Viral neuraminidase inhibitors show limited efficacy in mice infected with H7N9 influenza A viruses isolated from humans. Although baloxavir marboxil protected mice from lethal challenge infection with a low pathogenic avian influenza H7N9 virus isolated from a human, its efficacy in mice infected with a recent highly pathogenic version of H7N9 human isolates is unknown. Here, we examined the efficacy of baloxavir marboxil in mice infected with a highly pathogenic human H7N9 virus, A/Guangdong/17SF003/2016. Treatment of infected mice with a single 1.5 mg/kg dose of baloxavir marboxil protected mice from the highly pathogenic human H7N9 virus infection as effectively as oseltamivir treatment at 50 mg/kg twice a day for five days. Daily treatment for five days at 15 or 50 mg/kg of baloxavir marboxil showed superior therapeutic efficacy, largely preventing virus replication in respiratory organs. These results indicate that baloxavir marboxil is a valuable candidate treatment for human patients suffering from highly pathogenic H7N9 virus infection. Treatment of zoonotic influenza with neuraminidase (NA) inhibitors that target the viral sialidase activity of NA is thought to be effective. However, the efficacy of these inhibitors against H7N9 viruses is limited in mice ,2. In 20Human H7N9 virus infections were first reported in March 2013  and the To evaluate the efficacy of baloxavir marboxil in vivo infection with the H7N9 virus, baloxavir marboxil was orally administered to mice at 5 and 50 mg/kg twice a day for five days and was shown to have completely protected them from lethal challenge infection with a low pathogenic avian H7N9 human isolate, A/Anhui/1/2013 . Highly 10 PFU/mL at 24 and 48 h post infection  cells were infected with A/Guangdong/17SF003/2016 (H7N9) virus at a multiplicity of infection (MOI) of 0.001 and incubated for 24 or 48 h with Eagle’s minimal essential medium (EMEM) supplemented with 0.3% BSA, 1 μg/mL trypsin, and 0, 40, or 160 nM baloxavir acid. Virus titers were measured by means of plaque assays with MDCK cells. A/Guangdong/17SF003/2016 grew efficiently in MDCK cells in the absence of baloxavir marboxil, reaching a virus titer of approximately 7.8 lognfection . In contnfection . These r50; 104.3 PFU) of highly pathogenic A/Guangdong/17SF003/2016 (H7N9) possessing NA-294R (arginine at position 294 of NA indicates sensitive to NA inhibitors) [p = 0.009, log-rank test) but failed to protect them from the lethal challenge infection . These results show that a single dose of baloxavir marboxil with 15 mg/kg is sufficient to protect mice from infection with a highly pathogenic human H7N9 virus.Next, we assessed the efficacy of baloxavir marboxil in mice infected with the highly pathogenic human H7N9 virus. Six-week-old female mice  were anesthetized with isoflurane and intranasally infected with 10 mouse lethal dose 50 (MLDibitors) . Five innfection A. Oseltanfection A,B. In c50 of highly pathogenic A/Guangdong/17SF003/2016 virus during and after the various treatment regimens, we performed a standard plaque assay by use of MDCK cells. On day 3 post-infection, the virus titers in the nasal turbinates and lungs of mice treated with baloxavir marboxil were significantly decreased except for the virus titers in the lungs of mice treated with the single 1.5 mg/kg dose of baloxavir (To compare nasal turbinate and lung virus titers at three and six days post-infection of mice with 10 MLDaloxavir . A similaloxavir . These rEmergence of drug-resistant mutants after antiviral treatment is one of the major concerns for antiviral treatment . In factHere, we evaluated the in vivo therapeutic efficacy of baloxavir marboxil against a highly pathogenic human H7N9 virus. Baloxavir marboxil given as a single dose or as a 5-day treatment course improved the survival of mice infected with the highly pathogenic human H7N9 virus and suppressed its replication in the respiratory organs. Baloxavir marboxil also protected mice from a low pathogenic avian H7N9 human isolate, A/Anhui/1/2013 . Since t50 value by 10- to 50-fold [Treating with antivirals always encounters the potential problem of emergence of resistant mutants. Viruses resistant to baloxavir marboxil frequently emerged in patients within 3–5 days of treatment ,5 with t 50-fold ,5. In ouIn summary, baloxavir marboxil showed high therapeutic efficacy against a highly pathogenic human H7N9 virus. Since the risk of reemergence of H7N9 viruses persists, stockpiling baloxavir marboxil should be considered for pandemic preparation.


Arabidopsis pRb homolog RETINOBLASTOMA RELATED 1 (RBR1), similar to its animal counterparts, controls not only cell proliferation but is also implicated in developmental decisions, stress responses and maintenance of genome integrity. Although most functions of pRb-type proteins involve chromatin association, a genome-wide understanding of RBR1 binding sites in Arabidopsis is still missing. Here, we present a plant chromatin immunoprecipitation protocol optimized for genome-wide studies of indirectly DNA-bound proteins like RBR1. Our analysis revealed binding of Arabidopsis RBR1 to approximately 1000 genes and roughly 500 transposable elements, preferentially MITES. The RBR1-decorated genes broadly overlap with previously identified targets of two major transcription factors controlling the cell cycle, i.e. E2F and MYB3R3 and represent a robust inventory of RBR1-targets in dividing cells. Consistently, enriched motifs in the RBR1-marked domains include sequences related to the E2F consensus site and the MSA-core element bound by MYB3R transcription factors. Following up a key role of RBR1 in DNA damage response, we performed a meta-analysis combining the information about the RBR1-binding sites with genome-wide expression studies under DNA stress. As a result, we present the identification and mutant characterization of three novel genes required for growth upon genotoxic stress.Retinoblastoma (pRb) is a multifunctional regulator, which was likely present in the last common ancestor of all eukaryotes. The  Arabidopsis thaliana. We see prominent binding of RBR1 to the 5’ region of genes involved in cell cycle regulation, chromatin organization and DNA repair. Moreover, we also reveal extensive binding of RBR1 to specific classes of DNA transposons. Since RBR1 is involved in a plethora of processes, our dataset provides a valuable resource for researches from different fields. As an example, we used our dataset to successfully identify new genes necessary for growth upon DNA damage exerted by drugs such as cisplatin or the environmentally prevalent metal aluminum.The Retinoblastoma (pRb) tumor suppressor is a master regulator of the cell cycle and its inactivation is associated with many types of cancer. Since pRb’s first description as a transcriptional repressor of genes important for cell cycle progression, many more functions have been elucidated, e.g. in developmental decisions and genome integrity. Homologs of human pRb have been identified in most eukaryotes, including plants, indicating an ancient evolutionary origin of pRb-type proteins. We describe here the first genome-wide DNA-binding study for a plant pRb protein, i.e. RBR1, the only pRb homolog in  Retinoblastoma (pRb) was that of a transcriptional repressor controlling entry into S-phase. It was shown that pRb binds and inhibits the function of E2F-DP transcription factors and that phosphorylation of pRb by CDK-cyclin complexes disrupts this interaction, releasing E2F-controlled genes from repression [The first molecular function assigned to the human tumor suppressor pression .Since then, a wealth of additional functions in cell proliferation, differentiation, environmental response and genome stability have been discovered for the family of pRb related proteins in various organisms –5. To daC. elegans, i.e. lin-35, there are two homologs, Rbf1 and Rbf2, in Drosophila and in humans, pRb is member of a small gene family comprising pRb (p105), p107 and p130 [Arabidopsis thaliana, there is only one homolog, termed RBR1 (RETINOBLASTOMA RELATED 1) and its loss is female gametophytic lethal [The pRb–E2F module originated before the divergence of the plant and animal lineages, likely in the last common ancestor of all eukaryotes . While ting pRb p5, p107 ac lethal .In their function as transcriptional regulators of the cell cycle, pRb-type proteins not only control G1-S transition in proliferating cells but also operate at other phases, i.e. as repressors of G2 and M phase genes in response to DNA damage  as well DP, pRb-like (p130 or p107), E2F and the Multivulval class B (MuvB) core-complex, comprising five additional proteins, LIN9, LIN37, LIN52, LIN54, and RBAP48. When cells exit G0 and reenter the cell cycle, the DREAM-complex is disassembled upon phosphorylation of p130/p107 and the MuvB-core-complex sequentially associates with other transcription factors, like B-Myb and Fox-M1 to activate different sets of genes important for subsequent phases of the cell cycle [Drosophila, a homologous complex, termed dREAM , has been characterized but, in contrast to mammals, the fly pRb-type proteins and the only Myb transcription factor have been shown to concomitantly bind the MuvB-core complex [The human DREAM complex consists of Rb-like p0 or p107ll cycle . In Dros complex ,16.Arabidopsis DREAM-like complex were described by Kobayashi and co-workers and have been implicated in the control of mitotic genes [Arabidopsis contains five three-repeat MYBs (MYB3R), that function as transcriptional activators and/or repressors of mitosis [Recently, different versions of an ic genes . Arabido mitosis –19. Sinc mitosis .WUSCHEL [In addition to its role in controlling progression through the cell cycle, RBR1, like its animal counterparts , is invoWUSCHEL .Arabidopsis RBR1 has also been linked to chromatin modification [Like human pRb, which interacts with different chromatin modifiers, such as histone deacetylases and methyltransferases , the SWIfication . For exafication  as well