Tfap2a, Pax3 and other transcriptional regulator genes with transient function at earlier developmental stages. Tfap2a deletion in Ep400-deficient Schwann cells causes a partial rescue arguing that continued expression of early regulators mediates the phenotypic defects. Our results show that proper genomic distribution of variant histones is essential for Schwann cell differentiation, and assign importance to Ep400-containing chromatin remodelers in the process.Schwann cells ensure efficient nerve impulse conduction in the peripheral nervous system. Their development is accompanied by defined chromatin changes, including variant histone deposition and redistribution. To study the importance of variant histones for Schwann cell development, we altered their genomic distribution by conditionally deleting Ep400, the central subunit of the Tip60/Ep400 complex. Ep400 absence causes peripheral neuropathy in mice, characterized by terminal differentiation defects in myelinating and non-myelinating Schwann cells and immune cell activation. Variant histone H2A.Z is differently distributed throughout the genome and remains at promoters of  The Ep400 chromatin remodeler determines genomic distribution of variant histones. In the current study, the authors show that loss of Ep400 in Schwann cells leads to aberrant expression of developmental regulators, and a peripheral neuropathy phenotype. These include Tfap2a and Pax3 in the early neural crest and SC precursor stages, Sox2 in the immature, and Oct6 in the pro-myelinating stage as well as Krox20 as a central regulator of the final differentiation and myelination process2. Additionally, Sox10 is present and required throughout SC development from specification to maintenance of the mature myelinating state3.In the vertebrate peripheral nervous system (PNS), efficient conduction of nerve impulses requires Schwann cells (SCs) and their myelin. SC development from neural crest cells is coordinated by sets of transcriptional regulators with defined windows of expression4. In addition to histone modifications and alterations in nucleosome positioning, such chromatin changes usually involve region-specific exchanges of standard histones against variant histones that are performed by chromatin remodeling complexes of the SWR and INO80 type5. H2A.Z is such a variant histone that can replace H2A. H2A.Z has limited sequence identity to H2A so that its incorporation changes nucleosome properties and influences transcription. In cellular systems, activating and repressing effects on transcription initiation and elongation have been described for H2A.Z7. Very few studies exist on the role of variant histones in development9.SC development also depends on timely changes in chromatin structure11. It has also been reported to simultaneously replace the standard H3.1 by H3.3 in regulatory regions12. By deleting Ep400, the complex loses its chromatin remodeling activity, leading to defects in cell renewal, pluripotency, cell-cycle progression, cell survival, DNA repair, and apoptosis in cellular systems15.To address the importance of proper variant histone deposition for SCs and peripheral myelination, we deleted Ep400 at different times of SC development. Ep400 is the central ATP-hydrolyzing subunit of the prototypical SWR-type Tip60/Ep400 complex. This chromatin remodeler exchanges H2A.Z against H2A in nucleosomesEp400 in the mouse causes defects in late stages of SC development and peripheral myelination. Our results argue that altered genomic H2A.Z distribution leads to a failure to shut off early developmental regulators whose continued presence in differentiating SCs interferes with the maturation and myelination process.Here we show that SC-specific deletion of We generated antibodies against Ep400 to investigate its occurrence in SCs during development and PNS myelination. Starting at embryonic day (E) 12.5, Ep400 immunoreactivity was detected along spinal nerves in SCs marked by Sox10 expression. Ep400 remained present in Sox10-positive cells not only during prenatal development until E18.5  from approximately 0.65 in controls to 0.70 .In addition, we detected severe changes in myelin sheath length. Average internodal length in teased fibers was dramatically decreased to 56 µm in ols Fig. . Furtherker Fig. , we deteker Fig.  and a 19ice Fig. . Changes19 revealed normal SC numbers along the nerve of Ep400ΔPNS mice from birth until 2 months of age  analysis revealed that many markers of earlier phases of SC development exhibited comparable expression in sciatic nerves of control and Ep400ΔPNS mice at P9, P21, and 2 months , RNA samples from Ep400ΔPNS mice and controls clustered separately  analysis, upregulated genes mainly fell into one of three categories. A large fraction was associated with immune system, immune defense, and inflammatory response Fig. . Gene seEp400ΔPNS nerves revealed a substantial redistribution of promoter-associated H3K27ac modifications  on P9 sciatic nerves. H3K27ac-specific antibodies were used to identify transcribed genes as this histone modification is enriched in active promoters. By sorting genes according to H3K27ac presence around the transcriptional start site (TSS), we established a heatmap for gene transcription in control nerves Fig. , left. Aons Fig. , right. Ep400ΔPNS mice in which genes were ordered according to H2A.Z enrichment around the TSS in control samples  or enhancers (>1.5 kb away from the TSS). Among the 1654 promoters, 1588 were occupied by H2A.Z in both control and Ep400ΔPNS nerves . Closer ChIP-Seq data inspection revealed that H2A.Z peak changes in the vicinity of these genes were dramatic, correlated for all transcription factor genes with increased H3K27ac presence, and mostly affected the promoter regions as exemplarily shown for Tfap2a and Pax3  of the Krox20 gene was robustly activated by Sox10  mice were less affected than Ep400ΔPNS mice and survived longer  distribution in the vicinity of Krox20, the major myelin genes, and many other genes with expression in myelinating SCs exhibited little alteration in chromatin from Ep400ΔPNS nerves as compared to control arguing that they are not primarily affected by Ep400 deficiency. In contrast, H2A.Z distribution was dramatically and permanently changed in promoter regions of several transcriptional regulators that are normally active in neural precursors or SCs at early developmental stages  was provided via RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. To delete Ep400 in developing neural crest or SCs, floxed alleles for Ep40014 were combined in mice with a Sox10::Cre Bac transgene43 or a Dhh::Cre transgene17 to yield Ep400ΔNC and Ep400ΔPNS mice. In some Ep400ΔPNS mice, a floxed Tfap2a allele44 was additionally crossed in. Genotyping was as described44. Transgenic and control mice were on a mixed C3H x C57Bl/6J background and kept with continuous access to food and water under standard housing conditions in 12:12 h light–dark cycles. For bromodeoxyuridine (BrdU) or 5-ethynyl-2′-deoxyuridine (EdU) labeling, 100 µg BrdU  or EdU  per gram body weight were used. Mice were injected intraperitoneally with EdU 24 h and with BrdU 1 h before tissue preparation. Embryos were obtained at E12.5, E14.5, E16.5, and E18.5 and sciatic nerves at P0, P9, P15, P21, and at 2 months of age from male and female mice with relevant genotypes.Mouse housing and experiments were in accordance with animal welfare laws and all relevant ethical regulations and were approved by the responsible local committees and government bodies . The 45. Tissue fixation in 4% paraformaldehyde, transfer to 30% sucrose, and freezing in Tissue Freezing Medium (Leica)32 was followed by preparation of 10-µm transverse cryotome sections. In situ hybridization was performed with DIG-labeled antisense riboprobes specific for Tfap2a (positions 353–2056 acc. to accession number BC018226), Pou3f3 (positions 69–1582 acc. to accession number NM_138837), Pax3 (positions 396–1839 acc. to accession number NM_053710), and Mpz (positions 1–1864 acc. to accession number NM_001314068). The following primary antibodies were used in immunohistochemical stainings: rat anti-MBP monoclonal , rat anti-Pecam antiserum , rabbit anti-Caspr antiserum , guinea pig anti-Sox10 antiserum 46, rabbit anti-Ep400 antiserum , guinea pig anti-Krox20 antiserum , rabbit anti-Sox2 antiserum , rabbit anti-Oct6 antiserum , rabbit anti-Nav1.6 antiserum , rabbit anti-Iba1 antiserum , rabbit anti-cleaved caspase 3 antiserum , rabbit anti-Ki67 antiserum , rabbit anti-CD3 antiserum , rabbit anti-Desmin antiserum , rabbit anti-fibronectin antiserum , and mouse anti-α-smooth muscle actin antiserum . Secondary antibodies were coupled to Cy3 , Cy5 , or Alexa488  fluorescent dyes. Nuclei were counterstained with DAPI. Incorporated EdU was visualized by the Click-iT EdU Alexa Fluor 488 Imaging Kit  and incorporated BrdU by rat anti-BrdU monoclonal . TUNEL was performed according to the manufacturer’s protocol (Chemicon). Paraphenylene-2,6-diamine staining of sciatic nerve sections was as described47. Stainings were documented with a Leica DMI6000 B inverted microscope equipped with a DFC 360FX camera or a Leica DMIRE2 confocal microscope .Immunohistochemistry and in situ hybridization were performed on sections of the trunk region of mouse embryos and the sciatic nerve or on teased fibers from sciatic nervesg ratio of myelinated axons were determined.Sciatic nerves of control and genetically altered mice were dissected at P21 and at 2 months of age, followed by fixation in cacodylate-buffered fixative containing 2.5% paraformaldehyde and 2.5% glutaraldehyde, incubation in cacodylate-buffered 1% osmium ferrocyanide, dehydration, embedding in Epon resin, transverse or longitudinal sectioning, and staining with uranyl acetate and lead citrate. Fifty-nm sections were examined with a Zeiss Libra electron microscope . From electron microscopic pictures, the number of myelinated axons >1 µm and the Ep400ΔPNS mice. RNA samples were treated with DNase I to remove contaminating DNA. Quality and purity of samples were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies UK). Four hundred nanograms were used for library preparation (Ilumina Stranded mRNA Kit). Approximately 100 million reads per library were sequenced on an Illumina Hiseq 2500 platform  and aligned to mouse genome mm10 using STAR (version 2.5.1.b). Unique mappings to Ensembl Genes (Version 85) were counted using HTSeq. Differential expression analysis was carried out using the DESeq2 package (version 1.10.1) with default parameters in the R statistical environment. Gene expression values are deposited in GEO under accession number GSE119132.Total RNA was prepared from sciatic nerves obtained at P9 from control and Ep400ΔPNS sciatic nerve at P9 was performed using the GO enrichment, analysis, and visualization tool  or the Database for Annotation, Visualization and Integrated Discovery  with comparable results. In most cases, results from GORILLA are shown. GSEA from the Broad Institute was further used to determine whether the defined set of genes shows statistically significant, concordant differences between RNA samples from control and Ep400ΔPNS sciatic nerves (http://software.broadinstitute.org/gsea/index.jsp).GO analysis of genes upregulated and downregulated in SCs of the 48 with specific primers from the Tfap2a and Pax3 genomic regions . Sheared, quantified, and pre-cleared chromatin was incubated with rabbit antiserum against H3K27ac , rabbit antiserum against H2A.Z , or control rabbit IgG  and precipitated using bovine serum albumin-pretreated protein A sepharose beads. Crosslinks were reversed in an aliquot of input and the precipitated chromatin. DNA was purified by proteinase