C. elegans BMP-like “Sma/Mab” signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the  C. elegans, we have devised a highly specific and sensitive genetic screen to identify new modulators in the BMP pathway. Through this screen, we identified three conserved tetraspanin molecules as novel factors that function to promote BMP signaling in a living organism. We further showed that these three tetraspanins likely form a complex and function together with glycosphingolipids to promote BMP signaling. Recent studies have implicated several tetraspanins in cancer initiation, progression and metastasis in mammals. Our findings suggest that the involvement of tetraspanins in cancer may partially be due to their function in modulating the activity of BMP signaling.The bone morphogenetic protein (BMP) signaling pathway is required for multiple developmental processes during metazoan development. Various diseases, including cancer, can result from mis-regulation of the BMP pathway. Thus, it is critical to identify factors that ensure proper regulation of BMP signaling. Using the nematode  Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily of secreted polypeptides that regulate a variety of developmental and homeostatic processes , 2. The C. elegans, with its wealth of genetic and molecular tools and its well-defined cell lineage, provides an excellent model system to study the functions and modulation of TGFβ signaling during the development of a whole organism at single-cell resolution. There are at least three TGFβ-related pathways in C. elegans: one that controls dauer formation, one that regulates axon guidance and cell migration, and a third BMP-like “Sma/Mab” pathway that regulates body size and male tail formation, among its multiple functions , jjIs2437[CXTim50.19[pCXT51(5*RLR::deleted pes-10p::gfp) + LiuFD61(mec-7p::rfp)], sptl-1(ok1693); LG III: daf-4(m63), daf-7(m62), sma-2(e502), sma-3(e491), sma-4(e729), lon-1(e185), cup-5(ar465), ina-1(gm144), ina-1(gm39), bre-2(ye31), bre-3(ye26), bre-3(ye28), lin-12(n676n930ts), hT2[qIs48], ccIs4438[intrinsic CC::gfp]; LG IV: daf-1(m40), daf-1(m213), fat-2(wa17), fat-3(wa22), fat-6(tm331), tsp-12(ok239), nT1[qIs51]; LG V: dbl-1(wk70), fat-7(wa36), sma-10(wk89), sma-10(ok2224), sma-1(ru18), lon-3(ct417), crm-1(tm2218), him-5(e1467), bre-1(ye4), bre-5(ye17), cgt-1(ok1045), acs-1(gk3066)V/nT1[qIs51]IV;V; LG X: lon-2(e678), tsp-21(tm6269), sma-9(cc604), jjIs2433[RAD-SMAD: CXTim50.1[pCXT51(5*RLR::deleted pes-10p::gfp) + LiuFD61(mec-7p::rfp)]].The following mutations and integrated transgenes were used: Linkage group I (LG I): tsp-21 and sma-9 are located 0.79 map unit apart from each other on the X chromosome. We therefore separated the tsp-21(jj77) mutation from sma-9(cc604) via recombination. Specifically, progeny from tsp-21(jj77) sma-9(cc604)/+ + heterozygous parents were scored for the number of CCs. Animals with 6 CCs (jj77 cc604/+ + or + +/+ + or jj77 cc604/jj77 +) were genotyped for jj77 homozygosity by PCR. jj77 cc604/jj77 + animals were selected and their progeny were further genotyped by sequencing the sma-9 gene in order to obtain jj77 +/jj77 + animals. Four independent recombinants were obtained, #570, #778, #898 and #954. Each recombinant was then outcrossed with N2 three more times before further phenotypic analysis. All four recombinants behaved similarly regarding body size, RAD-SMAD and male tail patterning phenotypes.lon-2(e678) tsp-21(jj77) double mutant was generated from a lon-2(e678) egl-15(n484)/tsp-21(jj77) heterozygous worms by identifying Lon-non-Egl recombinants, and scoring for the presence of the tsp-21(jj77) and the lon-2(e678) mutations by PCR genotyping.The let-381(RNAi) was performed via feeding following the protocol described in  (jjIs3113 and jjIs3114) were generated using gamma-irradiation. pJKL840[sma-6p::nls::rfp::lacZ::unc-54 3’UTR] was used for co-localization of TSP-21::GFP and sma-6p::nls::rfp. pTAA1[hlh-8p::cgt-3.1a ORF::unc-54 3’UTR] was used to test for function of cgt-3 in the M lineage.The full-length tsp-21-specific sgRNA plasmid pZL10, which has GAAACTGACACGGTAGAAGATGG replacing the unc-119 sgRNA in plasmid 46169   . GFP knosma-6(jj69) worms using TRIzol Reagent (Invitrogen). Reverse transcription was performed with SuperScript III First-Strand Synthesis System (Invitrogen) following the manufacturer’s instructions. The primers used to detect the cDNAs of sma-6 and act-1 are: sma-6, MLF-34 and MLF-44; act-1, NMA-163 and NMA-164.Total RNA was isolated from mixed-stage N2 or http://www.graphpad.com/scientific-software/prism/).Body size measurement and RAD-SMAD reporter assay were carried out as described in Tian et al. . Dauer fGFP and RFP epifluorescence in transgenic animals was visualized either on a Leica DMRA2 compound microscope, where the images were captured by a Hamamatsu Orca-ER camera using the OPENLAB software, or on a Zeiss LSM 710 confocal microscope. Subsequent image analysis was performed using ImageJ and Photoshop CC.ftp:/ftp.ebi.ac.uk/pub/databases/reference_proteomes/QfO/QfO_release_2014_04.tar.gz). hmmsearch was run with the arguments '-E 1e-06—domE 1e-06—incE 1e-06—incdomE 1e-06-A [alignment]', which generated aligned regions of similarity to these core domains. Since the regions of homology were extracted from full-length proteins with an HMM, the specific residues extracted were generally a subset of the full protein; moreover, it was possible for two or more such regions to be independently extracted from a single protein chain, although this proved rare for tetraspanins.We identified tetraspanin homologs by running hmmsearch from HMMER 3.1b1  with the—localpair—maxiterate 1000'. The resulting alignments were purged of poorly aligned members by first running trimal v1.4.rev15 [-gt 0.5', and then running BMGE 1.1 [-t AA-h 1-g 0.5:1'. This purged the alignments of any columns in which over 50% of the columns' positions consisted of gaps rather than amino acid residues, and then any sequences in which over 50% of the residues were gapped, yielding global alignments that lacked excessive loops and gaps. From the filtered alignments, we computed protein maximum-likelihood phylogenies, with a WAG model of amino acid evolution [-pseudo-wag'. Confidence values for the branches of trees (ranging from 0.00 to 1.00) were automatically computed by FastTree with 1,000 internal replicates. We visualized the resulting trees with FigTree 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree). Branch lengths were measured in average substitutions (when comparing full sequences or their profiles) among non-gap positions in the aligned sequences, with distances derived from the BLOSUM45 matrix, a correction for multiple substitutions, and an allowed maximum of 3.0 substitutions per individual site [These regions were then realigned with MAFFT v7.158b  in L-INS.4.rev15  using thBMGE 1.1  using thvolution , using tual site .E. coli and confirmed by sequencing. The primers, cDNA templates, and the names of the resulting bait and prey constructs are summarized in MET25 promoter [The split-ubiquitin yeast two-hybrid experiments were carried out following the detailed method described in Grefen et al. . The baipromoter . Growth The plasmids KAT-1-Cub-PLV, NubG-KAT-1 (in pNX33 vector) and KAT-1-NubG (in pXN21 vector) , HMT-1-CS1 FigC. elegans tetraspanin proteins are colored in red, while their corresponding human homologs are colored in blue. Each subtree (clade) containing one or more C. elegans tetraspanin genes is displayed on one page and shaded in a different color. The human orthologs of tsp-21 are TSPAN4, TSPAN9, and CD53; while the human orthologs of tsp-12 and tsp-14 are TSPAN5, TSPAN10, TSPAN14, TSPAN15, TSPAN17 and TSPAN33.Identities of the sequences are given in Supplemental (PDF)Click here for additional data file.S2 Figsma-9(0);susm , sma-9(0) , lin-12(0) , and lin-12(0);sma-9(0)  animals. BWM: body-wall muscle, CC: coelomocyte, SM: sex myoblast. d: dorsal, v: ventral, l: left, r: right, a: anterior, p: posterior. M lineage-derived CCs are marked by blue arrowheads.Schematic representation of the M lineage (A-D) and diagrams showing the CC locations (E-H) in wild-type or (PDF)Click here for additional data file.S3 FigAn abbreviated glycosphingolipid metabolic pathway (based on ) showing(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Tablespecies of origin, their accession identifier names, and the amino acid residues from the full-length homolog that were aligned and used for the phylogenetic analysis. The accession names are from the SwissProt and TrEMBL divisions of UniProt (indicated by "sp|" and "tr|"), via the reference proteome set of the Quest for Orthologs consortium.The sequence names of 700 tetraspanin homologs phylogenetically analyzed in (XLSX)Click here for additional data file.


