Rationale: Abnormal Wnt/β-catenin signaling in the endometrium can lead to both embryo implantation failure and severe pathogenic changes of the endometrium such as endometrial cancer and endometriosis. However, how Wnt/β-catenin signaling is regulated in the endometrium remains elusive. We explored possible regulation of Wnt/β-catenin signaling by multi-drug resistance protein 4 (MRP4), a potential target in cancer chemotherapy, and investigated the mechanism.Methods: Knockdown of MRP4 was performed in human endometrial cells in vitro or in a mouse embryo-implantation model in vivo. Immunoprecipitation, immunoblotting and immunofluorescence were used to assess protein interaction and stability. Wnt/β-catenin signaling was assessed by TOPflash reporter assay and quantitative PCR array. Normal and endometriotic human endometrial tissues were examined. Data from human microarray or RNAseq databases of more than 100 participants with endometriosis, endometrial cancer or IVF were analyzed. In vitro and in vivo tumorigenesis was performed.Results: MRP4-knockdown, but not its transporter-function-inhibition, accelerates β-catenin degradation in human endometrial cells. MRP4 and β-catenin are co-localized and co-immunoprecipitated in mouse and human endometrium. MRP4-knockdown in mouse uterus reduces β-catenin levels, downregulates a series of Wnt/β-catenin target genes and impairs embryo implantation, which are all reversed by blocking β-catenin degradation. Analysis of human endometrial biopsy samples and available databases reveals significant and positive correlations of MRP4 with β-catenin and Wnt/β-catenin target genes in the receptive endometrium in IVF, ectopic endometriotic lesions and endometrial cancers. Knockdown of MRP4 also inhibits in vitro and in vivo endometrial tumorigenesis.Conclusion: A previously undefined role of MRP4 in stabilizing β-catenin to sustain Wnt/β-catenin signaling in endometrial cells is revealed for both embryo implantation and endometrial disorders, suggesting MRP4 as a theranostic target for endometrial diseases associated with Wnt/β-catenin signaling abnormality. The endometrium is a tissue layer of epithelial and stromal cells lining the uterus with its key physiological function in embedding the embryo/blastocyst for implantation The implanting embryos/placental cells are believed to share striking similarities with invasive cancer cells, and common pathways are activated in the endometrium during implantation and tumorigenesis 2). We have previously demonstrated that MRP4 is expressed in the endometrium and transports PGE22 is reported to regulate Wnt/β-catenin signaling in other cell types 2-transporter function, interacting with and stabilizing β-catenin, to sustain the Wnt/β-catenin signaling for endometrial receptivity during embryo implantation, as well as in pathogenic transformation of the endometrium, i.e. endometriosis and endometrial cancer.Multi-drug resistance protein 4 (MRP4), a member of the ATP-binding cassette (ABC) transporter subfamily C, is best known for its transporter function to exclude various drugs and therefore considered as a potential target to prevent drug-resistance, particularly in cancer chemotherapy 2-transporting function. To our surprise, despite successful blockage of PGE2 release  did not affect the level of active β-catenin either . The results showed that compared to cells treated with control shRNAs (shNC), shMRP4-treated cells exhibited significantly decreased TOPflash luciferase activity . Knockdown of MRP4 by siRNAs (siMPR4) in ISK cells reduced levels of the active form of β-catenin (non-phosphorylated), as compared to cells treated with control siRNAs (siNC), in either the presence or absence of Wnt3a 100 ng/mL), a Wnt ligand reported to activate endometrial Wnt/β-catenin signaling 00 ng/mL,per ce may affect the protein level of β-catenin in ISK cells. We suspected that such an effect of MRP4 on β-catenin might be mediated through protein-protein interaction, a well-documented mechanism to stabilize proteins, by preventing them from degradation in various cell types The observed effect of MRP4-knockdown, but not its transporter-functional inhibition, on β-catenin activity Figure A-D suggein vivo and thus sustain the Wnt/β-catenin signaling in the endometrium required for embryo implantation. Double-labeling for β-catenin and MRP4 showed co-localization of these two proteins in endometrial epithelial cells in uterine tissues collected from pregnant mice at 5 d.p.c. (days post coitum), the day right after implantation takes place at mid-night of 4 d.p.c.  at 3 d.p.c., shortly before implantation. Such operation led to successful knockdown of MRP4 at both mRNA  showed statistical significance  significantly boosted up the levels of active β-catenin  with MRP4 knockdown. 74 of total 84 genes in the array were successfully detected in the uterine tissues with most of them (61 genes) downregulated to different extend in siMRP4-treated uteri as compared to siNC-treated ones Figure . Downregr = 0.2488, p = 0.0073) and Wnt/β-catenin target genes including Birc5 , Bmp4 , Ccnd1 , Gdf5 , Id2 , Igf1 , Il6 , Nrp1  and Twist1   r = 0.7173, p = 0.0005) or active β-catenin  , and found significant correlations between MRP4 with β-catenin mRNA levels in endometrial cancers, in particular at stages I and IV  Figure A. We alsV Figure B. We wenV Figure . These r in vitro and in vivo tumorigenesis with ISK cells in conjunction of MRP4 knockdown by designed shRNAs. As shown in Figure To investigate the role of MRP4 in endometrial tumorigenesis, we next performedTaken together, the present study has revealed a role of MRP4 in sustaining Wnt/β-catenin signaling in the endometrium through a previously unidentified mechanism, which is independent of its transporter function but instead via interacting with and stabilizing β-catenin Figure . Such a 2-transport function also inhibits embryo implantation in mice 2 to stroma, a key event necessary for inducing stromal decidualization for embryo implantation 2-independent manner. Although PGE2 has been reported to regulate Wnt/β-catenin signaling in other cell types, it does not seem to exert detectable effect on the Wnt/β-catenin signaling in the endometrial epithelial cells. The importance of MRP4-dependent Wnt/β-catenin signaling for embryo implantation is evidenced by the fact that the retrieval of active β-catenin levels by blocking β-catenin degradation can rescue the MRP4 knockdown-induced implantation defects. This is further supported by the significant correlation between MRP4 and β-catenin, as well as MRP4 and Wnt/β-catenin downstream target genes, in human endometrial biopsy samples collected at mid-secretary phase during IVF treatment.The present observation of impaired decidualization and embryo implantation rate in the MRP4 knockdown mice suggests that MRP4 is required for adequate endometrial receptivity for a successful pregnancy, which is consistent with previously reported smaller litter size in MRP4 knockout mice The present study has demonstrated a previously unsuspected protein-protein interaction between MRP4 and β-catenin, which is shown to be important for sustaining the level of active β-catenin in the endometrium. The level of β-catenin inside the cell is a key to the canonical Wnt/β-catenin signaling In the present study, an enhanced stability of β-catenin by MRP4 is also observed as evidenced by 1) more rapid degradation of β-catenin (increase in degrading form and decrease in active form) in MRP4-knockdown cells; and 2) reversed β-catenin protein level by blocking either protein degradation in general (MG132 or chloroquine) or β-catenin degradation specifically (CHIR). Therefore, the observed interaction of MRP4 with β-catenin may contribute to the stability of β-catenin and thus underlie the capacity of MRP4 in sustaining Wnt/β-catenin signaling.in vitro and in vivo results confirms a role of MRP4 in endometrial tumorigenesis. While both the expression of MRP4 and the involvement of Wnt/β-catenin signaling have been reported in endometriosis and endometrial cancer Apart from its demonstrated role in endometrial receptivity, the presently discovered MRP4-dependent Wnt/β-catenin signaling appears to be involved in pathological transformation of the endometrium, i.e. endometriosis and endometrial cancer, as well. A correlation of MRP4 with β-catenin is consistently found in clinical samples of endometriosis and endometrial cancer, as demonstrated in the present study or from big databases. Although databases are limited to mRNA level analysis, interaction and correlation between MRP4 and β-catenin proteins in cancer tissues are still possible. Importantly, The non-transporter role of MRP4 discovered in the present study raises an interesting possibility that ABC transporters might play an important role in signal transduction independent of their transporter function. The roles of ABC transporters expressed in different tissues may have to be re-examined in light of the present finding. How MRP4 or multi-drug resistance family proteins should be targeted/inhibited in cancer chemotherapy needs to be re-considered, since simple transport-blockage may not be effective to attenuate Wnt/β-catenin signaling, a key pathway activated in tumorigenesis. Moreover, given the versatile roles of Wnt/β-catenin signaling in a variety of different cellular processes and the wide distribution of MRP4, as well as other ABC transporters, the presently discovered novel role of MRP4 in the regulation of Wnt/β-catenin signaling may have far-reaching implications beyond embryo implantation and tumorigenesis.oC warmer till woken up from the anesthesia. The mice were closely monitored for 1 to 4 consecutive days after surgery. Mice were sacrificed by CO2 asphyxiation on 4, 5 and 7 d.p.c.Institute of Cancer Research (ICR) mice were obtained from the Laboratory Animal Service