Prl7d1 male mice exhibited subfertility as reflected by reduced sperm counts and litter sizes. The testes from Prl7d1 transgenic mice appeared histologically normal, but the frequency of apoptotic germ cells was increased. Prl7d1 transgenic mice also had lower testosterone concentrations than wild-type mice. Mechanistic studies revealed that Prl7d1 transgenic mice have defects in the testicular expression of steroidogenic acute regulatory protein (STAR) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B). Further studies revealed that PRL7D1 overexpression affected the expression of transferrin (TF) in Sertoli cells. These results suggest that PRL7D1 overexpression could lead to increased germ cell apoptosis and exert an inhibitory effect on testosterone production in Leydig cells by reducing the expression of certain steroidogenic-related genes. In addition, PRL7D1 appears to have important roles in the function of Sertoli cells, which, in turn, affects male fertility. We conclude that the expression level of PRL7D1 is associated with the reproductive function of male mice.Prolactin family 7, subfamily d, member 1 (PRL7D1) is found in mouse placenta. Our recent work showed that PRL7D1 is also present in mouse testis Leydig cells, and the expression of PRL7D1 in the testis exhibits an age-related increase. In the present study, we generated transgenic mice with Leydig cell-specific PRL7D1 overexpression to explore its function during male reproduction.  Prl) family genes are located on chromosome 13 in mice and chromosome 17 in rats and are expressed mainly in the pituitary gland, uterus and/or placenta [Prl family members have been identified, including prolactin-like proteins, placental lactogens, prolactin family 2, subfamily c, member 2 (Prl2c2) and prolactin family 7, subfamily d, member 1 (Prl7d1). The majority of these genes are associated with female reproduction [Prl receptor or other signaling pathways, Prl family members have been divided into classical and non-classical groups.Prolactin  and so named because of its close relationship to another Prl-related hormone, Prl2c2  [ as Plf) ,7. PRL2C as Plf) . PRL2C2  as Plf) , while P as Plf) ,10. PRL7 as Plf) .Prl7d1 mRNA and protein expression during the developmental stage of rat testis increased in an age-dependent manner. In addition, Prl7d1 silencing did not affect basal testosterone production in the TM3 Leydig cell line, but did attenuate the increase of testosterone production in response to stimulation with human chorionic gonadotropin.Expression of PRL7D1 was first identified in mouse placenta . Howeverin vitro studies suggest that PRL7D1 might play roles in male reproduction, its physiological significance in vivo requires further study. Because the expression level of PRL7D1 reached its peak in aged testis, we speculated that a high expression level was associated with the age-related decline in male reproductive function. Thus, in the present study, we explored the potential biological activities of PRL7D1 in male reproduction in vivo by generating transgenic mice with Leydig cell-specific overexpression of PRL7D1. Male mice overexpressing Prl7d exhibited alterations of testosterone secretion and spermatogenesis. This demonstrated that an appropriate level of PRL7D1 expression was critical for the development and function of mouse testis.Although Lhr), located on the surface of Leydig cells of testis, plays a crucial role in the regulation of testosterone production [Lhr is also expressed in several non-gonadal tissues [Lhr upstream promoter had been previously validated to be sufficient for the expression of transgene in Leydig cells of testis [Flag-Prl7d1-SV40 transgene under control of the Lhr promoter , ransgene B. Additiransgene C,D. TherPrl7d1 transgene, the FLAG-epitope protein was detected by Western blotting in the transgenic mouse testes at a molecular weight appropriate for PRL7D1, but not in the wild-type littermates  mice (n = 6). In addition, testes and epididymides weights were not significantly different between wild-type and transgenic mice .No abnormal behavioral characteristics or anatomical changes were noted in either wild-type or transgenic mice. Body weights did not differ significantly between wild-type (29.2 ± 2.64 g) and Prl7d1 transgenic mice was markedly lower than that of wild-type mice (p < 0.01). The presence of a vaginal plug was observed in all wild-type females mated with wild-type or Prl7d1 transgenic mice. However, only five of 12 females became pregnant after mating with Prl7d1 males, whereas 11 of 12 females were pregnant after mating with wild-type males. Furthermore, the litter size resulting from wild-type females mating with Prl7d1 males was significantly smaller (p < 0.01)  . TogethePrl7d1 transgenic and wild-type mice  assay. We observed more apoptotic cells in Prl7d1 than in wild-type tubules  and environmental factors can trigger germ cell apoptosis, and increased germ cell apoptosis has been linked to a variety of adverse reproductive outcomes, including impaired fertility ,22. To e tubules A–C, and Prl7d1 testis    A. To exp < 0.01) B. These Prl7d1 transgenic mice was associated with the disruption of GnRH and LH levels, serum GnRH and LH levels were analyzed by ELISA. As shown in Prl7d1 transgenic mice, which demonstrated that PRL7D1 overexpression exerted its inhibitory effect on testosterone synthesis not through directly affecting GnRH or LH levels.The hypothalamic-pituitary-gonadal (HPG) axis plays important roles in regulating the male reproductive functions. GnRH, a hormone produced by specific neurons in the hypothalamus, triggers the release of LH from the pituitary gland. LH can increase testosterone production by affecting Leydig cells in the testes ,26. To fPotential molecular mechanisms underlying the PRL7D1-mediated decrease in testosterone production in Leydig cells were investigated by analyzing the protein levels of the LHR and several enzymes involved in the conversion of cholesterol to testosterone. Western blot analyses showed that the expression of STAR and HSD3B was decreased by PRL7D1 overexpression, while no significant difference was observed in the expression of the LHR, translocator protein (TSPO) or cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) .Prl7d1 transgenic mice. However, TF levels were significantly downregulated in Prl7d1 transgenic mice testis (p < 0.01) , tight junction protein 1 (TJP1) and claudin-11 (CLDN11) in testis lysates between wild-type and  < 0.01) , suggestPrevious studies demonstrated that testes have two functions: steroidogenesis (the biosynthesis of steroid hormones) and spermatogenesis (the production of spermatozoa) ,25. ThesBcl2 [Identifying key molecules that contribute to testis function often involves the use of genetically-modified (transgenic and knockout) mouse models. For example, mice that overexpress insulin , Bcl2 [2Bcl2 , spermatBcl2  or SRY . ELISA kits for testosterone, GnRH and LH were purchased from USCN . The pMD18-T vector was purchased from Takara . The remaining chemicals were purchased from Sangon Biotech .Lhr upstream promoter. The transgenic construct contained the following components: the upstream promoter region of the Lhr, Flag tags, the complete open reading frame of mouse Prl7d1 and the simian virus 40 (SV40) polyadenylation sequence. The Lhr promoter fragment was amplified from C57BL/6 mice genomic DNA using the primers 5′-TGTGCGGCAAGGCATCTAT-3′ and 5′-TGATGCCGTCTTCCTTTTC-3′. To clone Prl7d1 from the C57BL/6 mice and add the FLAG epitope (DYKDDDDK) to the PRL7D1 protein, the primers 5′-TGGCGGGCCATGGACTACAAGGACGACGATGACAAGCTCCCTTCTTTGAT-3′ and 5′-TGATCATTACTTATCTAGATCAAAATTCAGAGTAG-3′ were used. The SV40 fragment was amplified from the pcDNA3.1 vector using the primers 5′-GAATTTTGATCTAGATAAGTAATGATCATAATC-3′ and 5′-GATCCTCTGGAGATACAGACATGATAAGATACAT-3′. Subsequently, the Flag-Prl7d1-SV40 fragment was generated by in-fusion PCR using the primers 5′-TGGCGGGCCATGGACTACAAGGACGACGATGACAAGCTCCCTTCTTTGAT-3′ and 5′-GATCCTCTGGAGATACAGACATGATAAGATACAT-3′. The transgenic cassette, Lhr-Flag-Prl7d1-SV40, was generated by in-fusion PCR using the primers 5′-ATAATCAATGTCAACCATTCCAACCTTTCTCCTACA-3′ and 5′-GATCCTCTGGAGATACAGACATGATAAGATACAT-3′. The resultant fragment was cloned into the pMD18-T-EGFP vector for sequencing.Transgenic mice with Leydig cell-specific overexpression of PRL7D1 were generated using a fragment of the C57BL/6 mice were obtained from the Experimental Animal Center at Chongqing Medical University. Purified transgenic constructs were microinjected into the pronuclei of fertilized oocytes from these mice. To identify founder (F0) transgenic mice, tail biopsies were collected from two-week-old mice for the isolation of genomic DNA using the Universal Genomic DNA Extraction Kit . The resulting genomic DNA samples were screened by PCR using the primers F1 (5′-CAAGTGGGCAGTTTACC-3′), R1 (5′-GTACTGAGTGGCTCGTCA-3′), F2 (5′-TCAAACCGCTATCCACG-3′) and R2 (5′-TTTGTCCCGAGCCATCC-3′). The amplified products were resolved using electrophoresis in 1.5% agarose gels.Prl7d1 transgenic mice used littermates or age-matched animals derived from the same breeding colony. All animal studies were approved by the Animal Care and Use Committee of Chongqing Medical University.Animals were maintained on a 12-h light/dark cycle with free access to food and water. All experiments comparing wild-type and Blood and tissue samples were collected between 9:00 and 10:30 a.m. Briefly, mice were anesthetized with 6% chloral hydrate and exsanguinated by closed cardiac puncture. Sera were collected and stored frozen at −80 °C until further use. Testes and epididymides were removed rapidly and weighed. Tissues were fixed with formalin for routine histology or kept frozen at −80 °C for subsequent analyses.et al. [Formalin-fixed testes were embedded in paraffin, and 4-μm sections were cut. The sections were de-waxed and then re-hydrated through a descending series of alcohol concentrations to distilled water. Sections were stained with hematoxylin and eosin. The samples were analyzed for changes in testicular morphology and structure using light microscopy. Assessment of the diameter and circumference of seminiferous tubules was conducted using ImageJ software . Round or nearly round tubules were chosen randomly for this measurement according to the protocol developed by Neves et al. .2O2 solution, sections were incubated with 50 μL of a biotin-labeled solution at 37 °C for 60 min. Following washing with phosphate-buffered saline, 50 μL of a streptavidin-HRP working solution were added for 30 min