Drosophila, miR-92a and miR-92b are embedded in the intron and 3’UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b are highly expressed in neuroblasts of larval brain where Jigr1 expression is low. Genetic deletion of both miR-92a and miR-92b demonstrates an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. We also show that miR-92a and miR-92b directly target jigr1 in vivo and that some phenotypes due to the absence of these miRNAs are partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development.Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. Here we show that in  Drosophila stem cells in the brain as a model system to investigate the interactions between miR-92a and miR-92b and their host gene jing interacting regulatory protein 1 (jigr1). Our studies reveal that these miRNAs prevent premature differentiation of neural stem cells and they do so in part through directly targeting and suppressing their host gene, jigr1. Our results reveal a novel function of the miR-92 family and identify another negative feedback loop as an essential regulator in neural stem cell development.Animal development is regulated by many genes including a class of small RNAs called microRNAs (miRNAs). Nearly half of the miRNAs are located in the protein coding genes but functional importance of this genomic organization is unclear. Here we use  MicroRNAs (miRNAs) are short ~21–23 nt) noncoding RNAs that regulate gene expression post-transcriptionally in many physiological and pathological processes –23 nt no. Many inDrosophila neuroblasts as a model system to examine the expression and function of specific miRNAs. Drosophila neuroblasts form during embryonic development and enter a proliferative quiescent state at the end of embryogenesis  w[*]/DpG, P{hs-hid}Y; P{70FLP}11 P{70I-SceI}2B noc[Sco]/CyO, P{hs-hid}4, y[1] w[67c23] P{y[+mDint2] = Crey}1b; D[*]/TM3, Sb[1], hs-flp70; Tub-Gal4 UAS-mCD8:GFP; FRT82B Tub-Gal80, FRT82B. UAS-jigr1-RNAi (#100970) was obtained from the Vienna Drosophila Resource Center. Insc-GAL4 and Pros-GAL4 were kind gift of Chris Doe (University of Oregon).Fly stocks were maintained at 25°C on standard medium. The following fly stocks were obtained from the Bloomington Drosophila RNAi Screening Center, and UAS-miR-92b was generated by amplifying the 500–base pair pri-miR-92b from genomic DNA and cloning it into the pTW vector. Primers for PCR are listed in UAS-miR-92a and UAS-miR-92b transgenic flies were generated by Rainbow Transgenics.The UAS-dsRed-miR-92a vector was from the e04431, e04047, e00089, d03337, e01786, and e03251 (Harvard Exelixis Collection) were used to generate FLP-FRT-based deletions, as described. Deletions were confirmed by PCR and sequencing.The FRT-bearing transposon lines Del #1, miR-92-/- lines, which have one loxP site at miR-92a locus and two additional loxP sites at miR-92b locus, were used. Deletions were made as described in [In order to generate ribed in .Ends-out gene targeting was performed as described. Primers used to clone the 5’ and 3’ homology arms of miR-92a and miR-92b into pGX-attp are listed in hs-flp70, Tub-Gal4, UAS-mCD8:GFP; FRT82B, Tub-Gal80 flies with FRT82B flies to generate control clones and with FRT82B, miR-924 flies to generate mutant clones. Clones were induced at 24 ± 6 hr ALH by heat shock in 37°C water bath for an hour and analyzed 72 hr after clone induction. The size of the clones was determined by counting the GFP+ cells in single clones. Type II clones were distinguished by presence of several small GFP+ Dpn+ cells (Intermediate progenitor cells) in addition to single large GFP+ Dpn+ neuroblast.MARCM clones were generated as described before by crossing rp49 primers were used for normalization, and relative expression levels were calculated with the comparative CT method. The oligonucleotides used for qRT-PCR are listed in For qRT-PCR, cDNA was synthesized from total RNA with random primers and superscript-II reverse transcriptase (Invitrogen). SYBR Green PCR Master Mix (Applied Biosystems) was used for real-time thermal cycling. The reactions were performed with a StepOne Plus Real-Time PCR System (Applied Biosystems) and StepOne software v2.1. For quantification of mature miR-92a and miR-92b, miRNA Taqman assay was used (Applied Biosystems assay number 000285 for miR-92a and 000286 for miR-92b). U6 was used as an internal reference.Total RNA was extracted from larval brains with miRNeasy (Qiagen). 3’RACE was performed as described . Two speFor western blot, 25 larval brains were dissected and lysed in 200 μl of RIPA buffer. Larval lysate (20 μl) was mixed with 2X Laemmli buffer, boiled, and separated by 12% SDS-PAGE. The blot was probed with rabbit anti-jigr1 (1:5000), generated against the N-terminal 110 amino acids of jigr1 protein by SDIX. Mouse anti-tubulin  was used as a loading control. Horseradish peroxidase–conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories.Drosophila pri-miR-92a and pri-miR-92b were amplified from genomic DNA and cloned into pSuper-GFP vector. MiR-124 used as a control miRNA. A pri-miR-124 expression plasmid was generated by amplification of 500 bp of primary sequence from mouse genomic DNA and cloning into pSuper-GFP vector.To generate miR-92a and miR-92b expression vectors, jigr1 expression constructs, jigr1 coding region containing short 3’UTR and long 3’UTR are amplified from cDNA and cloned into pUASt vector.To generate jigr1 3’UTR sensor, jigr1 short and long 3’UTR sequences were amplified from genomic DNA and cloned into psiCHECK-2 downstream of Renilla luciferase. The target sites were mutated by site-directed mutagenesis with QuickChange multi and the QuickChange II site-directed mutagenesis kit (Stratagene). For the Jigr1-5’UTR sensor, oligonucleotides containing wild type and mutated miR-92a and miR-92b binding sites were synthesized, annealed, and cloned at the 5’ of the Renilla luciferase gene using the NheI site in psiCHECK-2 vector. The sequences of the wild type and mutant oligonucleotides are listed in To generate a Drosophila third instar larvae were fed on food containing 1 mg/mL BrdU for 3 hours. Larval brains were immediately dissected, fixed and stained with the addition of a 2N HCl treatment for 30 min prior to BrdU staining.hs-flp70, Tub-Gal4, UAS-mCD8:GFP; FRT82B, Tub-Gal80 /FRT82+ and, hs-flp70, Tub-Gal4, UAS-mCD8:GFP; FRT82B, Tub-Gal80/ FRT82B, miR-924 larvae at 24 ± 6 hr ALH. 48 hr later the larvae were first transferred to food containing 1mg/mL BrdU for 4 hours then to normal food for 24hr. Larval brains were dissected, fixed and stained with the addition of a 2N HCl treatment for 30 min prior to BrdU staining.For BrdU chase experiments, first clones were induced in Northern blot for miRNAs was performed as described . The fol+ , and crosslinked to the membrane with UV crosslinker. Hybridization was done overnight (16 hours) after 1 hour of pre-hybridization in DIG easy Hyb granules (Roche). For detection, DIG wash and block buffer set (Roche) were used according to the manufacturer’s protocol.Total RNA was extracted with the miRNAeasy mini kit (Qiagen). RNA (10–20 μg) was loaded onto a 15% polyacrylamide gel , transferred to Amersham Hybond-Njigr1, 10 μg of total RNA was loaded onto an agarose gel  and transferred to a nylon membrane overnight by capillary transfer. Hybridization and detection were done as described above. To generate probes for jigr1 and rp49, we amplified a ~500-bp region from genomic DNA by PCR. A T7 promoter sequence was added 5’ of the reverse primer. The probes were synthesized by in vitro transcription with T7 RNA polymerase (Roche). The oligonucleotides used to synthesize the jigr1 and rp49 probes are listed in For northern blot analysis of Third instar larval brains were dissected and fixed in 4% paraformaldehyde overnight at 4°C. The brains were transferred into 30% sucrose and incubated at 4°C overnight. Then, they were frozen and stored at -80°C until further use. Then 15 um sections were made. 5’ and 3’ DIG-labeled LNA-modified miR-92a and miR-92b probes (Exiqon) were used as recommended by the manufacturer. Anti-DIG-POD primary antibody  was used and detected with the TSA Plus Cyanine 3 System (Perkin Elmer).Larval brains were dissected and fixed in 4% paraformaldehyde in phosphate-buffered saline for 20 minutes. The primary antibodies used were rabbit anti-Dpn (1:500) from Y.-N. Jan , mouse anti–Dlg 4F3 (1:20) from the Developmental Studies Hybridoma Bank (DSHB), rat anti-Miranda (1:500) from Chris Doe (University of Oregon), mouse anti-Prospero MR1A (1:20) from DSHB, rat anti-BrdU (1:500) from Abcam, goat anti-HRP-Cy3 from Jackson ImmunoResearch Laboratories, and mouse anti-nc82 (1:5) from DSHB. Secondary antibodies were from Life Technologies. Confocal images were taken with a Nikon D-Eclipse C1 and processed with Image J and Adobe Photoshop.HEK293T cells were transfected with wild type or mutant miR-92 sensor plasmids or empty psiCHECK-2, pSuper-GFP-pri-miR-92a, or pSuper-GFP-pri-miR-92b expression vectors (described above) or pSuper-GFp-miR-124 using Fugene6 (Promega). 48 hours after transfection, cells were lysed in 1x passive lysis buffer (Promega). The Dual-Glo Luciferase Assay System from Promega was used according to the manufacturer’s protocol. Renilla luciferase activity was normalized to firefly luciferase activity. All the experiments were done in triplicate in three independent experiments.S1 FigDrosophila by miRNA Taqman assay.(A) Sequence alignment of miR-92a and miR-92b mature sequences from different species (miRBase). (B) Quantification of mature miR-92a and miR-92b at different developmental stages of (TIF)Click here for additional data file.S2 FigmiR-92b-/- and miR-92a-/- third instar larval brains. Enlarged view of dashed box shows expression of miR-92a in Dpn+ neuroblasts. Scale bar: 20 μm and 5 μm. (C) miR-92b expression (red) in wild type, miR-92a-/- and miR-92b-/- third instar larval brains. Enlarged view of dashed box shows expression of miR-92b in Dpn+ neuroblasts. Scale bar: 20 μm and 5 μm.(A) Coexpression of miR-92a (red) with the neuroepithelial marker Discs large (green) in third instar larval brains. Scale bar: 20 μm. (B) miR-92a expression (red) in wild type, (TIF)Click here for additional data file.S3 Figjigr1 locus generated in this study. (B) Schematic representation of jigr1 locus indicating the location of the primers used for 3’RACE. (C) DNA gel electrophoresis of RACE nested PCR products. (D) RNA-seq data obtained from modEncode project showing the expression of jigr1 extended 3’ UTR in L3 CNS neuroblasts.(A) Summary of all the deletions in the (TIF)Click here for additional data file.S4 FigmiR-92a–/–(n = 100), miR-92b–/–(n = 100) and miR-92–/–(n = 100) mutants.Survival curve of male flies of the wild type (n = 100), (TIF)Click here for additional data file.S5 FigmiR-92–/– third instar larval brain. Immunostaining of neuroblasts for Dpn (red) and cell cortex for Dlg (green). Scale bar: 20 μm. (B) Number of brain neuroblasts at 96 hr ALH. 1: w1118 (n = 12); 2: miR-92a-/-; 3: miR-92b-/-; 4: miR-924/4 (n = 11); 5: miR-921/4 (n = 16); 6: Insc-GAL4/UAS-miR-92a; miR-924/4 (n = 10); 7: Insc-GAL4/UAS-miR-92b; miR-924/4 (n = 12); 8: Insc-GAL4/UAS-miR-92a; miR-921/4 (n = 8); 9: Insc-GAL4/UAS-miR-92b; miR-921/4 (n = 8). Statistical significance was determined by one-way ANOVA. (C) Quantification of apoptotic neuroblasts in the brains of wild type (n = 20) and miR-92–/–(n = 18) third instar larvae. Statistical significance was determined by one-way ANOVA. (D) Neuroblast number in the brains of miR-921/4 and Insc-GAL4/UAS-p35 (n = 16); miR-921/4 (n = 10) flies. Student’s t test was used for statistical analysis. Values are mean ± s.e.m. in all graphs. *: p < 0.05, **: p < 0.005, ***: p < 0.001, ****: p < 0.0001. (E) Immunostaining of wild type and miR-92–/– mutant larval brains for Prospero (green) and Dpn (red) at 96 hr ALH. Single confocal section is shown. Scale bar: 10 μm.