N-glycosides in Arabidopsis, which was tentatively elucidated as nicotinate N-pentoside (NaNP) and NA N-rhamoside (NaNRha), using liquid chromatography triple-quadrupole mass spectrometry (LC-QQQ-MS) with precursor ion-scanning (PreIS). We further quantitatively profile the NAD+-related metabolites in 24 tissues of Arabidopsis. Biochemical assays of UGT76C family revealed that UGT76C5  was a multiple functional nicotinate N-glycosyltransferase, with high preference to UDP-xylose and UDP-arabinose. The deficiency of NaNP and NaNRha in ugt76c5 mutant suggested that UGT76C5 is responsible for biosynthesis of NaNP and NaNRha in planta. We also identify one amino acid difference in PSPG (plant secondary product glycosyltransferase) motif is responsible for the divergence of NaNGT (UGT76C4) and UGT76C5. Taken together, our study not only identifies a novel nicotinate N-glycosyltransferase but also paves the way for investigations of the in planta physiological functions of various NA conjugations.The Preiss-Handler pathway, which salvages nicotinate (NA) for NAD synthesis, is a conserved biochemical pathway in land plants. We previously demonstrated that various NA conjugations (mainly methylation and glycosylation) shared the NA detoxification function in all tested plants. It remains unclear whether other NA conjugates with low abundance exist in plants. In this study, we discovered at least two additional NA  In Arabidopsis, NA was catalyzed sequentially by nicotinate phosphoribosyltransferase , nicotinate mononucleotide adenylyltransferase , and NAD synthase  (14C]-nicotinamide ([14C]-NAM) as a tracer, showed that NA in plants, in addition to being a precursor for NAD replenishment, can also be converted into a variety of NA conjugations (N-position or carboxyl group of NA) had been detected in plants. We had demonstrated that NA O-glucosylation catalyzed by nicotinate O-glucosyltransferase , a process likely restricted to the Brassicaceae, functions to detoxify NA and involve into seed germination under stress conditions  were also cloned and functional characterized  might have facilitated the retention of the Preiss-Handler pathway in land plants  Figure 11g55090) . Feedingugations . We prevnditions . Two gencterized . Howeverd plants . More rethaliana .Arabidopsis thaliana. Given the low resolution of TLC analysis used in previous studies on NA metabolism, the discovery of unknown NA conjugates, especially for those which are in low abundance, is really limited. Recently, the mass spectrometry with precursor ion-scanning (PreIS) becomes an alternative choice to discover a series of derivatives with a common substructure (such as the NA moiety in NA conjugates)  . The QQQN-glycosides in Arabidopsis thaliana by using LC-QQQ-MS with PreIS; one peak was identified as NA N-pentoside, and another one was tentatively identified as NA N-rhamoside. Quantitative NAD+ metabolomics of 24 Arabidopsis tissues were determined using LC-QQQ-MS with the optimized MRM (multiple reaction monitoring) conditions. We further determine UGT76C5 (encoded by At5g05890) as a multiple functional NA N-glycosyltransferase with sugar-donor promiscuity. One amino acid in PSPG (plant secondary product glycosyltransferase) motif is responsible for the biochemical difference of UGT76C4 (NaNGT) and UGT76C5.In this study, we first discovered two types of NA Arabidopsis thaliana (Col-0 ecotype) lines were grown on soil at 22°C under a 16-h light (120 µmol m−2 s−1)/8-h dark cycle, 60% relative humidity. To harvest young seedlings, the sterilized seeds were sown on ½ MS plates (0.8% agar). The plates were incubated at 4°C for 3 days and then transferred into growth chamber with long-day condition . All chemicals used in this study were purchased from Sigma-Aldrich (USA) except the radiolabeled (carboxyl-14C)-NA (55 mCi/mmol) were purchased from American Radiolabeled Chemicals (USA). UDP-xylose and UDP-arabinose were purchased from Sugars Tech company .The wild-type and transgenic Arabidopsis are carried out as described previously  was used as the nebulizer  and sheath gas . The parent ion ranges from m/z 50 to 800. The precursor ion was m/z 124, fragment was 380 v, and collision voltage was 20 v. Data were analyzed by MassHunter Software B07.01 (Agilent).The extraction and measurement of NA, its conjugates, and other NAD-related chemicals in eviously . An AgilUGT76C4 and UGT76C5 were generated by using a previously described PCR-mediated method , the m/z 124.0 fragment  was thus chosen as signature ion for PreIS to screen unknown NA conjugates , and NaNG by comparison with authentic compounds. The chemical structures of peaks 3 and 4 were further elucidated by product ion mode. The [M+H]+ ions for peaks 3 and 4 display monoisotopic m/z at 270.1 (270.0975 on qTOF-MS) and 256.1 (256.0817 on qTOF-MS), respectively. The lost neutral fragment for peak 3 has a predicted molecular weight of 146.0 (C6H10O4) consistent with a rhamnose moiety (5H8O4), consistent with a pentose moiety  and commonly detected in plants (N-arabinoside and NA N-xyloside could not be well separated on our LC-QQQ-MS system (N-pentoside (NaNP) to express the sum of NA N-arabinoside and NA N-xyloside for further analysis.Based on mass spectrometry cleavage rules of known NA conjugates , which adenylates both NMN and NaMN in vitro  was barely expressed in pollen  plays an important role in pollen germination and pollen tube elongation. Further experiments are needed to reveal the mechanism of this phenomenon.There are three other intriguing results from the chemical analysis. First, NMN is highly accumulated in all tested tissues except the mature seeds, although the physiological function is unclear thus far. NMN could be re-utilized for NAD biosynthesis catalyzed by NaMNAT , which is homologous to UGT76C4 and UGT76C5, without a known function thus far, was also included in the NA glycosyltransferase assays using [14C]-NA and UDP-arabinose or UDP-xylose as co-substrate. The assays showed that UGT76C5, rather than UGT76C3 and UGT76C4, catalyzed the NA pentosylation with a high efficiency. Consistent with previous results, both UGT76C4 and UGT76C5 could utilize UDP-glucose and NA as substrate , UDP-GlcA (UDP-glucuronic acid), and UDP-GlcNA (UDP-N-acetylglucosamine), are not included in this study, as both UGT76C4 and UGT76C5 showed little activity toward these sugar donors , which had been characterized in our previous study  activity in Arabidopsis and remove of NaNP and NaNRha in ugt76c5 mutant increase the UGT76C4’s activity to some extent.Using the above-mentioned LC-QQQ-MS method, we further analyzed the knock-out mutants and overexpression lines of us study . The rese plants . One posNP or/and NaNRha could be secreted from plants, mainly from root tissue. The results clearly showed that all nicotinate conjugates, except MeNA, can be detected in the liquid medium , 23 (S in UGT76C5 vs. N in UGT76C4), and 32 (A in UGT76C5 and G in UGT76C4) in a total 44 amino acids in the common , while G32A had a slight decrease  compared to wild-type UGT76C4 activity. It is noteworthy that S23N mutation in UGT76C5 led to around five-fold increase NaNGT activity, and D6E and A32G mutants had little effect on the NaNGT activity of UGT76C5  or S (serine). Altogether, N23 is important for the maximal catalytic efficiency of glucosyltransfer activity, whereas G or S at the same position is probably required for recognition of other sugar donors  and UGT76C5  prompted us to further study the mechanism underlying this phenomenon. PSPG motif  is highly conserved in all glycosyltransferases involved in a plant specialized metabolism, as this motif is thought to correspond to a UDP-binding site . Variati UGT76C5 Table 1.r donors , like thMoreover, other residues located outside the PSPG motif also played important role the recognition of UDP-sugars . FurtherArabidopsis thaliana, which provided fundamental knowledge for studying the physiological functions of NA conjugates and the NAD salvage pathway. UGT76C5 is the first multiple functional N-glycosyltransferase with sugar-donor promiscuity characterized from plant species. One amino acid in PSPG motif is identified for sugar-donor specificity of UGT76C4 and UGT76C5. These results increase our understanding of the relationship between NAD biosynthesis and NA conjugations and should also allow for additional experiments to provide insights into NAD homeostasis when plants response and adapt to (a)biotic stresses. Two recent studies demonstrated that the plant specialized metabolites (both focus on terpenoids) played a key role to modulate the root microbiota , the “Priority Research Program” of the Chinese Academy of Science (ZDRW-ZS-2019-2-0202), and the State Key Laboratory of Plant Genomics of China (SKLPG2016A-13 and SKLPG2016B-13) to G.W.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chinese wheat mosaic virus (CWMV) has a bipartite, single-strand positive RNA genome, and can infect both wheat and Nicotiana benthamiana, and the optimal temperature for systemic infection in plants is 17°C. To assess the potential of the virus as a vector for gene silencing at low temperature, a fragment of the N. benthamiana or wheat phytoene desaturase (PDS) gene was expressed from a modified CWMV RNA2 clone and the resulting photo bleaching in infected plants was used as a reporter for silencing. Downregulation of PDS mRNA was also measured by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). In experiments using fragments of PDS ranging from 500 to 1500 nucleotides, insert length influenced the stability and the efficiency of VIGS. The CWMV induced silencing system was also used to suppress miR165/166 and miR3134a through expression of miRNA target mimics. The relative expression levels of mature miR165/166 and miR3134a decreased whereas the transcript levels of their target genes increased. Interestingly, we also found the CWMV-induced silencing system was more efficient compare with the vector based on Barley stripe mosaic virus (BSMV) or Foxtail mosaic virus (FoMV) in wheat or the vector based on TRV in N. benthamiana at 17°C. In summary, the CWMV vector is effective in silencing endogenous genes and miRNAs at 17°C, thereby providing a powerful tool for gene function analysis in both N. benthamiana and wheat at low temperature.Virus-induced gene silencing (VIGS) is an important tool for functional genomics studies in plants. With this method, it is possible to target most endogenous genes and downregulate the messenger RNA (mRNA) in a sequence-specific manner.  