Centre of the Chinese University of Hong Kong. All animal experiments were conducted in accordance with the university guidelines on animal experimentation, and approval by the Animal Ethics Committee of the Chinese University of Hong Kong was obtained for all related procedures. The day a vaginal plug was found after mating was identified as 1 d.p.c. The intrauterine injection surgery under general anesthesia was performed on 3 d.p.c. after mating as previously described 2 incubators at 37 oC. The cell line was recently authenticated by STR profiling at the Department of Anatomical and Cellular Pathology, Faculty of Medicine, The Chinese University of Hong Kong The human endometrial epithelial cell line (adenocarcinoma line), Ishikawa (ISK), was purchased from ATCC  and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (v/v) and 1% penicillin-streptomycin (v/v) in 5% COforward 5'-TCCCTTGTTCTGGCGAAGAC-3' and reverse 5'-CGAAGACGATGACTCCCTCG-3'), HoxA10 (Primers: forward 5'-AATGTCATGCTCGGAGAGCC-3' and reverse 5'-CTTCATTACGCTTGCTGCCC-3'), Igf2 (Primers: forward 5'-GTACAATATCTGGCCCGCCC-3' and reverse 5'-GGGTATGCAAACCGAACAGC-3'), Lif (Primers: forward 5'-GCCCAACAACGATGGTGTCA-3' and reverse 5'-CCCGTGTTTCCAGTGCAGA-3'), Pparg (Primers: forward 5'-GTCACACTCTGACAGGAGCC-3' and reverse 5'-ATCACGGAGAGGTCCACAGA-3') and Gapdh (Primers: forward 5'-TCTCCTGCGACTTCAACAGC-3' and reverse 5'-AGTTGGGATAGGGCCTCTCTT-3'). Quantitative PCR with SYBR Green Master Mix  was carried out in triplicate on a 96-well plate using a QuantStudio 7 Flex Real-time PCR system (Applied Biosystems). Gapdh was used as an internal control for normalization and data were calculated using the CT method.Total RNA of cells or uterine tissues were extracted using TRIzol reagent (Invitrogen Life Technologies) according to manufacturer's instructions. 1 μg total RNA was used for reverse transcription reaction using M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. SYBR green assay was used for mouse genes: MRP4 (Primers: 2 First Strand Kit (Qiagen), and followed with the RT2 Profiler PCR array for Wnt signaling target genes (SABiosciences array PAMM-243Z), according to manufacturer's guide. The array was performed using SYBR Green PCR Master Mix (Applied Biosystems) in QuantStudio 7 Flex Real-time PCR system (Applied Biosystems). Data were analyzed using the online SABioscience Array data analysis software.1 µg total RNA extracted from each mouse uterine tissue sample was used for reserve transcription by RTISK cells with or without MRP4 knockdown were incubated with cycloheximide (10 μM), a protein synthesis blocker, for 0, 2, 4, 6 or 8 h before proteins were extracted and analyzed by immunoblotting. In some experiments, cells were treated with MG132  or chloroquine  or CHIR99021 , an inhibitor of glycogen synthase kinase 3 β (GSK3β), which blocks degradation of β-catenin. oC. Antibodies against MRP4 ; Non-phospho (Active) β-catenin , β-catenin , p-β-catenin , β-tubulin  and β-actin  were used. After three washes in TBS containing 0.1% Tween 20 (TBST), membranes were further incubated with HRP-conjugated antibodies and visualized by the enhanced chemiluminescence assay  following manufacturer's instructions. Densitometry of Western blots was performed by Image J software.The cells or tissues were lysed in ice-cold RIPA lysis buffer  with protease and phosphatase inhibitor cocktail  for 30 min on ice. Supernatant was collected after centrifugation at 13,000 rpm for 30 min. Equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis and electro-blotted onto equilibrated nitrocellulose membrane. After blocking in Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were immunoblotted with primary antibody of target proteins overnight at 4oC, supernatants were collected as protein extracts, which were re-suspended in 1 ml immunoprecipitation binding buffer  with protease and phosphatase inhibitor cocktail and incubated with primary anti-β-catenin  or mouse IgG  as the control in conjunction with protein A/G beads  on rotator overnight at 4 oC. The protein-antibody-bead complexes were washed three times with binding buffer using a magnetic separator. In the end, proteins complexes were eluted from the beads by SDS sample buffer (Invitrogen) with 4% β-mercaptoethanol, and further analyzed by immunoblotting.Cells were lysed with 1 ml of ice-cold lysis buffer  with protease and phosphatase inhibitor cocktail for 30 min to 1 h on ice. After centrifugation at 13,000 rpm for 30 min at 4 oC. After three times washed in PBS, uterine tissues were cryoprotected in 30% sucrose in PBS at 4 oC for 24 h, mounted in OCT embedding media , and cryo-sectioned into 5 μm sections. Sections were rehydrated in PBS for 5 mins and microwaved in citrate buffer (pH 6.0) for 20 mins to retrieve antigens. After cooled down to room temperature and treated with 1% SDS in PBS for 4 mins, sections were blocked with 1% bovine serum albumin in PBS for 15 mins, incubated with primary antibody  and/or β-catenin  overnight at 4 oC and subsequently fluorochrome-conjugated secondary antibody (invitrogen) for 1 h at room temperature. DAPI was used to stain cell nuclei. Images were acquired with a confocal microscope .Uterus tissues were harvested and fixed by immersion in 4% paraformaldehyde overnight at 4 107 TU/ml) were infected into ISK cells with Polybrene (5 μg/ml). The cells were cultured for three passages with puromycin (4 µg/ml) and selected stable clones for further experiments. For tumorigenesis assays, additional 4 designs of shRNAs  together with the shMRP4_1 were purchased. To package the shRNAs into lentivirus, envelope vector pMD2.G  and packaging vectors psPAX2  together with the shRNAs were transfected into 293T cells using lipofectamine 2000 (Invitrogen). Packaged virus was harvested 72 h afterwards and used for next transfection into ISK cells for 48 h. Virus-transfected ISK cells were grown in the presence of puromycin (4 µg/ml) for 14 days to select cells with stable expression of shRNAs.siRNAs against MRP4  and a negative control Med GC Duplex  were used for knockdown of MRP4. For in vivo knockdown, 50-100 pmole siRNAs together with Lipofectamine 2000 (5 μl) in 10 μl Opti-MEM were injected into each uterine horn. In cells, 100 nM siRNAs were transfected with Lipofectamine 2000. Lenti-virus (LV3) packaged shRNAs targeting human MRP4  and scrambled non-coding shRNAs  were purchased from GenePharma (China). The viruses  and Renilla-Luc as an internal control, respectively. Cells were harvested 48 h after transfection in passive lysis buffer (Promega) and assayed by the Dual Luciferase Reporter Assay System (Promega) using GloMax luminometer (Promega). Renilla luciferase activity was used for normalization.2 content was collected and measured using an EIA kit .ISK cells were incubated with 1 % FBS in DMEM culture medium for 8 h to synchronize the cells before FBS-free DMEM medium was used for all the treatments. Cell-free supernatant with PGEWomen at the Second People's Hospital of Shenzhen diagnosed of ovarian endometriosis were recruited for the study. Tissues were collected at the proliferative phase from walls of the ovarian endometriomas during gynecological laparoscopic surgery. All samples were collected with informed consent from each woman and approval by the Institutional Review Board of the Second People's Hospital of Shenzhen . Individuals receiving hormone therapy or anti-inflammatory agents were excluded. Clinical data are shown in Table Cells were seeded in 96-well plates at a density of 2000 cells per well and incubated for 1-4 days at 37 °C. Directly after removal of the medium, 200 μl of MTT  was added in each well for incubation at 37 °C for 4 h. The medium and MTT solution were removed from each well and formazan crystals were dissolved in 100 μl of DMSO before the absorbance was measured at 570 nm using a plate spectrophotometer.Cells were seeded into 6-well plates at a density of 500 cells per well. The growth medium was replaced with fresh ones every 2 days. After culturing for 10 days, cells were fixed with methanol and stained with crystal violet. Colonies comprising > 50 cells were counted.6 of cells mixed with an equal volume of matrigel (BD Pharmingen) were injected subcutaneously at the back of 6-week-old female Ncr-nu/nu-nude mice. Tumor sizes were monitored every 3 days using digital vernier calipers, and tumor volumes were calculated using the formula [sagittal dimension (mm) × cross dimension (mm)2]/2 and expressed in mm3.Ncr-nu/nu-nude mice were provided by Laboratory Animal Unite, the University of Hong Kong. The protocols were performed after approval by the Animal Ethics Committee, the University of Hong Kong according to issued guidelines. 3 × 10http://www.ncbi.nlm.nih.gov/geo). Datasets of endometrial carcinoma (ID: UCEC) and colon carcinoma (ID: COAD) were retrieved from The Cancer Genome Atlas . Data from normal or undetermined biopsy samples were excluded for analyzing cancer datasets. R2 platform (http://r2.amc.nl) was used for data retrieval and analysis. Correlation analysis was performed using Log2 transformed microarray data.Human gene expression datasets of IVF endometrium (accession number: GSE58144), colorectal cancer , prostate cancer (GSE46691 and GSE21034), bladder cancer (GSE57933) and breast cancer (GSE12093) were retrieved from the Gene Expression Omnibus (GEO, The software GraphPad Prism 6.0 was used for graphing and statistical analyzing the data. Data are shown as mean ± SEM. Student's paired or unpaired t-test was used for two-group comparison. One-way or Two-way ANOVA was used for comparing three or more groups. Pearson test was used for correlation analysis. P < 0.05 was considered as statistically significant. The variance was calculated to be similar between comparison groups or otherwise corrected for particular tests.Supplementary figures.Click here for additional data file.