(A) Single confocal sections of wild type and (TIF)Click here for additional data file.S6 FigmiR-92–/– third instar larval brain neuroblast, stained with Miranda (green) and Numb (red). (B) Single confocal sections of wild type and miR-92–/– third instar larval brain neuroblasts, immunostained with Miranda (green) and aPKC (red). (C) Wild type and miR-92-/- neuroblast cells stained with Miranda (green) and phospho-histone H3 (red). Neuroblasts are encircled with dashed line. (D) Neuroblast clones of wild type control and miR-924 mutants. Clones are marked with CD8::GFP (green). Yellow arrows mark the neuroblast cell. Yellow dashed lines indicate the position of clones. Single focal planes are shown.(A) Single confocal sections of wild type and (TIF)Click here for additional data file.S7 FigPros-GAL4/+, Pros-GAL4/UAS-miR-92a and Pros-GAL4/UAS-miR-92b with Prospero (blue) and Jigr1 (red). Single confocal sections are shown. (B) Sequence alignment of predicted miR-92a and miR-92b binding sites with the 3’UTR of prospero mRNA. The seed sequences of miR-92a and miR-92b are shown in blue. (C) Dual luciferase assay of HEK 293T cell lysates cotransfected with miR-92a, miR-92b or empty vectors together with psicheck2 construct containing the short or long 3’UTR of prospero mRNA. Bar graph shows normalized mean luciferase activity of cells transfected with miR-92 expression plasmid to that of cells transfected with empty plasmid from two independent experiments. Statistical significance was determined by one-way ANOVA. Values are mean ± s.e.m. in all graphs. *: p < 0.05, **: p < 0.005, ***: p < 0.001, ****: p < 0.0001.(A) Staining of third instar larval brains of (TIF)Click here for additional data file.S8 Figw1118 (red), miR-92-/- (green), Def(3R)BSC321/miR-924 (orange), Def(3R)BSC321/miR-921 (yellow), Elav-GAL4/UAS-jigr1RNAi;miR-921/4 (blue), Del #1/miR-921 (brown) flies.Survival curve of (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Wound-inducible and ER-located amidohydrolases with overlapping substrate specificities for IAA– and JA–amino acid conjugates regulate the production and destruction of active auxin and JA signals in wounded leaves. Jasmonate (JA) and auxin are essential hormones in plant development and stress responses. While the two govern distinct physiological processes, their signaling pathways interact at various levels. Recently, members of the Arabidopsis indole-3-acetic acid (IAA) amidohydrolase (IAH) family were reported to metabolize jasmonoyl-isoleucine (JA-Ile), a bioactive form of JA. Here, we characterized three IAH members, ILR1, ILL6, and IAR3, for their function in JA and IAA metabolism and signaling. Expression of all three genes in leaves was up-regulated by wounding or JA, but not by IAA. Purified recombinant proteins showed overlapping but distinct substrate specificities for diverse amino acid conjugates of JA and IAA. Perturbed patterns of the endogenous JA profile in plants overexpressing or knocked-out for the three genes were consistent with ILL6 and IAR3, but not ILR1, being the JA amidohydrolases. Increased turnover of JA-Ile in the ILL6- and IAR3-overexpressing plants created symptoms of JA deficiency whereas increased free IAA by overexpression of ILR1 and IAR3 made plants hypersensitive to exogenous IAA conjugates. Surprisingly, ILL6 overexpression rendered plants highly resistant to exogenous IAA conjugates, indicating its interference with IAA conjugate hydrolysis. Fluorescent protein-tagged IAR3 and ILL6 co-localized with the endoplasmic reticulum-localized JA-Ile 12-hydroxylase, CYP94B3. Together, these results demonstrate that in wounded leaves JA-inducible amidohydrolases contribute to regulate active IAA and JA-Ile levels, promoting auxin signaling while attenuating JA signaling. This mechanism represents an example of a metabolic-level crosstalk between the auxin and JA signaling pathways. Hormones co-ordinate developmentally programmed processes as well as responses that are induced by external stimuli. Although different hormones govern distinct biological processes, the final outcome is often the result of complex interactions among multiple hormone pathways. Deciphering the molecular mechanism of this crosstalk has been an important trend in plant hormone research . This inPLETHORA (PLT1) and PLT2, which are key transcription factors (TFs) of the auxin-regulated root meristem specification and maintenance (ANTHRANILATE SYNTHASE a1 (ASA1) and ASB1 encoding enzymes in the l-tryptophan biosynthetic pathway, which provides precursors for auxin biosynthesis  (iar3 and ill6 mutants (Signs of connection between -JA-Ile) . ConsistHere we describe the further characterization of ILL6, IAR3, and a third JA-inducible IAH family member, ILR1, for their function in JA and auxin metabolism and signaling. Catalytic activities of recombinant proteins were tested for a variety of different JA and IAA conjugates. ILR1, ILL6, and IAR3 were overexpressed in Arabidopsis and the resulting plants were analyzed for their endogenous hormone profile and for phenotypes associated with JA and auxin signaling. Higher order mutants were created to examine functional redundancies between IAHs. Subcellular localization was determined for ILL6 and IAR3 proteins. These results support a hormone crosstalk model where wound-inducible amidohydrolases simultaneously regulate the levels of JA and auxin to co-ordinate stress responses.Arabidopsis thaliana ecotype Col-0 was used as the wild type (WT) for all experiments, except for ill6-2iar3-5 double mutant was made by a genetic crossing between the T-DNA insertion lines CS852193 (ill6-2) (iar3-5) obtained from the Arabidopsis Biological Resource Center. ilr1-1, ilr1-5 . Hormone treatments for gene expression studies were carried out either by spraying an indicated amount of hormones evenly onto the surface of fully expanded leaves of soil-grown plants or by growing seedlings on plates containing the hormone. Wounding was conducted on fully expanded rosette leaves of 4- to 5-week-old soil-grown plants by crushing across the midrib twice using a hemostat with serrated tips -Jasmonic acid, methyl jasmonic acid (MeJA), coronatine, IAA, IAA-Ala, and deviously  except tO-SO3Na-JA-Ile) was synthesized as follows. To a stirred mixture of 12OH-JA-Ile methyl ester  in dry pyridine (6ml), a solution of sulfur trioxide pyridine complex  in dry pyridine (6ml) was added and stirred for 2h at room temperature. A 45ml aliquot of methanol:water (2:1) was added and the mixture was neutralized by 1M KOH. The volatile component of the reaction mixture was removed under reduced pressure to give an oil, which was purified using Si gel column chromatography  to produce 12-sulfonyl JA-Ile methyl ester . 12-Sulfonyl JA-Ile methyl ester  in ethanol (2ml) was stirred with an aqueous solution of 1M NaOH (0.7ml) for 2h at room temperature. Following neutralization with Amberlite IR-120, the volatile component of the reaction mixture was removed under reduced pressure to give an oil, which was purified using Si gel column chromatography  to produce 12-sulfonyl-JA-Ile sodium salt . Physical data for 12-O-SO3Na-JA-Ile are as follows. Electrospray ionization (ESI)-MS m/z  419 (15), 418 , 344 (22). High-resolution (HR)-ESI-MS: m/z 418.1544 [M-Na]− . 1H-Nuclear magnetic resonance (NMR)  δ: 5.37 , 4.15 , 3.90 , 2.49 , 2.36-1.73 , 1.12 , 0.81 , 0.75 .12-Sulfonyl-JA-Ile sodium salt (12-in planta overexpression of ILL6 (At1g44350) (ILL6-OE) and IAR3 (At1g51760) (IAR3-OE) was carried out by amplifying the full-length open reading frame (ORF) of each gene by reverse transcription–PCR (RT–PCR) from a total RNA from wounded WT leaves using the primer pairs indicated in Supplementary Table S2 at JXB online. The resulting PCR fragments were cloned into a pBI121-derived pBITS vector (XbaI and XhoI for ILL6 or XbaI and BamHI for IAR3. The resulting constructs with the respective genes placed behind the Cauliﬂower mosaic virus (CaMV) 35S promoter (35S:ILL6 or 35S:IAR3) were transformed into the C58C1 strain of Agrobacterium tumefaciens. Agrobacterium tumefaciens harboring each construct was transformed into Arabidopsis using a floral dip method. Seeds harvested from the resulting plants (T1) were screened for resistance to kanamycin (50 μg ml−1). A total of 55 and 69 seedlings each from 35S:ILL6 and 35S:IAR3 that survived the kanamycin selection were tested for transgene expression by quantitative (q)RT–PCR and for JA levels. Two selected lines—one each from ILL6-OE and IAR3-OE—were further propagated eventually to obtain T3 homozygous lines.Construction of binary vector constructs for Supplementary Table S2 at JXB online. The N-terminal fusions had CFP inserted behind the putative 23 and 24 amino acid signal peptide sequences of IAR3 and ILL6, respectively. The C-terminal fusion for IAR3–CFP preserved the IAR3’s ER retention ‘KDEL’ motif at the very end, whereas ILL6–CFP did not have any ER retrieval sequence at the end. The amplified fragments were cloned into a Gateway binary expression vector, pGWB2  fused to either their N- or C-terminus. ORFs of IAR3 and ILL6 with fused CFP sequence were amplified using an overlapping PCR method with the primers indicated in Supplementary Table S2 at JXB online) and cloned in between the EcoRI and SalI sites of the pGEX-6P-1 vector  to obtain pGEX-ILL6 and pGEX-IAR3, which placed a glutathione S-transferase (GST) tag on the N-terminus of ILL6 or IAR3. Sequence-verified pGEX-ILL6 and pGEX-IAR3 were each transformed into Escherichia coli strain BL21 (DE3). The pGEX-ILR1 plasmid was obtained from Dr Bonnie Bartel (Rice University)  and culturing for another 16–20h at 16 °C. GST·Bind™ Kits (Novagen) were used to purify GST-fused proteins following the manufacturer’s instructions. The purity of recombinant proteins estimated by SDS–PAGE was >90%.ORFs of ILL6 and IAR3 lacking 24 and 23 amino acids, respectively, of predicted N-terminal signaling sequences were PCR amplified  and 0.5 μM d5-IAA as internal standards. A 5 μl aliquot of the centrifugation-cleared supernatant was directly injected for LC-MS.A previously described 20 primers and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega). A 5ng aliquot of the resulting cDNA was used as template for subsequent PCR steps. Phusion Taq polymerase (Thermo Scientific) and Taq-Pro Red Complete (Denville) were used as DNA polymerases for cloning purposes and for semi-quantitative RT–PCR experiments, respectively. qRT–PCR was performed on a CFX96 Touch™ real-time PCR detection system (Bio-Rad) using SsoFast™ EvaGreen® Supermix (Bio-Rad) as per the manufacturer’s instructions. Relative transcript abundance was calculated after normalization with ACTIN8 (AT1G49240) as an internal reference gene. For time course experiments, fold change compared with the 0h time point of the WT was plotted. All other graphs show transcript levels relative to ACTIN8.RNA was extracted using TRIzol reagent (Invitrogen) from 50–100mg of frozen tissue ground into a fine powder. First-strand cDNA was synthesized from 1 μg of total RNA treated with DNase I (Qiagen) using oligo(dT)m/z, 176>130), d5-IAA (181>135), and ESI negative mode for JA , dhJA (211→59), 12OH-JA (225→59), 12COOH-JA (239→59), 12-HSO4-JA (305→97), 12-O-Glc-JA (387→207), JA-Ile (322→130), [13C6]JA-Ile (328→136), 12OH-JA-Ile (338→130), 12COOH-JA-Ile (352→130), 12-HSO4-JA-Ile (418→130), and 12-O-Glc-JA-Ile (500→130). Data analysis was carried out using MassLynx 4.1 and TargetLynx software (Waters).Hormone metabolites were quantified using ultra-performance liquid chromatography–tandem MS (UPLC-MS/MS)  based on methods described previously . Mature rosette leaves of 4-week-old Arabidopsis plants were either mechanically wounded or sprayed with a 100 μM MeJA solution, and sampled at 0, 0.5, 1, 4, 8, and 12h after treatment. Transcripts of three genes, IAR3, ILL6, and ILR1, were strongly induced