USTB-05-A and USTB-05-C genes from Sphingopyxis sp. USTB-05, which are responsible for the biodegradation of MCs, the pathway for biodegrading NOD by these two enzymes was investigated in this study. The findings showed that the enzyme USTB-05-A converted cyclic NOD (m/z 825.4516) into its linear type as the first product by hydrolyzing the arginine and Adda peptide bond, and that USTB-05-C cut off the Adda and glutamic acid peptide bond of linearized NOD (m/z 843.4616) and produced dimeric Adda (m/z 663.4377) as the second product. Further, based on the homology modeling of enzyme USTB-05-A, site-directed mutants of USTB-05-A were constructed and seven crucial sites for enzyme USTB-05-A activity were found. A complete enzymatic mechanism for NOD biodegradation by USTB-05-A in the first step was proposed: glutamic acid 172 and histidine 205 activate a water molecule facilitating a nucleophilic attack on the arginine and Adda peptide bond of NOD; tryptophan 176 and tryptophan 201 contact the carboxylate side chain of glutamic acid 172 and accelerate the reaction rates; and histidine 260 and asparagine 264 function as an oxyanion hole to stabilize the transition states.Biodegradation is efficient for removing cyanobacterial toxins, such as microcystins (MCs) and nodularin (NOD). However, not all the microbial strains with the microcystin-biodegrading enzymes MlrA and MlrC could biodegrade NOD. Studies on genes and enzymes for biodegrading NOD can reveal the function and the biodegradation pathway of NOD. Based on successful cloning and expression of the  Nodularia spumigena was firstly reported in the literature as the toxic cyanobacterium for livestock +. The molecular ion at m/z 843.4616 which was m/z 18 more than NOD indicated that product A was hydrolyzed NOD [M+H2O+H]+  was m/z 151 less than hydrolyzed NOD (m/z 843.4616), corresponding to the loss of the amino NH2 group (Molecular Weight (MW): 16) and the phenylethymethoxy group PhCH2CHOCH3 (m/z 135.0805) via random fragmentation. However, all bonds and ions could be found through major peak analyses, except for the Adda and Arg bond. This analysis showed that enzyme USTB-05-A hydrolyzed the Adda–Arg bond in the first step of NOD biodegradation, converting cyclic NOD to the linear type. The protonated product B was detected at m/z 663.4377 (m/z 315.1953 and m/z 135.0804 were generated through losing the amino NH2 group (MW: 16) and the PhCH2CHOCH3 group form Adda, respectively. The m/z 283.1690 [Adda−NH3−CH3OH+H]+, 91.0544 [PhCH2]+, 179.1065 [Adda−PhCH2CHOCH3−NH3+H]+ ions also confirmed the structure of Adda, which was inferred as product B.The LC-MS/MS . The MS +H2O+H]+ b. In the+H2O+H]+ c, six ma663.4377 d, coinci663.4377 e, the ioAs shown by the NOD biodegradation kinetics in mlrA, mlrB, mlrC, and mlrD from Sphingomonas sp. strain MJ-PV [mlrA is crucial for opening the cyclic structure of MCs by cleaving the Adda–Arg peptide bond. After, linearized MCs are biodegraded by the enzyme MlrB and Mlr C. Studies about biodegradation genes or pathways of MC degredation can be found, but less information is available on NOD, which is also a typical type of cyanobacterial toxin in natural water bodies [Sphingomonas genus are the most reported strains to biodegrade MCs. The promising strain Sphingopyxis sp. USTB-05 has a strong ability to biodegrade MCs, and has at least three enzymes encoded by biodegradation genes [Sphingopyxis sp. USTB-05 was also found to have the ability to biodegrade NOD and two biodegradation products were observed by HPLC at a wavelength of 238 nm. The pathway for NOD biodegradation by Sphingopyxis sp. USTB-05 was also speculated [Sphingopyxis sp. USTB-05 was a consecutive process, not step by step.Biodegradation is essential for the reduction of NOD and MCs in water. Bourne et al. have detected the gene cluster for MC biodegradation, which includes in MJ-PV ,35. The r bodies ,46. Mostr bodies ,47,48, br bodies ,28,29 aron genes ,49. Sphieculated  but not USTB-05-A and USTB-05-C we previously studied on MCs [m/z 825.4516) (m/z 843.4616) (2 group of Adda and linking one hydroxyl on the carboxyl group of arginine. The linearized NOD was the product in the first step of NOD biodegradation (m/z 663.4377) in the second step was observed as the dimeric ion of Adda (MW: 331), which was due to the catalysis of the linearized NOD by the USTB-05-C enzyme (m/z 663). However, for the formation of dimeric Adda and the rest of the molecule, there were no explanations in the literature. With the MS/MS spectrum for the second product  a into pr43.4616) b. Based 43.4616) c, it couradation . The resC enzyme d. A prev product e, it cou product .USTB-05-A  in Sphingopyxis sp. USTB-05 [260AIH263NE265, USTB-05-A was assumed to bear an abortive infectivity (ABI) domain composed of four transmembrane helices TM4–TM7. The ABI domain was surrounded by the other four helices, which together form a large cavity accessible to substrates such as NOD. The critical catalytic residues  of MmRce1 were found to be well conserved in the USTB-05-A enzyme . Two conserved Trp residues of W144 and W169 in MmRec1 (W176 and W201 in USTB-05-A) were found to face the carboxylate side chain of E140 (E172 in USTB-05-A) for a better activation of catalytic water. Then, these residues were verified by following docking- and site-directed mutation experiments [Based on our previous work on the cloning of  USTB-05  and homoeriments . By comperiments . HoweverUSTB-05-A is likely a glutamate protease belonging to type II CAAX prenyl endopeptidases. Combined with the biodegradation of NOD by USTB-05-A and its mutants, a complete enzymatic mechanism for NOD biodegradation by USTB-05-A is proposed: Glu172 and His205 activate a water molecule facilitating a nucleophilic attack on the Adda–Arg peptide bond of NOD; Trp176 and Trp201 contact the carboxylate side chain of Glu172 and accelerate the reaction rates; and His260 and Asn264 function as an oxyanion hole to stabilize the transition states. Sphingopyxis sp. USTB-05 was clarified in this study. The enzyme USTB-05-A converted cyclic NOD into its linear type as the first product by hydrolyzing the Arg–Adda peptide bond, and USTB-05-C cut off the Adda–Glu peptide bond of linearized NOD and produced Adda as the second product. A complete enzymatic mechanism for NOD biodegradation by USTB-05-A was also proposed. The active site Glu172 and His205 activated a water molecule, facilitating a nucleophilic attack on the Adda–Arg peptide bond of NOD. Trp176 and Trp201 contacted the carboxylate side chain of Glu172 and accelerated the reaction rate. His260 and Asn264 functioned as an oxyanion hole to stabilize the transition states. The findings provide good foundations for investigating the biodegradation mechanism of both MCs and NOD in the future.The enzymatic pathway for biodegrading nodularin by USTB-05-A/BL21(DE3) and pET30a(+)/USTB-05-C/BL21(DE3) were previously studied on MCs and used in this study for NOD biodegradation [E. coli TOP10 and E. coli BL21(DE3) were purchased from Sangon Biotech . The recombinant and host strains were cultured in Luria–Bertani (LB) medium [BamHI, XhoI, SacI and NotI, the plasmid mini-prep kit and the polymerase chain reaction (PCR) kit were obtained from Sangon Biotech . Standard NOD was purchased from Enzo Science Inc., Farmingdale, NY, USA. All other chemicals were analytical grade. Recombinant pGEX-4T-1/radation . E. coli) medium  on a shaUSTB-05-A/BL21(DE3) and pET30a(+)/USTB-05-C/BL21(DE3) were grown in LB medium with 100 μg mL−1 ampicillin or 50 μg mL−1 kanamycin for 3 h, respectively. The inoculated culture was grown at 37 °C and 200 rpm to an optical density at 600 nm (OD600nm) of 0.6, induced with isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mmol L−1 and incubated at 30 °C for 3 h. The flask culture was grown in the same conditions above with an incubation amount of 10% and culture volume of 50 mL per 500 mL shake flask. One liter of recombinant cell culture was harvested by centrifugation , washed thrice with PBS  and resuspended in 20 mL PBS to a final concentration of 20–30 g dry cell weight per liter. The cells were disrupted by sonication  in an ice bath, and centrifuged . The supernatant was collected as CE. Protein content was determined according to Bradford [The expression of recombinants was performed as previously described ,49. The Bradford .Control groups for treatment A and treatment C: the CEs of recombinant bacteria pGEX-4T-1/BL21(DE3) without protein USTB-05-A and recombinant bacteria pET30a(+)/BL21(DE3) without protein USTB-05-C were added into two tubes with PBS containing NOD, respectively.USTB-05-A/BL21(DE3) containing crude protein USTB-05-A was added into PBS containing NOD.Treatment A: the CE of recombinant bacteria pGEX-4T-1/USTB-05-C/BL21(DE3) containing crude protein USTB-05-C was added into PBS containing NOD.Treatment C: the CE of recombinant bacteria pET30a(+)/Treatment AC: at 0 h, the CE containing crude protein USTB-05-A was added into PBS containing NOD. At 12 h, the CE containing crude protein USTB-05-C was added to the above solution.Control group for treatment A and treatment C: at 0 h, the CE containing crude protein USTB-05-A was added into the test tubes with PBS containing NOD. At 12 h, the CE of pET30a(+)/BL21(DE3) without protein USTB-05-C was added to the above solution.The experiments were carried out with five treatments as following:−1. The protein concentrations of CEs containing USTB-05-A and USTB-05-C were 158 mg L−1 and 165 mg L−1, respectively. All test tubes were placed in the incubator at 30 °C with shaking at 200 rpm. Samples of 200 μL were taken at 0 h, 3 h, 12 h, 13 h, 18 h and 24 h, respectively. Concentrated hydrochloric acid (2 µL) was added immediately to stop the reaction. Samples were centrifuged  and the undiluted supernatants were taken for the detection by HPLC. In each reaction system of 4 mL, the initial concentration of NOD was 13.7 mg L18 solid-phase extraction cartridge . Methanol (40 μL) was added at a rate of 1 mL min−1. The treated sample was collected and analyzed by liquid chromatography tandem-mass spectrometry  to determine the m/z ratios of NOD and the biodegradation products.For product identification, samples at several reaction time points were taken and purified through the CUSTB-05-A  in Sphingopyxis sp. USTB-05 [mlrAE172A, mlrAW176A, mlrAW201A, mlrAH205A, mlrAH260A, mlrAN264A, and mlrAE265A was performed by overlap extension PCR as previously reported [USTB-05-A mutants and preparation of CEs were in accordance to the steps for recombinant USTB-05-A mentioned above. For enzymatic activity comparison of recombinant USTB-05-A and mutants, the initial concentration of NOD was 18.7 mg L−1 and the protein concentration of CEs was 350 mg L−1. Samples of 200 μL were taken at 0 h, 6 h, 9 h, 12 h and 24 h, respectively.Based on our previous work involving the cloning of  USTB-05  and homoreported . The exp18 column (4.6 × 250 mm)  was used. Chromatographic grade acetonitrile and water containing 0.05% (v/v) trifluoroacetic acid  was used as the mobile phase. The flow rate was 1.0 mL min -1, and the injection amount was 20 μL. The spectra of NOD and its biodegradation products were detected at the time of HPLC analysis.The concentration of NOD was detected by high performance liquid chromatography (HPLC)  with a UV diode array detector at 238 nm. An Agilent TC–C2) pressure of 35 psi, desolvation gas flow rate of 10 L min−1 and desolvation gas temperature of 300 °C.For MS detection, an analytical column OOD-4622-ECLC  was used at a maintained temperature of 30 °C. Precursor ions for samples and internal standards were determined from mass spectrum obtained during infusion into the Agilent 6530 Accurate-Mass Q-TOF LC/MS. An electrospray ionization (ESI) source was used. The MS detection conditions were: capillary voltage of 4000 V, nebulizer gas (N