K treatment, phenol/chloroform extraction, and ethanol precipitation, and then used for standard qPCRq value <0.05). Single gene profiles were visualized in the Integrated Genome Viewer . Heatmaps and plotprofiles were created with the deeptools software. For illustrating average profiles over all genes, the computeMatrix scale region mode (version 2.5.0.0) was used by averaging genes with 2.5 kb upstream and downstream of the TSS. To detect ChIP-Seq peaks with differences in signal intensity in the control and Ep400ΔPNS condition, differential binding analysis of ChIP-Seq peak data was performed for all called peak regions identified with MACS2 in control and Ep400ΔPNS conditions using DiffBind (version 2.6.6). Gene coordinates were downloaded from the UCSC Table Browser (UCSC genes GRCm38/mm10) and used to create a genome-wide promoter list .Differential H2A.Z enrichment at gene promoters was determined by comparing the enriched or depleted peak regions in the Ep400ΔPNS condition  with GWPL using intersect (version 1.0.0). To detect ChIP-Seq peaks with differences in peak width in the control and Ep400ΔPNS condition ; all called peak regions identified by MACS2 in the control and Ep400ΔPNS conditions were separately combined with GWPL using join (version 1.0.0). Comparison of peak regions in control and Ep400ΔPNS conditions using the Venn diagram tool of the PBE homepage (http://bioinformatics.psb.ugent.be/webtools/Venn/) generated 3 different gene lists: genes shared in control and Ep400ΔPNS conditions, genes with peaks only in the Ep400ΔPNS condition, and genes with peaks only in the control condition. The list of genes shared in control and Ep400ΔPNS conditions was subsequently divided in genes with broader and higher peaks in the Ep400ΔPNS condition, genes with narrower and lower peaks in the Ep400ΔPNS condition, and genes with unaltered peaks between control and the Ep400ΔPNS condition. For further analysis, the lists of genes enriched in the Ep400ΔPNS condition and containing broader and higher peaks were merged, as were the list of genes depleted in the Ep400ΔPNS condition and containing narrower and lower peaks. GO analysis of genes differently occupied by H2A.Z in the control and Ep400ΔPNS condition was performed using GORILLA or DAVID with comparable results.Reads were mapped to the mouse reference genome (GRCm38/mm10) using Bowtie (version 2.3.4.2). After removing PCR duplicates with RmDup (version 2.0.1), mapped reads were converted to BAM format via SAMtools (version 1.1.2). BAM files were further processed using the BamCoverage tool within the deeptools software (version 3.0.2.0) and normalized to 1×. Experiments with anti-H2A.Z antibodies were performed in biological quadruplicates. ChIP-Seq with anti-H3K27ac antibodies were performed in duplicates. For peak calling, MA of ChIP-Seq (MACS2) (version 2.1.1.20160309.0) was used with input samples set as background and default parameters  using the primers listed in Supplementary Table 48. In pTATA-luc or pGL2 (Promega) based plasmids, luciferase reporters were under control of SC-specific regulatory regions from the Krox20 (MSE), Pmp22, Periaxin, Mag, Connexin32, and Mpz genes32. The pCMV5-based effector plasmids carried open-reading frames for Sox10, Krox20, Tfap2a, Pax3, Sox2, and Sox349. An amount of 0.5 µg was used for each reporter plasmid and 0.1 µg of effector plasmid per well on a 24-well plate. Overall amounts of plasmid were kept constant by adding empty pCMV5 where necessary. Whole-cell extracts were prepared 48 h posttransfection and luciferase activities were determined by detection of chemiluminescence as described48.Mouse Neuro2a neuroblastoma  were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and transfected with various combinations of luciferase reporter and effector plasmids using Superfect reagent (Qiagen)n ≥ 3 for all molecular biology experiments and experiments with mice as common for this kind of study except for ChIP-Seq studies with anti-H3K27ac antibodies (n = 2). No data were excluded from the analysis. Randomization was not possible. Investigators were not blinded in animal experiments. GraphPad Prism7  was used to determine whether differences in cell numbers, g ratios, transcript levels, or immunoprecipitated DNA were statistically significant by analysis of variance or unpaired, two-tailed Student’s t tests . The data met the assumptions of the chosen test. Variance between statistically compared groups was similar.Results from independent animals, experiments, or separately generated samples were treated as biological replicates. Sample size was Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


In this study we examined silencing of the distant imprinted gene Slc22a3 by the lncRNA Airn in the Igf2r imprinted cluster in mouse. Previously we proposed that imprinted lncRNAs may silence distant imprinted genes by disrupting promoter-enhancer interactions by being transcribed through the enhancer, which we called the enhancer interference hypothesis. Here we tested this hypothesis by first using allele-specific chromosome conformation capture (3C) to detect interactions between the Slc22a3 promoter and the locus of the Airn lncRNA that silences it on the paternal chromosome. In agreement with the model, we found interactions enriched on the maternal allele across the entire Airn gene consistent with multiple enhancer-promoter interactions. Therefore, to test the enhancer interference hypothesis we devised an approach to delete the entire Airn gene. However, the deletion showed that there are no essential enhancers for Slc22a2, Pde10a and Slc22a3 within the Airn gene, strongly indicating that the Airn RNA rather than its transcription is responsible for silencing distant imprinted genes. Furthermore, we found that silent imprinted genes were covered with large blocks of H3K27me3 on the repressed paternal allele. Therefore we propose an alternative hypothesis whereby the chromosome interactions may initially guide the lncRNA to target imprinted promoters and recruit repressive chromatin, and that these interactions are lost once silencing is established.Long non-coding (lnc) RNAs are numerous and found throughout the mammalian genome, and many are thought to be involved in the regulation of gene expression. However, the majority remain relatively uncharacterised and of uncertain function making the use of model systems to uncover their mode of action valuable. Imprinted lncRNAs target and recruit epigenetic silencing factors to a cluster of imprinted genes on the same chromosome, making them one of the best characterized lncRNAs for silencing distant genes  Airn silences genes in the Igf2r imprinted cluster, focusing primarily on silencing of the distant imprinted gene Slc22a3. We found that Airn expression blocks chromosome interactions between the Slc22a3 promoter and the Airn gene locus. By making a large genomic deletion including the Airn gene we showed that these interactions are not essential enhancer/promoter interactions, but may help to guide the Airn RNA to target genes to recruit epigenetic silencing. Our study adds to the understanding of how lncRNAs may act to silence distant genes.Long non-coding (lnc) RNAs are numerous in the mammalian genome and many have been implicated in gene regulation. However, the vast majority are uncharacterised and of uncertain function making known functional lncRNAs valuable models for understanding their mechanism of action. One mode of lncRNA action is to recruit epigenetic silencing to target distant genes on the same chromosome. A well-characterized group of lncRNAs that act in this way to silence genes are imprinted lncRNAs. In this study we examined how the imprinted lncRNA  Long non-coding (lnc) RNAs are a diverse and numerous group of non-protein-coding RNA species longer than 200 nucleotides, some of which have been shown to be involved in gene regulation ,2. A groIgf2r cluster expands from 120kb in most embryonic and adult tissues to almost 10Mb in placenta, while the Kcnq1 cluster expands from 250kb in embryonic tissues to 690kb in VYS . By. ByAirn RSDel region is correct, deletion of these enhancers on the maternal allele where Airn is not expressed should prevent expression of Slc22a3 on this chromosome. Slc22a3 silencing should also be maintained on the paternal allele when these enhancers are deleted  to distant imprinted genes in extra-embryonic tissues [Igf2r cluster in extra-embryonic tissues. Therefore, we performed H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) on VYS endoderm from FVB x CAST reciprocal crosses to determine the allele-specific distribution of this mark in the Igf2r cluster and throughout the genome. Using the Allelome.PRO pipeline to analyze the data [Igf2r cluster, despite imprinted expression in VYS endoderm being limited to a 450kb region from Slc22a3 to Airn  is not the same as an interaction between the Airn genomic locus and the Slc22a3 promoter on the maternal allele (detectable by 3C).Previously it has been shown in placenta using a technique derived from RNA FISH called RNA TRAP (Tagging and Recovery of Associated Proteins) that promoter . SurprisSlc22a3 and Slc22a3 in the Igf2r cluster are covered by a broad enrichment of the PRC2 mark H3K27me3, as are imprinted genes in the Kcnq1 cluster and in other imprinted clusters. Imprinted lncRNAs including Airn, Kcnq1ot1 and Meg3 have been reported to directly interact with PRC2 and EHMT2 [Airn-PRC2 interaction was reported in embryonic stem (ES) cells where Airn is very lowly expressed and no genes in the Igf2r cluster show imprinted expression [Kcnq1 cluster that show extra-embryonic specific imprinted expression [Dlk1 by Meg3 requires PRC2 [Igf2r imprinted silencing by Airn does not require PRC2 or EHMT2 [Slc22a3 and other members of the Igf2r cluster has not been tested, loss of EHMT2 has been shown to lead to loss of imprinted silencing of Slc22a3 [Here we show in VYS endoderm that the repressed alleles of nd EHMT2 ,20,21, apression ,20. PRC1pression ,38, and res PRC2 . Igf2r ior EHMT2 , but whiAirn may silence distant imprinted genes like Slc22a2, Slc22a3 and Pde10a by recruiting and targeted PRC1, PRC2 and EHMT2 to these genes to deposit repressive chromatin modification and cause silencing. This indicates that Airn silences imprinted genes in the Igf2r cluster by two different mechanisms: Airn transcription silences Igf2r by transcription interference that does not require repressive chromatin modifying complexes [Airn RNA product recruits repressive chromatin modifying complexes and targets them to distant, non-overlapped genes like Slc22a2, Slc22a3 and Pde10a to cause silencing. Importantly, the mechanism for targeting silencing remains unknown.Although it is technically difficult to exclude a role for transcription of the lncRNA versus the RNA product, together these results indicate that imprinted lncRNAs like omplexes , and theAirn gene body and the Slc22a3 promoter are enriched on the maternal allele because Airn expression represses these interactions on the paternal allele. We showed that these interactions are not required to upregulate Slc22a3 expression on the maternal allele, indicating that they are not essential promoter-enhancer interactions, but they may serve to place the Airn locus and Slc22a3 promoter in close proximity in the nuclear space. Chromosome interactions can exist in the ground state or be formed during development [Airn locus and the promoter of Slc22a3 , are present on both alleles  Protocol on the Protection and Welfare of Animals. The Animal Research is covered by Federal Austrian legislation  and the overriding EU and international legislation and codes of conduct. No experimental procedures were