by MeJA. The induction was transient, rising within a few hours of treatment and peaking at 4h, following typical ‘early gene’ kinetics , indicating a strict requirement for the functional JA perception pathway. The promoter regions of all three genes contained canonical MYC2-binding cis-elements (or G-box) according to the Arabidopsis Gene Regulatory Information Server (http://arabidopsis.med.ohio-state.edu/), and earlier microarray data indicated that their expression is regulated by MYC2 and MYC3 . Unlike the two IAA-responsive marker genes, IAA5 and GH3.1, which were strongly induced by IAA, none of the IAH transcripts responded to IAA treatment. This is in contrast to the GH family genes encoding the IAA–amino acid-conjugating enzymes, which were strongly induced by IAA . A linear increase of JA was maintained for at least 3h for both GST–IAR3 and GST–ILL6  under the chosen assay conditions . GST–IAR3 displayed at least several times higher hydrolytic activity compared with GST–ILL6 under all tested conditions, while purified GST protein alone showed no significant hydrolytic activity . Next, the three proteins were tested against 10 different conjugates of JA . GST–IAR3 hydrolyzed all six amino acid-substituted conjugates comparably well. Although hydroxylation at C12 had no effect on GST–IAR3 activity, further modifications to 12COOH-, 12-O-Glc-, or 12-HSO4- almost completely eliminated the cleavage activity.To test their catalytic activities, ILR1, IAR3, and ILL6 were expressed in activity . GST–ILRes of JA . Among t12 position eliminated the activity. GST–ILL6 showed essentially no activity against IAA-Ala or IAA-Leu, making ILL6 a specialized enzyme for JA conjugates. Even though GST–ILR1 did not show any activity for JA-Ile, it was active against JA-Leu and JA-Phe. This stark difference in preference for JA-Leu over JA-Ile was somewhat surprising, but a similar strict preference of ILR1 for -Leu over -Ile conjugates was observed with the IAA conjugates . Among 55 T1 lines of ILL6-OE resistant to antibiotic selection, eight lines  had markedly increased ILL6 transcripts. The same eight lines were also severely reduced in JA-Ile content, consistent with increased JA-Ile hydrolysis by ILL6 overexpression. IAR3-OE lines also displayed strong correlation between the increased IAR3 transcripts and reduced JA-Ile levels, and were established following a similar procedure to ILL6-OE . Two lines each from ILL6-OE (lines 5 and 47) and IAR3-OE (lines 4 and 42) were further propagated to generate T3 homozygous lines. Reduced JA-Ile levels were maintained in these homozygous lines, with levels in each representative line comparable with one another for both ILL6-OE and IAR3-OE progeny .ILL6-OE (line 5) and IAR3-OE (line 42) lines, along with a previously reported ILR1-OE line (ILL6-OE and IAR3-OE plants throughout the time course (ILL6-OE performed surprisingly well considering the lower hydrolytic activity (10 times less than GST–IAR3) that GST–ILL6 had shown in the in vitro assays  or lower than (4h) the level in the WT . ILR1-OE did not show any change in the major JA metabolite profile, consistent with the in vitro assay results.A detailed time course JA profiling was carried out on one each of these homozygous -OE line . JA-Ile e course , D. ILL6o assays . In addi IAR3-OE , E, H, wn the WT , F. The iar3-5ill6-2  and iar3-2ilr1-1  in the iar3-5ill6-2 double mutant compared with the WT or the two single mutants, indicating that the JA-Ile turnover was strained in this double mutant . These gene expression results show that JA-Ile hydrolysis by IAR3 and ILL6 can attenuate JA-responsive gene expression and, at the same time, IAA-Ala and IAA-Leu hydrolysis by IAR3 and ILR1 can activate IAA-responsive gene expression in plants.Constitutive activation of the JA-Ile turnover pathway in T leaves , B. JAZ8n the WT . This isn the WT , D. Cons, two independent lines (lines 5 and 47) displayed partial fertility defects, which were heritable in the following generations (Supplementary Table S1). Developing flowers of these plants exhibited short anther ﬁlaments and delayed dehiscence .We next investigated whether biochemical and molecular changes caused by ectopic expression of IAHs result in any visible plant symptoms. Among the eight erations , B. Overhiscence , which ahiscence . Increashiscence . Auxin a ILR1-OE , B. LackILL6-OE and IAR3-OE exhibited resistance to exogenous JA-induced growth inhibition (jar1), or increased turnover of JA-Ile (e.g. CYP94B3 overexpression) can make plants resistant to exogenous JA  or IAA-Ala (50 μM) (iar3-5) and enhanced by overexpressing IAR3, consistent with IAR3’s dominant function in plant sensitivity to IAA-Ala  , B. All u plates , demonstu plates . Surpris-5 roots . This wa-5 roots . Further-5 roots .ILL6-OE-conferred resistance to IAA conjugates is observed in leaves. For this, expression of IAA response marker genes, IAA5 and GH3.1, was measured 2h after spraying the mature rosette leaves of WT and ILL6-OE plants with solutions containing the mock treatment, IAA (5 μM), or IAA-Ala (50 μM). Both IAA5 and GH3.1 transcripts in the WT increased significantly upon IAA treatment and, to a lesser extent, upon IAA-Ala treatment compared with the mock treatment . In addition, we obtained indirect evidence that ILL6 could bind to IAA conjugates. As noted earlier, when GST–ILL6 is incubated with JA-Ile in in vitro hydrolysis assays, it hydrolyzes JA-Ile. However, when IAA-Ala was added along with JA-Ile, the JA-Ile hydrolysis by GST–ILL6 was strongly inhibited , indicating that IAA-Ala was somehow interfering with GST–ILL6’s activity against JA-Ile, most probably through competition for the active site. It is possible then, that, in ILL6-OE plants, the highly abundant ILL6 proteins are competing with the relatively small number of endogenous IAR3 or ILR1 proteins for IAA conjugate substrates, preventing their hydrolysis by IAR3 or ILR1. In this case, exceeding concentrations of IAA conjugates in the root assay medium may eventually saturate the ILL6-binding sites and restore the root sensitivity to IAA conjugates. Supporting this hypothesis, a dose-dependent inhibition of root growth was observed with the ILL6-OE seedlings grown on increasing concentrations of IAA-Ala .We tested whether the reatment , E. In IN. benthamiana leaves by syringe infiltration of Agrobacterium harboring each construct. A second culture of Agrobacterium carrying the CYP94B3–mRFP (monomeric red fluorescent protein) construct was co-infiltrated to be used as an ER marker , such as the CYP94-mediated ω-oxidation pathway  indicates its expression in flowers, which coincides with reduced fertility of ILL6-OE plants. The remaining three members of IAHs , even though they are not inducible by JA or wounding, could also potentially function as JA amidohydrolases in tissues where they are expressed. Catalytic activities of these other IAHs on JA conjugates remain to be tested.Regarding the physiological impacts of IAA conjugate hydrolysis by IAHs, IAA conjugate content in mature Arabidopsis leaves is quite low  and is e12 position of JA-Ile did not diminish the hydrolysis by GST–IAR3. On the other hand, further modification of the -OH group by oxidation (to form -COOH), glucosylation, or sulfation abolished their hydrolysis by GST–IAR3 , but these conjugates occur at much lower levels than JA-Ile in Arabidopsis seedlings and wounded leaves  and werein vitro and in vivo results are consistent with ILL6 being a JA-specific amidohydrolase. Even though GST–ILL6’s in vitro hydrolytic activity for JA-Ile was an order of magnitude lower than that of GST–IAR3, overexpression of ILL6 in plants (ILL6-OE) resulted in a severe depletion of JA-Ile. This stark difference between in vitro and in vivo activities illustrates the importance of proper contextual requirements for optimal ILL6 activity whether that is protein folding, cofactors, or post-translational modification. Surprisingly, even though GST–ILL6 enzyme displayed no in vitro hydrolytic activity against IAA-Ala or IAA-Leu, overexpression of ILL6 made plants strongly insensitive to these IAA conjugates, suggesting the potential function of ILL6 as a negative regulator of IAA–amino acid hydrolysis in vivo. IAA perception and signaling in ILL6-OE was normal, and no sign of changes in IAR3 or ILR1 transcription was detected to explain ILL6-OE’s insensitivity to IAA conjugates. We found out that the JA-Ile-hydrolyzing activity of GST–ILL6 was strongly inhibited by the addition of IAA-Ala (a non-suitable substrate for ILL6) to the reaction mixture along with JA-Ile substrates . This suggests that even though ILL6 cannot hydrolyze IAA-Ala, it may still bind IAA-Ala, which, by an unknown mechanism, interferes with the JA-Ile hydrolysis by ILL6. It can be speculated that in the ILL6-OE seedlings, high levels of ILL6 proteins may compete with the less abundant native IAR3 or ILR1 proteins for IAA conjugate substrates, preventing their hydrolysis by IAR3 or ILR1, and thus making plants appear resistant to the IAA conjugates. Our data showing restoration of ILL6-OE’s sensitivity to the increasing concentrations of IAA-Ala are consistent with this hypothesis. More study is needed to investigate this novel function of ILL6 as a negative regulator of IAA conjugate hydrolysis.Both iar3ill6 double mutant (IAH genes in wounded leaves and the lack of 12OH-JA-Ile hydrolytic activity by ILR1, a contribution of other members seems less likely. Recently, a monooxygenase from rice blast fungus was reported to catalyze direct 12-hydroxylation of JA (12OH-JA that forms as a result of 12OH-JA-Ile hydrolysis can be considered an inactive by-product of the JA catabolic pathway; however, specific signaling roles for 12OH-JA have been reported in other plant species . Howevere mutant  indicateIAR3-OE and ILL6-OE plants are consistent with JA-Ile signal being destroyed through hydrolysis rather than being futile. Differential partitioning between subcellular compartments may also play a role in physical separation of the cleaved JA from JA-Ile. In contrast to the cytosolic location of the JA-Ile-conjugating enzyme, JAR1 (One question regarding IAH’s contribution to the attenuation of JA signaling is: ‘What is the net effect of the JA-Ile hydrolysis to JA, which could potentially be conjugated back to JA-Ile by JAR1?’ We do not have direct evidence as to whether or how much of the hydrolyzed JA is recycled back to synthesize JA-Ile. However, the contribution by this recycling to the overall JA-Ile level is expected to be far less than JA-Ile hydrolysis, resulting in a net decrease of JA-Ile over time. This is partly because only a fraction of bulk JA, typically ~10%, is converted to JA-Ile in leaves. JA-Ile-deficient phenotypes of me, JAR1 .Additional studies are needed to understand the biological function of IAA and JA conjugate hydrolysis and the advantage provided by the simultaneous regulation of auxin and JA levels by IAHs. This may be too subtle to be detected easily. However, strong evolutionary conservation across higher plants of the amidosynthetase and amidohydrolase family genes as well as the ubiquitous occurrence of amino acid conjugates of auxin and JA  bear witJXB online.Supplementary data are available at Table S1. ILL6-OE plants have reduced fertility.Table S2. Primers used in this study.Figure S1. Arabidopsis IAH family gene expression.Figure S2. In vitro hydrolysis activities of GST–IAR3, GST–ILL6, and GST–ILR1.Figure S3. Generation of ILL6-OE plants.Figure S4. Generation of IAR3-OE plants.Figure S5. JA-Ile in the homozygous T3ILL6-OE and IAR3-OE plants.Figure S6. Jasmonic acid and 12COOH-JA-Ile accumulation in IAH-overexpressing lines.Figure S7. Molecular characterization of iar3-5ill6-2.Figure S8. JAR1 and GH3.1 gene expression in IAR3-OE and ILL6-OE.Figure S9. Possible mechanism of ILL6-OE-conferred insensitivity to IAA conjugates.