In rice, gene body-localized H2A.Z plays a prominent role in repressing stress-responsive genes under non-inductive conditions, whereas H2A.Z at the transcription start site functions as a positive or negative regulator of transcription. Oryza sativa) upon exposure to phosphate (Pi) deficiency and in response to RNAi knockdown of OsARP6, which encodes a key component of the H2A.Z exchange complex. Both Pi deficiency and OsARP6-knockdown resulted in similar, profound effects on global H2A.Z distribution. H2A.Z in the gene body of stress-responsive genes was negatively correlated with gene expression, and this was more apparent in response to Pi deficiency. In contrast, the role of H2A.Z at the transcription start site (TSS) was more context dependent, acting as a repressor of some stress-responsive genes, but an activator of some genes with housekeeping functions. This was especially evident upon OsARP6-knockdown, which resulted in down-regulation of a number of genes linked to chloroplast function that contained decreases in H2A.Z at the TSS. Consistently, OsARP6-RNAi plants exhibited lower chlorophyll content relative to the wild-type. Our results demonstrate that gene body-localized H2A.Z plays a prominent role in repressing stress-responsive genes under non-inductive conditions, whereas H2A.Z at the TSS functions as a positive or negative regulator of transcription.The H2A.Z histone variant plays a role in the modulation of environmental responses, but the nature of the associated mechanisms remains enigmatic. We investigated global H2A.Z deposition and transcriptomic changes in rice ( Saccharomyces cerevisiae), H2A.Z is necessary for proper expression of nutrient-related genes , which encodes a key component of the Swi/Snf2-related (SWR1) complex that exchanges H2A.Z/H2B dimers for H2A/H2B dimers (FLOWERING LOCUS C (FLC) floral repressor locus, resulting in down-regulation of FLC and an early flowering phenotype  indicated distinct H2A.Z genic profiles between callus and seedling samples, suggesting a role for H2A.Z in tissue development  cultivar Nipponbare and OsARP6-RNAi transgenic lines were surface-sterilized and pre-germinated for 1 d at 37 °C followed by 2 d at 28 °C. Seeds were germinated under 12/12 h light/dark, 30/22 °C. At 2 weeks old, seedlings were transferred to a Yoshida Rice culture modified solution in a hydroponic system (2PO4 for a 24-h Pi-deficiency treatment.Seeds of the rice (OsARP6-RNAi lines of rice (cv. Nipponbare) were made with the vector pFGC1008 , which targeted 489 bp within exons 2–7 of OsARP6 (LOC_Os01g16414) and was mobilized into Agrobacterium tumefaciens. Multiple lines showed varying degrees of knockdown of OsARP6, and line #19 was selected for ChIP-Seq and RNA-Seq analysis.OsOsARP6-RNAi plants using the RNeasy Plant Mini kit (Qiagen) and was DNase-treated using RNase-Free DNase (Qiagen). cDNA was prepared from 1 ug total RNA using an oligo(dT) primer by the SuperScript III synthesis kit (Invitrogen) according to the manufacturer’s instructions. RT-qPCR was performed with a ViiA7 real-time PCR system using SYBR Green detection chemistry (Applied Biosystems) and gene-specific primers (IDT). Expression data were normalized to the expression level of the OsActin gene.RNA was extracted from 0.1 g frozen shoots of wild-type and OsARP6-RNAi were used for physiological studies carried out as described by P<0.05 level. Student’s t-test was used to analyse the morphological parameters.Wild-type and transgenic lines of JXB online).Based on amino acid identity among the three rice H2A.Z sequences at the N terminus (http://www.ncbi.nlm.nih.gov/sra) under accession number SRP102661. Sequenced reads were quality-checked using FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Bowtie was used to uniquely align the reads to the reference genome (MSU Rice Genome Annotation Release 7.1) with up to two mismatches allowed (1–k3) (P≤0.05 (The chromatin immunoprecipitation (ChIP) experiments were carried out as described previously  on rice  allowed . Regionsn=2002) with FDR<0.001 obtained from DESeq2 were used for clustering. The fuzzy k-means clustering was done with the Aerie tool . The RNA-Seq libraries were prepared with a TruSeq Stranded mRNAseq Sample Prep kit (Illumina). The libraries were quantitated by qPCR and sequenced for 101 cycles from one end of the fragments on a HiSeq2500 using a HiSeq SBS sequencing kit version 4. Fastq files were generated and demultiplexed with the bcl2fastq v2.17.1.14 Conversion Software (Illumina). Illumina reads of all samples have been submitted to the Sequence Read Archive at the NCBI under accession number SRP102661. RNA-Seq reads for each sample were mapped to the reference genome (MSU Rice Genome Annotation Release 7.1) using the Bowtie2 tool . To quanrie tool .r=0.89; The primary aim of this study was to examine the separate and combined impacts of a major environmental stress and knockdown of a key component of the SWR1 H2A.Z exchange complex on H2A.Z localization. We first examined global H2A.Z distribution by carrying out ChIP-Seq experiments on shoots of 36-d-old rice plants. Two biological replicates were examined to ensure reproducibility (Pearson correlation coefficient 1) and Q2 had a prominent H2A.Z peak near the TSS and a lesser peak near the transcription termination site (TTS). Reciprocally, low-expressed genes in Q4 and Q5 exhibited relatively higher H2A.Z deposition, particularly in the gene body (GB). Interestingly, genes in Q0 (i.e. not expressed) could be placed in two divergent sub-groups  and a minor group . In k1, H2A.Z distribution showed broad peaks inside the gene body (GB) with three distinct sub-patterns  enriched with transcription factor genes and genes related to secondary metabolism and stress-responses, whereas k2 genes were over-represented in primary metabolism and biosynthesis GO terms. Genes in the smaller k3 cluster comprised a combination of GO terms enriched in k1 and k2. Based on these results, we assigned the k1 genes as ‘stress-related’ and the k2 genes as ‘housekeeping’. Consistent with this, 82% of a group of rice genes (n=4147) considered as housekeeping genes in a previous study  of genes containing a decrease in H2A.Z in WTP exhibited the decrease in the TSS region . These g2 group , and sho2 group . Genes wgroup . Reciprocally, down-regulated DEGS were enriched among genes with an increase in H2A.Z at the GB . These results revealed that differential expression in response to Pi deficiency was negatively correlated with H2A.Z levels in the GB. For example, genes encoding a WRKY transcription factor, glycosyl transferase, and receptor-like kinase, which showed up-regulation here as well as in 24-h Pi-deprived rice seedlings in a previous study (P<0.001). These observations are consistent with a repressive role for H2A.Z at the TSS, as was recently proposed in Arabidopsis , and membrane stability index  contained similar H2A.Z changes in response to Pi deficiency  gene group compared to the shared genes, whereas those unique to Pi deficiency were enriched in the k1 (stress-related) group  group . For genOsARP6-RNAi shoots (OsARP6-RNAi grown under control conditions. We identified 796 up-regulated and 1473 down-regulated DEGs in OsARP6-RNAi compared to the WT (FDR<0.001). Enriched GO categories for these DEGs were similar to those for DEGs in Arabidopsis arp6 mutants . However, unlike WTP, whose up-regulated DEGs were under-represented among genes with increases in TSS H2A.Z, OsARP6-RNAi up-regulated genes were under-represented among genes containing decreases at the TSS. These results suggested that changes in H2A.Z at the TSS in response to Pi deficiency were reflective of H2A.Z having a repressive role, but that disruption of H2A.Z deposition in OsARP6-RNAi revealed a more complex role for H2A.Z at the TSS in which it could act to promote or repress transcription of different subsets of genes.As with Pi-deficient WT (WTP), we identified correlations between OsARP6-RNAi compared to WTP, but the overlap was less  were also up-regulated in WTP, and 41% (n=607) of the 1473 down-regulated genes were also down-regulated in WTP  exposed to 24 h of Pi deficiency  in response to Pi deficiency (i.e. arp6C vs. arp6P). This suggested that knockdown of OsARP6 or a 24-h Pi-deficiency treatment had dramatic but similar impacts on H2A.Z localization, and that the combined perturbations led to only a moderate additive disruption of H2A.Z localization. To test this possibility, we compared the overlap in differences in H2A.Z deposition between each of the ‘stress’ samples  to WTC  to WTC . For eac) to WTC . This coOsARP6-knockdown and Pi deficiency, we employed clustering analysis in the deseq2 software package on RNA-Seq data for WTC, WTP, arp6C, and arp6P (n=2002) were obtained for downstream clustering (FDR<0.001), which resulted in 14 distinct clusters. Most of the DEGs fell into one of six major clusters . Also of note in this cluster was the presence of three core plant PSR genes despite the lack of differential expression in response to Pi deficiency, which may have been due to an insufficient duration of Pi deficiency in this study. Cluster C6 genes exhibited an obvious down-regulation in arp6C and arp6P compared to WTC and WTP (2 group (housekeeping). Enriched GO terms in this cluster included plastid and thylakoid (OsARP6 led to decreased H2A.Z at the TSS and a corresponding reduction in transcript abundance. The specific reductions in transcription of the genes implicated in chlorophyll biosynthesis were consistent with the decreased chlorophyll content observed in OsARP6-RNAi plants (To further investigate the interactions among nd arp6P . PCGs wis C1–C6; . Genes isporters . These cd to WTC . Genes fd to WTC . That thi status . Althoug and WTP , and thehylakoid . Interesi plants .14 and C9, contained genes that were up- and down-regulated by Pi deficiency, respectively, but unaffected by OsARP6-knockdown. In contrast to the OsARP6-specific genes described above, these Pi-specific DEGs were fewer in number and were not enriched in any GO terms. Of the remaining clusters, three yielded enriched GO terms despite containing small numbers of genes. Genes in C13 were enriched in growth-related GO terms, which included a kinesin and several expansins. The transcript response of this cluster was similar to clusters C3 and C4, in which kinesins and expansins were also