H3F3A and H3F3B). The identification of these driver mutations has led to development of novel diagnostic tools to distinguish between chondroblastoma, giant cell tumour of bone and other giant cell containing tumours. From a biological perspective, these mutations induce several global and local alterations of the histone modification marks. Similar observations are made for central cartilaginous tumours, which frequently harbour specific point mutations in the metabolic enzymes IDH1 or IDH2. Besides an altered methylation pattern on histones, IDH mutations also induce a global DNA hypermethylation phenotype. In all of these tumour types, the mutation-driven epigenetic alterations lead to a highly altered transcriptome, resulting for instance in alterations in differentiation. These genomic alterations have diagnostic impact. Further research is needed to identify the genes and signalling pathways that are affected by the epigenetic alterations, which will hopefully lead to a better understanding of the biological mechanism underlying tumourigenesis.Recently, specific driver mutations were identified in chondroblastoma, giant cell tumour of bone and central cartilaginous tumours , sharing the ability to induce genome-wide epigenetic alterations. In chondroblastoma and giant cell tumour of bone, the neoplastic mononuclear stromal-like cells frequently harbour specific point mutations in the genes encoding for histone H3.3 ( The genetic make-up of tumour cells alone is insufficient to explain differences in cellular behaviour. One factor that can explain these differences is epigenetics: the stable and heritable change of gene function caused by other factors than alterations in the DNA sequence . This inTo control DNA accessibility, several enzymes such as DNA methyltransferases, histone acetyltransferases, ubiquitin ligases and histone methyltransferases make modifications  on DNA itself or on certain amino acid positions on histone tails . At anotRecently, it was shown that epigenetic regulatory genes are frequently mutated across several tumour types, leading to deregulation of normal gene expression patterns (e.g. silencing of tumour suppressor genes and activation of oncogenes) and thereby promotion of tumourigenesis . EpigeneBone and soft tissue tumours are a rare, heterogeneous group of mesenchymal tumours which frequently harbour epigenetic alterations. For instance, the promoter of the tumour suppressor gene PTEN is frequently hypermethylated in soft tissue sarcomas, while loss-of-function mutations in PTEN are rare in these tumours . FurtherSMARCB1 is the hallmark of malignant rhabdoid tumours and epithelioid sarcomas [SMARCB1 is a core subunit of the SWI/SNF chromatin remodelling complex: a group of proteins involved in positioning the nucleosomes on the DNA. Furthermore, approximately 80% of all malignant peripheral nerve sheath tumours have mutations in the EED or SUZ12 subunits of the polycomb repressive complex (PRC) 2 [18-SSX fusion in synovial sarcomas, affect epigenetics. The SS18 gene is involved in the SWI/SNF complex, while SSX1 and SSX2 are subunits of the PRC complexes [SMARCB1 subunit, resulting in transcriptional repression of tumour suppressor genes (e.g. EGR1) and transcriptional activation of oncogenes (e.g. SOX2) [Deregulation of chromatin remodelling complexes is also commonly seen in sarcomas. For instance, loss of sarcomas , 10. SMA (PRC) 2 . This co (PRC) 2 . Moreoveomplexes . Fusion g. SOX2) , 16.H3 histone family member 3A and 3B (H3F3A and H3F3B) mutations in giant cell tumour of bone and chondroblastoma, respectively, and isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) mutations in central cartilaginous tumours.Mesenchymal tumours can also harbour mutations in histones themselves or genes indirectly related to epigenetic regulation. This review will focus on a group of bone tumours that share mutations in genes involved in epigenetic regulation: Giant cell tumour of bone (GCTB) is a locally aggressive and rarely metastasizing neoplasm Table . These tGCTB is histologically characterized by three types of cells: the multinucleated osteoclast-like giant cells, the mononuclear macrophage-like osteoclast precursor cells and the mononuclear spindle-shaped stromal cells. The latter are considered as the neoplastic component of GCTB; these cells have the ability to form tumours in mice and can be maintained in vitro , 35. TheThe current treatment of GCTB is curettage combined with local adjuvant therapy to fill the bone cavity (e.g. polymethylmethacrylate or cancellous bone grafts) or, if necessary, en bloc resection. If tumours are unresectable or recur, patients can be treated with denosumab, a monoclonal antibody against RANK ligand , or bispChondroblastoma is a benign, cartilage-forming tumour which accounts for less than 1% of all primary bone tumours (Table H3F3A gene (92% of cases), while H3F3B was predominantly affected in chondroblastoma . H3F3A and H3F3B are paralogous genes located on different chromosomes  and have a slightly different DNA sequence, but both encode for the exact same histone H3.3 protein. The altered amino acids in these genes are remarkably specific for each tumour type. GCTB specifically harbours G34W mutations or, less common, G34L variants in H3F3A, while chondroblastoma exclusively shows K36M alterations in both H3F3A and H3F3B [H3F3A (up to 60%) [H3F3A and H3F3B, or immunohistochemistry using G34W and K36M mutation-specific antibodies, to distinguish between chondroblastoma, GCTB and other giant cell-rich tumours [Recently, a study was published which described the presence of histone H3.3 mutations in both chondroblastoma and GCTB . Interesnd H3F3B . Histone to 60%) . Of note to 60%) , 43. Thi tumours , 31, 44  tumours  Histone H3.3 is a well conserved replacement histone that is highly structurally related to the canonical histone H3 protein. H3.3 is the most predominant form of histone H3 in non-dividing cells, and incorporation is independent of DNA synthesis. Usually, histone H3.3 replaces the canonical histone H3 protein in the nucleosome of active genes (e.g. promoter and gene bodies), suggesting that it may be an epigenetic regulator of transcriptionally active chromatin . The mosH3F3A and H3F3B (H3K36M) causes genome-wide reduction of methylation in chondroblastoma, specifically loss of di-methylation of H3K36 (H3K36me2) at intergenic regions and H3K36me3 at gene bodies [BMP2 and RUNX2, respectively) and homologous recombination (i.e. downregulation of BRCA1 and ATRX) [Several research groups have started to elucidate the epigenetic alterations caused by H3.3 mutations and the mechanisms underlying these changes in chondroblastoma and GCTB. The K36M mutation in e bodies , 47. Mete bodies . Pathwaynd ATRX) .The reduction in methylation is caused by the inhibition of histone methyltransferases (HMTs) such as NSD1, NSD2 and SETD2. The active SET domain within these HMTs contains tyrosine residues which normally bind to a lysine residue on histone tails to deposit a methylation mark. However, in the case of a H3K36M mutation, the lysine is mutated into a methionine which results in stronger binding of HMTs to the histone tail. This causes a global reduction in the HMT levels and its enzymatic activity, leading to a genome-wide alteration of the H3K36me2/3 landscape. This higher affinity of HMTs for H3K36M was confirmed by solving the crystal structure of the SETD2-H3K36M complex and by performing pull-down assays showing an enriched interaction between HMTs and H3K36M , 49, 50.Wnt6 and Sox6, genes involved in blocking mesenchymal differentiation. Thus, H3K36M mutations change the K27 and K36 methylation landscape, resulting in a highly altered transcriptome.Additionally, H3K36M mutations cause redistribution of the repressive H3K27me3 histone mark and its reader complex PRC1. Upregulation of H3K27me3 has been observed in intergenic regions, while loss was observed at gene loci . ConsequH3F3A (H3F3A G34) is less clear. Although histone modifications cannot be directly made on the G34 residue, H3F3A G34 mutations may affect the deposition of marks on the K36 position, and possibly also other modification sites, due to steric hindrance. An interaction study showed that the G34 position is involved in binding SETD2 [MYCN) [In contrast, the mechanism underlying the observed epigenetic changes in cells harbouring G34 mutations in ng SETD2 . SETD2 hng SETD2 . The inhng SETD2 . Howeverng SETD2 . Moreoveng SETD2 . This su2 [MYCN) .For GCTB, the genes undergoing a change upon redistribution of the H3K36me3 marks as a result of the H3F3A G34W/L mutations remain to be identified. Changes that are observed in primary GCTB cells harbouring a G34W mutation are increased proliferation, migration and colony formation capacity as compared to wild-type counterparts , 56. FurCurrently, there are limited reports on in vitro or in vivo studies describing promising epigenetic therapeutic strategies for GCTB. Therapies directly targeting the mutated histones are lacking, and reversal of genome-wide epigenetic alterations is not feasible without major side-effects on the healthy, normal epigenetic landscape. However, the use of histone demethylase inhibitors, such as GSKJ4, may counteract the observed reduction or redistribution of H3K36me3 and H3K27me3. On the other hand, reduction of H3K36me3 may be a promising therapeutic target and recently it was shown that WEE1 inhibition in GCTB results in a global reduction of H3K36me3 and a decrease in proliferation . NeverthEnchondroma is a benign, cartilage producing tumour which accounts for 10–25% of all benign bone tumours  , which probably become more important drivers of tumourigenesis than IDH mutations [Eighty-seven percent