at room temperature. Tissue sections were treated with 3,3′-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Finally, the sections were observed under a light microscope. At least 50 tubular cross-sections were selected randomly from each section, and two sections from each animal were examined as described by Kim et al. [Apoptosis was evaluated using the TUNEL Apoptosis Assay Kit (Beyotime Biotechnology) following the manufacturer’s instructions. Briefly, testis sections were incubated in proteinase K solution (20 g/mL) for 15 min and rinsed in phosphate-buffered saline. After endogenous peroxidase activity was inhibited using a 3% Hm et al. .To verify whether FLAG-tagged PRL7D1 was expressed in Leydig cells, the cellular co-localization of FLAG with PRL7D1 or HSD3B was examined in mouse testes using a double immunofluorescence staining. Briefly, sections were stained overnight with a mixture of rabbit anti-FLAG (1:100) IgG together with rat anti-PRL7D1 (1:100) or goat anti-HSD3B IgG (1:100). Slides were washed with phosphate-buffered saline and then incubated with a mixture of tetramethylrhodamine-conjugated anti-rabbit IgG and fluorescein isothiocyanate-conjugated anti-rat or anti-goat IgG for 1 h. Fluorescent images were obtained using a fluorescence microscope.Spermatogenesis was determined by counting the cauda epididymal sperm. Epididymides were minced in Dulbecco’s Modified Eagle Medium to release the sperm. Sperm were counted in a hemocytometer under a light microscope.Serum testosterone, LH and GnRH levels and intratesticular testosterone levels from testis homogenates were measured using an ELISA kit according to the manufacturer’s instructions. The testosterone, LH or GnRH value of each sample was extrapolated from a standard curve prepared with known concentrations of testosterone, LH or GnRH.To assess male fertility, two wild-type female mice were introduced per male. Mated females were checked daily for vaginal plugs, which is a marker of successful mating. After eight days, the male was removed and housed separately. Females were checked for pregnancy, and the number of offspring per pregnant female was recorded.The right testis was removed from each animal for protein extraction using radioimmunoprecipitation assay lysis buffer. Protein concentrations were determined using the bicinchoninic acid assay. Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. The membranes were then hybridized with diluted polyclonal antibodies . Each met-test. Fertility rates in adult animals from the two groups were compared using Fisher’s exact test. p-values less than 0.05 were considered statistically significant.Data are expressed as the means ± standard deviations. Significant differences were determined by Student’s Prl7d1 and found that increasing the expression of this protein decreases male fertility and testicular steroidogenesis by reducing certain steroidogenic-related proteins. Importantly, our findings suggest that PRL7D1 is most likely involved in Sertoli cell functions that are critical for normal spermatogenesis. Thus, we speculate that the increased expression of PRL7D1 plays a negative role in reproductive function.In the current study, we generated mice overexpressing


Fbxl3 knockout extremely lengthened the circadian period, and deletion of Fbxl21 gene in Fbxl3-deficient mice partially rescued the period-lengthening phenotype, suggesting a key role of CRY protein stability for maintenance of the circadian periodicity. Here, we employed a proteomics strategy to explore regulators for the protein stability of CRYs. We found that ubiquitin-specific protease 7  associates with CRY1 and CRY2 and stabilizes CRYs through deubiquitination. Treatment with USP7-specific inhibitor or Usp7 knockdown shortened the circadian period of the cellular rhythm. We identified another CRYs-interacting protein, TAR DNA binding protein 43 (TDP-43), an RNA-binding protein. TDP-43 stabilized CRY1 and CRY2, and its knockdown also shortened the circadian period in cultured cells. The present study identified USP7 and TDP-43 as the regulators of CRY1 and CRY2, underscoring the significance of the stability control process of CRY proteins for period determination in the mammalian circadian clockwork.Mammalian Cryptochromes, CRY1 and CRY2, function as principal regulators of a transcription-translation-based negative feedback loop underlying the mammalian circadian clockwork. An F-box protein, FBXL3, promotes ubiquitination and degradation of CRYs, while FBXL21, the closest paralog of FBXL3, ubiquitinates CRYs but leads to stabilization of CRYs.  The lysate was incubated with a precipitating antibody solution for 2 hr at 4°C, followed by incubation with 20 μl Protein G-Sepharose beads for 1 hr at 4°C. The beads were washed three times with IP Buffer and then subjected to immunoblotting.Transfected NIH 3T3 cells were lysed for 30 min in ice-chilled IP Buffer ) for 1 hr at 37°C, and then incubated overnight at 4°C with a primary antibody in the blocking solution. The signals were visualized by an enhanced chemiluminescence detection system (PerkinElmer Life Science). The blot membrane was subjected to densitometric scanning and the band intensities were quantified by using ImageJ software v.10.2.Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride membrane (Millipore). The blot membranes were blocked in a blocking solution  for 30 min at 37°C, followed by western blotting analysis.HEK293T/17 cells expressing FLAG-CRY2 proteins were treated with 10 μM MG132 for 6 hr and then lysed in IP Buffer for 30 min. The cell lysate was incubated with 20 μl of anti-FLAG M2 beads (Sigma Aldrich) for 2 hr at 4°C. FLAG-CRY2 was eluted with 150 μg/ml FLAG epitope peptide solution (Sigma Aldrich). Eluted FLAG-CRY2 was incubated with human recombinant protein of USP2 catalytic domain (Life Sensors) or human USP7 (Life Sensors) in USP reaction buffer (10 mM NaHFLAG-CRY2 proteins were co-expressed with Myc-USP7 or with Myc-USP7-C223A in HEK293T/17 cells. The cells were treated with 10 μM MG132 for 6 hr and then lysed for 30 min in IP Buffer. The cell lysate was incubated with 1 μg of anti-FLAG M2 antibody (Sigma Aldrich) for 2 hr at 4°C, followed by incubation with 20 μl Protein G-Sepharose beads for 1 hr at 4°C. The beads were washed three times with IP Buffer and then subjected to immunoblotting.HEK293T/17 cells were transfected with an expression vector for CRY1-LUC, CRY2-LUC or LUC (control)  and cultReal-time monitoring of the luciferase expression rhythm was performed as previously described  with a mS1 FigA. Interaction of TRIM28 with CRY1. HEK293T/17 cells expressing Myc-CRY1 and/or FLAG-TRIM28 were cultured in the presence of 10 μM MG132 for 6 hours and lysed with IP Buffer. The cell lysates were subjected to immunoprecipitation using anti-Myc (left panel), anti-FLAG (right) antibody, or normal mouse IgG (negative control) as precipitating antibodies. B. Interaction of KCTD5 with CRY1 protein. Co-immunoprecipitation was performed as described in A. C. Interaction of DDB1 with CRY1 or CRY2 proteins. Co-immunoprecipitation was performed as described in A.(EPS)Click here for additional data file.S2 Figt-test).HEK293T/17 cells were transfected with indicated expression vectors and cultured for 48 hours. Then the cells were lysed with SDS-PAGE sample buffer, and the lysate was analyzed by Western blotting. GFP was used for transfection and loading controls. Quantified data were shown by means + SEM (n = 3). n.s. represents non-significant change (p > 0.05 by Student’s (EPS)Click here for additional data file.S3 FigHEK293T/17 cells were transfected with expression vectors for LUC and Myc-USP7 (or the empty vector) and cultured for 48 hours. The culture medium was changed to the recording medium containing 0.1 mg/ml cycloheximide. Bioluminescence signals were recorded continuously at 10-min intervals and normalized to the value at time 0. Half-life of LUC was calculated by fitting an exponential decay curve to the bioluminescence signals and shown as means + SEM (n = 3).(EPS)Click here for additional data file.S4 FigHEK293T/17 cells were transfected with expression vectors for CRY2-LUC and cultured for 24 hours. The culture medium was changed to the recording medium containing 20 μM HBX 41108 and 0.1 mg/ml cycloheximide. Bioluminescence signals were recorded continuously at 10-min intervals and normalized to the value at time 0.(EPS)Click here for additional data file.S5 FigA. HEK293T/17 cells were transfected with Myc-USP7 and shRNA vectors targeting human Usp7, and cultured for 72 hours. The cells were lysed with SDS-PAGE sample buffer, and the cell lysate was analyzed by western blotting. The band intensities of Myc-USP7 were normalized to GFP levels. The quantified data are shown as means + SEM (n = 3). B. Knockdown of endogenous USP7 in HEK293T/17 cells. HEK293T/17 cells were transfected with the shRNA expressing vectors, and the transfected cells were selected by puromycin. C. HEK293T/17 cells were transfected with the shRNA expressing vectors. The cells were cultured for 72 hours, and the culture medium was changed to the recording medium containing 0.1 mg/ml cycloheximide. Bioluminescence signals were recorded continuously at 10-min intervals and normalized to the value at time 0 (upper panel). Half-life of LUC was calculated by fitting an exponential decay curve to the bioluminescence signals  and shown as means + SEM . The starting levels of CRYs-LUC bioluminescence signals were shown as means + SEM . n.s. represents non-significant change (p>0.05 by Tukey’s test). D. HEK293T/17 cells were transfected with FLAG-FBXL3 and Fbxl3 shRNA expression vectors, and cultured for 72 hours. The cells were then lysed with SDS-PAGE sample buffer, and the cell lysate was analyzed by western blotting. The band intensities of FLAG-FBXL3 were normalized to GFP levels, and shown as means + SEM (n = 3).(EPS)Click here for additional data file.S6 FigA. The cellular rhythms of PER2::LUC MEF were synchronized by 30-min treatment of 0.1 μM dexamethasone (Dex). The culture medium was changed to the recording medium including HBX 41108, and the bioluminescence signals of PER2::LUC were recorded continuously (left panel). The calculated period lengths are shown as means + SEM . B. NIH3T3 cells were transfected with Myc-USP7 and Usp7-targeting shRNA expression vectors, and cultured for 72 hours. The cells were lysed with SDS-PAGE sample buffer, and the cell lysate was analyzed by western blotting. The band intensities of Myc-USP7 were normalized to β-actin levels and the quantified data are shown as means + SEM (n = 3).