present. Finally, two clusters, C11 and C12, exhibited complex interactions between OsARP6-knockdown and Pi deficiency. Cluster C11 genes, which exhibited down-regulation by Pi in the WT but not in the OsARP6-RNAi line, were enriched in response to stimulus, and one gene was a core plant PSR gene. Genes in cluster C12 were up-regulated by both OsARP6-knockdown and Pi deficiency, and their expression was even higher in Pi-deficient OsARP6-RNAi shoots. These genes were enriched in transcription factor genes and highlighted a unique group of transcription factors that may respond to both Pi deficiency and OsARP6-knockdown perturbations, but via different mechanisms.In addition to the six major clusters described above, the interaction analysis resulted in a number of minor clusters . Two of OsARP6-knockdown did not result in a significant additive effect. Because Pi deficiency is both an acute environmental stress and deprivation of a major essential nutrient, it elicits transcript changes in numerous housekeeping and stress-responsive genes. Our findings are consistent with a role for H2A.Z in the induction and/or maintenance of this transcriptional response. Accordingly, disruption of H2A.Z exchange via OsARP6-knockdown mimicked the onset of Pi-deficiency responses under control conditions. Although extensive overlap was observed for Pi deficiency and OsARP6-knockdown, there were also notable differences. Pi deficiency resulted in a biased impact on differential expression of stress-responsive genes that was negatively correlated with H2A.Z in the GB. In contrast, OsARP6-knockdown was biased toward differential gene expression of housekeeping genes linked to differential H2A.Z peaks at the TSS. Our results indicate that GB-localized H2A.Z plays a prominent role in repressing stress-responsive genes under non-inductive conditions. In contrast, the role of H2A.Z at the TSS appears to be more context-dependent in that it behaves as a repressor of particular stress-responsive genes, but acts to promote expression of some genes with housekeeping functions, such as those linked to translation and chloroplast functions. This may reflect a role for H2A.Z in promoting DNA accessibility, which can be followed by recruitment of active or repressive complexes according to target gene function.Our results demonstrated that a 24-h Pi-deficiency treatment or knockdown of a SWR1c component had similar, profound effects on both the rice transcriptome and global genic distribution of H2A.Z. This suggests that these perturbations affected similar chromatin-remodeling and/or transcriptional regulatory mechanisms. Indeed, combining Pi deficiency with JXB online.Supplementary data are available at Table S1. Total reads, mapped reads, and peak counts for ChIP-Seq samples.Fig. S1. Number of H2A.Z enrichment peaks in each replicate and the overlap between replicates.Fig. S2. The average profiles of H2A.Z deposition among gene annotation types in wild-type shoots.Fig. S3. PCA plot of RNA-Seq samples.Fig. S4. The average H2A.Z deposition profiles of differentially expressed genes.OsARP6-RNAi transgenic lines.Fig. S5. Development of Fig. S6. Proportion of common and unique differential H2A.Z deposition genes.Fig. S7. Average H2A.Z profiles and overlap between differential H2A.Z peaks and differentially expressed genes for all samples.Fig. S8. Cluster analysis of differentially expressed genes.Dataset 1. Significantly enriched GO terms for three distinct H2A.Z genic profile gene groups.Dataset 2. Significantly enriched GO terms for genes with differential H2A.Z peaks or differential expression between wild-type grown under control and Pi-deficient conditions.OsARP6-RNAi and wild-type grown under control conditions.Dataset 3. Significantly enriched GO terms for genes with differential H2A.Z peaks or differential expression between Dataset 4. Significantly enriched GO terms for differential H2A.Z peaks and differentially expressed genes common or unique to WTC versus WTP or WTC versus arp6C.Dataset 5. Significantly enriched GO terms for genes within clusters based on interactions among differentially expressed genes in WTC, WTP, arp6C, and arp6P.Dataset 6. ChIP protocol and determination of OsH2A.Z antibody specificity.supplementary_Table_S1_figures_S1_S8Click here for additional data file.supplementary_dataset_S1Click here for additional data file.supplementary_dataset_S2Click here for additional data file.supplementary_dataset_S3Click here for additional data file.supplementary_dataset_S4Click here for additional data file.supplementary_dataset_S5Click here for additional data file.supplementary_dataset_S6Click here for additional data file.


V600E-mutated cancer cells. Last, we investigated whether 14-3-3 binding to the C terminus of CRAF is required for CRAF catalytic activity and observed that it was dispensable in vivo. Altogether, our study unravels the molecular mechanism by which 14-3-3 regulates dimerization-driven RAF activation and identified AMPKi as a potential agent to counteract drug resistance and adverse effects of RAF inhibitors in cancer therapies.The dimerization-driven paradoxical activation of RAF proto-oncogene Ser/Thr kinase (RAF) is the predominant cause of drug resistance and toxicity in cancer therapies with RAF inhibitors. The scaffold protein 14-3-3, which binds to the RAF C terminus, is essential for RAF activation under physiological conditions, but the molecular basis is unclear. Here we investigated whether and how 14-3-3 regulates the dimerization-driven transactivation of the RAF isoform CRAF by RAF inhibitors and affects drug resistance and toxicity by virtue of the dominant role of CRAF in these processes. We demonstrated that 14-3-3 enhances the dimerization-driven transactivation of CRAF by stabilizing CRAF dimers. Further, we identified AMP-activated protein kinase (AMPK) and CRAF itself as two putative kinases that redundantly phosphorylate CRAF's C terminus and thereby control its association with 14-3-3. Next, we determined whether the combinatory inhibition of AMPK and CRAF could overcome the paradoxical effect of RAF inhibitors. We found that the AMPK inhibitor (AMPKi) not only blocked the RAF inhibitor–driven paradoxical activation of ERK signaling and cellular overgrowth in Ras-mutated cancer cells by blocking phosphorylation of Ser-621 in CRAF but also reduced the formation of drug-resistant clones of BRAF Ras–RAF–MEK–ERK1–5–8–12–18–Dimerization-driven transactivation of RAF kinase is regulated by many factors. Canonical upstream Ras activation has been shown to facilitate RAF dimerization, probably through relieving the intramolecular interaction between the Ras-binding domain and kinase domain . SimilarXpS/TXP or RXXXpS/TXP motifs . These data demonstrated that 14-3-3 binding to the C terminus of CRAF indeed plays a critical role in the dimerization-driven transactivation of CRAF.Previous studies have suggested that 14-3-3 binding to the C terminus of RAF kinase is critical for its dimer-dependent activation . To confA). To do this, Nluc (the N terminus of luciferase) and Cluc (the C terminus of luciferase) were fused, respectively, to the CRAF kinase domain  with or without the S621A mutation or deletion of the 14-3-3 binding motif (hereafter referred to as ΔC). Then Nluc-CKD was co-expressed with either Cluc-CKD/S621A or Cluc-CKDΔC in 293T cells (B). The Cluc-fused R401H mutant (Cluc-CKD/R401H), which has a disrupted dimer interface, was used as a control. The RAF inhibitor PLX4720 (vemurafenib) induced much less luciferase signaling in 293T transfectants that express either Cluc-CKD/S621A, Cluc-CKD ΔC, or Cluc-CKD/R401H, in contrast to those expressing the WT counterpart (C), suggesting that CKD/S621A and CKDΔC have much less ability than CKD/R401H to associate with CKD upon inhibitor loading. To further confirm this finding, we next carried out a co-immunoprecipitation assay. As shown in D and E, treatment of 293 transfectants that express CKD with PLX4720 induced robust dimerization that is impaired by S621A mutation or deletion of the 14-3-3 binding motif (ΔC) as was done by R401H mutation. Taken together, these data indicate that 14-3-3 binding to the C terminus of CRAF improves the dimerization-driven transactivation of CRAF by facilitating its dimerization or stabilizing its dimers.To understand how 14-3-3 regulates the dimerization-driven transactivation of CRAF, we measured the dimer affinity of CRAF mutants with either deletion or mutation of the C-terminal 14-3-3 binding motif by complementary split luciferase assays A. To do 3T cells B. The Clnterpart C, suggesXSXP motif. Previous studies have suggested that the phosphorylation of Ser-621 is mediated by PKA, AMPK, or CRAF in different contexts , a direct target of PKA, whereas treatment with the AMPK inhibitor Compound C completely blocked the phosphorylation of acetyl-CoA carboxylase (ACC), a putative effector of AMPK, suggesting that the activity of PKA or AMPK, respectively, is inhibited in these cells. However, only the kinase-dead A373F mutant lost phospho–Ser-621 upon AMPK inhibition with Compound C , whereas PKA inhibition with H89 slightly enhanced phospho–Ser-621 in both WT and kinase-dead A373F CRAF . These data indicated that Ser-621 in the 14-3-3 binding motif of CRAF is phosphorylated redundantly by AMPK and CRAF itself, although the mechanism for the slight increase of phospho–Ser-621 in the presence of the PKA inhibitor is unclear and requires further investigation in the future. Further, we demonstrated by co-immunoprecipitation assay that the blockage of phospho–Ser-621 in kinase-dead A373F CRAF by Compound C impaired the association of 14-3-3 with this mutant (C and D).It is well-known that the binding of 14-3-3 to the C terminus of CRAF requires the phosphorylation of Ser-621 in the RScontexts –28. To cV600E-harboring cancers (–A–F). This paradoxical effect of vemurafenib on H1299 and Sk-mel-2 cancer cell lines was blocked by the AMPK inhibitor Compound C (A–F) or the shRNA-mediated knockdowns of AMPKα1, the predominant isoform of AMPKα in H1299 cells (G–I). Furthermore, the paradoxical stimulation of cell growth by vemurafenib in H1299 and Sk-mel-2 cancer cell lines was also inhibited by Compound C or AMPKα1 knockdown (A–G). Noteworthy, the concentrations of Compound C used in our experiments could effectively block the phosphorylation of ACC by AMPK (H) but had no apparent toxicity in these cell lines , which excludes the potential artifacts arising from this AMPK inhibitor. Altogether, these data further support that AMPK- and CRAF-mediated phosphorylation of the C-terminal 14-3-3 binding motif plays a critical role in the dimerization-driven transactivation of CRAF.The paradoxical activation of RAF–MEK–ERK signaling driven by RAF inhibitors is not only responsible for the intrinsic resistance of Ras-mutated cancers but also one of the important causes that lead to acquired resistance in BRAF cancers . Moreove cancers –20. As icancers –, A–F. Thmpound C , A–F or 99 cells , G–I. Funockdown , A–G. No by AMPK H but hadV600E-harboring cancers with RAF inhibitors. Hence we examined whether AMPKi would enhance the