Furthermore, BAF250a deletion abolished the interaction of OCT4 and BRG1 in mesoderm, suggesting that BAF250a is the key component in SWI/SNF complex that determines the interaction of Oct4/β-catenin in mesoderm. In contrast, we found that BAF250a did not regulate the OCT4/β-CATENIN interaction during neuroectoderm differentiation. Altogether, our results suggest that BAF250a specifically controls proper cardiac mesoderm differentiation by reorganizing the binding of OCT4/β-CATENIN and regulates both key lineage differentiation genes and cell cycle genes that coincided in response to WNT/β-CATENIN signal.Dissecting epigenetic mechanisms controlling early cardiac differentiation will provide insights into heart regeneration and heart disease treatment. SWI/SNF complexes remodel nucleosomes to regulate gene expression and play a key role in organogenesis. Here, we reported a unique function of BAF250a in regulating the physical interaction of OCT4 and β-CATENIN during cardiac lineage differentiation from human ESCs. BAF250a deletion greatly reduced the physical interaction between OCT4 and β-CATENIN but did not alter the expression of β-CATENIN and OCT4 in the mesodermal progenitor cells. BAF250a ablation led to decreased recruitment of OCT4 and β-CATENIN at promoters of key mesodermal lineage genes, such as MESP1 and EOMES. Subsequently, the expression of lineage-specific genes was downregulated, whereas the expression of pluripotent genes was upregulated. In parallel, BAF250a ablation also altered recruitments of OCT4 and β-CATENIN to the promoter of CCND2 and CCND3, two key genes for S phase entry during cell cycle. Consequently, BAF250a deletion led to prolonged S phase in Mesp1 Dissecting the molecular mechanisms underlying the differentiation of pluripotent stem cells (PSCs) into tissue-specific progenitors and terminal differentiated cell types is key to understanding organ development and regeneration. Within heart, embryonic cardiac progenitor cells (CPCs) are promising cell sources for cell-based therapies for heart disease. Direct differentiation of CPCs from PSCs has been achieved based on the knowledge of embryonic development . Each stSWI/SNF ATP-dependent chromatin remodeling complex modulates the chromatin accessibility by reorganizing nucleosomes to regulate chromatin accessibility and are essential in organogenesis . BAF comCell cycle control is another key regulatory process during differentiation. Recent studies have identified cross-talk between cell cycle and cell fate determination of human ESCs (hESCs) . The hES+ CPC cells. In addition, we found that BAF250a was dispensable for the interaction of OCT4 and β-CATENIN during neuroectoderm differentiation and BAF250a deficiency did not affect neuroectoderm differentiation. These results suggest an important role of BAF250a in reorganizing the binding of OCT4/β-CATENIN and regulating both key genes for lineage differentiation and cell cycle phases for proper cardiac mesoderm differentiation in response to WNT signal.In this study, we define a unique function of BAF in regulating cardiac lineage differentiation in human ESCs by examining a key regulatory subunit BAF250a. By taking advantage of conditional BAF250a knockout human ESCs we generated , we obseHuman ESCs were maintained on Matrigel-coated dishes with mTseR1 medium as described . The difg for 5 min at 4°C. Laemmli sample buffer was added before boiling for 5 min. Primary antibody OCT4 , BRG1 , BAF250a , T , active β-CATENIN , and total β-ACTIN  were applied to the blots at 4°C overnight. Secondary antibody from LiCOR was used for detection.Cells were lysed in 0.25% Triton X-100, 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 1 × protease inhibitors (Roche) and then spun at 16000 × The co-immunoprecipitation was performed as previously described . BrieflyEmbryos were fixed in 4% paraformaldehyde at 4°C overnight and embedded in paraffin. Sections were collected using microtome. Sections were blocked with 10% horse serum in PBS buffer containing 0.1% Tween 20 and 0.1% Triton X-100 for 1 h at room temperature and incubated with goat anti-T (sc-17745 1:100), mouse anti-OCT4 , and rabbit active β-CATENIN  at 37°C for 1 h. Then, sections were washed three times with PBS buffer with 0.1% Tween 20 before being incubated with a donkey anti-goat Alexa 488-conjugated, donkey anti-mouse Alexa 594-conjugated, and donkey anti-rabbit Alexa 647-conjugated antibodies at room temperature for 1 h. After washes, section slides were mounted and analyzed by fluorescent microscopy.®In Situ Red kit (DUO92101) according to Sigma’s protocol. Briefly, E7 embryo sections were dewaxed with xylene, and rehydrate with descending alcohol. Antigen retrieval was performed by microwaving section in pH 9 Tris–EDTA buffer for 15 min. The section was permeabilized with 0.2% Triton X-100 and blocked in 5% horse serum for 1 h. Incubation with primary antibodies OCT4 and β-CATENIN was performed at 4°C overnight. Sections were washed three times in PBST. The ligation and detection of OCT4/β-CATENIN by proximity probes were performed according the manufacturer’s protocol.Proximity ligation assay was performed using DuolinkChIP experiments were performed as previously described . Briefly+ cell (mesoendoderm) was not affected after BAF250a deletion (+) cell and ISL1+ CPCs  and 1.0% ± 0.4% Edu+/ISL1+ cells (p = 0.04) from the differentiation of BAF250a KO hESCs (+ cells are largely derived from non-proliferating mesoderm cells and BAF250a could contribute to the differentiation of ISL1+ cells by regulating cell cycle of mesoderm cells.To further determine how the disassociation of OCT4/ β-CATENIN contributed to the later cardiac differentiation defects shown in KO hESCs , suggestWe next examined the cell cycle genes regulated by BAF250a, together with OCT4 and WNT/β-CATENIN. We found that expression of G1/S transition protein CCND2 and CCND3 was significantly higher in BAF250a KO cells . FurtherIn this study, we show that BAF250a regulates cardiac lineage specification through regulating the interaction of OCT4 and β-CATENIN and their recruitment to target genes. BAF250a-mediated OCT4/β-CATENIN interaction not only fine-tunes the MESP1 and EOMES gene expression but also regulates the cell cycle progression of mesoderm cells. We further demonstrate that BAF250a regulates G1/S transition in mesoderm cells, which is critical for the differentiation into cardiac lineage. Therefore, by modulating the interaction and recruitment of OCT4 and β-CATENIN, BAF250a guides the direct cell-type-specific gene expression and the cell cycle machinery in mesodermal cells to promote cardiac lineage commitment. Importantly, our data indicate that BAF250a is a key subunit regulating SWI/SNF activity. We show that BAF250a is required for BRG1 interaction with OCT4 in mesoderm cells. In addition, the observed key role of BAF250a in regulating cardiac mesoderm differentiation appears lineage specific, as BAF250a is dispensable for the interaction of OCT4 and β-CATENIN during neuroectoderm differentiation and BAF250a deficiency does not affect neuroectoderm differentiation.+ cells for proper cardiac mesodermal differentiation. Direct differentiation of cardiomyocytes from human ESCs is based on sequential stimulation and inhibition of WNT/β-CATENIN signals identified during cardiogenesis (We report here that BAF250a is required for efficient interaction of OCT4 and β-CATENIN in Togenesis . Dynamicogenesis  and the ogenesis . In thisogenesis . In our Indeed, our studies show that the aberrant recruitment of Oct4 and β-CATENIN leads to altered expression of several key groups of genes that negatively affect cardiac lineage differentiation. Particularly, the expression of key mesodermal lineage gene MESP1 and EOMES is decreased. In contrast, we have identified upregulation of pluripotent genes, such as SOX2, NANOG, ESRRB, and DPPA2, in BAF250a knockout cells during differentiation. Our finding is consistent with reported studies that the interaction of OCT4 and β-CATENIN is essential for the repression of SOX2 and activation of mesoderm genes . Importa+ cells are derived from non-proliferating mesoderm cells reveals a significant role of cell cycle in regulating cell differentiation. Importantly, BAF250a deletion reduces the cardiac differentiation by promoting cell proliferation, as indicated by lower cardiac differentiation potential of S stage mesoderm cells in BAF250a knockout cells. Moreover, we find that BAF250a is also required for proper recruitment of OCT4/β-CATENIN to CCND2/3 promoters in mesoderm cells. Thus, our results showed that BAF250a coordinates Oct4/β-CATENIN to regulate the chromatin structure in both differentiation and proliferation genes and therefore control the balance of cell cycle exit and cardiac differentiation.Our findings on the connection of cell cycle regulation and direct differentiation in mesodermal cells highlight the importance of cell cycle regulation in direct differentiation in the multipotent cells. We find that BAF250a regulates the cell cycle of mesoderm cell through controlling the expression of cell cycle gene CCND2/3. This is consistent with previous studies that BAF complexes could act as transcriptional repressor of positive cell cycle factors in G1/S transitions . As CyclIn summary, we demonstrate that the physical interaction of BAF250 with Oct4 and WNT/β-CATENIN complex is critical for cardiac differentiation by directly regulating cardiac mesodermal lineage gene expression and cell cycle phases. Our study also indicates that manipulating the cell cycle state of mesoderm cell may further enhance the cardiac lineage specification. Further studies in understanding how BAF complex interplays with developmental signals in maintaining proliferation and differentiation balance may provide key molecular mechanisms for therapies against heart disease.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.IL, YZ, and ZW designed the study and wrote the manuscript. IL, ST, and VC performed the experiments. ZW gave final approval of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To apply a systems pathology-based approach to the quantification of nuclear Active β-catenin and human leukocyte antigen class I, and assess the biomarker involvement in a cohort of prostate tumor patients.The systems pathology approach applied allows a precise quantification of the marker expression in the different cell compartments as well as the determination of the areas that coexpress two markers. Our data shows that the accumulation of β-catenin in the nuclear compartment is significantly decreased in the adjacent normal areas when compared to tumor of the same patients (p < 0.001). In conclusion, the application of this novel multiple immunofluorescence assay demonstrates that the upregulation of Active β-catenin is a relatively common feature of prostate tumor development, and further supports the activation of the Wnt/β-catenin pathway in prostate cancer progression.The online version of this article (10.1186/s13104-019-4100-z) contains supplementary material, which is available to authorized users. The origin of the data is a summary of single observations derived from a more comprehensive study encompassing seven protein biomarkers that were assembled in two multiplex assays and applied to a cohort of prostate cancer patients (n = 505). Quantitative and cellular attributes representing biomarkers from both multiplex assays containing Active β-catenin (ABC), HLA-1 and CK18, among others, were correlated with clinical variables including biochemical recurrence (BCR). One of the objectives of the comprehensive work (manuscript in preparation) was to describe correlations between clinicopathological data and results from the quantification and colocalization of individual marker expressions. When we started analyzing the data we observed that the expression of Active β-catenin was higher in tumor areas than in adjacent normal tissue. In this research note we describe for the first time how a systems biology-based methodology yields a precise quantification of the nuclear Active β-catenin (effector fraction) and thus unequivocally demonstrates the implication of this marker in prostate tumor formation.Prostate cancer is the most common epithelial malignancy after skin cancer in men from western countries . SystemsHLA class I expression on the cell surface of cancer cells is critical for an appropriate anti-tumor immune response by presenting antigenic peptides to cytotoxic T lymphocytes . One of The Wnt/β-catenin signaling pathway is an evolutionarily conserved developmental pathway involved in several physiological processes . AberranProstate cancer samples were obtained from 505 patients  with localized and locally advanced disease that underwent a radical prostatectomy . The Gleason breakdown of the tumors was as follows: 81 tumors Gleason < 6, 130 tumors Gleason 6, 293 tumors Gleason > 6. Whole tissue sections from each patient were first stained and three tumor cores and a normal core previously selected were included in tissue microarrays (TMAs). Blank 3.5 µM thick consecutive sections of the TMA were used for IF analysis.® software (Molecular Devices). Multispectral images were acquired at 20× magnification with the Nuance imaging system using the selected filter combination and exposition time, and analyzed with the Nuance® software to unmix and quantify individual biomarker expressions, as well as determining biomarker co-localization in selected regions within each core. The individual expression of each marker is obtained as the result of the quantification of positive pixels for the marker that are included in the tumor area. All expression and coexpression results were obtained as an average of up to three cores from each patient in the case of tumor samples, and directly from a single core of their normal counterpart. Statistical tests were performed after all values were obtained from the multispectral images (see “An Immunofluorescent (IF) multiplex reaction was assembled for the study of the following biomarkers: Active β-catenin (ABC), HLA-1 and CK18, described in Table The antibody for Active β-catenin (ABC) is able to detect nuclear, cytoplasmic and membranous ABC. The values for nuclear β-catenin, accounting for the effector fraction of the transcription factor, were deduced from the co-localization between DAPI and ABC in cells that were expressing CK18  as expected according to the results described in previous papers [HLA class 1 is a critical component of the immune response and a candidate marker to show differences in expression between normal and tumor areas. HLA class 1 downregulation in prostate cancer has been described in previous articles , 11. HLAs papers , 11. A mNuclear Active β-catenin expression was found associated with prostate tumorigenesis according to the Wilcoxon signed-rank test calculation (p < 0.001). This correlation was obtained comparing the median values of normalized nuclear ABC in the tumor cores with the values of the same normalized ABC fraction in normal cores . The same level of significance (p < 0.001) was obtained when total ABC expression was assessed and compared in tumor cores vs. normal cores Table .In the current study, we present nuclear accumulation of Active β-catenin in prostate malignancy development. This finding is in agreement with the published literature , 12–14. Interestingly, the correlation between tumor progression and nuclear Active β-catenin is also found with total ABC . However, when average expression values are compared, the difference between tumor cores and normal cores is bigger for nuclear Active β-catenin than for total Active β-catenin. Our data further supports a role for the Wnt/β-catenin signaling pathway in prostate cancer formation and as a potential therapeutic target. Furthermore, applications which interrogate biomarkers at the intact tissue-cellular level will continue to advance our understanding of basic tumor biology.To summarize, our multiplex-based systems pathology approach is a novel tool for the precise quantification of CK18 epithelial nuclear Active β-catenin through colocalization of DAPI and ABC. The additional evaluation of HLA provides insight into the biology underpinning prostate cancer progression. Thus, the use of the multiplex approach is essential for a more comprehensive analysis of various markers, including DAPI, HLA class I, Active β-catenin and CK18. Our results show a trend for HLA Class I downregulation, and strongly support the implication of Active β-catenin, both nuclear and total, in prostate cancer development.The study needs an expanded cohort of patients and a more robust assessment of HLA and nuclear ABC to further characterize such discrete cell populations.Additional file 1: Figure S1. Subcellular localization of ABC. Magnified images demonstrating subcellular localization of ABC.