(EPS)Click here for additional data file.S7 Figt-test)HEK293T/17 cells were transfected with expression vectors for LUC and FLAG-TDP-43. The cells were cultured for 48 hours, and the culture medium was changed to the recording medium containing 0.1 mg/ml cycloheximide. Bioluminescence signals were recorded continuously at 10-min intervals and normalized to the value at time 0. Half-lives of LUC were calculated by fitting exponential decay curves to the bioluminescence signals and shown as means + SEM (n = 4). n.s. represents non-significant change (p>0.05 by Student’s (EPS)Click here for additional data file.S8 FigA. HEK293T/17 cells were transfected with expression vectors for FLAG-TDP-43 and shRNA targeting Tdp-43, and cultured for 72 hours. The cells were lysed with SDS-PAGE sample buffer, and the cell lysate was analyzed by Western blotting. The band intensities of FLAG-TDP-43 were normalized to GFP levels and averaged data are shown as means + SEM (n = 3). B. Knockdown of endogenous TDP-43 in NIH3T3 cells. NIH3T3 cells were transfected with shRNA expression vectors targeting Tdp-43, and the transfected cells were selected by puromycin.(EPS)Click here for additional data file.S1 TableProteins with Mascot score above 100 in CRY1 or CRY2 interactome analysis were listed.(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Cervical cancer is one of the leading causes of cancer death in women. Human papillomaviruses (HPVs) are the major cause in almost 99.7% of cervical cancer. E6 oncoprotein of HPV and E6-associated protein (E6AP) are critical in causing p53 degradation and malignancy. Understanding the E6AP regulation is critical to develop treating strategy for cervical cancer patients. The COP9 signalosome subunit 6 (CSN6) is involved in ubiquitin-mediated protein degradation. We found that both CSN6 and E6AP are overexpressed in cervical cancer. We characterized that CSN6 associated with E6AP and stabilized E6AP expression by reducing E6AP poly-ubiquitination, thereby regulating p53 activity in cell proliferation and apoptosis. Mechanistic studies revealed that CSN6-E6AP axis can be regulated by EGF/Akt signaling. Furthermore, inhibition of CSN6-E6AP axis hinders cervical cancer growth in mice. Taken together, our results indicate that CSN6 is a positive regulator of E6AP and is important for cervical cancer development. Cervical cancer is the leading cause of women's cancer death. Most of the cervical cancers are caused by the infection of the human papillomavirus (HPV). HPV E6 and cellular E6-Associating Protein E6AP/UBE3A) proteins target p53 for proteasome-mediated degradation, thereby facilitating cellular transformation , 2. E6APA proteinCsn2, Csn3, Csn5, and Csn8 caused defective embryo development [Csn6 gene in mice and found that Csn6–/– mice developed until 7.5 days post-coitus [Csn6+/– mouse tumor experiment we showed that Csn6 haplo-insufficiency mitigated the development of cancer [The COP9 signalosome (CSN) is a protein complex involved in protein degradation, transcriptional activation , 18, sigelopment –33. We pt-coitus . In a Csf cancer , which if cancer , 34, 35,In the present study, we found that CSN6 is overexpressed in cervical cancer and is an important positive regulator of E6AP. We also characterized that CSN6-E6AP axis is regulated by EGF signaling. Our studies provide important insight into the signaling of the CSN6-E6AP axis in the tumorigenesis of cervical cancer and help elucidate CSN6’s potential as a target for therapeutic design for cervical cancer.To understand the gene status of CSN6 and E6AP in cancers, we performed a cross-cancer genomic analysis of CSN6 and E6AP using The Cancer Genome Atlas (TCGA) database. We found that both CSN6 and E6AP are overexpressed in many types of cancer, including cervical cancer Figure . On the de novo protein synthesis inhibitor cycloheximide  data showed a positive correlation between CSN6 overexpression and genes involved in p53 down-regulated pathway Figure , suggestTo investigate the role of the CSN6-E6AP axis in cervical cancer tumorigenesis, we injected HeLa containing CSN6 ShRNA cells into athymic nude mice. Treatment with ShRNA-CSN6 inhibited HeLa cells-mediated tumor growth Figures  and 7B, Our mechanistic studies showed that EGF/Akt-mediated CSN6 S60 phosphorylation and subse quent stabilization of E6AP thereby causing p53 downregulation and promoting cell proliferation, cell transformation as well as tumorigenesis Figure . Our stuIn the present study, we found that CSN6 is elevated in cervical cancer. Both CSN6 and E3 ligase E6AP are involved in ubiquitination regulation and elevated in cervical cancer, suggesting that there is a mutual regulatory relationship involved in targeting protein degradation. In biochemical studies, high levels of CSN6 expression resulted in stabilization of E6AP in HPV positive cervical cancer cells, indicating that CSN6 has a critical role in HPV-mediated cervical cancer development. CSN6 is a critical ubiquitination regulator involved in cell cycle regulation , 22, butin vivo. Therefore, the effect of CSN6 in keeping the E6AP proteins stable has an impact on promoting transformation activity of HPV.Our studies showed that CSN6 functions in connection with E6AP in a circuit, resulting in the negative regulation of p53 target genes including p21 and Bax. We found that CSN6 associates with E6AP and reduces E6AP autoubiquitination, thereby stabilizing E6AP. The steady-state protein level of the E6AP protein is important in determining the ubiquitin–mediated degradation of p53, and E6AP tends to have autocatalytic degradation Our data show that N-terminal CSN6 is interacting with E6AP. This N-terminal CSN6 includes CSN6’s MPN domain, which is conserved in the N-terminus of yeast Mpr1 and Pad1 proteins –42. AlthPapillomavirus E6 oncoprotein can target several cellular interacting partners for proteasome-mediated degradation. Also, E6/E6AP complex can associate with S5a, the major ubiquitin-accepting proteasome subunit , 47. HerEGF functional polymorphism or EGF signaling is involved in influencing cervical cancer prognosis , 49. EGFTogether, our findings demonstrate that CSN6 overexpression can facilitate E6AP stabilization in cervical cancer and provide important insight into the mechanisms underlying EGF pathway deregulation in cervical cancer. That CSN6 functions as a positive regulator of E6AP prompts the idea that blocking the CSN6 signaling axis is an efficient therapeutic approach in E6AP-deregulated cervical cancer.HeLa and Caski , C33a  and HEK-293T (human embryonic kidney cell line) obtained from the ATCC. Cells were maintained in Dulbecco's modified Eagle's medium/nutrient F12 media (in-house supplier) supplemented with 10% (v/v) fetal bovine serum. LY294002 was obtained from Cell Signaling (#9901). Epidermal growth factor (EGF) was obtained from Calbiochem (#324831). MG132 C2211), cycloheximide (C4895) and Puromycin (P7255) were purchased from Sigma. Ni-NTA Argrose was obtained from Invitrogen (#R901-15). Antibodies: CSN6 (ENZO life sciences), E6AP (ENZO life Sciences), p53 (sigma), Flag , Myc (abcam), Akt , pAkt , Actin (Sigma). His-Ubiquitin (UW 8610) was purchased from BioMol International. Flag-CSN6, Myc-CSN6 were previously described . ShCSN6 11, cyclo5 cell/dish 18 h before transfection. Transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For the generation of CSN6 knockdown cells, HeLa cells were infected with lentiviral shRNA transduction particles  containing either control shRNA or CSN6 shRNA. After infection, the cells were selected with 2 μg/ml puromycin for 2 weeks according to the manufacturer's protocols. The antibiotic-resistant colonies were then picked, pooled, and expanded for further analysis under selective conditions.Cells were plated in 60-mm dishes at a density of 4 × 10Total-cell lysates were processed as previously described . Cell lyUbiquitination was performed as previously described –58. BasiThe cells were transfected with the indicated plasmids and incubated at 37°C with 5% (vol/vol) CO2 for 24 h. Then cycloheximide was added into the media to a final concentration of 100 μg/ml. The cells were harvested at the indicated times after CHX treatment. The protein levels were analyzed by immunoblotting.3 per well) were plated in 96-well culture plates. MTT (20 μl) was added to the cells, and the cells were incubated for 2 h. Dimethyl sulfoxide (100 μl) was then added to the cells. After incubation, the absorbance was measured at 570 nm.A 3--2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the cell growth rate as previously described . Cells  were used. Filters (8-μm pore size) precoated with fibronectin (Roche) were used for estimating cell migration. Cells were placed into the upper chamber in 0.5 ml of serum-free DMEM (1 × 105 cells per filter). DMEM supplemented with 10% fetal bovine serum was placed in the lower chamber as a chemoattractant. Migration was scored at 24 hours. Cells were fixed in methanol for 5 min at room temperature, stained with crystal violet for 5 min, and counted under microscopy. The mean numbers of cells per microscopic field over five fields per filter were calculated for triplicate experiments. Experiments were repeated at least three times.TRizol (Invitrogen) was used to extract total RNAs from cells or tissues. The iScript cDNA Synthesis Kit  was used to produce cDNA from 1 μg of RNA. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed using a 7500 Real-Time PCR System (Applied Biosystems), and SYBR Green PCR Master Mix (BioRad) as previously described .http://pga/mgh.harvard.edu/primerbank/). And ACTIN was used for normalization.Primers for qRT-PCR of CSN6  and p53 target genes, p21, GADD45A , BAX , CCNG1 . were as referred by Primer Banker . Apoptosis was determined using propidium iodide (PI) and FITC-conjugated anti-Annexin V (BD Pharmingen). Cells were stained with PI and FITC-conjugated anti-Annexin V and analyzed with a FACScalibur flow cytometer.6) in 0.1 ml PBS were injected subcutaneously into the flanks of mice. Tumor volumes were measured and recorded three times a week from day 5 of the cell inoculation. At the end of the experiment, the mice were sacrificed and the tumors were removed and weighted.4–5 weeks old nude mice were maintained in the animal facility at the University of Texas MD Anderson Cancer Center. Mice were randomly divided into experimental groups, five for each. Different stable transfectants of HeLa cells  and Gene Expression Omnibus databases. These cohorts were analyzed by Gene Set Enrichment Analysis (Broad Institute) as described , 38, 60.