Pax5 or Ebf1 recapitulates lineage reprogramming occurring during CD19 CAR pressure. Our findings establish lineage switch as a mechanism of CAR resistance exposing inherent plasticity in genetic subtypes of pre-B-cell ALL.Adoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells targeting the CD19 B lineage receptor has demonstrated marked success in relapsed pre-B-cell acute lymphoblastic leukaemia (ALL). Persisting CAR-T cells generate sustained pressure against CD19 that may drive unique mechanisms of resistance. Pre-B ALL originates from a committed pre-B cell or an earlier progenitor, with potential to reprogram into other hematopoietic lineages. Here we report changes in lineage markers including myeloid conversion in patients following CD19 CAR therapy. Using murine ALL models we study the long-term effects of CD19 CAR-T cells and demonstrate partial or complete lineage switch as a consistent mechanism of CAR resistance depending on the underlying genetic oncogenic driver. Deletion of  CAR-T targeting CD19 have been successfully used in a variety of B-cell malignancies but patients may eventually relapse. Here, the authors show that CD19 CAR-T resistance in pre-B cell ALL can be due to the induction of a myeloid lineage switch through an epigenetic alterations in master regulators of B cell development. Acute leukaemia is a heterogeneous group of clonal malignancies, classified as lymphoblastic (ALL), myeloid (AML) or mixed phenotype (MPAL)2356891089101112131415161718Use of adoptively transferred T cells armed with chimeric antigen receptors (CAR-T) is a promising new cancer therapy2122242526272826272831in vivo has been limited in xenograft models due to lethal xenogeneic graft-versus-host disease precluding long-term studies. Furthermore, lack of an intact host immune system could impact in vivo behaviour of both CAR-T cells and leukaemia. To overcome these limitations and to study leukaemia resistance in the setting of CD19 CAR pressure, we used in vivo murine ALL models in which initial clearance of leukaemia by CD19 CAR-T cells is achieved with long-term persistence of CAR-T. Here we describe lineage switch as a mechanism of CD19 CAR-T resistance. Using genomic analysis of myeloid lineage switched leukaemias generated under CD19 CAR pressure and gene-editing techniques, we show this phenomenon is not simply due to alterations of CD19 but rather from a global reprograming of ALL with inherent lineage plasticity.Analysis of the impact of CAR-T on leukaemia in vivo persistencein vivo CAR-T expansion, but persistent leukaemia at day 28 despite presence of CD19 CAR T cells . Under transient CAR pressure post-CAR leukaemia demonstrated stable expression of CD19 and increased CD11b compared to pre-CAR leukaemia  transgenic mouse crossed to a CD3−/− mouse with spontaneous pre-B ALL developmentin vitro and in vivo34Next we tested the impact of CD19 CAR-T in two murine ALL models. The E2a:PBX murine B-lineage ALL line was propagated from splenocytes of an Cd19 resulting in lack of expression of the targeted epitope and cytosolic localizationCd19 exon-specific primers, we identified a similar mechanism for early post-CAR CD19-negative relapse of E2a:PBX with isolated loss of an exon 1–3 junctional transcript indicating lack of exon 2 expression but maintained expression of other exons , but were essentially absent in myeloid or mixed B/myeloid samples  into murine hematopoietic cells, the resultant leukaemic phenotype can vary and often may emerge as T or myeloid leukaemiaE2a:PBX x CD3−/− transgenic mouse4153in vivo CD19-CAR immune pressure or in vitro deletion of Ebf1 and Pax5.When attempting to model human leukaemia in mice by introducing pre-B-ALL-associated transgenes  previously reported−/− was generously provided by Dr Janetta Bijl. These cells were developed into a stable cell line expressing pre-B markers. Leukaemia cells were cultured in 10% complete mouse media (CMM) containing RPMI 1,640 with 10% heat-inactivated fetal calf serum, 1% glutamin, 1% HEPES, 1% nonessential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin, (Invitrogen) and 50 μM 2-mercaptoethanol. Eμ-RET cell lines 289 and 309 were provided by Dr Alix SeifA murine pre-B ALL carrying the human E2a:PBX transgene crossed to a CD3ɛ−1) and IL7 (10 ng ml−1). On days 2 and 3 of culture, retronectin-coated plates were spun for 2 h with retroviral supernatant, followed by incubation of the T-cell-enriched product for 24 h. On day 4 of culture, T cells were removed from retronectin-coated plates and from activating beads, and expanded for an additional 24–36 h in the presence of the aforementioned cytokines.Murine CD19 CAR construct with a CD28 costimulatory domain on an MSGV retroviral backbone was provided by Dr James Kochenderferb CD45.2+), B6-Ly5.2 (H-2b CD45.1+) and Balb/C  mice were purchased from the National Cancer Institute (NCI) animal production program. Mice were used between 6 and 10 weeks of age. All animals were kept in a pathogen-free facility, and were treated under protocols approved by the Animal Care and Use Committee at the National Cancer Institute. Overall, mice were injected with leukaemia on day 0, conditioned with radiation (500 cGy) on day 4, followed by adoptively transfer of CD19-CAR or mock T cells on day 5. Alternatively, conditioning of mice included cyclophosphamide (4 mg per mouse) instead of radiation, prior to adoptive cell therapy or additional chemotherapy, which included two doses of ARA-C (2.5 mg per dose per mouse) on days 5 and 10. All mice were monitored three times a week for survival, and were killed when paralysed or moribund, and bone marrow and spleen were harvested for analysis.C57BL/6 , FITC-anti-CD9 , PE-Cy7-anti-CD45.2 , PE-anti-CD127 , efluor450-anti-CD19 , efluor-anti-CD45.2 , PECy7-anti-CD117 , efluor-anti-CD34 ; APC-Cy7–anti-B220 , APC-anti-CD22 , PE-Cy7-anti-CD93 ; PE-anti-CD22 , APC-anti-CD11b , PE-anti-BP1 , FITC-anti-GR1 . CAR detection was performed using protein-L with PE-Streptavidin (BD Biosciences). The following conjugated anti-human antibodies were used: efluor45-anti-CD19 , FITC-anti-CD19 ), efluor-anti-CD34 , PE-Cy7-anti-CD11b , FITC-anti-CD14 , APC-anti-CD117 , PE-anti-CD33 , PerCP-Cy5.5-anti-CD45 ; PE/Cy7-anti-CD10 ; PE-anti-CD22  and PE-anti-HLA-DR . Samples were analysed on a BD LSR-Fortessa (BD Biosciences), data collected using FACS Diva software and analysed using FLowJo version 9.6.4 (Treestar).http://cufflinks.cbcb.umd.edu/) was used to assemble and estimate the relative abundances of transcripts mapped with TopHat2 at both the gene and transcript level (FPKM). FPKM values were log2 transformed and Z-scored.Nucleic acid extraction was performed using Qiagene AllPrep kit. PCR primers for DNA and cDNA are reported in 6–20 × 106 cells using the ChIP-IT High Sensitivity Kit as per the manufacturer's protocol (Active Motif). Shearing occurred under the following conditions using the EpiShear Probe Sonicator-Amplitude: 0.3/Pulse: 30 s ON, 30 s OFF/Cycles: 56. Antibodies for Histone H3K27ac (Active Motif) were used for the immunoprecipitation reactions at 10 μg per reaction. Library preparation and sequencing utilized the TruSeq ChIP Sample Prep Kit and NextSeq 500 High Output Kit (75 cycles) according to the manufacturer's instruction (Illumina). The ChIP-IT qPCR Analysis Kit validated results using primers for CD19 and PAX5 (Active Motif).Chromatin was prepared from 15 × 10Pax5+/Ebf1+ specific, Pax5−/Ebf1− specific, or single lineage-specific clusters. Each set of enhancer peaks were analysed for motif enrichment with the HOMER package scanning sequence for known motifs within 500 bp of either side of the peak centre.H3K27ac peaks >2,500 bp from the nearest TSS in mouse genome build mm9 were classified as enhancers. Using Bedtools intersect, we identified peaks found in naive and relapsed cell types that were classified as either lineage un-specific, and http://crispr.mit.edu/, cloned into LentiCRISPR v2 plasmid , and finally transformed as previously published. Plasmids were co-transfected with packaging plasmids RRE, pMD-G and REV were transfected into HEK293T cells. After 2 days, CRISPR supernatants were harvested and filtered through a 0.45 μm low protein binding membrane (Millipore) and concentrated using Lenti-X concentrator (Clontech), resuspended in PBS and used immediately or stored at −80 °C. For viral transduction, 105 leukaemia cells were incubated with 10 μl of concentrated viral supernatant for 2 days, followed by expansion in CMM. Cell phenotype was assessed by flow cytometry, followed by sorting of cells with phenotypic alterations and single-cell cloning. Sequencing was performed on single-cell clones to confirm genotypic alterations.Guide-RNAs  were optn<30 per group). Statistical analysis was performed using GraphPad Prism version 6 for Macintosh (Graphpad Software). P values <0.05 were considered significant.Survival of mice was analysed through the Kaplan–Meier method, using Wilcoxon rank test. For continuous variables, the Mann–Whitney method was used to compare sample groups (www.ncbi.nlm.nih.gov/geo (accession numbers GSE83205 and GSE83203).The data reported in this study have been deposited in the Gene Expression Omnibus (GEO) database, How to cite this article: Jacoby, E. et al. CD19 CAR immune pressure induces B-precursor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity. Nat. Commun. 7:12320 doi: 10.1038/ncomms12320 (2016).Supplementary Figures 1-10 and Supplementary Tables 1 & 2.


Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3′UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage.In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a  Drosophila spermatogenesis provides an ideal research model to unravel the genetic network coordinating proliferation and differentiation. In a genetic screen, we identified a male-sterile mutant whose germ cells are under-differentiated and overproliferating. The responsible gene encodes an RNA-binding protein whose target belongs to a tumor suppressor family. We demonstrate that this and two other RNA-binding proteins form a physical and functional unit to ensure the proper differentiation and accurate proliferation of germline stem cell lineage.In regenerative tissues, the successive differentiation of stem cell lineage is well controlled and coordinated with proper cell proliferation at each differentiation stage. Disruption of the control mechanism can lead to tumor growth or tissue degeneration. The germline stem cell lineage of  Adult stem cells divide to replenish differentiated, dead, or damaged cells in regenerative tissues. To produce sufficient number of differentiated progeny for tissue homeostasis and to avoid the accumulation of oncogenic mutations derived from frequent cell divisions, stem cell daughters undergo multiple rounds of transit-amplifying (TA) divisions prior to terminal differentiation Drosophila spermatogenesis is a highly stereotyped and accessible system to study the control mechanisms of accurate TA divisions in adult stem cell lineage. At the apical tip of testis, germline stem cells (GSCs) and somatic cyst stem cells form a rosette surrounding the hub  is at the center of the picture not only because it was the first player identified in this process, also because in germ cells Bam protein accumulation signals the stop of TA division and/or the start of further differentiation In the past decades, many intrinsic and extrinsic factors regulating TA divisions have been found in Drosophila S2 cells, Bam and Bgcn repress the expression of a reporter coupled with the 3′ untranslated region (3′UTR) of DE-Cadherinnos 3′UTR mei-P26 3′UTR directly to repress Mei-P26, whose initial expression in early TA cells is required for Bam accumulation bam or bgcn mutant phenotype Benign gonial cell neoplasm (Bgcn) is an ‘intimate’ partner of Bam, given that they have the same mutant phenotype  mei-P26, bam, or bgcn. This mutation disrupts CG32364 which we named tumorous testis (tut). We found by genetic and biochemical methods that Tut, Bam, and Bgcn act in a complex to accurately coordinate TA division and differentiation of germline stem cell daughters.From a mutagenesis screen combined with germline clonal analysis, we isolated a mutant showing spermatogonial over-proliferation, a phenotype similar to that of CG32364, and designated it as tumorous testis (tut). tut encodes a protein containing a putative RNA recognition motif (RRM). The EMS-induced point mutation in 1tut generates a premature stop codon . Compared to the wild-type , from which both long and short isoforms were detected when the reporter was expressed (detected by 3′RACE specific for the reporter). The reporter expression driven by bam-Gal4 was repressed in ∼80% of Bam-positive cysts in the presence of mei-P26 3′UTR  did not show any spermatogonial tumor growth . Consistently, removing one copy of bgcn dramatically enhanced tut knockdown phenotype  in tut mutant v.s. wild-type background. As expected, ectopic expression of Bam in GSCs eliminated all germ cells , Zip-GFPdj-GFPHrb98DE-GFPnos-Gal4 and UAS-Flp were ordered from Bloomington Drosophila Stock Center; UAS-tutRNAi (v26044) was ordered from Vienna Drosophila RNAi Center. 