B-Raf genetic locus as one of the critical events in melanomagenesis. B-Raf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of B-Raf is tightly regulated and is required for cell growth and survival. B-Raf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of B-Raf mutated melanoma cells are stringently dependent on B-Raf-MEK-ERK activation, the downstream effector targets that are required for oncogenic B-Raf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic B-Raf (B-RafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of B-Raf wild-type melanoma cells, miR-10b silencing decreases B-RafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for B-RafV600E-mediated anchorage independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic B-RafV600E activity in melanoma.Recent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the  B-Raf [V600E is the most common mutation and accounts for nearly 80% of them [V600E cause a sustained activation of ERK signaling pathway in melanoma, it is also critical for the malignant process and is one of the few identified driver mutations essential for melanoma proliferation and survival. The transformation of melanocytes to melanoma by B-RafV600E requires activation of the MEK-ERK kinases cascade with multiple downstream components [V600E mutation remains undefined.Melanoma is the most aggressive of all the skin cancers. B-Raf is a serine/threonine protein kinase that activates the MEK/ERK-signaling pathway. About 25–70% of malignant melanomas harbor gain-of-function mutations in the oncogene B-Raf . Among s of them . Not onlmponents . The mecV600E oncogenic transformation program. In this study, we identified for the first time, significant positive correlation between B-RafV600E mutation and microRNA-10b (miR-10b) expression. Furthermore, we show that miR-10b is a novel downstream effector of B-RafV600E and that B-RafV600E plays a causal role in the induction of miR-10b in melanoma cell lines. Our results suggested that B-Raf V600E increased miR-10b expression by increasing the expression levels of helix-loop-helix transcription factor Twist1. We also show that miR-10b induced by B-RafV600E is able to increase invasive capacity and anchorage independent growth of melanoma cells.MicroRNAs (miRNAs) are small, non-protein coding RNA molecules and they regulate gene expression through a combination of translational repression and mRNA destabilization . Each mi2 and 6 cm2 cell culture plates were from Sarstedt. All the melanoma cell lines were cultured in Dulbecco’s modified Eagle’s medium with 10% FBS and 1% penicillin streptomycin and were grown in a humidified tissue culture incubator at 37°C in 5% CO2. Mel 505 [All cell culture media were from HyClone. Fetal bovine serum (FBS) was from Atlanta Biologicals, and newborn calf serum was from Lonza. 10 cm Mel 505 , sk-mel- Mel 505 , sk-mel- Mel 505  cells we Mel 505  cells wePLX4032 (Vemurafenib) and 4-OHT (4-hydroxy tamoxifen) were purchased from Selleck chemicals and Sigma, respectively. ERK (1:1000), phospho ERK (1:1000), B-Raf (1:1000) primary antibodies and horseradish peroxidase secondary antibodies were from Santa Cruz Biotechnology, Twist1 (1:500) antibody was from R&D systems, and tubulin (1:1000) antibody was from Sigma.Mammalian expression vectors, MDH1-PGK-GFP 2.0-miR-10b and pBABE-puro-miR-10b sponge, were purchased from Addgene (Plasmids were deposited by Weinberg lab) . Melanom4 of sk-mel-28 or M249 cells were plated on these inserts. 700mL of chemo-attractive medium  was added to the lower chambers . After 24 hours of incubation, the insert bottoms were dipped in 1X PBS and stained in Calcein AM reconstituted in DMSO to 1mg/mL- 1ml in 700 ml 1X PBS (BD Biosciences). Digital images were captured on EVOS inverted microscope along with manual cell count and fluorescence reading (485-538nM).The polycarbonate membrane (8μ pore size) of FluoroBlok cell culture inserts (BD Biosciences) was coated with 60 uL of Matrigel (1:26 in serum-free medium) (BD Biosciences) and incubated at 37°C for 2–3 hours. 1x10Chromatin Immunoprecipitations (ChIPs) were performed as described previously . BrieflyRNA extraction was carried out using Qiazol reagent (Qiagen). TaqMan miRNA qRT-PCR was performed according to manufacturer’s instructions (TaqMan). RNU6 or RNU44 were used as the internal control. Briefly, in the reverse transcription (RT) step, cDNA was reverse transcribed from total RNA samples using specific looped miRNA primers from the TaqMan miRNA assays and reagents from the TaqMan. In the PCR step, PCR products were amplified from cDNA samples using the TaqMan miRNA assay together with the TaqMan universal PCR master mix.3 of sk-mel-28 cells or 1x104 of Mel 505 cells or 1x104 of sk-mel-197 cells were plated. Number of colonies formed were stained with MTT and counted under bright field microscope at 4X objective. Cells were plated in triplicates and four fields were counted per 35 mm plate.1mL layer of 1:1 mixture of 1.2% agar and 2X medium  was evenly spread on 35mm plates. It was allowed to set for 30 minutes at RT. Cells were counted and plated in a mixture of 1.2% agar and 2X medium (1:4) to give a final agar concentration of 0.3%. 3x10https://tcga-data.nci.nih.gov/. TCGA melanoma cases (TCGA-SKCM) annotated for B-Raf WT or V600E mutation were extracted from the GDC portal . Among those, 407 samples were profiled for both mRNA expression and miRNA expression. Fragments per Kilobase per Million mapped reads (FPKM) from the RNA-sequencing data and reads per million miRNA mapped from the miRNA data were used as normalized read counts. Pearson correlation coefficients and P-values for miR-10b expression with Twist1 and MYC expression were calculated, respectively.The publicly available TCGA data were directly downloaded from the TCGA Data Portal at Cell extracts were prepared and western blotting was carried out. Briefly, cells were lysed with 20 mM Tris, pH 7.4, 150mM NaCl, 2mM EDTA, and 1% Triton X-100. Samples (10–50 μg protein) were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and then electrophoretically transferred from the gel to polyvinylidene fluoride membranes (Millipore). All the primary antibodies were diluted in phosphate buffered saline, pH 7.4, 0.2% Tween-20, 5% bovine serum albumin and 0.002% sodium azide. Following three washes, blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Proteins were visualized with enhanced chemiluminescence with the BioRad ChemiDoc EQ system.All experiments were performed in triplicates. All the statistical analyses were performed using the two-tailed student’s t-test (GraphPad Prism software).V600E in primary melanocytes [V600E, we chose miR-10b for further study because its expression positively correlates with V600E mutation in B-Raf in established melanoma cell lines , a pharmacological inhibitor of B-RafV600E -binding domain of mutated estrogen receptor (ERT2) [202 and Tyr204 indicating that the kinase activity of B-RafV600E was temporally turned on by 4-OHT treatment (V600E is better at activating miR-10b expression than sustained expression of the constitutively active kinase (Compare V600E dependent.We reasoned that if the effect of knocking down of B-Raf control . The drar (ERT2) . As expe Compare . Taken tmiR-10b gene resides upstream from the developmental gene Hoxd4. It has been shown that the basic helix-loop-helix transcription factor Twist positively regulates miR-10b expression in breast cancer cells by directly binding to the proximal E-box present in the miR-10b gene promoter. The expression levels of Twist are often deregulated in a vast majority of melanoma tumors and cell lines [V600E mutation in human melanoma cells [V600E may increase miR-10b expression by activating Twist. Indeed, hyper activation of B-Raf signaling pathway increases Twist1 transcript in established melanoma cell lines Click here for additional data file.S1 FigV600E mutation was determined by IHC staining with B-RafV600E specific Ab. Lesions were demarcated from normal tissues with white dotted line.(n = 4). The B-Raf(TIF)Click here for additional data file.S2 FigUpper panel shows the relative Twist1 mRNA expression in sk-mel-24 cells expressing two different Twist1 specific siRNAs or siGFP control as assessed by qRT-PCR. Lower panel shows the relative mR-10b expression in the indicated siRNA expressing sk-mel-24 cells as assessed by TaqMan qRT-PCR.(TIF)Click here for additional data file.


Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation. Several members of the NKL subclass are deregulated in T-cell progenitors and support leukemogenesis. We have recently described particular expression patterns of nine NKL homeobox genes in early hematopoiesis and T-cell development. Here, we screened NKL homeobox gene activities in normal B-cell development and extended the NKL-code to include this lymphoid lineage. Analysis of public expression profiling datasets revealed that HHEX and NKX6-3 were the only members differentially active in naïve B-cells, germinal center B-cells, plasma cells and memory B-cells. Subsequent examination of different types of B-cell malignancies showed both aberrant overexpression of NKL-code members and ectopic activation of subclass members physiologically silent in lymphopoiesis including BARX2, DLX1, EMX2, NKX2-1, NKX2-2 and NKX3-2. Based on these findings we performed detailed studies of the B-cell specific NKL homeobox gene NKX6-3 which showed enhanced activity in patient subsets of follicular lymphoma, mantle cell lymphoma and diffuse large B-cell lymphoma (DLBCL), and in three DLBCL cell lines to serve as in vitro models. While excluding genomic and chromosomal rearrangements at the locus of NKX6-3 (8p11) promoter studies demonstrated that B-cell factors MYB and PAX5 activated NKX6-3 transcription. Furthermore, aberrant BMP7/SMAD1-signalling and deregulated expression of chromatin complex components AUTS2 and PCGF5 promoted NKX6-3 activation. Finally, NKL homeobox genes HHEX, HLX, MSX1 and NKX6-3 were expressed in B-cell progenitors and generated a regulatory gene network in cell lines which we propose may provide physiological support for NKL-code formation in early B-cell development. Together, we identified an NKL-code in B-cell development whose violation may deregulate differentiation and promote malignant transformation. Hematopoietic stem cells (HSCs) generate common myeloid and lymphoid progenitor (CMP/CLP) cells which, respectively, initiate the differentiation into myeloid and lymphoid cell lineages. The latter produces all types of lymphocytes comprising B-cells, T-cells, and NK-cells. Early B-cell development begins with the B-cell progenitor (BCP) and takes place in the bone marrow. The final differentiation steps to memory B-cells and plasma cells via naïve and germinal center (GC) B-cells occur in lymph nodes and in the spleen –3. LymphHomeobox genes encode TFs containing a homeodomain which mediates both sequence-specific DNA-binding and contacts to cofactors. Most of these TFs regulate basic developmental processes in both embryos and adults. The clustered homeobox genes are expressed within the embryo in a particular pattern termed the HOX-code. This code determines the anterior-posterior differentiation of the branchial and head region . In the Homeobox genes are arranged into classes and subclasses according to sequence differences of the conserved homeobox . The humTo date, 24 aberrantly activated NKL homeobox genes have been described in T-cell acute lymphoid leukemia (T-ALL), representing the most prolific group of oncogenes in this malignancy ,16. ThesAberrant activities of NKL homeobox genes are reported in B-cell malignancies as well, comprising HLX in Hodgkin lymphoma (HL), MSX1 in mantle cell lymphoma (MCL), NKX2-1 in diffuse large B-cell lymphoma (DLBCL), and NKX2-3 in splenic marginal zone lymphoma (SMZL), mucosa-associated lymphoid tissue (MALT) lymphoma, and DLBCL –25. Howewww.ncbi.nlm.nih.gov). Of note, this type of profiling gene chip contains 37 probes of 48 known human NKL homeobox genes. We exploited datasets GSE56315 and GSE12366 for analyses of developing B-cells [www.broadinstitute.org/ccle) and from Astra Zeneca (GSE57083). For analysis of NKX6-3 expression in cell lines these data were transformed as follows: after RMA-background correction and quantile normalization of the spot intensities, profiling data were expressed as ratios of the sample mean and subsequently log2 transformed. Data processing was performed via R/Bioconductor using Limma and Affy packages.Public expression profiling datasets used in this study were generated by U133 Plus 2.0 gene chips from Affymetrix and obtained from Gene Expression Omnibus  at 350 V for 10 ms. Electroporated cells were harvested after 20 h cultivation. Cells were treated for 16 h with 20 ng/ml recombinant BMP7 , with BMP receptor inhibitor dorsomorphin (DM)  dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 5 μM.DLBCL cell lines, HL cell line L-540, and NIH-3T3 cells were obtained from the DSMZ  and cultivated as described elsewhere . To modiTotal RNA was extracted from cultivated cell lines using TRIzol reagent . Primary human total RNA was commercially obtained. We used RNA from peripheral blood mononuclear cells (PBMC), thymus, lymph node (LN), spleen, and bone marrow (BM) obtained from Biochain/BioCat , and RNA from peripheral CD19-positive B-cells and CD3-positive T-cells obtained from Miltenyi Biotec . cDNA was synthesized using 5 μg RNA, random priming and Superscript II (Invitrogen). RQ-PCR analysis was performed using the 7500 Real-time System and commercial buffer and primer sets . Quantification of MSX1 was performed as described recently . For norTo examine NKX6-3 protein expression we used primary antibody anti-NKX6-3 . Immuno-cytology was performed as follows: cells were spun onto slides and subsequently air-dried and fixed with methanol/acetic acid for 90 s. The antibody was diluted 1:20 in PBS containing 5% BSA and incubated for 30 min. Washing was performed 3 times with PBS. Preparations were incubated with secondary antibody (diluted 1:100) for 20 min. After final washing the cells were mounted in Vectashield  containing DAPI for nuclear staining. Documentation of subcellular protein localization was performed using an Axio-Imager microscope  configured to a dual Spectral Imaging FISH system .Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail . Proteins were transferred onto nitrocellulose membranes  and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), and SMAD1 . For loading control blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL . Documentation was performed using the digital system ChemoStar Imager .For genomic profiling genomic DNA of DLBCL cell lines was prepared by the Qiagen Gentra Puregene Kit (Qiagen). Labelling, hybridization and scanning of Cytoscan HD arrays was performed at the Genome Analytics Facility, Helmholtz Centre for Infection Research , according to the manufacturer´s protocols . Data were interpreted using the Chromosome Analysis Suite software version 2.0.1.2 (Affymetrix).Chromosomal analysis by FISH was performed as described previously . BAC cloHindIII/BamHI and EcoRI sites, respectively, of the expression vector pcDNA3 downstream of the CMV enhancer. The oligonucleotides used for the amplification of the PAX5-site were obtained from Eurofins MWG . Their sequences were as follows: NKX6-3-for1 AAGCTTCCTGATCCTTCCTGGAGCAGG-3´5´-AA, NKX6-3-rev1 GGATCCTTTTACAGCAGAGAAAACTGAC-3´5´-CT. The oligonucleotides used for the amplification of the MSX1/HLX-site were obtained from Eurofins MWG. Their sequences were as follows: NKX6-3-for2 AAGCTTGGCTGCACGCTCCGGCTGCTAG-3´5´-GC, NKX6-3-rev2 GGATCCGAATGGTCTATACATCAGAGTAGG-3´5´-TG. Introduced restriction sites used for cloning are underlined. Transfections of plasmid-DNA into NIH-3T3 cells were performed using SuperFect Transfection Reagent (Qiagen). Commercial HOXA9 and TBP assays were used for RQ-PCR to quantify the spliced reporter-transcript, corresponding to the regulator activity.For creation of reporter gene constructs we combined a reporter with two different regulatory genomic fragments derived from the upstream region of NKX6-3, containing consensus binding sites for PAX5 and BACH2 and a binding site for MSX1 and HLX, respectively. The PAX5 site was visualized at the UCSC Genome Browser, the consensus binding sites for MSX1 and HLX are published elsewhere . We clonS1 Fig). To analyze physiological expression patterns of NKL homeobox genes in subsequent stages of B-cell development we here exploited two public expression profiling datasets, GSE56315 and GSE12366, each comprising samples of naïve, germinal center (GC), memory B-cells, and plasma cells. From the NKL homeobox genes analyzed, corresponding activities at significant levels were detected for only HHEX and NKX6-3 in particular developmental stages of B-cells . The results of this screening are shown in Table 1 and Fig 1. Accordingly, naïve B-cells, GC B-cells, and memory B-cells expressed significant levels of HHEX. GC B-cells expressed NKX6-3 as well while plasma cells expressed NKX6-3 but not HHEX. Thus, these data extended the knowledge of the physiological activity of NKL homeobox genes during lymphopoiesis, showing an alternative NKL-code for the B-cell lineage. In contrast to mature T-cells, mature B-cells were positive for NKL homeobox gene activity, expressing either HHEX or NKX6-3.Recently, we identified specific expression patterns of NKL homeobox genes in early hematopoiesis, lymphoid progenitors and T-cell development . These dS3 Fig). Dataset GSE53786 contains 119 samples of DLBCL patients , and GSE16455 contains 22 samples of MCL, 5 hairy cell leukemia (HCL), 4 SMZL, 19 chronic lymphocytic leukemia (CLL), and 7 FL patients patients . Finally, dataset GSE21452 contains 64 samples of MCL patients . Seven of the nine expressed NKL homeobox genes in hematopoietic cells showed aberrant overexpression in subsets of B-cell malignancies, including HHEX, HLX, MSX1, NKX2-3, NKX3-1, NKX6-3 and TLX2 (Table 1). Furthermore, six NKL homeobox genes normally inactive in hematopoiesis were ectopically expressed. Accordingly, in HL patients we identified DLX1, EMX2, NKX2-2 and NKX3-2, in DLBCL we found NKX2-1, and HCL patients expressed BARX2 (Table 1). Some of these identified genes have been previously shown to be deregulated in subsets of particular B-cell lymphomas, including HLX in HL, MSX1 in MCL, NKX2-1 in DLBCL, and NKX2-3 in SMZL, MALT lymphoma and DLBCL [To identify aberrantly transcribed NKL homeobox genes in primary malignant B-cells we analyzed four expression profiling datasets including GSE12453 which contains 17 samples of HL, 4 T-cell rich B-cell lymphoma (TCRBL), 5 follicular lymphoma (FL), 5 Burkitt lymphoma (BL), and 11 DLBCL patients (nd DLBCL . CollectTable 1), indicating considerable deregulation in B-NHLs. Therefore, we focused on deregulated activities of this B-cell specific NKL homeobox gene. To identify cell line models for NKX6-3 we screened one FL cell line, four MCL cell lines and 14 DLBCL cell lines by expression profiling (S1 Table). This procedure revealed one DLBCL cell line (DOHH-2) which significantly overexpressed NKX6-3. Subsequent expression analysis of a similar set of 14 DLBCL cell lines via RQ-PCR confirmed enhanced NKX6-3 transcription in DOHH-2 and additionally in OCI-LY1 and WILL-1 as well . In these three cell lines NKX6-3 expression was further demonstrated at the protein level by immunocytology. The signals were located both in the nucleus and in the cytoplasm as shown for DOHH-2 , indicating functional relevance of NKX6-3 expression. RQ-PCR analysis of NKX6-3 in primary hematopoietic cell/tissue samples and DOHH-2 demonstrated expression in lymph nodes and mature B-cells. In contrast, the expression in bone marrow, thymus, spleen, peripheral blood cells, T-cells, and hematopoietic stem cells was nearly undetectable . These results confirmed NKX6-3 overexpression in DLBCL cell line DOHH-2 which together with OCI-LY1 and WILL-1 represent, therefore, appropriate models to analyze upstream factors and downstream effects of NKX6-3 expression in B-cells.Clinical data indicated that 14% of FL, 4% of MCL, and 2% of DLBCL patients showed aberrant overexpression of NKX6-3 . Furthermore, FISH analyses of DOHH-2, OCI-LY1 and WILL-1 excluded chromosomal translocations targeting the NKX6-3 gene . Of note, OCI-LY1 contains a translocation at 8p12 but this breakpoint mapped 5 Mbp telomeric of NKX6-3 at UNC5D. Thus, these data excluded genomic rearrangements underlying enhanced NKX6-3 expression in DLBCL cell lines.In T-ALL several NKL homeobox genes are deregulated via chromosomal rearrangements . To analwww.genome.cse.ucsc.edu) revealed potential sites for the B-cell associated TFs MYC, MYB and PAX5 . For these three factors/genes we performed RQ-PCR expression analyses and siRNA-mediated knockdowns in DLBCL cell lines . While the data for MYC showed no significant effect, those for MYB and PAX5 indicated an activating role. Furthermore, knockdown of NKX6-3 resulted in increased expression of PAX5 demonstrating mutual regulation of these genes. Finally, we performed a reporter-gene assay for the indicated PAX5-site , confirming that the activating impact of PAX5 on NKX6-3 transcription