Virus-induced gene silencing (VIGS) is a particularly powerful tool for studying plant functional genomics, especially for those organisms where genetic transformation is difficult . VIGS isTobacco mosaic virus (TMV) (Tobacco rattle virus (TRV) (Potato virus X (PVX) -based VIGS has been used as a vector in wheat, barley, oats, N. benthamiana, and Brachypodium (Foxtail mosaic virus (FoMV)-based VIGS has been applied for functional genomics in barley, wheat, and foxtail millet   , Tobaccous (TRV) , and Pot X (PVX)  used forhypodium  and Foxt italic) . BSMV-bain vivo. Plant miRNAs are important in controlling plant development and responses to abiotic or biotic stress  activity c stress . Increasc stress . Their fc stress , artificc stress , transcrc stress , and shoc stress . STTM waucleases .Chinese wheat mosaic virus is a member of the genus Furovirus, family Virgaviridae  hosts more effectively than a vector based on BSMV, FoMV, or TRV. We also demonstrate that the CWMV vector is effective in silencing endogenous miRNAs using STTM. Thus, the new vector is an economical and convenient tool that can be used to knockdown miRNAs and verify the function of “genes-of-interest” in plants in a low temperature environment.In this study, we developed a CWMV-based VIGS vector that silenced the phytoene desaturase (PDS) gene at 17°C in both monocot (wheat) and dicot  described previously  together Figures . Followi Figures , the infs Figure . Norther Figures . The resNicotiana benthamiana is the most widely used experimental host plant for VIGS because of its susceptibility to many plant viruses and the rapidity with which visible phenotypes appear  and T. aestivum  were inserted into pCB-35S-R3 in the sense orientation to generate pCB-35S-R3:NbPDS and pCB-35S-R3:TaPDS, respectively  caused systemic infection and induced visible photobleaching in the non-inoculated leaves of N. benthamiana at 27 dpi  and BSMV:TaPDS treatments caused systemic infection and symptoms when rub-inoculated to wheat. After 30 days, the PDS gene silencing phenotype was also observed in wheat  by the 500 bp NbPDS insertion  developed typical CWMV symptoms on their upper non-inoculated leaves  plants used for VIGS experiments were grown in a greenhouse until the four leaf-stage, then inoculated with CWMV and transferred to a climate chamber at 17°C until evaluation.Escherichia coli DH5α was used for the construction of all plasmids and all plasmids constructed in this study were verified by sequencing. The primers used in this paper are detailed in Supplementary Table pCB-35S-R1 and pCB-35S-R2 are full-length cDNA clones of RNA1 and RNA2 of CWMV developed and stored in our lab. Spe I/Sal I and ligated into the similarly digested plasmid pCB-35S-R2:M5 to obtain pCB-35S-R3 containing a MCS.pCB-35S-R3 was constructed as follows. The mutant construct pCB-35S-R2:M5 which abolished the translation of the N-terminal extension of CP and the RT domain of the CP was developed and stored in our lab and was selected as template. The primer pair P1F/P1R was used to amplify the segment S1 from pCB-35S-R2:M5. This 550 bp PCR product was digested with Chinese wheat mosaic virus gene silencing constructs were prepared as follows. The N. benthamiana 300, 500, 800, 1000, and 1500 bp PDS fragments  were obtained by RT-PCR using the P2F/P2R, P3F/P3R, P4F/P4R, P5F/P5R, and P6F/P6R primer pairs  were amplified by RT-PCR with P7F/P7R primer pairs (Supplementary Table STTM165/166 (for silencing of miR165/166 using the STTM strategy) was constructed as follows. A primer with restriction enzyme cutting site, corresponding to the target mimic of miR165/166 and 48 nt STTM spacer (5′-GTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAAT-3′) were used to amplify STTM165/166by PCR using primers P8F/P8R Figure . The res+ membranes (Amersham Bioscience) using 20× SSC buffer. The RNAs were cross-linked to membrane matrix by UV for 50 s. Northern blotting for assays of CWMV genomic RNAs was carried out using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). The DNA oligo nucleotides complementary to the 3′ terminus of the CWMV genome were labeled with digoxigenin (DIG) at their 3′ ends using the DIG Oligo nucleotide Tailing Kit (Roche) and then purified using a G25 Sephadex column (GE). Membranes were pre-hybridized for 2 h and hybridized overnight at 42°C using the DIG Luminescent Detection Starter kit for nucleic acids (Roche). The hybridization signals were visualized by Amersham Imager 600 (GE). All these procedures were performed according to the manufacturers’ instructions.Northern blot assays were performed as previously described . Total RA. tumefaciens strain GV3101. For agroinfiltration, single colonies were cultured overnight at 28°C with constant shaking in 10 ml of LB containing kanamycin (100 μg/ml) and rifampicin (25 μg/ml). Then, 0.1 ml aliquots were used to inoculate 10 ml LB containing the same antibiotics and grown at 28°C for 10 h. Bacterial cells were pelleted at 5000 rpm for 5 min, resuspended in infiltration buffer containing 10 mM 2-(N-morpholino) ethanesulfonic acid (MES), pH 5.2, 10 mM MgCl2, and 0.1 mM acetosyringone to 0.6–0.7 OD600 and incubated at room temperature for 2–3 h. For agroinfiltration, equal amounts of bacteria harboring pCB-35S-R1 and pCB-35S-R3 (or its derivatives) were mixed, and infiltrated into four to six N. benthamiana leaves immediately above the cotyledons with a 1 ml needleless syringe. After maintenance in a growth chamber for 7–10 days post infiltration (dpi), the infiltrated leaves were harvested, ground in 20 mM Na-phosphate buffer (pH 7.2) containing 1% celite, and the sap was mechanically inoculated onto wheat seedlings at the two-leaf stage.The pCB-35S-R3 derivatives were transformed into N. benthamiana plants, uninoculated wheat plants, CWMV-infected N. benthamiana plants, and CWMV-infected wheat plants with TRIzol reagents as described by the manufacturer (Invitrogen). Real-time quantification of microRNAs was performed by stem-loop RT-qPCR as previously reported . Relative expression was calculated using the comparative cycle threshold method  and then reverse transcribed to generate cDNA using an oligo (dT) primer and a Primescrip RT reagent kit (Takara) according to the manufacturer’s instructions. The primer pair P14F/P14R (see Supplementary Table T method . The valpression .H-MZ and J-PC were responsible for study conception, design, and coordination. JiY, LH, T-YZ, J-BL, Q-SL, and JL carried out the experiments for vector construction, northern blotting, real-time RT-PCR, and VIGS assays. LH, T-YZ, and JaY performed the virus inoculation, agro-infiltration, sample collection, and RNA isolation for gene expression and blotting assays. H-MZ, JaY, and J-PC were responsible for data collection and analysis and drafted the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila melanogaster is accompanied by major reorganisation of chromatin in post-meiotic stages, involving a nearly genome-wide displacement of histones by protamines, Mst77F and Protamine-like 99C. A proposed prerequisite for the histone-to-protamine transition is massive histone H4 hyper-acetylation prior to the switch. Here, we investigated the pattern of histone H3 lysine acetylation and general lysine crotonylation in D. melanogaster spermiogenesis to elucidate a possible role of these marks in chromatin reorganisation. Lysine crotonylation was strongest prior to remodelling and the deposition of this mark depended on the acetylation status of the spermatid chromatin. In contrast to H4 acetylation, individual H3 acetylation marks displayed surprisingly distinct patterns during the histone-to-protamine transition. We observed that Nejire, a histone acetyl transferase, is expressed during the time of histone-to-protamine transition. Nejire knock down led to strongly reduced fertility, which correlated with misshaped spermatid nuclei and a lack of mature sperm. protA and prtl99C transcript levels were reduced after knocking down Nejire. ProtB-eGFP, Mst77F-eGFP and Prtl99C-eGFP were synthesized at the late canoe stage, while histones were often not detectable. However, in some cysts histones persist in parallel to protamines. Therefore, we hypothesize that complete histone removal requires multiple histone modifications besides H3K18ac and H3K27ac. In summary, H3K18 and H3K27 acetylation during Drosophila spermatogenesis is dependent on Nejire or a yet uncharacterized acetyl transferase. We show that Nejire is required for male fertility since Nejire contributes to efficient transcription of protA and prtl99C, but not Mst77F, in spermatocytes, and to maturation of sperm.Spermatogenesis in many species including  One of the most dramatic processes of chromatin remodelling occurs during the post-meiotic phase of spermatogenesis. The previously histone-based chromatin becomes a highly compact structure due to the nearly genome-wide replacement of histones, first by transition proteins, which are then replaced by small basic proteins called protamines. The general aspects and other characteristic features of this large-scale chromatin compaction are conserved from fly to man . These data clearly demonstrate that hyper-acetylation is tightly linked to histone replacement. Simultaneously with H4 hyper-acetylation, H3K9 acetylation, lysine crotonylation and H3K4, H3K9 and H3K79 methylation are described to increase prior to the transition from histones to protamines [D. melanogaster histones are successively replaced by protamines (ProtA and ProtB) and in Drosophila by two additional abundant sperm chromatin components, Mst77F [One mechanism proposed to be a prerequisite for the histone-to-protamine transition is a genome-wide hyper-acetylation of histones occurring in the total absence of transcription . In mouse and radation –7. A lacradation , 9. Furtradation . Similarotamines –13. As i, Mst77F –17 and P, Mst77F .D. melanogaster, blocking histone acetylation specifically in the post-meiotic phase by inhibitors led to loss of protamine deposition. As in mice, D. melanogaster histone hyper-acetylation cannot be the sole inducer of the switch since premature histone hyper-acetylation does not lead to a premature protamine-based chromatin structure [How H4 hyper-acetylation and other histone modifications regulate the histone-to-protamine transition in elongating spermatids is poorly understood. According to the histone code hypothesis, modifications could define specific signals and serve as an interface language between histones and chromatin modifying activities . As parttructure .D. melanogaster with the focus on histone H3 acetylation. In addition, we searched for enzymes mediating histone modifications, such as histone acetyl-transferases, which may function as “writer factors” in the histone-protamine-transition phase. We show that histone H3 acetylation displays a highly diverse pattern compared to H4 acetylation. Remarkably, after meiosis H3K18ac and H3K27ac are limited to the early canoe stage just prior to the histone-to-protamine transition. We postulate Nejire or an enzyme with the same specificity as a putative “writer” of these post-meiotic H3 histone acetylations. Germ line-specific knock down of Nejire led to a lack of mature sperm, demonstrating a function of Nejire in proper spermatid differentiation.Here, we identified post-translational histone modifications accompanying histone-to-protamine transition in D. melanogaster strains were maintained on standard medium at 25°C. w1118 was used as the wild-type strain. Protamine B-mCherry (ProtB-mCherry) transgenic flies were generated by modifying the ProtamineB-eGFP construct of [nejire functional studies the RNAi line v105115 (Vienna Drosophila Resource Center) was combined with ProtB-GFP, Mst77F-eGFP [nejire knock down, v105115 protB-GFP males were crossed against virgin females of bam-Gal4/bam-Gal4; Sp/CyO; bam-Gal4-VP16/MKRS. Crossings for knock down experiments were maintained at 30°C.truct of . For nejPupal testes cultures of transgenic flies carrying ProtB-mCherry were established as described in . In shorTestes were treated with anacardic acid  and trichostatin A  in appropriate dilution in culture medium. Control cultures with solvent alone were analysed in parallel. Cultures were incubated for 24 h at 25°C.One adult male of each genotype was placed together with two wild-type virgin females in a vial for 3 days at 25°C. After 3 days, the parental generation was removed from the vials. After 2 weeks, offspring in each vial were counted.For immunofluorescence analysis, adult testes and cultured pupal testes of transgenic flies carrying ProtB-mCherry were squashed and treated essentially as described in Hime et al.. DNA was2O, 0.5 μl (10 μM) gene-specific primer A, 0.5 μl (10 μM) gene-specific primer B and 5 μl cDNA (1:25 dilution). qPCR was performed in three technical replicates on the Mx3000P qPCR. Ct values were normalized to the mRNA expression level of Rpl32. Mean value and SEM of three biological replicates was calculated using GraphPad PRISM version 5.03. For statistical analysis one-sample t-test with a hypothetical value of 1 was used.RNA from 300 Bam>>v105115 testes or v105115 undriven testes was extracted using TRIzol (Invitrogen). Extracted total RNA was treated with RNase-free Turbo DNase (Ambion) and purified using an RNeasy mini kit (Qiagen). 