2bgcn was generated in our lab and contains the deletion of TGACG in the 2nd intron of the gene.The fly strains used: 4tut homozygous and bam heterozygous phenotype varies at different temperatures, age, or nutritions. For 4tut, 2bgcn, and bam/+ related experiments, flies were cultured at 24°C, fed with fresh yeast daily, and dissected within 12 hours after eclosure.Fly stocks were maintained under standard culture conditions and all flies were dissected 0–2 days after eclosure unless otherwise indicated. For RNAi experiments, flies were cultured at 25°C for 6 days and transferred to 29°C for another 6 days before dissection. For germline clonal analysis, flies were heat-shocked in 37°C water bath for 1 hour at late pupal stage and dissected 4–5 days after clone induction. FRT and UAS-Flp were fed with EMS overnight. The progeny of EMS-treated flies were crossed to flies carrying FRT-GFP and nos-Gal4. Their male offspring were dissected and stained with DAPI 1tut was one of the mutants with germline over-growth phenotype, and was mapped by deficiency screen and candidate gene sequencing.Isogenized flies bearing 1tut bears a point mutation (3L: G8203128A) that creates a new stop codon. Wild-type Tut protein is 230 amino acids in length, and 1tut is expected to produce only the N-terminal 174 amino acids. 3tut and 4tut alleles were generated by genomic engineering tut was replaced with attP and loxP, generating the null allele 3tut. tut genomic region was introduced back into the tut locus of in 3tut background via attP-attB incorporation. However, after this manipulation, 91 bp (attR and vector sequence) sequences were inserted upstream of tut start codon and 68 bp (loxP and vector sequence) downstream of stop codon, generating the weak allele 4tut.1118w and p51D stocks were chosen as the hosts for P-element and attB-attP mediated transgenesis, respectively tutP-tut-GFP contains 1.5 kb sequence upstream of tut start codon, tut genomic region (introns included and stop codon removed) tagged with GFP at its C-terminus, and 2.2 kb sequence downstream of tut stop codon. tutP-tut-TAP and tutP-GFP contain the same regulatory sequences as tutP-tut-GFPUAS-GFP-mei-P26 3′UTR was generated by cloning GFP coding sequence and mei-P26 3′UTR (2 kb downstream of stop codon) to replace the SV40 element in pUAST vector.The E. coli), rat anti-Vasa .Fly testes were prepared and immunostained as previously described act-Gal4 construct was co-transfected with pUAST expression vectors for all transfection experiments except for pAFMW-bgcn. 48 hours after transfection, cells were lysed in Default Lysis Buffer  for 30 minutes on ice. Then the supernatants were incubated with corresponding beads for 4 hours at 4°C. The beads were washed 4 times with washing buffer , followed by Western analyses. To assess the RNA dependence of protein-protein interaction, S2 cell lysates were incubated with 0.5 µg/uL RNaseA for 30 min at room temperature S2 cells were cultured in SFM serum free medium . Transfection was performed using Cellfectin Reagent  according to the manufacturer's instructions. An The beads used in co-immunoprecipitation: IgG-beads , anti-Flag-beads , anti-Myc-beads , and anti-GFP-beads . The primary antibodies used in Western analyses: mouse anti-Flag , mouse anti-GFP , mouse anti-HA , mouse anti-Myc , mouse anti-SBP , rabbit anti-Vasa (1∶8000), mouse anti-Bam .Two-step co-immunoprecipitation was performed according to the procedures described previously mei-P26 3′UTR (549 bp and 844 bp from stop codon) was cloned into pUAS-GFP vector between GFP stop codon and SV40 3′UTR. S2 cells transfected with TAP-Tut and mei-P26 3′UTR or TAP-GFP and mei-P26 3′UTR were lysed in polysome lysis buffer according to 5′-CGTTGATAGGGGACTATACA, 5′-TTTGTTGCATTTTGTTTATC, 5′-TCAAGTCGCATTCAACGCAT, 5′-TTTTTTTAGTAGTAGCGCTAATTG) complementary to mei-P26 3′UTR, and quantified by real-time PCR with primers 5′-TCTTGGCAAGGAGTCAACAC and 5′-CTGTCGATGAGGCAAATGTT. Oligo-dT primer was used for reverse transcription to examine the actin5C mRNA bound to TAP-Tut or TAP-GFP.RNase inhibitor (Takara 2313A) was used for all RIP-related experiments. Yeasts were cultured on SD/-Ade/-His/-Leu/-Trp medium supplemented with Aureobasidin A and X-α-Gal (QDO/X/A) to test protein-protein interactions or on SD/-Leu/-Trp medium (DDO) to confirm the transformation of testing plasmid DNA.Yeasts were cultured on SD/-His/-Leu/-Ura medium supplemented with X-β-Gal (TDO/X) to test protein-RNA interactions or on SD/-Leu/-Ura medium (DDO) to confirm the transformation of testing plasmid DNA 1118w testes and 3′RACE was performed by following the manufacturer's instructions . Outer primer (5′-TCCGAGGGCTATGTGGTTAC-3′) and inner primer (5′-GTTCTAGTCCTGAACACCCT-3′) were used to amplify mei-P26 3′UTR. PCR products were loaded into 2% argarose gel and electrophoresed at 100 V for 1.5 h on ice.Total RNA was extracted using TRIzol  from 10 pairs of Figure S1tut acts in germline to restrict spermatogonial proliferation. (A) Blue and purple bars indicate the fragments of tut gene selected for hairpin constructs in UAS-tut-RNAi and for qPCR of tut mRNA, respectively. Deficiency stock (Bloomington 24400) was designated as dftut. (B) Relative tut mRNA level determined by real-time PCR, normalized to rp49, and presented as fold changes relative to 3tut. Error bars indicate SD. (C–D) 3 Hrb98DE-GFP/+tut (C) and 3 Hrb98DE-GFP/tutdftut (D) testes stained for GFP, germline marker Vasa, and DNA (DAPI). Yellow arrowheads point to cyst cells (Vasa negative) expressing Hrb98DE-GFP. Cyan arrowheads point to spermatocytes  expressing Hrb98DE-GFP. (E–F) 3 dj-GFP/+tut (E) and 3 dj-GFP/tutdftut (F) testes. Dj-GFP labels spermatid bundle (E), which is absent in tut mutant testis (F). (G–I) DAPI staining of bam-Gal4/Y; UAS-dcr2/+ (G), bam-Gal4/Y;UAS-tut-RNAi/UAS-dcr2 , and UAS-tut-RNAi/tj-Gal4; UAS-dcr2/+ . (J–K) bamP-GFP/Y (J) and 1bamP-GFP/Y; tut (K) testes stained for GFP, FasIII, and DNA (DAPI). (L–M) esgP-lacZ/+ (L) and 1esgP-lacZ/+; tut (M) mutant testes stained for LacZ, Vasa, and FasIII. (N–O) Immunofluorescence images of 1118w (N) and 1tut (O) testes. (P–Q) 3/+tut (P) and 3tut (Q) ovarioles stained for Vasa, α-Spectrin, and DNA (DAPI). Scale bars: 25 µm; 200 µm (E–I); and 50 µm (J–O).(Related to (TIF)Click here for additional data file.Figure S2mei-P26 3′UTR. (A) Schematic illustration of mei-P26 3′region. Blue box and grey arrow represent the last exon and the 3′region of mei-P26, respectively. The 3′ end of long (844 nt) and short (549 nt) isoforms of mei-P26 3′UTR are indicated by blue and magenta arrows. Red arrow indicates the fragment (2 k nt in length) selected for mei-P26 3′UTR reporter. (B) 3′RACE of mei-P26 3′UTR from 1118w (wt), tut, bam, bgcn mutant testes. The 844 bp (purple arrow) and 549 bp (blue arrow) bands were determined by sequencing. (C) 3′RACE of mei-P26 3′UTR from 1118w testes. PCR products were loaded into 2% agarose gel and electrophoresed at 100 V for 1.5 h on ice. (D) Schematic drawings of the full length Tut protein and the construct deleted of RRM. (E–F) Yeast 3-hybrid assay. The combination of AD-IRP&IRE-MES or AD-IRP&M3US-MS2 served as positive or negative control, respectively. M3US or M3UL symbolizes the short or the long isoform of mei-P26 3′UTR, respectively. TDR is the construct described in D. For higher stringency assay, yeasts were cultured on SD/-His/-Leu/-Ura medium supplemented with X-β-Gal (TDO/X). For lower stringency assay, yeasts were cultured on SD/-Leu/-Ura medium, transferred to filter paper, permeabilized and soaked in solution containing X-β-Gal (DDO/X).(Related to (TIF)Click here for additional data file.Figure S3mei-P26 expression via mei-P26 3′UTR. (A–C) The expression pattern of bam-Gal4 in different mutant testes. (D–D′) A 3/tutdfbgcnP-bgcn-GFP tut testis stained for GFP, 1B1, and DNA (blue). Bgcn was expressed in tut mutant germ cells. (E&E′–F&F′) Bam is required for the full expression of Tut-GFP. (G–I) Immunostaining of Mei-P26 in different genetic background. All images were scanned at the same confocal settings. The signal in mfs1mei-P26 mutant served as a negative control. (J–J″) Genotype: QS2/bgcn20093;UAS-GFP-meiP26-3′UTR (2k)/+bam-Gal4/Y;bgcn. Yellow dots outline Bam-expressing spermatogonia. GFP was de-repressed in bgcn mutant even though Bam was expressed. Scale bars: 25 µm  and 5 µm(G–I).(Related to (TIF)Click here for additional data file.Figure S4tut and bam. (A) Schematic showing the regulatory sequences for tut expression in tutP-tut-GFP and tutP-tut-TAP constructs. (B–C) DAPI-stained testes of wild-type appearance (B) or with spermatogonial tumor (C). (D) Genetic interaction between tut and bam. Y-axis: tumor rate . Green bar represents the portion of normal testes while the red bar represents the portion of testes with spermatogonial tumors. (E–F) 1bam-Gal4/Y; UASp-bam-GFP/+; tut (D) and BGbam-Gal4/Y; UAS-Flag-tut/+; bam (E) testes stained with DAPI. (G) Yeast 2-hybrid test of Bam and Tut. Yeasts were cultured on SD/-Ade/-His/-Leu/-Trp medium supplemented with Aureobasidin A and X-α-Gal (QDO/X/A) or SD/-Leu/-Trp medium (DDO). (H–H″) Localization of Myc-Tut and Flag-Bam in transfected S2 cells. Scale bars: 200 µm (B–C); 100 µm (E–F); 5 µm (H).(Related to (TIF)Click here for additional data file.Figure S5tut, bam, and bgcn. (A–C) Genetic interaction tests between tut and bgcn. (A–B) Representative DAPI-staining images showing testes with normal appearance (A) and with over-proliferating cysts (B). (C) Bar chart showing tumor rate. Dicer2 was not included in this experiment. (D) Testis extracts from 1118w and bamP-bam-HA/+; bgcnP-bgcn-GFP/+ flies were immunoprecipitated with anti-GFP beads. Western blots were performed with anti-HA and anti-GFP antibodies to analyze the presence of Bam-HA and Bgcn-GFP, respectively. (E–F) Genetic interaction between bam and bgcn. DAPI staining is shown. (G–G″) QS2/+; tut4bgcn testis stained for 1B1 (red), Vasa (green), and DAPI (blue). Note the branched fusome. (H–H′″) A tutP-tutTAP/Y; tutP-tutTAP/+;bamP-bgcnGFP/+ testis stained for TutTAP, Bam, and BgcnGFP. Arrowhead points to the cell focused for this confocal scan. Scale bars: 50 µm (A–B); 200 µm (E–F); 25 µm (G–H).(Related to (TIF)Click here for additional data file.Figure S6(Related to (TIF)Click here for additional data file.Figure S720093 UASp-bam-GFP/bgcnQS2; nos-Gal4/+bgcn testis. Yellow arrowhead points to the hub-adjacent germ cell expressing Bam-GFP. (B–B″) Immunofluorescence images of 20093 UASp-bam-GFP/bgcnQS2; nos-Gal4/+bgcn testis. Yellow arrowhead points to the hub-adjacent germ cell expressing Bam-GFP and containing the dot-shape spectrosome. (C–C″) Immunofluorescence images of 20093 UASp-bam-GFP/bgcnQS2; nos-Gal4/+bgcn testis. Yellow arrowhead points to the hub-adjacent germ cell expressing both Stat92E and Bam-GFP. (D–D′) 10 day old 20093 UASp-bam-GFP/bgcnQS2; nos-Gal4/+bgcn testis labeled with BrdU for 1 hour. Yellow arrowhead points to the hub-adjacent germ cells expressing Bam-GFP and positive for BrdU. (E) 10 day old 20093 UASp-bam-GFP/bgcnQS2; nos-Gal4/+bgcn testis. Yellow arrowhead points to the hub-adjacent germ cells expressing Bam-GFP and positive for pH 3. Scale bars: 25 µm.(Related to (TIF)Click here for additional data file.Figure S8A model depicting the relationship between Tut-Bam-Bgcn complex formation and germ cell differentiation. (A) Dynamic expression patterns of Tut, Bam, Bgcn, and their target Mei-P26 correspond to the different state of germline differentiation. (B) Germline differentiation is blocked at TA stage in the absence of Tut. (C) Ectopic expression of Bam in GSCs drives all GSCs to differentiate and leads to GSC loss. Such function of Bam requires the activities of Tut and Bgcn. See more details in (TIF)Click here for additional data file.