efficacy of RAF inhibitors by impairing the drug resistance in BRAFV600E-harboring cancers. To this end, we treated A375 and A101D, two BRAFV600E-positive melanoma cell lines, with vemurafenib alone or plus Compound C and identified the formation of drug-resistant clones by crystal violet staining. As shown in m) effectively blocked the phosphorylation of ACC by AMPK and dramatically reduced the formation of drug-resistant clones from both melanoma cell lines.As described above, the paradoxical activation of RAF–MEK–ERK signaling contributes significantly to acquired resistance in the treatment of BRAFin vitro upon purification, which can be restored by GST fusion–enhanced dimerization . These data suggest that the CRAF/DDEE/L397M/S621A and CRAF/DDEE/L397M/ΔC mutants have a lower dimer affinity than their parental protein, although they do not associate with 14-3-3, and further support that 14-3-3 binding to the C terminus of CRAF elevates its dimer affinity or stabilizes CRAF dimers.Dimerization of RAF kinase is required for both activation and catalytic activity , 33. Ourrization . In thisrization , 34 and nterface , these mT fusion , C and Din vivo, these dimers are so weak that they dissociate during purification and thus lose catalytic activity in vitro. This is consistent with our recent findings from oncogenic RAF mutants with β3-αC loop deletions that suggest that strong dimerization drives transactivation of RAF kinase, whereas weak dimerization is required for catalytic activity following its activation , anti-phospho-MEK1/2 (9154), and anti-MEK1/2 (9124) ; anti-phospho–Ser-621 ; anti-FLAG ; anti-HA ; anti-ERK2 ; anti-ERK1/2 ; and horseradish peroxidase–labeled secondary antibodies . PLX4720 (A-1131) was purchased from Active Biochem, H-89 (S1582) and Compound C (S7306) from Selleckchem; and D-luciferin (LUCK-2KG) from Gold Biotechnology. All other chemicals were obtained from Sigma.All expression vectors used in this study were either purchased from Addgene or synthesized by Integrated DNA Technologies, as noted in the text. All mutants were generated by PCR, tagged with either FLAG or HA or His, and cloned into vectors by Gibson assembly. The pCDNA3.1(+) expression vector (Invitrogen) was used for transient expression in mammalian cells and pET-28a (Novagen) for bacterial expression. To knock down AMPKα, shRNAs that target 5′-GAGGAGAGCTATTTGATTA-3′ or 5′-GCTTGATGCACACATGAAT-3′ in human AMPKα1 were cloned into the lentiviral vector with the U6 promoter by using traditional molecular methods. The shRNA against luciferase  was used as a control.6-tagged MEK1 (K97A) was expressed in bacterial BL21(DE3) strains and purified using a nickel column (Qiagen) and following our previous protocol . The AMPKα knockdown H1299 cell lines were constructed by lentiviral transduction as described before –38. For –protocol .5 cells/well. 24 h later, D-luciferin (0.2 mg/ml) and PLX4720  were added to the culture; the incubation was allowed to proceed for 30 min before the luciferase signals were measured by Promega GloMax® Multi Detection System.293T transfectants that express different pairs of Nluc- and Cluc-fused CRAF or CRAF mutants were plated in 24-well Krystal black image plates at a seeding density of 2 × 10in vitro kinase assays, the immunoprecipitants were washed once with kinase reaction buffer ) and then incubated with 20 μl of kinase reaction mixture (2 μg of substrate and 100 mm ATP in 20 μl of kinase reaction buffer) per sample at room temperature for 10 min. The kinase reaction was stopped by adding 5 μl/sample of 5× Laemmli sample buffer. Immunoblotting was carried out as described before  was added to the cell cultures with or without Compound C (0.62 μm). The culture medium was changed every other day, and the concentration of PLX4720 was increased 2-fold every time until it reached 0.8 μm. After 2 weeks of culturing, cells were fixed with 4% paraformaldehyde and stained with crystal violet solution. The clones were counted manually, and the graphs were generated using GraphPad Prism 6.Cells were seeded in 60-mm dishes at a density of 2 × 10t test in all experiments, and error bars represent standard deviation to show variance between independent experiments.All statistical analyses in this study were performed using GraphPad InStat (GraphPad Software). Statistical significance was determined by one-sample J. Yuan, W. H. N., J. Yap, B. C., X. H., and J. H. investigation; M. W. writing-review and editing; J. H. conceptualization; J. H. data curation; J. H. supervision; J. H. methodology; J. H. writing-original draft.


Escherichia coli K-12 strains, was able to be propagated on Citrobacter rodentium strain ICC169 and a range of Shigella sonnei strains with high efficiency of plating (EOP). It represents the first lytic bacteriophage originating from rabbit and the first infectious P2-like phage of animal origin. In the three characteristic moron-containing regions of P2-like phages, R18C contains genes with unknown function that have so far only been found in cryptic P2-like prophages.Here, we report a novel virulent P2-like bacteriophage, R18C, isolated from rabbit faeces, which, in addition to The online version of this article (10.1007/s00705-019-04424-5) contains supplementary material, which is available to authorized users. Myoviridae . They a viruses , and theHere, we report the characterization of bacteriophage R18C isolated from rabbit faeces , which proved to be a new member of viable P2-like viruses.E. coli K-12 strain MG1655 [E. coli K-12 (the base titer was 2 × 105 plaque-forming units per milliliter (PFU/ml)), phage R18C was propagated with an 104 times higher efficiency of plating (EOP) on Citrobacter rodentium strain ICC169 [Shigella sonnei strains. S. sonnei 75/02 is a recently characterized Shiga-toxin-producing strain [S. sonnei 866-F [3. Enteroinvasive E. coli (EIEC) strain 548 of the O124 serogroup [The cell-free supernatant of the culture was spotted on layered soft agar plates containing n MG1655 . A singlMorphological investigation was performed by electron microscopy as described earlier . The morC. rodentium ICC169 according to the protocol described by Bassiri [A one-step growth experiment was performed in three parallel runs on  Bassiri . The latd0 distance formula [Phage DNA was isolated by the phenol-chloroform method  with the formula . The gen formula . The genPstI restriction endonuclease as well .Homology searches and comparisons showed that R18C is a close relative of the P2-like bacteriophages, and its genome organization and order of ORFs with known function strictly corresponds to those of the prototype phage P2 and other viable phages of the P2-like viruses group. The end positions of the genome were also determined based on homologies, and they were later confirmed by the electrophoretic pattern of the genomic DNA after digestion with E. coli, and even E. albertii, that are more closely related to R18C than either of the viable P2-like phages .Phylogenetic analysis of the whole genome  indicated that the closest lytic relative of phage R18C within the group is bacteriophage WPhi [In P2-like phage genomes there are three regions that are known to carry morons, genes that are unnecessary for the life cycle of the phage . These tin and old genes, which occupy this position in the prototype P2 phage and encode phage immunity genes against T-even and lambdoid phages, respectively. It is noted for carrying the gene cluster encoding the cytolethal distending toxin (CDT) type V in multiple P2-like prophages, which are harbored by E. coli strains of the O157 serogroup [Salmonella strains, in one of which (GenBank no. CP032816.1) it is annotated as a pseudogene termed ‘cell filamentation protein Fic.’The first such region is the TO region, between the genes encoding the replication protein A and capsid packaging protein Q (ORFs 44 and 1), named after the erogroup , 19. In E. coli strains, in all cases annotated as ‘hypothetical protein.’Instead of the short, 261-bp-long ORF30 of the original P2 phage, there is a 741-bp-long ORF annotated as a hypothetical protein between baseplate assembly protein V and the phage tail completion protein S  phage immunity-protein-encoding gene, two short ORFs  annotated as encoding hypothetical proteins were found , flanked by relatively long non-coding regions. This location currently has 15 homologues in GenBank. In the cases where there is an annotation available, ORFs with the same length and similar annotations are found, with ORF24 annotated as ‘DUF3606-domain-containing protein’ in some cases.In the case of all these moron ORFs, a protein motif search with PSI-BLAST did not yield any additional information regarding their possible structure or function.C. rodentium and Shigella sonnei with a high EOP. Phage R18C is a member of the P2-like viruses within the subfamily Peduovirinae, carrying a set of rare morons with unknown functions in its insertion sites. To our knowledge, R18C is the first infectious bacteriophage isolated from rabbit, and the first P2-like phage of animal origin. It is only the fifth viable phage characterized from this group, and the first P2-like phage described that is capable of lysing C. rodentium, and to our knowledge, R18C is the first P2-like phage that has had its burst size measured. As C. rodentium is frequently used as a model organism for the pathogenesis of enteropathogenic and enterohemorrhagic E. coli , a new Supplementary Table 1 List of ORFs identified in the genome of bacteriophage R18C (GenBank accession no. MN016939), their closest homologues, and their assigned functions (XLSX 13 kb)Supplementary Figure 1 One-step growth curve of bacteriophage R18C on Citrobacter rodentium strain ICC169. The initial number of phage particles was 2×106, and samples were collected every 10 minutes. The PFU value is given for the whole volume of the culture, which was 50 ml. (TIFF 1536 kb)Supplementary Figure 2 Agarose gel electrophoresis of PstI digested R18C phage DNA (A) and HindIII-digested lambda phage DNA (B). Electrophoresis was performed in a 1.7% agarose gel with a constant current of 4 V/cm for 2.5 hours. Arrows point to the 3.5- and 4.5-kb fragments of the R18C genome, which resulted from PstI cutting sites at positions 4574-4575 and 27764-27765, which are closest to the 5’ and 3’ end of the genome, respectively. (TIFF 411 kb)Supplementary Fig. 3 Phylogeny of lytic P2-like bacteriophages based on the whole-genome sequence, made with VICTOR. GenBank accession numbers: P2, AF063097.1, WPhi: AY135739.1; L-413C, AY251033.1; fiAA91-ss, KF322032.1; R18C, MN016939 (TIFF 1093 kb)Supplementary Fig. 4 Whole-genome-based phylogeny of P2-like bacteriophages and prophages made with VICTOR. Ten of the first prophages among the whole-genome-based BLAST hits with the highest BLAST score to R18C were chosen and included in the tree. GenBank accession numbers of the prophages are given in the figure, and those of the lytic phages are as follows: P2, AF063097.1; WPhi, AY135739.1; L-413C, AY251033.1; fiAA91-ss, KF322032.1; R18C, MN016939. (TIFF 2068 kb)Below is the link to the electronic supplementary material.