performed on the animals so no extra permissions were required.Thp) mouse (EM:09898) contains an minimum 5.56Mb deletion on chromosome 17 that includes the Igf2r imprinted cluster allowing parental allele-specific analysis [AirnT) mouse (EM:09895) has a polyadenylation cassette inserted into the Airn gene, 3kb downstream from its start site causing it to be truncated and non-functional [R2Δ) mouse (EM:09897) has a deletion that includes the Airn promoter and the imprint control element (ICE) of the Igf2r imprinted cluster [Thp, AirnT and R2Δ mice have been cryopreserved by the EMMA mouse repository (EMMA ID indicated). The Sod2-flox mice contain loxP sites flanking the exon 3 of Sod2 and were made in a 129 ES cell line [FVB/NJ (FVB) mice were obtained from Charles River. CAST/EiJ (CAST) mice were obtained from the Jackson Laboratory. The FVB.AK-Del(17)T<hp>  was isolated from E9.5 and E12.5 embryos under a dissection microscope. The whole VYS was used for the chromosome conformation capture (3C experiments), while for RNA isolation and for chromatin immunoprecipitation (ChIP) the VYS endoderm was mechanically separated away from the rest of the VYS after 1–2 hours of DispaseII digestion at 4°C, as previously described .Igf2r imprinted locus we used reciprocal crosses of Thp and FVB mice [Airn on interactions we used Thp x AirnT cross, where AirnT mice have a truncated and non-functional Airn [Igf2r imprint control element (ICE). Around 28 VYS were then pooled per genotype and thawed on ice and then incubated with 8ml lysis buffer for 15 minutes on ice (1 tab Complete Protease Inhibitor (Roche) per 25ml lysis buffer ). The samples were then dounced in a 15ml glass dounce (Wheaton) with a loose pestle about 30 times, and then 30 times with a tight pestle, before centrifuging for 5 minutes at 2000g at 4°C. The supernatant was then removed leaving a nuclear pellet, which was then resuspended in 1 x EcoRI buffer (Fermentas). Samples were then subject to EcoRI digestion and ligation as previously described [Chromosome Conformation Capture (3C) was performed following established protocols with minor modifications ,49. To aFVB mice . To detenal Airn . We collnal Airn . Brieflyescribed . The forSlc22a3 promoter) and a “prey” primer was designed near the EcoRI site for fragments in the target region (e.g. Airn gene body). All 3C interactions detected in the Igf2r imprinted cluster (Thp deletion region on chromosome 17) were normalized by dividing by the mean of 2 interactions with the H19/Igf2 ICE, an independent locus on chromosome 7. To correct for technical and biological variation between experiments the highest interaction level was then set to 1. The primer/probe combinations used are given in We detected 3C interactions by Taqman quantitative PCR following a previously published protocol with minor modifications , and by Tissue from VYS endoderm or placenta was collected and homogenized in TRI reagent, and total RNA isolated according to the manufacturers protocol (Sigma-Aldrich).RSDel x FVB reciprocal crosses was DNase treated using the DNA-free kit (ThermoFisher Scientific), and then converted to cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was then conducted using either a Taqman or SYBR Green system using the standard curve method to analysis the data, and normalization to a house keeping gene (cyclophillin A). The RT-qPCR results in Total RNA from E9.5 VYS endoderm and E12.5 placenta collected from RSDel x CAST reciprocal crosses using the TruSeq RNA Sample Prep Kit v2 (Illumina) modified as previously described [RSDel x CAST, maternal deletion cross) and 3x WT and 3x Del . Native ChIP was performed using an H3K27me3 antibody  on E12.5 VYS endoderm from FVB x CAST reciprocal crosses  as previously described [Strand-specific polyA enriched RNA-seq libraries were generated from E12.5 placenta and VYS endoderm from escribed . For eacescribed . ChIP-seescribed .RSDel mouse was backcrossed to FVB, but the region around the Igf2r cluster is likely to have a 129 background as both the R2Δ and Sod2Δ alleles from which the RSDel mouse is derived were made in 129 ES cells [Allele-specific expression and histone modification enrichment was detected from RNA-seq and ChIP-seq data using the Allelome.PRO program . The SNPES cells ,30. TherWe used the following Allelome.PRO parameters for our analysis:RNA-seq: minread 2 , RefSeq annotation.H3K27me3 ChIP-seq enrichment: FDR 1%, allelic ratio cutoff 0.7, minread 1, 20Kb sliding windows.Note: minread = minimum number of reads that must cover a SNP for it to be included in the analysis.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableChromosome Conformation Capture (3C) quantitative PCR (qPCR) raw data and analysis for (XLSX)Click here for additional data file.S4 TableChromosome Conformation Capture (3C) quantitative PCR (qPCR) raw data and analysis for (XLSX)Click here for additional data file.S5 TableReal time quantitative PCR (RT-qPCR) raw data and analysis for (XLSX)Click here for additional data file.S6 TableReal time quantitative PCR (RT-qPCR) raw data and analysis for (XLSX)Click here for additional data file.


MYC translocations and overexpress MYC but both differ significantly in their clinical outcome. In DLBCL, MYC translocations are associated with an aggressive behavior and poor outcome, whereas MYC-positive BL show a superior outcome.MYC is a heterogeneously expressed transcription factor that plays a multifunctional role in many biological processes such as cell proliferation and differentiation. It is also associated with many types of cancer including the malignant lymphomas. There are two types of aggressive B-cell lymphoma, namely Burkitt lymphoma (BL) and a subgroup of diffuse large cell lymphoma (DLBCL), which both carry To shed light on this phenomenon, we investigated the different modes of actions of MYC in aggressive B-cell lymphoma cell lines subdivided into three groups: (i) MYC-positive BL, (ii) DLBCL with MYC translocation (DLBCLpos) and (iii) DLBCL without MYC translocation (DLBCLneg) for control. In order to identify genome-wide MYC-DNA binding sites a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed. In addition, ChIP-Seq for H3K4me3 was used for determination of genomic regions accessible for transcriptional activity. These data were supplemented with gene expression data derived from RNA-Seq.ADGRE5  - a member of the EGF-TM7 subfamily of adhesion G protein-coupled receptors - as a MYC target gene, which is specifically expressed in BL but not in DLBCL regardless of MYC translocation.Bioinformatics integration of all data sets revealed different MYC-binding patterns and transcriptional profiles in MYC-positive BL and DLBCL cell lines indicating different functional roles of MYC for gene regulation in aggressive B-cell lymphomas. Based on this multi-omics analysis we identified MYC breaks in DLBCL. Since ADGRE5 plays an important role in tumor cell formation, metastasis and invasion, it might also be instrumental to better understand the different pathobiology of BL and DLBCL and help to explain discrepant clinical characteristics of BL and DLBCL.Our study describes a diverse genome-wide MYC-DNA binding pattern in BL and DLBCL cell lines with and without MYC translocations. Furthermore, we identified ADREG5 as a MYC target gene able to discriminate between BL and DLBCL irrespectively of the presence of The online version of this article (10.1186/s12885-019-5537-0) contains supplementary material, which is available to authorized users. MYC translocations in almost all cases. The tumor is predominantly found in male children but may also occur in adults especially with a compromised immune system. Treatment of BL is mainly based on high dose chemotherapy with usually favorable clinical outcome  using titanium rods combined with a Bioruptor Sonicator . After addition of 300 μL 10% (vol/vol) Triton X-100 and centrifugation the supernatant was removed, 50 μL of which were stored as input DNA sample. 1.5 mL of the supernatant was incubated with 10 μg MYC antibody or 5 μg H3K4me3 antibody at 4 °C overnight. For ChIP antibody information, refer to Additional file United States). Single-read NGS was done on an Illumina HiSeq 1500 system (50 cycles). Illumina adapters were trimmed from the raw sequence data and low quality bases and reads were removed with trimmomatic (LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36) [MYC peaks were called with MACS2 [https://sites.google.com/site/anshulkundaje/projects/blacklists). Differential peaks between DLBCL with and without MYC break and BL were estimated using DiffBind [Chromatin immunoprecipitation (ChIP) was done according to published protocols , 12 withNLEN:36) . SequencNLEN:36) . H3K4 anth MACS2 , keepingDiffBind  tool.6 lymphoma cells, which were previously spiked in with 1 × 105 insect cells (Schneider cells) for data calibration [United States). 500 ng RNA per sample were processed using the Illumina TruSeq Stranded mRNA LT Sample Prep Kit following the manufacturer’s instructions to generate libraries for RNA sequencing. Samples were sequenced on a Hi-Seq 4000  using the Illumina HiSeq 3000/4000 SBS 150 cycle kit. Sequence reads were aligned to a concatenated genome that consisted of the human (GRCh38) and the Drosophila melanogaster (BDGP5) reference genome, using STAR alignment tool [Drosophila genes. Before differential gene expression analysis, we calculated DESeq2 sample sizeFactors [Drosophila gene counts and applied them to the human sample data. This way, gene expression levels of the cell lines were calibrated to the number of sample cells. Then, gene expression levels were modeled with a generalized linear model assuming negative binomial distributed data and categorical variables for the lymphoma type (BL or DLBCL) and MYC status (MYC break positive or negative). Gene expression changes were tested for significance with the Wald test and fold changes with an associated False Discovery Rate (FDR) below 0.05 were considered significant differentially expressed.Total RNA was isolated from 1 × 10ibration . The quaent tool  with defent tool  with defeFactors  on the DThe SWATH-MS-based quantification of the proteins ADGRE5, BYSL and NPM1 was obtained from previously published data . SWATH-Mn = 12) and primary tissue samples (n = 38). The use of human primary tissue samples was approved by the Institutional Review Board of the Charité – Berlin (EA4/104/11). The immunostaining carried out using the Leica Bond-maX autostainer  according to the manufacturer’s protocol. After heat-induced epitope retrieval, the sections were incubated with anti-c-myc and anti-CD97 (ADGRE5) rabbit antibodies, respectively (dilution 1:200). Horseradish peroxidase-labeled Anti-rabbit-IgG using the Bond Polymer Refine Detection Kit  was employed to convert the chromogen substrate. Staining was performed with appropriate positive and negative controls.Immunohistochemical staining was performed using sections derived from formalin-fixed paraffin-embedded cell line blocks  were carried out in order to locate genomic areas with open chromatin as indicators for potential transcriptional activity of nearby genes , 29. To MYC break positive  than MYC break negative (DLBCLneg) cells  cell lines compared to MYC break negative (DLBCLneg) cell lines.MYC-binding is not the sole factor for activating of gene expression and associated functional consequences. In order to gain a deeper insight into the transcriptional landscape and the impact of the various MYC-binding patterns, we performed RNA-Seq and correlated the results with the presence of ZAP-70, ADGRE5, CDK20, GPAM, SMAD1 and TERT were the most interesting. Genes lacking differentially expression such as LARS, FARSA and already