBS data on cases with histone H3.3-G34R. These data not only consolidated the findings in a previous study using the methylation array12 but extend the characteristics of G34-mutated gliomas, including a specific signature shared by G34-mutated bone tumors.We used the enhanced PBAT protocol12. In contrast to global hypomethylation  in the same nucleosome particle, when overexpressed in 293 T and HeLa cells, respectivelyIn this study, we used KNS-42 cells to examine the effects of G34V-allele disruption on the methylome and to reveal genomic distribution of G34V-substituted histone H3.3. These approaches are straightforward but have inevitably limitations. First, considering the context-dependent and feedback-rich nature of epigenetic regulation, we should note that simple disruption experiments allow us to examine only immediate effects on the maintenance, but not the generation, of methylome patterns. Molecular mechanisms for de novo formation of a methylome pattern can be dispensable for its maintenance, and the well-known recurrence of G34 mutations in distinct tumor types may be a reflection of developmental context-dependence of their epigenetic effects. Second, ChIP-seq results could be confounded if wild-type and mutant proteins are located at the same loci, because no antibody was available to distinguish wild-type histone H3.3 from G34-substituted proteins. Despite these limitations, our results collectively suggested a role for the deposition of G34-substituted histone H3.3 in elevating methylation levels of a subset of CGIs Fig. . There sH3F3A mutations in gliomas25, we included 11 H3F3A-mutated primary gliomas comprised of four G34R and seven K27M cases. As a control, we included three cases of age-matched glioblastomas, in which genetic analyses confirmed the absence of driver mutations, such as those in IDH1/2, H3F3A, and BRAF (V600E), in addition to 1p/19q codeletion37. These cases are summarized in Table From our previous comprehensive study analyzing H3F3A with CHOPCHOP (New England Biolabs). A template oligonucleotide was designed with EnGen sgRNA Template Oligo Designer (New England Biolabs), synthesized by Eurofins genomics, and used to prepare the single guide RNA (sgRNA). A ribonucleoprotein complex formed between the sgRNA and EnGen Cas9 NLS, S. pyogenes (New England Biolabs) was electroporated into KNS-42 cells using NEON Transfection System (Thermo Fisher Scientific). Following the cloning by limiting dilution, the targeted region was amplified by PCR, cloned with Topo TA cloning kit (Thermo Fisher Scientific), and sequenced using BigDye Terminator v1.1 cycle sequencing kit and SeqStudio genetic analyzer (Thermo Fisher Scientific). Loss of G34V-substituted histone H3.3 was confirmed by Western blotting with anti-histone H3.3 G34V rabbit monoclonal antibody .The G34V-mutated glioma cell line KNS-42 was obtained from Japanese Collection of Research Bioresources Cell Bank. For CRISPR/Cas9-mediated disruption of G34V allele, a target DNA sequence (5′- TTC TTC ACC ACT CCA GTA GA-3′) was selected from the second exon of 24 to prepare PCR-free WGBS libraries from 100 ng of genomic DNAs and subjected them to paired-end sequencing (150 nt × 2) using the Illumina HiSeq X Ten platform at Macrogen Japan Corp. . WGBS reads were processed as described previously24. The statistics are summarized in Table We used an improved PBAT protocol based on a highly efficient method for single-stranded DNA ligationH3F3A (EGAS00001000578), respectively, and eight normal cerebellum samples (EGAS00001000561) from the International Cancer Genome Consortium (ICGC). Methylation array data on GCTBs and osteosarcomas15 were obtained from the authors.We obtained WGBS data on six and five high-grade gliomas bearing K27M and no mutation in 38. For clinical samples, DMRs were filtered to retain those containing at least 20 CpGs, with P-values less than 0.01 and with a methylation difference larger than 30%. For the comparison between KNS-42 with and without G34V allele, we used DMRs with P-values less than 10−5. ICA was performed using the fastICA package in R as described previously39. Outputs of ICA were used to generate Euclidian distance matrices for hierarchical clustering by Ward’s method. All heatmaps were generated using the ComplexHeatmap library40. Motif enrichment analysis was carried out using the findMotifsGenome.pl of HOMER41. All functional enrichment analyses with the Gene Ontology (GO) biological process terms were performed using the topGO package in R.Autosomal CpG sites covered at least 10 times were used for downstream analyses. DMRs were identified using the program metilene with default parametersTotal RNAs were prepared from frozen tissues of five glioma cases and KNS-42 cells using the RNeasy Mini Kit (Qiagen) with DNase treatment. RNA-seq libraries were prepared using the TruSeq stranded mRNA Kit (Illumina) and sequenced using NovaSeq6000 at Macrogen Japan Corp. RNA-seq reads were processed using the CLC Genomics Workbench (Qiagen).42.KNS-42 cells were fixed with 1% formaldehyde and incubated at room temperature for 15 min. The cells were collected with centrifugation and re-suspended in ChIP-lysis buffer  sodium deoxycholate, 1% (v/v) NP-40, 0.1% (w/v) sodium dodecyl sulfate) containing 1 × protease inhibitor cocktail . The chromatin was sheared by sonication with Focused-ultrasonicator S220 (Covaris) using milliTUBE 1-ml AFA Fiber. For ChIP, anti-histone H3.3 rabbit monoclonal antibody  and anti-histone H3.3 G34V rabbit monoclonal antibody were used as primary antibodies with pre-washed Dynabeads Protein A . The epitope of the former antibody lies between the 50th and C-terminal residues of human histone H3.3. The ChIP-ed DNA was purified using the DNeasy Blood and Tissue kit (Qiagen) with some modifications, and 10 ng of the DNA was used for library preparation with the SMARTer ThruPLEX DNA-seq Kit (Takara Bio) according to the manufacturer's instruction. Sequencing was performed using the HiSeq X Ten or NovaSeq 6000 at Macrogen Japan Corp. ChIP-seq reads were mapped to the reference human genome GRCh37 (hg19) and peak calling was performed using MACS2Supplementary information.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.