Glioblastoma multiforme (GBM) is the most common type of malignant gliomas, characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior. Disappointing results in the treatment of gliomas with surgery, radiation and chemotherapy have fueled a search for new therapeutic targets and treatment modalities. Here we report new approach towards RNA interference therapy of glioblastoma multiforme based on the magnetic nanoparticles delivery of the double-stranded RNA (dsRNA) with homological sequences to mRNA of tenascin-C (TN-C), named ATN-RNA. The obtained nanocomposite consisted of polyethyleneimine (PEI) coated magnetic nanoparticles conjugated to the dsRNA show high efficiency in ATN-RNA delivery, resulting not only in significant TN-C expression level suppressesion, but also impairing the tumor cells migration. Moreover, synthesized nanomaterials show high contrast properties in magnetic resonance imaging (MRI) and low cytotoxicity combining with lack of induction of interferon response. We believe that the present work is a successful combination of effective, functional, non-immunostimulatory dsRNA delivery system based on magnetic nanoparticles with high potential for further application in GBM therapy. Although accounting for less than 2% of adult cancers, gliomas are the most common form of malignant primary brain tumor in adults. Glioblasin vitro method can cleave the targeted mRNA and silenced the gene of interest expression.[Among recently developed new methods towards GBM treatment, a lot of attention has been drawn to gene therapy.,7,8 One pression.,11 We usIn the last decade, nanotechnology has been widely applied in synthesis of nanoscale materials with main focus on cancer therapy.,13,14 EsIn the paper, we demonstrate application of magnetic nanoparticles as a multifunctional carrier for a double-stranded RNA with contrast properties in MRI. We used a previously reported ATN-RNA sequence shown to significantly reduce the expression level of TN-C. The conjugation of nanoparticles with ATN-RNA showed the more effective TN-C downregulation followed by the substantial impairment of the migration properties of cancer cells regarding the routinely used transfection agent. Furthermore, obtained nanocomposites and their complexes with ATN-RNA did not show the toxic properties and consequently, they do not stimulate the interferon induction and innate immune response. Therefore, our results shed new light on application of multifunctional magnetic nanoparticles in gene therapy of GBM utilizing RNAi approach and point promising candidate for further studies.−1) was used.All reagents and solvents were of reagent-grade quality. For all experiments, Milli-Q deionized water (resistivity 18 MΩ×cm3·6H2O (Sigma-Aldrich)  were mixed with FeCl2·4H2O (Sigma-Aldrich)  in water (5 mL) and degassed with N2. The mixture was heated up to 80°C followed by addition of 1 mL of working solution (0.5 g of 25-kDa branched polyethyleneimine (PEI-25Br), 250 μL of Capstone FS-65 fluorosurfactant (Du Pont) and 2.5 mL of NH4OH dissolved in 10 mL of Milli-Q water) and constant heating was maintained for 120 min. After cooling down the mixture of nanoparticles was washed with water (2 x 150 mL) and collected by an external magnet. Finally, the NPs were redispersed in 10 mL of water. The Mag@PEI concentration was determined spectrophotometrically in terms of the iron content in an aqueous suspension of the stock nanomaterial by complexation with 1,10-phenanthroline (Sigma-Aldrich) as described previously.[Magnetic nanoparticles coated with PEI (Mag@PEI) were synthesized according to a previously reported protocol with small modifications. Briefly, FeCl50 mg, 0. mmol in Nanoparticles conjugated with fluorescent dye ATTO 550 were prepared as described elsewhere  Briefly,2 relaxation experiment (at 18°C) we employed MEMS protocol. Each echo is acquired after an excitation pulse with an increasing echo time. The parameters of the data acquisition were as follows: field of view 15 mm (FOV), matrix size 128 × 128, Gaussian-shaped pulse with 2048 μs length. The echo time was set to 4 and 10 ms in two separate experiments. In total, 16 consecutive images with varied echo time were acquired for each sample. The initial analysis was performed in VnmrJ 4.2 revision software . The raw relaxation data points were collected from an average intensity obtained from circular Region of Interests (ROIs) and then analyzed using Origin 8.5 software  using simple single exponential decay function. In order to prepare MRI agarose-based phantom, nanoparticles were suspended in a hot 2% agarose solution. The hot solution was finally transferred to 10 mm plastic vials and left for full agarose gelation.For transmission electron microscopy (TEM) a small amount of the sample was placed on a copper measuring grid (Formvar/Carbon 200 Mesh made by TedPella (USA)) after 5 minutes of sonication in deionized water. Then, the sample was dried in a vacuum desiccator for 24 hours. TEM images were recorded on a JEM-1400 microscope made by JEOL (Japan) at an accelerating voltage of 120 kV. The ImageJ software  was used to process TEM micrographs in order to analyze the size of nanoparticles. Magnetic measurements were performed on SQUID magnetometer at 5 K and 300 K. FT-IR spectra were recorded on Bruker Tensor 27 spectrometer in KBr pallets. Multiple light scattering (MLS) measurements were performed on Turbiscan Lab produced by Formulaction SA in 10 mL vials. Zeta (ζ) potential measurements were carried out using Zetasizer Nano-ZS ZEN 3600 produced by Malvern Instruments Ltd (UK). The experimental setup for magnetic resonance imaging studies consisted of an Agilent 9.4 T MRI preclinical scanner equipped with a 120 mm gradient coil (2 mT/m/A) and 30 mm millipede coil. In order to perform a quantitative Tin vitro as it has been already published.[ATN-RNA was synthesized ublished. ATN DNA Sequence of ATN-RNA 5’ CAAGCGACAGAGUGGGGUGAACGCCACCCUGCCAGAAGAGAACCAGCCAGUGGUGUUUAACCACGUUUACAACAUCAAGCUGCCAGUGGGAUCCCAGUGUUCGGUGGAUCUGGAGUCAGCCAGUCCCCUCUUCUGGACCGGGCGGAAGUCUCGGGCGCU 3’3’ GUUCGCUGUCUCACCCCACUUGCGGUGGGACGGUCUUCUCUUGGUCGGUCACCACAAAUUGGUGCAAAUGUUGUAGUUCGACGGUCACCCUAGGGUCACAAGCCACCUAGACCUCAGUCGGUCACCCCUCUUCUGGACCGGGCGGAAGUCUCGGGCGCU 5’The binding ability of ATN-RNA to Mag@PEI was performed by the gel retardation assay and UV-Vis spectrophotometry. To prepare Mag@PEI/ATN-RNA complexes, 1 μg of ATN-RNA was mixed with Mag@PEI with a series of Fe weight ratios  in the nuclease-free water, and incubated for 30 min at RT, allowing for sufficient binding of dsRNA molecules with the Mag@PEI. The Mag@PEI/ATN-RNA complexes were loaded onto 1% agarose gel for electrophoresis in TAE buffer at a constant voltage of 70 V for 30 min to visualize the ATN-RNA bands using a Digital Imaging and Analysis System II (Serva). SimplySafe fluorescent stain was used for detecting RNA in agarose gel. Meanwhile, the formed Mag@PEI/RNA complexes suspension was centrifuged  and the prepared supernatant was analyzed spectrophotometrically  at A260 nm to reveal ATN-RNA content.2.The study was performed on a human U-118 MG cell line (ATTC) derived from a glioblastoma multiforme. Adherent cell growth was carried out in a Dulbecco's Modified Eagle Medium  High Glucose supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin antibiotic (Sigma-Aldrich). Cells were grown at 37°C in a 95% humidified chamber with 5% COATN-RNA transfection was performed with Mag@PEI or Lipofectamine (Invitrogen) as a carrier. The procedure was carried out on the day-old cell culture seeded on 12 or 96-well plates in the supplemented medium. In a case of the 12-well plate, 150,000 cells were seeded in 1 mL of medium, for 96-well it was 10,000 cells in 200 μL. Once the cells have reached 75–90% confluency, the medium was removed, cells were washed with phosphate-buffered saline  buffer and an unsupplemented culture medium in the amount reduced by the volume of the transfection mixture was added. Transfection complexes containing Mag@PEI nanoparticles were prepared by mixing Mag@PEI suspension (100 μg Fe/mL) with ATN-RNA solution (100 ng/μL) in serum and supplement-free DMEM with an iron-to-RNA ratio of 3:1 (w/w). The mixture was further incubated at RT for 30 min to allow the components to assemble. After this time, the volumes of the prepared complexes corresponding to the proper ATN-RNA final concentration  were transferred to the wells.Lipofection was performed according to the manufacturer's recommendations. Two separate mixtures, OptiMEM (Gibco) with Lipofectamine and OptiMEM with ATN-RNA, were incubated for 5 min at the room temperature. After that, both solutions were combined. The prepared reaction mixture was incubated 20 min at the room temperature and then transferred to the appropriate wells. In addition, cells treated with Lipofectamine or Mag@PEI carriers only were used as controls.4 cells/well) were plated onto an 8-well Nunc Lab-Tek Chamber Slide (Thermo Fisher Scientific) and cultured for 24 h. Next, 50 μL of Mag@PEI/ATN-complexes (100 nM) was added and incubated with the cells at 37°C for 24 h.To track the magnetic complexes in the cell, ATN-RNA was fluorescently labelled with fluorescein, according to the manufacturer’s protocol . The final concentration of the fluorescein containing RNA was quantified on a spectrophotometer (NanoDrop 2000). The fluorescently labelled Mag@PEI nanoparticles were then used to assemble complexes with fluorescently labelled ATN-RNA, as described in the previous section. U-118 cells  in phosphate-buffered saline (PBS) for 15 min. The cell membranes were stained with ConcanavalinA-FITC (Life Technologies) at a concentration of 50 μg/mL, the F-actin fibers were stained with Oregon Green 488 phalloidin (Molecular Probes) at a concentration of 165 nM and the cell nuclei were stained with Hoechst 33342 (Molecular Probes) at a concentration of 8 μM. Cells were imaged using a confocal laser scanning microscope . Image acquisition and analysis were performed with a 60x objective, a 1.4 oil immersion lens and FV10-ASW software (Olympus). Images of the Mag@PEI were visualized using 559 nm excitation and 570–590 nm emission filters, whereas ATN-RNA was visualized using 488 nm excitation and 495–545 nm emission filters. To visualize the cell