of the solitary and multiple enchondromas, ~ 50% of the primary central conventional chondrosarcomas and 86% of the secondary central chondrosarcomas harbour heterozygous point mutations in and IDH2 , 26. The in mice . Furtherin vitro , 60. Thuutations .IDH1 and IDH2 mutations also frequently occur in for instance acute myeloid leukaemia (AML), glioma and cholangiocarcinoma [IDH1 and the R140/R172 positions in IDH2. Some of these variants can be detected with the use of immunohistochemistry and mutation-specific antibodies, displaying the mosaicism for IDH mutations in central cartilaginous tumours , glioma has mainly R132H mutations in IDH1 (90%) and AML has most often R140Q mutations in IDH2 (30–50%) [IDH1 and IDH2 mutations on tumourigenesis.arcinoma . Most ofosarcoma . Interes(30–50%) . However2. Due to the arginine point mutations, the IDH enzymes acquire a gain-of-function, leading to additional conversion of α-KG into the oncometabolite D-2-hydroxyglutarate (D-2-HG). Of note, different IDH1 and IDH2 mutations produce variable levels of D-2-HG: R132C is a strong D-2-HG producer, while R132H and R140Q produce lower levels of the oncometabolite [IDH1 and IDH2 are enzymes with a similar function in the tricarboxylic acid cycle (TCA cycle) where they convert isocitrate into α-ketoglutarate (α-KG) and COtabolite . This coIDH mutations also affect other processes within the cell, such as metabolism, cell growth signalling pathways and DNA damage repair [D-2-HG and α-KG have a high structural similarity, leading to competitive binding of α-KG-dependent enzymes by D-2-HG. Some of these α-KG-dependent enzymes are involved in maintaining the epigenetic landscape of cells, such as DNA demethylases (family of TET enzymes) and histone demethylases (family of Jumonji enzymes). e repair , but theIDH-mutated tumours, including enchondroma and central chondrosarcoma [IDH mutation status [IDH1 mutant inhibitor AGI-5198 does not alter the DNA methylation phenotype, suggesting that the epigenetic alterations might have become independent of the IDH1 mutation [IDH1 mutant in astrocytes [It has been shown that D-2-HG inhibits the activity of Tet Methylcytosine Dioxygenase 2 (TET2) in vitro . Normallosarcoma , 66. Hown status . Additiomutation . Anothertrocytes . To concIDH1 or IDH2 mutation in HEK293T cells leads to elevated levels of the H3K4me3, H3K9me2/3, H3K27me3, H3K36me3 and H3K79me2 histone marks [IDH1 mutation [IDH1 mutant knock-in mice show that none or only a subset  of the histone methylation marks are aberrant [IDH mutation alone is not enough to alter the histone methylation landscape. Similar conflicting results are also observed in enchondroma and central chondrosarcoma: an immunohistochemistry study on 101 primary tumours shows high levels of the H3K4me3, H3K9me3 and H3K27me3 histone modification marks, irrespective of the IDH1 or IDH2 mutation status [Another group of enzymes that are inhibited by D-2-HG is the Jumonji-C domain-containing histone lysine demethylases (KDM enzymes), including the KDM4 family members which are also affected by histone H3.3 mutations . Inhibitne marks . An indumutation . Howeveraberrant , 71. Thin status . Addition status . Both stIDH mutation-induced epigenetic alterations remain to be elucidated, most studies point towards reduced differentiation capacity. Both neural and haematopoietic differentiations are impaired due to aberrant histone methylation marks or DNA hypermethylation [IDH1-mutated cholangiocarcinoma, reduced expression of H3K4me3 at the HNF4A promoter inhibits hepatocyte differentiation [IDH mutation or addition of D-2-HG impairs osteogenic differentiation and induces chondrogenic differentiation in mesenchymal stem cells [IDH mutations also reduce the expression of DNA repair protein ATM [NAPRT1 promoter seems to correlate to NAMPT inhibitor sensitivity in IDH-mutated glioma [NAPRT1 promoter, although this phenotype is independent of the IDH mutation status [Although the specific genes affected by hylation , 72. In ntiation . Furtherem cells , 60. IDHtein ATM , althougd glioma . Chondron status .IDH1 or IDH2 mutant enzyme and thereby prevention of the formation of the oncometabolite. However, in vitro experiments do not show a beneficial effect in chondrosarcoma cell lines: proliferation, migration and colony formation capacity are not affected, only at high concentrations [IDH mutant inhibitors in patients, but the results for chondrosarcoma patients have not yet been published. Since several in vitro studies indicate that the observed changes might have become static and independent of the IDH mutation over time, it may be more promising to directly target these changes. To counteract the DNA hypermethylation phenotype, the use of DNA methyltransferase inhibitors, such as decitabine, could be a promising therapeutic strategy. An in vitro study shows that decitabine induces the re-expression of several epigenetically silenced tumour suppressor genes and reduces the proliferation and migration of chondrosarcoma cells [The most straightforward way to target the epigenetic alterations is direct inhibition of the trations , 77. Sevma cells . Contradma cells . These cIDH and H3.3 mutations are considered drivers only in benign  and locally aggressive (GCTB) neoplasms. This is in line with the fact that these epigenetic changes predominantly affect differentiation, instead of proliferation. In the case of malignant chondrosarcoma, the IDH mutation is no longer associated with tumourigenic behaviour and probably other mutations have taken over the driver function of the IDH mutation [Chondroblastoma, GCTB and central cartilaginous tumours all harbour driver mutations that induce wide-spread epigenetic alterations Fig. . Interesmutation . Similarmutation . FurtherH3F3A and H3F3B both support pathologists to differentiate between chondroblastoma, GCTB and other giant cell containing neoplasms [IDH1 R132H mutation-specific antibody is commonly used in neuropathology [IDH1 R132C mutation in central cartilaginous tumours is not yet available but would be very helpful in distinguishing enchondroma and chondrosarcoma from their histologic mimics [The identification of specific histone H3.3 variants in both chondroblastoma (i.e. H3K36M) and GCTB (i.e. H3F3A G34) has led to development of novel diagnostic tools. Mutant protein-specific immunohistochemistry and/or sequencing of eoplasms , 31, 44 athology , an antic mimics   and mutant cells. A similar relation between wild-type and mutant cells has been observed for central cartilaginous tumours, which display intra-neoplastic mosaicism for the IDH1 or IDH2 mutations, alternative driving mechanisms should be considered. It remains to be elucidated whether these alternative driver mechanisms induce similar epigenetic alterations as observed in IDH mutant chondrosarcomas. Also, one could speculate that the IDH wild-type cells may overgrow the IDH mutant cell population over time. While IDH mutations are essential for the initiation of enchondroma, the additional acquired mutations that take over the driver function of IDH mutations in chondrosarcoma may occur in the wild-type cells which might explain the observed IDH wild-type genotype in half of the chondrosarcomas.As only ~ 50% of the chondrosarcomas harbour


Gallbladder cancer (GBC) is an extremely malignant tumor with a high mortality rate. Little is known about its invasion and metastasis mechanism so far.To identify the driver genes in GBC metastasis, we performed a mRNA microarray of metastatic GBC and paired non-tumor samples, and found PLEK2 was markedly upregulated in GBC tissues. Next, the expression of PLEK2 in GBC were examined in a larger cohort of patients by qRT-PCR, western blot and IHC staining. The clinicopathologic correlation of PLEK2 was determined by statistical analyses. The biological involvement of PLEK2 in GBC metastasis and the underlying mechanisms were investigated.In this study, we found that PLEK2 had higher expression in GBC tumor tissues compared to non-cancerous adjacent tissues and cholecystolithiasis tissues. The clinicopathologic analyses showed PLEK2 expression was positively correlated with tumor TNM stage, distant metastasis and PLEK2 was an independent predictor of overall survival (OS) in GBC patients. The cellular function assays showed PLEK2 promoted GBC cells migration, invasion and liver metastasis in mouse model via the regulation of epithelial-mesenchymal transition (EMT) process. Our mass spectrum and co-immunoprecipitation (co-IP) assays demonstrated that PLEK2 could interact with the kinase domain of EGFR and suppress EGFR ubiquitination mediated by c-CBL, leading to constitutive activation of EGFR signaling. Furthermore, RNA-sequencing and qRT-PCR results demonstrated chemokine (C-C motif) ligand 2 (CCL2), a target gene downstream of PLEK2/EGFR signaling, mediated the motility-promoting function of PLEK2.On the basis of these collective data, we propose that PLEK2 promotes the invasion and metastasis of GBC by EGFR/CCL2 pathway and PLEK2 can serve as a potential therapeutic target for GBC treatment.The online version of this article (10.1186/s13046-019-1250-8) contains supplementary material, which is available to authorized users. Gallbladder cancer (GBC) is the most common tumor in biliary tract disease. The median survival is less than 1 year and overall survival rate is about 17.8–21.7% , 4. GBC − subsets melanoma patients from healthy people. Transcriptome profiling of PLEK2 expression in whole blood cells could be used as early detection of melanoma [To identify the driver genes in GBC metastasis, we performed a mRNA microarray analysis of metastatic GBC and paired non-tumor samples and found several genes upregulated, including pleckstrin-2 (PLEK2). PLEK2 is a 353 amino acid protein with 2 pleckstrin homology (PH) domains and a disheveled–Egl-10–pleckstrin (DEP) domain . Unlike melanoma . AltogetOur protein–protein interaction analysis suggests that PLEK2 can interact with EGFR. Whether PLEK2 is involved in EGFR activation process is what we deal with in this study. EGFR is a 170 kDa receptor tyrosine kinase and widely expressed in numerous tumors . EGFR abIn this study, we provided evidences both in vitro and in vivo that PLEK2 promoted GBC cells migration, invasion and liver metastasis via the regulation of epithelial-mesenchymal transition (EMT) process. Additionally, PLEK2 had higher expression in GBC tumor tissues compared to cholecystolithiasis tissues and high PLEK2 expression was positively correlated with liver metastasis and prognosis in GBC patients. Mechanical investigations verified that PLEK2 could combine with EGFR and suppress EGFR ubiquitination mediated by c-CBL, leading to constitutive activation of EGFR signaling. Furthermore, we found chemokine (C-C motif) ligand 2 (CCL2) mediated the motility-promoting function of PLEK2. In conclusion, our study demonstrated that PLEK2 promoted the invasion and metastasis of GBC by EGFR/CCL2 pathway.Tumor samples and paired normal samples were collected from GBC patients who underwent surgical resection and postoperative adjuvant chemotherapy. Total149 GBC tumor and 149 cholecystolithiasis samples were collected at the Department of Pathology, Renji Hospital, from January 2004 to February 2015. The 149 GBC tumor and 149 cholecystolithiasis tissues were used for tissue microarray, the 29 pairs of the fresh primary GBC tissues and paired non-tumorous tissues were used for qRT-PCR and the 14 pairs of tissues were used for western blotting assays. Medical records and Follow-up data were obtained from the questionnaires and patients’ medical records of the hospital. This project was approved by the Ethical Committee of Renji Hospital, Shang Hai Jiao Tong University School of Medicine.Total 149 GBC tumor samples were stained with the PLEK2 (Proteintech), CCL2 (Proteintech) and EGFR (Santa Cruz) antibody. The staining was scored as the intensity of the positive staining  multiplied by the staining areas . These scores were independently determined by two pathologists.Human GBC cell line GBC-SD and HEK 293 T cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Human GBC cell line NOZ was obtained from Xinhua hospital . HEK 293 T and GBC-SD cells were cultivated in RPMI-1640 medium  supplemented with 10% fetal bovine serum (GIBCO) in an atmosphere consisting of 5% CO2 and 37 °C. Willian’s E medium was used in NOZ cells. The PLEK2 knockdown and overexpression cells were all constructed as stable cell lines. The Flag-tagged PLEK2 were cloned into into pCDH-CMV plasmid . The PLKE2 shRNA was constructed by Shanghai GenePharma Medical Biotechnology Company. PCDH-flag-PLEK2 sense: 5′- TGCTCTAGAGCAATGGATTACAAGGA TGACGACGATAAGGAGGACGGCGTGCTCAAGGA -3′; PCDH-flag-PLEK2 antisense: 5′- CCGGAATTCCGGTCATGTTAGCTTTTTGATAGCTTCAATC − 3′. The sense sequence of PLEK2 shRNA was: TGCTGAGAGCTACAAAAAG; The sequence of the PLEK2 shRNA was the following: 5′-TGCTGTTGACAGTGAGC GCTTGCTGAGAGCTACAAAAAGATAGTGAAGCCACAGATGTATCTTTTTG TAGCTCTCAGCAAATGCCTACTGCCTCGGA-3′. The detailed methods of transfection and infection were previously described .Total RNA was isolated from GBC tissues and cell lines using Trizol RNA isolation reagent (Invitrogen) and reversely transcribed to cDNA with a cDNA Synthesis kit . qRT-PCR was used to detected the gene expressions with SYBR Premix Ex Taq . The primers were as follows:PLEK2; β-actin; EGFR; CCL2; ARHGDIB;CNN3; FGD4; NEXN;WIPF1; ARRB1; TWIST1; SLUG; ZEB1;ZEB2; SNAIL.Cells were transfected with the indicated constructs for 48 h and then cells were dissolved in IP lysis buffer  with protease inhibitor cocktail (Sigma) and PMSF (Sigma) for 1 h at 4 °C. After centrifugation at full speed for 20 min, cleared supernatant were gently rotated with antibodies and protein A beads (Invitrogen) for 4 h at 4 °C. Then beads were washed four times with IP lysis buffer. The beeds were eluted in 1X SDS buffer. Primary antibodies were as followings: anti-PLEK2 (Proteintech), anti-EGFR , anti-IgG (Abcam), and anti-Flag (Sigma).The analyses were performed in an HPLC system  and mass spectrometer . A 15-cm dish of Flag-PLEK2 overexpression NOZ cells and control cells were collected. The sample were prepared as described in the method of coimmunoprecipitation. Eluates were subjected to western blot and then stained by coomassie brilliant blue.104 cells in 100 μl serum-free medium with 8-μm pore size polycarbonate membrane . 600ul medium containing 10% FBS was added to the lower chamber. 4% paraformaldehyde was used to fix the migrated cells and coomassie brilliant blue was used to stain the cells after 16 h. For invasion assay, 8 × 104 cells were maintained in the matrigel  coated chamber for 48 h.Three independent experiments were carried out.GBC cells were performed transwell assay. For migration assay, GBC cells were seeded into the 24-well transwell chamber at a density of 4 × 4 GBC-SD cells were plated in 24-well plates covered with sterile coverslips. After12 hours, cells were starved 16 h prior to 50 ng/ml EGF incubation with 10 min. In the control group, cells were not treated with EGF. Then the primary and matched secondary antibody diluted in PBS containing 2% BSA were used to stain PLEK2. Next, we washed the cells with PBS three times and stained cell nuclei with 4, 6-diamidino-2-phenylindole (DAPI). Three independent experiments were carried out.2× 106 NOZ cells diluted in 100 μl PBS were inoculated subcutaneously into the right lower regions of 4-week-old male nude mice. The mice were sacrificed after 7 weeks from the inoculation. The subcutaneous xenografts and livers were dissected and made into sections for haematoxylin and eosin (H&E) staining. All procedures were performed in accordance with the regulations of Renji Hospital, School of Medicine, Shanghai Jiao Tong University. All the procedures were according to the regulations of the RenJi Hospital of Shanghai Jiao Tong University.4 × 10P < 0.05 was considered statistically significant.Data are expressed as mean ± SEM. An unpaired two-tailed Student’s t test and Pearson’s Χ2-test was used to analyze the variance of each experimental group. Kaplan–Meier method and log-rank test were used to estimate the survival probabilities. Cox proportional hazard regression model was performed in univariate and multivariate analysis. http://www.broadinstitute.org), we found that PLEK2, one of the most upregulated genes in GBC compared with paired non-tumor tissues, had a relative high expression in biliary tract cancer . Altogether, these results suggested PLEK2 expression was elevated in GBC tissues. Besides, higher levels of the PLEK2 protein was found to positively correlate with TNM stage and liver metastasis , also PLEK2 overexpression NOZ and GBC-SD cells  . (PDF 145 kb)Additional file 2:Figure S2A. Cells construction of PLEK2 down-regulation NOZ and GBC-SD cells , also PLEK2 overexpression NOZ and GBC-SD cells . (PDF 95 kb)Additional file 3:Figure S2B. Proliferation ability of NOZ and GBC-SD cells with stable PLEK2 knockdown and controls cells were measured by CCK-8 assay. (PDF 91 kb)Additional file 4Figure S2C. EMT markers of NOZ and GBC-SD cells with stable PLEK2 knockdown and overexpression were detected by qRT-PCR. (PDF 186 kb)Additional file 5:Figure S4A. EGFR expression of PLEK2 overexpression and control cells after 50 ng/ml EGF treatment were detected by IF staining. (PDF 218 kb)Additional file 6:Figure S4B. EGFR mRNA levels of NOZ and GBC-SD cells with stable PLEK2 knockdown and overexpression were detected by qRT-PCR. (PDF 118 kb)Additional file 7:Figure S4C. GBC cells were treated with 100 μM Chloroquine for 8 h, followed by 50 ng/ml EGF stimulation for 5 m. Alterations of EGFR expression in PLEK2 knockdown cells were detected by western blot. (PDF 139 kb)Additional file 8:Figure S4D. Protein levels of EGFR in PLEK2 overexpression cells with increasing ectopic c-CBL expression were detected by western blot. (PDF 93 kb)Additional file 9:Figure S5. Representative images of H&E staining of mouse model. Figure S5A and S5B were the representative images of H&E staining of metastatic focuses in livers, Figure S5C was a representative image of the H&E staining of subcutaneous xenografts. (PDF 1697 kb)