Ionizing radiation (IR) exerts deleterious effects on the developing brain, since proliferative neuronal progenitor cells are highly sensitive to IR-induced DNA damage. Assuming a radiation response that is comparable to mammals, the chick embryo would represent a lower vertebrate model system that allows analysis of the mechanisms underlying this sensitivity, thereby contributing to the reduction, refinement and replacement of animal experiments. Thus, this study aimed to elucidate the radiation response of the embryonic chick retina in three selected embryonic stages. Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development. Unlike cell cycle control, radiation-induced apoptosis (RIA) showed strong variations between its extent, dose dependency and temporal occurrence. Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age. For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points. In summary, our results are in good agreement with data from the developing rodent retina, strengthening the suitability of the chick embryo for the analysis of the radiation response in the developing central nervous system. The transfer of unrepaired DNA damage into prospective daughter cells by DNA duplication during S-phase or mitosis represents an important risk for the organism. Thus, accurate detection and repair of DNA damage occurring after genotoxic stress are preconditions for the maintenance of genomic integrity of dividing stem and progenitor cells during embryonic development. To minimize the risk of DNA damage propagation, cells must rely on cell cycle checkpoints, giving damaged cells ample time to repair the damage before they enter these critical cell cycle phases. Besides this, damaged cells might become eliminated via apoptosis when the degree of damage exceeds a specific threshold. In the developing central nervous system (CNS) of rodents, elimination of damaged cells by apoptosis is a major component of the response to ionizing radiation (IR) ,2, whereThe retina represents a comparably simple tissue of the CNS with only few cell types, whose spatio-temporal genesis during development is well characterized. Mitosis is primarily confined to the apical side, whereas proliferating cells exhibit interkinetic nuclear migration in the developing tissue, with DNA synthesis taking place at the inner  margin . Cell cyWhen exposed to ionizing radiation, the response of the retinal tissue is comparable to other parts of the brain: recently, distinct apoptotic waves were detected after irradiation of rodents, with proliferating and postmitotic cells dying at different times ,10. As dBy comparing the radiation response in the CNS of chick embryos with previous data from rodents, this study aimed to establish the chick embryo as a new animal model for the analysis of radiation effects on neural development. The chick not only represents a lower vertebrate, but also offers some further advantages over mammals. Most importantly, the mother has not to be sacrificed in order to gain access to the embryos. Additionally, the possibility to "window" fertilized eggs allows the determination of an embryonic stage before the experiment and makes the embryo highly accessible to experimental manipulations. Thus, the establishment of the chick embryo as a model system in the research of radiation effects on development allows us to address the National Institute of Health 3R policy to Reduce, Refine and Replace animals in research.Here we present a detailed analysis of the radiation-induced activation and maintenance of cell cycle arrests via checkpoints and the time-, dose- and region-dependent occurrence of apoptotic events after irradiation in three selected developmental stages of the chick retina. Our data show the lack of a radiation-induced G1/S checkpoint in retinal progenitor cells, allowing cells to replicate their damaged DNA. In contrast, a rapid G2/M checkpoint activation efficiently inhibited the progression of irradiated cells into mitosis. Noticeably, cells that were released from this G2/M arrest still harbored unrepaired DNA damage. IR further induced apoptosis, whereby the dose-dependency as well as the temporal and spatial occurrence of apoptotic events drastically changed during development. In summary, our data are in good agreement with previous results obtained from rodent retina. Thus the chick embryo represents a suitable lower vertebrate model system for the research of the radiation response of the developing CNS.Eggs from the white leghorn chick  were incubated at 37°C and 65% humidity for 2–3 days. Then, a window was cut into the egg shell for Hamburger Hamilton (HH) stage determination and—if necessary—5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2’-deoxyuridine (EdU) application. Before treatment, HH stages were determined; the following stages were used: E3 HH 16–18, E5 HH 26–28, E7 HH 30–32. Irradiation was performed with 135 kV and 19 mA, using a Philips MCN 165/796704 X-Ray machine, equipped with a Tungsten anode and a Beryllium window. Doses of 0.5 Gy, 1 Gy, 2 Gy and 4 Gy were estimated by using an ion chamber (PTW) that was placed on top of the egg; the dose rate was 0.5 Gy / min. Dependent on the experiment, chick embryos were sacrificed by decapitation at 30 min, 1, 3, 6, 12, 24, 48 or 72 hours (hrs) after the treatment. For tissue isolation, eyes were collected in F-12 medium (Gibco) on ice. For immunohistochemistry, eyes were fixed in PBS containing 4% formalin (pH 7.3) for 16 hrs. Formalin-fixed tissues were embedded in paraffin and sectioned at a thickness of 3 μm.Animal experiments were performed in strict accordance with German Animal Welfare legislation and were approved by the animal welfare representative of the TU Darmstadt (Tierschutzbeauftragte) and the Regierungspräsidium Darmstadt .After dewaxing in xylene and rehydration, sections were incubated in citrate buffer for 1 h at 95°C. Sections were encircled with a liquid blocker  and incubated with primary antibodies for 5 hrs at 37°C or overnight at room temperature. Antibodies used were: BrdU from mouse  1:500, Cleaved Caspase-3 from rabbit ((Asp 175) 5A1E Rabbit mAb #9664 Cell Signaling Technology Inc.) 1:1000, PCNA from mouse  1:200, phospho-Histone H2A.X from mouse (Ser 139) clone JBW301  1:500, phospho Histone H3 (Ser-28) from goat  1:200, Rad51 (Ab-1) from rabbit  1:500, Visinin from mouse  1:500. Primary antibodies were diluted in blocking solution  following Alexa488 or Alexa594-conjugated secondary antibodies  for 2.5 hrs at room temperature and staining of DNA with DAPI.In order to analyze radiation-induced G1/S checkpoint activation, BrdU and EdU incorporation studies were performed. For BrdU studies alone, 100 μl of a 25 mM BrdU solution  were pipetted on the top of the embryo at various time points after irradiation. To check S-phase entry of irradiated cells at early times after irradiatin, BrdU incorporation was allowed to take place from 3 to 6 hrs after irradiation. To check for a late activation of the G1/S checkpoint, BrdU exposure was started 6 hrs after irradiation and lasted till 12 hrs post irradiation. Fixation was done as described above. For EdU-BrdU double labeling, 250 μl of a 500 μM EdU solution  was added directly after irradiation, followed by the administration of 100 μl of 25 mM BrdU (solved in PBS) at 3 hrs post irradiation.6 hrs after irradiation of E5 and E7 embryos with 2 Gy, eyes were collected in F-12 medium (Gibco) on ice. Retinae were isolated in Hank’s balanced salt solution  and dissociated by accutase (pAA) digestion for 30–45 minutes at 37°C. Digestion was stopped by adding DMEM incl. 10% FCS, followed by mechanical dissociation with a fire polished Pasteur pipette. Preceding FACS analysis, single cells were fixed in 70% ice cold ethanol, stained with propidium iodide (50 μg / ml in PBS) and incubated with 0.5 mg / ml RNase A (Epicentre) for 30 min in PBS. FACS analysis was performed on a FC500 (Beckman Coulter).Images of retinae were taken on a confocal microscope (Leica TCS SP5 II) with LAS AF Lite software (Leica). Foci determination of the phosphorylated form of the H2AX histone (γ H2AX), phospho-Histone H3 positive mitotic cells, pyknotic nuclei and cleaved caspase 3 positive cells was performed on captured images. Localization studies of pyknotic nuclei were carried out in a standard sector of the retina next to the optic nerve head. Sectors of comparable thickness were divided into 5 equally sized bins in its horizontal dimension. The sector was aligned such that the first bin was at the presumptive photoreceptor layer of the retina, with its long axis parallel to the apical border of the tissue. The images were arranged using ImageJ and GraphPad Prism. All images are arranged such that the apical part of the retina (referred to the presumptive outer nuclear layer (pONL)) faces up- and the basal part downwards.t-test was used for testing of γ H2AX foci and BrdU quantifications after in ovo irradiations. One-way ANOVA with Tukey-test was used for significance testing of pH3-positive cell determination and the quantification and localization analysis of pyknotic nuclei. For all analyses a 95% confidence interval with *P < 0.05, **P < 0.01 and ***P < 0.001 was defined. The number of each experiment is indicated in the figure legends.Statistics were performed using GraphPad Prism statistical software (version 6.07). If not indicated otherwise, at least three independent experiments were carried out. Unpaired two-tailed student’s Previous studies have shown a direct correlation between the radiation dose and the number of induced DNA double strand breaks (DSBs) in irradiated cells . To confin vitro [Recent studies have shown the existence of a radiation-induced G2-arrest by the G2/M-checkpoint but no blockade of S-phase entry by a G1/S-checkpoint in the developing CNS of mice ,4. To anin vitro , BrdU waTo rule out earlier changes in S-phase entry, E7 embryos were double-labeled with EdU and BrdU. Hereby, EdU was administered directly after irradiation and BrdU was added in excess at 3 hrs. Fixation was done at 6 hrs after irradition . No signTo check for later radiation-induced changes in S-phase entry, BrdU was applied to E5 and E7 embryos from 6 to 12 hrs after irradiation. Again, irradiation had no impact on the ratio of BrdU+ cells .In a final step we analyzed the radiation-induced activation of the G2/M checkpoint by staining against the phosphorylated histone H3 (pH3), allowing us to determine the numbers of mitotic events in untreated and irradiated retinae. To check whether G2/M checkpoint released retinal progenitor cells still harbour unrepaired DSBs, we analyzed those cells by staining against γ H2AX. At 30 min after irradiation with 2 Gy, residual mitotic cells showed massive γ H2AX signals . 3 hrs a2 with a dose of 1Gy (2). This result correlated well with the quantification of cells positive for cc3 .Radiation has been described to induce apoptotic waves in progenitor cells of the developing CNS 4;9;10). In order to characterize the temporal occurrence and the dose dependency of the apoptotic response in different developmental stages of the retina, this tissue was analyzed for the occurrence of dying cells at various time points after treatment. Hereby, pyknotic nuclei and cells positive for cleaved caspase 3 (cc3+) were used as established markers for RIA [;9;10. Ine of 1Gy . Similar for cc3 . A dose 2 by increasing the dose from 1 to 2 Gy  Rad51 (red) and Visinin (green) staining of E7 retina at 1 h after 2 Gy irradiation. Nuclei were counterstained with DAPI (blue). (B) Rad51 (red) and PCNA (green) staining of E7 retina at 1 h after 2 Gy irradiation. Nuclei were counterstained with DAPI (blue). (C) Rad51 (red) and BrdU (green) staining of E7 retina at 1 h after 2 Gy irradiation. Nuclei were counterstained with DAPI (blue). Note that Rad51 foci are not present in postmitotic, Visinin-positive photoreceptor precursors but highly abundant in cells positive for the proliferation markers PCNA and BrdU. Scale bar = 5 μm.(PDF)Click here for additional data file.S2 Fig(A) Represenative FACS blots of the cell cycle distribution in retinal cells of E5 controls and embryos at 6 hrs after irradiation with 2 Gy. (B). Representative FACS blot of the cell cycle distribution in retinal cells of E7 controls and embryos at 6 hrs after irradiation with 2 Gy.(PDF)Click here for additional data file.S3 FigBrdU was added at 6 hrs after 2 Gy irradiation. Fixation was done at 12 hrs after irradiation. (B) Staining against BrdU (green) in retinae of E5 controls and embryos irradiated with 2 Gy. Nuclei were counterstained with DAPI (blue). (C) Scheme of experimental design and quantification of BrdU+ cells in E7 embryos. BrdU was added at 6 hrs after 2 Gy irradiation. Fixation was done at 12 hrs after irradiation. (D) Staining against BrdU (green) in retinae of E7 controls and embryos irradiated with 2 Gy. Nuclei were counterstained with DAPI (blue). Data are presented as means  ± SEM. Scale bar = 10 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.(PDF)Click here for additional data file.S4 FigpH3 staining (red) in control and 2 Gy irradiated retinae of E5 and E7 embryos. Nuclei were counterstained with DAPI (blue). No differences in the amount of pH3 positive cells were detected, although irradiated E7 retinae revealed high amounts of pyknotic nuclei. Scale bar = 50 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.(PDF)Click here for additional data file.S5 Fig(A) Representative γH2AX (red) staining of a mitotic cell in E7 retina at 30 min after 2 Gy irradiation. Nuclei were counterstained with DAPI (blue). (B) Representative γH2AX (red) staining of a mitotic cell in E7 retina at 3 hrs after 2 Gy irradiation. Nuclei were counterstained with DAPI (blue). Note that cells that enter mitosis 3 hrs after the treatment still harbor unrepaired DSBs. Scale bar = 2 μm.(PDF)Click here for additional data file.S6 Fig+ cells  12 hrs after irradiation with 0.5, 1 and 2 Gy in central parts of E3 retinae. Note strong increase of apoptotic events after dose doubling from 0.5 to 1, but no further increase after doubling the dose from 1–2 Gy. Data are presented as means  ± SEM (*P<0.05 **P< 0.01 *** P<0.001). Scale bar = 25 μm. RPE, retinal pigmented epithelium; pONL presumptive outer nuclear layer.(A-H) cc3 staining (green) in control and 2 Gy irradiated E3 retinae at 3, 6, 12 and 24 hrs after treatment. Nuclei were counterstained with DAPI (blue). Note highest abundance of apoptotic cells at 12 hrs after treatment. (I-J) Quantification of cc3(PDF)Click here for additional data file.S7 Fig+ cells in central retina, and also their strong increase from 1 to 2 Gy in intermediate and central parts of the retina. (J) Quantification of cc3+ cells in central E5 retina at defined time points after irradiation with 1 and 2 Gy. RIA peak 12 hrs after irradiation; note correlation between doses and RIA, and also between appearance of cc3+ and pyknotic nuclei. Data are presented as means (n = 2) ± SEM (*** P<0.001). Scale bar = 50 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.(A-I) cc3 staining (green) in control, 1 and 2 Gy irradiated E5 retinae at 12 hrs after treatment. Nuclei were counterstained with DAPI (blue). Note absence of cc3(PDF)Click here for additional data file.S8 Fig(A-I) cc3 staining (green) in control, 1 and 2 Gy irradiated E7 retinae at 3 hrs after treatment. Nuclei were counterstained with DAPI (blue). Note absence of RIA after 1 Gy, and—after 2 Gy —higher numbers in peripheral than in central parts of the retina. (J) Determination of cc3 activity in lysates of whole E7 retinae from controls and embryos irradiated with 2 Gy at defined time points after treatment. Data are presented as means (n = 3) ± SEM (*** P<0.001). Scale bar = 50 μm. RPE, retinal pigmented epithelium; pONL, presumptive outer nuclear layer.(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.