described as MYC target genes like BYSL [NMP1 [To validate the data derived from genome-wide DNA-binding and gene expression, we performed additional gene-specific ChIP (MYC and H3K4me3) and RT-PCR experiments . Figure Here we describe the impact of MYC in three types of aggressive B-cell non-Hodgkin lymphomas: BL and DLBCL with and without MYC break . More MYC-binding sites were found by MYC ChIP-Seq in BL and DLBCLpos as compared to DLBCLneg. Interestingly, MYC was found to be bound to different target genes in BL and DLBCLpos, which is also reflected by their gene expression differences. By combined analyses, ADGRE5 (CD97) was identified as an interesting differentially expressed MYC target gene, an observation confirmed by immunohistochemistry of primary FFPE patient samples. Its expression, in particular that of the largest ADGRE5 isoform , was significantly higher in BL than DLBCL. Based on the reported function of the EGF4 repeat as a receptor for chondroitin sulfate, we hypothesize that this might contribute to some peculiar features of BL, namely macrophage attraction and angiogenesis, and potentially to the very good responsiveness to treatment. In addition, we suggest ADGRE5 as a marker to discriminate between BL and DLBCL (regardless of the presence of MYC breaks) in patient stratification.Additional file 1:Table S1. Compilation of antibodies used for Chromatin Immunoprecipitation (ChIP), Western Blot (WB) or Immunohistochemistry (IHC) and list of TaqMan Assays used for RT-PCR analysis. *indicated endogenous control. (PDF 42 kb)Additional file 2:File S2. ChIP-Seq data of differentially bound MYC and H3K4 target genes derived from comparison of BL vs. DLBCL, BL vs. DLBCLneg, DLBCLneg vs. DLBCLpos and BL vs. DLBCLpos. (XLSX 1673 kb)Additional file 3:File S3. RNA-Seq data of differentially expressed genes derived from comparison of BL vs. DLBCL, BL vs. DLBCLneg, DLBCLneg vs. DLBCLpos and BL vs. DLBCLpos. (XLSX 13003 kb)Additional file 4:Figure S4. ADGRE5 IHC staining of cell line and patient tissue samples. (A) Burkitt Lymphoma (BL): Cell lines  and primary tumor tissues (7–14) are manly ADGRE5 positive. (B) Diffuse large B cell lymphoma without MYC break (DLBCLneg): Cell lines  and primary tumor tissues (5–19). (C) DLBCLpos: Cell lines  and primary tumor tissues (4–6). DLBCLpos and DLBCLneg are manly negative for ADGRE5. Strong positive staining in some tissue sections results from macrophages or T-cells. (PDF 4769 kb)


Chromatin upstream from fbp1 is progressively altered into an open configuration, as a cascade of transcription of three overlapping mlonRNA species  occurs with transcriptional initiation sites progressing 5′ to 3′ upstream of the fbp1 promoter. Initiation of the shortest mlonRNA (mlonRNA-c) induces chromatin remodeling around a transcription factor-binding site and subsequent massive induction of fbp1. We identify the cis-element required for mlonRNA-c initiation, and by changing the distance between mlonRNA-initiation site and the transcription factor-binding site, we show that mlonRNA-initiation effectively induces chromatin remodeling in a limited distance within 290 bp. These results indicate that mlonRNAs are transcribed across the fbp1 promoter as a short-range inducer for local chromatin alterations, and suggest that strict chromatin modulation is archived via stepwise mlonRNA-initiations.Long noncoding RNAs (lncRNAs) transcribed across gene promoters have been detected. These regulate transcription by mechanisms that have not been fully elucidated. We herein show that the chromatin configuration is altered into an accessible state within 290 bp downstream from the initiation site of metabolic-stress-induced lncRNAs (mlonRNAs) in the promoter of the fission yeast  Various functions of such RNAs have been identified in a range of biological processes3. lncRNAs transcribed within gene promoters play a role in the regulation of neighboring genes3. Several lncRNAs interact with polycomb repressive complex 2 (PRC2) and recruit it to target genes, leading to methylation of histone H3K27 following chromatin compaction4. Intergenic noncoding transcription at the budding yeast SER3 gene promoter represses the expression of this gene5, and this repressive activity was observed even when >90% of the lncRNA sequence was replaced6. These data indicate that RNA polymerase II (RNAPII) transcription in the regulatory region is sufficient to mediate repression, and that lncRNA itself does not play a direct role. In Schizosaccharomyces pombe (fission yeast), upon glucose starvation, stepwise expression of lncRNAs at the fbp1 gene promoter plays a critical role in chromatin modulation and subsequent gene activation7. This activation is mediated through two distinct mechanisms: (1) the lncRNA itself interacts with Tup1-like corepressors and thereby antagonizes the repressive function of the Tup1-like corepressors and facilitates binding of the Atf1 transcription factor8, and (2) RNAPII-mediated transcription of the lncRNAs mediate chromatin remodeling and further enhance Atf1 transcription-factor binding9.Recent transcriptome analyses have revealed that most regions in the human genome are transcribed into RNAs, of which RNAs longer than 200 nucleotides possessing mRNA-like structure (carrying cap-structure and poly-A tail) without protein-coding potential are referred to as long noncoding RNA (lncRNA)fbp1 gene as a result of glucose starvation stress is mediated by two transcription factors: Atf1 and Rst211. Upon glucose starvation, these transcription factors bind to critical cis-acting binding sequences far upstream from the fbp1 promoter (upstream-activating sequences 1 [UAS1] and 2 [UAS2])13 13 Fig. . In thisfbp1 transcriptional activation upon glucose starvation, the chromatin state far upstream from the fbp1 promoter is progressively altered into an open configuration. In this process, several species of mlonRNAs are transcribed in a stepwise manner with transcriptional initiation sites progressing in a 5′ to 3′ direction, thus inducing chromatin remodeling along the same tract9. However, how such stepwise mlonRNA transcriptions mediate chromatin remodeling is not well-understood. To investigate the significance of these stepwise mlonRNA transcriptions, we sought to selectively disrupt one of the transcripts in a cascade of transcription of three overlapping mlonRNAs. Since the initiation sites of mlonRNA–a and –b are close to essential cis-element, UAS1, and selective disruption of cis-elements for these mlonRNAs was difficult, we focused our efforts on disrupting the shortest mlonRNA-c transcription. To this end, we searched for the cis-element required for mlonRNA-c initiation by replacing 10–15 bp segments with act1 ORF sequences in the region upstream from the mlonRNA-c transcription initiation site   exhibit a drastic reduction in fbp1 mRNA transcription  Fig. . Moreoveion Fig. . These dmlon-c-replacement/mlon-b(190 bp); Fig. mlon-c-replacement cells described in Fig. mlon-c-replacement/mlon-b(190 bp) cells, the distance of the corresponding region is 190 bp, a distance similar to that of mlonRNA-c and UAS2 in wild-type cells (197 bp)  Fig. . By placred Fig. .Figure 2mlon-c-replacement cells exhibit less prominent MNase sensitive region around UAS2-TATA box ), MNase sensitivity around UAS2-TATA box region is completely restored to wild-type level )  to measure sensitivity to MNase reflecting open chromatin configuration. In wild-type cells, MNase sensitive sites appear near UAS1 followed by sites in the UAS2-TATA box region Fig. . In markbox Fig. . Howevervel Fig. . Consistmin Fig. . This hip)) Fig. .Figure 3mlon-c-replacement cells exhibit delayed and reduced histone acetylation in this region  cells  and this cell line shows very similar kinetics to those of the wild-type cells , mlon-c-replacement/mlon-b(290 bp), mlon-c-replacement/mlon-b(340 bp), and mlon-c-replacement/mlon-b(390 bp) cells  cells exhibit higher or wild-type levels of fbp1 mRNA induction, whereas the mlon-c-replacement/mlon-b(340–509 bp) cells show significantly lower levels of induction compared to wild-type cells . The higher level of fbp1 expression in the mlon-c-replacement/mlon-b(190 bp) strain might be attributable to the alteration of genome geometry in this region by sequence deletion, since fbp1 activation is also affected by the local DNA-loop that brings UAS1 and UAS2 into close spatial proximity16. We next evaluated histone disassembly at 60 min after glucose starvation in the cell lines with various segment lengths between the mlonRNA-b initiation site and UAS2. In the wild-type cells, histone H3 binding is reduced to below 30% of the original state. In the mlon-c-replacement cells with 190 bp, 240 bp, and 290 bp segments between the mlonRNA-b initiation site and UAS2, histone disassembly is very similar to that of the wild-type. In marked contrast, the mlon-c-replacement cells with the 340, 390, and 509 bp segments exhibit histone binding levels of 50%, 60%, and 75%, respectively . These data suggest that mlonRNA initiation efficiently induces histone disassembly within a limited distance of ~290 bp from the initiation site, while chromatin remodeling gradually decreases as the distance from the mlonRNA initiation site increases beyond 290 bp.We next sought to identify the limitation in the distance from mlonRNA initiation to induce chromatin remodeling at UAS2. To this end, we examined various distances  between the mlonRNA-b initiation site and UAS2 by generating lls Fig. . We founses Fig. . The mlofbp1 gene is preceded by RNAPII-mediated transcription of a series of lncRNAs, referred to as mlonRNAs, in a stepwise manner. Transcription of the mlonRNAs induces chromatin remodeling in their transcribed regions, thereby contributing to the access of transcription factors to the fbp1 regulatory elements7. In this study, we identified the cis-acting sequence required for transcriptional initiation of the mlonRNA-c transcript, which is the transcript nearest to the fbp1 promoter. Loss of mlonRNA-c transcription due to inactivating this sequence resulted in a critical defect in chromatin remodeling at the Rst2 transcription-factor binding site located downstream from the mlonRNA-c initiation site. These results indicate the pivotal role played by mlonRNA transcriptional initiation in chromatin remodeling, as the mlonRNA-b transcript overlaps with the mlonRNA-c transcript, showing that transcriptional elongation through this region is not sufficient to promote chromatin remodeling. An interesting possibility is that the RNAPII complex that transcribes mlonRNA might be distinct from the usual RNAPII complex associated with the TATA box, and such mlonRNA transcribing RNAPII may play a role in inducing chromatin remodeling. Should this be true, this mlonRNA-RNAPII would function locally, around the initiation site, since RNAPII accumulates around its transcription-start site and dissociates from the initiation complex after promoter clearance17. This is indeed the case, as we found that mlonRNA initiation efficiently induces chromatin remodeling only within 290 bp of the initiation site  strain  the mlonRNA-c initiation site and the cis-element (UAS2) required for fbp1 mRNA are 192 bp and 197 bp, respectively. Stepwise transcriptional activation of several species of mlonRNA may be responsible for converting chromatin into an open configuration upstream from fbp1, thereby contributing to the strict binding-order regulation of transcription factors. The transcription factor Rst2 binding is mediated by a local DNA-loop that brings UAS1 and UAS2 into close spatial proximity16. The formation of an open chromatin configuration upstream from fbp1 is a prerequisite for the formation of this