membranes or cytoskeleton, 488 nm excitation and 495–545 nm emission filters were applied. The Hoechst fluorescence was detected using 405 nm excitation source and 425–475 nm emission filters. The 3D-scan of the sample was performed using Z-stack mode measurements and analyzed with Imaris software (Bitplane).The WST-1 cell proliferation assay, as well as fluorescent cell viability assay, were carried out to assess the cytotoxicity of the nanoparticles and complexes with ATN-RNA.In WST-1 assay (Takara), U-118 cells were seeded at the density of 10,000 cells per well in the 96-well plate. After 24 hours, medium containing an increasing concentration of tested Mag@PEI/ATN-RNA complexes (in terms of RNA concentration) was added to each well and the cells were incubated for 24 h. After incubation, 10 μL of the WST-1 Cell Proliferation Reagent was added to each well and incubated for 4 hours. After this time, the absorbance at 450 nm (reference wavelength 620 nm) was recorded against the background control, using a multiwell plate reader  and the cell viability was expressed as the respiration activity normalized to untreated cells.In Live/Dead assay, the U-118 cells were seeded in black polystyrene 96-wells flat bottom plate with the transparent bottom (Greiner Bio-One GmbH) at densities of 10 000 cells per well. After 24 h, medium containing an increasing concentration of tested Mag@PEI/ATN-RNA complexes (in terms of RNA concentration) was added to each well. Following 24 h exposure to the complexes, cells were incubated with 2 μM calcein AM (Thermo Fisher Scientific), 2 μM ethidium homodimer-1 (Thermo Fisher Scientific) and 8 μM Hoechst 33342 containing PBS (100 μL/well) during 30 minutes at 37°C. Finally, the cells were analyzed with the IN Cell Analyzer 2000 . Viable cells were imaged using the FITC/FITC excitation/emission filters while for the dead cells the TexasRed/TexasRed ex/em filter combination was applied. DAPI/DAPI was applied to detect the Hoechst 33342 blue signal. A minimum of 20 fields was imaged per well with a 20x magnification. Analysis of the collected images was performed with the IN Cell Developer Toolbox software  using in-house developed protocol. First, the total cell number was retrieved from the DAPI images by means of defining and counting the nuclei. Subsequently, the number of viable cells from the FITC images and the number of dead cells from the TexasRed images were determined.Total RNA was extracted from cell lines with TRIZOL solution (Invitrogen) according to the Chomczynski's procedure. Obtainedhttps://qpcr.probefinder.com/organism.jsp). Primers sequences are given in The quantitative reverse transcriptase real-time PCR (qRT-PCR) was performed with the LightCycler480 using LightCycler480 Probes Master and Universal Probe Library (UPL) Set for Human (Roche). Primers with probes were designed by the Universal Probe Library Assay Design Center (Transfected cells were lysed by sonication. 150 μg and 10 μg of denaturated protein extracts were separated on 12% and 15% SDS-PAGE (SDS-polyacrylamide gel electrophoresis) for tenascin-C and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) detection respectively. Spectra Multicolor Broad Range Protein Ladder (Thermo Fisher Scientific) was used as the size marker. Subsequently the wet transfer onto PVDF membrane with 0.45 μm pore size  was performed in transfer buffer . The membranes were placed in the SNAP i.d. 2.0 apparatus (EMD Milipore), where it was blocked with a 0.5% solution of skimmed milk in PBS. For detection of tenascin-C the TNC polyclonal antibody  was used, while GAPDH was detected using monoclonal antibody . Antibodies were diluted 1:500 in 3% BSA (Sigma-Aldrich). Membranes were incubated with the primary antibody for 10 min or overnight,. Afterwards, secondary anti-rabbit IgG and anti-mouse IgG antibodies conjugated with horseradish peroxidase (HRP) (Sigma-Aldrich) were used. Detection of a protein was carried out with WesternBright Sirius Chemiluminescent Detection Kit (Advansta). Intensity of individual bands was analyzed qualitatively by Multi Gauge ver. 2.0 (Fujifilm).μL of medium. Next, the impedance was measured at 15-minute intervals for 72 h. The transfection was performed 24 h after cell seeding. For migration assays, the 16-well plate consisting of two chambers with a microporous polyethylene terephthalate (PET) membrane containing microfabricated gold electrode arrays on the bottom side of the membrane between them (CIM-plate) was used. 10,000 cells per well were seeded in an upper chamber in unsupplemented medium and moved towards a bottom chamber filled with supplemented medium. The impedance was measured at 15-minute intervals for 96 h. The seeded cells were transfected 24 h before experiment. Obtained cell index (CI) values were entered to the GraphPad Prism ver. 5.1  software and used to calculate half maximal inhibitory concentrations (IC50) of effecting cell proliferation. In case of transwell migration assay, obtained CI values from each experimental condition were plotted against time, fitted to four-parameter logistic non-linear regression model and the half-time of migration  was calculated.Real-time cell proliferation and migration were monitored by measuring changes in electrical impedance using xCELLigence RTCA DP system (ACEA Biosciences). In the first step, the background impedance of a culture medium in plates was measured. In proliferation experiments, the 16-well plate with incorporated sensor electrode arrays (E-plate) were seeded with 10,000 cells per well in a final volume of 200 Cell migration scratch assay was performed on 12-well plate, with 200,000 cells per well seeded in a supplemented medium. Cells were grown for 24 h under optimal growth conditions. Next, the transfection was performed and the plate was incubated for another 24 h. After this time the medium was replaced and "wound" was created in the monolayer of cells covering the well. The scar effect was obtained by scraping cells in a straight line using a 200 μL tip. From that moment, images of the cultured cells at 12 h intervals were taken for 48 h using a fluorescence microscope at 5x magnification. The analysis of the degree of scarring of individual "wounds" was carried out by computer using the Tscratch program (CSElab).Experimental results were subjected to statistical evaluation using GraphPad Prism ver. 5.1. Values presented are an average of three biological along with three technical replicates as mean values ± standard error of the mean (SEM). Differences between mean values of tested samples and controls were compared with the analysis of variance (ANOVA) extended by Tukey or Bonferroni post-hoc tests. Statistically significant results were obtained at the level of: * for p<0.05; ** for p<0.01; *** for p<0.001; no statistical significance for p≥ 0.05.via previously reported procedure in one step protocol from iron III and II chlorides in the molar ratio 2:1 in the presence of polyethyleneimine and surfactant.[In the first step , the PEIrfactant.-1 which was assigned Fe-O bond. Characteristic signals at 1080 cm-1 and 1330cm-1 were attributed to C-N stretching vibration from the PEI. The peak observed at 1575cm-1 corresponds to N-H bending vibration from amine moieties present in polyethylenimine structure. Moreover, the signals from CH2 groups were observed at 2820 cm-1 and 2950 cm-1, respectively. Thus, FT-IR proved attachment of PEI at the surface of magnetic nanoparticles , Resovist (186 mM-1s-1) and Combidex (65 mM-1s-1).[Furthermore, contrast properties of synthesized magnetic nanocarriers and their potential application in magnetic resonance imaging were assessed. In order to avoid drift of magnetic nanomaterial in the high magnetic field, the suspensions of nanoparticles in an agarose gel (2 mg/mL) were prepared, according to recently reported protocol. The spinmM-1s-1). Thus, ouThe characterized nanocarrier was further used to determine its binding capability towards ATN-RNA. In this experiment, ATN RNA was mixed with Mag@PEI NPs for 30 minutes at different weight ratios varied from 1:1 to 10:1. We expected that negatively charged ATN-RNA will interact with positively charged Mag@PEI NPs resulting in polyplexes formation. This type of structure is often postulated in literature when PEI is used as a carrier for nucleic acids delivery.[The DLS measurements were performed to investigate the size of synthesized nanoparticles and prepared complexes in water. The Mag@PEI NPs had the hydrodynamic diameter of ~150 nm while the hydrodynamic diameter of Mag@PEI/ATN-RNA complexes increased slightly to around 200 nm after nucleic acid binding .The uptake of Mag@PEI/ATN-RNA complexes by U-118 cells was investigated by means of confocal microscopy. First, Mag@PEI nanoparticles were labelled with ATTO 550 dye. As illustrated in A colocalization analysis was also performed to further demonstrate transfection of Mag@PEI/ATN-RNA complexes into glioblastoma cells. As shown in Cytotoxicity of both Mag@PEI NPs and polyplexes bearing ATN-RNA on GBM U-118 cells were determined by two cytotoxicity tests, namely WST-1 and LIVE/DEAD assays. In the first step, the toxicity of Mag@PEI NPs without bounded ATN-RNA at the concentrations of NPs corresponding to the concentrations used in further transfection experiments  and trigger an interferon (IFN) response.[Taking into account the possible use of nanoparticles as the therapeutic tool in GBM treatment, the analysis of expression level of the genes involved in innate immune response was performed. We examined whether our nanoparticles delivery system would elicit an innate immune response invasion. For examresponse. Additionresponse. Thus, thThe highest activation with ATN-RNA both with lipofectamine and using Mag@PEI nanocarrier was observed for TLR3 and INFγ genes. The ATN-RNA delivered with lipofectamine caused the TLR3 gene expression activation at the level from 16% to 37%, starting from the 25 nM ATN-RNA concentration (p<0.005). The Mag@PEI NPs used as the ATN-RNA carrier resulted in the lowest activation of the TLR3 gene expression with the observed 8–15% level, for 25–100 nM ATN-RNA concentrations (p<0.01).INFγ gene was significantly upregulated both by the lipofectamine and Mag@PEI NPs. However, with the same ATN-RNA concentration, the gene expression was elevated at the higher level by the lipofectamine-mediated ATN-RNA delivery. With the already lowest concentration, we observed 10–12% overexpression by the lipofection and Mag@PEI NPs (p<0.01). However, starting from the 25 nM ATN-RNA concentrations we noticed the growing differences with the effect of lipofectamine versus Mag@PEI NPs. With this concentration, we observed the INFγ gene overexpression of about 27 and 42% for Mag@PEI NPs and lipofection, respectively. The highest concentrations caused –for 50 nM: 28 and 63%, for 100 nM: 44 and 75% in case of use of Mag@PEI NPs and lipofection, respectively.To achieve down-regulation of TN-C expression in U-118 glioblastoma cell line, lipofection along with Mag@PEI NPs with various concentration of ATN-RNA– 10, 25, 50 and 100 nM was performed. 