fication , and a Rfication .Arabidopsis RBR1 is its role in DNA damage response (DDR) [Another facet of se (DDR) ,35, whicse (DDR) ,35. On tse (DDR) ,35, and se (DDR) . In addise (DDR) .Arabidopsis RBR1 is a multi-functional protein. However, despite several studies on specific aspects, a comprehensive view of RBR1 action has been precluded by the lack of genome-wide DNA binding data. Using an optimized ChIP protocol on Arabidopsis cell culture material, we now generated the first comprehensive collection of direct RBR1 targets. These data allowed us to obtain insights into the properties of RBR1 binding in mitotically active cells revealing the localization of RBR1 to genes and transposable elements (TEs). Furthermore, we then used this dataset to identify three new genes to be required for growth under genotoxic stress providing functional evidence for the power of our genome-wide study.Thus, like its animal homologs, Arabidopsis [One likely reason for the lack of genome-wide RBR1-ChIP data is the indirect binding of RBR1 to DNA, i.e. via E2F and other transcription factors/chromatin modifiers. To close this gap, we introduced several changes to our standard plant ChIP protocol . Startinbidopsis , we founArabidopsis cell culture  in the second intron, nor AT1G60020, where a Simplehat2 transposon (AT1TE72980) is inserted into the upstream-region, showed upregulation in hypomorphic mutants of RBR1 VANDAL21.wildtype . So far,Arabidopsis E2F-transcription factor family, i.e. WTTSSCSS, where W stands for A or T and S stands for C or G [A motif analysis of the RBR1 bound domains associated with TEs using the MEME-Chip software  detectedr C or G   including all three overrepresented motifs. A total of 218 clusters were identified in the RBR1-marked TE associated domains 22 of which contained only TE-motif 1, while pure TE-motif 2 or TE-motif 3 clusters occurred merely once. In most clusters however all 3 motifs were present in a variety of different layouts .PCNA1, ORC3, ORC1A, MCM5 and MCM2, the DNA repair genes RAD51 and BRCA1 as well as the cyclin-dependent kinases CDKB1;1 and CDKB1;2 have been shown to be RBR1 targets by gene specific RBR1-ChIP-qPCR experiments [The S-Phase genes eriments –35,50,51cell cycle, DNA repair, DNA replication and chromatin organization , by RBR1-only and by MYB3R3-only . This rely class . Furthere mutant , we saw  [KNOLLE  , we performed a search for overrepresented motifs in our RBR1-ChIP data. A MEME-ChIP analysis with standard settings identified six motif-clusters. The most significantly enriched motif of each cluster is shown in To estimate the genome-wide frequency of these motifs as well as the distribution among the MYB3R3- and/or RBR1-marked protein coding genes, we performed a FIMO-analysis   using 40Arabidopsis telomere repeat, are enriched in promoters of components of the translational machinery and can be bound by AtPurα [Arabidopsis [This analysis showed that sequences matching the repetitive motifs 1 and 3 occur at high frequency within 400 bp upstream of the translational start of genes on a genome-wide level. Gene-motif 1 strongly resembles the so-called GAGA-motif ..rbr1-2 mAT2G45460 grew like the wildtype on bleomycin, cisplatin as well as HU containing media, the loss of any of the other genes resulted in different patterns of hypersensitivity to these three DNA damaging drugs (AT1G04650 was shown to participate in meiotic recombination and designated MEICA1 (Meiotic Chromosome Association1) [Arabidopsis and therefore named FLIP (FIDGETIN-LIKE-1 INTERACTING PROTEIN) [AT5G46740 will be referred to as UBP21 (Ubiquitin-specific protease 21), according to the nomenclature introduced by Yan et al. [AT3G20490 will be called KNOTEN1  since the mutant shows an accumulation of DNA lesions upon genotoxic stress (see below).Whereas plants mutant for ng drugs , S11 Figiation1) . More rePROTEIN) . While Fn et al.  and AT3GKNO1 showed a strong growth inhibition on cisplatin, were only mildly affected by HU and displayed no significant growth reduction on bleomycin at the concentrations used in our assay  is a toxin plants are frequently exposed to on acidic soils [kno1 mutants from day three after treatment onwards. Also the flip mutant lines showed a clear trend towards growth reduction on aluminum. However, the result was statistically significant only for line flip-3 after 4 days. In contrast, the ubp21 mutants did not show any obvious reduction in recovery growth after aluminum treatment at the conditions tested.We further tested all hypersensitive lines for recovery growth after treatment with 1.5 mM aluminum . Bioavaiic soils  and previc soils . Signifikno1, flip and ubp21 plants to genotoxic agents is indeed due to enhanced DNA damage, we monitored γH2AX foci as a marker for double strand breaks after short term incubation in media with and without cisplatin or bleomycin [kno1-1 and flip-2 on cisplatin was slightly more severe than the damage on bleomycin at the conditions tested, the opposite was true for ubp21-1, which showed a stronger accumulation of γH2AX-foci on bleomycin than cisplatin in agreement with its slightly higher sensitivity towards bleomycin in the root growth assay.To analyze if the observed hypersensitivity of leomycin . While wSOG1 targets CYCB1;1 and RAD51, known to be transcriptionally upregulated upon DNA lesions , and on Ku70/Ku80, a key player in non-homologous end-joining (NHEJ), but also that Sleeping Beauty transposase directly interacts with the Ku70/Ku80 hetero-dimer [Arabidopsis, we and others have seen that RBR1 is involved in DNA repair at the site of the lesion [Our ChIP data indicates that RBR1 is recruited to E2F-sites that have been picked up and amplified by TEs in  species . Interes species , one posro-dimer . Conversro-dimer ,103. Alse lesion ,35, parte lesion . It is ke lesion ,105. ThuHiun, which belongs to the VANDAL21 family, we see a different example of interplay between the host machinery and the transposon, since one of the Hiun-localized ORFs is transcriptionally controlled by RBR1 and therefore potentially activated during G1/S phase, the moment when DNA transposons excise and mobilize [Additionally, in case of the non-MITE transposon mobilize .KNO1 (AT3G20490) and FLIP (AT1G04650) were described as genes of unknown function. With the new set of mass annotation provided by Araport11 [KNO1 became annotated as putative Rho GTPase-activating protein and FLIP as Holliday junction resolvase, but both annotations are still lacking experimental support in Arabidopsis. Holliday junction resolvases function in meiotic as well as somatic HR in different organisms [To make further use of the information derived from our genome-wide study, we combined the core-set of RBR1-bound genes with transcriptional data from DNA stress experiments. This led to the identification of three genes with so far unknown function in protection against DNA damage. At the beginning of this study, only two of the four genes analyzed had a functional annotation based on homology, i.e. AT5G46740 as ubiquitin-specific protease (UBP21) and AT2G45460 as SMAD/FHA domain-containing protein, while raport11 , KNO1 berganisms  and FLIPrganisms . A role KNO1 as well as UBP21 are among 146 recently identified direct targets of the major DNA damage related transcription factor SOG1 [Arabidopsis is a ubiquitin carboxyl-terminal hydrolase, has been shown to de-ubiquitinate and stabilize BRCA2 to promote efficient RAD51 loading at DNA double-strand breaks [ubp21 mutants. Further studies are needed to unravel the exact molecular function of Arabidopsis KNO1, FLIP and UBP21 in somatic cells to fully understand their here discovered requirement under DNA damaging conditions.Our results show that KNO1 is needed after DNA damage to efficiently up-regulate and probably also localize components of the HR repair machinery like RAD51. In humans genotoxin-induced DNA damage stimulates nuclear Rac1, a Rho GTPase required for the activation of stress kinases . Howevertor SOG1 , adding d breaks  and it icell cycle, replication, chromatin and DNA repair in actively dividing cells. However, previously described developmental RBR1 targets remain unmarked. To investigate RBR1's proposed role as a potential integrator of cell cycle regulation with developmental processes, genome-wide RBR1 distribution at specific developmental time points and in defined cell types will be beneficial and can be achieved by applying our optimized ChIP protocol in combination with FACS or INTACT methods [Here we have presented the first genome wide RBR1-binding study in plants. We show, that RBR1 associates with TEs, especially MITEs, and with genes highly enriched for GO-terms like  methods ,114.Further, our results demonstrate a vast commonality of genes bound by E2Fa, MYB3R3 and RBR1. In this respect, it will be valuable to gain and integrate information on genome wide binding of the different MYB3R and E2F transcription factors as well as RBR1 in distinct cell types as well as upon short and long term DNA damage. Recently, an involvement in DDR has been shown for repressive MYB3R proteins, but so far only the impact on G2/M genes has been analyzed in detail . A comprFinally, the here presented set of RBR1-controlled genes is a valuable resource that can be exploited to identify new genes involved e.g. in cell cycle control, chromatin remodeling and DNA repair as exemplified by our successful approach to reveal new DNA damage regulators.Landsberg erecta [Triplicates of the Arabidopsis MM2d cell culture, ecotype g erecta , were cog erecta  and antig erecta , respectArabidopsis thaliana genome assembly using Bowtie  and genomic (INPUT) DNA were used to prepare libraries with the MicroPlex Library Preparation kit (Diagenode). Quality of libraries was validated using 2100 Bioanalyzer (Agilent). Multiplexed libraries were sequenced using a HiSeq 2000 system (Illumina) with single-end 50-bp reads. Following a FASTQC (version 0.11.5) quality control, reads were mapped onto the TAIR10 sion 0.3 ) run in