operated directly. Thus, this promoter analysis demonstrated that NKX6-3 is activated by the TFs MYB and PAX5 –the latter represents a key master gene for B-cell development [To identify candidate TFs involved in NKX6-3 (de)regulation we screened the promoter region of this NKL homeobox gene for consensus binding sites. The UCSC Genome Browser . The top 250 most differentially expressed genes included SMAD1 and BMP7—both components of the BMP-signalling-pathway [S8 Fig). The gene encoding BMP7 lay out with the top 250 differentially expressed genes in this dataset but, nevertheless, BMP7 also showed statistically significant overexpression in NKX6-3 positive patients . Furthermore, these patient data revealed significantly reduced expression levels of PCGF5 encoding a component of polycomb repressor complex (PRC)1.5 [To identify alternative mechanisms and factors involved in NKX6-3 activation we performed comparative expression profiling of DLBCL cell lines DOHH-2 and OCI-LY1 and six NKX6-3 negative controls using dataset GSE42203 (-pathway . Compari(PRC)1.5 . Thus, aFig 5A). SiRNA-mediated knockdown of SMAD1 in DOHH-2 resulted in decreased expression levels of NKX6-3 , demonstrating that SMAD1 mediated NKX6-3 activation. SiRNA-mediated knockdown of SMAD1-cofactor SMAD4 resulted in decreased NKX6-3 expression as well , supporting the activatory impact of this SMAD-tandem. RQ-PCR analysis of BMP7 confirmed elevated expression levels in NKX6-3 positive cell lines . Furthermore, primary hematopoietic cell/tissue samples showed low/absent BMP7 expression indicating ectopic activation of this gene in selected DLBCL cell lines . Treatment of DOHH-2 and WILL-1 with recombinant BMP7 protein enhanced NKX6-3 expression , while siRNA-mediated knockdown of BMP7 in DOHH-2 resulted in reduced expression of NKX6-3 . Thus, autocrine BMP7/SMAD1-signalling activated NKX6-3 in DLBCL.RQ-PCR and Western blot analyses of SMAD1 confirmed elevated expression levels in the NKX6-3 positive cell lines . In contrast RQ-PCR analysis of AUTS2 indicated elevated expression levels in these cell lines . AUTS2 interacts with PCGF5 to convert this repressive factor into an activator [Fig 6C), indicating that NKX6-3 is activated by AUTS2 as well. Surprisingly, the expression level of MSX1 increased after AUTS2 knockdown . This observation indicated mutual regulation of NKX6-3 and MSX1 (see below). Furthermore, forced expression of PCGF5 in OCI-LY1 resulted in reduced expression of NKX6-3 . Thus, NKX6-3 transcription is regulated via the repressive chromatin complex PRC1.5 which is antagonized by AUTS2.PRCs are involved in the suppression of many homeobox genes notably clustered HOX genes . Howeverctivator . This efctivator . Here, sFig 6C). RQ-PCR analysis of MSX1 transcripts in DLBCL cell lines showed varying expression levels . While DOHH-2 expressed high levels, OCI-LY1 and WILL-1 expressed very low MSX1 RNA levels. SiRNA-mediated knockdown of MSX1 in DOHH-2 resulted in reduced NKX6-3 expression while knockdown of NKX6-3 activated MSX1 expression . Thus, MSX1 is an activator of NKX6-3 which in turn inhibits MSX1. To see if MSX1 regulates NKX6-3 directly, we screened the upstream-region of NKX6-3 for potential binding-sites. This exercise identified a consensus site for MSX1 at position –6.011 bp. Subsequently we performed a reporter-gene assay for the corresponding region , demonstrating that MSX1 activated NKX6-3 transcription via this site.Our data obtained for AUTS2/PCGF5 indicated a potential regulatory role for NKX6-3 in MSX1 expression . Furthermore, this treatment boosted MSX1 expression strongly in DOHH-2 but only moderately in WILL-1 . Consistently, treatment of DOHH-2 and WILL-1 with BMP7 resulted in inhibition of MSX1 expression in both cell lines . These data indicated that NKX6-3 and MSX1 were respectively activated and repressed by BMP7/SMAD1-signalling. However, the activating impact of MSX1 on NKX6-3 was only effective in DOHH-2 which expresses significant levels of both genes.Treatment of DOHH-2 and WILL-1 cells with BMP-receptor inhibitor dorsomorphin (DM) resulted in increased NKX6-3 levels in DOHH-2 but decreased levels in WILL-1 . Accordingly, forced expression of HHEX in OCI-LY1 cells resulted in reduced NKX6-3 expression levels , indicating that HHEX repressed NKX6-3 transcription. Forced expression of HHEX in DOHH-2 resulted in reduced transcription of MSX1 as well, indicating that HHEX repressed MSX1 in addition to NKX6-3 . However, siRNA-mediated knockdown of NKX6-3 in DOHH-2 cells showed no significant alteration in HHEX expression levels , discounting mutual regulation.To see if in addition to MSX1 the remaining B-cell associated NKL homeobox genes HHEX and HLX also influence NKX6-3 expression, we examined their gene activities and impact. RQ-PCR analysis of HHEX demonstrated low transcript levels in NKX6-3 positive cell lines DOHH-2, OCI-LY1 and WILL-1 . As a control we additionally quantified HLX transcripts in HL cell line L-540 which has been shown to aberrantly express this NKL homeobox gene at a level similar to HSCs [Fig 7I). Forced expression of MSX1 in WILL-1 resulted in reduced transcription of HLX, indicating mutual repression . Finally, we reused the reporter-gene assay for HLX as described above for MSX1 because both factors bind the same consensus site. These data showed that HLX repressed NKX6-3 transcription directly . Thus, MSX1 activated while HLX repressed NKX6-3 transcription, competing for the same binding site. Taken together, these data demonstrated that NKX6-3 is regulated by B-cell associated NKL homeobox genes MSX1, HHEX and HLX, forming a gene network.RQ-PCR analysis of HLX showed low/absent expression levels in all DLBCL cell lines examined . Additional results of our work are summarized in Fig 8, which shows a gene regulatory network surrounding NKL homeobox gene NKX6-3. TFs MYB and PAX5, BMP7/SMAD1-signalling, and PRC1.5-mediator AUTS2 all activate expression of NKX6-3.In this study we analyzed physiological activities of NKL homeobox genes in B-cell development. Considered together with previously published data, this examination detected expression patterns which complete the NKL-code for early hematopoiesis and lymphopoiesis  and in other hematopoietic cells, suggesting aberrant reactivation of SMAD1 via ectopically expressed BMP7. Deregulated BMP-signalling has been shown to activate MSX1 in T-ALL [Then, we identified TFs MYB and PAX5 as  upstream regulators of NKX6-3. While MYB represents a general hematopoietic TF PAX5 is a master factor for B-cell development ,45, highin T-ALL , highligS1 Fig), supporting the idea of oncogenic reactivation of regulatory factors normally operating in progenitor cells. Chromatin complexes and modified histones are common regulators of homeobox genes [Deregulation via AUTS2/PCFG5 represents another concordance in oncogenic activation between NKX6-3 in DLBCL and MSX1 in T-ALL . AUTS2 iox genes ,46. Bivaox genes . Accordiox genes . Therefoox genes ,24.Transcriptional analyses of all four B-cell associated NKL homeobox genes revealed mutual regulation. MSX1 repressed HLX but activated NKX6-3. Furthermore, HHEX and HLX acted as repressors of NKX6-3 and MSX1. In HL, aberrantly expressed HLX represses the transcription of MSX1 as well, supporting the importance of this regulatory connection in developing B-cells . Due to In conclusion, we identified particular NKL homeobox gene activities in B-cell development, thus completing the NKL-code for lymphopoiesis. According to this code we were able to reveal aberrantly expressed NKL homeobox genes in B-cell malignancies. Although less frequent when compared to T-ALL, NKL homeobox gene deregulation plays a considerable role in B-cell cancers as well. NKX6-3 is physiologically expressed in B-cell differentiation and aberrantly overexpressed in B-cell lymphomas. These data may contribute to the understanding of normal and aberrant B-cell differentiation.S1 FigRNA-seq data from samples of early hematopoiesis and of T-cell development (GSE69239) were used to analyze expression of particular genes including NKL homeobox genes and regulators of NKX6-3. Expression levels are indicated in FPKM (fragments per kilobase of mappable gene length and million reads). HSPC: hematopoietic stem and progenitor cells, LMPP: lymphoid and myeloid progenitor, CLP: common lymphoid progenitor, BCP: B-cell progenitor, DN: double negative T-cell progenitor, DP: double positive T-cell progenitor, SP4: single positive CD4+ T-cell progenitor, SP8: single positive CD8+ T-cell progenitor. The samples of CLPs and BCPs were of special interest and are highlighted in red.(TIF)Click here for additional data file.S2 FigExpression profiling datasets GSE56315 and GSE12366 contain samples from B-cell developmental stages including naïve B-cells, GC B-cells, memory B-cells and plasma cells. Significant positive expression were revealed by cutoffs defined at 6 (GSE56315) and at 200 (GSE12366). Using these criteria just two NKL homeobox genes, HHEX and NKX6-3, were identified to be consistently expressed in both datasets in any of these types of B-cell.(TIF)Click here for additional data file.S3 FigExpression profiling dataset GSE12453 contains patient samples for Hodgkin lymphoma (HL), classical HL (cHL), nodular lymphocyte-predominant HL (NLPHL), T-cell rich B-cell lymphoma (TCRBL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), in addition to samples from normal B-cells including naïve (N), memory (M), germinal center (GC) and plasma cells (P). Boxplots were performed for expression levels from indicated types of lymphomas in comparison to normal B-cells. Outliers were defined as aberrant overexpression and highlighted by red arrowheads. Accordingly, DLX1, EMX2, HLX, NKX3-2 and TLX2 were each overexpressed in 1/7 (6%) and NKX2-2 in 2/7 (12%) of HL patients. MSX1 was overexpressed in 1/5 (20%) of FL patients, NKX2-3 was overexpressed in 1/11 (9%) of DLBCL patients. The expression level for MSX1 in normal memory B-cells (sample GSM312686) was interpreted as outlier and excluded (X).(TIF)Click here for additional data file.S4 FigExpression profiling dataset GSE53786 contains patient samples from 119 diffuse large B-cell lymphoma (DLBCL). Boxplots were performed for expression levels and outliers were defined as aberrant overexpression, highlighted by red arrowheads. Accordingly, NKX2-1 was overexpressed in 8/119 (7%) of DLBCL patients, NKX2-3 in 3/119 (2%) of patients, and NKX6-3 in 2/119 (2%) of DLBCL patients.(TIF)Click here for additional data file.S5 FigExpression profiling dataset GSE16455 contains patient samples for mantle cell lymphoma (MCL), hairy cell leukemia (HCL), splenic marginal zone lymphoma (SMZL), chronic lymphocytic leukemia (CLL), and follicular lymphoma (FL). We set the cutoff at 100 for positive gene activity. Boxplots were performed for expression levels and outliers were defined as aberrant overexpression, highlighted by red arrowheads. Accordingly, BARX2 and HHEX were each overexpressed in 1/5 (20%) of HCL patients. BARX2 was overexpressed in 3/22 (14%) of MCL patients. HHEX and HLX were each overexpressed in 1/4 (25%) of SMZL patients. HLX, NKX3-1 and NKX6-3 were each overexpressed in 1/22 (4%) of MCL patients. HLX and NKX6-3 were each overexpressed in 1/7 (14%) and MSX1 in 2/7 (28%) of FL patients.(TIF)Click here for additional data file.S6 FigExpression profiling dataset GSE21452 contains 64 patient samples for mantle cell lymphoma. We defined a cutoff at 8 to reveal positive gene activity. Boxplots were performed for expression levels and outliers were defined as aberrant overexpression, highlighted by red arrowheads. Accordingly, MSX1 was overexpressed in 2/64 (3%) of MCL patients. Of note, in this dataset no overexpression of HLX and NKX3-1 was detectable–overexpression of NKX6-3 was not significant.(TIF)Click here for additional data file.S7 FigExpression profiling dataset GSE42203 contains two DLBCL cell line samples  showing overexpression of NKX6-3. Expression levels of NKX6-3, SMAD1 and BMP7 were shown in comparison to six control DLBCL cell lines . The statistical significances are indicated as p-values.(TIF)Click here for additional data file.S8 FigExpression profiling dataset GSE53786 contains two DLBCL patient samples showing overexpression of NKX6-3 (blue background). Expression levels of NKX6-3, SMAD1, BMP7 and PCGF5 are shown in comparison to 10 control DLBCL patients (red background). The statistical significances are indicated as p-values.(TIF)Click here for additional data file.S1 TableExpression profiling data for NKX6-3 of 19 B-cell lines are indicated in addition to the source of data and the derived malignancy (left). Representation of the data in a boxplot demonstrates the value of DOHH-2 as outlier, demonstrating NKX6-3 overexpression in this cell line (right).(TIF)Click here for additional data file.