1 μg of DNAse treated and purified RNA was used for cDNA synthesis with the Transcriptor First Strand cDNA Synthesis kit (Roche) according to the manual in a 20 μl reaction. qPCR reactions were set up with 10 μl iTaq™ Universal SYBR® Green Supermix (Bio-Rad), 4 μl ddHD. melanogaster, the histone-to-protamine transition takes place during the so-called canoe stage. Here, the early canoe stage is defined by the start of histone removal, and the late canoe stage by the start of protamine accumulation [In mulation . Hyper-amulation . HoweverD. melanogaster using specific commercially available antibodies against acetylated lysine residues K4, K9, K14, K18, K23 and K27 in the N-terminal domain of histone H3 and K36, K56 and K64 in the globular core domain , was reported to show intense labelling during elongating spermatid steps 9–11  and histone deacetylase (HDAC) on H3 lysine acetylation and lysine crotonylation specifically in the post-meiotic phase, we isolated pupal testes of ProtB-mCherry transgenic flies at around 24 h APF. To block histone acetylation, these testes were subjected in culture to 150 μM of the HAT inhibitor anacardic acid (AA) for about 24 hours. A known inhibitor of several HAT families . AA neitansition .D. melanogaster spermatogenesis. In control testes, Kcr was visible at the early canoe stage up until the late canoe stage was reached  and three day old males (72–78 h after hatching) . At bothWe hypothesized that Nejire/dCBP acetylates the histone lysine residues shortly before the time of histone-to-protamine transition. Alternatively, acetylations might persist from the spermatocyte phase and become visible due to the enormous reduction in the nuclear volume . To testImmunofluorescence staining with anti-H3K18ac antibody revealed that in contrast to the untreated control testes  H3K18ac Based on these findings we propose that Nejire/dCBP-mediated H3K18 and H3K27 acetylation in spermatids is a specific post-meiotic activity.nejire knock down  to quantify nejire transcripts. We determined the relative transcript level of mst77F, protA, prtl-99C, nejire/dCBP and tbrd-1 as controls  and Mst77F and Prtl99C  [Using a combination of a Dot1l) , 38. StrSince histone acetylation seemed crucial for progressing to histone-to protamine transition, we aimed to identify acetyl-transferases, in particular those expressed at the canoe stage. H3 acetylation appears later than H4 hyper-acetylation and very specific in the early canoe stage (H3K18ac and H3K27ac). H3K18ac and H3K27ac acetylation marks are set by the aceyl-tranferease Nejire/dCBP.protamine mRNA synthesis. Hyper-acetylation of H3 at K18 and K27 during the histone-to-protamine transition likely is responsible for efficient histone degradation, since inhibition of histone acetyltransferases in the post-meiotic phase abolished H3K18 and H3K27 acetylation. These data are in agreement with de novo acetylation by Nejire/dCBP, if the detection in the early canoe stage indeed failed because of accessibility failure for the antibody. Alternatively, we propose that a so far unknown acetyl transferase with the same target specificity exists and that Nejire/dCBP targets other nuclear proteins during the canoe stage. While in vitro cultures of testes allowed us to selectively address the consequence of inhibition on post-meiotic stages, RNAi knock down experiments are only possible in the highly transcriptionally active spermatocyte stage. Thus, the distortions seen after knock down of ISWI [Here, we present evidence that H3K18 and H3K27 acetylation depends on Nejire. Nejire is essential for fertility and responsible for efficient  of ISWI  and NejiExpression of Nejire during the late canoe stage and the loss of H3K18 and H3K27 acetylation after stage-specific inhibitor application led us to hypothesise that Nejire has dual functions in transcription in spermatocytes and H3 acetylation shortly before and during the histone-to-protamine transition. Nejire/dCBP contains a bromodomain, a protein motif known to specifically bind acetyl-lysine residues . Here, ale mice . In contIn our view, Nejire likely regulates H3K18 and H3K27 acetylation during transcription in spermatocytes. H3K18 and H3K27 are prominent also during the histone-protamine transition. We hypothesise that male sterility is directly and indirectly dependent on Nejire and caused by failure H3K18 and H3K27 acetylation in both stages.In conclusion, with respect to the histone-to-protamine transition we propose that Nejire regulates transcript levels for sperm relevant proteins in spermatocytes and in directly or indirectly acetylation of H3K18 and H3K27 for fully efficient histone degradation. We propose that the numerous different histone modifications are required for histone degradation.S1 FigSquashed preparations of testes from ProtB-eGFP expressing wild-type flies (A-Aʹʹ) in comparison to testes of ProtB-eGFP males after Nejire/dCBP knock down (B-Bʹʹ). DNA was visualized with Hoechst dye .  H3K18ac detected with an anti-H3K18ac antibody,  ProtB-GFP was used to visualize protamine expression. In the wild-type, H3K18ac characterizes the spermatocyte and early canoe stage (Aʹ), ProtB-eGFP characterizes nuclei at the late canoe stage and in individualized sperm (Aʹʹ), Knock down of Nejire/dCBP led to a complete absence of individualized sperm (B-Bʹʹ) and the H3K27 acetylation signal in spermatocytes and in post-meiotic spermatid stages (Bʹ). After Nejire/dCBP knock down, residual ProtB-eGFP positive spermatid nuclei are observed (Bʹʹ). Scale bar: 5 µm.(JPG)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Scientific Reports 10.1038/s41598-017-10694-3, published online 11 September 2017Correction to: This Article contains typographical errors in the Acknowledgements section.“This work was supported by the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology from Baylor Scott & White, a VA Research Career Scientist Award and a VA Merit award to Dr. Alpini (5I01BX000574), a VA Merit Award (5I01BX002192) to Dr. Glaser, a VA Merit Award (1I01BX001724) to Dr. Meng, and the NIH grants DK058411, DK076898, DK095291 and DK062975 to Drs. Alpini, Meng and Glaser.”should read:“This work was supported by the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology from Baylor Scott & White, a VA Research Career Scientist Award and a VA Merit award to Dr. Alpini (5I01BX000574), a VA Merit Award (5I01BX002192) to Dr. Glaser, a VA Merit Award (1I01BX001724) to Dr. Meng, and the NIH grants DK058411, DK076898, DK107310 and DK062975 to Drs. Alpini, Meng and Glaser.”


CD163 is edited so as to remove the viral interaction domain while maintaining protein expression and biological function, averting any potential adverse effect associated with protein knockout. This research demonstrates a genetic-control approach with potential benefits in animal welfare as well as to the pork industry.Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the etiological agent of PRRS, causing late-term abortions, stillbirths, and respiratory disease in pigs, incurring major economic losses to the worldwide pig industry. The virus is highly mutagenic and can be divided into two species, PRRSV-1 and PRRSV-2, each containing several subtypes. Current control strategies mainly involve biosecurity measures, depopulation, and vaccination. Vaccines are at best only partially protective against infection with heterologous subtypes and sublineages, and modified live vaccines have frequently been reported to revert to virulence. Here, we demonstrate that a genetic-control approach results in complete resistance to PRRSV infection  in vitro. Here, ΔSRCR5 pigs were challenged with a highly virulent PRRSV-1 subtype 2 strain. In contrast to the wild-type control group, ΔSRCR5 pigs showed no signs of infection and no viremia or antibody response indicative of a productive infection. Histopathological analysis of lung and lymph node tissue showed no presence of virus-replicating cells in either tissue. This shows that ΔSRCR5 pigs are fully resistant to infection by the virus.Porcine reproductive and respiratory syndrome virus (PRRSV) has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 protein is expressed at high levels on the surface of specific macrophage types, and a soluble form is circulating in blood. CD163 has been described as a fusion receptor for PRRSV, with the scavenger receptor cysteine-rich domain 5 (SRCR5) region having been shown to be the interaction site for the virus. As reported previously, we have generated pigs in which exon 7 of the CD163 gene has been deleted using CRISPR/Cas9 editing in pig zygotes. These pigs express CD163 protein lacking SRCR5 (ΔSRCR5 CD163) and show no adverse effects when maintained under standard husbandry conditions. Not only was ΔSRCR5 CD163 detected on the surface of macrophage subsets, but the secreted, soluble protein can also be detected in the serum of the edited pigs, as shown here by a porcine soluble CD163-specific enzyme-linked immunosorbent assay (ELISA). Previous results showed that primary macrophage cells from ΔSRCR5 CD163 animals are resistant to PRRSV-1 subtype 1, 2, and 3 as well as PRRSV-2 infection IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the etiological agent of PRRS, causing late-term abortions, stillbirths, and respiratory disease in pigs, incurring major economic losses to the worldwide pig industry. The virus is highly mutagenic and can be divided into two species, PRRSV-1 and PRRSV-2, each containing several subtypes. Current control strategies mainly involve biosecurity measures, depopulation, and vaccination. Vaccines are at best only partially protective against infection with heterologous subtypes and sublineages, and modified live vaccines have frequently been reported to revert to virulence. Here, we demonstrate that a genetic-control approach results in complete resistance to PRRSV infection in vivo. CD163 is edited so as to remove the viral interaction domain while maintaining protein expression and biological function, averting any potential adverse effect associated with protein knockout. This research demonstrates a genetic-control approach with potential benefits in animal welfare as well as to the pork industry. Arteriviridae family and the order Nidovirales. Infected pigs of all ages may present with symptoms involving inappetence, fever, lethargy, and respiratory distress. However, the most devastating effects of PRRSV infection are observed in young piglets and pregnant sows. In pregnant sows, full abortions or death and mummification of fetuses in utero are observed, and live-born piglets from an antenatal infection are often weak and display severe respiratory symptoms (–Porcine reproductive and respiratory syndrome (PRRS) is arguably the most economically important infectious disease affecting pigs worldwide. The causative agent of PRRS is PRRS virus (PRRSV), a member of the symptoms –3. Pigleymptoms –. Due to ymptoms –, 6.There are two different species of PRRSV with distinct geographic distributions: PRRSV-1 is found primarily in Europe and Asia, overlapping the range of PRRSV-2, which is found in Asia and the Americas. PRRSV-1 can be further divided into at least three subtypes, currently based on open reading frame 7 (ORF7) sequences and geographical distribution, with subtype 1 being pan-European and subtypes 2 and 3 currently being limited to eastern Europe   as both a soluble and a cell-bound protein.Recent P-PRRSV) , 23. GenP-PRRSV) , in effelleagues . This atlleagues , making lleagues . Therefoin vitro . The aimFounder-generation (F0) animals carrying a deletion