sion 0.3 ) was usemakeMetaGeneProfile.pl of the Homer Software [VENN diagram generator designed by Tim Hulsen at http://www.biovenn.nl [phyper function in R [The MetaGene Profile was generated using the tool Software . Venn diovenn.nl . The tesion in R . Data soion in R , Fisher’ion in R  for Motiion in R , includiion in R  and DREMion in R  for motiion in R  for calcion in R  to countion in R  to perfoion in R  was usedt-test: *, p < 0.05.Plants were germinated and grown on vertical plates containing half Murashige and Skoog (1/2 MS) medium under long day light conditions (16h) at 22°C for 6 days. Chemicals used in this study are bleomycin , cisplatin  and hydroxyurea (Sigma-Aldrich). Seedlings were transferred to medium with or without 0.3 μg/ml bleomycin, 20 μM cisplatin, 2 mM hydroxyurea or 2.5 mM hydroxyurea and grown for 6 days further. The position of the primary root tip was marked daily for each plant. After 6 days, plates were photographed and root length was measured using ImageJ software. Data are presented as mean ± SD (n > 30). Significant differences from wildtype were determined by Student’s 3, 0.2 mM KH2PO4, 2 mM MgSO4, 0.25 mM (NH4) 2SO4, 1 mM Ca(NO3)2, 1 mM CaSO4, 1 mM K2SO4, 1 μM MnSO4, 5 μM H3BO3, 0.05 μM CuSO4, 0.2 μM ZnSO4, 0.02 μM NaMoO4, 0.1 μM CaCl2, 0.001 μM CoCl2) prepared as previous described [t-test: *, p < 0.05.For the aluminum recovery growth assay, plants were germinated and grown on vertical plates containing ½ MS medium for 6 days. Seedling were then transferred to 1.5 mM Al-containing hydroponics (water solution (pH 4.2) consisting of 1 mM KNOescribed ,129 and 10-day-old seedlings were transferred to ½ MS liquid medium containing 3μg/ml bleomycin or 50μM cisplatin. Incubation time was 3 hrs. Root tip spreads and immunostaining was subsequently performed as described earlier in Friesner et al . γH2AX iArabidopsis seedlings or inflorescence material using the RNeasy Plant Mini Kit from Qiagene according to the instructions of the manufacturer. cDNA synthesis was performed using a Transcriptor First-Strand cDNA Synthesis kit for RT-PCR according to the manufacturer’s instructions (Roche). The cDNA produced was used in semi-quantitative PCR experiments to test for presence of mRNA. Quantitative PCR was performed with a Roche LightCycler 480 SYBR Green I Master with 0.5 μM specific primers and 0.1 μg of first-strand cDNAs. PCR reactions were conducted with the LightCycler 480 Real-Time PCR System (Roche) under the following conditions: 95°C for 5 min; 45 cycles of 95°C for 10 sec, 60°C for 10 sec and 72°C for 15 sec. Cq calling was done using the second derivative maximum method. Target-specific efficiencies were calculated as the mean of all reaction-specific efficiencies for a given target. Reaction-specific efficiencies were deduced using LinRegPCR 2015.2 [RNA was extracted from 10-day-old R 2015.2 ,132. DatR 2015.2 . PrimersSequence data for genes characterized in this article can be found in the EMBL/GenBank data libraries under accession numbers AT3G12280 (RBR1), AT3G20490 (KNO1), AT1G04650 (FLIP), AT5G46740 (UBP21) and AT2G45460.The RBR1-ChIP-seq data generated in this publication have been deposited in NCBI's Gene Expression Omnibus  and are http://publiclines.versailles.inra.fr). They are part of the SALK line collection [http://publiclines.versailles.inra.fr), respectively.The mutant lines used in this study were provided by the Nottingham Arabidopsis Stock Centre (NASC ) and thellection  or the Fkno1-1, SALK_023527 is kno1-2)AT3G20490 –note, that we renamed our mutant lines to be congruent with the numbering used by Fernandes et al. [AT1G04650 AT5G46740 .AT2G45460  Flow cytometry profiles of propidium iodide-stained nuclei from MMd2 culture cells 3 (3D) and 7 days (7D) after inoculation. 3D cultures were used for the RBR1-ChIP experiment since flow cytometry showed a higher number of cells in 4n, i.e. more cells progressing through S-phase, indicative of proliferation. (C) ChIP-qPCR results for the previously confirmed E2Fa and RBR1 targets MCM5, ORC3 as well as RBR1 (E2F-consensus site) and negative controls, i.e. CDKA;1 as well as 5’- and 3’ locations of the RBR1_E2F-consensus site. ChIP was performed using an anti-RBR1 or an anti-E2Fa antibody, respectively. Bars show the average of enrichment of IP vs. input of three biological replicates and its standard deviation. (D) Genomic representation of the RBR1 locus and the corresponding qPCR fragments analyzed in (C). One bar equals 100nt length, transcripts are shown as boxes and exons as blue arrows, marking the direction of the transcript. The E2F-consensus site is indicated by an orange circle.(A) Workflow of an (PDF)Click here for additional data file.S2 FigMCM5, PCNA1, RBR1, KNO1). (B) Examples for RBR1-ChIP signals over transposable elements (AT3TE91990 (Simpleguy 1), AT2TE23725 (Simplehat 2), AT5TE47555 (Simplehat 1)). (C) No significant RBR1 signal was detected upstream of the mitotic gene KNOLLE (KN). WTTSSCSS sites are indicated.(A) Examples for RBR1-ChIP signals associated with genes ((PDF)Click here for additional data file.S3 Fig(A) Domains identified as origins of replication by Costas et al.  were com(PDF)Click here for additional data file.S4 FigMotif alignments using the TOMTOM software . The P v(A) TE-motif 1 aligned to the E2F consensus WTTSSCSS, P value = 1.11e-02. (B) Gene-motif 1 aligned to the GAGA-element , P value(PDF)Click here for additional data file.S5 FigVenn diagrams showing overlap of RBR1-marked genes with genes involved in (A) replication, (B) cell cycle, (C) DNA repair and (D) chromatin. In each case, the overlap is highly significant.Overlap of RBR1-ChIP withGO:0006260 (Replication): P(X> = 61) = 1.37E-56Core replication (Shultz): P(X> = 45) = 5.16E-57GO:0007049 (Cell cycle): P(X> = 125) = 4.15E-79Core cell cycle (Vandepoele): P(X> = 24) = 8.18E-22GO:0006281 (DNA repair): P(X> = 70) = 6.00E-47DNA repair (Dohmann/Britt/Girard): P(X> = 51) = 1.27E-35GO:0006325 (Chromatin organization): P(X> = 48) = 1.23E-20ChromDB/manual collection: P(X> = 65) = 5.49E-24See (PDF)Click here for additional data file.S6 FigRBR1-bound genes were compared with E2F targets identified by different techniques. In addition, the number of genes having an E2F-binding site within 400 bp upstream of the START codon (upstream of the gene for non-protein coding genes) is indicated in each comparison. Overlap of RBR1-bound genes with (A) E2Fa-bound genes identified by ChIP, ChAP and TChAP  or the tiling GeneChip (C). For comparison, RBR1-ChIP data were therefore reduced to genes present on the Ath1-Chip or GeneChip, respectively.Transcriptional upregulation was monitored using the *P values marked by an asterisk (*) were below the calculation limits of the software (highly significant).(PDF)Click here for additional data file.S7 Fig(A) RBR1-ChIP fragments were mapped with respect to the center of E2Fa-binding sites identified by ChIP, ChAP and TChAP . (B) RBR(PDF)Click here for additional data file.S8 Figmyb3r1/3/5 triple mutants with MYB3R3-bound genes  Overlap of genes upregulated in leaves of 15 day old i et al. , P(X> = (B) GO-term enrichment analysis of genes that show binding by MYB3R3 and RBR1 together (overlap), by RBR1 only and by MYB3R3 only.(PDF)Click here for additional data file.S9 FigBRCA1, RAD51) and candidate genes (AT3G20490 (KNO1), AT1G04650 (FLIP), AT5G46740 (UBP21) and AT2G45460) in wildtype and rbr1-2 mutant seedlings. Significant differences to the wildtype are marked by an asterisk (p<0.05).qRT-PCR results showing relative expression of known DDR genes ((PDF)Click here for additional data file.S10 FigAT2G45460 (B) AT3G20490 (KNO1), (C) AT1G04650 (FLIP) and (D) AT5G46740 (UBP21). The location of the T-DNA insertions and the primers to test for mRNA expression are indicated. We tested for full-length as well as transcript before and after the insertion site. While transcript 5’ of the insertion site was present in all of the cases, in none of the insertion lines, full length mRNA or transcript 3’ of the insertion site could be detected by RT-PCR. Two independent insertion lines were used per gene. Note that the exact position of the T-DNA in kno1-2 was not determined due to masking of the T-DNA borders by vector backbone and inverted T-DNA fragments. However, we provide PCR-results from genomic DNA of kno1-2 showing that amplification the UP and DOWN fragments was possible while only the UP-fragment could be amplified from kno1-2 mutant cDNA. Primers used in this study are listed in Overview of the genomic region of (A) (PDF)Click here for additional data file.S11 Figkno1-1, flip-2, ubp21-1 and at2g45460-1 mutant lines on hydroxyurea-containing media (A) Pictures were taken after 6 days of growth. (B) Root length per day. Statistically significant differences to the wildtype are indicated by asterisks (p < 0.05).Root growth of the wildtype and (PDF)Click here for additional data file.S12 Figkno1 (at3g20490), flip (at1g04650) and ubp21 (at5g46740), represented by 2 mutant alleles each, on MS medium after treatment with 1.5mM aluminum for 12 hours. (A) Pictures after 5 days of recovery growth. (B) Root length per day. Statistically significant differences to the wildtype are indicated by asterisks (p < 0.05).Recovery root growth of the wildtype and (PDF)Click here for additional data file.S13 FigVenn diagrams showing comparison of the RBR1-ChIP data with H3K27me3 and FIE ChIP data from different publications  Overlap of RBR1-ChIP with H3K27me3, 14 d seedlings (Bouyer): P(X< = 79) = 4.58E-14.(B) Overlap of RBR1-ChIP with H3K27me3, 12 d seedlings (Deng): P(X< = 30) = 2.26E-18 and with FIE, 12 d seedlings (Deng): P(X< = 28) = 0.1122245, P(X> = 28) = 0.9194621.(C) Overlap of RBR1-ChIP with H3K27me3 in germinating embryos (Xiao): P(X< = 74) = 2.32E-11 and with FIE in germinating embryos (Xiao): P(X< = 40) = 0.04813777, P(X> = 40) = 0.9656176.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S1 Appendix(PDF)Click here for additional data file.S2 Appendix(HTML)Click here for additional data file.