Scientific Reports 10.1038/s41598-019-54167-1, published online 28 November 2019Correction to: This Article contains an error in Figure 2 where a duplicate of Fig. 2C (Ki67 staining) was inadvertently copied into Fig. 2D (cleaved caspase-3 staining) of NSC at day 1, during reassembly of the manuscript. The correct Figure 2 appears below as Figure


Caenorhabditis elegans pharynx, acetylcholine (ACh) released from the MC and M4 motor neurons stimulates two different types of contractions in adjacent muscle cells, termed pumping and isthmus peristalsis. MC stimulates rapid pumping through the nicotinic ACh receptor EAT-2, which is tightly localized at the MC NMJ, and eat-2 mutants exhibit a slow pump rate. Surprisingly, we found that eat-2 mutants also hyperstimulated peristaltic contractions, and that they were characterized by increased and prolonged Ca2+ transients in the isthmus muscles. This hyperstimulation depends on cross talk with the GAR-3 muscarinic ACh receptor as gar-3 mutation specifically suppressed the prolonged contraction and increased Ca2+ observed in eat-2 mutant peristalses. Similar GAR-3-dependent hyperstimulation was also observed in mutants lacking the ace-3 acetylcholinesterase, and we suggest that NMJ defects in eat-2 and ace-3 mutants result in ACh stimulation of extrasynaptic GAR-3 receptors in isthmus muscles. gar-3 mutation also suppressed slow larval growth and prolonged life span phenotypes that result from dietary restriction in eat-2 mutants, indicating that cross talk with the GAR-3 receptor has a long-term impact on feeding behavior and eat-2 mutant phenotypes.Precise signaling at the neuromuscular junction (NMJ) is essential for proper muscle contraction. In the  COMMUNICATION between motor neurons and their target muscles is crucial for proper muscle contraction and function . This communication is mediated by the neurotransmitter acetylcholine (ACh), which is released from the motor neuron and binds receptors embedded in the muscle cell membrane. In vertebrate skeletal muscle, these receptors are nicotinic ACh receptors (nAChRs), which are homo- or hetero-pentameric, ligand-gated ion channels , VC657 gar-3(gk305), FX863 acr-7(tm863), RB918 acr-16(ok789), VC2661 acr-10(ok3118), RB2294 acr-6(ok3117), and VC2820 eat-2(ok3528)/mT1; mT1+/.The following strains were generated by the acr-14(ok1155) (eat-2(ad1116) (eat-2(ad465) (ace-3(dc2) (ace-2(g72); ace-1(p1000) (eat-18(ad820) (unc-119(ed3); leIs2702(ceh-19bProm::unc-119(+))gfp,  , DA1116 2(ad465) , PR1300 -119(+)) .cuIs36[myo-2Prom::GCaMP3], OK1062 gar-3(gk305); cuIs36, OK1063 eat-2(ok3528); cuIs36, OK1064 eat-2(ok3528); gar-3(gk305), OK1075 eat-2(ok3528); gar-3(gk305); cuIs36, OK1023 eat-2(ok3528), OK1081 ace-3(dc2); gar-3(gk305), OK1082 eat-2(ok3528); unc-119(ed3); leIs2702Prom::gfp, unc-119(+)); cuEx828.The following strains were constructed in this study: OK1020 myo-2 promoter from plasmid pPD96.48 was cloned into a HindIII- and MscI-digested str-2::GCaMP3 plasmid (ceh-19b promoter was amplified from N2 genomic DNA using primers PO1452 (5′-CGAGCATGCGAAAAACAGGAAAGTCTCG-3′) and PO1453 (5′-GACCCGGGATGTAGAGTTGAGAAGTTGCCA-3′), digested with SphI and XmaI, and inserted into SphI- and XmaI-digested Promser-7b::snb-1::gfp plasmid pOK219.08 to generate the Promceh-19b::snb-1::gfp plasmid pOK361.01 , gar-3(gk305), and ace-3(dc2) alleles by PCR (eat-2(ok3528) were PO1423 (5′-TGCGTGGTAGAGGGATAGTG-3′), PO1424 (5′-TCTCGACGAGACCTACGTTG-3′), and PO1425 (5′-ACAGCTACAGTACCTCGCAC-3′). Primers for genotyping gar-3(gk305) were PO1426 (5′-TAATAGGTTCGGCCCAGAGC-3′), PO1427 (5′-GTGATCGTTTGCTGGGAAGC-3′), and PO1428 (5′-CGAAGCTCAGAATGTCAGTAACG-3′). Primers for genotyping ace-3(dc2) were PO1435 (5′-CAAGGATACAGAGTACACGGCA-3′), PO1436 (5′-CAAGCCCGCAAATTGAACTGA-3′), and PO1437 (5′-GCAAGTGGCAAGCGAGAATA-3′).Individual animals were genotyped for s by PCR . PrimersOP50 and imaged on a 2% agarose pad under a coverslip. For drug treatments, either arecoline  or nicotine  was included, and animals were treated for 15 min prior to adding a coverslip. Individual N2 or mutant animals that pumped were recorded at 25 frames/sec for 1 min (cha-1 mutants were recorded for 5 min) using a Zeiss  AxioImager microscope with an MRm camera and ZEN Software. For each genotype and drug treatment, the feeding behavior was analyzed in at least five animals . Time-lapse images were exported and processed using Fiji/ImageJ  for 10 min on a 2% agarose pad, and then immobilized using 1.5 μl polystyrene 0.10 μm microspheres and a coverslip . GCaMP3 imaging was performed on a Zeiss AxioImager microscope using a Q-Imaging Rolera EM-CPromceh-19b::gfp expression, Z-series images were collected. Maximum intensity Z projections were produced using Fiji/ImageJ  and eat-2(ok3528); gar-3(gk305) animals were allowed to grow for an additional day to quantify slow-growing adults. For each genotype, two plates were set up with 50 embryos each.Adult hermaphrodites were allowed to lay eggs for 8 hr at 25°, and embryos were transferred to freshly seeded plates and incubated at 20° for 16 hr. Unhatched embryos were counted and hatched L1s were allowed to grow for an additional 3 days. Adult animals were counted and removed on day 5. OP50Escherichia coli at 20°, and dead or missing animals were replaced by animals of the same age. For each experiment, triplicate plates of each genotype were assayed, and representative results of three or more experiment are shown. Kaplan–Meier survival analysis was used to generate survival plots and calculate median life span, and for statistical analysis (GraphPad Prism 5).Life span assays were performed according to a previously published protocol . Assays C. elegans. Table S1 contains measurements of pharyngeal muscle contractions in acr mutant C. elegans. Tables S2–S52 contain raw data of pharyngeal muscle contractions (Tables S2–S13) and GCaMP3 fluorescence intensities (Tables S14–S52) derived from time-lapse images. Supplemental material available at Figshare: https://doi.org/10.25386/genetics.6160355.Strains and plasmids are available upon request. Supplemental Material, Figure S1 contains measurements of baseline GCaMP3 fluorescence intensity in wild-type and mutant strains. Figure S2 contains tracings of GCaMP3 fluorescence intensity from representative wild-type and mutant animals. Movies S1–S9 include time-lapse images of wild-type and mutant cha-1  mutant L1 animals. cha-1(ok2253) is a previously uncharacterized null allele containing a 1.7-kb deletion that eliminates the catalytic histidine (His341) required for ACh synthesis, and homozygous mutants arrest as paralyzed L1s with a coiled appearance (C. elegans Deletion Mutant Consortium 2012). Untreated cha-1(ok2253) homozygotes completely lacked pharyngeal muscle contractions, but mutant animals treated with either nicotine or arecoline exhibited both pumping and peristalsis  null mutants (gar-3(gk305) mutants are viable, look healthy, and grow normally, and pharyngeal muscle contractions in gar-3(gk305) L1s were similar to those in wild-type worms (gar-3(gk305) animals. In comparison, gar-3(gk305) animals were almost completely insensitive to exogenously applied arecoline (gar-3(gk305) mutants were largely unaffected by arecoline. The pump rate was unaffected in gar-3(gk305) and, although these animals did exhibit a small decrease in the duration of peristaltic contractions when treated with arecoline, we note that this change is the opposite of the prolonged peristalses observed in arecoline-treated wild-type animals. Thus, GAR-3 mediates the response to arecoline, but loss of this receptor does not have a strong effect on pharyngeal muscle contractions in the absence of this drug.To examine the role that mAChR signaling plays in pharyngeal muscle contractions, we characterized these contractions in pe worms . Neitherrecoline . Wild-tyeat-2, which encodes the only nAChR subunit known to function in the pharyngeal muscles  mutants treated with nicotine at the L1 stage. Like cha-1 mutants, wild-type animals treated with increasing concentrations of nicotine exhibited a dose-dependent decrease in pharyngeal muscle contractions  and eat-2(ad1116) are point mutations introducing an early stop codon and affecting a splice site, respectively (eat-2(ok3528) contains a 614-bp deletion predicted to cause a frameshift mutation upstream of the transmembrane domains (C. elegans Deletion Mutant Consortium 2012). As expected, the rate of pumping in all three mutants was significantly reduced compared to in wild-type worms  mutants exhibited the strongest phenotypes, which, together with a molecular lesion in this allele, suggests that eat-2(ok3528) is a null allele.To understand how pe worms  (Avery 1eat-2(ok3528) mutants using a Promceh-19b::gfp reporter (eat-2(ok3528) adults  mutants, synapses were visible in the MC process beginning near the junction of the pm4 and pm5 muscle cells, and extending into the anterior region of the isthmus (eat-2(ok3528) mutants exhibited a small but not statistically significant decrease in the number of vesicle clusters compared to wild-type  4.9 ± 1.0; P = 0.09, n = 13). Thus, the prolonged peristalses in eat-2 mutants do not result from morphological or synaptic defects in the MCs. Notably, using both of these reporters, we observed the MC axon extending into the anterior isthmus further than has been previously reported  adults . The MC ic cells . In both isthmus . eat-2 animals also exhibit prolonged peristalses with almost all pumps followed by peristalses (acr-6(ok3117), acr-7(tm863), acr-10(ok3118), acr-14(ok1155), and acr-16(ok789) ; gar-3(gk305) double mutants.Pharyngeal muscle contractions in recoline , suggestgar-3 mutation partially suppressed the prolonged peristalses observed in eat-2 single mutants (eat-2(ok3528); gar-3(gk305) double mutants was strongly reduced compared to eat-2(ok3528) single mutants, although they were still longer than those in wild-type animals or gar-3(gk305) single mutants. In contrast, the reduced pumping frequency and increased frequency of peristalsis observed in eat-2(ok3528) single mutants was not suppressed in eat-2(ok3528); gar-3(gk305). Thus, eat-2 loss-of-function affects pumping and peristalsis by different mechanisms, and only the prolonged peristalsis phenotype depends on cross talk with the GAR-3 receptor.We found that  mutants . The durmyo-2 promoter . While Ca2+ waves have not been previously described in the C. elegans pharynx , the width of each peak at 50% maximum fluorescence (peak duration), and the time that it took for fluorescence to reach its maximum (rise time) (To compare Case time) . In addieat-2(ok3528) mutants, which have prolonged peristalses, also exhibited significantly increased ∆F/Fo, peak duration, and rise time, in both the center and posterior isthmus. In addition, the peak delay was increased twofold (Movies S8 and S9). In comparison, gar-3(gk305) mutants, which have normal duration of peristalses, exhibited ∆F/Fo and rise times that were similar to those of wild-type animals, with a small decrease in ∆F/Fo only in the posterior isthmus, as well as a small decrease in peak duration in both the center and posterior isthmus. However, the most striking change in these animals was a strongly decreased peak delay between the center and posterior isthmus. Finally, eat-2(ok3528); gar-3(gk305) double mutants, which suppress the prolonged peristalses observed in eat-2(ok3528) single mutants, exhibited decreases in ∆F/Fo, peak duration, rise time, and peak delay compared to eat-2(ok3528) single mutants, although only ∆F/Fo was reduced to wild-type levels. Taken together, these results show that the increases in level, duration, and rise time of Ca2+, in both the center and posterior isthmus, best correlate with the duration of peristaltic contractions. Loss of eat-2 increases these Ca2+ signals and produces prolonged peristalses that depend on wild-type gar-3.We found changes in GCaMP3 fluorescence in the pharyngeal isthmus that paralleled many of the defects we observed in peristaltic contractions in various mutants . eat-2 mutants exhibited significantly prolonged peristalses, which were similar to those of eat-2 mutants (ace-2(g72); ace-1(p1000) double mutants exhibited only mildly prolonged peristalses. Both of these strains also exhibited a small decrease in the pump rate, but this was not significantly different from wild-type, indicating that MC can effectively stimulate pumping in ace mutants.It is surprising that  mutants . In compgar-3 mutation, we examined ace-3(dc2); gar-3(gk305) double mutants and found that these animals exhibited peristalses that were similar to wild-type  single mutants reached adulthood after 5 days at 20°, eat-2 mutants grew much more slowly and, even after 6 days, only 30% had reached adulthood (eat-2(ok3528); gar-3(gk305) double mutants also grew slower than wild-type and gar-3(gk305), but they grew faster than eat-2(ok3528) single mutants and 100% of these animals reached adulthood within 6 days.While nearly all wild-type animals and dulthood . eat-2 and eat-2(ok3528) mutants exhibited significantly extended life spans  (gar-3(gk305); eat-2(ok3528) double mutants was indistinguishable from that of wild-type animals and gar-3(gk305) single mutants .Likewise,  animals , and we ctively) . In compgar-3(gk305) suppresses both the slow larval growth and extended life span phenotypes of eat-2(ok3528) mutants, and we suggest that this suppression is the result of improved isthmus peristalsis and feeding.Thus, EAT-2 and the mAChR GAR-3 affects peristaltic muscle contractions in the isthmus of the C. elegans pharynx. Under normal circumstances, GAR-3 has a relatively minor role in these contractions, but in eat-2 mutants, GAR-3 can stimulate increased cytoplasmic Ca2+ levels and peristalses that are prolonged compared to those of wild-type animals. We suggest that this GAR-3-dependent stimulation of peristalsis results from ACh spillover from synapses between MC and the pharyngeal muscles in eat-2 mutants, and, consistent with this suggestion, acetylcholinesterase mutants similarly exhibit prolonged peristalses that are dependent on GAR-3. Cross talk with GAR-3 contributes to the slow larval growth and extended life span phenotypes observed in eat-2 mutants, indicating that this cross talk has a long-term impact on C. elegans feeding.In this work, we show that cross talk between the nAChR eat-2 mutants have a reduced pump rate, indicating that EAT-2 plays an excitatory role in pumping (eat-2 mutants exhibit prolonged peristalses, indicating that EAT-2 does not excite isthmus muscle contractions. Rather, it is necessary for rapid relaxation. These peristalsis phenotypes are similar to previous observations demonstrating that eat-2 mutation leads to prolonged depolarization and contraction of the terminal bulb muscles during pumping ( pumping .EAT-2 receptor is specifically localized at the sites where MC forms synapses near the junction of the pm4 and pm5 pharyngeal muscles, and it is necessary for MC to directly stimulate rapid pumping (EAT-2-containing receptors.The eat-2 mutants depend on the GAR-3 receptor. gar-3 mutation partially suppressed the prolonged peristaltic contraction of eat-2 mutants, as well as the increased and prolonged Ca2+ transients in the isthmus muscles of these animals. This suppression demonstrates that GAR-3 receptor activation contributes to the eat-2 mutant peristaltic phenotypes. Previous studies demonstrated that gar-3 mutation similarly suppressed the prolonged contraction of the terminal bulb muscles in eat-2 mutants (gar-3 single mutants have relatively minor defects in peristalsis. The duration of peristalsis was similar to wild-type animals. However, these mutants exhibited a sharp decrease in the peak delay between Ca2+ transients in the center and posterior isthmus, and a decreased ∆F/Fo in the posterior isthmus. Thus, in wild-type animals, GAR-3 contributes to the spatially and temporally dynamic changes in Ca2+ concentration in the isthmus muscles, but this did not produce recognizable changes in the duration of peristalsis. These observations demonstrate that GCaMP3 may be a more sensitive assay for muscle excitation.The prolonged peristalses in  mutants . In compgar-3 mutation did not suppress the reduced pump rate observed in eat-2 mutants (eat-2 mutants arise via different mechanisms. The reduced pump rate results directly from a loss of EAT-2 excitatory activity stimulated by the MC neuron, while the prolonged peristalsis results from excitation that depends on GAR-3.In contrast to its effect on peristalsis,  mutants . Thus, tGAR-3 has previously been shown to respond to humoral ACh (eat-2 mutants, ACh spillover from synapses between MC near the junction of the pm4 and pm5 muscles similarly activates GAR-3 receptors in pm5. Activation of slow-acting, metabotropic GAR-3 stimulates increased and prolonged Ca2+ transients in the isthmus, which lead to prolonged peristalsis. GAR-3 is known to regulate Ca2+-dependent processes in the pharyngeal muscles and increased GAR-3 signaling produces prolonged pharyngeal muscle contractions during pumping (gar-3 mutation only partially suppresses the prolonged peristalsis in eat-2 mutants, other mechanisms must also contribute to it. For example, voltage-activated K+ channels such as EXP-2 might not be efficiently activated in eat-2 mutants for rapid repolarization of the isthmus muscles (Extrasynaptic  pumping . Because muscles .ace-3C. elegans  mutants with reduced acetylcholinesterase activity produce prolonged peristalses that are dependent on GAR-3.At cholinergic synapses, unbound ACh is normally degraded by acetylcholinesterases located at the synapse [reviewed in eat-2 mutants, we cannot rule out that other defects in synapse formation or remodeling occur in these animals. Interestingly, loss of the γ-subunit of the nAChR leads to a diffuse distribution of acetylcholinesterase clusters during the development of mouse diaphragm muscles (eat-2 mutants.While MC morphology and synapse positioning is normal in 2+ levels in muscle cells have been observed in a number of human neuromuscular disorders, including myotonic dystrophies, Duchenne muscular dystrophy, as well as in ColQ and slow-channel myasthenic syndromes [reviewed in eat-2 and other mutants. Thus, eat-2 mutants may provide a new model to characterize the immediate effects of Ca2+ dysregulation in muscle cells.Increased cytoplasmic Ca


In contrast, USP17 depletion attenuates ELK-1-dependent gene expression and slows cell proliferation. The reduced rate of proliferation upon USP17 depletion appears to be a direct effect of ELK-1 ubiquitination because it is rescued by an ELK-1(K35R) mutant refractory to ubiquitination. Overall, our results show that ubiquitination of ELK-1 at K35, and its reversal by USP17, are important mechanisms in the regulation of nuclear ERK signalling and cellular proliferation. Our findings will be relevant for tumours that exhibit elevated USP17 expression and suggest a new target for intervention.ELK-1 is a transcription factor involved in ERK-induced cellular proliferation. Here, we show that its transcriptional activity is modulated by ubiquitination at lysine 35 (K35). The level of ubiquitinated ELK-1 rises in mitogen-deprived cells and falls upon mitogen stimulation or oncogene expression. Ectopic expression of USP17, a cell cycle-dependent deubiquitinase, decreases ELK-1 ubiquitination and up-regulates ELK-1 target-genes with a concomitant increase in  The ETS transcription factor ELK-1 is acutely stimulated by mitogens to establish a gene expression programme commensurate with cell proliferation . ELK-1 iLatent, nuclear ELK-1 acquires activity upon phosphorylation by MAPKs and deSUMOylation by PIASx . Target Developmental regulators are subject to multiple levels of control. Mechanisms that attenuate key transcription factors include binding of specific repressor proteins , post-trHere, we report a new mode of ELK-1 regulation involving its reversible ubiquitination. Mono-ubiquitin or low molecular weight ubiquitin species are conjugated to the ETS domain of ELK-1, predominantly to lysine 35 (K35). For convenience, we refer hereafter to these ELK-1-ubiquitin conjugates collectively as mono-ubiquitinated ELK-1 (ELK-mUBQ). In mitogen-stimulated or oncogene-expressing cells, i.e. when ERK activity is elevated, levels of ELK-mUBQ decline. We show that ELK-1 is a client of the ubiquitin-specific protease 17 (USP17) and that the two proteins interact directly. When expressed ectopically USP17 markedly decreases levels of ELK-mUBQ, augments ELK-1 activity and increases expression of ELK-1 target genes. Conversely, USP17 knockdown leads to the accumulation of ELK-mUBQ and attenuates ELK-1 transcriptional activity.USP17 expression is cell-cycle regulated, but its elevated expression is a hallmark of multiple cancers and has been linked to aggressive tumour phenotypes . We findl-glutamine, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. DU145 cells were grown in Dulbecco’s MEM (high glucose) with the same supplements. Whole cell extracts (WCEs) were prepared in a modified RIPA buffer; HeLa nuclear extracts were prepared as described earlier  supplemented with 10% foetal calf serum (FCS), 2 mM  earlier .Plasmids and sources are listed in the Cells were lysed in buffer containing 6 M guanidinium–HCl and protein–ubiquitin conjugates were captured by immobilized metal affinity chromatography (IMAC) on Nickel-Agarose beads (Qiagen) and washed in 8 M urea. After release from the beads conjugates were resolved by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by immunoblotting. The antibodies used are listed in In vitro ubiquitination assays were performed as described elsewhere (35S-labelled ELK-1 was generated by cell-free expression using the coupled TNT reticulocyte lysate system (Promega). After removal of unincorporated 35S-methionine by gel filtration , radio-labelled ELK-1 was incubated with UBQ (10 μg), rE1 (500 ng), rE2 , ATP (4 mM), DTT (1 mM), ubiquitin aldehyde (5 μM) and HeLa Nxt (15–30 μg) for 1 h at 30°C. Reactions were resolved on 8% SDS-PAGE gels, dried and visualized by phosphor-imaging (Fujifilm).lsewhere . Briefly−1) before harvesting and protein enrichment by denaturing IMAC. On-bead samples were reduced (50 mM dithiothreitol), alkylated (100 mM chloroacetamide) and digested with sequencing-grade trypsin (0.02 mg/ml). They were then submitted to tandem mass spectrometry (MS/MS) on an LTQ-Orbitrap-Velos spectrometer with nano-flow liquid chromatography (LC). Identification of peptides was conducted in data-dependent mode. The raw data file obtained from each LC-MS/MS acquisition was run against the Uniprot human database. Data were analysed and di-gly modified peptides (+114) identified using Scaffold proteome software, combining Mascot and X! tandem search engines to validate assigned spectra.HEK293T cells transfected with a vector encoding His-tagged ELK-1 were starved for 24 h or starved and stimulated by addition of 15% FCS and tetradecanoylphorbol acetate (TPA) (100 ng mlElectrophoretic mobility shift assays (EMSAs) were performed as described previously . SouthweFor reporter assays, cells in 24-well plates were lysed 36 h post transfection in passive lysis buffer; firefly and renilla luciferase expression were analysed with the Stop and Glow system (Promega). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses were performed as previously described . Gene-spCo-immunoprecipitations were performed as described previously . GST-USPn = 48).Nuclear and cytosolic fractions of HEK293T cells transfected with vectors for ELK-1 and control shRNA or shUSP17#1 were isolated as described previously (3 cells per well. Cell proliferation was monitored daily using an MTT assay (4 cells per well and counted after 4 days using the Moxi Z Mini Automated Cell Counter (Orflo).One day post transfection HEK293T cells were trypsinized, counted and re-seeded into multiple 96-well plates at a density of 2 × 10TT assay . Alternat-test. Proliferation (MTT) assays were analysed using ANOVA with Tukey’s multiple comparisons test. Significance is reported in figures by *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.Reporter assay, qRT-PCR, cell counting and high content imaging data are expressed as mean ± SEM. Statistical analyses were performed with Student’s ELK-1 and its neuronal-specific, truncated isoform sELK can be poly-ubiquitinated to mediate their degradation by the proteasome  29). Ho. Ho29). mapk inhibitor SB202190 did not  within the ETS domain, we made multiple lysine substitutions in ELK-1 or re-introduced lysines into the R18 mutant Figure . Only WTTo corroborate the functional mapping data, we used LC-MS/MS to identify modified lysines directly by di-glycine remnant mapping  37). We. We37). CFOS SRE   to increCFOS, an established ELK-1 target gene, when ELK-1(K35R) was expressed instead of ELK-1  with an active RAF-1 that mimics mitogen signalling and this activation was blunted by alanine substitutions at key ERK phosphorylation sites [ELK-13A)]. The transcriptional activity of ELK-1(K35R) (i.e. mutant NTR1) was significantly elevated over ELK-1 Figure , whereas]. The tr1 Figure . These fCFOS. USP17 expression markedly increased endogenous CFOS mRNA levels whereas catalytically inactive USP17 and other DUBs did not  , so ectot Figure . In agret Figure . In compt Figure ,45  but not control genes , whereas expression of USP22 had no such effect  mRNA were also observed in cells transfected with USP17 but not with USP22  appeared to have little effect Figure  and D. HIn response to mitogens, ELK-1 up-regulates a set of target genes to promote and support cell proliferation . While tin vitro mono-ubiquitination of ELK-1 seen with SRE duplex DNA. Ubiquitination of proteins associated with DNA is known and exemplified by RNF8 acting in the DNA damage response  responsible. Moreover, scrutiny of lysines involved in DNA binding  was compresponse ,50. Alteresponse .LC-MS/MS-based di-glycine remnant mapping  unequivoThe increase in ELK-mUBQ and partial redistribution of ELK-1 to the cytoplasm upon USP17 depletion is consistent with impaired DNA binding, as noted earlier for DNA-binding-defective ELK-1 mutants . HoweverApproximately 95 mammalian DUBs serve to reverse the actions of ubiquitin E3 ligases . Severalin vitro and USP17 expression caused ELK-1 de-ubiquitination, correlating with increased transcriptional activity, the specific expression of multiple ELK-1 target genes and the downstream cell cycle regulator CCND1 (cyclin D1)  . Conversclin D1) , and expclin D1) , this isUSP17 gene (DUB3) has a highly variable copy number within the human genome  substantially rescued the loss of proliferation associated with USP17 depletion. The effectiveness of ELK-1(K35R) in these experiments is perhaps surprising given that ELK-1 is just one of several USP17 clients and, arguably, may be functionally redundant to ELK-3 and ELK-4 . The impSupplementary DataClick here for additional data file.


HIV-1 Cap-RNA58 induced potent type I IFN responses in monocyte-derived DCs, monocytes, macrophages and primary CD1c+ DCs. Compared with RLR agonist poly-I:C, HIV-1 Cap-RNA58 induced comparable levels of type I IFN responses, identifying HIV-1 Cap-RNA58 as a potent trigger of antiviral immunity. In monocyte-derived DCs, HIV-1 Cap-RNA58 activated the transcription factors IRF3 and NF-κB. Moreover, HIV-1 Cap-RNA58 induced DC maturation and the expression of pro-inflammatory cytokines. HIV-1 Cap-RNA58-stimulated DCs induced proliferation of CD4+ and CD8+ T cells and differentiated naïve T helper (TH) cells toward a TH2 phenotype. Importantly, treatment of DCs with HIV-1 Cap-RNA58 resulted in an efficient antiviral innate immune response that reduced ongoing HIV-1 replication in DCs. Our data strongly suggest that HIV-1 Cap-RNA58 induces potent innate and adaptive immune responses, making it an interesting addition in vaccine design strategies.Strong innate and adaptive immune responses are paramount in combating viral infections. Dendritic cells (DCs) detect viral infections via cytosolic RIG-I like receptors (RLRs) RIG-I and MDA5 leading to MAVS-induced immunity. The DEAD-box RNA helicase DDX3 senses abortive human immunodeficiency virus 1 (HIV-1) transcripts and induces MAVS-dependent type I interferon (IFN) responses, suggesting that abortive HIV-1 RNA transcripts induce antiviral immunity. Little is known about the induction of antiviral immunity by DDX3-ligand abortive HIV-1 RNA. Here we synthesized a 58 nucleotide-long capped RNA (HIV-1 Cap-RNA DDX3Tat mRNA and subsequent formation of translation initiation complexes  and a 5′cap, but lacking a poly A tail , 34. The58). Our data strongly suggest that HIV-1 Cap-RNA58 induces potent type I IFN responses in monocyte-derived DCs, macrophages as well as primary CD1c+ DCs. Furthermore, HIV-1 Cap-RNA58 is a potent stimulus that induces both innate and adaptive immune responses in monocyte-derived DCs. HIV-1 Cap-RNA58-dependent induction of DC maturation and cytokine secretion leads to TH2 differentiation. Notably, HIV-1 Cap-RNA58 responses inhibited ongoing HIV-1 infection. Our data further define the importance of sensing abortive HIV-1 transcripts to evoke strong antiviral immunity and provide a rationale for using HIV-1 Cap-RNA58 in vaccine design strategies.To characterize the role of abortive HIV-1 RNA in establishing innate and adaptive immune responses without interference due to DC-SIGN inhibition, we developed a synthetic 5′capped HIV-1 RNA of 58 nucleotides that mimics the naturally occurring abortive HIV-1 RNA  . HIV-1 Cap-RNA58 and HIV-1 control RNA58 structures were predicted with the MFOLD program.The synthetic abortive HIV-1 RNAs were designed based on the HIV-1 genome. HIV-1 Cap-RNA+ monocyte isolation and subsequent generation of monocyte-derived DCs was performed as previously described  O/N at 37°C 5% CO2 for monocyte stimulations or cultured for 6 days in IMDM complete supplemented with GM-CSF  to obtain monocyte-derived macrophages. Stimulations were performed on day 1 (monocytes) or day 6 (monocyte-derived macrophages and DCs). CD1c+ DCs were isolated using human CD1c+ dendritic cell isolation kit (Miltenyi Biotec) according to manufacturer's instructions. Cells were stimulated with synthetic HIV-1 RNAs  complexed with transfection reagent lyovec (InvivoGen), polyinosinic:polycytidylic acid complexed with lyovec  and lipopolysaccharide (LPS) Salmonella enterica serotype typhimurium . Poly-I:C concentration series were performed with molecular weight ranges from 223801.1 to 895204.4 g/mol. Cells were pre-incubated with blocking IFNα/βR antibodies  and BAY 11–7082 (2 μM) for 30 min and 2 h, respectively.This study was performed according to the Amsterdam University Medical Centers, location AMC Medical Ethics Committee guidelines and all donors gave written informed consent in accordance with the Declaration of Helsinki. CD14escribed . CD14+ m2. Seventy-two hours after transfection, silencing of expression of target proteins in DCs was confirmed by quantitative real-time PCR and flow cytometry (RNA interference was performed using the Neon Transfection System according to manufacturer's protocol (Thermo Fisher). On day 4 of monocyte-derived DC cultures, cells were washed with PBS, resuspended in buffer R (Thermo Fisher) and divided according to the different short interfering (si) SMARTpool RNAs . siDDX3 (M-006874-01), siMAVS (M-024237-02) or siNon-Target as a control (D-001206-13) were added to DC-buffer R mixtures and transfection of DCs with the siRNAs was achieved by subjecting them to 1,500V for 20 ms. Transfected cells were seeded in 24-wells plates in RPMI 1640 with 10% FCS (Invitrogen) and 2 mM L-glutamine (Lonza), without antibiotics. After 48 h, viable cells were harvested, washed and seeded in a 96-wells round bottom plate and incubated overnight at 37°C, 5% COytometry  and cellGAPDH, according to the formula Nt = 2Ct(GAPDH)−Ct(target). For each donor, expression levels induced upon stimulation with HIV-1 Cap-RNA58 were set as 1.mRNA was extracted using an mRNA capture kit (Roche) and was reverse transcribed to cDNA using a reverse transcriptase kit (Promega). Quantitative real-time PCR was performed on an ABI 7500 Fast Real-Time PCR detection system (Applied Biosystems) using SYBR Green (Thermo Fisher), with primers that were designed using Primer Express 2.0  and stained with PE-conjugated anti-CD80 , allophycocyanin-conjugated CD83 , FITC-conjugated anti-CD86 , PE-conjugated anti-HLA-DR , or PE-Cy7-conjugated anti-CD40 . Expression levels after RNA interference were determined using anti-DDX3 or anti-MAVS  followed by PE-conjugated donkey anti-rabbit . HIV-1 infection levels were assessed using anti-p24 . Flow cytometric analysis was performed using the FACSCanto II (BD Biosciences) and FlowJo software v10.DCs were stimulated for 4 h, fixed with 4% pFA and permeabilized with 0.2% Triton X-100 in PBS. p65 was stained with anti-p65  and IRF3 with anti-IRF3 , followed by a secondary donkey anti-rabbit labeled with Alexa-546  and 1 μg/mL Hoechst (Invitrogen) and cellular localization was visualized with a 100x magnification, using Leica DM6 B upright microscope. Analysis was performed with LAS X Navigator software. Nuclear extracts (NE) were prepared 4 h after DC stimulation, using the NucBuster protein extraction kit (Novagen). Twenty micrograms of NE per sample was used to detect nuclear p65 or IRF3 using the TransAM NF-κB-p65 and IRF3 kits (Active Motif). OD450 nm values were measured using BioTek Synergy HT.DC supernatants were harvest 24 or 48 h after stimulation and secretion of TNF, IL-6, and IL-12p70 protein (eBiosciences) was measured by ELISA as described by manufacturer. OD450 nm values were measured using BioTek Synergy HT.