of exon 7 in the CD163 gene, which encodes SRCR5 of the protein, were generated by CRISPR/Cas9 gene editing as previously described (ΔSRCR5 pigs) . BrieflyAs described above, ΔSRCR5 animals express the ΔSRCR5 CD163 mRNA and protein at levels equivalent to CD163 transcript and protein levels in wild-type siblings. Furthermore, the native-structure ΔSRCR5 CD163 is recognized on the surface of pulmonary alveolar macrophages (PAMs) by a commercial antibody . We furtin vitro-differentiated peripheral blood monocytes (PBMCs) are resistant to infection with both PRRSV-1 and PRRSV-2. Now, we aimed to assess the resistance of these pigs to PRRSV-1 infection in vivo. We selected four homozygous ΔSRCR5 F2 animals and four wild-type controls that were cohoused from weaning  and are comparable to sCD163 levels in humans . The cohort was cohoused for the duration of the experiment and allowed to settle for 1 week prior to the initiation of the challenge. On day 0 of the challenge with PRRSV-1, a second serum sample was taken, and soluble CD163 (sCD163) serum levels were assessed using a commercially available enzyme-linked immunosorbent assay (ELISA) recognizing soluble porcine CD163. Serum CD163 levels were found to be 433.2 ± 69.57 ng/ml in wild-type pigs and 463.5 ± 68.99 ng/ml in ΔSRCR5 pigs . These r50), as determined by assessment on PAMs, of the virus isolate used. Rectal temperature, respiration, nasal discharge, coughing, and demeanor were recorded every day, and serum samples were collected on day 0  and on days 3, 7, 10, and 14 (prior to euthanasia). Weights were recorded on days 0, 7, and 14 (prior to euthanasia). People assessing the pigs clinically, conducting the challenge, and analyzing pathology were blind to the genotype status of the animals.At 7 to 8 weeks of age, the pigs were inoculated intranasally with PRRSV-1 subtype 2 strain BOR-57 . EasternP = 0.0465)  . One wil 0.0465) . The pre 0.0465)  but wereDuring necropsy, lungs were assessed initially, and detailed photographs of the dorsal and ventral sides were taken. Lungs were scored for the presence of lesions. An established scoring system, based on the approximate contribution of each lung section to the complete lung volume, was employed . On averin vitro results . This is a clear demonstration that ΔSRCR5 animals are resistant to PRRSV-1 infection, confirming our previous  results .CD163 is involved in the cytokine response to infection and immune stimuli as well as hemoglobin-haptoglobin (Hb-Hp) uptake. Hb-Hp stimulation has been shown to lead to increased levels of interleukin-6 (IL-6) and IL-10 as well as IL-1 and tumor necrosis factor alpha (TNF-α) downregulation , 32. SRCWe observed sequential cytokine responses to PRRSV-1 infection in wild-type animals, with an early increase in the levels of IFN-α, IL-17A, and the IL-1 receptor antagonist (IL-1ra)  to C, foThe results of this study show that ΔSRCR5 pigs are healthy under standard husbandry conditions and maintain the biological function of the CD163 protein while being resistant to PRRSV infection. So far, we have bred three generations of edited animals with over 10 litters and have not observed any abnormalities in breeding.CD163 CRISPR/Cas9. Here, we have shown that heterozygous and homozygous animals can be bred naturally and yield normal-litter-size offspring. Our previous data showed that primary PAMs and peripheral blood monocyte (PBMC)-derived macrophages from ΔSRCR5 pigs are fully resistant to PRRSV-1 subtype 1, 2, and 3 infection as well as to both typical and atypical PRRSV-2  was observed. This confirms that our previous in vitro results directly translate to the in vivo situation.ΔSRCR5 pigs were generated, as previously described , by usin PRRSV-2 . To confin silico using RaptorX predicts that posttranslational folding will yield a protein that closely mimics the structure of full-length CD163. The expression of ΔSRCR5 CD163 in animals has several advantages over previously described PRRSV-resistant CD163 knockout animals generated by the random introduction of a premature stop codon in exon 3 or exon 7 of the CD163 gene (AF488-Hp)  . All ΔSRF488-Hp) . Surprisin vitro , 41 in hin vitro , and Borin vitro , all of Staphylococcus aureus, which promotes the recognition, phagocytosis, and killing of this important livestock and human pathogen  Act of 1986. Humane endpoints were clearly defined.g. When necessary, red blood cells were removed using red cell lysis buffer  for 5 min before washing again with PBS. Cells were collected by centrifugation as described above and frozen in a solution containing 90% fetal bovine serum (FBS)  and 10% dimethyl sulfoxide (DMSO) (Sigma). Cells were frozen gradually at 1°C/min in a −80°C freezer before being transferred to −150°C.Primary pulmonary alveolar macrophages (PAMs) for the propagation of PRRSV-1 subtype 2 strain BOR-57 (isolated from a sample from a Belarusian pig in 2009 by T. Stadejek) were harvested from wild-type research animals aged 6 to 9 weeks as previously described . Briefly+/+).PAM cells were cultivated in a solution containing RPMI 1640, GlutaMAX (Invitrogen), 10% FBS , 100 IU/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) , blood and serum samples were taken from all animals by jugular venipuncture, and the blood samples were subjected to whole-blood-count analyses. Sera were screened by using the Idexx PRRSV X3 ELISA to confirm that none of the animals had previously been exposed to PRRSV. Animals were acclimated in the SPFU for 1 week prior to challenge.50 assay on PAMs immediately after production, prior to challenge, and on both challenge dates after administration. The BOR-57 inoculum was tested for the absence of mycoplasma and other major viral pathogens. Animals were inoculated intranasally in the left nostril with 5 ml of 1E6 TCID50/ml BOR-57 in RPMI+/+. Body weights of the animals were measured on day 0 prior to challenge and on day 7 and day 14 prior to euthanasia. Serum samples were collected on day 0 prior to challenge and on days 3, 7, 10, and 14 prior to euthanasia by jugular venipuncture into Vacutainer tubes. After clotting, samples were centrifuged at 2,000 × g for 10 min at 4°C, and samples were aliquoted and frozen at −80°C for further analysis. Clinical observations were recorded daily, making note of the rectal temperature, demeanor, nasal discharge, coughing, and respiration. Feeding, water consumption, and general health were observed and recorded daily. Humane endpoints were defined prior to challenge. No animal reached the criteria for premature termination during the challenge.Infectivity of BOR-57 stocks was assessed using a TCIDOn day 14 of the challenge, animals were euthanized according to a schedule I method. During necropsy, the lungs were removed and initially assessed, and detailed photographs were taken from the dorsal and ventral sides for detailed scoring of macroscopic lung lesions. An established scoring system, based on the approximate contribution of each lung section to the complete lung volume, was employed as previously described . BrieflyViral RNA was extracted from the sera collected on days 0, 3, 7, 10, and 14 using the QIAamp viral RNA minikit (Qiagen) according to the manufacturer's instructions. Viral RNA levels were assessed by reverse transcription-quantitative PCR (RT-qPCR) using the GoTaq 1-Step RT-qPCR system (Promega) and analyzed on a LightCycler II 480 instrument (Roche). Viral RNA extracted from the PAM cell culture supernatant with a known multiplicity of infection (MOI) and a custom-synthesized DNA fragment (Invitrogen) of a known concentration (GAGAGCGGCCGCTAATACGACTCACTATAGTCAGCTGTGTCAGCTGCTGGGAAAAATGATGAAATCCCAGCGCCAGCAACCCAGGGGAGGACAGGCCAACAAAAGGAAAAAGCCTGAGAAGCCTCATTTTCCCTTGGCTGCTGAAGATGACGTTCGGCACCATCTCACCGCAACTGAGCGTTCCCTCTGTCTGCAATCGATCCAGACAGCCTTCAATCAGGGTGCAGGAACTGCGTCGCTTTCACCCAGTGGGAAGGTCAGTTTTCAGGTAGAGTTCATGCTGCCCCTGCAGGGAGA) were used as standards. The primers used were BOR57_FWD (GAAATCCCAGCGCCAGCAAC) and BOR57_REV (TTCCCACTGGGTGAAAGCGA).Serum samples collected a week prior to and on day 0 of the challenge were analyzed for the presence and level of soluble CD163. A sandwich ELISA was performed using the porcine CD163 ELISA kit (Elabscience) according to the manufacturer's instructions and using serial dilutions of serum.Serum samples collected on day 0  and on days 3, 7, 10, and 14 of the challenge were analyzed for the levels of 20 different cytokines. Cytokine array analyses were performed using porcine cytokine antibody array A  and array B  (both from Abcam) according to the manufacturer's instructions.


Dear Editor:1. Base editors that can achieve targeted C-to-T conversions without generating DNA double-strand breaks, or requiring a donor template, represent significant advances in both disease modeling and gene therapy2. However, the currently used base editor 3 (BE3), derived from the rAPOBEC1 (rA1)-nCas9  fusion, is limited by low editing efficiency in GC contexts, and by high bystander activity when more than one C is present in the editing window3. Recently, human APOBEC3A (hA3A) was substituted for rA1 to produce new base editors 6. Another variant, hA3A-N57G, showed a striking preference for the TCR (A/G) motif and a reduction in bystander mutations7. In this study, we demonstrate the effectiveness of the hA3A-nCas9 fusion and its associated variants (Y130F and N57G) to induce efficient and precise base editing in rabbits.The largest class of known human genetic diseases are caused by point mutations or single-nucleotide polymorphisms (SNPs)ors Fig. . The ediTyr-1 and Tyr-3) or non-GC (Tyr-2) contexts are contained in the editing window , compared with that of BE3 (~8 nt), and a higher editing efficiency in the GC context at Tyr-1 and Tyr-3 in rabbit blastocysts  to perform base editing in rabbit embryos. A total of six single guide RNAs (sgRNA) containing GC and non-GC contexts were selected to evaluate the editing efficiency of hA3A-eBE-Y130F  , but not indels, when compared with BE3  Fig. . At all BE3 Fig. . The ave7. Thus, to further improve precision in the TC context, a N57G mutation was introduced into hA3A-eBE to create hA3A-eBE-N57G  motifs, but greatly reduced when cytidines are in other sequence contexts57G Fig. . Three s57G Fig. . The edites Fig. .Tyr gene is the major causal gene of human oculocutaneous albinism (OCA)12. The high frequency of bystander mutations (86%) was determined in the founder (F0) rabbits using BE3 in our previous study11. Thus, to generate a rabbit model to precisely mimic OCA, the mRNA encoding hA3A-eBE-N57G and sgRNA target to Tyr p.Q68Stop were injected into zygotes, making desired mutations in the TC context adjacent to multiple Cs  in founder rabbits   Fig. . Moreoveype Fig. . Furtherype Fig. . In addiype Fig. , demonstIn summary, we demonstrated that hA3A-eBE can induce efficient C-to-T conversions with a large editing window, and that hA3A-eBE-Y130F, having a narrowed editing window, can function as a generic version of the base editor, particularly at pathogenic SNPs in GC contexts. Furthermore, hA3A-eBE-N57G can induce efficient base editing in the TC context and minimize bystander mutations in vivo. Thus, these three APOBEC3A-nCas9 base editors expand the scope and improve the precision of the currently used BE3 system.Supplementary information


Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which εN-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in  The recent development of mass spectrometry (MS)-based proteomic analysis enables the discovery of a wide variety of posttranslational modifications (PTMs) in prokaryotes . AcetylaEscherichia coli, acetylation is introduced to lysine residues by a KAT-dependent reaction utilizing acetyl-CoA and a non-enzymatic reaction utilizing acetyl-phosphate  Ala , 5 µM transcribed tRNAAla, and 5 ng/µL (50 nM) His-tagged AlaRS or its variants. At time points of 2 min, 4 min, 8 min, and 10 min, 10 µL aliquots of the reaction mixture were spotted onto Whatman 3MM filter paper  and the reaction was immediately quenched with 5% trichloroacetic acid (TCA). After washing the filter paper with 5% TCA and drying, radioactivity on the filter paper was counted by a Beckman LS 6500 Scintillation Counter .Alanine tRNA  spectra of AlaRS and its derivatives were recorded on a J-805 CD Spectrometer . AlaRS WT, K73A, and K73Ac (NAM+) were respectively prepared at a concentration of 0.1 mg/mL (1.0 µM) in a buffer containing 5 mM Tris-HCl pH 7.5 and 5 mM MgClE. coli DH10B cells harboring pACYC184P-1218-cobB, pACYC184P-417-cobB, pACYC184P-30-cobB, pACYC184P-cobB-L, or pACYC184P-cobB-S were cultivated in LB medium at 37 °C overnight. The cells were broken in lysis buffer  by sonication. The protein concentration of the lysates was determined by an XL-Bradford . Ten micrograms of protein were subjected to 15% SDS-PAGE and Western blotting analysis using Anti-His-tag mAb-HRP-DirecT to detect the His-tagged CobB expression.+ , 0.5 µM AlaRS K73Ac (∆CobB), and 1.5 or 5 µM His-tagged CobB-S. The reaction was stopped by adding an equal volume of 2× sample buffer for SDS-PAGE. The resulting samples containing 0.2 pmol of AlaRS were separated by 7.5% SDS-PAGE and acetylation levels of the AlaRS K73Ac (∆CobB) were determined by Western blotting analysis using anti-AcK antibody.The assay was performed at 37 °C for 12 h in a reaction mixture (30 µL) containing 25 mM Tris-HCl pH 8.0, 100 mM NaCl, 27 mM KCl, 0.5 mM NADE. coli acetylome studies identified 15 lysine acetylation sites in AlaRS  showed higher acetylation signals than AlaRS K73Ac (NAM−), although AlaRS WT and its K73A mutant as negative controls did not give a signal of acetylation. The observation that acetylation of the AlaRS WT and K73A mutant could not be detected by Western blotting is thought to be due to the amount of the overexpressed enzymes being excess to a capability of endogenous lysine acetylation, resulting in reducing populations of acetylated AlaRS. We next examined the acetylation level of K73Ac purified from the ∆CobB strain. The acetylation level of K73Ac (∆CobB) was almost the same as that of K73Ac (NAM+). Quantification of the band intensity showed that the acetylation levels of K73Ac (NAM+) and K73Ac (∆CobB) were 3.3-fold and 4.0-fold higher than that of K73Ac (NAM−), respectively . Almost the same spectral curves were observed among the three proteins . The proSalmonella enterica, both of which are biologically active in the cell [E. coli yet. The cobB ORF in the public E. coli database encodes a 279-amino acid isoform (CobB-L), while a shorter 242-amino acid isoform (CobB-S) can be translated from the 112 nt-downstream second Met codon in the ORF. CobB-L contains a 37-amino acid N-terminal extension comprised of 15 basic and 13 hydrophobic residues, similar to CobB-L of S. enterica , we detected the candidates of the −10 and −35 consensus sequences between −347 and −319 for CobB-L and between +51 and +83 for CobB-S expression, respectively  in the cell . The exienterica a. We expli DH10B b. We detli DH10B c. Meanwhectively d. These +-dependent decrease in acetylation was observed and it was enhanced by increasing concentrations of CobB-S; 27% and 39% decreases in acetylation were observed when 1.5 µM or 5.0 µM of CobB-S were used, respectively . This observation supports our result that the acetylated K73 is a substrate of CobB. Our in vitro study showed the deacetylase activity of CobB-S for AlaRS K73Ac, but it seemed to be moderate. We consider the possibility that CobB-L may prefer to remove this acetylation or another factor like a chaperone might be required for the reaction.Our in vivo and in vitro studies indicate that the acetylation of K73 is reversed by CobB deacetylase. Systematic analysis to search for substrates of CobB has shown that CobB does not recognize a specific sequence, but tends to prefer aspartate (D), glutamate (E), alanine (A), glycine (G) and tyrosine (Y) on β-sheet or loop structures adjacent to an acetylated lysine . The K73Vibrio parahaemolyticus, mice, and humans [We clarified the CobB-dependent deacetylation mechanism of K73 in AlaRS in vivo, while the mechanism of K73 acetylation remains elusive. The K73 acetylation has been detected at the stationary phase of growth without glucose  and at td humans ,42. The E. coli was previously performed using an enhanced green fluorescent protein (EGFP)-based reporter system and real time quantitative PCR (RT-qPCR), showing that promoter activity for a nagK-cobB transcription was detected in the 5′-upstream region (−300 to +50) of nagK but no significant activity was detected in the upstream region (−400 to +151) of cobB [cobB: the one within nagK directs CobB-L expression and the other within cobB directs CobB-S expression (S. enterica, in which the upstream and downstream promoters correspond to P2 and P3 promoters, respectively [S. enterica that the extended N-terminal region may be involved in interaction with other molecules such as nucleic acids and acetylated protein substrates, and cellular localization [Promoter analysis for CobB expression in  of cobB . Contrarpression d. These ectively . Since Cectively ,36, the lization . Our faiAs aaRSs are essential enzymes, severe inhibition of their activities causes growth retardation and cell death . Therefo


Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.Reactivation of latent Human Cytomegalovirus (HCMV) in CD34 + HPCs proliferation and total hematopoietic colony formation. An HCMV miR-US22 mutant is unable to reactivate from latency suggesting that the ability of the miRNA to disrupt CD34+ HPC renewal in the bone marrow niche to initiate a differentiation pathway is critical for viral reactivation.Human cytomegalovirus (HCMV) is a widespread herpesvirus that persists in the host and remains a significant cause of morbidity and mortality in solid organ and stem cell transplant patients. HCMV latency is complex, and the molecular mechanisms for establishment, maintenance, and reactivation from latency are poorly understood. Quiescent stem cells in the bone marrow represent a critical reservoir of latent HCMV, and the mobilization and differentiation of these cells is closely linked to viral reactivation from latency. HCMV encodes small regulatory RNAs, called miRNAs that play important roles in the regulation of viral and cellular gene expression. In this study, we show that HCMV miR-US22 targets Early growth response gene 1 (EGR-1) a host transcription factor that is necessary for stem cell quiescence and self-renewal in the bone marrow. Expression of this miR-US22 down-regulates expression of EGR-1 that reduces CD34 HCMV latency is complex, and the mechanisms for establishment and maintenance of HCMV latency and reactivation of virus are poorly understood at the molecular level. HCMV reactivation is exquisitely linked to CD34+ HPC hematopoiesis and differentiation into myeloid lineage cells [+ HPC hematopoiesis program is considered a major determinant of HCMV latency and reactivation. Activation of growth factor receptor signaling that induces transcriptional reprogramming is necessary to both maintain CD34+ HPCs in a quiescent state and induce myelopoiesis. Viral regulation of these events determines whether the HCMV remains latent or initiates the reactivation program.Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in solid organ and hematopoietic stem cell transplant patients –3. CD34+ge cells . Viral r+ monocytes [in vivo is challenging, hence experimental models of cultured CD34+ HPCs have been instrumental in studying molecular models of latency and reactivation . Theonocytes –11. In iussed in ).+ HPCs promotes “stemness”  in the bone marrow niche [+ HPC differentiation and migration to the periphery [+ HPC myelopoiesis.Early growth response gene 1 (EGR-1) is a member of a family of sequence-specific zinc finger transcription factors that was originally characterized as an oncogene –16 but wow niche . Consequeriphery . Importaeriphery , while ieriphery . Therefo+ HPCs [+ HPCs and then is thought to induce an environment primed for the establishment of latency. Contrary to infection of fibroblasts that support virus replication, EGFR cell surface levels are transiently increased during infection of CD34+ cells [+ HPCs [EGFR and downstream PI3K signaling are important for establishing and maintaining a latent infection in CD34+ HPCs , 23. HCM4+ cells . The HCM4+ cells . These U [+ HPCs , 24.+ HPCs or stimulates cellular differentiation resulting in viral reactivation. HCMV miR-US25–1 was shown to target five cell cycle genes, including cyclin E2, and several genes that modify DNA chromatin [HCMV, similar to other herpesviruses, encodes multiple miRNAs , 26 exprhromatin . Lastly,hromatin , 41. The+ HPCs. These data indicate that miR-US22 regulation of EGR-1 expression is a necessary step in viral reactivation from latency.In the current study, we demonstrate that HCMV miR-US22 efficiently down-regulates EGR-1 expression that results in a decrease in total hematopoietic colony formation and progenitor cell proliferation. Additionally, mutation of miR-US22 in the virus significantly reduced the ability of HCMV to reactivate in CD34+ HPCs in the bone marrow niche, we examined whether HCMV miRNAs functionally target EGR-1 activity. For this experiment, several HCMV miRNA mimics were co-transfected with an EGR-1 luciferase reporter into HEK293 cells in the presence or absence of EGF to examine their effect on promoter activation. Several of the HCMV miRNAs significantly altered luciferase expression from the EGR-1 reporter  [+ HPCs (+ HPCs at latency (14 dpi) indicated that, the ΔmiR-US22 mutant had lower amounts of viral genomic DNA in comparison to WT HCMV at the time of reactivation  . WT HCMV [+ HPCs A. Quantitivation . These d+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation during latency. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV down-regulation of EGR-1 is a necessary step to block HPC proliferation and induce the cellular differentiation necessary to promote reactivation of virus. We propose a model of HCMV reactivation in CD34+ HPCs, in which latently infected cells initiate a process of reactivation that results in expression of miR-US22  multiple HCMV genes, from all the kinetic classes, are expressed in cells that are not detected after latency is established. All of the HCMV miRNAs are expressed in CD34+ HPCs at 48 hpi, including miR-US22. Therefore, we cannot rule out whether miR-US22 is necessary to establish an efficient latent infection or for early maintenance of the genome. Another potential explanation for the reduction of ΔmiR-US22 genomes in latently infected CD34+ HPCs is the ability of the miRNA to inhibit proliferation of infected progenitor cells —the latent/lytic switch for the virus to maintain latency . HCMV-en+ HPCs. While the functions of these miRNAs in CD34+ HPCs during latency are unknown, the miRNAs most likely play important roles in maintenance of the virus during latency. HCMV miR-US22 is not expressed during latency in CD34+ HPCs but represents a class of viral gene products that are unnecessary for replication in fibroblasts but are required to initiate viral reactivation in CD34+ HPCs through the induction of cellular proliferation and differentiation. Another member of this class of HCMV genes is UL7, an early-late gene that functions as an Flt-3 receptor ligand [Analysis of HCMV miRNA expression indicates that only subset of the 14 viral miRNAs  are expressed in latently infected CD34r ligand . Similar+ hematopoietic progenitor cells (HPCs) were isolated from de-identified human fetal liver obtained from Advanced Bioscience Resources or