A large proportion of the world is inflicted with health concerns arising from infectious diseases. Moreover, there is a widespread emergence of antibiotic resistance among major infectious agents, partially stemming from their continuous dialog with the host, and their enormous capacity to remodel the latter toward a secure niche. Among the several infection-driven events, moderation of WNT signaling pathway has been identified to be strategically tuned during infections to govern host-pathogen interactions. Primarily known for its role in arbitrating early embryonic developmental events; aberrant activation of the WNT pathway has also been associated with immunological consequences during diverse patho-physiological conditions. Here, we review the different mechanisms by which components of WNT signaling pathways are exploited by discrete bacterial agents for their pathogenesis. Furthermore, recent advances on the cross-talk of WNT with other signaling pathways, the varied modes of WNT-mediated alteration of gene expression, and WNT-dependent post-transcriptional and post-translational regulation of the immune landscape during distinct bacterial infections would be highlighted. Later it was shown that overexpression of int1 is sufficient to induce tumorigenesis in mice. At a similar time, int1 homolog Wingless was identified in a Drosophila mutant screen; wherein the gene was found to be essential for segment polarity and for the formation of wing tissue. By the end of 1980s, enormous screens were carried out to decipher other integration sites that may contribute to tumors . However, those tumorigenic integration events were diverse and had least homology with int1; necessitating the nomenclature to be revisited. The researchers working then with int1 and Wingless consented to the hybrid name “Wnt” (Wingless-related integration site), and int1 as the pioneer was called Wnt1; whereas int2 is known today as FGF3, int3 as NOTCH4 and int4 as Wnt3A  receptors (10 in humans) in an autocrine or paracrine manner. Fzd form a class of seven-pass transmembrane proteins, showing topological homology to G-protein coupled receptors (GPCRs). The ligand binding occurs at the N-terminal cysteine-rich extracellular domain, bringing about conformational alterations that subsequently activate the adaptor molecule Disheveled (Dsh/Dvl). The phosphoprotein Dvl is activated by kinases such as Casein Kinase1, Casein Kinase2, and Protein Kinase C (PKC) among others. Apart from Wnt and Fzd, co-receptors  are critical for tuning the transduction of signal through Wnt receptor. Physiological and biochemical characterization of Wnt ligands, their receptors, co-receptors and the corresponding signaling mechanisms have identified at least three distinguishable consequences of Wnt interaction with specific Frizzled (Fzd) receptors: the canonical β-CATENIN-dependent signaling pathway; and the non-canonical β-CATENIN-independent Planar Cell Polarity and Wnt/Ca2+ pathways. Further, the signaling cascades are kept under tight control by various negative regulators operating both intracellularly and extracellularly , secretory glycoproteins, upon being translated undergo palmitoylation by the ER-resident This forms the first pathway to be identified by genetic screens in Drosophila and validated in worms, frogs, fishes, and mice. The central objective of the pathway ensures the stabilization and activation of the transcription co-activator β-CATENIN. When unstimulated, β-CATENIN is trapped in a destruction complex composed of Axin, Adenomatous Polyposis Coli (APC), Casein kinase1α (CK1α), glycogen synthase kinase3 (GSK3). Here, GSK3/CK1α phosphorylates β-CATENIN, thereby priming it for ubiquitination-mediated proteasomal degradation. Upon signaling activation, Axin is translocated to the membrane to bind to LRP5/6, which leads to a series of relatively uncharacterized events activating Dvl and inactivating GSK3. These steps release the repression from β-CATENIN and allow it to translocate to the nucleus along with specific nuclear localization signal (NLS)-containing proteins. β-CATENIN, then effectuates Wnt-specific gene expression by interacting with the TCF/LEF (T cell factor/ lymphoid enhancer factor) family of DNA-binding transcription factors, while eliminating the negative regulator Groucho from the site.The non-canonical pathways activate Fzd and utilize specific domains of Dvl to transduce the signals. LRP5 and LRP6 do not participate in the non-canonical pathways, and are reportedly replaced by NRH1, Ryk, Ror as co-receptors. In the PCP pathway, activated Dvl adopts a route through GTPases Rho and Rac to activate ROCK and JNK, respectively. These events majorly culminate in cytoskeletal re-organization, and mechanisms of the pathway's contribution to transcriptional rewiring still require investigation.2+ release from the endoplasmic reticulum (ER) in a GPCR-dependent manner. Though the precise GPCR has not been elucidated, it is clear that the activated trimeric G proteins lead to Ca2+ release and activation of Ca2+-dependent kinases such as Calcium/ calmodulin Kinase II (CamKII) and PKC, and transcription factor NFAT. Interestingly, CamKII can also intercept β-CATENIN-driven regulation of gene expression, thereby altering the canonical arm of Wnt signaling.This pathway came to light with the observation that discrete Wnt signals can lead to CaConventionally, Wnt genes have been correlated with carcinogenesis, retrovirus-mediated tumorigenesis and developmental processes, however, its contributions in the context of various infections is manifold . SentineMycobacterium tuberculosis, Shigella flexneri, Salmonella typhimurium, Citrobacter rodentium etc. In this context, it was recently shown that canonical Wnt signaling is essential to maintain an inflammatory equipoise during S. flexneri infection. It was found that Shigella-induced inflammation is maintained by NOD2-driven Epidermal Growth Factor Receptor (EGFR)-dependent activation of Wnt/β-CATENIN axis, leading to the production of the anti-inflammatory molecule IDO1 . The perturbation of Wnt signaling compromised IDO1 expression and pharmacological inhibition of IDO1 in mice exacerbated Shigellosis by inducing intestinal hyper-inflammation  in the susceptible strains  infection via mitochondrial cytochrome c-driven Caspase3 activation. This involved the upregulation of the pro-apoptotic Bax and downregulation of anti-apoptotic Mcl1 proteins  with lysosomes. The authors suggest that the virulence factor Etf1 allows amphisome formation in order to acquire nutrients, while, another set of virulence determinants TRP120, 32 and 47 act in concert to activate Dvl/Wnt pathway to mediate mTOR-driven TFEB cytosolic retention and consequent inhibition of amphisome-lysosome fusion  to utilize the non-canonical Wnt pathway for its pathogenesis. The authors showed that the unusual bacterium upregulates several components of the Wnt pathway, including ligands Wnt6 and 10a; receptors Fzd5 and 9; co-receptor LRP6 and TCF7 early during infection. Interestingly, E. chaffeensis tandem repeat proteins (TRPs) were shown to assist bacterial internalization through non-canonical Wnt components, where they interacted with Wnt pathway regulators like ARID2, KDM6B, IRF2BP2, PPP3R1, and VPS29, thus influencing Wnt signaling activation and aiding in bacterial survival by both canonical β-CATENIN and non-canonical Ca2+-CAMKII-NFAT routes  and Wnt  signaling pathways. This pathogen-specific TLR2-dependent activation of Wnt and NOTCH relied on a Wnt/ β-CATENIN-driven expression of NOTCH pathway ligand Jagged1 in iNOS/NO-dependent manner. Critically, such studies broaden the repertoire of signals responsible for canonical β-CATENIN activation to include non-canonical Wnt ligands, such as WNT5a. Such an orchestration was observed to mediate regulatory T cell expansion during pathogenic bacterial infections  signaling through GSK3β, which acts as a negative regulator of both the pathways. In the context of mycobacterial infection in macrophages, miRNAs 31 and 155 were found to repress the expression of the phosphatase PP2A, thereby retaining the inhibitory phosphorylation of GSK3β, and consequently allowing the activation of Wnt and SHH through β-CATENIN and NUMB, respectively . Furtherfections . The sigal agent .S. flexneri infection-activated EGFR pathway. It was shown that the pathogen utilizes EGFR pathway to initiate a cascade comprising the engagement of c-Abl and HIPK2 to mediate the activation of the Wnt/ β-CATENIN axis and the subsequent expression of anti-inflammatory IDO1  and formation of colorectal cancers  and Pygopus (Pygo). The C-terminus is more diverse in the repertoire of chromatin binding proteins that include histone methyl transferases such as MLL, PRC2 complex (EZH2), demethylase LSD1, histone acetyl transferase like p300, and chromatin remodelers such as BRG1 and ISW1. A study by Yakulov et al. has compared the chromatin partners of β-CATENIN in the presence or absence of the canonical ligand, WNT3a , 27. AltC. rodentium infection in mice, Roy et al. proposed the activation of WNT signaling-driven crypt hyperplasia and tumorigenesis to result from EZH2-dependent downregulation of the negative Wnt regulator WIF1. They demonstrate an enhanced expression of EZH2 upon infection with Citrobacter (In a model of robacter . Howeverrobacter .Infectious diseases account for a vast proportion of disability associated life