+ and CD8+, CD3+ T cells. To examine TH cell differentiation, DCs were primed for 48 h with stimuli or LPS (10 ng/mL) in combination with IFNγ  or PGE2 , as positive controls for TH 1 and 2 skewing, respectively. DCs were cocultured in the presence of Staphylococcus aureus enterotoxin B  in a 1:4 ratio with allogeneic naïve T cells that were isolated from PBMCs or PBLs from buffy coats with a human CD4+ T cell isolation kit II (Miltenyi Biotec), with PE-conjugated anti-CD45RO (Dako) and anti-PE beads (Miltenyi Biotec) according to manufacturer's instructions. On day 5, IL-2 was added . After 11–13 days, resting T cells were restimulated with PMA  and ionomycin  for 6 h and treated with brefeldin A  for the final 4 h. Cells were fixed for flow cytometry analysis with 4% pFA, permeabilized using 0.1% saponin in PBS and stained with FITC-conjugated anti-IFNγ  and allophycocyanin-conjugated anti-IL-4  to determine TH 1 and 2 differentiation, respectively. Cells were analyzed using the FACS Canto II (BD Biosciences) and FlowJo software v10.To assess T cell proliferation levels, DCs were primed with stimuli and cocultured in a 1:4 ratio with allogeneic peripheral blood lymphocytes (PBLs) isolated from buffy coats of healthy donors (Sanquin) labeled with CellTrace Violet (Thermo Fisher). On day 3, IL-2 was added . After 5 days, cells were fixed with 4% pFA and stained with FITC-conjugated anti-CD3 , PerCP-Cy5.5-conjugated anti-CD4 , PE-Cy7-conjugated anti-CD8  and fixable viability dye eFluor780 . Proliferation was assessed in viable CD458 for 5 days, after which intracellular p24 levels were measured using flow cytometry to determine infection.DCs were infected with R5 HIV-1 strain NL4.3 BaL. NL4.3 BaL was produced as described previously , 37. Fort-test for paired  and unpaired observations  using GraphPad version 8. Statistical significance was set at P < 0.05.Statistics were performed using Student's 58 consists of the first 58 nucleotides common to all HIV-1 transcripts and contains a 5′cap while lacking a poly A tail (58 induced type I IFN responses in monocyte-derived DCs (DCs) by treating DCs with HIV-1 Cap-RNA58 complexed with transfection reagent lyovec (vehicle control) to facilitate cytoplasmic delivery   . Silencimulation . Both vepression . These d58 induces adaptive immunity, we first examined the expression levels of costimulatory molecules CD80, CD83, and CD86 after stimulation with HIV-1 Cap-RNA58, by flow cytometry. HIV-1 Cap-RNA58 induced expression of CD80, CD83, and CD86 compared to unstimulated DCs, albeit to a lesser extent than LPS . mRNA expression levels of IL6 and TNF peaked at 10 h, while the ISG IL27A, which encodes IL-27p28, a subunit of IL-27, peaked at 8 h  induced antiviral responses comparable to those induced by HIV-1 Cap-RNA58. Similar to HIV-1 Cap-RNA58, HIV-1 Cap-RNA630 induced IFNB 2 h after stimulation reaching a peak level after 10 h  transcription at similar levels as HIV-1 Cap-RNA58, peaking again at 10 h after stimulation  induced by coculture for 5 days with DCs that were treated with vehicle control, HIV-1 control RNA, HIV-1 Cap-RNA58 or LPS. Flow cytometry analysis showed that HIV-1 Cap-RNA58-activated DCs enhanced proliferation of both CD4+ and CD8+ T cells compared to untreated DCs and DCs treated with vehicle control or HIV-1 control RNA . Our data strongly suggest that synthetic HIV-1 Cap-RNA58 encapsulated in lyovec triggered DDX3 and MAVS-mediated IFNβ responses. It has been described that DDX3 has been found in complexes with RIG-I and MDA5 and might therefore induce IFNβ responses via a RIG-I or MDA5-MAVS-dependent way  cells  and subsequent differentiation of B cells in GCs (H2-induced IL-4 production can direct class switching from immunoglobulin G (IgG) to IgE antibodies  cells . Both TH viruses . TFH cels in GCs . Once a tibodies , 48. Recst HIV-1 –51. Alth+ T cell depletion. LPS as a potent immunostimulatory compound could be involved in inflammatory responses during chronic phase of infection (58 (58 in latent infected cells can also contribute to immune activation observed in HIV-1 infected individuals during chronic phase of infection.Microbial LPS is increased in serum of HIV-1 infected individuals due to intestinal damage upon CD4nfection , 53. Inttion (58 , 55. Our58 induces potent antiviral innate and adaptive immune responses in human DCs or directed by human DCs. Our data shows that DDX3 is a pattern recognition receptor and its synthetic ligands can be used as adjuvants to induce potent immune responses.In conclusion, DDX3 is a highly versatile protein involved in a multitude of cellular processes. During HIV-1 infection, the dual role of DDX3 in exerting both proviral and antiviral capacities provides insight in its complexity and to the various roles DDX3 might play in establishing immunity. Here we have identified the antiviral role of DDX3 upon sensing of a viral-derived RNA and how its ligands can be used as adjuvants. Our data strongly indicate that HIV-1 Cap-RNAAll datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by Amsterdam University Medical Centers, location AMC Medical Ethics Committee according to the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.MS designed, performed, and interpreted most experiments and prepared the manuscript. JS helped with study design. JH performed HIV-1 infections and subsequent FACS analyses. TK and EZ-W performed nuclear extract isolations. SG helped with study design and interpretation of data. TG supervised all aspects of this study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila to reveal secreted factors that influence systemic aging in distal tissues. Utilizing this model, we uncovered a cytokine—Diedel—that, when secreted from muscle or adipose, can attenuate age-related intestinal tissue degeneration by promoting proliferative homeostasis of stem cells. Diedel is both necessary and sufficient to limit tissue degeneration and regulate lifespan. Secreted homologs of Diedel are also found in viruses, having been acquired from host genomes. Focusing on potential mechanistic overlap between cellular aging and viral-host cell interactions, we found that Diedel is an inhibitor of apoptosis and can act as a systemic rheostat to modulate cell death during aging. These results highlight a key role for secreted antagonists of apoptosis in the systemic coordination of tissue aging.Aging is characterized by degeneration of unique tissues. However, dissecting the interconnectedness of tissue aging remains a challenge. Here, we employ a muscle-specific DNA damage model in  Aging in multicellular organisms is characterized by a progressive decline in the proper function of organs. This deterioration of organ function is a risk factor for many diseases. However, it is unlikely that organs age in isolation, as damage in one organ can presumably impact aging of other organs through either beneficial or detrimental cross-talk. Our work attempts to explore this aspect of aging using fruit flies as a model system. We uncovered that damaged fly muscle can protect against aging in other organs, such as the intestine, through the secretion of a blood-borne factor named Diedel. This blood-borne factor presumably allows damaged organs to communicate with each other during aging. Related factors are also found in certain viruses, which have been hijacked from insect genomes to promote viral spreading during infection. Using this information, we found that viral Diedel inhibits death of infected cells, allowing viruses to spread. Similarly, host (insect) Diedel also blocks cell death in organs during aging, thus limiting deterioration of organ function and extending the organism’s lifespan. In most metazoans, aging is characterized by a drastic decline in cellular and tissue function. This ultimate decline in function is driven by a multitude of complex, age-related cellular changes. The cellular and tissue changes that promote aging can be defined by (i) primary causes of cellular damage (such as genomic instability/DNA damage and telomere attrition), (ii) antagonistic responses to damage , and (iii) integrative responses that eventually promote tissue dysfunction . AddingAttenuation in regenerative potential is a defining characteristic of aged tissues and a primary driver of tissue dysfunction. This decline in regenerative potential originates from age-related changes in the regulation of stem cell function, which include stem cell exhaustion and/or deficient proliferation and differentiation, as well as stem cell hyperproliferation –3. ColleThe precise nature of communication between unique tissues with diverse functions, during aging, remains unclear. However, the systemic coordination of tissue aging and regeneration, or the ability of age-related changes in one tissue to alter aging in distal tissues, is constricted by both deleterious and beneficial consequences.Antagonistic mechanisms that respond to primary causes of age-related cellular damage also govern proliferative homeostasis to control tissue regeneration. This includes altered mitochondrial function and associated stress response pathways , cell senescence, altered programmed cell death/apoptosis, and changes in nutrient sensing , –13. CritThe utilization of these antagonistic stress response mechanisms is not limited to cell-autonomous/nonautonomous control of tissue function and regeneration. Similar signaling mechanisms play critical roles in pathogen-host cell interactions, especially those related to viruses. For example, host cells can employ mtDNA stress, ER/UPR stress, cell senescence, and/or apoptotic responses as antiviral mechanisms –17. In cDrosophila, we designed a model in order to characterize age-related systemic interactions between distal tissues that influence proliferative homeostasis, tissue degeneration, and longevity (described in detail in the section Hormetic responses from muscle-specific DNA damage attenuate systemic tissue