were isolated from de-identified medical waste following bone marrow isolations from healthy donors for clinical procedures at the Banner-University Medical Center at the University of Arizona.CD34+ hematopoietic progenitor cells (HPCs) were isolated from de-identified human fetal liver obtained from Advanced Bioscience Resources as previously described [HEK293T  and adult normal human dermal fibroblasts (NHDF)  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum , 100 units/ml penicillin, and 100ug/ml streptomycin (Invitrogen). Human aortic endothelial cells (AEC)  were cultured in EBM-2 basal medium with EGM-2 SingleQuots supplement excluding Heparin (Lonza), as well as 10% FBS, penicillin, and streptomycin. CD34escribed  or from escribed . All celWild type HCMV TB40/E-GFP bacterial artificial chromosome (BAC), in which the SV40-GFP cassette was introduced as a marker for infection was used to generate infectious virus , 58. MutGGTCTGGTCCGTCGTCTCCCATCTGGTCGGGTTCGGGGATGGGGAC CTC AAG CAA CGTGTGTCCGCGGGCGTGCATGGCTTTTGCTCGCCGGCCGCGCTG and CAGCGCGGCCGGCGAGCAAAAGCCATGCACGCCCGCGGACACACGTTGCTTGAGGTCCCCATCCCCGAACCCGACCAGATGGGAGACGACGGACCAGACC. All virus stocks were propagated and titered on NHDFs. For viral growth curves, NHDFs were infected at 3 pfu/cell for single step and 0.05 pfu/cell for multi-step for 2 hours. Both cell-associated and supernatant viruses were harvested at multiple timepoints, and titers were determined by plaque assay on NHDFs. For all other infections, NHDF and AEC were inoculated with 3 pfu/cell for 2 hours at 37C. Afterwards, the viral inoculum was removed and replaced with fresh medium. Samples were harvested as indicated for each experiment.Renilla luciferase gene in the pSICHECK2 plasmid (Promega) by XhoI and NotI restriction sites. Mutations in the seed sequence of the miR-US22 target site in Egr-1 were introduced by site-directed mutagenesis using the following primer pair: GAACTTGGACATGGCTGTTGGAGGCAGCTGAAGTCAAAGG and CCTTTGACTTCAGCTGCCTCCAACAGCCATGTCCAAGTTC. The mutated construct was verified by sequencing. Short-hairpin RNA (shRNA) targeting EGR-1 was cloned into pSiren expression plasmid via BamHI and EcoRI restriction sites using the following sequence: TGCTGTTGACAGTGAGCGATCCAGAATGTAAGAAAACAAATAGTGAAGCCACAGATGTATTTGTTTTCTTACATTCTGGAGTGCCTACTGCCTCGGA [The 3’UTR of human Egr-1 was amplified by PCR from genomic DNA extracted from NHDFs using DNAzol, and was cloned downstream of the GCCTCGGA . miR-US2For the EGR-1 3’ UTR reporter assays HEK293T cells are seeded into 96-well plates were co-transfected in triplicate with 100ng plasmid and 3pmol mimic per well using Lipofectamine 2000 (Invitrogen). Twenty hours later, the cells were harvested for luciferase assay with the Dual-Glo reporter assay kit (Promega). Luminescence was detected using Veritas microplate luminometer (Turner Biosystems). For EGR-1 reporter assays, the Cignal EGR-1 reporter assay was obtained from Qiagen. This vector expresses luciferase driven from a minimal promoter containing tandem repeats of the EGR-1 binding site. The EGR-1 reporter is co-transfected into HEK293T cells along with a constitutively-expressing Renilla luciferase plasmid and either negative control mimic, miRNA mimic or siRNA of interest as described above. At 16 hours post transfection, cells were serum-deprived in 0%FBS DMEM for 4 hours, and then treated with 5 ng/mL EGF for 4 hours. All experiments were performed in triplicate and results are shown as mean ± standard deviation.https://fiji.sc).Cells were harvested in protein lysis buffer . Cell lysates, along with loading buffer (4X Laemmli Sample Buffer with 2-mercaptoeathanol) were incubated at 95C for 5 minutes, loaded on 4–20% polyacrylamide gels (BioRad), and transferred to Immobilon-P Transfer Membranes (Millipore Corp). After visualizing protein levels with the specified antibodies, the relative intensity of bands was quantitated using Fiji software  using 100 ng total RNA and custom-designed miRNA hairpin-specific primers. Samples were incubated at 16°C (30min), 42°C (30 min), and 85°C (5 min). ABI StepOnePlus real time PCR machine was used with the following program: initial denaturation at 95°C (10 min), and 40 cycles at 95°C (15 sec), 60°C (1 min). The reaction was performed with Taqman Fast Advanced master mix (ABI). HCMV miRNA primers and probes were custom designed ). Viral miRNA expression was normalized to cellular miR-16 levels . For quantitation of miRNA copy number in infected CD34+ HPCs, purified oligos representing the mature form of each miRNA were included in an independent RT reaction in a known quantity. For qPCR, serial dilutions of the RT reaction were included to determine absolute miRNA copy number.Reverse transcription PCR (RT-PCR) was used to quantify viral microRNA expression in infected NHDF or CD34+ HPCs was extracted using the two-step TRIZOL (ThermoFisher) method according to the manufacturer’s directions. Total DNA was analyzed in triplicate using TaqMan FastAdvanced PCR master mix (Applied Biosystems), and primer and probe for HCMV UL141 and human β-globin as previously described [DNA from CD34escribed . Copy nu+ HPCs isolated from human bone marrow were infected at an MOI of 2 for 20h in IMDM supplemented with 10% BIT9500 serum substitute , 2 mM L-Glutamine, 20 ng/ml low-density lipoproteins , penicillin/streptomycin, and 50 μM 2-mercaptoethanol. Following infection, pure populations of infected CD34+ HPCs (>98% GFP-positive) were isolated by fluorescence-activated cell sorting  using a phycoerythrin-conjugated antibody specific to CD34 (BD Biosciences). Cells were sorted by the University of Arizona Shared Service at the University of Arizona Cancer Center. Pure population of infected HPCs were cultured in trans-wells above an irradiated  M2–10B4 and Sl/Sl stromal cell monolayer [+ cells at each dilution using software, Extreme limiting dilution analysis  [+ human hematopoietic cells (HPCs) were isolated from de-identified medical waste following bone marrow harvest from normal donors for clinical procedures at the University of Arizona Banner Medical Center.CD34onolayer  for 10–1onolayer . Brieflyre/elda) . For lat+ HPCs were thawed and recovered overnight in stem cell media (IMDM containing 10% BIT serum replacement (Invitrogen), penicillin/streptomycin and stem cell cytokines . Following recovery, HPCs were transfected using the Amaxa 4D system and the Primary Cell P3 solution according to the manufacturer’s instructions (Lonza). HPCs were transfected with 1ug pSIREN plasmid DNA per 106 cells using either program EH-100 or EO-100. HPCs were recovered in stem cell media for 48hrs, then isolated by FACS  for a pure population of viable, CD34+, GFP+ HPCs. Pure populations of sorted HPCs were plated either at 500 cells/mL in Methocult H4434 (Stem Cell Technologies) in 6 well plates in triplicate for myeloid colony assays, or at 104 cells/mL in stem cell media, 200uL/well in 96 well plates for proliferation assays. Total and specific myeloid colony types were enumerated at 7 and 14 days using a standard microscope and standard protocols as previously described [Primary CD34Data are shown as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism (v6 or v7) for comparisons between experimental groups using unpaired t test or two-way analysis of variance (ANOVA) with Tukey’s post-hoc test.


In contrast, inhibition of miR-125a suppressed stromal cell tumorigenicity and growth. Then, we found that miR-125a stimulates IL-17A by targeting TET2 and Foxp3, and it stimulates β-catenin expression by targeting APC and GSK3β in stromal cells. Furthermore, we identified that IL-17A stimulates miR-125a by activating nuclear factor κB (NF-κB) signaling in stromal cells. Finally, our data show that simultaneous inhibition of IL-17A signaling and miR-125a more significantly inhibits stromal cell growth than miR-125a inhibition alone. miR-125a stimulates the progression of GCTB, and it might represent a useful candidate marker for progression. Simultaneously blocking miR-125a and IL-17A might represent a new therapeutic strategy for GCTB.Giant cell tumors of bone (GCTBs) exhibit high recurrence and aggressive bone lytic behavior; but, the mechanism of GCTB progression is largely unknown. In GCTB, we detected abundant levels of miR-125a, which were associated with tumor extension, grade, and recurrence. miR-125a stimulates stromal cell tumorigenicity and growth  Giant cell tumor of bone (GCTB) is a benign, locally aggressive, osteolytic tumor that causes significant bone destruction in the epiphysis of long bones. The major challenge in the clinical treatment of GCTB is recurrence. The primary method for GCTB treatment in the clinic is surgery. However, according to one report, 20%–50% of patients with GCTB exhibit local recurrence following primary surgical treatments, up to 5% of patients develop pulmonary metastases, and 2% develop spontaneous malignant transformation.GCTB consists of three major cell types: osteoclast-like multinucleated giant cells (MNGCs), monocytic round-shaped macrophage-like cells, and spindle-shaped fibroblast-like stromal cells.Recent studies showed that aberrantly increased expression of interleukin-17A (IL-17A) and β-catenin are closely associated with GCTB progression, including tumor growth, bone destruction, and recurrence.To identify GCTB progression-associated miRNAs, we performed miRNAs array analysis using recurrent GCTB tissues and their matched normal bone tissues from 3 patients. As shown in in vitro using animal experiments. To investigate the effects of miR-125a on tumor growth and tumorigenicity in vivo, two doses of miR-125a-overexpressing stromal cells  in miR-125a-overexpressing stromal cells D, and thsilenced E and S1Fsilenced F. Notablsilenced F. These To investigate how miR-125a enhances IL-17A and β-catenin expression, we screened miR-125a target genes using computational algorithm analysis, and we identified Foxp3, TET2, APC, and GSK3β as potential target genes of miR-125a A. APC anTo confirm the negative regulation of miR-125a on these candidate target genes, we measured the expression level of the candidate target genes after overexpression of miR-125a in stromal cells. Our data showed that all candidate genes of miR-125a were significantly decreased in miR-125a-overexpressing stromal cells compared to control at both the mRNA and protein levels C and 4D.Next, we investigated whether these target genes directly contribute to miR-125a-induced expression and function of IL-17A and β-catenin. Our data showed that overexpression of TET2 or Foxp3 significantly abolished the miR-125a-induced IL-17A expression in stromal cells F. NotablAlthough not conducted in GCTB, previous studies showed that miR-17A activates nuclear factor κB (NF-κB) signaling, and activated NF-κB signaling is involved in the regulation of miRNAs, including miR-125a.Our previous study showed that IL-17A is expressed in both stromal cells and MNGCs and IL-17A can stimulate its self-expression in MNGCs.In this study, we found that the expression level of miR-125a was significantly increased in GCTB specimens compared to normal bone tissues. In addition, we showed that an increased expression level of miR-125a was closely correlated with GCTB grade, extension, and recurrence in patients. These findings suggest miR-125a is a tumor-promoting miRNA and can be a prognostic marker for GCTB progression.in vitro and in vivo experiments to demonstrate that aberrantly increased expression of miR-125a stimulates stromal cell growth and tumorigenicity. Importantly, the inhibition of miR-125a significantly inhibited stromal cell tumorigenicity and tumor growth in a xenograft model. Taken together, our findings indicate that miR-125a is a novel therapeutic target of stromal cells and inhibition of miR-125a is a novel strategy to suppress GCTB