Since 2008, avian influenza surveillance in poultry-related environments has been conducted annually in China. Samples have been collected from environments including live poultry markets, wild bird habitats, slaughterhouses, and poultry farms. Multiple subtypes of avian influenza virus have been identified based on environmental surveillance, and an H1N8 virus was isolated from the drinking water of a live poultry market.Virus isolation was performed by inoculating influenza A-positive specimens into embryonated chicken eggs. Next-generation sequencing was used for whole-genome sequencing. A solid-phase binding assay was performed to test the virus receptor binding specificity. Trypsin dependence plaque formation assays and intravenous pathogenicity index tests were used to evaluate virus pathogenicity in vitro and in vivo, respectively. Different cell lines were chosen for comparison of virus replication capacity.According to the phylogenetic trees, the whole gene segments of the virus named A/Environment/Fujian/85144/2014(H1N8) were of Eurasian lineage. The HA, NA, PB1, and M genes showed the highest homology with those of H1N8 or H1N2 subtype viruses isolated from local domestic ducks, while the PB2, PA, NP and NS genes showed high similarity with the genes of H7N9 viruses detected in 2017 and 2018 in the same province. This virus presented an avian receptor binding preference. The plaque formation assay showed that it was a trypsin-dependent virus. The intravenous pathogenicity index (IVPI) in chickens was 0.02. The growth kinetics of the A/Environment/Fujian/85144/2014(H1N8) virus in different cell lines were similar to those of a human-origin virus, A/Brisbane/59/2007(H1N1), but lower than those of the control avian-origin and swine-origin viruses.The H1N8 virus was identified in avian influenza-related environments in China for the first time and may have served as a gene carrier involved in the evolution of the H7N9 virus in poultry. This work further emphasizes the importance of avian influenza virus surveillance, especially in live poultry markets (LPMs). Active surveillance of avian influenza in LPMs is a major pillar supporting avian influenza control and response.The online version of this article (10.1186/s12879-019-4079-z) contains supplementary material, which is available to authorized users. Both animal and human infections with influenza A virus have been reported. Sometimes mammals, such as pigs, horses, and seals, and poultry can be infected under natural conditions. Wild aquatic birds are the major reservoir of avian influenza, harbouring 16 haemagglutinin (HA) and 9 neuraminidase (NA) subtypes of viruses , 3.Exposure to live poultry markets (LPMs) is an important risk factor for highly pathogenic avian influenza infection. Closure of LPMs has been reported to be efficient in blocking avian influenza transmission , 5. In CAlong with avian influenza control and prevention, regular LPM surveillance has been conducted in China annually since 2008. Specimens are sampled from relevant environments, and viruses are isolated and identified. The distribution and prevalence of influenza A virus subtypes in LPMs are then analysed. Multiple subtypes of avian influenza virus have been identified in the more than 10 years of surveillance. One avian influenza virus subtype, H1N8, which is uncommon among animals and animal-associated environments, was identified from poultry drinking water in a LPM in Fujian Province in 2014. To better understand its potential risk to human health, studies were conducted on the genetic and biological characteristics of the virus.A QIAamp Viral RNA Mini Kit  was used to carry out RNA extraction. Real-time reverse transcription polymerase chain reaction (RT-PCR) assays for the influenza A were performed on each of the samples. The reactions were carried out using an AgPath-ID™ One-Step RT-PCR Kit  under the following conditions: 10 min at 45 °C; 10 min at 95 °C; and 40 cycles of 15 s at 95 °C and 45 s at 60 °C. The sequences of the primers and probe are as follows: forward primer, 5’GACCRATCCTGTCACCTCTGAC3’; reverse primer, 5’AGGGCATTYTGGACAAAKCGTCTA3’; and probe, 5’FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ1–3′.Viruses or influenza A-positive specimens were inoculated into the allantoic cavity of 9-day-old embryonated chicken eggs for 48 h at 37 °C for virus propagation. The allantoic fluid was harvested, and haemagglutination assays were performed using 1% turkey red blood cells to detect the titres of the influenza viruses.PathAmp FluA Reagents  were used to amplify the full genome. The primer sequences were as follows: 5′-CTGGATACGCCAGCRAAAGCAGG-3′ (sense) and 5′-GACCTGATGCGGAGTAGAAACAAGG-3′ (antisense). Whole-genome sequencing was then performed on an Ion Torrent™ Personal Genome Machine™ platform (Thermo Fisher Scientific) with a read length of 200 base pairs following the instructions, and the data were analysed using CLC Genomics Workbench 7.5.1 software.Phylogenetic analysis was performed using MEGA7.0, and a phylogenetic tree was constructed using the maximum likelihood method. The bootstrap value was tested with 1000 replications for each gene segment. Homology analysis of nucleic acids was performed on the NCBI website with the BLAST platform.A solid-phase binding assay was used as described previously , 11. Syn4 cells/well were cultured at 37 °C overnight. Serial dilutions of virus were inoculated into MDCK cells. Two hours after virus absorption, overlay medium (2× DMEM and Avicel) with or without trypsin  was prepared and added. The cell plates were fixed with 4% paraformaldehyde 1 day later. ELISA was performed with a mouse monoclonal antibody against influenza type A (CDC-WHO kit used at 1:2000 in ELISA buffer) as the primary antibody and a goat anti-mouse IgG (H + L) HRP conjugate  as the secondary antibody. Finally, plaque formation was visualized by adding True Blue™ peroxidase substrate (KPL 50–78-02) and 0.03% H2O2 (1:1000 dilution of a 30% solution) [Virus pathogenicity in vitro can be reflected by plaque formation with or without trypsin. The plaque formation protocol was performed as follows: 96-well MDCK plates with 3 × 10olution) .Five-week-old specific pathogen-free (SPF) leghorn chickens were purchased from the company Beijing Vital River Laboratory Animal Technology. According to the OIE/WHO guidelines , the chi4 cells/well at 37 °C overnight, and the test viruses were added with a multiplicity of infection of 0.01. The plates were then incubated with DMEM containing 0.5% BSA and 2 μg/ml TPCK-treated trypsin at 37 °C. The supernatants were collected at 12, 24, 36, 48, 60 and 72 h post infection. End-point titration of the supernatants was performed with MDCK cells.The virus replication capacity in mammalian cells was evaluated. Human type II alveolar epithelial cell line (A549), chicken embryo fibroblasts cell line DF1 (DF1), and porcine kidney cell line (PK15) were inoculated into 96-well plates at a density of 3 × 10P < 0.05 was considered to indicate a significant difference.The statistical significance of the differences was determined using T tests, and The avian influenza virus subtype H1N8 identified in this study was named A/Environment/Fujian/85144/2014(H1N8); the full genome sequences of the virus have been uploaded to the Global Initiative on Sharing All Influenza Database (GISAID) with accession numbers EPI11315724-EPI11315731. The nucleotide homology analysis of A/Environment/Fujian/85144/2014 showed that it might be a reassortant virus. Three of the 8 segments, HA, NA and PB2, were derived from an A/Duck/Fujian/13/2013 (H1N8)-like virus; the PB2, PA, and NS genes showed the highest similarity with the H7N9 virus A/Environment/Fujian/S10058/2017(H7N9). NP and M were most closely related to the H1N2 viruses A/Environment/Fujian/FJ1273/2014 and A/duck/Guangxi/GXd-2/2012, respectively  of the Phylogenetic analysis indicated that the HA gene of A/Environment/Fujian/85144/2014(H1N8) was clustered with those of H1N8 viruses isolated from local ducks in Fujian Province named A/duck/Fujian/13/2013(H1N8), A/duck/Fujian/83/2013(H1N8), A/duck/Fujian/89/2013(H1N8) and A/duck/Fujian/84/2013(H1N8). These viruses belong to the avian H1 group of the Eurasian lineage; they are different from the avian H1N8 North American–lineage viruses and phylogenetically far from swine–origin H1 and seasonal human H1 subtype viruses. The N8 gene evolutionary tree indicated that the studied H1N8 virus was most likely derived from the local duck H1N8 viruses and clustered with the duck H6N8 virus isolated from Shantou, Guangdong Province. The N8 gene in the studied virus was clearly different from the N8 genes of avian influenza H1N8 North American–lineage viruses . The growth of the A/Environment/Fujian/85144/2014(H1N8) virus in the different cell lines was similar to that of the human-origin virus A/Brisbane/59/2007(H1N1) and lower than that of the control avian-origin and swine-origin viruses, A/duck/Alberta/35/1976(H1N1) and A/swine/Iowa/15/1930(H1N1), respectively , A/duck/Alberta/35/1976(H1N1), and A/swine/Iowa/15/1930(H1N1), which are of human origin, avian origin, and swine origin, respectively. The mean peak titres of A/Environment/Fujian/85144/2014, A/Brisbane/59/2007 (H1N1), A/duck/Alberta/35/1976(H1N1), and A/swine/Iowa/15/1930(H1N1) in A549 cells were 6, 5.25, 9 and 7.5 logNo plaques formed when A/Environment/Fujian/85144/2014(H1N8) or the reference seasonal influenza virus A/Brisbane/59/2007(H1N1) was inoculated into MDCK cells without addition of TPCK-treated trypsin. However, clear plaques of both viruses were present when trypsin was added, and the sizes and shapes of the plaques were similar. This result showed that the studied H1N8 virus is a low-pathogenic avian influenza virus in vitro Fig. .Fig. 3PlA solid-phase binding assay was used to test six viruses, including 5 reference viruses with different origins. Four different types of sialylglycopolymers  were coated on the plates at concentrations of 0, 0.625, 1.25, 2.5, 5 and 10 μg/ml . As showDuring the 10-day observation period, only 