aging and enhance lifespan). Using this fly model, we uncovered that Diedel, when secreted from muscle or adipose, can attenuate age-related intestinal tissue degeneration by promoting proliferative homeostasis of stem cells and that Diedel is both necessary and sufficient to limit tissue degeneration and extend lifespan. Focusing on potential mechanistic overlap between cellular aging and viral-host cell interactions, we found that Diedel and its viral homolog act as functionally conserved inhibitors of apoptosis. For the virus, virokine Diedel can limit apoptosis to facilitate viral abundance and spread. For the host, cytokine Diedel acts as a systemic rheostat to limit apoptosis-induced degeneration of the aging intestine. These results highlight a key role for secreted antagonists of apoptosis, specifically those acquired by viral genomes, in the systemic coordination of tissue aging.To this end, we show here that a secreted cytokine named Diedel, which has also been acquired by viruses , 22, is Drosophila, genetic manipulations in multiple individual tissues and unique cell types have been shown to impact longevity. In regard to systemic aging, muscle and fat body (adipose) appear to be critical effector/modulator tissues, and the brain can function as a control center though the regulation of secreted hormones ) or a recombinant version in which the putative signal peptide from the viral protein was replaced with the Drosophila signal peptide sequence  leads to up-regulation of nuclear factor kappa B . Dufunction –20, 49. Drosophila S2 cells with vesicular stomatitis virus , inflammation, and growth-factor signaling , we, weDrosoV5-conditioned media, Drosophila, as various apoptotic responses are mediated by initiator caspases  and effector caspases , as well as a family of inhibitor of apoptosis  antagonists  governed by Jun-N-terminal kinase (JNK) activity  controls) and (B) overexpression of Diedel using the Act88FGal4 driver (compare to Act88FG4>+[w1118] controls). (C) Mortality plots  associated with fat body specific inhibition of Diedel . These plots correspond to survival analysis found in Figs (TIF)Click here for additional data file.S3 FigRelated to Figs 1118) +RU486 compared with −RU486  sibling controls; (B) Act88FGS>UAS-Mei-9RNAi +RU486 compared with −RU486  sibling controls; (C) Act88FGS>UAS-Diedel +RU486 compared with −RU486  sibling controls. These plots correspond to survival analyses found in RNAi. (E) TubGS>+(w1118). (F) RU486 dose dependency of mortality in TubGS>Diedel female flies, utilizing 20 mM, 50 mM, and 100 mM doses of RU486. These plots correspond to survival analyses found in (TIF)Click here for additional data file.S4 FigRelated to Figs n = 4 independent samples) or (B) climbing  compared with Act88FG4>+(w1118) controls. (C-D) Knock-down of Mei-9 and ERRC1 concurrently  specifically in thoracic muscle has no effect on (C) lifespan  compared to Act88FG4>+(w1118) controls. (E-F) Knock-down of Mei-9 (UAS-Mei-9 RNAi) specifically in intestinal enterocytes  leads to tissue-autonomous (E) accumulation of DNA damage (measured by immunostaining with pH2AvD antibody in dissected midguts), (F) increases in intestinal stem cell mitoses , and (G) decreases in lifespan  controls. (H-I) (H) RPKM values for select, basally high genes that show no change upon muscle-specific depletion of MEI-9 in all of the unique tissue transcriptomes (thorax/muscle and intestine). Plotted on graph (I); log scale; each line represents a unique gene. (J-K) Intestinal immune gene induction during aging in Act88FG4>UAS-Mei-9RNAi flies, (J) Diptericin (“Dpt”) and (K) Drosomycin (“Drs”) measured in dissected midguts from young (10 d) and old (30 d) flies by qRT-PCR; bars represent mean ± SE, n = 3; compared with Act88FG4>+(w1118) controls. Underlying data can be found in (TIF)Click here for additional data file.S5 FigRelated to Figs n = 3; compared with Act88FG4>+(w1118) controls. (B) Diedel knock-down (UAS-Die RNAi) specifically in the thoracic muscle  has no effect on intestinal stem cell proliferation during aging  compared to Act88FG4>+(w1118) controls. (C) UAS-Diedel RNAi efficiency was evaluated in the fat body after sterile wounding. Diedel is induced after sterile wounding in the control flies (CGG4>+), and this induction is inhibited in CGG4>UAS-DieRNAi animals; bars represent mean ± SE; n = 3; whole flies were used in this analysis. (D) Knock-down of Mei-9 (UAS-Mei-9 RNAi) specifically in thoracic muscle  induces accelerated and age-related activation of a Diedel reporter; displayed by an increase in the proportion of Diedel-RFP-positive flies (DiedelP-RFP +) in Act88FG4>UAS-MEI9RNAi animals compared with Act88FG4>+(w1118) controls. (E) Knock-down of Mei-9 (UAS-Mei-9 RNAi) specifically in thoracic muscle  promotes elevated Diedel transcription in the carcass/fat body during aging  compared with Act88FG4>+(w1118) controls. (F) Diedel knock-down (UAS-Die RNAi) in the fat body  leads to premature intestinal stem cell hyperproliferation  compared to PplG4>+(w1118) controls. (G) Induction of Diedel (UAS-Diedel) or inhibition of Diedel (UAS-DieRNAi) specifically in the fat body (CGG4) or thoracic muscle (Act88FG4) has no effect on feeding behavior . (H-I) Diedel overexpression  inhibits some, but not all, IMD/NFkB pathway target genes (such as Dpt and Att but not Drs) and is correlated with a decrease in age-related commensal dysbiosis. (H) qRT-PCR of Drs, Dipt, and Att from young (10 d) and old (30 d) dissected midguts of flies with indicated genotypes; bars represent mean ± SE, n = 4; compared with Act88FG4>+(w1118) controls. (I) A 1/1,000 dilution of bacteria from dissected midguts from 30-d-old control flies (Act88G4>+[w1118]) or Act88FG4>UAS-Diedel flies plated on mannitol agar plates, 1 midgut per plate. Underlying data can be found in (TIF)Click here for additional data file.S6 FigRelated to Figs Drosophila Diedel (blue) identified in the genome of the indicated viruses (green). (B-C) Diedel is a secreted protein. Protein extracted from S2 cells transfected with empty plasmid (pAC5.1 V5-His) or a plasmid expressing tagged Diedel (DieV5), tagged ORF121 (ORF121V5), or tagged Recombinant ORF 121 (Rec-ORF121V5) was analyzed by western blot with anti-V5 antibody in either (B) cell lysate or (C) supernatant (conditioned media). (D-E) Protein extracted from S2 cells untransfected (Mock) or transfected with empty plasmid (pAC5.1), a plasmid expressing tagged Diedel (DieV5), tagged Upd1 (Upd1V5), tagged Upd2 (Upd2V5), or tagged Upd3 (Upd3V5) were analyzed by western blot with anti-V5 antibody in (D) cell lysate and (E) supernatant. (F-H) Drosophila S2 cells exposed to UV (100 mJ/cm2) and treated with mock or Diedel-V5 (+DieV5) conditioned media (F) Att, (G) Dipt, (H) Drs transcription (measured by qRT-PCR); bars represent mean ± SE, n = 4. (I-J) dsRNA targeted against RFP (control) or Diedel (“Die”) in S2 Cells exposed to UV (100 mJ/cm2) (I) Att and (J) Dipt measured by qRT-PCR; bars represent mean ± SE, n = 3. Underlying data can be found in (TIF)Click here for additional data file.S7 FigRelated toRNAi flies display elevated apoptosis at earlier time points during aging (20 d of age), monitored by Cas-3 immunostaining and TUNEL immunostaining in the posterior midgut compared with CGG4>+(w1118) and CGGal4>UAS-Diedel controls. (D) Representative images of Cas-3 immunostaining—Cas-3 (green), armadillo (red), and DAPI (blue)—(E) and TUNEL immunostaining (green), DAPI (blue). (F-H) PplGal4>UAS-DieRNAi flies display elevated apoptosis at earlier time points during aging (30 d of age) monitored by Cas-3 immunostaining and TUNEL immunostaining in the posterior midgut compared to PplG4>+(w1118) and PplGal4>UAS-Diedel controls. (F) Quantification of TUNEL immunostaining in the posterior midgut of flies with the indicated genotype; represented as dot plot ; n = 5–10. (G) Representative images of TUNEL immunostaining (green), DAPI (blue), and (H) Cas-3 immunostaining; Cas-3 (green), armadillo (Red), and DAPI (blue). Underlying data can be found in (TIF)Click here for additional data file.S8 FigRelated to2) and Diedel-V5 conditioned media (+DieV5) display an inhibition in JAK/STAT pathway activation. (A) Inhibition of Upd3 transcription and (B) Socs36e transcription  compared with cells exposed to mock-conditioned media (controls). (C) dsRNA targeting of Diedel (“Die”) in S2 cells leads to an increase in UV-induced (100 mJ/cm2) Upd3 transcription . (D) Schematic representation of Diedel action: Diedel inhibits apoptosis, which prevents Upd3 expression, leading to JAK/STAT pathway attenuation. (E) Induction of apoptosis in S2 cells leads to increases in Upd3 and Dronc transcription and JAK/STAT pathway activation (Soc36e) without JNK activation (Puc). dsRNA targeting RFP (Mock) or Diap1 in S2 cells; transcription of indicated genes measured by qRT-PCR, bars represent mean ± SE, n = 3. (F) Diedel does not attenuate JAK/STAT pathway activity directly (through influencing Upd/receptor binding). JAK/STAT pathway activation time course evaluated by Socs36e transcription measured by qRT-PCR in S2 cells exposed to indicated conditioned media; bars represent mean ± SE, n = 4. (G-J) Systemic Diedel inhibits JAK/STAT pathway activity in vivo, in the intestine, after Ecc15 infection and during aging by inhibiting Upd3 expression. JAK/STAT activation was evaluated with 10xSTAT GFP flies; (G) Act88FG4>UAS-Diedel, 10xSTAT GFP flies display an attenuation of JAK/STAT pathway activation 18 h after Ecc15 ingestion compared to controls . Representative images of posterior midgut; 10XSTAT-GFP (green), Armadillo (red), DAPI (blue). (H) Upd3 relative expression in dissected midguts of flies with indicated genotypes . (G) Act88FG4>UAS-Diedel, 10xSTAT-GFP flies display an attenuation of JAK/STAT pathway during aging (30 d of age) compared to controls . Representative images of posterior midgut; 10XSTAT-GFP (green), Armadillo (red), DAPI (blue). (H) Upd3 relative expression in dissected midguts of flies with indicated genotypes at indicated ages . (K) Inhibition of UV-induced proinflammatory cytokine by Diedel (conditioned media) in MEFs. IL-1β and TNFα transcription , compared with controls exposed to mock-conditioned media. Underlying data can be found in Drosophila inhibitor of apoptosis 1; Dronc, death regulator Nedd2-like caspase; dsRNA, double-stranded RNA; Ecc15, Erwinia carotovora carotovora 15; IL-1β, interleukin beta; JAK/STAT, Janus kinase/signal transducer and activator of transcription proteins; JNK, Jun-N-terminal kinase; MEF, mouse embryonic fibroblast; Puc, Puckered; qRT-PCR, quantitative real-time PCR; RFP, red fluorescent protein; Socs36e, suppressor of cytokine signaling 36E; TNFα, tumor necrosis factor alpha; Upd3, unpaired 3.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 TableRU486, mifepristone.(TIF)Click here for additional data file.S3 TableRU486, mifepristone.(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.