higher-order structure. Thus, the strict regulation of chromatin configuration by a cascade of mlonRNA transcriptions and the subsequent formation of a local DNA-loop structure should cooperatively control fbp1 transcription. Limiting the range in which mlonRNA can effectively induce chromatin remodeling may contribute to this strict chromatin modulation by prohibiting improper chromatin opening. This stepwise regulation via the cascade of mlonRNA transcriptions within the range limitation for each mlonRNA initiation may mediate this strict modulation of gene expression and the appropriate response to glucose starvation stress23.We note that the positional relationships between each of the mlonRNA initiation sites and their target elements are very similar, as evidenced by the fact that the distances between (1) the mlonRNA-b initiation site and the S. pombe core environmental stress response (CESR) gene, the first nucleosome downstream from the transcription-start site (+1 nucleosome) is removed during gene activation. This process is mediated in a histone H3 acetyltransferase, Gcn5 dependent manner24. Gcn5 is recruited through Atf1 binding at UAS1 in the fbp1 gene and is involved in the activation of this gene9. It is thus possible that chromatin eviction at UAS2 through mlonRNA-c initiation is mediated by a process involving Atf1 and Gcn5. This hypothesis is supported by the fact that expression of mlonRNA-c and subsequent chromatin remodeling is missing in atf1− cells7. Interestingly, loss of the Tup1-like corepressors in atf1− cells totally rescues the defect in fbp1 induction without recovering mlonRNA-c initiation7. These data suggest a possible counteractive regulation between mlonRNA-initiation-mediated chromatin opening and Tup1-like-corepressor-mediated chromatin compaction. Since mutant fission yeast cells lacking Tup1-like corepressors show inappropriate, nonspecific fbp1 transcription25, this counteractive regulation might be pivotal for precise fbp1 activation resulting from environment stresses.In the 26. Transformation was performed using the lithium-acetate method as previously described27.The fission yeast strains used in this study are listed in Supplementary Table The primer sequences used in this study are listed in Supplementary Table fbp1 promoter region including the mlonRNA-c upstream sequence was amplified using a primer set (p1 and p2) and cloned in pCR®-Blunt II-TOPO® (Invitrogen). Replacement of the region upstream from mlonRNA-c with act1 ORF was performed by PCR using the primer sets described in Supplemental Table ura4 marker gene in the fbp1 promoter were transformed using plasmid carrying the fbp1 upstream sequence, in which a part of the segment upstream from the mlonRNA-c initiation site was replaced with the act1 ORF sequence. To isolate the fbp1 upstream-sequence replacement cells, transformants were selected for uracil auxotrophy using SD plates containing 5-FOA and uracil.The  p1 and p and clonmlon-c-replacement cells with various deletion lengths in the region between the mlonRNA-c initiation element and UAS2, we deleted regions -937 to -619, -669, -719, -769, and -819 (relative to the first A in the ORF as +1) by PCR, using the primer sets p31 and p32 to p36 as described in Supplemental Table fbp1 upstream region cloned in pCR®-Blunt II-TOPO® (Invitrogen) as a template. PCR products were phosphorylated by T4 Polynucleotide Kinase (Takara Bio) and ligated using T4 DNA Ligase (TOYOBO). Fission yeast cells carrying the ura4 marker gene in the fbp1 promoter were transformed with these plasmid constructs and selected for uracil auxotrophy, as above.To construct rst2-3flag strains were constructed as described previously28.All 29. DNA probes for fbp1 and cam1 were amplified by PCR using primer sets p37/p38 and p39/p40, respectively. Anti-histone H3 (Abcam), anti-acetyl histone H3 (millipore), and anti- DYKDDDDK antibody (Wako) were used for the ChIP analysis. DNA concentrations were quantified using Thermal Cycler Dice Real Time (Takara Bio) and THUNDERBIRD® SYBR qPCR Mix (TOYOBO). Primer sets p41/p42 at UAS2 and p43/p44 at the prp3 locus were used for quantitative PCR analysis. MNase digestion assays were performed as described previously16.Northern blotting and chromatin immunoprecipitation (ChIP) were performed as described previouslySupplementary Information


In this paper, we describe the characterization of these isoforms by RT-PCR, northern blot, and western blot, their preferential expression in human mature testis and spermatozoa, and the elements that punctuate their proximal promoters and suggest cues for their expression in spermatogenic cells. Mechanistically, we show that i19VEGFR-1 has a strong ability to phosphorylate and activate SRC proto-oncogene non-receptor tyrosine kinases and a significant bias toward a decrease in expression in patients considered infertile by WHO criteria.The vascular endothelial growth factor receptor 1 (VEGFR-1) family of receptors is preferentially expressed in endothelial cells, with the full-length and mostly the soluble (sVEGFR-1) isoforms being the most expressed ones. Surprisingly, cancer cells (MDA-MB-231) express, instead, alternative intracellular VEGFR-1 variants. We wondered if these variants, that are no longer dependent on ligands for activation, were expressed in a physiological context, specifically in spermatogenic cells, and whether their expression was maintained in spermatozoa and required for human fertility. By interrogating a human library of mature testis cDNA, we characterized two new truncated intracellular variants different from the ones previously described in cancer cells. The new isoforms were transcribed from alternative transcription start sites (aTSS) located respectively in intron-19 (i During meiotic and post-meiotic stages of spermatogenesis, the genome is reprogrammed prior to packaging. Simultaneously, new mRNAs responsible for active and precise synthesis of proteins are transcribed with stage specific precision. Selected transcripts and proteins, essential for spermatozoa activity, fertilization or embryo development are retained in the spermatozoa , 2.Many spermatogenic transcripts are produced from alternative transcription start sites and alternative splicing of the 5′UTR. Although these mRNAs may code for proteins identical to their somatic counterparts, they often differ, from the corresponding somatic transcripts, in regulatory potential , 4.19VEGFR-1 and Tr-KIT.Protein tyrosine kinases (PTKs) are key factors regulating spermatogenesis, essential for differentiation, migration, adhesion, spermatid shaping, motility and capacitation. In addition to the general requirement for PTKs, normal spermatogenesis and spermiogenesis also seem to be dependent on the expression of truncated isoforms . TruncatVascular endothelial growth factor receptor 1 (VEGFR-1), also known as FLT-1 (fms related tyrosine kinase 1) is a high-affinity and low-activity (relatively to VEGFR-2) tyrosine kinase receptor of the vascular endothelial growth factor VEGF-A, and binds selectively to ligands VEGF-B and placental growth factor (PlGF) –13. ExprVEGFR-1 in humans consists of 30 exons spanning more than 193 kb . Two extIn mouse , the vas19VEGFR-1 and i28VEGFR-1, as they start transcription in intron 19 and 28, respectively, and we have characterized them by sequencing, RT-PCR, northern and western blot. Mechanistically, we have demonstrated, using binding experiments and transfection of recombinant plasmids in CHO and HEK293 cells, that i19VEGFR-1 is able to bind and strongly phosphorylate and activate SRC protein kinases.In this paper, we have identified two intracellular isoforms of the VEGFR-1 receptor in mature testis and spermatozoa. We have named them iAs a preliminary study, due to sample scarcity and the lack of confident parameters for fertility, we have analyzed by Real time-PCR, the expression of both isoforms in samples of astenozoospermic, oligozoospermic or oligoastenozoospermic patients relative to normozoospermic samples (WHO parameters) . We wantin silico approach, taking advantage of the huge quantity of available data in well-documented networks and testis cell lines, the chromatin landscape and the response element present in the corresponding proximal promoters around the TSS, of both isoforms. The reported results showed a relatively open chromatin configuration, punctuated by activating marks signatures and regulatory DNA sequence elements, which may allow for transcription in post-meiotic cells.We have also explored by an 19VEGFR-1 and i28VEGFR-1 isoforms described in this paper may represent haploid cell specifically expressed transcripts that function as convenient ligand-independent intracellular factors with interest in fertility and fertilization.Taken together, the two i19VEGFR-1 and i28VEGFR-1, the number indicating the intron where the aTSS is located (A search by Rapid amplification of cDNA ends (RACE5′ and RACE3′) for truncated intracellular isoforms of the VEGFR-1 receptor, failed to detect previously characterized intracellular isoforms  of the V located .19VEGFR-1) or the whole kinase domain, leaving only a sequence coding for a C-terminal tail of 85 amino acids in the case of i28VEGFR-1. Both isoforms incorporate new leader 5′UTR sequences  that jumps to the second exon acceptor site (ag), generating a small VEGFR-1 isoform of 3 exons, the last 2 corresponding to exons 29-30 of the full-length VEGFR-1. The 5′UTR of the new isoform is 135 nucleotides long. The putative i28VEGFR-1 encodes a protein of 85 amino acids, from amino acid 1,254 to amino acid 1,338 of the full-length VEGFR-1 receptor, with a molecular weight of 9.5kD. The i28VEGFR-1 isoform lacks the entire kinase domain , and human spermatozoa.We analyzed the expression of i19VEGFR-1 and i28VEGFR-1 transcript isoforms, having different 3′UTR lengths, but preferentially using the short 3′UTR of 675 nucleotides obtained by RACE3′ .Quantitative real-time PCR of i samples . Express19VEGFR-1 and i28VEGFR-1 transcripts were translated into proteins.Western blotting was employed to determine whether i19VEGFR-1 isoform. It was detected in spermatozoa using an anti-VEGFR-1 antibody from the C-terminal of the protein  kinase by cell co-transfection we detected more Src (or Fyn) protein bound to the anti-flag resin beads when Flag tagged-i19VEGFR-1 protein was present  in introns 19 and 28, respectively, of the full-length VEGFR-1 receptor.The two isoforms, iin silico for potential DNA binding elements and chromatin cues that allowed i19VEGFR-1 and i28VEGFR-1 transcription in testis  by another palindrome with the stalk of the RGGTCA sequence just mentioned (We took advantage of the huge amount of available information (Encode/Jaspar/Consite) and searched n testis . The seqn testis , with a entioned .19VEGFR-1 and i28VEGFR-1 are shown in 19VEGFR-1 and i28VEGFR-1. Also the Spz1 transcription factor, located in both i19VEGFR-1 and i28VEGFR-1 promoter regions, that functions as a spermatogenic Zip Protein relevant for spermatogenesis.The transcription start sites, Hot Spots for transcription activation, CpG islands, DNase hypersensitive peak clusters, and Histone H3 Lys27 acetylation (H3K27ac) marks around the aTSS of i19VEGFR-1 and i28VEGFR-1, of the VEGFR-1 receptor, missing the extracellular and transmembrane domains and therefore intracellular and independent of ligands, are predominantly expressed, over the complete transmembrane transcript and soluble variants of the VEGFR-1 gene, in mature testis and human sperm cells. The two isoforms start TATA-less transcription in introns 19 and 28, respectively, in rich polypyrimidine stretches, a characteristic shared by transcripts without a TATA-box  (ATCCeBrowser .