In vivo siRNA-AEG-1 administration to rats implanted with C6 glioma cells inhibited tumor growth and increased the incidence of apoptosis among tumor cells. These findings shed light on the mechanisms underlying the invasiveness and progression of malignant gliomas.Autophagy is a tightly regulated process activated in response to metabolic stress and other microenvironmental changes. Astrocyte elevated gene 1 (AEG-1) reportedly induces protective autophagy. Our results indicate that AEG-1 also enhances the susceptibility of malignant glioma cells to TGF-β1-triggered epithelial-mesenchymal transition (EMT) through induction of autophagy. TGF-β1 induced autophagy and activated AEG-1 via Smad2/3 phosphorylation in malignant glioma cells. Also increased was oncogene cyclin D1 and EMT markers, which promoted tumor progression. Inhibition of autophagy using siRNA-BECN1 and siRNA-AEG-1 suppressed EMT. In tumor samples from patients with malignant glioma, immunohistochemical assays showed that expression levels of TGF-β1, AEG-1, and markers of autophagy and EMT, all gradually increase with glioblastoma progression.  Malignant glioma originates in the cerebral glia of brain tissue. No obvious boundary between glioma and brain tissue exists due to its invasive growth . RadiothAEG-1 has been first identified in research on Alzheimer's disease or migraines caused by HIV virus and then found at high expression levels in a variety of tumors, participating in tumor occurrence and development –5. AEG-1Tumor cells must adapt to different survival pressures during the complex and multistep biological process of tumor invasion and progression. Autophagy (here referring to macroautophagy) is a highly conserved process of cell self-eating . One of EMT refers to a transformation whereby epithelial cells with polarity become freely moving mesenchymal cells in the intercellular space under specific physiological and pathological situations . EMT hasTransforming growth factor beta (TGF-β) super-family is polypeptide cytokines that are extensively involved in the regulation of cell biological processes such as proliferation, differentiation, invasion and migration . TGF-β1 However, the correlation between autophagy activation and EMT mechanisms of TGF-β1 has not been elucidated. Furthermore, AEG-1 and EMT have not been studied in the context of glioma. Therefore, our objective is to study the mechanism underlying the variation in the tumor environment and the consequent progression of malignant glioma. We hypothesized that AEG-1 might act beneficially during autophagy activation. This hypothesis has been validated by our experiments. Our results demonstrated that autophagy flux is activated within 3 hours and accompanied by the up-regulation of AEG-1 in malignant glioma cells following TGF-β1 (5 ng/ml) treatment for 1, 3, 6, 12 and 24 hours. The expression of the oncogene cyclin D1 and EMT markers increased with AEG-1, suggesting that this biological process may promote tumor progression. Autophagy inhibition by siBECN1 and siAEG-1 pretreatment reversed the expression levels of the above markers. Thus, our data support the hypothesis that AEG-1 that stimulated glioma cell autophagy and enhanced EMT and progression of gliomas.TGF-β1 has been reported to be the most important regulator of EMT and cell migration in human lung tumor cells . In our To identify these effects at the molecular level, western blot analyses are performed to determine the expression of AEG-1/MTDH and EMT hallmarks. First, the expression of AEG-1 and its downstream cyclin D1 are increased in both U251 and U87 malignant glioma cells by treatment with TGF-β1 (5 ng/ml) for 1, 3, 6, 12 and 24 hours Figure . The expNext, we determine whether TGF-β1 could induce autophagy during EMT in malignant glioma cells. The conversion of the soluble form of MAP-LC3 (LC3-I) to the lipidated and autophagosome-associated form (LC3-II) is considered as one of the hallmarks of autophagy ; thus, wTo explore whether BECN1 is involved in TGF-β1-induced malignant glioma cell autophagy, we examine the effect of siRNA BECN1 on malignant glioma cell autophagy. We observe a decrease in the occurrence of GFP-LC3-positive dots, as well as the down-regulation of LC3-II and LAMP-1 in TGF-β1-stimulated cells Figure . BecauseAs a gold standard to gain insight into the morphological changes induced in malignant glioma cells upon TGF-β1 administration, we perform transmission electron microscopy analysis of U87 cells. Notably, TEM images reveal an accumulation of numerous large autophagy-related structures in the cytoplasm of TGF-β1-treated cells Figure  comparedTransmission electron microscopy analysis shows the morphological changes in U87 cells induced by AEG-1/MTDH overexpression. Double-membrane vacuolar structures with morphological features of autophagosomes are observed in pcDNA3.1-AEG-1/MTDH-transfected cells compared to pcDNA3.1-transfected cells Figure . Then, wTo confirm the contribution of AEG-1 in the TGF-β1-induced autophagy and EMT, we further study the effect of siRNA AEG-1/MTDH on malignant glioma cells. In this study, we use chemically modified AEG-1/MTDH siRNA oligonucleotides for the suppression of direct sites. The inhibition of autophagy by siRNA AEG-1/MTDH weakens autophagy and EMT Figure . MAP-LC3To further confirm that AEG-1 is essential for autophagy, EMT and invasion in response to TGF-β1 treatment, confocal fluorescence microscopy are used to assess the autophagy dynamics. As shown in Figure P < 0.001, Figure P < 0.001, Figure We analyze the expression of TGF-β1, AEG-1, hallmarks of autophagy and EMT in patient-derived brain tumor samples to investigate the relationship between AEG-1-sensitized autophagy, EMT and glioblastoma multiforme development and progression. Biopsies are taken from anaplastic astrocytoma (grade II), oligodendroglioma and oligoastrocytoma (grade III), glioblastoma multiforme (grade IV), and normal brain tissue according to the glioma grading classification of the World Health Organization (WHO). We use immunohistochemical (IHC) staining to evaluate TGF-β1, AEG-1, MAP-LC3, BECN1, E-cadherin and N-cadherin expression in formalin-fixed, paraffin-embedded sections of normal brain and brain tumors with glioma grading. As shown in Figure in vivo, C6 glioma cells are first transfected with modified siRNA-AED-1/MTDH and implanted into rat striatum. Glioma-bearing rat are intracranially injected with 2 nM modified AED-1/MTDH siRNA-1 and AED-1/MTDH siRNA-2 seven days after initial tumor cell injection once every other day for two weeks (n = 5 per group). The H & E staining results reveal that the glioma-bearing rats treated with AED-1/MTDH siRNA exhibit better tumor margins and fewer invasive cells to the contralateral striatum compared with the untreated control rats  AEG-1 participates in TGF-β1-triggered EMT and invasion in malignant glioma cells; (b) autophagy is enhanced by AEG-1 over-expression involved in TGF-β1 treatment; (c) autophagy mediates AEG-1-sensitized EMT and invasion; (d) siRNA AEG-1 prevented TGF-β1-induced autophagy and EMT; (e) the coordination of AEG-1 expression and autophagy and EMT activation is associated with the development of anaplastic astrocytoma and glioblastoma; (f) systemically delivered siRNA AEG-1/MTDH reduces tumor volume and induced apoptotic cell death in the rat C6 glioma model. Due to the change of microenvironment and pathways by TGF-β1 activation, the level of AEG-1 increases, causing activation of autophagy. Autophagy promotes EMT via degradation and utilization of intracellular macromolecules and organelles including epithelial markers. This triggers the development of malignant glioma invasion o Figure .Both the loss of the epithelial phenotype and the acquisition of mesenchymal characteristics are the primary features of EMT . TransfoThe typical progression of autophagy includes the following aspects , 23, 24:AEG-1 is considered as a cancer promoter and stimulator of apoptosis , autophain vitro. In summary, our results show that the up-regulation of AEG-1 by TGF-β1 activates protective autophagy and promotes EMT to promote malignant glioma cell proliferation, invasion and metastasis. Moreover, down-regulating the AEG-1 level in malignant glioma by pharmacological inhibition or genetic interference may contribute to the prevention and reduction of glioma invasion and progression. Our present studies are focusing on the relationship between autophagy and EMT. The selective autophagy receptor and signaling adaptor sequestosome-1 (SQSTM1/p62) is a multifunctional protein. The presence of an LC3-interacting region (LIR) enables SQSTM1/p62 to bind to the autophagy protein LC3, and the ubiquitin-associated domain (UBA) binds to ubiquitin to mediate the selective degradation of ubiquitinated cargo by autophagy .Animals are deeply anesthetized and perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains are removed from the skulls and post-fixed overnight at 4°C in 4% paraformaldehyde. Next day, the brains are transferred to 30% sucrose in PBS solution for 48 h at 4°C. Coronal sections with a thickness of 10 mm are cut using a cryostat microtome . Hematoxylin and eosin (H & E) staining is used to visualize the tumor area and tumor necrosis. For evaluating cell proliferation, immunostaining for proliferating cell nuclear antigen (PCNA) is used. Antibody against PCNA (Abcam) is diluted 1: 300 in blocking solution containing 0.3% Triton X-100 and incubated overnight at 4°C. After being washed in PBS, the sections are incubated with a biotinylated secondary antibody for 2 h. They are washed and further incubated with a streptavidin-biotin-peroxidase complex (Vector Laboratories). Apoptosis is examined using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method . 3-methyladenine  is diluted to 5 mM. Cholesterol-conjugated siRNA with chemical modification of 2′-O-Me for in vivo RNA interference and its negative control are from GenePharma, Inc. Recombinant human transforming growth factor β1  is applied to induce malignant glioma cells to undergo EMT. This reagent is dissolved in triple-distilled water at a concentration 100 μg/ml and stored at −80°C. A stock solution is diluted to the appropriate concentrations with growth medium immediately before use. Lipofectamine™ 800-conjugated anti-mouse IgG ; (2) anti-rabbit IgG: Alexa Fluor 680 goat anti-rabbit IgG ; (3) anti-goat IgG: Alexa Fluor 680 rabbit anti-goat IgG ; (4) goat anti-mouse IgG HRP ; (5) goat anti-rabbit IgG HRP .Primary antibodies directed against AEG-1/lyric/metadherin (CST-9596), MAP-LC3 (CST-12741), TGF-β1 (CST-3711), LAMP-1 (CST-9091) and BECN1 (CST-4122) are purchased from Cell Signaling Technology, Inc. . Primary antibodies directed against E-cadherin (BS-1097), N-cadherin (BS-2224), vimentin (BS-1855), mTOR (BS-3611) and p-mTOR (BS-4706) are purchased from Bioworld Technology Co., Ltd. Primary antibody directed against β-actin (BM0627) is purchased from Boster Biological Technology, Ltd. Primary antibodies directed against AEG-1/MTDH (13860-1-AP) Snail (26183-1-AP), Twist1 (25465-1-AP), Slug (12129-1-AP) are purchased from Proteintech Technology, Inc. . Primary antibodies against Smad2/3 (sc-6202), p-Smad2/3 (Ser 423/425) (sc-11769) and Smad2/3 siRNA (sc-37238) are purchased from Santa Cruz Technology, Ltd. . The secondary antibodies used are as follows: (1) anti-mouse IgG: IRDye™ 2000  according to the manufacturer's recommendations. The expression vector is transfected 24 hours before treatment with TGF-β1.The pcDNA3.1-GFP-LC3 fusion plasmid is kindly provided by Dr. Rong Mu . The pcDNA3.1-AEG-1/MTDH plasmid is kindly provided by Dr. Kunmei Liu . For transfection, the cells are trypsinized and seeded in 6-well plates at a density of 5000 cells/well. Plasmids are introduced at 2 μg/well or 1 μg/well into the cells using Lipofectamine™ 2000  according to the manufacturer's instructions. Briefly, the cells that reached 50% confluence are transfected with serum-free DMEM medium containing 50 nmol/l scrambled siRNA, AEG-1, or BECN1 siRNA for 6 hours, followed by recovery in medium containing serum and other special treatments as necessary.The human AEG-1/MTDH siRNA oligonucleotides , Beclin-1 (BECN1) siRNA oligonucleotides  and the rat AEG-1/MTDH siRNA oligonucleotides  are synthesized by GenePharma, Inc. . A universal negative control siRNA is used. We perform siRNA transfection using Lipofectaminein vitro   is used for co-localization analysis .U87 cells are pretreated with TGF-β1 (5 ng/ml) for 24 hours or transfected with pcDNA3.1/pcDNA3. 1-AEG-1/MTDH for 24 hours. The cells are fixed immediately in 1% glutaraldehyde and post-fixed in 2% osmium tetroxide. Then, the cell pellets or sections are embedded in epon resin. Representative areas are chosen for ultrathin sectioning and viewed using a FEI Tecnai G2 Spirit Bio TWIN transmission electron microscope  at an acceleration voltage of 120 kV.IHC staining of tumor tissue using AEG-1/MTDH-specific rabbit polyclonal antibody (#13860-1-AP), MAP-LC3 (CST-12741), TGF-β1 (CST-3711), E-cadherin (BS-1097) and N-cadherin (BS-2224) antibodies is performed as described previously . BrieflyP < 0.05, #P < 0.05 and **P < 0.01, ##P < 0.01.All data are expressed as mean ± SD and statistically compared by one-way ANOVA with Dunnett's test and post-hoc tests are undertaken using the GraphPad Prism 5.0 software. The details of each statistical analysis used are presented in the figure legends. Significance is indicated as *


Glioblastoma (GBM) is a poorly immunogenic neoplasm treated with local radiation. Despite the standard of care, median survival remains low. Immunotherapy has synergized with stereotactic radiosurgery (SRS) in murine GBM, as radiation promotes a pro-inflammatory tumor microenvironment amenable to the anti-tumor effects of immune modulation. Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a co-stimulatory receptor expressed constitutively on regulatory T cells and inducibly on effector T cells. We tested the hypothesis that anti-GITR monoclonal antibody (mAb) and SRS combination therapy would confer immune-mediated survival benefit in murine glioma.Mice were implanted with GL261-luc murine glioma cells and began SRS and anti-GITR IgG1 treatment after 10 days. Mice were randomized to four treatment groups: control, SRS only, anti-GITR only, anti-GITR+SRS. SRS was delivered to the tumor in one fraction; mice were given mAb thrice i.p. Mice were euthanized on day 21 to analyze the immunologic profile of tumor, spleen, and tumor draining lymph nodes.Anti-GITR mAb plus SRS conferred significantly improved survival over either treatment alone . The increased survival required CD4+T cells but not CD8+T cells or regulatory T cells (Tregs). There was elevated intratumoral CD4+ effector-cell infiltration (CD4+/Foxp3-/IFNγ+) relative to Treg infiltration (CD4+/Foxp3+) at day 21 in mice treated with anti-GITR+SRS, and significantly elevated IFNγ and IL-2 production by CD4+T cells and elevated IFNγ and TNFα production by CD8+T cells. Intratumoral mononuclear cells demonstrated increased mRNA expression of pro-inflammatory M1 markers and decreased expression of immunosuppressive M2 markers.In all, anti-GITR mAb synergizes with SRS to significantly prolong survival in murine orthotopic glioma in a potentially CD4+ Th1-dominant anti-tumor mechanism with M1 polarization. These findings provide preclinical evidence for the use of anti-GITR IgG1 non-depleting antibodies alongside SRS in human GBM.