2 chickens presented mild signs and cough as early as 2 days after infection. The other chickens, including those in the test and control groups, appeared normal until the end of observation. The IVPI in the chickens was 0.02, which indicated that A/Environment/Fujian/85144/2014(H1N8) is a low-pathogenic avian influenza virus.H1 subtype influenza viruses are known for their wide range of hosts  and rapid evolution as a result of antigenic drift and antigenic shift, which can cause seasonal influenza epidemics and even influenza pandemics , 20. TheResearch on avian influenza virus ecology in LPMs in China has demonstrated that H5, H9, and H7 are not the only subtypes of avian influenza viruses; subtypes such as H3, H4, H6, H1, and H2 are also present . A few sReceptor binding preference plays an important role in influenza replication and transmission. The ability of avian influenza virus to bind to the human receptor is the basis for efficient human-to-human transmission. The relationship between human receptor binding specificity and HA gene molecular markers has been extensively revealed in H5, H7 and H9 subtype viruses , 30. BasPlaque formation assays are some of the most quantitative and useful biological methods for virus research and can be used for quantification of infectivity and identification of individual virus particles with specific biological features. We intended to determine the pathogenicity of the H1N8 avian influenza virus in vitro using this assay. If a virus is highly pathogenic, a plaque can be formed without trypsin present. This property is based on the presence of multiple basic amino acids at the cleavage site of the HA protein . We founVirus growth curves in different cell lines can be used to describe virus replication and proliferation ability. In our study, the H1N8 virus grew better in A549 and PK15 cells than in DF1 cells, which indicated the possibility of a growth preference for mammalian cells. A sharp increase in virus replication was detected 12 h post infection in the A549 and DF1 cells. However, the virus growth in PK15 cells increased to a peak at 36 h post infection. A/Environment/Fujian/85144/2014 displayed avian receptor binding characteristics, indicating that the virus retains avian influenza binding capacity. Despite its great similarity with other avian influenza viruses and its low pathogenicity to poultry, this H1N8 subtype avian influenza virus presented efficient replication in mammalian cells, indicating its potential risk for poultry and even mammals. Further exploration will be beneficial to enhance understanding of the mechanism of H1N8 infection in birds and mammals.The PB2, PA, and NS genes of the H1N8 virus studied showed the highest similarity with a highly pathogenic avian influenza (HPAI) H7N9 virus, A/Environment/Fujian/S10058/2017(H7N9), which was reported in 2018 . AlthougThe H1N8 influenza virus in this study contained a gene constellation of avian origin and might have been involved in the evolution of H7N9 viruses in poultry. In addition, an avian receptor binding preference was present, and low pathogenicity to poultry was confirmed both in vitro and in vivo. This H1N8 virus can also grow well in mammalian cells. Although this subtype of virus is not frequently detected, it still contributes to the diversity of avian influenza ecology and provides some insights for virus surveillance. The results of this study also emphasize the forecasting function of LPMs regarding the trends in avian influenza activity in China.Additional file 1:Figure S1. Phylogenetic analysis of six internal genes of A/Environment/Fujian/85144/2014(H1N8). Phylogenetic analysis of six internal genes, including PB2, PB1, PA, NP, M, and NS. All sequences were downloaded from GenBank. The maximum likelihood method was used to construct the phylogenetic trees. The virus strains are marked with different colours. Red represents the A/Environment/Fujian/85144/2014(H1N8) strain in this study, blue represents other H1N8 subtype viruses, pink represents all available previous H1N8 subtype viruses sequence from China, black represents viruses of other subtypes, and the black star indicates the HPAI H7N9 viruses isolated in 2017 and 2018. (PDF 1538 kb)


Rb1-null osteosarcoma tumors. Using genetically engineered mouse models of osteosarcoma, we found that loss of activator E2Fs, E2F1 and E2F3, significantly delays tumor progression and increases the overall survival of the p53/Rb1-deficient osteosarcoma mouse model. We also studied the role of helicase, lymphoid specific (HELLS), a chromatin remodeling protein identified as a critical downstream effector of the RB-E2F signaling pathway in various cancers. In this study, we confirmed that the RB-E2F pathway directly regulates HELLS gene expression. We also found that HELLS mRNA is upregulated and its protein overexpressed in osteosarcoma. Using loss-of-function assays to study the role of HELLS in human osteosarcoma, we observed that HELLS has no effect on tumor proliferation and migration. Further, we pioneered the study of Hells in developmental tumor models by generating Hells conditional knockout osteosarcoma mouse models to examine the role of HELLS in osteosarcoma tumor development. We found that loss of Hells in osteosarcoma has no effect in tumor initiation and overall survival of mice. This suggests that while HELLS may serve as a biomarker for tumorigenesis and for RB-E2F pathway status, it is unlikely to serve as a relevant target for therapeutics in osteosarcoma.Osteosarcoma is the most common primary bone malignancy in children and adolescents. Among the various molecular mechanisms implicated in osteosarcomagenesis, the RB-E2F pathway is of particular importance as virtually all cases of osteosarcoma display alterations in the RB-E2F pathway. In this study, we examined the transcription factor E2F family members that are associated with increased malignancy in  TP53 and RB1 tumor suppressor genes [RB1 gene in osteosarcoma are associated with poor therapeutic outcome, as defined by increased mortality, metastasis, and poor response to chemotherapy [Osteosarcoma is a mesenchymal tumor that is histologically characterized by the presence of malignant mesenchymal cells and the production of a bone stroma. Osteosarcomas are commonly characterized by an appendicular primary tumor with a high rate of metastasis to the lungs . The mosor genes , 3. Lossotherapy –8. Unforotherapy –12.RB1 gene encodes the tumor suppressor protein RB. RB forms a transcriptional repression complex with the E2F family of transcription factors and thereby negatively regulates G1/S transition during the cell cycle through the suppression of E2F target genes. There are six E2F family members that bind to the RB family and are classified as activator E2Fs  and repressor E2Fs  [1/S transition. In addition to E2Fs, RB associates with a large number of nuclear proteins, including a variety of chromatin-associated proteins that have diverse activities [RB1 mutations are present in up to 70% of osteosarcoma cases [RB1 loss is correlated with poor prognosis for patients with osteosarcoma [RB1 function is associated with increased risk of osteosarcoma metastasis and poor response to chemotherapy, compared with osteosarcoma patients with intact RB1 [RB1, indicated that retinal tumorigenesis was driven by E2F1- and E2F3-transcribed genes following the lack of transcriptional repression due to RB loss [The nd E2F5) . Of thestivities –16. RB1 ma cases . Furtherosarcoma . Specifitact RB1 , 19. A p RB loss .HELLS  is a protein that belongs to the SNF2 family of chromatin-remodeling ATPases . HELLS iRb1. Since HELLS was identified as aE2F target gene and critical contributor of tumorigenesis in retinoblastoma, we evaluated the role of HELLS in osteosarcoma tumorigenesis. We demonstrated that activator E2Fs, E2F1 and E2F3, directly regulate HELLS expression. We also showed that RB-E2F pathway inactivation, present in most osteosarcomas, results in increased HELLS expression. Further, we evaluated the role of HELLS in osteosarcoma cell survival, proliferation, migration, and tumorigenesis both in vitro and in vivo. We showed that, unlike what has been observed in other malignancies, targeting HELLS in human osteosarcoma has a modest effect in osteosarcoma survival and no effect on migration. Further, using a Hells conditional knockout mouse model, we found that loss of Hells has no effect on osteosarcoma tumor incidence and overall survival. This suggests that HELLS is not critical for tumor initiation and progression in osteosarcoma.In this study, we generated a series of genetically engineered mouse models of osteosarcoma to study the role of E2Fs in tumor development. We focused on E2F1 and E2F3, two aE2F previously described as critical for Rb-mediated tumorigenesis, and E2F5 as a rE2F control . SimilarTp53 in conditional knockout mice driven by osterix-cre recombinase (Osx-cre), a transgene that is actively expressed in more differentiated pre-osteoblasts, results in osteosarcoma with high penetrance  [Rb1 , recapitulating the negative impact of RB1 mutations in human osteosarcoma [E2f1 and E2f3, but not E2f5, prevents retinoblastoma formation in Rb1-deficient mice [Rb1-deficient mice, aE2Fs (E2F1 and E2F3) constitutively facilitate transcriptional activation of downstream targets that associate with poor outcome. To determine if loss of these E2F family members can revert the increased malignancy associated with loss of Rb1, we developed lox/lox; Rb1lox/lox; E2f1Osx-cre; p53−/− (p53/Rb1/E2f1 TKO), lox/lox;Rb1lox/lox;E2f3lox/loxOsx-cre; p53 (p53/Rb1/E2f3 TKO), and lox/lox;Rb1lox/lox;E2f5lox/loxOsx-cre; p53 (p53/Rb1/E2f5 TKO) triple-knockout mice. To characterize the tumor incidence in these mouse models, we monitored mice weekly until advanced tumor burden required euthanasia. 