24 hours after transfection the expression level of tenascin-C was examined by qRT-PCR analyses. The downregulation of TN-C mRNA expression was observed compared to controls treated with the transfection agent or with scrambled RNA. The level of TN-C was decreased from 2% at a concentration of 10 nM ATN-RNA to 34% for cells treated with 100 nM ATN-RNA in comparison to the untreated cells (p< 0.005) in case of samples where ATN-RNA was delivered with lipofectamine . At the The qRT-PCR analysis was also supported by the direct analysis of the protein expression level. The Western blot detection reveled again with the more significant drop of the TN-C expression caused by the nanoparticles-mediated ATN-RNA delivery. We observed already 40% decrease of the protein expression with the 10 nM ATN-RNA. The highest concentration used caused the dramatic drop of the protein expression measured as the 85% of the decrease. The lipofectamine mediated ATN-RNA delivery at the same time caused only about 40% decrease of the expression level with ATN-RNA concentrations 50 and 100 nM . These oThese results clearly support the idea of nanoparticles mediated delivery in order to achieve the significant biological effect along with the lowest concentration of therapeutic agent. The Mag@PEI mediated ATN-RNA delivery was already efficient with the lowest- 10 nM concentration- 20% of TNC downregulation, while the lipofectamine at the same time was not able yet to deliver the RNA. During the whole experiment, we observed about 30–50% higher efficiency of nanomediated delivery compared to the lipofectamine-driven one.In order to investigate the delivery efficacy of the carriers, we measured the cells proliferation rate. U-118 glioblastoma cell line was treated with Mag@PEI NPs and Lipofectamine combined with ATN-RNA followed by the real-time cell proliferation assay with the xCELLigence system. The cell's ability for proliferation was measured for 72 h. We noticed ATN-RNA concentration-dependent decrease of proliferation rate both for Lipofectamine carrier with the significant decrease of proliferation with 100 nM–ATN-RNA concentration. The proliferation rate dropped for ATN-RNA delivered with the cationic carrier from 40–61% . HoweverTo get more insight into the down-regulation of TN-C expression by ATN-RNA on the mobility of glioblastoma cancer cells, real-time measurements of migration was carried out. In this approach, U-118 cells were treated again with Mag@PEI NPs and Lipofectamine and ATN-RNA and allowed to migrate through microporous polyethylene terephthalate (PET) membrane towards chemoattractant. The number of cells that crossed the membrane was continuously recorded.We found that down-regulation of TN-C expression by ATN-RNA delivered by Mag@PEI NPs significantly impaired the cell migration in GBM cells . The resNotably, the infection did not change substantially also the viability of the cells, what was observed in terms of lipofection  Cytotoxicity SRB of Mag@PEI for various nanoparticles concertation,  Schratch test. Destabilization kinetic of Mag@PEI recorder by Turbiscan apparatus over 24 h,  Representative high-content images of U-118 cells exposed to Mag@PEI nanoparticles at various concentrations. 10% DMSO was used as a positive control. Images were obtained using different filters to detect nuclei (DAPI), live cells (FITC), and dead cells (TexasRed). The scale bars denote 100 (DOCX)Click here for additional data file.S1 Video(AVI)Click here for additional data file.


Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about  Drosophila melanogaster, a popular model organism to study centromeric chromatin, CENP-A is incorporated during mitotic metaphase in S2 cells or anaphase in early embryos , which represents the most conserved part of the protein, while the N- and C-terminal regions show the strongest degree of divergence from H3 and are not conserved between CENP-A proteins from different species . CanonicDrosophila melanogaster CENP-A. We found S20, S75 and S77 to be phosphorylated and K105 to be acetylated in pre-nucleosomal CENP-A  containing 10% FBS (Gibco) and 1% Penicillin/Streptomycin. Drosophila S2 cell lines were engineered to contain stably integrated constructs for inducible expression of SNAP (S), Strep-Flag (SF) and GFP-tagged wt and mutant CENP-A fusion proteins. To this end, S2 cells were transfected with expression constructs made with a modified pMT that contains a puromycin resistance cassette  CENP-A fused at its N-terminus to a SNAP, Strep-Flag or GFP epitope tag was described previously . These cpSPNDDDTAFR) by Davids Biotechnology GmbH . Final sera were affinity purified against the immunizing peptide and depleted for non-phospho-specific antibodies by purification against the peptide in its non-phosphorylated form (Davids Biotechnology GmbH). Specificity tests were performed by ELISA and by western blotting. Detailed procedure in Supplementary Information.Antibodies specifically recognizing phosphorylated S20 in CENP-A were raised in rabbits against a peptide spanning amino acids 15–29 of CENP-A phosphorylated at S20  followed by elution for 1 h with rotation in 100 μl TBS buffer  containing 100 μg/ml Flag peptide .Anti-FLAG M2 Magnetic Beads  were equilibrated in the corresponding extract buffer and incubated with the protein extracts at ∼30 μl beads per 10 ml cytoplasmic or 5 ml nuclear extract overnight at 4°C on a rotating wheel. Subsequently, beads were washed 4 times with 1 ml Wash Buffer , rabbit α-H3 , mouse α-tubulin , rabbit α-CENP-A-S20p . Secondary anti-rabbit and anti-mouse IgG antibodies (Sigma) coupled to horseradish peroxidase (HRP) were used at 1:10 000. Raw images of western blots from Figures Protein samples were separated on 10–12% SDS-PAGE gels and blotted to nitrocellulose membrane. Antibody incubation and detection were performed as described in Supplementary Information. The following antibodies were used: mouse α-Flag , rabbit α S2 cells were settled down on cover slips for 30 min and fixed with 3.7% paraformaldehyde in PBS/0.3% Triton-X100 for 12 min. Cells were blocked in 5% bovine serum albumin (BSA) in PBS, washed and incubated with primary antibody diluted in 5% BSA/PBS, followed by three washing steps and secondary antibody incubation. DNA was stained with 5 μg/ml DAPI (Roche) in PBS followed by three washes in PBS. Cells were mounted in Vectashield and imaged on a Leica TCS SP5 instrument. To analyze mitotic chromosomes of S2 cells, cells were treated with 75 mM KCl before fixation and antibody incubation. The following primary antibodies were used: α-CENP-A , α-CENP-C , α-CENP-A-S20p , α-tubulin . Experimental details are described in Supplementary Information.® TMR Star (NEB) for 30 min. Non-reacted TMR Star was washed out by incubating the cells in fresh medium for 10 min before removing the medium and repeating the washing steps three times. Cells were settled on polylysine-coated coverslips and fixed in 3.7% paraformaldehyde/0.3% Triton-X100 in PBS for 12 min, nuclei were counterstained with 5 μg/ml DAPI and finally mounted in 8 μl Vectashield (Vector). Confocal fluorescence microscopy was performed on a Leica TCS SP5 instrument.To determine steady-state levels of SNAP-tagged CENP-A wt and mutant proteins at centromeres, cells were incubated with 3 μM SNAP-Cell6 cells/ml in 2 ml fresh culture medium in six-well plates coupled with gentle shaking. Cell concentrations were measured every 24 h for 3 days. Doubling times were calculated using the exponential growth function of Prism 7.0 (Graphpad), and statistical significance was determined by one-way ANOVA.Cells were counted by TC20™ cell counter (Bio-Rad) and diluted to 1 × 104 for 8 h. Then CuSO4 containing medium was removed, cells were washed 3 times for 10 min with fresh medium and left to grow for an additional 16 h (chase). Samples were collected after the expression pulse and after the chase period and processed for confocal fluorescence microscopy. GFP-CENP-A_wt expressing cells after 8 h of induction were used to define a threshold for overexpression that was applied to all images from all samples using ImageJ. The numbers of overexpressing cells relative to total cells  were calculated and statistical differences between the 8 and 16 h time points were calculated by unpaired t-test (Prism).GFP-CENP-A S2 cells were subjected to an expression pulse by addition of 0.5 mM CuSO4 for 16 h. Cells were allowed to attach to coverslips for 10 min, and subsequently overlaid with agarose sheets according to the method by  were imaged on a Leica TCS SP5 confocal fluorescence microscope with 60–85 and 10–20 z stacks, respectively, of 0.13 μm z-sections using a 63x/1.40 oil objective. Mitotic chromosomes were imaged on a Leica TCS SP8 confocal microscope with 30–40 z stacks of 0.13 μM using a 100×/1.40 oil objective. 