We performed a detailed ChIP-seq analysis in cells of lymphoid and endothelial origin and compared H3K4me3, H3K27me3, polII, and LANA occupancy. On viral episomes LANA binding was detected at numerous lytic and latent promoters, which were transactivated by LANA using reporter assays. LANA binding was highly enriched at H3K4me3 peaks and this co-occupancy was also detected on many host gene promoters. Bioinformatic analysis of enriched LANA binding sites in combination with biochemical binding studies revealed three distinct binding patterns. A small subset of LANA binding sites showed sequence homology to the characterized LBS1/2 sequence in the viral terminal repeat. A large number of sites contained a novel LANA binding motif (TCCAT)3 which was confirmed by gel shift analysis. Third, some viral and cellular promoters did not contain LANA binding sites and are likely enriched through protein/protein interaction. LANA was associated with H3K4me3 marks and in PEL cells 86% of all LANA bound promoters were transcriptionally active, leading to the hypothesis that LANA interacts with the machinery that methylates H3K4. Co-immunoprecipitation demonstrated LANA association with endogenous hSET1 complexes in both lymphoid and endothelial cells suggesting that LANA may contribute to the epigenetic profile of KSHV episomes.Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus associated with KS and two lymphoproliferative diseases. Recent studies characterized epigenetic modification of KSHV episomes during latency and determined that latency-associated genes are associated with H3K4me3 while most lytic genes are associated with the silencing mark H3K27me3. Since the latency-associated nuclear antigen (LANA) (i) is expressed very early after  KSHV is a DNA tumor virus which is associated with Kaposi's sarcoma and some lymphoproliferative diseases. During latent infection, the viral genome persists as circular extrachromosomal DNA in the nucleus and expresses a very limited number of viral proteins, including LANA, a multi-functional protein. KSHV viral episomes, like host genomic DNA, are subject to chromatin formation and histone modifications which contribute to tightly controlled gene expression during latency. We determined where LANA binds on the KSHV and human genomes, and mapped activating and repressing histone marks and RNA polymerase II binding. We found that LANA bound near transcription start sites, and binding correlated with the transcription active mark H3K4me3, but not silencing mark H3K27me3. Binding sites for transcription factors including znf143, CTCF, and Stat1 are enriched at regions where LANA is bound. We identified some LANA binding sites near human gene promoters that resembled KSHV sequences known to bind LANA. We also found a novel motif that occurs frequently in the human genome and that binds LANA directly despite being different from known LANA-binding sequences. Furthermore, we demonstrate that LANA associates with the H3K4 methyltransferase hSET1 which creates activating histone marks. Eukaryotic DNA is packaged into chromatin which plays a central role in the regulation of all DNA processes including replication, transcription, and repair. Chromatin contains nucleosomes with DNA wrapped around the core histones H2A, H2B, H3, and H4. Nucleosomes carry epigenetic information in the form of post-translational histone modifications. N-terminal histone modifications including acetylation, methylation, phosphorylation, and sumoylation are important in partitioning chromatin into transcriptionally active or repressive domains  that exist in protein complexes. A single enzyme can be responsible for the three states of methylation in a progressive manner, or different enzymes may be required for different methylation states. Mammalian cells contain 10 different H3K4 KMTs, which include the hSET complex, mixed lineage leukemia 1 to 5 (MLL1-5) complexes, Set7/9, Smyd1, Smyd3, and Prdm9, which are largely not redundant Kaposi's sarcoma-associated herpesvirus  is a γ is a named. In addition to Kaposi's sarcoma (KS) which targets endothelial cells, KSHV is associated with two lymphoproliferative disorders: primary effusion lymphoma (PEL) and a subset of multicentric Castleman's disease (MCD). Although the majority of cells in KS tumors are latently infected, both latent and lytic phases of KSHV infection contribute to pathogenesis and tumorigenesis LANA is a multifunctional protein with important roles during viral latency. LANA is the only viral protein required for episome maintenance by supporting KSHV latent DNA replication and tethering the viral episome to cellular chromosomes. Tethering is mediated by LANA via interactions with the viral terminal repeats and the core histones H2A/H2B We hypothesized that LANA plays a role in the establishment and maintenance of the KSHV epigenome. To address this question and to identify viral and cellular genes potentially regulated by LANA, we performed genome-wide ChIP-seq analyses for LANA, Pol II, and histone modifications. Our data confirm that during latency both active H3K4me3 and repressive H3K27me3 marks are associated with the viral episomes Histone marks on KSHV genomes have previously been mapped by PCR-based ChIP assays and ChIP-on-chip assays using tiling array hybridization Based on studies demonstrating that the patterns for acetylated H3K9 and H3K14 were almost identical with H3K4me3 on the KSHV epigenome ChIP-seq tags mapped to KSHV were used for peak analysis by CisGenome We focused in more detail on H3K4me3, H3K27me3, and Pol II occupancy at the +/−2kb region surrounding known transcription start sites (TSS) of selected viral genes . PromoteFor validation, quantitative ChIP-PCR assays confirmed ChIP-seq results for the promoter regions of LANA, vIRF1, vIL6, RTA, and K7, and the coding region of the late lytic gene ORF19, which was enriched with H3K27me3 . In summin vivoTo date, epigenetic modifications have only been mapped in cells of lymphoid and epithelial cells but not in endothelial cells, which give rise to KS. To address this gap in the literature, we performed ChIP-seq assays in long-term infected TIVE-LTC cells, which contain BCBL-1 derived episomes H3K4me3 occupancy showed significant differences in TIVE cells compared to BCBL-1 cells within the latency-associated region. A prominent H3K4me3 peak within the ORF73 (LANA) promoter  observed in BCBL-1, was demonstrably reduced in TIVE-LTC cells. Instead, a strong H3K4me3 peak appeared at nt 126,280 within the LANA coding region, which was not present in BCBL-1 cells  panel B.In addition to its role in latent DNA replication and episomal maintenance, LANA is a key regulator of host and viral gene expression. LANA binds to DNA directly in a site-specific manner, or indirectly through protein-protein interactions with multiple chromatin associated proteins including core Histone H2A and H2B, CREB2, mSin3, RING3, MeCP2, SSRP1, and P53  in vivo. Furthermore, this LANA peak completely overlaps with three CTCF binding sites (nt 127 514–127 693) in vitroLANA ChIP-seq revealed at least 17 distinct LANA peaks in both BCBL-1 and TIVE-LTCs and observed occupancy patterns are almost identical between both cell types . Howeverin vitro replication assays in vitro EMSA assays. Unexpectedly, numerous LANA peaks are located within promoters of IE, E, and late genes, including ORF16, ORF33, ORF39, ORF48, ORF58, ORF64, and vIRF-1 and -3 (LTI), which is RTA-responsive and induced during lytic replication LANA negatively regulates RTA expression and it was demonstrated that the RBP-Jk sites within the RTA promoter are critical for LANA-dependent regulation 1 and -3 . To deteAfter viral mapping, unmapped reads from LANA ChIP-seq were mapped to the human genome hg19, and 2180 and 2951 unique peaks were identified in BCBL-1 and TIVE-LTC, respectively. In agreement with immunofluorescence data on LANA binding to mitotic chromosomes A list of all gene loci enriched by LANA ChIP-Seq in BCBL-1 cells and TIVE-LTC cells is provided in the supplement  and S3. Gene Ontology (GO) analysis was performed by using DAVID  and S5. Recently, Lu et al. performed LANA ChIP-seq in BCBL-1 cells and reported 256 enriched genes To test LANA regulation of IQGAP3 in cells, we determined IQGAP3 transcript levels in BCBL-1 and LANA-inducible BJAB cells. IQGAP3 mRNA levels are about 2-fold higher in BCBL-1 cells and induction of LANA in BJAB-Tet on-LANA cells moderately induced IQGAP3 transcription . TogetheTCCAT)3 formed from overlapping the motifs in TCCAT)3 motif formed a complex with LANA-C that was visible on longer exposures comparable to those used to detect binding of LANA-C to LBS2. The complex was supershifted in the presence of V5 mAb, confirming specificity. This demonstrates that the 15 nt (TCCAT)3 sequence  and in TIVE-LTC cells 205 out of 2951 (6.9%) peaks contained sequence similarity to LBS1/2 . Hence, sequence  present Next, we asked whether LANA-bound promoters were enriched for specific host transcription factors. Genome-wide occupancy data for many transcription factors are available through the Encyclopedia of DNA Element (ENCODE) Consortium. As the closest available cell line to BCBL-1, we mined ChIP-seq data from GM12878 cells, a B cell lymphoma cell line, for which 43 genome-wide transcription factor binding profiles are available 2 = 0.9)  . This suin vitro translated LANA, but did not detect direct interaction with these proteins (data not shown). In summary, these data show that LANA interacts with endogenous hSET1 complexes either directly or through protein-protein interaction. Recruitment of hSET1 complexes to specific chromatin loci has been reported to be mediated by a number of transcription factors or co-activators, including E2F, NF-E2, and USF1 de novo infection by interacting with HCF-1, which subsequently binds to and recruits hSET1 The strong correlation of LANA and H3K4me3 peaks  raised tde novo infection or during long-term latency.Previous reports by several independent groups demonstrated the importance of H3K27me3 deposition for both the establishment and maintenance of KSHV latency As with two previous studies, we found that in BCBL-1 latency-associated genes are enriched with H3K4me3 and PolII but depleted of H3K27me3  that is highly prevalent in the human genome and was highly enriched in both cell types (TCCAT)3 motif was lower than LBS1 but comparable to LBS2, the low affinity site within the TR TCCAT)3 consensus sequence demonstrates that LANA can directly bind to host cellular DNA via two distinct sequence motifs. This interpretation is also supported by cluster analysis using seqMiner algorithms, which revealed a number of distinct binding patterns across all LANA-enriched promoters 3, which occurs with high frequency in the human genome.Mercier et al. Motivated by LANA's preferential association with H3K4me3 mark-containing promoters, we asked whether LANA interacts with KMTs and demonstrated that LANA efficiently immunoprecipitates with hSET1, the main H3K4 methylase in mammalian cells We propose a working model for the establishment of the viral epigenome which integrates recent findings affecting i) the epigenetic variation of KSHV episomes de novo infection an early burst of promiscuous transcription which includes both LANA and the RTA gene occurs which leads to co-transcriptional H3K4me3 deposition at many promoters After We envisage competition between PRC2 silencing telomerase-immortalized human umbilical vein endothelial cells (TIVE-LTC) 2 