100% (n = 35) of the p53 cKO mice developed tumors by 65 weeks with a median survival of 58.45 weeks  of the p53/Rb1 DKO mice developed tumors by 40.4 weeks with a median survival of 26.9 weeks  of the p53/Rb1/E2f1 TKO mice developed tumors by 48.6 weeks with a median survival of 37 weeks, significantly higher than p53/Rb1 DKO mice  of the p53/Rb1/E2f3 TKO mice developed tumors by 42.4 weeks with a median survival of 32.75 weeks, also significantly higher than p53/Rb1 DKO mice  of the p53/Rb1/E2f5 TKO mice developed tumors by 41.7 weeks with a median survival of 29.2 weeks, which was not significantly different from p53/Rb1 DKO mice (p = 0.3783)  . This diosarcoma . Previouent mice , 29. We s Figure . 100% (n) Figure . Further) Figure .RB1 loss in osteosarcoma patients is associated with poor prognosis, we hypothesized that loss of RB potentiates osteosarcoma through transcriptional deregulation of chromatin remodeling genes including HELLS. Initial analysis of The Cancer Genome Atlas (TCGA) data set of 263 sarcoma tumor samples indicated that HELLS is upregulated by 4.27-fold compared to normal controls, making HELLS an attractive target to study in osteosarcoma   Figure . At the ) Figure . Increasl Figure . We alsoin vitro study in gliomas [Evidence of direct regulation of HELLS by the RB-E2F signaling pathway is limited to a single  gliomas . To conf gliomas . PCR priHELLS in human osteosarcoma, we acquired lentiviral vectors encoding shRNAs targeting E2F1 (shE2F1) and transduced the four human osteosarcoma cell lines in which we had previously observed HELLS overexpression  and p53/Rb1/Hells triple knockout  were compared to their corresponding littermate controls  for the assessment of the role of HELLS in tumor initiation and promotion during osteosarcoma development. All genotypes lead to the development of osteosarcoma in mice with 100% penetrance. As seen in Figure Hells conditional knockout alleles compared to their littermate controls. The median survival in p53/Rb1/Hells TKO mice was 27.7 weeks (n = 23) compared to 26.9 weeks  in p53/Rb1 DKO mice , compared to 58.45 weeks in p53 cKO  . Western blot analysis of Hells wild-type (p53/Rb1 DKO and p53 cKO) and Hells-null (p53/Rb1/Hells TKO and p53/Hells DKO) tumors confirmed that HELLS was efficiently knocked-out in these osteosarcomas  Figure . Intrigus Figure .RB1 gene are associated with increased mortality, metastasis and poor therapeutic outcome in osteosarcoma [RB1 loss leads to poor prognosis remain to be elucidated. In this study, we demonstrate that poor clinical outcome associated with loss of RB1 is mediated, at least in part, by de-repression of aE2F family members. We also examined the role of HELLS, a chromatin remodeling protein transcriptionally regulated by E2F1 that promotes tumorigenesis in several cancers [Osteosarcoma is the most common primary cancer of bone and typically occurs in children and young adults. As a highly metastatic cancer, 15–20% of osteosarcoma patients are diagnosed after the cancer has already metastasized , which translates to 5-year survival rates of <40% . Thus, tosarcoma . However cancers , 27. Herp53 is sufficient in driving osteosarcoma tumorigenesis in mouse [Rb1, which we were able to recapitulate. Here, we tried to elucidate the mechanism through which RB inactivation contributes to poor clinical outcome in osteosarcoma. We found that loss of either E2f1 or E2f3 significantly increased the median survival age of p53/Rb1 DKO mice; whereas loss of E2f5 did not improve or worsen the outcome. These results are consistent with other studies reporting that loss of transcriptional control by aE2Fs, particularly E2F1 and E2F3, is responsible for tumorigenesis following Rb1 inactivation [Rb1 loss. This could be due, at least in part, to compensation by other E2F family members (discussed in the next section). The observation that inactivation of either E2F1 or E2F3 could significantly delay the onset of tumors and increase overall survival of osteosarcoma-bearing animals suggests that the increase malignancy upon Rb1 loss might be reversible, and that direct downstream targets of aE2Fs are valuable potential therapeutic targets.Preosteoblast-specific loss of in mouse . Diseasetivation , 36–38. HELLS is overexpressed in osteosarcoma, we blasted The Cancer Genome Atlas (TCGA) data set and found that HELLS is upregulated in sarcoma tumor samples compared to normal controls. We confirmed HELLS gene upregulation in the human osteosarcoma cell lines 143B, SJSA-1, and SaOS-2, as well as in four of the five independent patient-derived orthotopic xenografts used in this study. Despite the lack of mRNA upregulation in some cell lines and tumors, HELLS protein overexpression was observed in all the human osteosarcoma cell lines and xenografts analyzed in this study. The discordance between mRNA and protein levels in some of the samples suggests that while HELLS protein levels are predominantly transcriptionally regulated there are also translational and post-transcriptionally mechanisms of protein expression regulation. Further, the HELLS protein overexpression observed in human osteosarcoma is recapitulated in the p53/Rb1 DKO osteosarcoma mouse model, strengthening its validity as a study model for this disease.Others and we have identified HELLS as a transcriptional downstream target gene of aE2Fs that is overexpressed in cancer and contributes to tumor progression , 24, 27.in vitro study in gliomas [HELLS where putative E2F1 binding motifs reside. Interestingly, significant reduction of HELLS protein level was only achieved through combined knockdown of E2F1 and E2F3, and not by E2F1 or E2F3 knockdown alone. This suggests that E2F1 and E2F3 compensate for the loss of each other to regulate HELLS expression. This observation could begin to explain why losses of neither E2f1 (p53/Rb1/E2f1 TKO) nor E2F3 (p53/Rb1/E3f3 TKO) were sufficient to reverse the increased malignancy observed upon Rb1 loss (p53/Rb1 DKO) in osteosarcoma but rather provided only partial improvements. The observation that rescue of altered expression of RB-E2F transcriptional gene targets is orchestrated by more than one E2F family member is an important consideration for therapeutic approaches being developed that aim to inhibit E2Fs directly.Evidence of direct regulation of HELLS by the RB-E2F signaling pathway was limited to a single  gliomas . In thisRB1 loss, RB hyperphosphorylation through increased activity of CDK4/CDK6, or E2F hyperphosphorylation, all of which prevent repression of E2Fs from binding to downstream targets. Therefore, it is not surprising that HELLS upregulation and overexpression in osteosarcoma is not restricted to RB1-null cases, but rather a generalized event observed across samples that bear RB-E2F pathway inactivation. Of the osteosarcoma cell lines examined in this study, SaOS-2 harbors a deletion in the RB1 gene that leads to a non-functional truncated protein [CDK4 amplification [p16 null [RB-E2F signaling pathway is often inactivated in cancer due to various reasons including  protein , SJSA-1 fication , and U-2p16 null . Here, win vitro results in decreased proliferation of various cancer cell types including retinoblastoma [The expression of HELLS has been positively associated with proliferation in both normal and malignant cells , 27, 41.blastoma , gliomasblastoma , nasophablastoma  cells, ablastoma . In thisin vitro loss-of-function studies in human osteosarcoma models, we generated novel genetic engineered mouse models to study the role of Hells in osteosarcoma tumor formation. We observed that p53/Hells DKO and p53/Rb1/Hells TKO mice showed no improvement in tumor incidence or overall survival compared to p53 cKO and p53/Rb1 DKO controls, respectively. This result suggests that HELLS is not a critical driver of Rb1-mediated malignancy in murine osteosarcoma tumors.Beyond Taken together, our studies and others indicate that HELLS may be a reliable biomarker for RB-E2F pathway inactivation. However, HELLS upregulation may not always be synonymous to a critical role in tumorigenesis or tumor progression, or a warranted target for therapeutics in all malignancies where HELLS overexpression is observed.The five orthotopic xenografts used in this study: SJOS001105 (PDX1), SJOS001112 (PDX2), SJOS001107 (PDX3), SJSO010930 (PDX4), and SJOS001121 (PDX5) were obtained from the Childhood Solid Tumor Network . Athymiclox/loxp53 and lox/loxRb1 conditional-knockout mice were obtained from the Mouse Models of Human Cancer Consortium at the National Cancer Institute; E2f1-knockout and lox/loxE2f3 conditional knockout mice were obtained from Dr. Gustavo Leone (The Ohio State University); lox/loxE2f5 mice were obtained from Dr. Joseph Nevins (Duke University); the Osx-cre mice were obtained from The Jackson Laboratory. The Hells-conditional knockout mouse was generated from mice obtained from the European Mouse Mutant Archive, backcrossed to Flp mice for removal of the neo-cassette (tm1c conversion), and then backcrossed to C57BL/6N mice for Flp removal. Mice were monitored weekly for signs of osteosarcoma. Moribund status was defined as the point when tumors had reached 10% body weight or induced paralysis in the mouse. The University of California Irvine Institutional Animal Care and Use Committee approved all animal procedures. Survival curves were generated using GraphPad Prism. Mantel-Cox test was used for statistical analyzes of the curves.The 2. 143B were cultured in MEM (Gibco), with 10% BCS, penicillin/streptomycin, and 0.015 mg/ml BrdU. SJSA-1 were cultured in RPMI (Gibco), with 10% BCS and penicillin/streptomycin. SaOS-2 were cultured in McCoy's 5A (Gibco), with 15% BCS and penicillin/streptomycin. U-2 OS were cultured in McCoy's 5A (Gibco), with 10% BCS and penicillin/streptomycin. MSCs were cultured in Alpha-MEM (Gibco), with 10% FBS and penicillin/streptomycin. HEK293T cells were culture in high-glucose DMEM (Gibco), with 10% BCS, penicillin/streptomycin and sodium pyruvate. Cells were passaged every 3 to 4 days or when they reached 70–80% confluency. At the time of passage, cells were split to 20% confluence.Osteosarcoma cell lines 143B, SJSA-1, SaOS-2 and U-2 OS, as well as MSCs and HEK293T cells were acquired from the American Type Culture Collection (ATCC). Cells were cultured in a humidified atmosphere at 37° C and 5% COCt method. Primer sequence for PCR amplification are as follows: HELLS , CDK4 , CDK6 , GAPDH , 18S .Total RNA was isolated from cells or tumor tissues by homogenizing samples with TRIzol Reagent. RNA was isolated through chloroform extraction. 