C. elegans, disruption of the Integrator complex leads to transcription of genes located downstream of the snRNA loci via a non-conventional transcription mechanism based on the lack of processing of the snRNAs. RNAP II read-through generates long chimeric RNAs containing snRNA, the intergenic region and the mature mRNA of the downstream gene located in sense. These chimeric sn-mRNAs remain as untranslated long non-coding RNAs, in the case of U1- and U2-derived sn-mRNAs, but can be translated to proteins in the case of SL-derived sn-mRNAs. The transcriptional effect caused by disruption of the Integrator complex is not restricted to genes located downstream of the snRNA loci but also affects key regulators of signal transduction such as kinases and phosphatases. Our findings highlight that these transcriptional alterations may be behind the correlation between mutations in the Integrator complex and tumor transformation.Gene expression is generally regulated by recruitment of transcription factors and RNA polymerase II (RNAP II) to specific sequences in the gene promoter region. The Integrator complex mediates processing of small nuclear RNAs (snRNAs) as well as the initiation and release of paused RNAP II at specific genes in response to growth factors. Here we show that in  Caenorhabditis elegans model, we describe how the lack of snRNA processing leads to transcription of genes located downstream of the snRNA loci. This primary alteration is not restricted only to those genes but has a broad effect on the expression of other genes involved in the regulation of signaling pathways by protein phospho-modification.The gene transcription profile determines the developmental state of an organism. During embryogenesis, aging, starvation or any lifecycle stage, organisms express specific sets of genes that must be turned off at other stages to maintain the correct metabolic and differentiated state of the cells. Mutations that disrupt control of signaling pathways may give rise to certain types of tumors. This happens with mutations in genes coding for the Integrator complex, a multi-protein complex involved in the processing of small nuclear RNAs (snRNAs). Here, we uncover a major mechanism underlying gene expression changes in mutants affecting the Integrator complex. Using a  Drosophila S2 cells found two additional subunits that were renamed Ints13  and Ints14  has hast1903yo death .ints-6 promoter and the eft-3 promoter  under the control of its own promoter . Transgey CRISPR ,41,42. Cy CRISPR ,43, the ints-6 in 3’-end processing of the snRNAs, as suggested by its homology to the human Integrator complex subunit 6 and its nuclear localization, we performed deep-sequencing of total RNA obtained from ints-6 (t1903) mutant worms. Processing of the 3’ end of snRNAs was already defective at 15°C (permissive temperature) whereas mRNA termination appeared unaffected. Shifting of the ints-6 (t1903) mutant to 25°C (restrictive temperature) for 12h resulted in an increase in the amount of long transcripts derived from unprocessed snRNAs . Virtually all Integrator subunits except INTS-14 in C. elegans are conserved throughout evolution  further confirmed that the proteins identified as ion Figs  and S7. ion Figs .ints-6 (t1903) mutant , whereas genes not affected by RNAP II read-through are only slightly affected  to reaching adulthood but having reduced offspring and subsequent embryonic lethality , whereas the subunits that resulted in only slight snRNA processing defects , showed non-obvious phenotypes  and Integrator complex depleted worms  mutant worms revealed the existence of both types of transcripts: mRNA (in sense) derived from the endogenous promoter activity and antisense RNAs on the opposite strand, derived from the lack of processing of snRNAs located in antisense downstream of the gene. This suggests that these antisense RNAs might not be efficient at performing RNA silencing , tagged with FLAG and/or eGFP at the 3’-end of the coding gene in its genomic sequence. The 5 transgenes assayed contained two U1 snRNA genes (H27M09.8 and F08H9.10) in the 5’ upstream region of the tagged genes H27M09.5 and F08H9.3 respectively; two U2 snRNA genes (W04G5.11 and F08G2.9) in the respective 5’ region of the tagged W04G5.8 gene and ins-37, an insulin-like peptide; and finally, an SL-2 (sls-2.8) in the 5’ region of the tagged Y75B8A.23 gene. A single copy of these transgenes was integrated into the C. elegans genome by mosSCI  II; unc-119(ed3) IIIJCP301 jcpSi3 [pJC50 )] II; unc-119(ed3) IIIJCP341 jcpSi10 [pJC51 (unc-119(+))] II; unc-119(ed3) IIIJCP343 jcpSi12 [pJC55 )]II; unc-119(ed3)IIIJCP378 jcpSi19  IIJCP383 unc-119(+))]II; unc-119(ed3)IIIJCP387 jcpSi24 [pJC57 )]II; unc-119(ed3)IIIJCP394 jcpSi31 [pJC58 ]II; unc-119(ed3)IIIJCP405 jcpSi37 [pJC60 [ints-6::3xFLAG]JCP462 unc-119(+))] II; unc-119(ed3) IIIJCP479 jcpSi53  II; unc-119(ed3) IIIJCP504 jcpSi55 JCP590 ints-3(jcp8)[ints-3::3xFLAG]JCP614 ints-9(jcp10)[ints-9::3xFLAG]JCP625 ints-5(jcp21)[ints-5::3xFLAG]JCP626 ints-7(jcp22)[ints-7::3xFLAG]JCP630 ints-11(jcp31)[ints-11::3xFLAG]JCP643 ints-13(jcp33)[ints-13::3xFLAG]JCP645 MZ16FA). Gravid hermaphrodites were dissected. 2- to 4-cell stage embryos were mounted on 4% agar pads in water and sealed with Vaseline petroleum jelly. Imaging was performed at 25°C. Differential interference contrast microscopy (DIC) was performed on a motorized fluorescent Leica microscope (DM6000B) equipped with a Hamamatsu Orca-ER C10600 camera and fitted with DIC optics. The appropriate filters were selected for fluorescent microscopy. Images were captured using the open source Micro-manager software (www.micro-manager.org) and processed with XnView software and ImageJ software.Worms were monitored on NGM plates under a Leica dissecting microscope  for 5 min. Once eggs or worms were properly prepared, they were blocked for 15 to 30 min in 1% BSA PBST blocking solution. Subsequently, samples were incubated either O/N at 4°C or at RT using FLAG antibody in 1% BSA PBST  followed by 1 or 2 hours incubation with the secondary antibody Alexa Fluor 633 in 1% BSA PBST . Finally, samples were mounted using VECTASHIELD Antifade Mounting Medium (H-1000 Vector laboratories) with DAPI (1 μg/ml).Synchronized populations of 2+Mg2+ (1 mM) and fixed with 4% paraformaldehyde in PBS/Na+K+ (1 mM) for 30 min at RT with gentle agitation. Next, two washes with PBS (1 mM) were performed for 5 min each and then cells were permeabilized with 0.1% Triton X-100/PBS for 10 min at RT with gentle shaking.U2OS cells were cultured and incubated on pre-treated poly-L-Lysine coated coverslips (Sigma) to improve adhesion. Cells were washed with PBS/CaCyTM3-conjugated AffiniPure Goat Anti-Rabbit IgG, 115-165-003, Jackson ImmunoResearch Laboratories, 1:500 dilution) in 0.2% PBS-BSA for 30 min at RT in the dark. After this incubation, cells were washed with PBS  and incubated with DAPI (2 μg/ml) in PBS for 5 min, all in the dark. Finally, cells were washed with Milli-Q water (3 times) and coverslips were mounted on the slides using the SlowFade Antifade kit (Invitrogen).Next, cells were washed with 0.2% PBS-BSA for 10 min at RT. Subsequently, cells were incubated with Ints6 antibody  in 0.2% PBS-BSA for 1 h at RT in a humid chamber. Afterwards, cells were washed with PBS  and incubated with the secondary antibody )]II;unc-119(ed3)III) were used and extracts from N2 worms were the negative control. IPs/Co-IPs were performed with ANTI-FLAG M2 Magnetic Beads (Sigma).To immunoprecipitate INTS-6, and co-immunoprecipitate its interactors, protein extracts from the JCP378 strain . This step was repeated, leaving the beads with a small amount of buffer. Then, the protein extract was incubated with the equilibrated beads for 3–4 h or O/N, always at 4°C in a rotating rack with gentle mixing. Once the binding step was complete, the beads were collected and the supernatants were removed, followed by the washing steps. The beads were washed with TBS buffer (1500 μl per IP/Co-IP reaction) three sequential times for 10 min on a rotating rack at 4°C to remove all non-specifically bound proteins.INTS-6::3xFLAG::eGFP fusion protein and consequently its interacting proteins were eluted from the magnetic beads either by boiling samples in SDS-PAGE sample buffer (100 μl) or by competitive elution with 3xFLAG peptide (400 ng/μl 3xFLAG peptide in TBS) for 1 h at RT in a rotating rack with gentle mixing). Finally, eluates were precipitated using TCA.Mini-PROTEAN™ TGX™ Precast Gels, Any kDa). Next, gels were stained with Coomassie Blue and bands were excised. The CIC bioGUNE proteomics platform (https://www.cicbiogune.es/org/plataformas/Proteomics) performed the proteomic analysis. Proteins were digested with Trypsin from each gel band and analyzed by LC-MS/MS: Liquid Chromatography-Mass Spectrometry/Mass Spectrometry.Eluted IPs were run on SDS-PAGE  and 1x phosphatase inhibitors  was added to each worm pellet. Next, samples were ground in liquid nitrogen using a pre-chilled mortar and pestle. Ground worms were thawed on ice followed by centrifugation at 4°C  to eliminate any non-soluble tissue or cellular remains. The supernatants were transferred to fresh Eppendorf tubes.Worms  were harvested with M9 buffer and collected in 50 ml Falcon tubes. They were washed several times, allowing them to settle to the bottom between washes. After the last washing step, as much supernatant as possible was removed. Then, a double volume of lysis buffer , containing 1x protease inhibitors (Mini-PROTEAN TGX Precast Gels) after boiling for 5 min in Laemmli buffer . Samples were run using the Mini- PROTEAN Tetra Cell or the Criterion™ Cell electrophoresis system (Bio-Rad) at a constant voltage of 120 V in SDS-PAGE running buffer  SDS) until the tracking dye reached the bottom of the gel. Precision Plus Protein Dual Color Standards (Bio-Rad) were used as the size reference.30 μg of protein extract per sample were loaded onto the polyacrylamide gels  in transfer buffer  for 90 min at 4°C and a constant voltage of 90 V using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) or Criterion Blotter. After transfer, the membrane was blocked in TBS-T  Tween-20, pH 8) with 5% (w/v) non-fat dry milk (Sveltesse Nestlé) for 60 min with gentle rocking. To detect the protein of interest, the membrane was incubated with the primary antibody  sc-1616 Santa Cruz Biotechnology 1:1000) diluted in TBS-T milk for 120 min at RT or O/N at 4°C. Afterwards, the membrane was washed three times with TBS-T for 10 min each and then incubated with the respective horseradish peroxidase (HRP) conjugated secondary antibody in TBS-T milk  for 60 min at RT. The membrane was washed twice more with TBS-T for 10 min each time and then once with TBS only.For antibody-specific detection of proteins, samples were separated by SDS- PAGE and transferred to a 0.45 μm nitrocellulose membrane  and Amersham Hyperfilms ECL  were used according to the manufacturer’s instructions.To chemiluminescently detect the protein of interest, Films were