progression.Both MNGCs and stromal cells play important roles in GCTB progression. Notably, recent studies have suggested that stromal cells may be involved in GCTB development and recurrence. According to these reports, stromal cells are the only proliferating population in GCTB, and they express osteoblast factors,GCTB is characterized by a high recurrence rate, and studies have shown that aberrantly highly expressed β-catenin and IL-17A are closely associated with GCTB progression, including tumor growth and recurrence.In a previous study, we demonstrated that IL-17A was secreted by both stromal cells and MNGCs, thereby significantly promoting GCTB progression.In summary, our study, to the best of our knowledge, is the first to have delineated the correlation between miR-125a and the progression of GCTBs. miR-125a stimulates the progression of GCTBs through increasing the expression of β-catenin and IL-17A by directly targeting their negative regulators. Our results suggest that miR-125a is a useful progression marker of GCTB and simultaneously blocking miR-125a and IL-17A might represent a new therapeutic strategy for GCTB.Tumor samples were obtained from 107 patients with GCTB, and all patients received extended curettage, with no adjuvant therapy. The clinical characteristics of the patients with GCTB are summarized in The stromal cells were isolated from fresh tumor tissues of GCTB patients as described previously,Stromal cells were transfected with the indicated nucleotides  or lentiviral vectors. After 24 hr of transfection, the cells were trypsinized. For the cell viability assay, the cells were reseeded in 96-well plates at a density of 5,000/well, and then the cell viability was measured at the indicated times using Cell Counting Kit-8 , according to the manufacturer’s instructions.For the soft agar assay, cells were re-suspended in 0.5 mL 0.35% agar (Sigma) in growth medium at a density of 5,000 cells/well (6-well plates). Then, the agar-cell mixture was plated on the top of a solid layer of 0.8% agar in growth medium. Colonies were counted 14 days later.RNA was isolated from cells and human specimens using the TRIzol reagent  or miRNeasy Mini Kits , according to the manufacturer’s protocol. Microarray analysis was performed using LC MicroRNA microarray Chip_H11.0_081344, which detects miRNA transcripts listed in Sanger miRBase Release 11.0. Imaging and quantifying analysis were performed using an Affymetrix scanner and a fag software.−ΔCt method, and the miR-125a expression fold change in GCTB samples matched to nontumor control samples was evaluated using the 2−ΔΔCt method. Based on the mean fold change of miR-125a expression, patients were divided into high (fold change > mean) and low (fold change < mean) miR-125a expression groups.For miR-125a and RNU6B, reverse transcription and qPCR were performed with The TaqMan MicroRNA Reverse Transcription Kit and TaqMan Universal PCR Master Mix, respectively, using Ambion miRNA primers. The relative expression of miR-125a was normalized against RNU6 expression using the 23′ UTR segments of miR-125a target genes that were predicted to interact with miR-125a were amplified by PCR from human genomic DNA and inserted into the Hind III and Sac I sites of the miRNA Expression Reporter Vector (Life Technologies). The primer sequences are provided in Pre-miR-125a-encoding sequences were subcloned into the GV838 lentiviral vector  and lentiviral vectors. For the miR-125a sponge, synthetic oligonucleotides containing five tandem repeats of bulged miR-125a-binding sites were ligated into the GV369 lentiviral vector (contain EGFP sequences) (GENECHEM). The sequence of the control sponge was as follows: 5′-ACTAGTTAATGTTAGCTAAGAGAAAACTTCGATTGTTAGCTAAGAGAAAACTTCGATTGTTAGCTAAGAGAAAACTTCGATTGTTAGCTAAGAGAAAACTTCGATTGTTAGCTAAGAGAAAACTTCGATTGTTAGCTAAGAGAAAACTTCGCGGATCC-3′. APC and GSK3β expression constructs were purchased from GeneCopoeia . Small hairpin RNA (shRNA) of β-catenin and IL-17RA lentiviral vectors were obtained from Santa Cruz Biotechnology . The TET2 construct was generated as described by Ito et al.For the luciferase assay, stromal cells were cotransfected with the indicated reporter plasmids and either the miR-125a mimics or control oligonucleotides. These cells were also cotransfected with a renilla luciferase plasmid as an internal control. Luciferase activity was measured 72 hr post-transfection using a Dual-Luciferase Assay Kit , according to the manufacturer’s protocol, and the luciferase activity was normalized to the activity of renillar luciferase.Western blot analysis and IHC were conducted as previously described.The IL-17A in the media of primary cultured cells was measured using a human IL-17A ELISA kit (Abcam), according to the manufacturer’s instructions.The stromal cells were infected with the indicated lentiviral vectors. After 48 hr of infection, cells were injected subcutaneously into 6-week-old female nude mice. At 2 months after the cell injection, the mice were sacrificed.2 tests were used for calculating the recurrence rate of GCTB and the association of miR-125a expression with clinical parameters, respectively. All of the other results are expressed as the mean values ± SD. Statistical significance was analyzed by unpaired Student’s t tests or one-way ANOVA and Duncan’s multiple range tests using SAS statistical software package version 6.12 (SAS Institute); p ≤ 0.05 was considered to be statistically significant.Kaplan-Meier survival analysis and xH.J., M.X., and C.-X.X. contributed to the conception and design of the study. S.L., D.-W.L., Q.L., and M.X., collected the human samples and performed IHC. H.J., D.-W.L., and P.H. performed laboratory and animal experiments. H.J., S.-N.W., J.-M.W., and C.-X.X. analyzed the data. H.J. and C.-X.X. wrote the manuscript.The authors have no conflicts of interest.


In this study, Weng et al. provide new insight into the mechanism of piRNA transcription. They used functional proteomics approaches to identify an upstream sequence transcription complex (USTC) that is essential for piRNA biogenesis, and their findings demonstrate the repurposing of a general transcription factor complex, USTC, to aid in genome defense against transposons. Caenorhabditis elegans, most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. While a few genes have been shown to be required for piRNA biogenesis, the mechanism of piRNA transcription remains elusive. Here we used functional proteomics approaches to identify an upstream sequence transcription complex (USTC) that is essential for piRNA biogenesis. The USTC contains piRNA silencing-defective 1 (PRDE-1), SNPC-4, twenty-one-U fouled-up 4 (TOFU-4), and TOFU-5. The USTC forms unique piRNA foci in germline nuclei and coats the piRNA cluster genomic loci. USTC factors associate with the Ruby motif just upstream of type I piRNA genes. USTC factors are also mutually dependent for binding to the piRNA clusters and forming the piRNA foci. Interestingly, USTC components bind differentially to piRNAs in the clusters and other noncoding RNA genes. These results reveal the USTC as a striking example of the repurposing of a general transcription factor complex to aid in genome defense against transposons.Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and nonself nucleic acids and maintain genome integrity and are essential for fertility in a variety of organisms. In  Caenorhabditis elegans, piRNAs preserve genome integrity in the germline by recognizing and silencing “nonself” genomic loci such as transposons or other foreign nucleic acids and induce chromatin modifications  are a class of small (21- to 30-nucleotide [nt]) RNAs that associate with Piwi proteins, a highly conserved subclass of Argonaute proteins, and play significant roles in fertility and genome stability . piRNAs ications . In addial cells .C. elegans piRNAs are also referred to as 21U-RNAs, given that they are predominantly 21 nt in length and have a strong bias toward 5′ monophosphorylated uracil , the biogenesis of piRNAs is a Dicer-independent process . In Drosunctions . The mouunctions —pachytenunctions .C. elegans encodes two Piwi proteins: PRG-1 and PRG-2. Whereas the function of PRG-2 is unknown, mature piRNAs associate with PRG-1 to conduct their functions  that initiates precisely 2 nt upstream of the 5′ end of mature piRNAs to generate 25- to 29-nt capped small RNA (csRNA) precursors . Next, tunctions . The bin animals , and los animals . Interes animals .C. elegans. Type I piRNAs are predominantly transcribed from two broad regions on chromosome IV and contain an 8-nt upstream Ruby motif (CTGTTTCA) and a small YRNT motif in which the T corresponds to the first U of the piRNA. Type II piRNAs are present outside of chromosome IV and lack the Ruby motif . These proteins significantly separate together from the PRDE-1 knockout immunoprecipitation , assuming 1:1:1:1 stoichiometry . Altogether, these results raise strong evidence that PRDE-1, SNPC-4, TOFU-4, and TOFU-5 function as a complex during piRNA transcription.Our previous study identified  elegans . Later,  elegans . To furt elegans , were ampitation B. Thus, hiometry C. To idehiometry , we wanttofu-4 and tofu-5 mutant phenotypes, respectively, advocating that the tagged proteins could recapitulate the functions of endogenous proteins . Both TOFU-4::GFP and TOFU-5::GFP exhibit distinct foci in the germline nuclei . Previous work showed that PRDE-1 colocalizes with SNPC-4 and forms distinct foci in germline cell nuclei . To validate that PRDE-1, TOFU-4, and TOFU-5 function as a complex, we crossed the mCherry::PRDE-1 transgene into TOFU-4::GFP- and TOFU-5::GFP-expressing animals  transgenic strains using the Mos1-mediated single-copy insertion (MosSCI) technology . The TOFl nuclei . We immu animals D. ConsisSupplemental Fig. S3). We found that all four factors have similar genome-wide binding profiles, with strong enrichment on chromosome IV piRNA clusters , although SNPC-4 did not appear enriched on the smaller piRNA cluster (cluster I). However, this might be a reflection of lower signal to noise ratio of the SNPC-4 ChIP-seq experiments . Consistent with our previous finding that PRDE-1 is not required for type II piRNA transcription . However, SNPC-4 and TOFU-5 were enriched at type II piRNA genes . We observed that all four factors exhibit robust enrichment around type I piRNA genes  and prde-1(mj207) mutants and found that TOFU-5 failed to form the subnuclear foci in germline cells in young adult animals  balanced mutant animals and found that TOFU-5 fails to form the subnuclear foci in snpc-4(tm4568) homozygous mutant young adult offspring of these animals (snpc-4(tm4568) mutants, TOFU-5::GFP accumulates in the germline syncytium, further confirmed by knocking down snpc-4 by feeding RNAi . Unlike wild-type TOFU-5, the TOFU-5(*SANT)::GFP fails to form the piRNA foci and is instead enriched in the germline syncytium  mutants and found that PRDE-1 was required for the formation of TOFU-4 piRNA foci (snpc-4(tm4568/hT2) and tofu-5(tm6408/hT2) and found that TOFU-4 fails to form piRNA foci in snpc-4(tm4586) and tofu-5(tm6408) homozygous mutants, which was further confirmed by knocking down snpc-4 by feeding RNAi . These results suggest that, in addition to their ancestral functions, including ncRNA transcription, USTC components might have acquired other functions, such as promoting piRNA biogenesis  genes . We therogenesis .Supplemental Table S2; Supplemental Fig. S7A,B; Supplemental Table S2). However, we did not identify any forkhead transcription factors in this screen, which had been shown previously to recognize the Ruby motif .To further understand the function of the USTC in promoting piRNA transcription, we searched for factors that are required for the formation of TOFU-5 subnuclear piRNA foci. We selected