Aspergillus flavus produces mycotoxins especially aflatoxin B1 and infects crops worldwide. As a PHD transcription factor, there is no report on the role of Rum1 in the virulence of Aspergillus spp. yet. This study explored the biological function of Rum1 in A. flavus through the construction of rum1 deletion mutants and rum1 complementation strains with the method of homologous recombination. It was found, in the study, that Rum1 negatively regulates conidiation through abaA and brlA, positively regulates sclerotia formation through nsdC, nsdD, and sclR, triggers aflatoxin biological synthesis, and enhances the activity of amylase. Our findings suggested that Rum1 plays a major role in the growth of mycelia, conidia, and sclerotia production along with aflatoxin biosynthesis in A. flavus. Aspergillus flavus is a famous opportunistic soil-borne pathogen and its harmfulness to immunosuppressed patients is only second to A. fumigatus . Th. ThA. flbidopsis . PHD conbidopsis . In recebidopsis . It was gression . Some PHgression ,21. In agression . Since tdulation . HoweverA. flavus on the safety and development of global society and economy and in the light of the putative critical role of the PHD transcriptional factor Rum1 in epigenetic regulation, it is of great importance to explore the biological function of Rum1 in A. flavus. Through sequence alignment with rum1 sequence from U. maydis, we located a Rum1 ortholog (NCBI Gene bank No: AFLA_006240) in A. flavus. The study was carried out to explore the biological functions of Rum1 in the growth of mycelia, sporulation, sclerotial development, and aflatoxin production of A. flavus.In view of the serious impacts of A. flavus, A. niger, A. oryzae, A. nidulans, A. fumigatus, A. bombycis, A. nomius, P. digitatum, S. pombe, M. musculus, H. sapiens, and U. maydis) were obtained from NCBI (http://www.ncbi.nlm.nih.gov), and the evolutionary relationship of these homologs was analyzed using MEGA5.0. The result showed that Rum1 in Aspergillus spp. was classified into a cluster in which the highest identity was found between A. flavus and A. oryzae  and the lowest identity was found between A. flavus and A. nidulans   and the lowest identity was 30% . The protein domains in Rum1 were further analyzed with the use of software SMART, and IBS 1.0. A JmjN, an ARID, a JmjC, and two PHD fingers domains were found in almost all Rum1 homologs. Rum1 homolog from S. pombe has three PHD fingers domains, but lacks a JmjN domain, which is shown in The homologs of 12 Rum1 proteins (from ver 99%) A. Among rum1 deletion mutant (∆rum1) was constructed through homologous recombination  strains, a BP fragment (2.2 kb) was amplified from both ∆rum1 and ∆rum1-C strains, a 2.0 kb AP fragment was amplified from ∆rum1 strain, and a 5.5 kb AP fragment was amplified from ∆rum1-C strain  and complementation strain (∆rum1-C) were correctly constructed.The bination A and wasC strain B. Afterw1 mutant C. These analysis D. The re1 mutant E, which A. flavus, the WT, ∆rum1, and ∆rum1-C strains were incubated on PDA medium in the dark at 29 °C for 5 days, 37 °C for 5 days, and 42 °C for 10 days, respectively. The results showed that the ∆rum1 mutant displayed no significant difference in colony growth compared to WT and ∆rum1-C strains under 37 °C and 42 °C (data not shown), but the diameter of the ∆rum1 colony was significantly smaller than that of WT and the ∆rum1-C strains at 29 °C  were significantly up-regulated in the ∆rum1 mutant  at 37 °C in the dark for a week. We found that there was no sclerotia formed in the ∆rum1 mutant strain compared to WT and ∆rum1-C strains on both media, which indicated that the deletion of rum1 prominently declined the capability of sclerotia formation  were all transcriptionally down-regulated in the absence of Rum1 , which revealed that the expression levels of these genes were significantly decreased in the ∆rum1 mutant compared to findings from the WT and ∆rum1-C strains  . JmjC (Jumonji C) is a demethylase-related domain ,27 and JbrlA and abaA  in aflatoxin pathway were found to have a five-fold to 20-fold reduction. In addition, genes known as aflC and aflO catalyze the chemical reaction of acetate to norsolorinic acid and DMST to ST in the biosynthesis pathway of AFB1, respectively [aflR dramatically decreased by approximately five-fold in ∆rum1 mutant (p < 0.001) (aflR promoter were predicted with MEME online (http://meme-suite.org/tools/meme), which revealed that Rum1 might bind to the possible RE on the promoter of aflR to regulate the synthesis of aflatoxin  and crop colonization models (maize and peanut), the results showed that Rum1 dramatically enhanced the AFBand aflS C. As a 4< 0.001) C. Meanwhflatoxin . Above rA. flavus on crop kernels is critical and complicated. In the study, Rum1 was found to significantly repress conidiation and to initiate sclerotial development (p < 0.001), which seemed that Rum1 attempted to initiate sexual reproduction by depressing asexual activity to some degree in the colonization of A. flavus to crops. Similar to the results from YES liquid medium , YES medium , potato dextrose agar , potato dextrose broth , and GMM medium  were prepared for fungal cultivation in this study. For solid medium, agar was added at 15 g/L. For the auxotrophic marker (pyrG-), the medium was supplemented with 1 mg/mL uracil and 1 mg/mL uridine [All  uridine .A. flavus, A. niger, A. nidulans, A. oryzae, A. fumigatus, A. bombycis, A. nomius, P. digitatum, S. pombe, M. musculus, H. sapiens, and U. maydis were obtained from the National Center for Biotechnology Information . The phylogenetic tree was constructed for the above 12 Rum1 orthologs by MEGA 5.0 with an algorithm of 1000 times Neighboring comparison. In the study, SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1) was used to identify the domains in Rum1 and the framework of domains was mapped with software IBS 1.0  [Rum1 orthologs from , China) .rum1 mutants were prepared by a homologous recombination approach [rum1, 1411 bp 3′-UTR and 1890 bp pyrG) were amplified and overlapped by Overlap PCR and the primers used in the Overlap PCR were shown in ku70 ΔpyrG) was carried out, which was mediated by PEG (polyethylene glycol) [pyrG prototroph strain  . The pyrn ∆rum1,  was furtn ∆rum1, .rum1 mutants were constructed following the method provided by Yang [rum1 fragment amplified from A. flavus with rum1-p7/rum1-p8 primers were transformed into the protoplasts of ∆rum1 with 2 mg/mL 5-FOA (5-fluoroorotic acid) to replace pyrG [pyrg gene was removed from the first step of complemented strains (Pyrg gene was inserted behind the rum1 to produce the pyrG prototroph complementation strains (∆rum1-C) by using a homologous recombination. The complementation strain was tested by using PCR analysis. Both of the verified ∆rum1 mutant and ∆rum1-C strains were further confirmed by a q-PCR assay with an actin gene as the inner reference. The primers used in the analysis are listed in The complementation strains for the ∆ by Yang . First, ace pyrG . PCR ana strains . Lastly,The analysis was implemented according to the protocol used by Nie . RNA wasrum1, and ∆rum1-C strains were incubated onto PDA medium in the dark at 37 °C and the diameter of each colony was measured after 5 days. Conidia were homogenized in 5 mL water solution (0.05% Tween-20) and the conidia number was counted haemocytometrically from 1 mL conidia mixture. For sclerotia production analysis, sclerotia inducing WKM medium was prepared and all cultures were grown at 37 °C for a week in the dark. Lastly, 75% ethanol was used to spray the surface of each plate to wash conidia away. Then sclerotia were collected and the number of sclerotia was counted with the Asana microscope. Above experiments were completed with three repetitions.The analysis was carried out according to the method used by Lan . The WT,rum1, and ∆rum1-C strains were incubated into 15 mL PDB and YES media at 29 °C in the dark for 6 days, respectively. Then AFB1 was extracted from the culture, which was previously described by Yang [1 production was analyzed with thin layer chromatography (TLC)  and examined under a UV light. The pictures of the TLC plates were inverted by the PS software to get clearer data. The experiments were repeated three times.The WT, ∆ by Yang . AFB1 prrum1, and the ∆rum1-C strains were inoculated onto PDA agar with hyperosmotic stress mediators , oxidative stress agent (5–10 mM H2O2), DNA damaging agent (0.02% MMS (Methyl methanesulfonate)), and cell wall stress agents  or 300 μg/mL CR (Congo red)) at 37 °C in the dark for 3 days, respectively. To analyze the role of Rum1 in stress response of A. flavus, the relative inhibition rates were calculated, according to the formula listed in the brackets (diameter of colony without inhibitor—diameter of colony with inhibitor/diameter of colony without inhibitor). The experiments were performed in three repetitions.The WT, the ∆1 extraction and these tubes were placed on the 29 °C shaker and shaken at 180 r/min for 30 min. The lower chloroform layer (10 mL) was collected and dried by airing. After re-dissolving in 1 mL chloroform, 5 µL of each sample was allotted for the TLC assay. The experiments were repeated three times.To explore the role of Rum1 in crop colonization, the assay was carried out following the methods used by Lan with minor modification . Kernelsrum1, and the ∆rum1-C strains were incubated on the amylase screening medium  in the dark at 29 °C for 3 days. Iodine solution was added evenly in the plate and the diameter of the degradation zone was measured. The relative degradation rate equals the diameter of degradation zone/colony diameter.The activity of amylase was analyzed according to the protocol used by Li . The WT,p < 0.05.In this study, all data were presented with the means ± SD (standard deviation). The significant differences  among groups were calculated with ANOVA and LSD (least significant difference) tests. The analysis of statistical and significance was implemented with the software GraphPad Prism5  and the difference is regarded to be statistically significant when


Aedes aegypti, in addition to its role in immature stages, juvenile hormone III (JH) governs post-eclosion (PE) development in adult females, a phase required for competence acquisition for blood feeding and subsequent egg maturation. During PE, JH through its receptor Methoprene-tolerant (Met) regulate the expression of many genes, causing either activation or repression. Met-mediated gene repression is indirect, requiring involvement of intermediate repressors. Hairy, which functions downstream of Met in the JH gene-repression hierarchy, is one such factor. Krüppel-homolog 1, a zinc-finger transcriptional factor, is directly regulated by Met and has been implicated in both activation and repression of JH-regulated genes. However, the interaction between Hairy and Kr-h1 in the JH-repression hierarchy is not well understood. Our RNAseq-based transcriptomic analysis of the Kr-h1-depleted mosquito fat body revealed that 92% of Kr-h1 repressed genes are also repressed by Met, supporting the existence of a hierarchy between Met and Kr-h1 as previously demonstrated in various insects. Notably, 130 genes are co-repressed by both Kr-h1 and Hairy, indicating regulatory complexity of the JH-mediated PE gene repression. A mosquito Kr-h1 binding site in genes co-regulated by this factor and Hairy was identified computationally. Moreover, this was validated using electrophoretic mobility shift assays. A complete phenocopy of the effect of Met RNAi depletion on target genes could only be observed after Kr-h1 and Hairy double RNAi knockdown, suggesting a synergistic action between these two factors in target gene repression. This was confirmed using a cell-culture-based luciferase reporter assay. Taken together, our results indicate that Hairy and Kr-h1 not only function as intermediate downstream factors, but also act together in a synergistic fashion in the JH/Met gene repression hierarchy.Arthropod-specific juvenile hormones control numerous essential functions in development and reproduction. In the dengue-fever mosquito  Aedes aegypti female mosquitoes for blood feeding, egg development, and pathogen