years (DALYs) as well as mortality in the global context. This stems from the ability of major infectious agents to adopt strategies in order to co-evolve with their host, and exhibit immune evasion and subversion mechanisms as a part of their survival tactics. With the early reports of the implications of Wnt pathway in development and tumorigenesis, it could be foreseen that the pathway might be hijacked by infectious entities to disrupt homeostasis and establish infection. In this light, we discussed the many facets that are exploited by pathogens along the Wnt signaling axis, including Wnt ligands, receptors, coreceptors, transcription factors, and certain epigenetic alterations , which stands as a pre-requisite for β-CATENIN nuclear translocation and Wnt activation . FurtherP. aeruginosa and S. pneumoniae infections to execute the same (Finally, the extensive repertoire of Fzd receptors still require detailed investigation for their possible implications in pathogen uptake and ensuing microautophagy/macropinocytosis events. The endless possibilities of Wnt functions, given the apparent cross-talks and the overlaps of the canonical and non-canonical components, can levy contrasting immune outcomes during host-pathogen interactions as may be observed with the ability of Mtb to induce canonical Wnt/ β-CATENIN signaling to inhibit autophagy, while the non-canonical Wnt being downregulated by the same , 15. Wntthe same . Moreovethe same . GPX4 acthe same . These oA major proportion of research on Wnt pathway in the realm of infectious diseases limits to intestinal bacterial infections and subsequent inflammation. However, as may be noted now that the potential implications of a versatile and complex pathway like Wnt has so far not been appropriately exploited in distinct infections and associated cellular consequences. With the expanding dynamics of antibiotic resistance among globally relevant pathogens, host-directed therapeutics form an essential alternative , and we KB constructed the framework of the manuscript. TM wrote the initial draft. TM and KB finalized, edited, and proofread the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glycosylation, a common modification of cellular proteins and lipids, is often altered in diseases and pathophysiological states such as hypoxia, yet the underlying molecular causes remain poorly understood. By utilizing lectin microarray glycan profiling, Golgi pH and redox screens, we show here that hypoxia inhibits terminal sialylation of N- and O-linked glycans in a HIF- independent manner by lowering Golgi oxidative potential. This redox state change was accompanied by loss of two surface-exposed disulfide bonds in the catalytic domain of the α-2,6-sialyltransferase  and its ability to functionally interact with B4GalT-I, an enzyme adding the preceding galactose to complex N-glycans. Mutagenesis of selected cysteine residues in ST6Gal-I mimicked these effects, and also rendered the enzyme inactive. Cells expressing the inactive mutant, but not those expressing the wild type ST6Gal-I, were able to proliferate and migrate normally, supporting the view that inactivation of the ST6Gal-I help cells to adapt to hypoxic environment. Structure comparisons revealed similar disulfide bonds also in ST3Gal-I, suggesting that this O-glycan and glycolipid modifying sialyltransferase is also sensitive to hypoxia and thereby contribute to attenuated sialylation of O-linked glycans in hypoxic cells. Collectively, these findings unveil a previously unknown redox switch in the Golgi apparatus that is responsible for the catalytic activation and cooperative functioning of ST6Gal-I with B4GalT-I.  Yet, thi.e. transcription factors that regulate the expression of hundreds of genes affecting among others cellular metabolism and signaling networks ,33−pVHL)normoxia b. In conTherefore, we next focused on other potential defects by which hypoxia could attenuate terminal sialylation of N- and O-glycans. As our main targets, we chose N-glycan terminating ST6Gal-I sialyltransferase and its binding partner β1,4-galactosyltransferase  due to their co-operational functioning . Immunop2) hypoxia.Since hypoxia can potentially alter the redox state of a cell, compartment, or protein, we utilized next the ratiometric redox-sensitive fluorescent probe RoGFP2 a to exam3.3in vivo [Previous studies have shown that sequentially acting Golgi glycosyltransferases can form both homomeric and functionally more important heteromeric complexes in vivo . Since tin vivo . Quantif3.4® FL-conjugated maleimide (a thiol-specific labeling reagent). Quantification of the bound fluorescent dye to either enzyme protein in immunoprecipitated samples after separation with Blue-Native (BN)-PAGE -PAGE b revealeBN)-PAGE b. TherefTo reveal which one(s) of the disulfide bonds are affected by hypoxia, we replaced one cysteine in each disulfide bond with serine or alanine residue before testing whether the mutants still localize correctly in the Golgi membranes and can form ST6Gal-I homomers and heteromers with B4GalT-I. We found that only the C184A mutant mislocalized into the ER, likely due to improper folding of the mutant enzyme c. This iWT) was used as a control. We expressed both these enzyme constructs individually in the cells for 24 h and determined a-2,6-linked sialic acid content by staining cells with Alexa 488-conjugated SNA lectin and quantifying the bound dye with Operetta high content imaging system. We found that expression of wild type ST6Gal-I increased SNA lectin binding to cells by two-fold, when compared to non-transfected cells  has any effect on the enzymatic activity of the ST6Gal, we measured first its ability to add α-2,6-linked sialic acid to cell surface glycans in normoxic cells. The wild type ST6Gal-I  or the CysDM encoding plasmids, and followed possible changes in proliferation, migration and apoptotic resistance as well as changes in nuclear or cell morphology. We found that overexpression of the ST6Gal-IWT, but not that of CysDM  oxidative potential, reduced disulfide bond formation and the loss of catalytic activity of a relevant ST6Gal-I N-sialyltransferase and its ability to interact with its binding partner B4GalT-I. Thereby, hypoxia-induced silencing of the ST6Gal-I provides also a rational explanation for the attenuated sialylation of N-glycans in hypoxic cells. Intriguingly, its binding partner B4GalT-I appears not to be affected, consistent with increased levels of terminal galactoses in N-glycans. Similar surface exposed disulfide bonds are also present in ST3Gal-I sialyltransferase, an enzyme implicated in the synthesis of O-glycans, glycosaminoglycans and glycolipids. This suggests that other glycosylation pathways are also defective in hypoxic cells. Our findings also show that glycosylation can be, to a large extent, independent of the HIF system, and emphasizes the sensitivity of the glycosylation process to changing oxygen concentrations. Since the HIF system involves HIF-dependent transcription, followed by translation of a certain protein, i.e. represents a long-term response towards hypoxia, the involvement of a redox switch regulating directly glycosylation enzymes themselves indicates a short-term response. Hence, this system allows an immediate response towards hypoxia that helps cells to proliferate due to their altered glycosylation profiles.The data unveils a hitherto unknown regulatory circuit that relies on disulfide bond formation in the Golgi and is needed normally for the catalytic activation of ST6Gal-I in this cellular compartment. In support of this view, we showed that the loss of the two surface exposed disulfide bonds seen in hypoxic cells or obtained by mutagenesis of the selected cysteine residues in ST6Gal-I resulted in the loss of enzymatic activity and its ability to interact with GalT-I. The two surface-exposed disulfide bonds in ST6Gal-I are thus needed not only for the catalytic activity but also to facilitate ST6Gal-I/B4GalT-I complex assembly, a phenomenon that likely involves a subtle conformational change in ST6Gal-I and is mediated by the formation of the two disulfide bonds. The fact that the assembly of the complex takes place only after the enzymes arrive in the Golgi compartment , indicatSialylation regulates numerous cellular processes, including cell–cell communication, immune surveillance, defense against pathogens and cancer progression and metastasis ,53. In cS. cerevisiae [Our findings, however, do not explain why hypoxia alters Golgi redox homeostasis. The main obstacle preventing this is that only few proteins with clear regulatory role in setting up the high oxidative potential in the Golgi lumen are known. One of them is the Golgi-localized QSOX1a protein that has been shown to play a role in disulfide bond formation, possibly in the maturation of ECM components and the formation of higher order structures there . Two othrevisiae  and showrevisiae . These frevisiae . FurtherA.H., F.K-A., E.K., E.Y.D., D.M., D. H. and M.N performed research and analyzed the data, A.H-L. designed the sialyltransferase enzymatic assays, supervised the work of M.N and compiled the activity data. T.G. supervised the structural studies of. D.B. SK together with TK designed the experiments, compiled the data and wrote the manuscript. F.K-A., E.K., A.H-L, and T.G. read the manuscript and made suggestions to the text.The authors declare no conflict of interest regarding this publication.