3D images were reconstructed and analyzed by Imaris v5.1. Using the Arena function of Imaris, one image of the CENP-A wild type group was selected to define cells, nuclei and centromeric spots and those settings were applied to all images of the same experiment to ensure identical image processing. A total of 900–1713 centromeres per experiment were analyzed in this manner. For comparison of the intensity of S-CENP-A in the different cell lines, all spot intensities within one nucleus were summarized and plotted. Mean ± SEM TMR intensities per nucleus were calculated and statistical significance was determined by Mann-Whitney-test using Prism 7.0 (Graphpad) software.4 for 16 h. Then, cycloheximide  was added to a final concentration of 40 μg/ml for 4 h to inhibit protein synthesis. Subsequently, aliquots of ∼109 cells were removed from the flask at every 2 h and collected by centrifugation at 1000 × g for 5 min followed by a wash in PBS. WCE was prepared as described in Supplementary Information. For experiments testing the effect of the proteasome on CENP-A degradation, MG132 (Sigma) was added for 5 h to a final concentration of 20 μM; Protein signal intensities were analyzed using ImageJ software, and half-life and statistical calculations were done in Prism 7.0.S2 cells were grown in 50 ml round-bottom flasks containing 15 ml culture medium with gentle stirring. Overexpression of SF-tagged wt and S20-mutant CENP-A was induced by addition of 0.5 mM CuSOIn vitro kinase assays with CKII were performed in a reaction volume of 25 μl for 1 h at 30°C. 1000 units of recombinant CKII (NEB) were incubated in 1× CKII reaction buffer (NEB) with 200 μM ATP, 0.5 μl gamma-32P-ATP  and 10 μg of peptide p20 (ANNSKSPNDDDTAFR) spanning amino acids 15–29 of CENP-A. As negative controls, reactions containing a peptide phosphorylated at S20, p20P (ANNSKpSPNDDDTAFR) or reactions without peptide or CKII, respectively, were performed. Reactions were then spotted onto P81 filter paper (Whatman), filters were washed three times for 10 min each in 500 ml 0.75% phosphoric acid, once in 200 ml acetone for 5 min and dried at RT. 32P incorporation was measured in a scintillation counter (HITACHI AccuFLEX LSC-8000). In addition, reaction products were analyzed by nano-LC mass spectrometry.Drosophila kinases, Ppa and CENP-A were selected using the algorithm at DRSC/TRiP Functional Genomics Resources (https://fgr.hms.harvard.edu/fly-cell-based-rnai). In addition, nucleotide region 48–573 of the coding sequence of the bacterial Tet repressor (TetR) was chosen as a negative control. Generation of double-stranded RNA probes and RNAi treatment were performed exactly as described previously . To determine the percentage of mitotic cells in wt and mutant cell lines, 5 × 105 cells each were fixed in 1 ml ice-cold 70% ethanol. Cells were washed and incubated with 1% BSA/PBS containing Alexa Fluor® 488-coupled α-Histone H3 Phospho (Ser10) antibody  at room temperature for 60 min. DNA was stained by addition of 5 μg/ml DAPI. For live cell sorting, 3.2 × 106 cells were stained with 5 μM Hoechst prior to sorting according to their DNA content. Flow cytometric analysis or cell sorting of single cell suspensions of S2 cells were performed on an LSR Fortessa or a FACS-Aria-III, respectively, (both BD) instrument and analyzed using FlowJo® v10 software.To determine cell cycle profiles, 10Drosophila S2 cells  and one acetylation site (K105ac) in CENP-A preparations from cytoplasmic and nuclear extracts of S2 cells , yet theS2 cells  for analS2 cells . BecauseS2 cells  as well S2 cells , we focuS2 cells  and perfS2 cells . LikewisS2 cells . When weS2 cells  the relaS2 cells . These rS2 cells  suggestiS2 cells . Consists Figure , F.in vitro kinase assay using recombinant CKII and a peptide spanning S20 of CENP-A. CKII incorporated significant amounts of radiolabeled phosphate into the S20 peptide, whereas a pre-phosphorylated S20 peptide was not modified . Togeth4-inducible metallothioneine promoter. In the absence of CuSO4 induction, transgenic CENP-A was expressed at near physiological levels, restricting its incorporation to centromeres as demonstrated by colocalization with the centromeric protein CENP-C  in which S20 was mutated to alanine (S20A) to block phosphorylation or to aspartate (S20D) to mimic the phosphorylated state . All tran CENP-C . We firsn CENP-C , B. Becan CENP-C . Althougs CENP-A  but did s CENP-A  and stros CENP-A . Measuris CENP-A , C. Thes4 induction revealed that S20A and to a lesser extent S20D-expressing cell lines showed reduced proliferation rates compared to S-CENP-A_wt expressing cells  S20p is involved in CENP-A loading and maintenance at the centromere. This idea is supported by the enrichment in chromatin of phosphorylated CENP-A and of the phosphomimetic S20D mutant protein but depletion from chromatin of the non-phosphorylatable CENP-A_S20A. (ii) S20p is required for the degradation of soluble CENP-A. Reduced amounts of phosphomimetic CENP-A_S20D but increased levels of CENP-A_S20A in the soluble protein pool point towards this possibility. To determine which of these possibilities holds true, we examined the potential of S20p to affect CENP-A turn-over.4 followed by treatment of cells with cycloheximide (CHX) to inhibit new protein synthesis, and analysis of CENP-A protein abundance in whole cell extracts by western blot with α-Flag antibodies at different time points after addition of CHX , one of multiple adaptor subunits of E3-ubiquitin ligase SCF. Ppa was shown to interact with Drosophila CENP-A, and CENP-A gained increased stability when Ppa was knocked-down by RNAi , while SF-CENP-A_S20A half-life was largely unaffected . ImportPpa.Altogether, our data demonstrate that the phosphorylation of S20 by CKII sensitizes CENP-A for degradation in a manner that is dependent on the ubiquitin ligase SCFIt is well established that upon overexpression, CENP-A can be incorporated at ectopic sites which may lead to the formation of neocentromeres ,56,62,634 induction. In this way, CENP-A located at centromeres should constitute the predominant form  might be cleared more efficiently from certain sites than from others, which would allow the accumulation of CENP-C on the latter and result in the observed focal staining of CENP-C. To test this hypothesis, we first analyzed the stability of centromeric CENP-A by performing Western blot-based degradation assays of chromatin-bound CENP-A in the absence of CuSOant form ,65. The ant form , B. We aant form . In contant form .4 was washed out, and cells were collected for fluorescence microscopy right after CuSO4 removal and after additional 16 h cultivation (chase period). Comparison of GFP signals revealed a strong decrease of the fraction of cells showing diffuse nuclear CENP-A_S20D staining, intermediate decrease for CENP-A_wt and almost no decrease for the S20A mutant (Figure Next, we performed pulse-chase expression experiments to monitor protein clearance under overexpression conditions. To this end, GFP-tagged wt and mutant proteins were induced for eight hours, then CuSOt Figure . To moret Figure . When wet Figure . FRAP ant Figure , F corrot Figure , G. Moret Figure . These rIn summary, S20 phosphorylation appears to be necessary for fast removal of aberrantly incorporated CENP-A by proteasomal degradation.Ppa, and third, it controls the amount of aberrantly incorporated CENP-A in chromatin outside of centromeres.In this study, we examined the functional consequences of CENP-A phosphorylation at the N-terminally located serine 20. Our analyses revealed that though S20 phosphorylation is present in the soluble protein pool, it is particularly enriched in chromatin-bound CENP-A. We identified CKII as the enzyme responsible for S20 phosphorylation. Notably, we found three major functions of S20 phosphorylation: First, it is required for efficient loading/maintenance of CENP-A to the chromatin, second, it regulates the levels of soluble CENP-A by controlling proteolysis upstream of the ubiquitin ligase SCFCse4 was shown to be ubiquitylated by the E3 ligase Psh1 thereby preventing its ectopic incorporation (Drosophila, the ubiquitin ligase adapter F-box protein Ppa is required for ubiquitylation and proteolytic clearance of CENP-A from ectopic sites (Cdh1 complex has been identified as another E3 ligase regulating CENP-A protein degradation in the fly (The tight regulation of CENP-A abundance is a recurrent strategy by which different organisms ensure that CENP-A assembly is restricted to the centromere. Proteolysis of excess amounts of CENP-A by the proteasome has been detected in yeast and flies ,67 but hporation ,69. Likeic sites ,67. Very the fly .Ppa. Moreno-Moreno et al. have shown that Ppa interacts with both the histone fold domain of CENP-A and its N-terminal region (Ppa. Notably, when CENP-A is in complex with CAL1, SCFPpa cannot induce degradation, while APC/CCdh1 can modify CENP-A regardless of its presence in the CAL1 complex (Ppa. Thus, we conclude that S20 phosphorylation renders CENP-A a substrate for ubiquitylation by SCFPpa, thereby regulating the abundance of free pre-nucleosomal CENP-A. The role for S20 phosphorylation in protein turn-over is further supported by the fact that knock-down of CKII, which removes S20 phosphorylation, also leads to a stabilization of CENP-A (Figure Our studies showed that soluble CENP-A becomes substantially more labile when S20 is mutated to the phosphomimetic aspartate, while it displays increased stability compared to wild-type CENP-A when S20 is mutated to the non-phosphorylatable alanine Figure . Knock-dl region . Thus, i complex . Our deg complex  suggestiA Figure . CKII, wA Figure .et al. in Drosophila larvae (In contrast to the results obtained for soluble and therefore pre-nucleosomal CENP-A, incorporation into centromeric chromatin seemed to protect the phosphomimetic CENP-A_S20D from proteolysis. This is on one hand reflected in the relative higher abundance of chromatin-bound CENP-A_S20D versus endogenous CENP-A under non-overexpression conditions Figure  and on ta larvae . Centroma larvae , F suggea larvae , F, whica larvae . Our obsa larvae , F may sIn addition to the observed effects of S20 phosphorylation on CENP-A degradation, our results also suggest that it affects CENP-A loading to the chromatin. This conclusion is supported by the observations that the non-phosphorylatable CENP-A_S20A protein shows significantly lower centromeric steady-state levels in non-overexpression conditions Figure , and thaDrosophila. Regulation of the phosphorylation state of S20, e.g. by modulating CKII activity or by the action of an as yet unknown phosphatase, may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.In summary, our study provides multiple lines of evidence for an important role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in gkz809_Supplemental_FileClick here for additional data file.