developed manually: 1 min in developing solution (Agfa developer G153), 1 min in fixing solution (Agfa fixer G-345) and then rinsed in water.Colloidal Blue Staining Kit (Invitrogen) according to the manufacturer’s instructions. Polyacrylamide gels were silver stained using Silver Stain for Mass Spectrometry (Thermo Scientific) according to the manufacturer’s instructions.Polyacrylamide gels were stained with Coomassie blue using the ints-2), Y51A2D.7 (ints-5) T23B12.1 (ints-12) R02D3.4 (ints-13) or the Ahringer Library  II using antiFLAG antibodies (left panel) and DAPI (right panel). C. elegans INTS-6 shows a mainly nuclear localization in early embryos (1 and 2), middle-late embryos (3), and adults (head (4), gonad (5), gut (6) and tail (7)).Immunostaining of: JCP383 (TIF)Click here for additional data file.S5 Figints-6 (t1903) mutant at 15°C and, at a higher level, at the restrictive temperature, 25°C. Reads corresponding to the chimeric sn-mRNA are polyadenylated and therefore detected in the polyA assay. As expected for polyA RNAseq, the 3’ end of the gene is enriched.snRNAs are non-polyadenylated and therefore practically not detected in the polyA RNAseq analysis of WT N2 control (upper panel). Reads corresponding to the snRNA, the downstream intergenic region and the downstream gene are detected in the (TIF)Click here for additional data file.S6 FigC. elegans genome in the regions of SL snRNA genes, visualized on IGV software. N2 reads are shown in gray whereas ints-6 (t1903) mutant reads are in black. Underneath each graph, the C. elegans genome is represented in blue. The exons are shown as blue boxes and the introns as lines. (a) Shows a region of the C. elegans chromosome V where sls-1 genes cluster paired with rRNAs genes. (b) Shows the C. elegans chromosome II in the region of the gene sls-2.1. In the WT, SL snRNAs are processed and trans-spliced as short exons to coding mRNAs. Therefore, their reads do not fully match to the SL loci and the alignment is low. In contrast, unprocessed SL snRNAs reads align to their coding and downstream region and therefore are fully detected.RNA deep sequencing reads aligned to the (TIF)Click here for additional data file.S7 FigIn addition to members of the Integrator complex, other proteins were immunoprecipitated along with INTS-6::3xFLAG::GFP. Proteins found in the FLAG affinity eluate that also appeared in the control using WT N2 animals were discarded as nonspecific binding. RNAPII was not immunoprecipitated with INTS-6, suggesting an indirect interaction in the complex. In humans, the interaction between both complexes is mediated by INTS1 and the C terminal domain of the RNAPII.(TIF)Click here for additional data file.S8 Figa) RNA-dependent RNA polymerase (RDRP) mediated amplification of the different integrator members, shown as a red line, indicates efficient dsRNA interference. (b) RNAi Integrator subunits depletion was efficient in the 3 different transcriptional classes. Western blots of representative subunits of the Auxiliary, Holder and Catalytic classes tagged with 3xFLAG show a strong depletion of the proteins upon RNAi treatment. In all cases, the upper panel shows the tagged protein in the control L4440 RNAi vs specific RNAi, visualized with anti-FLAG. The lower panels show actin as the loading control.((TIF)Click here for additional data file.S9 FigC. elegans worms six days after treatment with RNAi L4440 (control), RNAi of ints-6, N2 and ints-6 (t1903) mutant grown o/n at 25Northern blot analysis for U1 snRNA (a) and U2 snRNA (b) from °C. Mature snRNA is detected after six days of ints-6 silencing. U6 snRNA is shown as a control. Knockdown of ints-6 and the t1903 mutation lead to generation of chimeric sn-mRNAs . Probes from either internal region of U1 snRNA and U2 snRNA or the 3’ region of snRNA are shown for each blot.ints-6 t1903 mutant or RNAi) versus the normal expression of gene mRNA (in an empty L4440 RNAi vector or a WT N2) .Capture of the corresponding RNA-seq alignment reads shows the contribution of chimeric sn-mRNA (in (TIF)Click here for additional data file.S10 Figa) or U2 (b) snRNAs are not significantly affected by RNAi of the different integrator subunits. Normalized counts for snRNA gene expression of the 3 replicas show no statistical differences between the control and the different RNAi integrator subunits. U1 and U2 are properly processed at their 3’ ends in the control (c and d). RNAi Knockdown of C. elegans Integrator subunits leads to no more than a 1.4% lack of U1 3’ end processing (c) and up to a 6.2% lack of U2 3’ end processing.Expression levels of U1 ((TIF)Click here for additional data file.S11 Figact-1/actin, atm-1/ATM ced-1/MEGF11, daf-16/FOXO, egl-1/BH3 and lit-1/NLK genes are shown for the control and the knockdown of different Integrator subunits. U1 and U2 read-through are absent in the control but reach a physiological level after Integrator subunit RNAi.Normalized expression data of the (TIF)Click here for additional data file.S12 Figa) Shows intron retention of genes located directly downstream of snRNA loci, and therefore transcribed as chimeric sn-mRNAs. Analyzed genes: F08H9.3, C15F1.5, H27M09.5, F58G1.7, F08H9.12, T08G5.3, W04G5.8, F15H9.3, F15H9.4, R05D7.3, F56H6.2, Y54G9A.4, F08G2.6, F08G2.8, Y57G11C.5. Expression of these genes in the WT was low and no intron retention was detected. As a control, (b) shows intron retention of genes located upstream of the snRNA loci, and therefore not transcribed as chimeric sn-mRNAs. Analyzed genes: F08H9.4, C15F1.6, H27M09.3, Y38F1A.1, F08H9.6, T08G5.5, W04G5.2, M01G12.9, R05D7.1, R05D7.4, F41D3.5, Y54G9A.5, F08G2.7, Y57G11.4. Significant differences in a T-student test are shown with asterisks.Intron retention was determined as the ratio between reads in the gene introns versus total reads of the gene (mean ± standard error of the mean). ((TIF)Click here for additional data file.S13 Figints-6 (t1903) mutant worms to the C. elegans reference genome. Reads on the + strand are shown in blue and reads on the–strand are shown in red. The black line marks the 3’ end of the snRNA.Directional RNAseq alignments of WT and ints-6 (t1903) mutant worms. Both types of transcripts are present: mRNA and antisense RNAs on the opposite strand, derived from the lack of processing of snRNAs located in antisense downstream of the gene. (a) Shows the gene glc-4, (b) the gene W06D4.2, (c) the gene rpt-1 and (d) the gene W09D6.5.For each case, the upper track shows the genomic region of snRNA loci located downstream and opposite to coding genes. The middle track shows the RNAseq alignment of WT worms. RNAseq shows only the mRNA and the mature snRNA. The lower track shows the RNAseq alignment of (TIF)Click here for additional data file.S14 Figa) Scheme of the plasmids made and integrated into chromosome II, using the mosSCI system. Plasmid pJC63 was used to generate the JCP479 transgenic strain. A genomic region amplified upstream of U1 F08H9.10 to the downstream region of the F08H9.3 gene is shown. A cassette with the HA, MYC (+1b) and TY (+2b) tags in each of the 3 ORFs was inserted into U1 F08H9.3 so that the HA tag and the MYC tag were in-frame respectively with the first and the second ATGs of the U1 F08H9.10. Plasmid pJC64 was used to generate the JCP504 strain. A genomic region amplified upstream of U2 W04G5.11 to the downstream region of the W04G5.8 gene is shown. The same cassette with the HA, MYC (+1b) and TY (+2b) tags in each of the 3 ORFs was inserted into the U2 W04G5.11. (b) WBs of protein extracts from the JCP479 and JCP504 transgenic strains after RNAi treatment of the C. elegans Integrator complex subunits ints-2, -9 and -11 and the empty L4440 vector. Expected molecular weights: JCP479/JCP504 for HA (1st ORF): 7 kDa; MYC (2nd ORF): 8.7 kDa; TY (3rd ORF): 5.5 kDa. Positive control HA: 28.4 kDa; positive control MYC: 61.5 kDa; positive control TY: 54.3 kDa.((TIF)Click here for additional data file.S15 FigSample-to-sample distance heatmap showing the Euclidean distances  between worm samples. Upper and left-side dendrograms show samples grouped by similarity of their transcriptional profiles.(TIF)Click here for additional data file.S1 TableAnalysis of the influence on the 3’-end processing when any of the Integrator complex subunits are knocked down. For each ncRNA type, the following information is shown: gene name, chromosome (Chr), genomic position, U Strand , 3’-end processing affected (yes/no), name of downstream gene affected, strand of downstream gene affected  and snRNA localization inside of an intron (yes/no).(XLS)Click here for additional data file.S2 Tablehttp://www.uniprot.org/) and their corresponding orthologs were searched for using the BLASTp tool with default parameters against the corresponding genome (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Results were corroborated at www.orthodb.org. Percentages of identity (in bold) and similarity (in brackets) are shown. Each cell in the table is colored depending on the percentage of identity between the human sequence and the organism being considered. Black means 100% homology and white means no significant homology. Asterisks indicate that significant homology was found on a portion covering less than 50% of the protein sequence.The Integrator subunit protein sequences were searched for in the Uniprot database ((XLSX)Click here for additional data file.S3 TableC. elegans. CLUSTAL multiple sequence alignment by MUSCLE (3.8) and consensus sequence of each snRNA type.Sequence of the different snRNAs in (DOC)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 TableThe cellular component and molecular function of the corresponding up-regulated and down-regulated genes are shown.(XLSX)Click here for additional data file.