This age-dependent decline in proliferation occurs despite evidence that the Wnt pathway is postnatally active and can be further enhanced by Wnt stimulators. Using an in vivo mouse model and RNA sequencing, we show that proliferation in the early neonatal cochlea is correlated with a unique transcriptional response that diminishes with age. Furthermore, we find that augmenting Wnt signaling through the neonatal stages extends the window for HC induction in response to Notch signaling inhibition. Our results suggest that the downstream transcriptional response to Wnt activation, in part, underlies the regenerative capacity of the mammalian cochlea.Lack of sensory hair cell (HC) regeneration in mammalian adults is a major contributor to hearing loss. In contrast, the neonatal mouse cochlea retains a transient capacity for regeneration, and forced Wnt activation in neonatal stages promotes supporting cell (SC) proliferation and induction of ectopic HCs. We currently know little about the temporal pattern and underlying mechanism of this age-dependent regenerative response. Using an  Summary: Canonical Wnt activation in the mammalian cochlea elicits a unique, age-dependent transcriptional response, which in part regulates the regenerative capacity of supporting cells during cochlear maturation. Noise-induced and age-related degeneration of sensory hair cells (HCs) is a leading cause of hearing deficits. Owing to a lack of regenerative capacity in the adult mammalian cochlea, damage to HCs is irreversible. In contrast to adults, neonatal mice have a limited capacity to regenerate HCs . ConsequBy birth, cellular organization of the cochlea is complete, with the sensory epithelium organized into one row of inner HCs, three rows of outer HCs and interdigitating non-sensory supporting cells (SCs). In response to modulation of specific signaling pathways, SCs in the neonatal cochlea function as endogenous stem cells and HC regeneration proceeds through either direct transdifferentiation or proliferation of SCs and subsequent transdifferentiation to HCs . Direct Two key pathways that can be modulated to stimulate these endogenous stem cells are canonical Wnt and Notch pathways . Canonicin vitro and in vivo can promote SC proliferation and induction of new HCs  3 . Similar new HCs , 2012. I new HCs . Underst+ prosensory cells exit the cell cycle at embryonic day (E) 12.5 and subsequently differentiate into HCs or SOX2+ SCs (in vitro (DIV) using CHIR99021 (CHIR), a selective inhibitor of GSK3β function, and supplemented the media with the proliferation marker BrdU for the entire culture period. Control explants were cultured with DMSO and BrdU. After fixation, explants were immunolabeled for SOX2 (early prosensory or late SC marker), BrdU (proliferation marker) and myosin VI (MYOVI) or myosin VIIA . Co-labelling with SOX2 and BrdU was used to quantify proliferation.We first sought to evaluate the temporal pattern of the regenerative response to canonical Wnt activation in the post-mitotic cochlea in terms of proliferation and HC induction. During mouse cochlear development, SOX2OX2+ SCs . We ther+ prosensory cells are typically quiescent . Live imaging of an E13.5 cochlear explant from a EGFP/+Sox2 reporter mouse further illustrates the effects of Wnt activation on prosensory cells. In contrast to controls . Furthermore, when we established cochlear explants at E13.5 and activated Wnt on E15.5 for 4 days, an expanded JAG1+ domain was observed at the apex .Additionally, we immunostained E13.5 cultured explants for the Notch-ligand jagged 1 (JAG1), another early prosensory and late SC marker. Wnt activation at E13.5 resulted in an expanded JAG1+ SCs at P0; however, this proliferative response was no longer observed by P5. By P0, HC and SC differentiation is complete but the cochlea continues to mature until the onset of hearing at P14. In control explants established at P0, BrdU incorporation by SOX2+ cells was not observed, indicating that SOX2+ cells remained mitotically quiescent .As 10 μM CHIR induced a more robust proliferative response at P0, we used this dose to activate Wnt at P5. However, Wnt activation at P5 with 10 μM CHIR for 5 DIV failed to induce proliferation in SOX2As Wnt activation promoted SC proliferation at P0 but not P5, we hypothesized that this age-dependent decline in proliferation could result from decreased β-catenin accumulation, diminished TCF/Lef complex activation and/or changes in the transcriptional response to Wnt by P5. First, in order to determine whether there is decreased β-catenin accumulation at P5 compared with earlier developmental stages, we quantified β-catenin in embryonic and postnatal cochleae following transient exposure to the Wnt activator. We treated E13.5, P0 and P5 cochleae with DMSO or 10 μM CHIR for 3 h and subjected whole-cochlear lysates to western blotting. Membranes were immunoblotted with active β-catenin, β-catenin and GAPDH (loading control) antibodies. Active β-catenin antibody selectively recognizes the stabilized form of β-catenin, which is dephosphorylated at Ser37 and/or Thr41.Compared with controls, transient exposure of E13.5 cochlear explants to CHIR resulted in increased accumulation of both active β-catenin and total β-catenin after 3 h A. This pTCF/Lef:H2B-GFP reporter mice  to validate changes in expression of Notch ligands tivation A. In agrr 5 days B,C. Exprtivation A. In thetivation A.Fig. 6These results demonstrate that maintaining high levels of Wnt activation in the neonatal stages alters the expression of specific ligands, receptors, modulators and downstream target genes of Wnt and Notch signaling pathways. This may play a role in SC proliferation and confer competency for HC induction in response to Notch inhibition.in vitro experiments demonstrated that Wnt activation using CHIR promotes SC proliferation at P0, but fails to produce the same effect at P5. As β-catenin accumulation and Wnt reporter activity appear to have a similar pattern at both P0 and P5, we sought to compare the broad transcriptional response to Wnt activation between early and late neonatal time-points using RNA sequencing. To exclude off-target effects of the GSK3β inhibitor, we chose to activate Wnt using our genetic mouse model flox(Exon3)/+Sox2-CreER; β-catenin. Exon 3 of Ctnnb1 contains the GSK3β phosphorylation site, which marks β-catenin for degradation. When Cre is activated with tamoxifen, exon 3 is excised and β-catenin is stabilized specifically in SOX2+ cells. We have previously shown that Wnt activation using genetic β-catenin stabilization promotes the proliferation of SOX2+ cells at P1 but not in the adult cochlea (Our  cochlea .flox(Exon3)/+Sox2-CreER; β-catenin pups at P1 or P4 (‘P1 or P4 treated’) and six Cre negative controls (‘P1 or P4 untreated/control’). Principal component analysis (PCA) outlined age as being responsible for 42% of the variation between samples, whereas treatment accounted for 17% of the variation. Pearson correlation heat-map of normalized counts was used to visualize the variability between samples. Samples were segregated by age and by treatment, presumably owing to differences in Cre activation between samples . We therefore chose to exclude this sample from further analysis.Altogether, 12 samples were treated with tamoxifen and subjected to RNA-sequencing, including six  samples A. AdditiAs the samples after β-catenin stabilization and the controls were genetically different, we first compared differentially expressed genes between P1 and P4 with and without β-catenin stabilization. 2914 genes were significantly differentially expressed in untreated samples at P1 compared with P4 and 3022 genes were significantly differentially expressed between P1- and P4-treated samples. Ingenuity pathway analysis   was subsWe next analyzed potentially enriched upstream regulators, whereby IPA clusters differentially expressed genes that are targets of a common regulator and observed further contribution of treatment to differential expression of proliferation-associated genes between P1 and P4. Higher activation and inhibition was observed for the 10 most significantly enriched regulators after treatment C. Additiin vitro results. Owing to the larger dataset available from RNA sequencing, we expanded the list used in the in vitro experiment to include additional Wnt components as well as targets from published sources . After treatment at P4, 304 genes were significantly differentially expressed, with 166 increased and 138 decreased (median fold change=1.73). In order to examine the effects of β-catenin stabilization, we first assessed Wnt signaling components using a list of Wnt pathway genes and targets from the RT² Profiler PCR Array (Wnt Pathway) and compared them with our  sources . At P1,  sources . Six of Lgr5, which encodes an R-spondin receptor. Lgr5 is a Wnt target gene and a Wnt activator that is associated with SC regeneration in the inner ear . Levels of the Notch target gene Cdkn1a were significantly increased in vitro and at P1  and supplementing it with MGI Gene Ontology database . Out of 113, 96 Notch-pathway associated genes remained expressed in P4-untreated samples  and only weakly enriched at P4 .IPA was further used to elucidate activated pathways. As expected, Wnt/β-catenin-signaling and planar cell polarity pathways were most prominently activated in treated samples . Nevertheless, when observing the differences at each time-point, 66 Erbb2 targets were differentially expressed in P1-treated samples and only 29 at P4 . It was also defined as a significantly enriched upstream regulator by itself at both time-points and estrogen-receptor signaling was among the activated pathways between P1 and P4   . These Eom birds . Pillar om birds . AlthougHebp1 suppresses oxidative stress as part of the Nrf2 pathway and can protect against cisplatin and noise-induced hearing loss  and CHIR-treated (in vitro) samples. The Notch target gene Cdkn1a was also similarly expressed. Distinctions between the expression of additional Wnt and Notch components are most likely due to differences between in vivo and in vitro experiments, as well as in vivo β-catenin stabilization compared with the use of a GSK3β inhibitor.We have shown previously that SOX2 levels influence Wnt and Notch-induced proliferation and differentiation of cochlear cells . One lim in vivo . Our in In summary, we show that exogenous Wnt activation following birth not only promotes the proliferation of SCs but also extends the window for HC induction up to P8, thus generating new HCs in the maturing cochlea. Exogenous Wnt activation potentially promotes an arrest of maturation progression or even partial dedifferentiation of SCs into a less mature state. Our results suggest that efforts to stimulate regeneration in the adult auditory organ may require recapitulating the transcriptional response of the early neonatal cochlea to exogenous Wnt activation and subsequent alterations of silenced state of Wnt target genes.EGFP/+Sox2 reporter mice   and  017592)  were maiflox(Exon3)/+Sox2-CreER; β-catenin mice were used to stabilize β-catenin in SOX2+ cells. Sox2-CreER (flox(Exon3)/+β-catenin 61Hadj/J, stock number 013752] were used to visualize Wnt activation. These strains were maintained and euthanized in accordance with Institutional Animal