atmosphere. BCBL-1 and BJAB cells were cultured in RPMI 1640 medium supplemented with 10% FCS and antibiotics at 37°C under 5% CO2 atmosphere.293 cells, the human embryonic kidney cells, and KSHV long term-infected 5′-GATCGGTACCACAACCCAGTCTCTAAACCAG-3′ and 5′-GACTACGCGTGTTCCTTAGGCTGCCCC), ORF16 (5′- CCGCTCGAGCGGTTGTCAACCAACCAGTCAATCACC and 5′- CCCAAGCTTGGGGCAAAACGTCCTCGTCCATT), ORF39 (5′- GGGGTACCCCGAATGATGTTTGTCTTCGCC and 5′- CCGCTCGAGCGGCGCATGTTTCTCGGTCTTTT), ORF48 (5′- GGGGTACCCCTCTACCATGGAAGCCGGCAA and 5′- CCGCTCGAGCGGGGATACACACCTCCATGTTC), and vIRF1 (5′- GGGGTACCCCATGGAAGCCGGCAACAGTCCT and 5′- CCGCTCGAGCGGACACCTCCATGTTCAGTCAC). Corresponding promoter regions were PCR amplified, and cloned into plasmid pGL3-basic at appropriate sites.Primer pairs were designed to amplify the promoter region (+/−2 kb relative to the transcription start site) for IQGAP3 , rat IgG , rabbit against H3K4me3 , H3K27me3 , rabbit anti Pol II , rat LANA monoclonal antibody , rabbit anti hSET1 , rabbit anti RbBP5 , rabbit anti ASH2L , and rabbit anti MLL1 .7 cells were lysed in 1 ml ice-cold Farnham lysis buffer  with protease inhibitors. The nuclei were spun down and resuspended in 1 ml RIPA buffer  with protease inhibitors. Chromatin was sheared to about 250 bp fragments with 5 sets of 30-second pulses using a Sonic Dismembrator (Fisher Scientific) set to 50% of maximum power. Chromatin from 6×107 cells was incubated with 10 µg primary antibody  and 100 µl magnetic beads  at 4°C overnight on a rotator. Beads were washed 5 times with LiCl wash buffer and once with TE. The immune complexes were eluted with 200 µl elution buffer twice at 65°C for 1 hour. The combined eluates were de-crosslinked at 65°C overnight. DNA was extracted once with phenol/chloroform and precipitated with ethanol. 20 µg glycogen was added as DNA carrier. DNA pellets were washed once with 70% ethanol and resuspended in 40 µl H2O.ChIP experiments were performed as described before with minor modifications 5′-pGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG and 5′- ACACTCTTTCCCTACACGACGCTCTTCCGATCT) were ligated to both ends of the DNA fragments. DNA within the range of 150 bp to 300 bp was gel-purified and PCR-amplified for 18 cycles with primer 5′ -AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT and 5′- CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT. The resulting libraries were gel-purified, quantified with QuantIT dsDNA Assay Kit (Invitrogen) and sequenced with Genome Analyzer IIx (Illumina) in Department of Molecular Genetics and Microbiology (MGM) at University of Florida (UF).100 ng ChIP-enriched DNA or control IgG ChIP DNA was blunt-ended with T4 DNA polymerase and Klenow DNA polymerase (NEB) and phosphorylated with T4 PNK (NEB). Addition of an “A” base to the 3′ ends of the blunt phosphorylated DNA fragments was performed in the presence of Klenow exo- (NEB). Subsequently, adapters . The captured DNA was eluted and purified. The DNA was re-amplified for 22 PCR cycles with primers (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT and 5′-CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT), and purified using Agencourt AMPure XP beads. The libraries were quantified with QuantIT dsDNA Assay Kit (Invitrogen) and sequenced as above. HTS sequencing generated 20.8 million, 17.1 million, and 20.5 million tags for H3K4me3, H3K27me3, and Pol II ChIP-seq, respectively. After enrichment 42.76%, 65.4%, and 87.58% of H3K4me3, H3K27me3, and Pol II ChIP-seq tags were mapped to the viral genome compared to 0.014%, 0.028%, and 0.007% without enrichment; hence enrichment efficiency was about 2,000- to 4,000-fold.The biotin-labeled RNA baits specific for KSHV genome was customized with eArray XD (Agilent) with help from Agilent. The RNA baits are 120 nt long with 4× tiling frequency. TIVE-LTC ChIP-seq libraries were constructed as above. After adapter ligation, DNA fragments between 150 bp and 300 bp were gel-selected and amplified with 10 cycles in first PCR. Samples were purified with Agencourt AMPure XP beads (Beckman Coulter). The KSHV-specific DNA sequences were enriched with SureSelect Enrichment System (Agilent) according to the manufacturer's instruction. The purified 1http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52421).After completion of Illumina sequencing, the raw output of BCL files were converted to the FASTQ format using the Illumina BCL Converter. Bowtie Peak finding was performed with CisGenome v1 In order to examine the reads that might align to the junction of two TRs and that might be unaligned to the reference genome with only one TR at the end, we attached the sequence of a half or an entire TR to the viral genome and ran the alignment pipeline against these new reference .Visualization in IGV indicated that the chromatin mark H3K4me3 usually existed near LANA binding signals. We applied CisGenome on H3K4me3 ChIP-seq data but did not obtain reasonable peaks. The finding was consistent with the consensus that most peak calling programs were not designed for histone data and therefore performed poorly in identifying these diffuse signals To study the potential regulatory function of LANA in gene expression, we mapped the reads aligned to the human genome to the promoter regions. We downloaded transcript information of Ensembl build 62 from UCSC Table Browser and defined promoters as the +/−2 Kb region of the transcription start site (TSS) of annotated transcripts. The genome positions of peaks were compared to the promoter positions using BEDTools intersectBed and any overlap was reported. The distances between the center of the peaks and their adjacent TSS's were plotted for each lane. Putative targets genes of LANA regulation were compared between BCBL1 and TIVE-LTC, as well as with previous publications.Similarly, the reads aligned to the KSHV genome were also mapped to KSHV genes, the genome positions of which were downloaded from the NCBI Nucleotide database (NC_009333.1) and parsed using a custom Perl script.GCCCCATGCCCGGGCGG , GCCGCAGGCCCCGGCGG , ATTGTCCCGGGCGCCGCG, and CCGGGTCTCCAGGGCGCGCCGCGTG. For this purpose, we used an EMBOSS tool FUZZNUC The peaks were scanned for LANA consensus LANA binding sites (LBSs): To study the tissue-specific co-occupancy of LANA and other TFs, we downloaded the peak files of all ENCODE TFBS ChIP-seq experiments in GM12878 . A total of 43 TFs were available in GM12878 The peak files were mapped to the hg19 promoter regions. A peak overlapping at least one base with the promoter was considered to be a regulator of the promoter, the same as we mapped our LANA peaks. The transcripts with both LANA and ENCODE TFBS peaks in their promoters were identified. The distance between the center of the LANA peak and the ENCODE peak within the same promoter was calculated and plotted (e.g. CTCF and STAT1 in GM12878 vs. BCBL1). In the cases that multiple peaks exist in a promoter, the smallest distance was retained.http://rsat.ulb.ac.be/rsat/) We used the “peak motifs” tools from Regulatory Sequence Analysis Tools  according to the manufacturer's instruction using the following program: Step 1: 95°C 20 sec; Step 2: 95°C 3 s, 60°C 30 sec, repeat 39 times; Step 3: melting curve from 60°C to 95°C. 32P]-ATP, and incubated with purified LANA-C protein as described previously DNA duplexes were created by annealing the oligonucleotide pairs listed in Supplementary 5 BJAB, BCBL-1, TIVE, or TIVE-LTC cells were harvested and washed once with PBS. Cells were lyzed with 100 ul RIPA buffer. 20 ul of the cells lysate was separated in 8% SDS-PAGE gels and transferred to PVDF membrane. Membranes were blocked for 2 hours in T-TBS buffer containing 5% fat-free milk. Primary antibodies against specific proteins were diluted according to manufacturers' instruction and hybridized with membrane at 4°C overnight. After washing, 1∶5000 diluted corresponding secondary antibodies conjugated with peroxidase were incubated with the membrane for 1 hour at room temperature. After final washing, the blots were developed with ECL substrates (Millipore) and exposed to films.Western blot analysis was performed as previously described 7 cells were harvested and resuspended in 1 ml hypotonic buffer . Nuclei were spun down and resuspended in hypertonic buffer . Cells were lyzed at 4°C for 15 minutes with rotation. Dilution buffer which had the same ingredients as hypertonic buffer except for NaCl was added to make the final NaCl concentration 150 mM. Cell lysate was centrifuged for 10 minutes and pre-cleared with 50 µl protein A/G beads. A small portion of the lysate was saved as input. Cell lysate was incubated with 10 µg antibody at 4°C overnight with rotation. 100 µl protein A/G beads were added and incubated for 2 hours. The beads were collected by pulse centrifugation and washed 5 times with 1 ml ice-cold PBS with 0.1% Tween-20. Proteins were eluted with 1× Laemmli buffer and subjected to Western blot analysis.IP experiments were performed as described before with minor modifications Figure S1Bland-Altman Plot to investigate the agreement between two replicates of the ChIP-seq experiments. RPKM was calculated for each sample after Bowtie alignment. The Y axis is the difference between two biological replicates and the X axis is the average between biological replicates. Green lines are the average of all differences +/−1.96× (standard deviation of the differences), indicating 95% confidence interval. The red line is drawn at zero.(EPS)Click here for additional data file.Figure S2Distribution of H3K4me3, H3K27me3, Pol II, and LANA within the latency-associated region. Detailed wiggle plots generated with UCSC genome browser illustrating nearly identical occupancy between latently infected BCBL-1 cells (A) and TIVE-LTC cells (B). Note distinct LANA, H3K4me3, and polII peaks within LANA ORF in TIVE-LTC cells (B).(EPS)Click here for additional data file.Figure S3LANA binding within the terminal repeat and ORF50 regions. Wiggle plots visualizing LANA occupancy on the viral TR and ORF50/RTA region. (A) Identical peak recovery from BCBL-1 and TIVE-LTC cells for binding to the LBS1/2 binding site within TR. Note very high recovery of LBS1/2 from BCBL-1 cells (y-axis). (B) Identical peak recovery within exonic sequences of RTA gene from both cell types. No LANA occupancy upstream of the RTA TSS was observed.(EPS)Click here for additional data file.Figure S4Examples for cell type-specific and common LANA peaks within host promoters. Wiggle plots generated with UCSC genome browser on selected LANA CHIP-seq-enriched promoters demonstrating cell type-specific (A–C) and (E–H) and common (D) occupancy between BCBL-1 and TIVE-LTC cells.(EPS)Click here for additional data file.Figure S5Flow diagram of LANA peak identification and annotation. Details about algorithms and programs used at each step are described in the Material and Methods Data analysis section.(PPTX)Click here for additional data file.Table S1Comparison of KSHV nts sequences in Accession number NC_009333.1 and U75698.(DOCX)Click here for additional data file.Table S2Potential cellular targets regulated by LANA in BCBL-1 cells.(XLSX)Click here for additional data file.Table S3Potential cellular targets regulated by LANA in TIVE-LTC cells.(XLSX)Click here for additional data file.Table S4GO analysis of LANA targets in BCBL-1 cells.(XLSX)Click here for additional data file.Table S5GO analysis of LANA targets in TIVE-LTC cells.(XLSX)Click here for additional data file.Table S6Oligonucleotides used in EMSA assays.(XLSX)Click here for additional data file.