transmission. JH acting through its receptor Methoprene-tolerant (Met) regulates the expression of large gene cohorts. JH mediated gene repression, unlike activation that is directly mediated by Met, is indirect and requires intermediate transcriptional repressors Hairy and Krüppel-homolog 1 (Kr-h1). Here, we demonstrate that Hairy and Kr-h1 can act synergistically in the JH-Met gene repression pathway in Aedes female mosquitoes. These interact directly with regulatory regions of the genes that have both Hairy and Kr-h1 binding sites. Thus, this study has significantly advanced our understanding of the complexity of the JH-mediated gene expression pathway. This research yields valuable information about the JH control of reproductive development of the mosquito A. aegypti, one of the most important vectors of human diseases.Juvenile hormone (JH) plays an essential role in preparing  The gengene set . Additiogene set .2H2 domain containing Kr-h1 is expected to bind to specific DNA elements. Kayukawa et al. (2016) have demonstrated that Kr-h1 binds directly to a Kr-h1 binding site (KBS) in the 5′ regulatory region of the Broad complex (BR-C) gene in silkworm Bombyx mori  ATP, as described previously [A. aegypti Kr-h1 antibodies were used to test the presence of the TF in the observed DNA-protein complex.The DNA oligonucleotides containing the core motif and flanking region were annealed, purified and end-labeled with [γ-eviously . NuclearA. aegypti Kr-h1 with a C-terminal Myc-tag was sub-cloned into the pAc5.1 vector (Kr-h1-Myc) . Previously cloned A. aegypti Hairy-Flag and Gro1-V5 were also used in this experiment [AAEL005093 and AAEL006978, both harboring KBSs and HBSs, was PCR amplified and cloned into reporter vector PGL3basic (Promega), followed by sequence confirmation (AAEL0050931kb-Luc and AAEL0069781kb-Luc). Kr-h1 or Hairy binding motifs in both the cloned promoters were mutated by incorporating restriction sites in place of the defined KBS (AAEL0050931kbΔKBS-Luc and AAEL0069781kb ΔKBS-Luc) or HBS (AAEL0050931kbΔHBS-Luc and AAEL0069781kb ΔHBS-Luc), respectively. Mutation of both KBSs and HBSs was achieved by sequential incorporation of restriction sites replacing the binding motifs (AAEL0050931kbΔKBSΔHBS-Luc and AAEL0069781kb ΔKBSΔHBS-Luc). Transient transfection of cultured Drosophila S2 cells was performed using the FuGENE HD reagent (Promega) following manufacturer’s instructions. 100ng of desired reporter plasmids and 10ng of the control Renilla luciferase reporter vector pCopia were co-transfected into the S2 cells along with 200ng each of the expression plasmids Kr-h1-Myc, Hairy-Flag and Gro1-V5, in various combinations. The total concentration of transfected plasmid in each well was normalized by adding the empty expression vector pAc5.1. Synthesized dsRNA samples for DmKr-h1, DmHairy and DmGroucho were supplemented in the transfection mixture as required, in order to negate the effects of endogenous factors on our experiment. Luciferase assays were performed using the Dual Luciferase Assay Kit (Promega), as previously described [periment . A 1-kb escribed .Drosophila S2 cells as previously described [6 transfected S2 cells were fixed with 1% formaldehyde in tissue culture media for 10 min at room temperature followed by addition of glycine (0.125M) in order to stop the reaction. After washing in ice cold PBS (1X) for 2 mins, cells were re-suspended in cell lysis buffer  followed by the addition of nucleus lysis buffer . After a 20 min incubation at 4°C, the nuclear extract was sheared by sonication on ice for 5 min  using Athena Ultrasonic Processor (Probe Sonicator) ATP-150; Athena Technology Inc. The chromatin solution was then clarified by centrifugation at 14,000 rpm for 10 mins at 4°C. For immunoprecipitation 2μl of each Aedes anti-Hairy and anti-Krh1 were incubated with the chromatin for 2h followed by overnight binding to 50μl pre-washed protein A agarose beads at 4°C with shaking. Anti-IgG antibody was used as mock control. The beads were washed three times with wash buffer 1 ; three times with wash buffer 2 ; and two times with wash buffer 3 . The immunoprecipitated DNA was eluted from the beads in 0.1 M NaHCO3 and 1% SDS followed by revrese cross-linking by incubating overnight at 65°C. DNA was extracted by phenol-chloroform method using ethanol precipitation. Quantifications were performed by qRT-PCR and represented as % of input DNA. A primer pair targeting the plasmid backbone was used as control. The primers used are mentioned in the ChIP assays were performed in escribed . BrieflyS1 FigKr-h1 (iKr-h1) in three different biological replicates, as identified by RNA-seq analysis of the female mosquito fat body. With 207 common transcripts, there was a high degree of overlap among the three replicates, indicating reproducibility of the parallel experiments.Venn-diagram showing the number of transcripts co-activated by the dsRNA-mediated depletion of (TIF)Click here for additional data file.S2 Fig(A) Hormonal application experiments showing the effect of JH on the expression of AAEL006978 (protein-glutamine gamma-glutamyltransferase). A clear repression of the tested gene was observed in JH-treated samples. NT, no treatment. (B) The necessity of intermediate factors for the JH-mediated repression of the target gene AAEL006978 as determined by in-vitro fat body culture experiments. JH-mediated repression was compromised by the addition of CHX into the tissue culture medium (JH+CHX). Solvent (acetone) and CHX-treated samples were used as controls. (C) qRT-PCR-based expression analysis of Met/Hairy/Kr-h1 target gene AAEL006978 in iMet, iHairy, iKr-h1 and iHairy+iKr-h1 samples. iLuc samples were used as controls. Error bars represent ± SD. *p < 0.05; ***p < 0.001.(TIF)Click here for additional data file.S3 FigMet, Hairy, and Kr-h1 were mapped to get an overview of the functional groups affected by the transcription factors. Two major categories Cellular Processes and Signaling (left) and Metabolism (right) are shown as pie diagrams. Functional groups with corresponding abbreviations and colors are indicated. The other major category- Information Storage and Processing (not shown in the graph) was not represented in the iMet/iHairy/iKr-h1-activated gene set, with no transcript mapped to the subcategories under this heading.All 130 transcripts activated in the fat body by dsRNA-mediated knockdown of (TIF)Click here for additional data file.S4 FigiMet/iKr-h1/iHairy upregulated transcripts. The 23 8-mers used are listed in The graph showing the frequency of hits for all the 8-mer sequences generated for the characterization of potential Kr-h1 binding site (KBS) from  in the r(TIF)Click here for additional data file.S5 Fig(A and B) Western blots showing the expression of tagged proteins Kr-h1-Myc, Hairy-Flag and Gro1-V5 in cell culture samples in (TIF)Click here for additional data file.S6 Fig(A) Luciferase reporter assays after co-transfection of expression vectors Hairy-Flag and/or Gro1-V5 and/or Kr-h1-Myc along with reporter construct AAEL0069781kb-Luc. Treatments with no input DNA and empty expression vector served as controls. Repression of promoter activity was observed when either Kr-h1-Myc or Hairy-Flag was overexpressed in the cell culture system. Co-repressor Gro1-V5 was required for the successful functioning of Hairy. However, overexpression of both Kr-h1-Myc and Hairy-Flag  dramatically enhanced the intensity of repression of luciferase activity. Error bars represent ± SD. *p < 0.05; ***p < 0.001. (B) Mutation analysis of AAEL006978 promoter by luciferase reporter assays. The Kr-h1 and Hairy binding sites  in the AAEL0069781kb-Luc reporter construct were mutated either separately (AAEL0069781kb ΔHBS-Luc and AAEL0069781kb ΔKBS-Luc) or together (AAEL0069781kb ΔKBSΔHBS-Luc) and co-transfected along with expression vectors Hairy-Flag and Gro1-V5 and Kr-h1-Myc. The repression in the luciferase activity observed with the AAEL0069781kb-Luc construct was partially compromised with the mutation of either KBS or HBS. A complete loss of repression in the promoter activity was observed when both KBS and HBS were mutated in the promoter upstream of the luciferase gene in reporter construct. Error bars represent ± SD. *p < 0.05; ***p < 0.001. (C) Predicted KBS and HBS along with their flanking regions harbored within 1kb of the AAEL006978 promoter and the various promoter mutations utilized in (A) and (B) are indicated.(TIF)Click here for additional data file.S1 DatasetiKr-h1 and in iMet mosquito fat body (overlapped transcripts between iKr-h1↑ and iMet↑) are provided as well. The dataset includes the list of 10,000 transcripts with the highest abundance, utilized for downstream analysis of iKr-h1 upregulated transcripts.The list of transcripts upregulated in (XLSX)Click here for additional data file.S2 DatasetiKr-h1 upregulated transcripts and the overlapped transcripts between iKr-h1↑ and iMet↑ for the biological replicate 2 and 3 are provided.List of 10,000 most abundant transcripts, (XLSX)Click here for additional data file.S3 DatasetiMet, iHairy and iKr-h1 adult female mosquito fat body.The list shows the three way overlap between upregulated transcripts in three different RNA-seq libraries prepared from (XLSX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


BAP1 is a histone deubiquitinase that acts as a tumor and metastasis suppressor associated with disease progression in human cancer. We have used the “Calling Card System” of transposase-directed transposon insertion mapping to identify the genomic targets of BAP1 in uveal melanoma (UM). This system was developed to identify the genomic loci visited by transcription factors that bind directly to DNA; our study is the first use of the system with a chromatin-remodeling factor that binds to histones but does not interact directly with DNA.piggyBac (PBase) was fused to BAP1 and expressed in OCM-1A UM cells. The insertion of transposons near BAP1 binding sites in UM cells were identified by genomic sequencing. We also examined RNA expression in the same OCM-1A UM cells after BAP1 depletion to identify BAP1 binding sites associated with BAP1-responsive genes. Sets of significant genes were analyzed for common pathways, transcription factor binding sites, and ability to identify molecular tumor classes.The transposase We found a strong correlation between multiple calling-card transposon insertions targeted by BAP1-PBase and BAP1-responsive expression of adjacent genes. BAP1-bound genomic loci showed narrow distributions of insertions and were near transcription start sites, consistent with recruitment of BAP1 to these sites by specific DNA-binding proteins. Sequence consensus analysis of BAP1-bound sites showed enrichment of motifs specific for YY1, NRF1 and Ets transcription factors, which have been shown to interact with BAP1 in other cell types. Further, a subset of the BAP1 genomic target genes was able to discriminate aggressive tumors in published gene expression data from primary UM tumors.The calling card methodology works equally well for chromatin regulatory factors that do not interact directly with DNA as for transcription factors. This technique has generated a new and expanded list of BAP1 targets in UM that provides important insight into metastasis pathways and identifies novel potential therapeutic targets.The online version of this article (10.1186/s12920-018-0424-0) contains supplementary material, which is available to authorized users. BAP1 is a histone deubiquitinase that remodels chromatin to regulate gene expression. The BAP1 polypeptide is the catalytic subunit of the polycomb-repressive deubiquitinase complex, which requires either ASXL1 or ASXL2 , 4; howeBAP1 gene in 80% of these tumors [Melanomas arising from the pigmented layers (uvea) of the eye are highly aggressive cancers: almost half of patients with uveal melanoma (UM) die from metastatic disease, even after the primary tumor is completely removed by surgical excision of the eye , becausee tumors , 10, ande tumors , which me tumors –17, suggTransposon integration by targeted transposases has been used to identify genomic regions in several contexts . The cooThe coding region of human BAP1 cDNA (NM_004656.2) from pReceiver-M12 BAP1  was fused to cDNA encoding the hyperactive piggyBac transposase derived from a pCMV-hyPBase plasmid, generously provided by Dr. Allan Bradley . Both N-2. Cells were transfected using TransIT-LT1 transfection reagent  according to manufacturer’s instructions. After 24 h, the medium was replaced with growth medium containing 1.4 μg/mL puromycin. Cells were maintained under selection for 2 weeks, at which point large visible colonies were formed. Colonies were harvested by trypsinization and centrifugation, and cell pellets were stored at − 80 °C.OCM-1A cells were originally derived by Dr. June Kan-Mitchell . Cells wThe following procedure was adapted and modified from . GenomicRaw reads were mapped back to the human genome hg19) with Bowtie2. Significant Calling Cards peaks were called with a modified version of the previously described algorithm [ with BowBAP1 (NM_004656.2-2658s1c1) was designed by the RNAi consortium (TRC) and obtained from the Children’s Discovery Institute / Genome Sequencing Center at Washington University. Lentiviral shRNA expression plasmids were cotransfected into HEK293T cells with the plasmids pCMV-dR8.2 dvpr and pCMV-VSV-G. After 72 h, viral particles were harvested. OCM-1A cells were infected with lentivirus in growth medium with 8 μg/mL protamine sulfate. After 24 h, medium was changed to fresh growth medium containing 1.4 μg/mL puromycin. OCM-1A cells were harvested for analysis of protein and RNA 6 days post infection.The following was modified from Mooren et al. . LentiviBAP1 and control (GFP) knockdowns were sequenced. RNA-Seq analysis was performed by the Genomic Technology Access Center at Washington University. Reads were aligned to the Ensembl release 76 top-level assembly with STAR version 2.0.4b. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.5. Transcript counts were produced by Sailfish version 0.6.3. Sequencing performance was assessed for total number of aligned reads, total number of uniquely aligned reads, genes and transcripts detected, ribosomal fraction known junction saturation and read distribution over known gene models with RSeQC version 2.3.RNA was isolated using the Qiagen RNeasy Mini Kit according to manufacturer instructions, including the optional DNase I step. Second-strand cDNA synthesis was performed using SuperScript IV  according to manufacturer instructions. Next-gen RNA sequencing was performed on the Illumina HiSeq2500 1 × 50 by the Genome Technology Access Center (Washington University). Three biological replicates for p-values less than or equal to 0.01. Gene ontology (GO) and pathway analyses was performed using Gene Set Enrichment Analysis (GSEA) [http://broadinstitute.org/GSEA) that showed significant overlap (p < 0.001) with response to BAP1 depletion.Gene-level and transcript counts were imported into the R/Bioconductor package. EdgeR and TMM normalization size factors were calculated to adjust for samples with differences in library size. Genes or transcripts not expressed in any sample were excluded from further analysis. The TMM size factors and the matrix of counts were imported into R/Bioconductor package Limma. Weighted likelihoods based on the observed mean-variance relationship of every gene/transcript and sample were calculated for all samples with the voomWithQualityWeights function. Generalized linear models were then created to test for gene/transcript level differential expression. Differentially expressed genes and transcripts were then filtered for FDR adjusted s (GSEA) . GO grouhttp://homer.ucsd.edu). Peaks were analyzed separately depending on whether they were associated with genes showing significant change (p < 0.01) in expression after BAP1 depletion, and whether the associated gene increased or decreased expression upon Bap1 depletion. HOMER was used to identify enriched motifs in genomic regions (findMotifsGenome.pl) using the given size of each peak.BAP1-PBase calling card peaks with five or more insertions were used for Hypergeometric Optimization of Motif EnRichment (HOMER) analysis (available at: We fused the piggyBac transposase (PBase) to BAP1 with a flexible 18-aa linker at either end of BAP1 Fig.  genomic U tests revealed that BAP1-PBase and PBase-BAP1 peaks were both significantly closer to transcription start sites (p < 0.0001 for both). One concern was that PBase alone might be predisposed to target transposons to open chromatin and thus near active transcription start sites. Comparing BAP1-fusion insertions with all background insertions, we found distances to transcription start sites as highly enriched for both fusions . Thus, BAP1 binds specifically near transcription start sites.Many genomic peaks with high numbers of insertions were narrow and centered close to transcription start sites. The distances of each BAP1-PBase or PBase-BAP1 genomic peak to the nearest transcription start site were determined and plotted as histograms Fig. . As a cop > 0.01; Fig. To ascertain whether genes targeted by BAP1 in the calling card analysis were functionally regulated by BAP1, we performed RNA-Seq on OCM-1A cells depleted of BAP1 by lentiviral shRNAs targeting BAP1. GFP-targeting shRNAs served as a negative control . BAP1 prp < 0.001) molecular signatures were identified by Gene Set Enrichment Analysis of BAP1 calling-card target genes associated with gene expression response to BAP1 depletion  were targets of BAP1 binding in UM cells, we found no significant response in gene expression to BAP1 depletion. Taken together, these observations suggest that BAP1’s role in differentiation depends on specific changes in gene expression defined by other lineage-specific regulators. This agrees with our observation that BAP1 is recruited by NRF1 and YY1 to both up- and down-regulated genes. Thus, the pathways regulated by BAP1 that link its loss to metastasis – as it is in UM tumors – also depend on the tissue and lineage contexts of the tumor.We found pathways related to Epithelial-to-Mesenchymal Transition (EMT) and embryonic stem cell differentiation affected by depletion of BAP1 in UM cells. Prior studies of gene expression in response to loss of BAP1 have been performed in two other cell types, which showed substantial differences from each other, suggesting that the targets of BAP1 vary depending on cell lineage. In melanocytic cells, BAP1 is important for expression of genes involved in melanoblast and neural crest differentiation . In contThe genomic loci identified by the calling card analysis represent bona fide, biologically valid, physiologically relevant targets of BAP1. First, genes adjacent to genomic loci targeted by BAP1 show differential expression when BAP1 is depleted: 70% of genes identified by calling-card analysis showed BAP1-sensitive expression; and conversely, 45% of all BAP1-sensitive genes were adjacent to genomic loci targeted in the calling-card analysis. Second, the genomic insertions identified by calling-card analysis clustered as narrow peaks near gene promoters with motifs known to bind transcription factors that interact with BAP1. Third, and most important, the subset of BAP1-responsive, Calling Card genes identified here are able to discriminate aggressive, class 2 primary tumor samples from human patients.Identification of class 2 tumors is vital for clinical prognosis of the individual patient , and losWe used the calling card methodology to identify the genomic regions occupied by the chromatin-remodeling factor, BAP1, despite its inability to bind directly to DNA. This both expands the possible uses of the calling card technique to other chromatin-remodeling factors – as well as transcriptional co-activators and co-repressors – and identifies for the first time the genomic targets of BAP1 in UM cells. Far from targeting broad regions of modified histones, we found that BAP1 interacts with the genome at narrow regions around transcription start sites and is targeted to these sites by a specific set of DNA motifs. We suggest that BAP1 is recruited by NRF1, YY1, and Ets factors to specific sets of genes that are co-regulated by other tissue and lineage determinants. Importantly, several BAP1 genomic targets were able to discriminate aggressive class 2 UM tumors, demonstrating the first functional link between the role of BAP1 in chromatin remodeling and the phenotypic switch to metastasizing tumors. Moreover, this link suggests that the class 2 gene expression profile may serve as a functional readout for loss of BAP1 activity in UM tumors. Understanding the roles of these class-discriminating BAP1 genomic targets in the broader context of tumor progression will provide new insights into the cellular mechanisms of UM metastasis.Additional file 1: BAP1-PBase genomic peaks. List of genomic regions of transposon insertion from the BAP1-PBase construct giving location, number of insertion events (hops), significance, and nearest gene (Feature). (XLSX 636 kb)Additional file 2: PBase-BAP1 genomic peaks. List of genomic regions of transposon insertion from the PBase-BAP1 construct giving location, number of insertion events (hops), significance, and nearest gene (Feature). (XLSX 619 kb)Additional file 3: Immunoblot to confirm knockdown of BAP1. Immunoblot of protein collected from the three independent experiments that were used for RNA-seq expressing control or BAP1-specific shRNAs in OCM-1A cells. Specific bands for BAP1 and GAPDH are indicated. (PDF 278 kb)Additional file 4:p-value, and linear fold change. (XLSX 1392 kb) Table summarizing the RNA-seq results. Differential gene expression results in BAP1-knockdown compared to control OCM-1A cells are shown from the RNA-seq data. Each row gives the unique Ensembl identifier, gene name, and description for each gene, as well as the log of the fold change (logFC), average expression, adjusted Additional file 5: BAP1-sensitive calling-card target genes. List of the 784 genes identified by the combination of > 2-fold BAP1-sensitive RNA expression changes and calling-card transposon targeting. Each row gives the gene name, unique Ensembl identifier, and description for each gene. Linear fold change and adjusted p-value in gene expression are from RNA-seq data. Experimental hops, p-value, and fusion construct data are from the calling card experiments. (XLSX 135 kb)Additional file 6: Pathways associated with BAP1 target genes. The ten most significant pathways associated with BAP1 calling-card target genes that were up-regulated (positive response) or down-regulated (negative response) in response to BAP1 depletion. Pathways were identified by GSEA. (PDF 49 kb)Additional file 7:p-value, false discovery rate (FDR), family-wise error rate (FWER), and rank-at-max value calculated by the GSEA software. (XLSX 23 kb) GSEA terms associated with BAP1 target genes. Gene set enrichment analysis based on gene expression values for the 784 genes identified by the combination of > 2-fold BAP1-sensitive RNA expression changes and calling-card transposon targeting. Each row gives the name of the curated GSEA term, and the nominal enrichment score, Additional file 8: DNA motifs associated with BAP1 binding. HOMER was used to identify DNA motifs that were significantly enriched in genomic regions containing BAP1 calling card insertions. Calling card loci were divided into three categories based on response of associated genes to BAP1 depletion, and analyzed separately. (PDF 25 kb)Additional file 9:p-values, false discovery rate (FDR), Bonferroni correction, family-wise error rate (FWER), fold change, means and standard deviations were all calculated by the comparative marker selection software. The original expression values from the published data [ Heatmap data. Gene expression values from published microarray data of primary UM tumors depicted in heatmap in Fig. hed data  used to Additional file 10: GSEA terms associated with top BAP1 target genes. Gene set enrichment analysis on the subset of 79 BAP1 targets that were able to discriminate molecular class in primary tumors. Each row gives the name of the curated GSEA term, the number of overlapping genes (size), enrichment score (ES), nominal enrichment score (NES), nominal p-value, false discovery rate (FDR), family-wise error rate (FWER), and rank-at-max value calculated by the GSEA software. (XLSX 12 kb)