Adventitious roots (AR) play an important role in the vegetative propagation of apple rootstocks. The potential role of hormone, wounding, and sugar signalling pathways in mediating AR formation has not been adequately explored and the whole co-expression network in AR formation has not been well established in apple. In order to identify the molecular mechanisms underlying AR formation in ‘T337’ apple rootstocks, transcriptomic changes that occur during four stages of AR formation  were analyzed using high-throughput sequencing. A total of 4294 differentially expressed genes were identified. Approximately 446 genes related to hormones, wounding, sugar signaling, root development, and cell cycle induction pathways were subsequently selected based on their potential to be involved in AR formation. RT-qPCR validation of 47 genes with known functions exhibited a strong positive correlation with the RNA-seq data. Interestingly, most of the candidate genes involved in AR formation that were identified by transcriptomic sequencing showed auxin-responsive expression patterns in an exogenous Indole-3-butyric acid (IBA)-treatment assay: Indicating that endogenous and exogenous auxin plays key roles in regulating AR formation via similar signalling pathways to some extent. In general, AR formation in apple rootstocks is a complex biological process which is mainly influenced by the auxin signaling pathway. In addition, multiple hormones-, wounding- and sugar-signaling pathways interact with the auxin signaling pathway and mediate AR formation in apple rootstocks. Malus domestica Borkh.) is one of the most commercially important fruits in the world. In terms of nutritional value and economic importance, apple, grape, orange, and banana are the most predominant fruit crops globally. Among these four fruit crops, apple is the most widely consumed and China is the world’s largest apple producer. Apple rootstocks play an important role in regulating the environmental adaptability and growth management of apple trees. The ‘T337’ dwarfing apple rootstock is widely used and confers early fruiting and high yields. Although the development of dwarfing, disease-resistant apple rootstocks is a major pursuit of the global apple industry, apple rootstock breeding programs in China have not been well established [Apple  levels in the basal region of stem cuttings are required for competent cells in the cambium to resume proliferation and to initiate a root-specific developmental program ,7,8,9. IAUX/LAX) and efflux (PIN-Formed-PIN) carriers that play a role in the basipetal transport of auxin; allowing for the local accumulation of auxin at the stem base [ARF6 (AUXIN RESPONSE FACTOR 6) and ARF8 (AUXIN RESPONSE FACTOR 8), have been identified as positive regulators of AR formation in Arabidopsis [Arabidopsis and other plants. Several gene families in the auxin-related pathway are among the genes that have been shown to mediate adventitious rooting in many plants. The identified auxin-related genes include members of the auxin inducible ARF family [GH3 (GRETCHEN HAGEN 3) family [YUCCA gene family [PIN (PIN-FORMED) and ABCB/PGP (ATP BINDING CASSETTE-TYPE B/P-GLYCOPROTEINs) [AUX1/LAX (AUXIN/IAA) [Prior studies have suggested that auxin is the main hormone associated with AR formation ,16,17,18bidopsis . A varieF family ,20, membe family , the auxROTEINs) ,23,24, aXIN/IAA) ,23. AlthXIN/IAA) . For exaWhile extensive research on a variety of physiological aspects of apple rootstocks has also been conducted, characterization of the underlying biochemical and cellular mechanisms responsible for the regulation of root regeneration is lacking. Therefore, in order to further understand the molecular mechanism of AR formation in apple rootstocks, a transcriptomic approach was used in the present study to identify genes associated with AR formation in ‘T337’ apple rootstocks. A high throughput sequencing analysis was used to provide the first global monitoring of changes occurring at the gene expression and whole co-expression network levels during AR formation in ‘T337’ apple rootstocks. The results obtained in the present study contribute to our basic understanding of the molecular events underlying AR formation in apple rootstocks. A perspective on future research on AR formation in tree species is also provided.The ‘T337’ apple rootstock cultivar was chosen for this study due to its rooting performance relative to other rootstock genotypes, and its differential response to a mild IBA treatment during AR rooting . In the Under IBA-treatment, however, the ‘T337’ apple rootstocks formed callus and divided into roots in the fourth stage . The curHormone levels and their ratios to each other were analyzed in ‘T337’ cuttings at four time points during AR formation A,B. UndeThe ratios of AUX, CTK, GA, and ABA to each other were also determined over the time course of the experiment B. Comparp-value < 0.05 was used to filter the data and approximately 446 genes among the annotated genes were selected based on their potential to be involved in AR formation  at the different stages of AR formation. Venn diagrams illustrating the DEGs in all the stages are presented in The numbers of DEGs categorized into the three main GO categories  are presented in Although the exact fold change of the DEGs at several data points varied between RNA-Seq and qPCR, the differential expression trends detected by the two approaches were largely consistent, indicating the reliability of the RNA-Seq results.The above-described DEGs and their corresponding RT-qPCR results were sorted and classified into seven categories. The results of the categories are as follows:IAA3/SHY2, AXR3/IAA17, IAA19, IAA29); auxin efflux transport gene (PIN1); auxin response factors ; auxin-responsive gene family member ; auxin polar transport-related gene (PAT1) and a GRAS family transcription factor (SCR1)  A,B. A grr (SCR1) .ARF9 (MDP0000634433) significantly increased from S1 to S3 (ARF1 (MDP0000564492) and ARF11 (MDP0000268306) were significantly down-regulated from S2 to S3 (PIN1 (MDP0000138035) was decreased by 57% from S1 to S2, but no significant difference was observed from S2 to S4 (IAA19 (MDP0000296324) was up-regulated during AR induction and then plateaued (IAA29 (MDP0000543718) exhibited a reduction from S1 to S2 but then decreased in expression by 91% from S2 to S4 (IAA17 (MDP0000253285) exhibited its highest expression level at S4 (IAA3/SHY2 (MDP0000303142) was up-regulated 7.8-fold from S1 to S3, but then decreased by 55% from S3 to S4 (GH3:1 (MDP0000121609) was significantly up-regulated from S1 to S2 and exhibited its highest expression level at S2 (PAT1 (MDP0000152670) increased by 12% from S1 to S2 and then decreased by 74% from S2 to S3 (SCR1 (MDP0000203826) was up-regulated 1.6-fold from S1 to S2 and then plateaued , CTK oxidase gene (CKX7), histidine kinase 1 (AHK1), and the CTK-responsive gata factor 1 (GATA1), Isopentenyl adenosine transferase gene (IPT) etc. These genes were subjected to a clustering analysis (CRF4 (MDP0000308379), CKX7 (MDP0000238100), GATA1 (MDP0000190038), GATA5 (MDP0000172464), IPT1-2 (MDP0000189484), and IPT5 (MDP0000220668) during AR formation  increased 2.4-fold from S2 to S3, while at S1, S2, and S4 were similar to each other. CKX7 (MDP0000238100) was down-regulated from S1 to S2 and then plateaued. Interestingly, CKX (MDP0000223673), GATA1 (MDP0000190038) and GATA5 (MDP0000172464) exhibited similar expression profiles with CKX7 (MDP0000238100). Specifically, their highest levels of expression were detected at S1 and subsequently decreased by 55–75% from S1 to S4. IPT1-2 (MDP0000189484) decreased by 70% from S1 to S2 and then increased approximately 1.6-fold from S2 to S4. IPT5 did not exhibit any difference between S1 and S2, and then increased from S2 to S4. Consistent with the data pertaining to auxin-related genes, the CTK-related genes also exhibited a variable pattern of expression during AR formation. Collectively, the RT-qPCR results indicated that the expression patterns of representative CTK-related genes were significantly affected by exogenous IBA-treatment in relative comparison to the control , gibberellins 3-oxidase 1 (GA3OX1), and gibberellin-regulated family protein genes (GA2OX2 (MDP0000145827) and GA2OX4 (MDP0000161181) were largely consistent with the RNA-seq data (The expression patterns of GA-related genes were selected from the DEGs database that were examined during AR formation. These genes included gibberellin 2-oxidases (in genes . In respseq data .GA2OX2 was highest at S1 and lowest at S4 (GA2OX4 (MDP0000161181) was different from GA2OX2 (MDP0000145827). The lowest expression value for GA2OX4 was observed at S1 and then increased 3.7-fold from S1 to S2 and then plateaued (GA2OX2 (MDP0000145827) and GA2OX4 (MDP0000161181) were significantly affected by the exogenous IBA-treatment in comparison to the control , ethylene responsive element binding factors , and ethylene-forming enzyme genes (EFE1 and EFE2); JA-related genes included the jasmonate-zim-domain protein genes ; BR-related genes included BR-signaling kinase (BSK1), and brassinosteroid-6-oxidase 2 (BR6OX2)  . The exp(BR6OX2) A. Under (BR6OX2) .ACO1 (MDP0000200896) increased 1.5-fold from S1 to S2, and then increased 1-fold from S3 to S4. No significant differences were observed between S2 and S3 (EFE1 (MDP0000251295) was highest at S1, after which it was down-regulated by 89.9% from S1 to S3. No further changes in expression were observed between S3 and S4 (EFE2 (MDP0000200737) was significantly up-regulated from S1 to S2, and then down-regulated during the extension phase; with