Pseudomonas fluorescens strains PA4C2 and PA3G8 and Pseudomonas putida strain PA14H7 were isolated from potato rhizosphere and show an ability to inhibit the growth of Dickeya phytopathogens. Here, we report their draft genome sequences, which provide a basis for understanding the molecular mechanisms involved in antibiosis against Dickeya. Pectobacterium and Dickeya genera are causative agents of the blackleg and soft rot diseases on potato crops , and contigs >2,000 bp were collected. Scaffolding was performed using SSPACE basic V2.0 , and GapAXDA00000000 (P. fluorescens PA4C2), JBOO00000000 (P. fluorescens PA3G8), and JBOP00000000 (P. putida PA14H7).These whole-genome shotgun projects have been deposited in DDBJ/ENA/GenBank under the accession numbers


Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90.Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of  Plasmodium falciparum strains displaying delayed clearance by the current treatment of choice, artemisinin and its derivatives, and it is considered inevitable that artemisinin resistance will eventually develop and spread  is an abundant protein expressed in many stages of the ed mRNAs . In the ed mRNAs , 11. Itsin vitro  and thatin vitro –14, thergene out . This successful ; howeverccessful .P. falciparum growth, has been shown by affinity purification to bind to CDPK1  in the subnanomolar range (–50) of 12 nM for killing P. falciparum in culture, the compounds performed disappointingly in a rodent model of malaria infection. In addition, one puzzling feature of these inhibitors was the poor correlation between their ability to inhibit the enzyme and their ability to block parasite growth, which was suggestive of off-target activity contributing to their inhibitory effects -dependent protein kinase (PKG) with a T618Q substitution has been described previously . All drug treatments were carried out in duplicate at a final dimethyl sulfoxide (DMSO) concentration of 0.05%. After incubation, a modification of a previously described SYBR green assay was performed . The compounds 1--3--1H-pyrazolopyrimidin-4-amine (NA-PP1) and 1--3--1H-pyrazolopyrimidin-4-amine (NM-PP1) were obtained from Merck.Aliquots of 100 μl of erformed . Cells wP. falciparum 3D7 genomic DNA at 194 bp upstream of the ATG to bp 435 of the open reading frame using primers 1 and 2 for the wild-type (WT) version and primers 1 and 3 for the glycine version. Each of these fragments was cloned via XmaI and EcoRI sites into a Geneart vector containing a recodonized gene fragment from bp 436 to 1572 of the P. falciparumcdpk1 (Pfcdpk1) open reading frame. Together, the native and recodonized cdpk1 sequences were cloned between XmaI and AvrII sites of the pHH4-HA plasmid (P. berghei dihydrofolate reductase [DHFR]-thymidylate synthase) and human DHFR (hDHFR) drug selection cassette. Primers were the following: primer 1, ACACCCCGGGGTATACAACGTATAAGACAAATTACTTTTCTTTC; primer 2, ATATGAATTCGGTTACTAAATAAAAATATTTCTTATCTTCAAAAACATCAAAC; primer 3, ATATGAATTCGCCTACTAAATAAAAATATTTCTTATCTTCAAAAACATCAAAC.Parasites expressing CDPK1 T145G and CDPK1 T145T were generated as described previously . Briefly plasmid , which aP. falciparum 3D7 parasites were transfected with pHH4-CDPK1-HA plasmids (T145T or T145G) using standard methods . Transfer to a nitrocellulose membrane was performed using an iBlot system (Life Technologies). Antibodies against HA  and CDPK1 . After centrifugation for 20 min at 15,000 × PfCDPK1 and PfCDPK1 T145G proteins were produced using methods described elsewhere . The PfHEscherichia coli Rosetta(pLysS) cells, and protein was expressed by induction with 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37°C for 2 h. PfHSP90 proteins were purified using Ni-nitrilotriacetic acid (NTA) affinity chromatography (Qiagen) as described in the manufacturer's protocol.The PCR product was cloned into pRSET-A (Life Technologies) between the BamHI and XhoI sites. Positive clones were confirmed by restriction digestion and sequencing. The clone was transformed into 50s were determined in kinetic mode using a rhodamine-labeled ParM (Rh-ParM) ADP sensor  to a final concentration of 100 nM and mixed with 10 μl of Rh-ParM at a final concentration of 100 nM in black 384-well plates (Corning). Compounds were diluted in half-log series in DMSO, and 2-μl volumes of diluted compound, or DMSO alone, were added to the enzyme and incubated for 30 min at room temperature. A further control reaction had DMSO but no enzyme. The reaction was initiated with 5 μl of 20 mM MgCl2 and ATP at the appropriate previously determined mK values .ICP sensor . For them values  of 30 μME. coli Rosetta2(DE3). Single colonies were grown in LB rich broth (supplemented with 50 μg/ml carbenicillin and 34 μg/ml chloramphenicol) at 37°C. Protein expression was induced with 1 mM IPTG once an optical density of 0.6 to 0.7 had been reached. Subsequent overnight growth was at 16°C. PKGs were purified via the histidine tag on HiTrap Talon columns  according to the manufacturer's instructions and then concentrated on 10-kDa-molecular-mass-cutoff concentrators (Amicon). Purified proteins were stored in 50% glycerol at −80°C in single-use aliquots. Final buffer composition of the purified product was 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 150 mM NaCl, 0.1% β-mercaptoethanol, 50% glycerol, 0.03% Brij-35, 1 mM benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride (PMSF).Full-length PfPKG and PfPKG T618Q were prepared as described previously , 34. Bri50s were determined for test compounds using a microfluidic fluorescent shift assay (unpublished data). Briefly, compounds were prepared over a 10-well half-log dilution series in DMSO in duplicate in 50-μl volumes using 96-well polypropylene U-bottomed plates . The reaction mix for each well consisted of 20 μl of enzyme/peptide mix , 20 mM β-glycerophosphate, 2 mM DTT, 10 μM cGMP, 0.01% (wt/vol) bovine serum albumin (BSA), 0.01% (vol/vol) Triton X-100]) plus 5 μl of compound. Samples were preincubated at room temperature for 30 min, and reactions were initiated by addition of 25 μl of ATP mix . Reactions were terminated at approximately 10% substrate conversion by addition of 50 μl of stop solution (25 mM EDTA in water). Samples were analyzed by electrophoretic separation of substrate and product peak and fluorescence detection using a Caliper LabChip EZ Reader  with a 0.2-s buffer sip time, downstream voltage of 500 V, upstream voltage of 1,950 V, and pressure of 0.5 to 1.5 lb/in2. IC50s for the compounds were determined using a four-parameter logistical fit of the data (GraphPad Prism).ICThe chemical synthesis of compound D with biotin attached to the R1 or R2 group is described in Information S1 in the supplemental material.2, 250 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 1× protein phosphatase inhibitor (Sigma-Aldrich), 1× complete protease inhibitors (Roche), and 0.1% Triton X-100 (Sigma-Aldrich). Insoluble proteins were removed by centrifugation at 15,000 × g for 20 min, and protein was quantitated using a detergent-compatible (DC) protein assay (Bio-Rad). The resin was extensively washed in lysis buffer to remove unbound proteins, and proteins were eluted by resuspending the resin in 100 μl of 2× reducing lithium dodecyl sulfate (LDS) sample buffer (Life Technologies) and heated at 95°C for 5 min.A total of 200 μl of high-capacity streptavidin agarose (Thermo Scientific) was resuspended in 1 ml of 250 μg/ml biotinylated compound D solution or a DMSO control. After incubation with mixing for 1 h at room temperature, unbound compound was removed by extensive washing with phosphate-buffered saline (PBS). The resin was incubated with trophozoite lysate containing 2 mg of protein in a total volume of 1.5 ml overnight at 4°C. The lysate was prepared by resuspending trophozoites in 20 mM Tris-HCl, pH 8.0, 10 mM MgCl18 column, coupled to a linear trap quadrupole (LTQ) Orbitrap Velos Pro equipped with a Nanoflex electrospray source . A gradient of 6 to 32% acetonitrile–0.1% formic acid over 48 min was used at a flow rate of 0.3 μl/min. The Orbitrap was operated in data-dependent acquisition mode with a survey scan at a resolution of 60,000, and up to the 10 most intense ions were selected for MS/MS. Raw files were processed using Proteome Discoverer (PD), version 1.3 (Thermo Scientific) with Mascot, version 2.4 , as the search engine against the appropriate protein database. A decoy database of reversed sequences was used to filter the results at a false detection rate of 1%.Proteins were run 4 mm into a 10% NuPAGE Bis-Tris gel (Life Technologies) and then excised using a clean scalpel blade. Proteins were reduced and alkylated prior to overnight trypsin digestion. The resulting digests were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Ultimate 3000 nanoRSLC high-performance liquid chromatograph (HPLC), equipped with a 50-cm by 75-μm Acclaim Pepmap CN-allylamino-17-demethoxygeldanamycin (17-AAG) or compound D ranging from 0 to 60 μM. The binding buffer used for the reaction contained 50 mM Tris-HCl, pH 7.4, and 1 mM EDTA. Samples were excited at 280 nm, and tryptophan fluorescence measurements were carried out by scanning the emission spectrum in the wavelength range 300 to 400 nm. A maximum wavelength (λmax) of 346 nm was selected for all the calculations. The slit widths of excitation and emission were set at 2.5 and 5 nm, respectively. The difference in fluorescence intensity between protein alone and protein with various concentrations of 17-AAG or compound D was calculated and plotted against the different concentrations of the respective ligand used. The resultant hyperbolic curve was analyzed with GraphPad Prism software, using nonlinear regression analysis with single-site-specific binding as described previously  was incubated with concentrations of 17-eviously .3K60)  of DockingServer by Virtua Drug  . The PfH3K60) . Chain A3K60) . An affi3K60) . Test an3K60) . Here, GiK), and frequency of probable binding sites for ranking our docking results. Frequency shows the percentage of the local searches with similar geometry having a root mean square tolerance (rmstol) of 2 Å. Docked structures with the lowest free energy of binding and iK values and structures having two or more hydrogen bond interactions between ligand and protein and a frequency of 10% or more were used for prediction of the probable binding configuration for compound D. The analyzed PDB format of the docking file was downloaded and analyzed using Discovery Studio Visualizer, version 4.0.The prepared molecular structures were subjected to docking simulation using the Lamarckian genetic algorithm (LGA) and the Solis and Wets local search method  which is50s and are extremely effective at killing parasites in culture, with the most potent compound having an EC50 of 12 nM  killed parasites at the mature schizont stage late in the cycle, while others (class 2 compounds) killed parasites at the early trophozoite stage  and 96 h (EC5096), and the ratio of the EC50s was calculated; an EC5048/EC5096 ratio greater than 5 was a strong indication that a compound acted after DNA synthesis. When the structure of each compound was examined, a determinant of the stage at which the compound acted was the presence or absence of a pyrimidine at the aromatic linker position . Of the 25 compounds with nonpyrimidine linkers, 24 had an EC5048/EC5096 ratio less than 5, suggesting that their action was prior to DNA synthesis (class 2). Full details are provided in Table S1. This classification was again confirmed by microscopic examination of Giemsa-stained blood smears