1 μg of total RNA was used for cDNA synthesis with the SuperScript™III First-strand synthesis system (Invitrogen) according to manufacturer's protocol at a reaction volume of 20 μl. Quantitative PCR amplification was performed using 1 μl of reverse-transcribed product in Power SYBR Green PCR Master Mix . Primers were designed using IDT Real-Time PCR tool (Integrated DNA Technologies). Reaction was carried out using 7500 Real-Time PCR system (Applied Biosciences). Data were normalized to those obtained with endogenous control 18S and GAPDH mRNA, and analyzed using ΔΔCell pellets or tumor tissues were homogenized by pipetting or pellet pestle in RIPA buffer  supplemented with protease inhibitor . Samples were allowed to lyse for 30 min on ice before centrifugation at 14000 RPM at 4° C for 30 min. Protein concentration was measured using BCA protein assay (Pierce™ BCA Protein Assay Kit). 40 μg of total protein were resolved in 4–15% SDS-PAGE gel (Mini-PROTEAN TGX Gels (4–15%), Bio-rad), and transferred onto PVDF membrane  using semi-dry transfer apparatus (Bio-rad). Ponceau S stain was used to validate successful transfer. Non-specific binding was prevented by incubating the membrane in 3% non-fat dry milk in TBS-0.25% Tween (TBS-T) for 1 hour at room temperature, shaking. Primary antibodies were diluted in 0.5% non-fat dry milk in TBS-T as follows: 1:1000 anti-HELLS , anti-actin , anti-E2F1 , anti-E2F2 , anti-E2F3 , 1:1000 anti-Rb . Membranes were incubated in primary antibody overnight, shaking, at 4° C. Membranes were then rinsed 3 times with TBS-T on shaker and incubated with secondary antibody for 40min at room temperature, shaking. Secondary antibodies were diluted in 0.5% non-fat dry milk in TBS-T as follows: 1:1000 peroxidase labeled anti-mouse IgG , 1:1000 peroxidase labeled anti-rabbit IgG . Membranes were again rinsed 3 times with TBS-T on shaker. Chemiluminescence was detected using SuperSignal™ West Pico Chemiluminescent Substrate . Relative band intensity was analyzed using ImageJ software.ChIP assays were essentially performed as previously described , 43. ChIOsteosarcoma cells were seeded at a density of 500000 cells/well of 6-well plate and allowed to adhere overnight. Two concentrations of Palbociclib, 0.5 μM and 1 μM, were used to treat cells for 24 h prior to collection. DMSO was used as drug vehicle control.Lentiviral particles were produced by co-transfecting the envelope plasmid pCMV-VSV-G (Addgene), packaging plasmid pCMV-dR8.2 dvpr (Addgene), and GIPZ shRNA vectors (GE Dharmacon): E2F1 shRNA (V3LHS_393591), E2F2 shRNA (V3LHS_324068), E2F3 shRNA (V3LHS_325936), HELLS shRNA (V2LHS_155497), or GIPZ lentiviral empty vector shRNA control into HEK293T cells using calcium phosphate transfection method. Supernatants containing lentiviral particles were harvested at 24 and 48 hours post-transfection. Cell debris were cleared by centrifugation at 1600 × g for 10 min at 4° C. Supernatants were then filtered through 0.45μm PES filter , and concentrated by ultracentrifugation at 23000 RPM for 2 hours at 4° C. Lentiviral particles were resuspended in ice-cold PBS and stored at −80° C. Transduction of target cells were achieved by exposing cells to viral particles in serum-free condition for 6 hours. Puromycin selection was carried out at a concentration of 2 μg/ml.All cells were seeded into 96-well black assay plates (Costar) at a density of 3,000 cells per well. Cell viability was determined at 24, 72, and 96 hours after seeding using CellTiter-Glo (Promega) according to manufacturer's instructions. Luminescence readings were normalized to the 24 h post-seeding reading. Survival curves were generated using GraphPad Prism.2) as single cells onto 6-well cell culture plates in the appropriate cell culture medium. Fresh medium was supplemented every 4 days. Cells were incubated at 37° C with 5% CO2 until cells in control dishes have formed sufficiently large colonies (~50 cells). Cells were washed once in PBS before fixing and staining in a mixture consisting of 6% glutaraldehyde and 0.5% crystal violet for 30 min. Stains were washed out with tap water and the plates left to dry at room temperature. Colonies were counted from triplicate experiments.Lentiviral transduced cells were seeded at a low-density (20 cells/cm6/well) were plated on 6-well plates. The day after, once cells were grown to a confluence of about 80%, the monolayer was scratched using a 10 μl sterile pipette tip. Images were captured immediately and 8 h after the wound. The exact location of the image was marked to identify the same gap. The distances between the boundaries of the wound at 0 and 8 h at 10 different locations were measured in pixels using Zen software (Zeiss).Scratch-wound assay was performed as previously described . Cells (


Scientific Reports 10.1038/srep43941, published online 06 March 2017Correction to: In this Article, the U-20S Bicalutamide image in Figure 5C is a duplication of the KHOS control image in Figure 4F. The correct Figure 5 appears below as Figure


RECQL4, a RecQ helicase family member. In vitro studies have identified functions for the ATP-dependent helicase of RECQL4. However, its specific role in vivo remains unclear. To determine the physiological requirement and the biological functions of Recql4 helicase activity, we generated mice with an ATP-binding-deficient knock-in mutation (Recql4K525A). Recql4K525A/K525A mice were strikingly normal in terms of embryonic development, body weight, hematopoiesis, B and T cell development, and physiological DNA damage repair. However, mice bearing two distinct truncating mutations Recql4G522Efs and Recql4R347*, that abolished not only the helicase but also the C-terminal domain, developed a profound bone marrow failure and decrease in survival similar to a Recql4 null allele. These results demonstrate that the ATP-dependent helicase activity of Recql4 is not essential for its physiological functions and that other domains might contribute to this phenotype. Future studies need to be performed to elucidate the complex interactions of RECQL4 domains and its contribution to the development of RTS.Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by skin rash (poikiloderma), skeletal dysplasia, small stature, juvenile cataracts, sparse or absent hair, and predisposition to specific malignancies such as osteosarcoma and hematological neoplasms. RTS is caused by germ-line mutations in  DNA helicases unwind double-stranded nucleic acids using energy from ATP to access genetic information during cell replication. In humans, several families of helicases have been described and one of particular importance is the RecQ family, where mutations in three of five members cause human disease. RECQL4 is a member of this family and its mutation results in Rothmund-Thomson syndrome (RTS). Prior studies have shown that defects in the helicase region of RECQL4 may contribute to the disease, but no studies have specifically assessed the biological effects of its absence in a whole animal model. In this study, we generated a mouse model with a specific point mutation resulting in a helicase-inactive Recql4 protein. We found that an absence of ATP-dependent helicase activity does not perturb the physiological functions of Recql4 with the homozygous mutants being normal. In contrast, when we assessed point mutations that generate protein truncations these were pathogenic. Our results suggest that the helicase function of Recql4 is not essential for its physiological functions and that other domains of this protein might account for its functions in diseases such as RTS. RECQL4 [RECQL4 mutations are located in its helicase domain, yet the physiological role of this domain remains unclear [Rothmund-Thomson syndrome (RTS) OMIM #268400) is a rare autosomal recessive disorder characterized by skin rash (poikiloderma), skeletal dysplasia, small stature, sparse or absent hair, gastrointestinal complications, and high predisposition to specific malignancies such as osteosarcoma (OS) and hematological neoplasms , 2. RTS 68400 is RECQL4 –5. The mRECQL1, RECQL4, RECQL5, BLM and WRN. Mutations in three are associated with syndromes that present with premature aging and cancer-predisposition: WRN in Werner’s syndrome, BLM in Bloom’s syndrome, and RECQL4 in RTS [2+-binding domain (R4ZBD) and, importantly, an N-terminal region of homology with the S. cerevisiae DNA replication initiation factor Sld2 [Helicases are enzymes that unwind double-stranded or more complex DNA and RNA structures using energy from ATP hydrolysis. The unwinding of double-stranded DNA (dsDNA) is necessary to allow access to the DNA during replication, repair, recombination and transcription . In huma4 in RTS , 9. RecQ4 in RTS , 9; the 4 in RTS ; and the4 in RTS . RECQL4 tor Sld2 , 13. Sldtor Sld2 , and RECRECQL4 gene is located at the long arm of chromosome eight (8q24.3) and consists of 21 exons and 13 relatively short introns (<100 bp in length), yielding a full-length transcript of 3,627bp [RECQL4 gene have been found in the majority of RTS patients, and also in the related RAPADILINO and Baller-Gerold Syndromes [The human ght 8q24. and consIn vitro, RECQL4 ATPase and helicase activity is completely abolished by point mutations in the canonical Walker A and B motifs [in vitro [Recql4K525A) and compared this to two different truncating mutations Recql4G522Efs and Recql4R347*. Here we show that mice with a specific deficiency in Recql4 helicase activity are strikingly normal, in contrast to pathogenic effects of truncating mutations that remove the entire helicase domain and downstream part of the protein.RECQL4 has been well characterized biochemically in terms of its single-stranded DNA (ssDNA) binding, ATP hydrolysis and DNA unwinding ability –17. In vB motifs . Howeverin vitro , 20. In in vitro assays for Recql4 function. Both