Care and Use Committee regulations at the Massachusetts Eye and Ear Infirmary.Male and female x2-CreER  and β-caExon3)/+  were kinin vitro (DIV). Cochlear explants of littermates were cultured with vehicle (DMSO) as controls. Proliferation under both conditions was assayed by supplementing culture media with BrdU . The Notch pathway was inhibited with DAPT . Each experiment was performed with a minimum of three independent litters and a minimum of seven cochleae per condition.Cochlear ducts were dissected from embryos of timed-pregnant mice at embryonic day (E) 13.5 and neonatal pups at P0 and P5, and the roof of the duct was removed to expose the sensory epithelium. Cochleae were established as explant cultures as previously described . The WntTCF/Lef:H2B-GFP reporter mice were established at P0 and P5 and incubated with CHIR99021  for 13 h. Cochlear explants from littermates were cultured with DMSO as controls.In order to visualize Wnt activation, cochlear explant cultures of EGFP/+Sox2 reporter mice were established and imaged as previously described using an Olympus VivaView FL Incubator Microscope (Cochlear explant cultures from E13.5 croscope . The WntTCF/Lef:H2B-GFP mice were prepared using a z-projection from a stack covering the entire depth of the explant. The top segment (7.25 μm thick) was defined as the HC layer according to apparent MYOVIIA expression in the image annotation; the bottom segment (4.75 μm thick) was defined as the SC layer according visualized SOX2. Samples from a specific time-point were harvested, treated and analyzed simultaneously, and the GFP signal gain was determined using the control sample and then applied to the treated sample.Immunofluorescence detection was performed as previously described . The fol+BrdU+ cells among all SOX2+ cells within a 200×200 μm box positioned over the HC domain, at the 50% position from the base of cochlea. The number of differentiated HCs in E13.5 explants were assessed by counting MYOVIIA+ cells within a 200 μm segment along the length of the cochlea and the entire width of the sensory epithelium at the 50% position from the base of the cochlea. Proliferation in P0 and P5 cochlear explants treated with control media or CHIR99021 was assessed by calculating the percentage of SOX2+BrdU+ cells among all SOX2+ cells within a 200 μm segment along the length of the cochlea and the entire width of the sensory epithelium at positions 25%, 50% and 75% from the base of cochlea. HC induction in response to Notch inhibition in control or CHIR99021-treated samples was assessed by counting MYOVI+BrdU+ cells per 200 μm segment along the length of the cochlea and the entire width of the sensory epithelium at positions 25%, 50% and 75% from the base of cochlea. Cell counts for each experiment were obtained from a minimum of three independent litters and a minimum of seven cochleae per condition. Statistics were performed using a two-tailed Student's t-test with the significance threshold set to P<0.05.Proliferation in E13.5 cochlear explants treated with control media or CHIR99021 was assessed by calculating the percentage of SOX2g for 20 min at 4°C and supernatant was collected. Proteins were separated using Bolt Mini Gels (Life Technologies) according to the manufacturer's instructions. Proteins were blotted onto polyvinylidene difluoride membranes and immunoblotted as previously described ]. Lysate was centrifuged at 14,000 escribed . The fol2 First Strand Kit (Qiagen). Quality of reverse transcription reaction was assessed with RT² PCR Array Mouse RNA QC plates . Amplification of genomic DNA was not detected. PCR was performed according to manufacturer's instructions using RT² SYBR Green ROX qPCR Mastermix (Qiagen) and RT² Profiler PCR Array Mouse Wnt Signaling Pathway  or RT2 Profiler PCR Array Mouse Notch Signaling pathway . These arrays were pre-designed to detect 84 Wnt pathway genes and 84 Notch pathway genes, respectively. Thermal cycling was performed using StepOnePlus Real-Time PCR system (Applied Biosystems) as previously described (t-test with the significance threshold set to P<0.05) was performed using RT2 Profiler PCR Array Data Analysis Workbook, Version 4 . A complete list of genes assessed can be accessed through Qiagen for both RT² Profiler PCR Array Mouse Wnt Signaling Pathway (www.qiagen.com/ca/shop/pcr/primer-sets/rt2-profiler-pcr-arrays/?catno=PAMM-043Z#geneglobe) and RT2 Profiler PCR Array Mouse Notch Signaling pathway (www.qiagen.com/ca/shop/pcr/primer-sets/rt2-profiler-pcr-arrays/?catno=PAMM-059Z#geneglobe).Cochlear explants established on P0 were cultured with DMSO or 10 μM CHIR99021 for 5 DIV. RNA was extracted from three independent litters and five cochleae per condition using RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. Concentration of RNA was determined using a Qubit Fluorometer (Thermo Fisher Scientific) and cDNA was prepared using the RTescribed . QualityGapdh (Mm99999915_g1) as control, Jag1 (Mm00496902_m1), Jag2 (Mm01325629_m1), Notch1 (Mm00435249_m1), Notch2 (Mm00803077_m1), Notch3 (Mm01345646_m1), Notch4 (Mm00440525_m1), Fzd4 (Mm00433382_m1) and Wnt11 (Mm00437328_m1). Statistics were obtained using a two-tailed Student's t-test with the significance threshold set to P<0.05.RNA was prepared as described above and cDNA was transcribed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). The following primers (Applied Biosystems) were used for RT-qPCR gene expression assays: t-test with the significance threshold set to P<0.05.For H2B-GFP analysis, cDNA was transcribed using the qScript XLT cDNA Synthesis Kit (QuantaBio) and qPCR was conducted using SYBR Select SYBR Mix (Applied Biosystems) with Gapdh PrimePCR SYBR Green Assay (Bio-Rad) and the following primers for H2B-GFP: forward, CAAGATCCGCCACAACATCG; reverse, GACTGGGTGCTCAGGTAGTG. Statistical analyses were carried out using a two-tailed Student's flox(Exon3)/+Sox2-CreER; β-catenin mice were exposed to tamoxifen at P1 or P4 through the mother's milk and sacrificed 72 h later at P4 or P7, respectively. To verify Cre activation, brain tissue was collected from neonatal mice and genotyped using Kapa Mouse Genotyping Kit (Kapa Biosystems) with the following primers: forward, GGT AGG TGA AGC TCA GCG CAG AGC; and reverse, ACG TGT GGC AAG TTC CGC GTC ATC C. PCR amplified samples were separated on a 1% agarose gel. A 900 bp band was used to identify Ctnnb1 wild-type allele and a 700 bp band was used to identify the exon3 excision. Littermates carrying flox(Exon3)/+β-catenin but without Sox2-CreER were used as controls.For Cre activation in neonatal mice, mothers were given 100 μl tamoxifen  via intraperitoneal injection 1 or 4 days after delivery. Thus, Once dissected, cochleae were incubated in Cell Recovery Solution (Corning) for 45 min to separate HCs and SCs from the underlying mesenchyme and neurons. RNA was extracted using RNeasy Micro Kit (Qiagen) from three individual mice per condition, separately and without pooling.Library preparation and RNA sequencing was performed at the Dana Farber Cancer Institute Molecular Biology Core Facility. In brief, cDNA was synthesized from 2.5 ng of RNA using SMARTer V4 Kit (Clontech). Following fragmentation using M220 Focused-Ultrasonicator (Covaris), 2 ng of sheared cDNA was taken for library preparation using ThruPLEX DNA-seq kit (Rubicon Genomics). NextSeq500 Single-End 75 bp (SE75) Sequencing (Illumina) was performed on all 12 samples in one lane after they were indexed and pooled in equimolar amounts to ensure 20-30 million reads per sample.www.bioinformatics.babraham.ac.uk/projects/fastqc/), Qualimap (P-adjusted (FDR) (Xist), which pointed out the potential bias that might occur due to the random segregation of female and male mice in our dataset . We therefore excluded from our results any gene on chromosome X with the same pattern of differential expression as Xist, which could account from gender association rather than age or treatment.Reads were aligned to mm10 augmented with Ensembl gene build 75 with STAR v2.5.2b . Reads aQualimap  and custQualimap . For quaQualimap . TranscrQualimap . All 12 Qualimap , fittinged (FDR)  was smal


Fusarium wilt process, by comparing the endophytic bacterial and fungal community in banana roots and shoot tips during growth and wilting processes. The keystone bacterial species were isolated and further engineered to improve banana wilt resistance.Plant microbiome highlights the importance of endosphere microbiome for growth and health of the host plant. Microbial community analysis represents an elegant way to identify keystone microbial species that have a more central position in the community. The aim of this study was to access the interactions between the keystone bacterial species and plants during banana Enterobacteriaceae family with different relative abundances were detected in all the samples. The Klebsiella spp. might be the keystone bacteria during the growth of banana plantlets. The relative abundance of Fusarium associated with the wilt disease did not increase during the wilting process. The endophytic Enterobacteriaceae strains Enterobacter sp. E5, Kosakonia sp. S1, and Klebsiella sp. Kb were isolated on Enterobacteriaceae selective medium and further engineered by expressing 1-aminocyclopropane-1-carboxylate (ACC) deaminase on the bacterial cell walls . Pot experiments suggested that plants inoculated with strains E5, E5P, S1, and S1P increased resistance to the Fusarium wilt disease compared with the controls without inoculation, whereas the Klebsiella inoculation (Kb and KbP) did not increase the wilt resistance. Compared with the inoculation with the wild strains E5 and S1, the inoculation with engineered strains E5P and S1P significantly increased wilt resistance and promoted plant growth, respectively. The results illustrated that the keystone species in the banana microbiome may not be dominant in numbers and the functional role of keystone species should be involved in the wilt resistance.Banana endosphere microbiome structure varied during plant growth and wilting processes. Bacterial and fungal diversity in the shoot tips and roots increased with the development of the banana plantlets. The bacterial groups belonging to the Fusarium wilt resistance and growth promotion of banana plants. Engineering keystone bacteria in plant microbiome with ACC deaminase on the cell walls should be a promising method to improve plant growth and disease resistance.The ACC deaminase activity of engineered bacteria was essential to the The online version of this article (10.1186/s40168-019-0690-x) contains supplementary material, which is available to authorized users. Musa spp.) is an important fruit and cash crop in the sub-tropic and tropic regions [Fusarium wilt is one of the most destructive diseases of banana [Fusarium oxysporum f. sp. cubense (FOC) infects banana roots, and the infection progresses into vascularized portions of the rhizome in susceptible banana cultivars [Fusarium infection, and the affected xylem becomes reddish and eventually pugged, impeding water and nutrient transport [Fusarium wilt, resistant cultivars are the only consistently effective tool for controlling the disease in pathogen infested soils. Nevertheless, resistant cultivars are commercially unacceptable, scarce, and nonproductive [Banana  and