of parasites treated with a subset of 10 compounds  have EC5048/EC5096 ratios greater than 500.We analyzed 41 compounds in this manner in order to define the molecular features that determine their modes of action . For each compound, the ECin vitro, and this binding was blocked by replacing the gatekeeper residue with the larger glutamine, which blocks access to the enzyme's binding pocket, rendering it relatively insensitive to specific inhibitors . We have shown previously that the correlation between the IC50 for CDPK1 and the P. falciparum EC50 for these compounds was poor (50 and parasite-killing EC50 when the aromatic linker was a pyrimidine (class 1)  , panel i50 of six class 1 and five class 2 compounds against 3D7 wild-type (WT) and PKG T618Q parasites, using the SYBR green I assay described previously, with a 96-h incubation time. We also included compound 1, a trisubstituted pyrrole previously shown to be an apicomplexan PKG inhibitor that is also sensitive to the size of the gatekeeper residue (90 (0.65 μM) showed that the parasites were arrested at the schizont stage, suggesting that it behaves like other class 1 compounds in terms of its mode of parasite killing. Compound 1 showed a 7-fold decrease in potency against the PKG T618Q parasite, in line with previously published data  in vitro was confirmed by measuring changes in intrinsic tryptophan fluorescence values upon binding , which had 24 unique peptide hits . HSP90 is a protein chaperone with an ATP binding site in its N-terminal domain. Using e energy . The Ki  binding . The Kd nhibitor  . Kinases with large gatekeeper residues tend to be insensitive to these inhibitors, whereas those with small gatekeeper residues are more sensitive , 47. Theae cells . CDPK1 Tectively . We used50s for each compound were calculated . Both of the BKIs used in this study have been demonstrated to be cell permeable in several other systems  at almost undetectable levels in asexual blood stages was used, there was no effect on parasite development during these stages .Our view that CDPK1 may not be essential in e stages . More reelopment . There iP. falciparum parasites accumulate in late schizogony, with a block of merozoite egress (Purfalcamine, a 2,6,9-trisubstituted purine, has been identified as an inhibitor of PfCDPK1, and in its presence e egress . While te egress . It willPlasmodium infections. While CDPK1 may represent a target for transmission-blocking treatment, owing to its proven role in the sexual stages of the parasite (Plasmodium.Target validation for drug discovery can be a complex process, and extrapolating from genetic knockout studies to the consequences of inhibition by small molecules can be problematic . Where tparasite , it seem


Domestic ducks are natural reservoirs of avian influenza viruses and serve as reassortant hosts for new virus subtypes. We isolated 2 novel influenza A(H5N8) viruses from domestic ducks in eastern China, sequenced their genomes, and tested their pathogenicity in chickens and mice. Circulation of these viruses may pose health risks for humans. Orthomyxoviridae and contain 8 segments of single-stranded RNA with negative polarity  viruses, A/duck/Zhejiang/W24/2013(H5N8) (W24) and A/duck/Zhejiang/6D18/2013(H5N8) (6D18), from domestic ducks. To better understand genetic relatedness between these viruses, we sequenced all gene segments of these 2 viruses and compared them with influenza virus sequences in GenBank. We also determined the virulence of the 2 isolates in chickens and mice.For virus isolation, cloacal swab specimens from domestic ducks were inoculated into embryonated chicken eggs as described  according to the manufacturer’s instructions. All gene segments were amplified with primers, and fragments were sequenced and analyzed as described  of viruses W24 and 6D18 showed 99.9%–100% sequence similarity . ResultsSequence analysis suggested that these H5N8 subtype viruses were most closely related to isolates from poultry in countries in eastern Asia. Previous studies have shown that H5 subtype viruses within clade 2.3.2 have been circulating widely in poultry and wild birds in China since 2007 , panel BLee et al. . On the On the basis of deduced amino acid sequences of HA genes, we found that the HA cleavage site pattern (PLREKRRKR) of the 2 novel H5N8 subtype viruses indicated that these viruses were highly pathogenic. In this study, amino acid sequences of these H5N8 subtype viruses at positions 236–241 and 146–150 were NGQRGR and GVSAA, respectively. Receptor-binding sites (Gln226 and Gly228) of H5N8 subtype viruses were similar to those of the 2 novel H5N8 subtype viruses, which suggested that these 2 viruses would preferentially bind to avian-like receptors  in NS1 proteins had been observed more frequently in H5N1 subtype viruses, which indicated possible adaptation of these viruses to avian species  and 100% (5/5), respectively, for each virus during 14 days postinfection  viruses were isolated from domestic ducks in eastern China in 2013. Results of phylogenetic analysis showed that these viruses were reassortant viruses that derived their genes from different virus subtypes. These reassortant H5N8 subtype viruses and their 3 possible parent viruses, A/duck/Jiangsu/k1203/2010 (H5N8), A/environment/Jiangxi/28/2009 (H11N9), and A/duck/Hunan/8–19/2009 (H4N2), were isolated in China. Both H5N8 subtype isolates were highly pathogenic for chickens and showed moderate pathogenicity for mice. Domestic ducks are considered the natural reservoir of avian influenza viruses and serve as reassortant hosts for creation of new virus subtypes. Continued circulation of these viruses may pose health threats for birds and humans.


V600E inhibitor-resistant melanoma cells showed about 80% decreased invasion in the DTX3L-depleted cells compared to that in the DTX3L-intact cells. Thus, DTX3L is clinically a potential therapeutic target as well as a potential biomarker for melanoma.Deltex-3-like (DTX3L), an E3 ligase, is a member of the Deltex (DTX) family and is also called B-lymphoma and BAL-associated protein (BBAP). Previously, we established RFP/RET-transgenic mice, in which systemic hyperpigmented skin, benign melanocytic tumor(s) and melanoma(s) develop stepwise. Here we showed that levels of Dtx3l/DTX3L in spontaneous melanoma in RFP/RET-transgenic mice and human melanoma cell lines were significantly higher than those in benign melanocytic cells and primarily cultured normal human epithelial melanocytes, respectively. Immunohistochemical analysis of human tissues showed that more than 80% of the melanomas highly expressed DTX3L. Activity of FAK/PI3K/AKT signaling, but not that of MEK/ERK signaling, was decreased in Dtx3l/DTX3L-depleted murine and human melanoma cells. In summary, we demonstrated not only increased DTX3L level in melanoma cells but also DTX3L-mediated regulation of invasion and metastasis in melanoma through FAK/PI3K/AKT but not MEK/ERK signaling. Our analysis in human BRAF Previous studies showed that the incidence of melanoma, which is known as an aggressive cancer with high metastatic ability, is increasing at a greater rate than that of any other cancer , 2. MelaMetastatic dissemination of a primary tumor to a secondary site is the major cause of deaths from solid tumors , 5. The in vitro study showed the effect of DTX3L via STAT1 and IRF-1 in prostate cancer cells [in vitro studies on Dtx3l/DTX3L functions are limited to solid tumors, and in vivo studies are further limited.Deltex-3-like (DTX3L), an E3 ligase, is a member of the Deltex (DTX) family and is also called B-lymphoma and BAL-associated protein (BBAP). DTX3L was originally identified as a binding partner of B aggressive lymphoma 1 (BAL1), a risk-related gene and protein in diffuse large B cell lymphoma (DLBCL) , 14. Exper cells , in vitrRFP/RET-transgenic mice of line 304/B6 (RET-mice), in which systemic hyperpigmented skin, benign melanocytic tumor(s) and melanoma(s) develop stepwise [in vitro and in vivo study newly clarified a function as well as expression level of DTX3L in murine and human melanomas.Previously, we established stepwise . RET-micstepwise , 2. Our stepwise  showed iAfter selection of Dtx3l from our previous results of DNA microarray analysis , we firsDTX3L transcript expression levels in all of the melanoma cell lines  were significantly higher than the level in NHEM cells , primary melanomas (n = 54) and metastatic melanomas (n = 20)  Figure  and 3B. We next tried to clarify the function of Dtx3l in murine B16F10 melanoma cells. Invasion activity Figure  and invaWe next examined the function of DTX3L in human G361 melanoma cells. Invasion activity in DTX3L-depleted G361 melanoma cells was about 30% of that in control G361 melanoma cells. Corresponding to the murine melanoma cells, phosphorylation levels of FAK, PI3K and AKT in DTX3L-depleted cells were decreased compared to those in control cells  was reduced compared to that in control cells (Sh-Control) in our macroscopic analysis for fluorescence intensity on the surface of the lung  cells  were cultured in HMGS medium. Human melanoma cell lines of SK-Mel28 and G361 were obtained from Riken Bio Resource Center. A MNT1 cell line was a kind gift from Dr. VJ Hearing . Human melanoma cell lines of A375P and A375 and a murine nontumorigenic immortalized melanocyte (melan-a) cell line were kindly provided by Dr. Dorothy C Bennett, St George's, UK. Murine melanoma cell lines of B16, B16F1, B16F10 and B16BL6 were obtained from Cell Resource Center for Biomedical Research in Tohoku University. Benign melanocytic tumors and melanomas in transgenic mice of line 304/B6 (RET-mice) carrying constitutively activated RET [Total RNA was prepared from a frozen tumor sample and from murine and human cell line samples using a High Pure RNA Kit (Roche Diagnostics) according to the method previously described . cDNA waImmunoblot and immunohistochemical analyses were performed according to the method described previously , 34. RabSilencing vector pRNAT-U6–1-Neo (Invitrogen) was used for construction of the DTX3L silencing vector. A double-strand DNA fragment including a knockdown sequence for mouse Dtx3l was inserted into BamHI and HindIII sites. Control and DTX3L silencing vectors were transfected into B16F10 cells, and stable cell clones were selected with 1 mg/ml neomycin (Wako). Oligonucleotide sequences of the DNA fragment are 5′-GATC CGCATGGAGGGTAG TGATGGAATTAATTCA AGAGATTAATTCCATCACTACCCTCCATGCTTTTT TA-3′ and 5′- AGCTTAAAAAAGCATGGAGGGTAGTG ATGGAATTAATCTCTTGAATTAATTCCATCACTAC CCTCCATGCG-3′. Expression vector pCMV-c-Fa-Puro3 (Invitrogen) was used for construction of the DTX3L expression vector. The human DTX3L coding region fused with a FLAG sequence was inserted into BamHI and XhoI sites. Empty and DTX3L expression vectors were transfected into G361 cells, and stable cell clones were selected with 1 mg/ml puromycin (Wako).in vitro invasion assay according to the method previously reported [in vivo analysis of metastasis was performed by the method previously reported [6; n = 5) and control B16F10 cells  in 50 μl serum-free RPMI medium had been injected into tail veins of 6–8-week-old C57/BL6 mice, metastatic foci in the lung 14 days after inoculation were evaluated by fluorescence intensity.Cell invasion ability was evaluated by an reported , and in reported . After aThe Animal Care and Use Committee , the recombination DNA Advisory Committee  and the ethical committee  in Nagoya University and Chubu University approved this study.t-test.Statistical analysis in this study was performed according to the method previously described . Results