no further significant differences observed during the initiation phase (ERF1 (MDP0000235313) was significantly decreased during the AR induction phase, and then decreased further during the initiation and extension phases of AR formation (JAZ12 (MDP0000901967) were observed between S1 and S2; but then increased by 1.68-fold from S2 to S4 (JAZ26 (MDP0000565690) decreased by 23% from S1 to S2, and then increased by 48% during the extension phases of AR formation (BR6OX2 (MDP0000286141) increased 1.5-fold from S1 to S2 and then plateaued   and Wd-Id-8 (MDP0000228919) showed largely similar trends between transcriptomics and RT-qPCR (Wound-induced-related genes were selected from among the DEGs and their expression profiles were examined during the four stages of AR formation. The selected genes included members of the wound responsive family genes (Wd-Id-8) . The expWd-Id-8) A. RT-qPC RT-qPCR .Wd-Id-4 (MDP0000836784) did not exhibit any significant changes during AR induction and initiation phases but then increased 7-fold during the extension phase of AR formation (Wd-Id-8 (MDP0000228919) decreased by 60% and 38% during AR induction and initiation phases, however, no significant changes in expression were observed during the extension phase , sucrose-phosphate synthase family protein gene (SPS4F), cellulose synthase (CSLA2 and CSLC4), hexokinase 2 (HXK2), polyol/monosaccharide transporter 5 , cellulose synthase , cellulose synthase family protein gene (IRX1 and IRX3), cellulose-synthase-like (CSLC4 and CSLC12) and a pectinlyase-like superfamily protein gene (QRT3). The expression patterns of several of the selected genes were also validated by RT-qPCR. Under the IBA-treatment, most of the expression profiles for the above-mentioned genes that were derived from the RNA-seq data were similar to the profiles obtained by RT-qPCR  and PMT5 (MDP0000688348) decreased from S1 to S2, but no significant differences were observed from S2 to S4 (SUS4-3 (MDP0000250070) was up-regulated 1.7-fold from S1 to S2 and no further changes were observed during subsequent stages (SPS4F (MDP0000288684) was similar at S1 and S2, but then decreased by 83% from S2 to S4 (WINV4 (MDP0000275150) decreased by 93% from S1 to S2 and then plateaued  gene, root hair specific 19 (RHS19), cell cycle-related genes , cell wall/vacuolar inhibitor of fructosidase 1 (C/VIF1) gene, WRKY family transcription factor (WRKY), peroxisomal NAD-malate dehydrogenase (MDH), Integrase-type DNA-binding superfamily protein gene (PLT2), S-adenosylmethionine synthetase family protein (SAMS3) gene, and a flavonol synthase1 (FLS1). The expression pattern of several of the selected genes  were also validated by RT-qPCR. Under the IBA-treatment, although the exact fold change of the DEGs at several data points varied, the expression trends were largely similar between transcriptomics and RT-qPCR  increased 5.5-fold from S1 to S2, and then plateaued (RHS19 (MDP0000488361) was similar to LRP1 (MDP0000171430). In addition, the relative expression of SHI (MDP0000147890) increased 8.2-fold during the induction phase and 12-fold during the initiation phase; followed by a 29% decrease during the extension phase of AR formation , CYCD1:1 (MDP0000809276), CYCD3:1 (MDP0000286130), and CYCP4:1 (MDP0000139550) exhibited their highest level of expression at S4. The relative expression of C/VIF1 (MDP0000305934) was down-regulated by 51% from S1 to S2 and then increased 6-fold from S3 to S4 (CYCD1:1 (MDP0000809276) was also significantly up-regulated during the extension phase, but no further differences were observed between S2 and S3 (CYCD3:1 (MDP0000286130) increased by 69.7% from S3 to S4, but no further changes were observed from S1 to S3 (CYCP4:1 (MDP0000139550) decreased by 84% during the induction phase and then increased 2.3-fold during extension phase; no further changes were observed between S2 and S3  decreased during the induction phase, and was further down-regulated from S2 to S4 (SAMS3 (MDP0000302980) was unaffected during the initiation phase of AR formation but then increased 2.5-fold from S3 to S4 (WRKY (MDP0000602139) decreased by 51% during the induction stage but then increased by 77% during the extension phase (FLS1 (MDP0000294667) was down-regulated during the induction phase and then plateaued (PLT2 (MDP0000871080) significantly increased from S1 to S3, with no further changes observed during the extension stage of AR formation  which can occur in both juvenile and mature plants . In the To further characterize the differential expression of genes related to AR formation, gene expression under exogenous IBA-treatment was examined and revealed the induction of genes related to hormones-, wounding- and sugar-signaling related pathways etc. Numerous authors ,32 have The interaction of different hormone networks plays an important role in plant development ; and in IAA19 (MDP0000296324), IAA29 (MDP0000543718), IAA3/SHY2 (MDP0000303142), ARF9 (MDP0000634433) and GH3.1 (MDP0000121609) are change during the AR induction phase (PAT1 (MDP0000152670) significantly decreased during the initiation phase of AR formation , and poplar [IPT1-2 (MDP0000189484) and IPT5 (MDP0000220668) exhibited high levels of expression but cytokinin oxidase genes CKX (MDP0000223673) and CKX7 (MDP0000238100) exhibited lower expression during the extension phase (SHY2 (MDP0000303142) and PIN1 (MDP0000138035) [SHY2 and PIN1  appears to play a role in AR formation . CTK levd poplar ,46,47,48on phase . In addi0138035) . These dand PIN1 . CollectPAT, and thus changes the ability to accumulate auxin in different plant tissues [PINs; and further resulted in decreased expression levels of PAT which consequently affected the accumulation of auxin , apple, mung bean (Vigna radiata), and petunia (Petunia sp.) [Arabidopsis [Other hormones, including Eth, JA, and BR also appeared to affect AR formation. ARs often form directly from sites in cuttings that were wounded or indirectly from wounded vascular tissues ,14,54. Enia sp.) ,57. JA hbidopsis . In the ACO1 (MDP0000200896), EFE1 (MDP0000251295), EFE2 (MDP0000200737), ERF1 (MDP0000235313), JAZ12 (MDP0000901967), JAZ26 (MDP0000565690), and BR6OX2 (MDP0000286141) were associated with AR formation  and Wd-Id-8 (MDP0000228919), are involved in regulating AR formation  and SPS4F (MDP0000288684) were high during the induction stage (SUS) cleaves sucrose in a reversible reaction, producing UDP-glucose and fructose [SUS activity has been related to sink strength determination and storage functions [SUS4-3 (MDP0000250070) and SPS4F (MDP0000288684), which would result in the localization of high level of soluble sugar, were high during the induction phase of AR formation. This finding was in accordance with reports showing that soluble sugars levels play an important role during the induction phase of AR and that fructose content is closely related to rooting ability [PMT5 (MDP0000688348) was high during the induction phase and was ultra-low during the extension phase; indicating that PMT5 may also play an important role in regulating AR formation. HXK has been identified as a glucose sensor, independent of its enzymatic role in converting glucose to glucose 6-phosphate in Arabidopsis [HXK2 (MDP0000181206) expression may have resulted from the accumulation of soluble sugars during the induction phase of AR formation; which may have induced the up-regulation of HXK2 expression in order to increase the sugar signal response sensitivity. HXK2 regulates the transcription of glucose-related genes in a glucose-dependent manner, and also participates in hormone signaling. In poplar, WINV4 plays a key role in sucrose metabolism in various tissues and organs [WINV4 (MDP0000275150) also played a role in the induction phase of AR formation (SUS4-3 (MDP0000250070), SPS4F (MDP0000288684), HXK2 (MDP0000181206), and WINV4 (MDP0000275150), is likely transported into the basal portion of the rootstock stems undergoing AR formation; thus providing sufficient energy and signal activity required for adventitious rooting to occur , CYCD3:1 (MDP0000286130), CYCP4:1 (MDP0000139550), and C/VIF1 (MDP0000305934) exhibited their highest expression level at S4  is a marker gene for the induction stage of AR formation in Arabidopsis [RHS19 (MDP0000488361) is known to regulate root hair development [LRP1 and SHI were significantly higher during the extension phase than the induction phase of AR formation. Additionally, RHS19 expression gradually increased by IBA-treatment in comparison to the control; especially at S2  represents stem cuttings treated with 1 mg·L−1 IBA at 0 day ; stage 2 (S2) represents stem cuttings in which AR formation was induced at 3 day ; stage 3 (S3) represents stem cuttings in which callus formed at the base of the stem at 9 day ; and stage 4 (S4) represents stem cuttings in which AR broke through the epidermis and AR emerged at 16 day . Based on previous research, the S1 to S2 period of growth was considered as the AR induction phase, S2 to S3 was defined as the AR initiation phase, and the S3 to S4 stages were defined as the AR extension phase. The experimental errors were managed by using a complete randomized design for three biological replicates, each including at least 60 stem cuttings. Samples consisted of basal portions of the stems (approximately 0.5 cm) containing the AR zone. The collected samples were immediately immersed in liquid nitrogen and stored at −80 °C until used for further processing. Cuttings of IBA treated material  were used for RNA-seq with three biological replicates and 12 libraries were sequenced. Cuttings of both the IBA treatment and control were used