healthy and wilting mature banana plants. Based on comparative analyses of the banana endosphere microbiomes, subsequent functions of engineered crucial Banana samples were collected from a banana plantation in the suburbs of Guangzhou, China . These domesticated banana plants are dependent on propagation via cloning, either by using suckers and cuttings taken from the underground stem or through modern tissue culture. Most banana plantlets are produced by modern tissue culture. The transmission of microbiota to the progeny represents a way to ensure the presence of beneficial symbionts within the habitat. The banana symbiotic microbiota may transmit from shoot tips to explants and to mature plants. The samples in different growth stages  were collected and analyzed simultaneously to avoid the sequencing deviation from different sequencing condition.Musa sp., AAA, Giant Cavendish cv. Baxi) on Murashige and Skoog (MS) basal medium supplemented with 5 m/L of benzyl aminopurine (BAP)  from Guangzhou Academy of Agricultural Sciences were collected for DNA extraction. Suckers of healthy and wilting mature plants  were collected from the banana plantation in the suburbs of Guangzhou, China . The banana suckers were uprooted, the external leaf sheaths were removed, and the shoot tips (10–15 mm) with almost half shoot and corm tissues were excised aseptically. The shoot tips (0.1 g) were used for DNA extraction. The plant roots were collected in plastic bags, washed with tap water, and surface sterilized within 4 h [n = 5), shoot tips and roots of wilting plants , and shoot tips and roots of plants adjacent to the wilting plants but without wilting symptoms  were employed for total DNA extraction using E.Z.N.A. HP Plant DNA Kit (Omega) according to the manufactures’ protocol [To determine crucial microbial members proliferated in plant tissues during the growth stages and wilting development, the pseudostems and roots of banana explants  and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) targeting ITS2 ommunity . The bacanalysis . The seqanalysis . The libRaw sequences were merged by overlapping paired-end reads using FLASH software (v 1.2.7) . The cle(EMB) medium with 2.0% agar. The shoot tip samples were incubated at 28 °C for 7 days for bacterial colony formation. The colonies were purified, identified, and transformed with surface displaying plasmids. The 16S rRNA gene sequences of isolated strains have been deposited in the NCBI GenBank database under the accession numbers of KY800390, KY8003901, and KY8003902. The surface displaying plasmids, consisting of promoter sequences from Enterobacter cloacae, ice nucleation protein-N gene inaK-N, ACC deaminase gene (acdS JQ646055), and pUC57, were constructed with the previous methods [Enterobacteriaceae strains using BTX ECM 399 electroporators under the following conditions: 2.0 kV, 25 μF, 200 Ω, and 2.5 ms. Cells harboring the recombinant plasmid were grown in Luria-Bertani (LB) medium with 500 μg mL−1 ampicillin for 2 days. The cells were collected for determination of ACC deaminase activity by measuring the amount of α-ketobutyrate released [The surface sterilized shoot tips were cut into small pieces (about 5 × 5 mm) and placed onto selective Eosin-Methylene Blue  methods , 30 (Addreleased . Toluene−1) was determined using the colorimetric method with Salkowski reagents [Fusarium wilt pathogen was evaluated from plate confronting tests on banana pseudostem extract medium [−1) was rated to be tolerant, moderately tolerant, and intolerant according to the inhibition percentages of fusaric acid by 0–40%, 41–80%, and 81–100%, respectively [The indole-3-acetic acid (IAA) produced by endophytic bacteria in LB medium or LB medium supplemented with tryptophan (200 μg mLreagents . The antt medium . Endophyectively . StrainsFusarium wilt pathogen Fusarium oxysporum f. sp. cubense race 4 (FOC4) were prepared from potato dextrose broth and the soil was infested with 1% of the inoculums [Soil samples were collected from the banana plantation of Guangzhou, air dried, and sieved (5 mm). The soil was light gravel soil with texture of sandy loam . Inoculums of banana noculums .6 cfu mL−1) for 2 h. Explants soaked in the sterile LB medium were used as the controls. The inoculated and control plantlets were planted in pots with the soil. One plantlet was sown in each pot and five pots were set up for each treatment. The soil moisture was kept at > 95% of saturation. The banana plants were kept in a glasshouse at 25 °C and day/night cycle of 16:8 h. The biocontrol effect was evaluated after 90 days of planting by measuring plant height, leaf area, pseudostem girth, and fresh and dry weights [Banana explants were inoculated with endophytic bacteria by soaking in bacterial suspension . For the biocontrol experiments, analysis of variance (ANOVA) was conducted to compare the inoculation results among the different symbiotic bacteria. All statistical tests were subjected to ANOVA and the significance threshold was set up at rDNA V3-V4 region and fungal ITS2 sequencing, 20,012 bacterial processed reads were obtained with an average length of 430 bp and 113,780 fungal processed sequences with an average length of 338 bp for the samples  declined to 7. The shoot tips shared 3, 5, and 6 bacterial OTUs with Btip, Sroot, and Sstem, respectively. Only one OTU was shared within the four types of samples    Fig. . The proem) Fig. . After tem) Fig. . At the em) Fig. . Only onem) Fig. . The shaem) Fig. . A totalem) Fig. . The banCaulobacter (15%) and Paracoccus (2.9%) were the abundant bacterial genera in the banana plants. Cladosporium (12%) and Eurotium (4.2%) were the predominant fungal genera in the plants. Erwinia, Salmonella, Klebsiella, Brenneria, Stenotrophomonas, and Lactococcus were the most detected bacterial genera in the banana explants (Sroot and Sstem) , and Amaricoccus, Paracoccus, Kocuria, and Micrococcus were most frequently detected in the plant roots (Broot) , and Caulobacter, Staphylococcus, and Comamonas dominated in the roots (Hroot) , and Caulobacter and Comamonas were the dominant bacterial genera in the roots (Wroot)  Fig. . In the Eurotium .2% were obacter 1% and Parot) Fig. . KlebsieCladosporium was the most frequently detected fungal genera in the healthy banana shoot tip (Btip). The fungal OTUs affiliated with Fusarium were not detected in the wilting plant roots, whereas Cladosporium, Penicillium, Aspergillus, Eurotium, and Cryptococcus were the dominant fungal genera in the roots (Wroot) . During the infection process, Fusarium and Eurotium were detected in the roots of plants infested by the Fusarium pathogen (Hroot). With different relative abundance, Eichleriella was the crucial fungal taxa because it was detected in the pseudostems (shoot tips) and roots of explants and mature plants with different wilting stages  Fig. . Cladospges Fig. .Fig. 7PhKlebsiella belonged to the Enterobacteriaceae. Colonies grown from the shoot tip pieces were picked out, transmitted, and purified on selective EMB agar. The purified bacterial isolates were identified as Kosakonia sp. S1 (S1), Enterobacter sp. E5 (E5), and Klebsiella sp. Kb (Kb) by 16S rRNA gene sequencing analysis. The constructed surface display plasmids with ACC deaminase genes were transferred into E5, S1, and Kb cells with electroporation. The engineered strains of E5, S1, and Kb with plasmids were designated as E5P, S1P, and KbP, respectively. Klebsiella sp. Kb produced > 150 mg L−1 IAA, while E5 and S1 produced < 30 mg L−1 IAA in the media with and without tryptophan (Trp) addition. Plate confronting tests indicated that the strains of E5, E5P, S1, S1P, Kb, and KbP did not show any antagonism to the wilt pathogen FOC4 on the banana pseudostem extract medium. The growth of the six strains was not influenced by the addition of 100 μg mL−1 fusaric acid in vitro. These strains could tolerate the fusaric acid concentration of 400 μg mL−1 in the banana pseudostem extract medium. The bacterial strains could survive in banana plant tissues after the wilting symptoms appeared.The keystone endophytic bacterial genera P > 0.05)  were similar, and the differences among the concentrations of α-ketobutyrate released from ACC were not significant (5) Table . The ACCFusarium wilt disease index by 10%  of plants inoculated with the wild and engineered strains were compared. The growth parameters of plants inoculated with the six strains were improved compared with the controls without inoculation  of banana explants and plants in the different wilting processes. Endophytic Enterobacteriaceae including Klebsiella genera have been detected from different tissues of banana with different methods [Enterobacteriaceae (Enterobacter spp. and Klebsiella spp.) may proliferate in plant tissues and transmit from generation to generation in banana growth and proliferation. Klebsiella and Enterobacter are more beneficial to banana plants than other microbial taxa [Enterobacteriaceae bacteria were considered as the keystone bacteriome in banana plants, whereas the keystone bacteria may not be dominant in numbers [Plant core microorganisms establish consistently in plants and are not influenced by different spaces, time, and plant organs . Cauloba methods , 40–45. ial taxa . Therefo numbers .Although the core bacterial and fungal communities in banana plants have been elucidated, there is still a lack of knowledge about the relationships between endophytes and host plants . In planKlebsiella and Enterobacter strains did not show any ACC deaminase activity in vitro. The Klebsiella and Enterobacter strains were further engineered by expressing ACC deaminase activity on the bacterial cells because the ACC produced by plants can contact with ACC deaminase on the endophytic bacterial cells in planta. Compared with the engineered Klebsiella strains without ACC deaminase activity, the engineered Enterobacter strains with ACC deaminase activity could reduce the plant ethylene levels. The engineered Enterobacter strains without IAA production showed the potentials to promote plant growth and wilt resistance.It remains difficult to correlate microbial OTUs with physiology nowadays . In thisEnterobacter sp. E5 did not show any activity against pathogenic fungi FOC4 in vitro, engineered Enterobacter sp. E5 with ACC deaminase activity on cells could promote banana plant growth and increase resistance to banana Fusarium wilt. Our results suggested that regulation of ethylene levels of banana plantlets by engineering plant core bacterial function may be of great interests to breeding banana resistant cultivars to Fusarium wilt. The novel strategies of engineering the interactions between endosphere microbiome with host plants provided valuable methods to build next-generation suitable agriculture.In this study, the variation of endosphere bacteriome and mycobiome of the banana plant during plant growth and wilting development suggested that the banana plant contained core bacterial and fungal taxa despite of plant physiological variation. The outgrowth of wilt pathogen did not occur during wilt development. The wilting symptom development was related with ethylene levels induced by pathogenic fungi. Although the keystone species Additional file 1:Figure S1, S2, and Table S1 Supplementary Table and Figures. (DOC 226 kb)