a number of candidate genes from our proteomic experiments and previous genome-wide RNAi screens and carried out a focused candidate RNAi screen for TOFU-5 foci  and a small YRNT motif. Type II piRNAs lack the Ruby motif and are present outside of chromosome IV . We speculate that tbp-1 and the Ruby motif may be required together for USTC binding to piRNA promoters. Here, TBP-1 may act as a bridge to bend the DNA so that the USTC comes closer to the piRNA promoter sequences. Further investigation of the roles of TBP-1 in piRNA biogenesis will shed light on the specific mechanistic understanding of the transcription regulation of piRNAs.The function of Both SNPC-4 and TOFU-5 contain SANT domains . SequencDrosophila, transcription of piRNA clusters is enforced through RNA Pol II preinitiation complex formation within repressive heterochromatin, in which the heterochromatin protein-1 variant Rhino recruits Moonshiner  and set-16 (a gene encoding a putative H3K4 methyltransferase) were identified. However, these two proteins and other chromatin modification factors were not required for the formation of piRNA foci . Therefore, we speculate that either the biogenesis of piRNAs is independent of chromatin modification processes or certain chromatin factors act together to promote the formation of piRNA foci. Further analysis of the properties of chromatin, including histone modifications, in the germline will be necessary to directly explore the relationship between the chromatin state, the USTC, and piRNA expression.Chromatin modifications play important roles in small RNA biogenesis. In onshiner . In C. e regions . In our Supplemental Table S3.Bristol strain N2 was used as the standard wild-type strain. All strains were grown at 20°C unless specified. The strains used in this study are listed in Antibodies against PRDE-1 and TOFU-5 were produced at the Laboratory Animal Resources . For this, New Zealand White female rabbits were immunized with the recombinant full-length proteins together with complete Freund's adjuvant. After the priming immunization, four further booster immunizations were performed every 3 wk. The harvested serum was purified by affinity purification using the recombinant full-length PRDE-1 or TOFU-5, respectively.3VO4) and lysed in a FastPrep-24 5G homogenizer. The supernatant of lysate was incubated with homemade anti-GFP beads for 1 h at 4°C. The beads were then washed three times with cold lysis buffer. Proteins were eluted with chilled elution buffer (100 mM glycine-HCl at pH 2.5). Eluates were precipitated with TCA or cold acetone and dissolved in 100 mM Tris (pH 8.5) and 8 M urea. The proteins were reduced with TCEP, alkylated with IAA, and finally digested with trypsin overnight at 37°C. The liquid chromatography-tandem MS (LC-MS/MS) analysis of the resulting peptides and the MS data processing approaches were conducted as described previously ::GFP were resuspended in an equal volume of 2× lysis buffer  and frozen in liquid nitrogen. Qproteome cell compartment kit (Qiagen) was used to purify nuclear fractions that were diluted with PBSCM buffer . Protein concentration was measured with BCA protein measurement kit (Pierce). Anti-PRDE-1 antibody KSOJ (0.88 mg/mL) was coupled to 5 µg of Dynabeads per 1 mg of beads according to the manufacturer's instructions. Uncoupled IgG beads were used as a control. Beads were incubated with nuclear extracts overnight at 4°C and washed eight times with PBSCM, and proteins were eluted in 80 µL of elution buffer  by boiling for 30 min at 60°C. The elutes were precipitated in TCA or cold acetone and dissolved in 100 mM Tris (pH 8.5) and 8 M urea. The proteins were reduced with TCEP and alkylated with IAA. The samples were digested with trypsin overnight at 37°C, and each sample was run separately.The wild-type strain was var. Bristol N2 , and theC. elegans TOFU-4 protein fragment (amino acids 1–223), the full-length C. elegans TOFU-5 protein (amino acids 1–311), the full-length C. elegans PRDE-1 protein (amino acids 1–526), and the full-length C. elegans SNPC-4 protein (amino acids 1–928) (NCBI sequence reference NM_060011.4) were PCR-amplified and cloned into pB27 as a C-terminal fusion to LexA-DBD . Constructs were checked by sequencing the entire insert and used as bait to screen a random-primed C. elegans mixed-stage cDNA library constructed into pP6. pB27 and pP6 were derived from the original pBTM116  and L40:Gal4 (MAT) yeast strains as described previously  using a fully automated procedure. A confidence score  was attributed to each interaction as described previously , was injected into a Superose6 xK 16/70 . One-milliliter fractions (starting after the void volume of 40 mL) were collected, and proteins were precipitated with TCA, separated by gel electrophoresis, and transferred to a nitrocellulose membrane using a semidry transfer system (Bio-Rad).TOFU-4::GFP, a TOFU-4 promoter and CDS region were PCR-amplified with the primers 5′-GCAGATCTTCGAATGCATCGAATTGAAAATGAGAAAAAACTG-3′ and 5′-ATAGCTCCACCTCCACCTCCGAATTCTGCGTCTACCATCA-3′ from N2 genomic DNA. A GFP::3xFlag region was PCR-amplified with the primers 5′-GGAGGTGGAGGTGGAGCTATGAGTAAAGGAGAAGAAC-3′ and 5′-TCACTTGTCATCGTCATCCT-3′ from plasmid pSG085. A TOFU-4 3′ UTR (untranslated region) was PCR-amplified with the primers 5′-AGGATGACGATGACAAGTGAAAGAATTTTTATCCGAGTCAC-3′ and 5′-AGATATCCTGCAGGAATTCCGAAACTTGATTTTCAAAATTTA-3′ from N2 genomic DNA. The ClonExpress MultiS one-step cloning kit  was used to insert the TOFU-4::GFP::3xFlag fusion gene into the pCFJ151 vector. The transgene was integrated onto the C. elegans chromosome II by the MosSCI method  was used to insert the TOFU-5::GFP::3xFlag fusion gene into the pCFJ151 vector. The transgene was integrated onto the C. elegans chromosome II by the MosSCI system.For TOFU-5(*SANT)::GFP, plasmid was PCR-amplified with the primers 5′-TCGCAGCTTCATCGAAGAGATAAAGTAAGT-3′ and 5′-TCTCTTCGATGAAGCTGCGACCTTCTGCGA-3′ from TOFU-5::GFP::3xFlag plasmid. The transgene was integrated onto the C. elegans chromosome II by the MosSCI system.For RNAi experiments were conducted as described previously . Images ChIP experiments were performed as described previously with hypochlorite-isolated embryos or young adults . AnimalsThe DNA samples from ChIP experiments were sent to in-house sequencing for library preparation and sequencing. Briefly, 10–300 ng of ChIP DNA was combined with End Repair Mix and incubated for 30 min at 20°C followed by purification with a QIAquick PCR purification kit (Qiagen). The DNA was then incubated with A-tailing mix for 30 min at 37°C. The 3′ end adenylated DNA was incubated with the adapter in the ligation mix for 15 min at 20°C. The adapter-ligated DNA was amplified by several rounds of PCR amplification and purified using a 2% agarose gel to recover the target fragments. The average molecule length was analyzed on the Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents). The library was quantified by qPCR (TaqMan probe). The libraries were further amplified on cBot to generate the clusters on the flow cell and sequenced with a single-end 50 method on a HiSeq1500 system.C. elegans genome using BWA version 0.7.7 . The same summed inputs were used for BEADS normalization and peak calls. We observed that despite using input files for MACS2  and filtq-value cut-off and a fragment size of 150 bp against summed ChIP-seq input. To generate sharp peak call sets, we obtained peak summits and extended them 150 bp upstream and downstream, creating 300-bp regions around summit calls. Furthermore, we combined ChIP-seq replicates by intersecting these regions and setting the final peak size back to 300 bp. Finally, peaks overlapping nonmappable (GEM mappability <25%) or blacklisted regions were discarded. This produced sharp uniform peaks suitable for further quantitative analyses.ChIP-seq peaks were called using MACS2 version 2.1.1  with a pPRDE-1, SNPC-4, TOFU-4, and TOFU-5 peak calls were classified using selected annotations from Ensembl version 92. The peak was assigned to a given class if it directly overlapped with annotated loci. The assignments were exclusive (a peak could be assigned to only a single class), giving the following order of priority: snRNA, snoRNA, transfer RNA (tRNA), miRNA, large intergenic ncRNA (lincRNA), ncRNA, promoter, and gene body. Promoters were defined as 500 bases upstream of annotated TSSs. The overlap significance was estimated using a hypergeometric test for overrepresentation.SeqPlots  software2-scaled signal in 1-kb bins divided into piRNA clusters and chromosomes I, II, III, and V. The signal was obtained using the bigWigSummary utility from the Kent library . Genomic coordinates of piRNA genes were obtained by SAMtools against the C. elegans ce10 genome assembly. Type II piRNA genes were obtained from a previous publication (piRNA annotations were downloaded from the piRBase online database (lication . Type I


Vibrio fischeri, which colonizes the squid host Euprymna scolopes.The type VI secretion system (T6SS) facilitates lethal competition between bacteria through direct contact. Comparative genomics has facilitated the study of these systems in  Vibrio fischeri, which colonizes the squid host Euprymna scolopes. Here, we report the draft genome sequences of two lethal V. fischeri strains that encode the T6SS, FQ-A001 and ES401.The type VI secretion system (T6SS) facilitates lethal competition between bacteria through direct contact. Comparative genomics has facilitated the study of these systems in  Vibrio fischeri strains during colonization of Euprymna scolopes juvenile squid  is a membrane-embedded syringe-like structure that delivers effectors to bacterial and, in some cases, eukaryotic cells . A role le squid . V. fisc FQ-A001  and ES40petition . Experimpetition . Draft gE. scolopes adult female (21-mm mantle length) from Kaneohe Bay, Oahu, HI, in 2015, and was cultivated in the laboratory on LB salt (LBS) medium (8 bp (MiSeq) and 1.1 × 109 bp (RS II) of sequence data were obtained, yielding approximately 470-fold coverage of the FQ-A001 genome. De novo assembly of the FQ-A001 genome was conducted using SPAdes version 3.13.0 (with the parameters “-careful -k 127”) (–Strain FQ-A001 was isolated from an ) medium . For Ill-k 127”) –8. For eE. scolopes juvenile (3.5-mm mantle length) from Maunalua Bay, Oahu, HI, in 1990 and can be cultivated in the laboratory on LBS medium (8 bp (MiSeq) and 1.8 × 109 bp (RS II) of sequence data were obtained, yielding approximately 450-fold coverage of the ES401 genome. Assembly, annotation, and GenBank deposition of the resulting 6 contigs of >1,000 bp were conducted as described above for FQ-A001 but with the SPAdes parameters “-careful -k 125” and with PGAP (accessed 2 April 2019).Strain ES401 was isolated from an http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Upon submission to the PGAP, an additional fifth contig in the FQ-A001 sequence contained only the PhiX phage sequence and was excluded from the assembly and further analysis. PGAP additionally identified an Illumina adapter sequence at one location in the ES401 genome; we conducted Sanger sequencing of this region, which clarified the correct sequence. The correct sequence was resubmitted to PGAP, and the surrounding genomic context was not affected.For quality control, the original Illumina and PacBio base calling resulted in 0 sequences flagged as poor quality by FastQC  revealed genomes that are colinear with MJ11 and the presence of the lethal strain-specific T6SS on chromosome II in both FQ-A001 and ES401  2, 11, . DefaultSJSX00000000 , FQ-A001 read accession numbers SRR8647323 and SRR8647324, ES401 genome accession number SRJG00000000 , and ES401 read accession numbers SRR8708068 and SRR8708069.The data for this paper are available at the NCBI as FQ-A001 genome accession number