for analysis of paraffin section, hormone levels and gene expression.Stem cuttings of ‘T337’ apple rootstock were cultivated in tissue culture at the Northwest Agriculture and Forestry University, Yangling, China. The tissue culture cuttings were maintained under a 16/8 h light/dark cycle at 25 ± 1 °C, followed by 8 h 15 ± 1°C. Under these conditions, the relative humidity was approximately 70~80%. The rootstock cuttings were treated with indole-3-butyric acid (IBA), which is widely used to promote adventitious rooting. Separate rootstock cuttings served as controls and were not treated with IBA. The cuttings were randomly divided into two groups; IBA treatment and control material. The cuttings of the IBA treatment group were transferred into a medium composed of 1/2 MS, 1 mg·Ln = 10 in each replicate.Samples collected for fixation, paraffin embedding and sectioning were processed using previously published protocols ,84. All v/v) methanol and 1 mM butylated hydroxytoluene overnight for about 12 h at 4 °C. In order to collect extracts, the mixture was centrifuged , passed through a Sep-Pak C18 cartridge and dried under N2. The resulting residues were dissolved in phosphate buffer. The ELISAs used to analyze AUX, CTK, ABA, JA, BR and GA3 contents were completed in 96-well microtitration plates. The coated plates were incubated for 40 min at 37 °C after adding standard hormone, sample extracts and antibodies. After rinsing plates, 100 μL peroxidase-labeled goat antirabbit immunoglobulin was added to each well, and the plates were incubated for 40 min at 37 °C. Colored substrate (O-phenylenediamine) was added to each well, for enzymatic reaction at 37 °C for 15 min, and the reaction was halted by the addition of 3 M H2SO4. Absorbances at 490 nm were determined with an ELISA spectrophotometer and used to calculate AUX, CTK, ABA, JA, and GA3 concentrations. All antibodies against each hormone were monoclonal and were obtained from the Center of Plant Growth Regulator, China Agricultural University. Three biological replicates were used for each hormone analysis (200 mg per replicate).Auxin (AUX), Cytokinin (zeatin riboside) (CTK), Gibberellic acid 3 (GA), Brassinolide (BR), Jasmonic acid (JA), and Abscisic acid (ABA) contents between stem cuttings harvested from IBA-treatment and control were determined using an enzyme linked immunosorbent assay (ELISA). ,86. Apprβ-mercaptoethanol pre-heated to 65 °C just prior to use), inverted for 5 min, and then incubated at 65 °C for 5 min. The samples were then centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was collected and extracted twice with an equal volume of chloroform/isoamyl alcohol (24:1 v/v). The RNA was precipitated with 3 M LiCl  and resuspended in 500 μL of SSTE buffer  pre-heated to 65 °C. Then, an equal volume of chloroform/isoamyl alcohol was added to each tube which was subsequently centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was collected and the RNA was precipitated with 2.5 volumes of ethanol at −80 °C for at least 30 min. Finally, the RNA was pelleted by centrifugation at 12,000× g for 20 min at 4 °C, washed with 70% ethanol, dried and resuspended in DEPC–water. The integrity of the total RNA was verified by running samples on 2% agarose gels. cDNA was synthesized using a PrimeScript RT Reagent Kit with gDNA Eraser .Total RNA was extracted using a CTAB-based method , with slStatistical Analysis of Clean ReadsTotal RNA was extracted from stem cutting samples that were collected at 0, 3, 9 and 16 days from IBA-treated cuttings. These extracts were then used for the construction cDNA libraries and subsequent sequencing. The library construction was performed according to a previously published protocol . A totalMapping Rate StatisticsPaired-end DNA sequencing results contained low-quality reads and the end contained the unknown base N reads. In order to ensure the quality of downstream data generation and subsequent analyses on clean data, we filtered the next generation sequencing machine data using Trimmomatic (version 0.32) software . For triftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF_000148765.1_MalDomGD1.0/GCF_000148765.1_MalDomGD1.0_genomic.fna.gz) (Default parameter) using HISAT2 (version 2.0.1-beta) software [The Distribution of Reads in The Reference GenomeThe sample reads were compared with the reference genome , Introns (intron) and transcription initiation sites; in proximity to 1 k, 5 k and 10 k regions near the termination site. Under normal circumstances, the Exon (exon) region of the sequencing positioning percentage should be the highest, and the other regions of the sequencing positioning percentage should be smaller than the Exon (exon) region.Gene Expression Abundance StatisticsStringtie software was used to assemble reads and cuffcompare enabled the comparison of the assembled results with known genes in the genomic annotation document. A total of 11,833 isolates were identified , the new predictive transcription site sequence was extracted and CPC software  predicteDifferentially Expressed Genes (DEGs)Gene expression levels are estimated by counting the reads that are localized to exon regions of the gene. The reads count is proportional to the true expression level of the gene and is positively correlated with gene length and sequencing depth. In order to compare expression levels between different genes and experiments, the concept of FPKM (Fragment Per Kilo bases per Million) reads was introduced. FPKM is the number of PEs per kilobase length per million reads from a gene. FPKM also considers the effect of sequencing depth and gene length on the counting of reads. The FPKM values can reflect the gene expression. In the current study, FPKM ≥ 1 was used as a standard for identifying gene expression. The weakly expressed genes were filtered out by this standard.p-value < 0.05. Two strategies were used to analyze the DEGs. The first was gene ontology functional enrichment and the second was pathway enrichment. GO enrichment analysis of functional significance applied a Fisher’s Exact Test for mapping all DEGs to terms in the GO database. topGO (version 2.18.0) software was used for the GO enrichment analysis and screening of differentially expressed genes [p-value was corrected by the Bonferroni test and a corrected p-value < 0.05 was chosen as the threshold to define a significantly enriched GO term. This analysis allowed us to identify the major biological functions of the DEGs. The KEGG Pathway enrichment analysis identifies significantly enriched metabolic pathways or signal transduction pathways. KOBAS (kobas2.0-20150126) analysis software [p-values < 0.05 were defined as significantly enriched. In addition, this study also combined previous research backgrounds to screen for DEGs.For differential expression analyses, the documented transcripts within the reference genomic annotation file, as well as the newly assembled transcripts from String Tie, were analyzed by ballgown (version 2.2.0) software . Screenied genes . The p-vsoftware  was usedMalus × domestica) that were published in GenBank using Primer 6.0 (Genetyxv. 10) software. The sequences of the utilized gene-specific primers are listed in 2O. The RT-qPCR assay was conducted on an iCycler iQ Real Time PCR Detection System  with an initial denaturation of 3 min at 95 °C, followed by 40 cycles of 15 s at 94 °C, 20 s at 60 °C and 20 s at 72 °C. An apple ACTIN gene was used for normalization. Each of the analyzed samples consisted of three biological and technical replicates. The 2Ct−ΔΔ method was used to calculate the relative expression of the analyzed genes [In this study, the expression patterns of candidate genes were validated by RT-qPCR. Specifically, genes involved in hormone metabolism , sugar, cell cycle, root development, and enzyme activity were analyzed. The identification of gene functions was based on reports that were derived from other species, and the sequences of the corresponding genes were used as the query in GenBank to identify the gene homologue and MDP number in apple. The sequences of the designed primer pairs were selected based on apple data (http://www.tm4.org/mev.html) and the log2 (FPKM) values of each gene. The Pearson correlation with complete centroid linkage was adopted for all clustering analyses.The time-series cluster method was used to analyze the expression profiles of the DEGs  and the t-test at the 5% level. Graphs were generated in Excel 2007 and SigmaPlot10.0 .Statistical processing of plant phenotype data, hormone content, and RT-qPCR data was performed in Excel 2007. Differences among means were evaluated with the Statistical Program for Social Science 19  using a two-tailed The current study proposes a preliminary model which describes the important biological processes occurring during AR formation in apple rootstocks. AR formation involves the regulation of hormones , carbohydrate metabolism, and the regulation of cell-cycle-related genes and suites of genes related to AR differentiation. Many sugar-, wounding-, root development-, and cell cycle-related genes interact with auxin signaling pathway-related genes to ultimately control AR formation . Our stuAR formation is a complex process involving various regulatory pathways. Our work provided a transcriptomic overview pertaining to IBA-induced AR formation. However, owing to technical limitations, our study did not provide any post-transcriptional or (post)-translational evidence for AR formation, which are also important regulatory pathways for AR formation. Whether these pathways are also involved in the IBA-induced AR formation still needs further analysis.