Helicoverpa armigera SNPV-AC53 (KJ909666).Wild-type baculovirus isolates typically consist of multiple strains. We report the full genome sequences of seven alphabaculovirus strains derived by passage through tissue culture from  Helicoverpa armigera were infected with HaSNPV-AC53. Strains were isolated from hemolymph of infected larvae either by plaque purification in HzAM1 cells, one passage through tissue culture, and one passage through larvae (“C-strains”), or by passage through tissue culture cells and then one passage of occlusion bodies produced through larvae, followed by plaque purification, passage in cell culture, and one passage through larvae as above (“T-strains”). Viral DNA was extracted from occlusion bodies using a Bioline Isolate II Genomic DNA kit  following published methods  and the Genome Analysis Toolkit version 3.4-46 (–k-mer size of the mapped data and then assembled de novo using Tadpole (BBMap 35.59 package)  were merged into a single fasta file and mapped against the HaSNPV-AC53 reference using the Geneious R9 mapper with medium-low sensitivity and 5× iterations  and sequenced using the Illumina NextSeq 500 with 150-bp paired-end reads. Trimming was completed using the FASTX-Toolkit version 0.0.13 . An eighn 3.4-46 –13. The  3.4-46 – and loadpackage) . The maperations . The finThe HaSNPV-AC53 sequence produced had 100% sequence identity to the published HaSNPV-AC53 reference sequenced on the Ion Torrent PGM . One strKU738896, KU738897, KU738898, KU738899, KU738900, KU738901, and KU738904, respectively.The complete sequences of HaSNPV-AC53C1, HaSNPV-AC53C3, HaSNPV-AC53C5, HaSNPV-AC53C6, HaSNPV-AC53C9, HaSNPV-AC53T2, and HaSNPV-AC53T5 were deposited to GenBank under the accession numbers


Plasmodium chabaudi in mice. Mice deficient either in IL-21 or the IL-21 receptor fail to resolve the chronic phase of P. chabaudi infection and P. yoelii infection resulting in sustained high parasitemias, and are not immune to re-infection. This is associated with abrogated P. chabaudi-specific IgG responses, including memory B cells. Mixed bone marrow chimeric mice, with T cells carrying a targeted disruption of the Il21 gene, or B cells with a targeted disruption of the Il21r gene, demonstrate that IL-21 from T cells signaling through the IL-21 receptor on B cells is necessary to control chronic P. chabaudi infection. Our data uncover a mechanism by which CD4+ T cells and B cells control parasitemia during chronic erythrocytic-stage malaria through a single gene, Il21, and demonstrate the importance of this cytokine in the control of pathogens by humoral immune responses. These data are highly pertinent for designing malaria vaccines requiring long-lasting protective B-cell responses.Interleukin-21 signaling is important for germinal center B-cell responses, isotype switching and generation of memory B cells. However, a role for IL-21 in antibody-mediated protection against pathogens has not been demonstrated. Here we show that IL-21 is produced by T follicular helper cells and co-expressed with IFN-γ during an erythrocytic-stage malaria infection of  Plasmodium, was first described when immune serum, passively transferred into Plasmodium falciparum-infected children, reduced parasitemia. This was later confirmed in experimental models in which mice deficient in B cells were unable to eliminate erythrocytic-stage infections. The signals required to activate these protective long-lasting B cell responses towards Plasmodium have not been investigated. IL-21 has been shown to be important for development of B-cell responses after immunization; however, a direct requirement for IL-21 in the control of infection via B-cell dependent mechanisms has never been demonstrated. In this paper, we have used mouse models of erythrocytic P. chabaudi and P. yoelii 17X(NL) infections in combination with IL-21/IL-21R deficiency to show that IL-21 from CD4+ T cells is required to eliminate Plasmodium infection by activating protective, long-lasting B-cell responses. Disruption of IL-21 signaling in B cells prevents the elimination of the parasite resulting in sustained high parasitemias, with no development of memory B-cells, lack of antigen-specific plasma cells and antibodies, and thus no protective immunity against a second challenge infection. Our data demonstrate the absolute requirement of IL-21 for B-cell control of this systemic infection. This has important implications for the design of vaccines against Plasmodium.The importance of antibody and B-cell responses for control of the erythrocytic-stage of the malaria parasite,  Malaria is the leading parasitic cause of morbidity and mortality worldwide; about half of the world's population is at risk of infection . There iPlasmodium falciparum transmission, there are associations between Plasmodium-specific antibody responses and protection against infection  source and reconstituted less than 24hr after irradiation by i.v. injections of different combinations of donor BM cells (The BM cells . Recipie5P. chabaudi chabaudi AS–infected or 103P. yoelii 17X(NL)-infected rbc by i.p. injections, and parasitemias monitored by Giemsa-stained blood films, as previously described  and viable cells were counted using trypan blue exclusion (Sigma) and a hemocytometer. Cells were then resuspended in PBS containing 2% FBS, 0.1% N2  to bloc2 Fragment Goat Anti-Human IgG (Jackson ImmunoResearch). For the study of cytokines co-expressed with IL-21, cells were further stained with PE/Cy7 anti-IFN-γ, FITC anti-IL-10, alexa fluor 647 anti-IL-4  or FITC anti-IFN-γ (eBioscience). Intranuclear staining of Bcl-6 was performed using PE anti-Bcl-6 (eBioscience) and the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), following manufacturer’s manual. Cells were acquired on CyAn ADP (Beckman Coulter), BD FACSVerse, BD LSRII or BD LSRFortessa X-20 (BD Biosciences) flow cytometers. Dead cells were excluded by staining with LIVE/DEAD Fixable Aqua or Blue stain (Invitrogen). Singlets were selected based on FCS-A vs FCS-W and further based on SSC-A vs SSC-W. “Fluorescence minus one” (FMO) controls, in combination with isotype controls, were used to set the thresholds for positive/negative events. Analysis was performed with FlowJo software version 9.6 or higher (Tree Star).For intracellular cytokine staining, cells were stimulated for 5h in complete IMDM with PMA , Ionomycin , and Brefeldin A , and surface stained as described above. Intracellular staining for IL-21-producing Tfh cells was performed as described –60. Brieex-vivo frequencies of P. chabaudi-specific ASC in BM and MBC in spleen were performed as previously described ; Gene ID: 102641210; Uniprot ID: P01872Tcra T-cell receptor alpha chain C region [Mus musculus]; Gene ID: 21473; Uniprot ID: P01849Rag2 V(D)J recombination-activating protein 2 [Mus musculus]; Gene ID: 19374; UniProt ID: P21784Il21 Interleukin-21 [Mus musculus]; Gene ID: 60505; Uniprot ID: Q9ES17Il21r Interleukin-21 receptor [Mus musculus]; Gene ID: 60505; Uniprot ID: Q9JHX3Bcl6 B cell leukemia/lymphoma 6 [Mus musculus]; Gene ID: 12053; Uniprot ID: P41183CD16 Low affinity immunoglobulin gamma Fc region receptor III [Mus musculus]; Gene ID: 14131; Uniprot ID: P08508CD32 Low affinity immunoglobulin gamma Fc region receptor II [Mus musculus]; Gene ID: 14130; Uniprot ID: P08101CXCR5 chemokine (C-X-C motif) receptor 5 [Mus musculus]; Gene ID: 12145; UniProt ID: Q04683IFNg Interferon gamma [Mus musculus]; Gene ID: 15978; Uniprot ID: P01580Il10 Interleukin-10 [Mus musculus]; Gene ID: 16153; Uniprot ID: P18893TLR7 Toll like receptor 7 [Mus musculus]; Gene ID: 170743; Uniprot ID:Q548J0Il4 Interleukin-4 [Mus musculus]; Gene ID: 16189; Uniprot ID: P07750PD1 Programmed cell death 1 [Mus musculus]; Gene ID: 18566; Uniprot ID: Q02242CD38 antigen [Mus musculus]; Gene ID: 12494; Uniprot ID: P56528CD19 antigen [Mus musculus]; Gene ID: 12478; Uniprot ID: P25918B220 protein tyrosine phosphatase, receptor type, C [Mus musculus]; Gene ID: 19264; Uniprot ID: P06800Ter119 lymphocyte antigen 76 [Mus musculus]; Gene ID: 104231S1 FigP. yoelii 17X(NL) infection in WT C57BL/6 (black circles), Il21-/- (open circles) and Il21r-/- (open squares). Statistical significance was obtained using Mann Whitney U test .Course of primary (A) and secondary (B) (TIF)Click here for additional data file.S2 FigIl21r-/- and WT C57BL/6 mice at day 32 post-infection. (B) Flow cytometry gating strategy applied to identify the different cell populations. Statistical significance was obtained using Mann Whitney U test .(A) Numbers of different cell populations in the spleen of (TIF)Click here for additional data file.S3 Fig(A) Surface staining. (B) Intranuclear staining of Bcl-6.(TIF)Click here for additional data file.S4 Fig+ B cells. (C) Frequencies of CD3+CD4+ T cells. (D) Frequencies of CD3+CD8+ T cells. No significant differences between the experimental groups and their corresponding control groups were obtained using Mann Whitney U test (P≤0.05).Peripheral blood was obtained from all mixed BM chimeric mice used for experiments and analyzed by flow cytometry before infection. (A) Individual examples showing the gating strategy. (B) Frequencies of CD19(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.