WT and K525A proteins had equivalent affinity for ssDNA binding, using an electrophoretic-mobility shift assay (EMSA)  . In ATPad levels . Finallyd levels . Robust wind DNA . Togethein vivo knock-in model of the K525A mutation. Sequencing of the Recql4 locus in targeted mice confirmed the change in nucleotides encoding lysine (AAG) to alanine (GCA), and the resultant introduction of a unique MslI restriction enzyme site in the mutant allele . The K525A mutant protein has the same predicted molecular mass (~133kDa) as wild-type Recql4 and neither size nor abundance of the protein were affected by the mutation when assessed in thymocyte derived protein samples   . Heteroz 0.6255) . Further 0.6255) . Both maanalysis . Collect alleles , 22, theRecql4 resulted in a fully penetrant bone marrow (BM) failure [Recql4K525A/+ and Recql4K525A/K525A mice. Analysis of the peripheral blood (PB) revealed no changes in leukocyte or platelet numbers , the Pre-B, Pro-B, and Pre-Pro-B subpopulations were all unaltered in Recql4K525A/+ and Recql4K52A/K525A compared to WT littermates (- c-kit+ Sca-1+ CD34/Flt3)) fractions, nor in their myeloid committed subpopulations (-c-kit+Sca-1-CD34/FcγRII) and colony forming unit-erythroid  in the Recql4K525A/+ animals, however the absolute change was very small and not statistically significant in the Recql4K525A/K525A mice. The basis for this mild elevation in RBC counts and committed erythroid progenitors is currently undefined. In summary, unlike for complete deletion of Recql4, the abrogation of Recql4 helicase activity does not substantively perturb hematopoiesis in vivo.Within the BM, the total numbers of leukocytes were comparable across genotypes . Similartermates , as weretermates . The freulations . There win vitro B and T cell development from murine Recql4Δ/Δ hematopoietic cells [in vivo, T and B cell development was assessed from thymocytes and splenocytes respectively in 10-week-old mutant and WT mice. There was no difference in thymus cellularity or in the numbers of double positive CD4+/CD8+ nor the mature single positive CD4+ and CD8+ cells . No shift of B cells in the follicular or marginal zone compartments was apparent  was able to rescue ic cells . To dete8+ cells . Analysitermates . In the termates . To deteapparent . TherefoRecql4+/K525A and Recql4K525A/K525A spleens and stimulated them in vitro using bacterial lipopolysaccharide (LPS) and rmIL-4 to induce proliferation and immunoglobulin gene (Ig) class switch recombination. The proliferation of the cells in response to LPS was the same irrespective of Recql4 helicase status as measured by cell-trace violet dilution kinetics  by immortalization with HoxB8 [Recql4fl) allele. This resulted in the cells either becoming Recql4 heterozygous (Recql4Δ/+) or expressing only the K525A mutant allele (Recql4Δ/K525A). Isogenic cells from each genotype (pre-treated with tamoxifen for 4 days or untreated) were seeded in 96-well plates and the response to four different genotoxic agents was assessed: doxorubicin (topoisomerase II inhibitor), hydroxyurea (ribonucleotide reductase inhibitor), 4-nitroquinoline (oxidative DNA damage) and topotecan (topoisomerase I inhibitor). Cell viability was measured after 48 hours using the CellTiter-Glo Luminescent assay. As shown in R26-CreER Recql4Δ/K525A had a comparable IC50 to Doxorubicin, Hydroxyurea, 4-Nitroquinoline, and Topotecan as the non-tamoxifen treated isogenic controls  mutagenesis induced truncated mutation R347* (R355 in humans). Three RTS patients have been reported with p.Arg350GlyfsX21 mutations, mapping closely to this allele [Recql4 locus as this mutation was predicted to be benign. We crossed all the individual point mutant alleles  to the R26-CreER Recql4fl/fl line that we previously described and assessed the R26-CreER Recql4fl/PM in parallel with the R26-CreER Recql4fl/+ and R26-CreER Recql4fl/fl allele. [Recql4 floxed allele broadly throughout the body. Using this experimental design, the tamoxifen treated R26-CreER Recql4fl/fl animals (completely Recql4 deficient) developed fully penetrant BM failure [Recql4 deletion was confirmed by PCR for genomic recombination in vivo.  allele. . At 8–10 failure . The effbination .Recql4Δ/Δ). The K525A and M789K mutation, as well as the heterozygous (Recql4fl/+) control, did not show any significant change in leukocyte or erythroid indices. In addition, analysis of individual lineages within the PB showed a similar pattern across granulocytes, B lymphocytes, and CD4+ and CD8+ T lymphocytes in the truncating and null mutant (+Ter119+) from R26-CreERki/+Recql4Δ/G522Efs and Recql4Δ/R347* mice showed a dramatic decrease, consistent with the BM failure phenotype we had previously described in Recql4Δ/Δ , it was mutants . Similarextracts . Murine extracts . When thextracts  and a trextracts . Since ain vitro studies have pointed to the importance of the N-terminal Sld2-like region. Lethality of RECQL4-depleted chicken cells was rescued by expression of the N-terminal region only [Xenopus laevis and human cell extracts [in vivo experiments however, we have shown that one copy of the N-terminal region alone (R347* or G522Efs) is insufficient for viability, indicating that a certain level of expression or localization of full-length Recql4 protein is required even if it has no ATP-ase or unwinding capacity.A range of prior ion only . In addiextracts , 33. In The high frequency of chromosome abnormalities found in cells from RTS patients and the increased cancer incidence rates, suggest that RECQL4 may have a role in maintaining genome stability through DNA repair . Prior sin vivo analysis we did not detect any defect in T cell maturation at the CD4+/CD8+ double positive to CD4+ and CD8+ single positive transition or any earlier stage, nor did the mice develop any T cell lymphomas as a result of chromosomal anomalies. In addition, in vitro B cell activation and class switch recombination in helicase-dead splenic B lymphocytes was indistinguishable from that in WT cells, arguing that the helicase activity is not required for either physiological checkpoint activation or DNA damage repair.A recent study showed that RECQL4-depleted U2OS cells were also deficient in ATM dependent checkpoint activation in response to drug induced DNA DSBs. Complementation assays using helicase-inactive point mutants in Walker A (K508G) or Walker B motif (D605A and E606A) further indicated that this was the result of a lack of helicase activity . The ATMRECQL4 gene [Recql4 null. This was not seen in the helicase-dead K525A, the M789K or the WT heterozygous null mice. Furthermore, when we assessed BM, PB, thymus, and spleen from heterozygous and homozygous K525A helicase-dead mutants, we did not find any significant change. A similar observation was made for the human WRN helicase. A naturally occurring single nucleotide polymorphism (R834C) was shown to have less than 10% of the WT helicase activity, but normal exonuclease activity. None of the heterozygous or homozygous carriers of this mutation developed Werner Syndrome , clearly separating the WRN helicase function from other WRN functions [in vivo in mammals.RTS patients, however, usually present with compound heterozygous mutations. It was reported that in 46% of RTS patients compound heterozygous mutations were present in the unctions . Our finCollectively, this study has demonstrated the ATP-dependent helicase activity of Recql4 is not physiologically essential for murine embryonic development or adult homeostasis, cellular replication and physiological DNA damage repair. However, mutations that create truncating proteins are not tolerated. Further studies will have to be performed to elucidate the complex interactions of Recql4 mutations, their role in RTS and the contribution of the individual Recql4 domains to its normal physiological function.2 asphyxiation or cervical dislocation.All animal experiments were approved by the Animal Ethics Committee, St. Vincent’s Hospital, Melbourne, Australia (#007/14 and 011/15). Animals were euthanized by COg 30 minutes. Anti-Flag M2–Affinity Gel (Sigma-Aldrich) resin was added and incubated for 60 minutes. Resin was extensively washed with TNG (without PI or EDTA), then washed overnight in TNG containing Benzonase nuclease (Sigma-Aldrich). Further washes were performed to remove the nuclease. Subsequently Recql4 was eluted with 100ug/ml Flag peptide in TNG.The full-length WT and K525A mutant codon optimized cDNA sequence of the mouse Recql4 containing 3xFLAG tag at the C-terminus were cloned into vector pFL-EGFP and transferred to the Multibac expression system to generate baculovirus . Baculov2, 0.5mM TCEP) were assayed in an EnSpire 2300 microplate reader (Perkin Elmer) at 25°C. The DNA substrate  contained a 3′-15nt polyT ssDNA loading site and a 15nt dsDNA part with a generic sequence. After recording baseline fluorescence for 60s (Excitation 530nm / Emission 580nm), the helicase reaction was initiated by adding ATP to a final concentration of 1.25mM and the increasing Cy3-fluorescence as the quencher-labelled bottom-DNA strand is separated from the Cy3-labelled top-DNA strand, was recorded for 5 min. Measurements using H2O in place of ATP as well as reactions with buffer instead of protein served as blank and were subtracted from the ATP-data.Helicase assays were performed according to Kaiser et al . 80μl re2, 0.5mM TCEP). Color change was measured at Abs650nm in an EnSpire 2300 microplate reader (Perkin Elmer) at 25°C.PiColorLock phosphate detection reagent (Expedeon) was used to measure the presence of inorganic phosphate (Pi) release from ATP as a measure Recql4 ATPase activity. The proteins were assayed at 115nM in the presence of 1mM ATP and DNA in assay buffer  was used to measure the relative binding of WT versus K525A mutant Recql4 to DNA binding. Protein was serially diluted from 300nM to 19nM and bound to 25nM single stranded DNA oligo XOm1 conjugated to IRDye680. Bound Protein-DNA complex was separated on a 6% TBE/Acrylamide gel. The gel was directly imaged on a Li-Cor Odyssey CLx near-infrared fluorescence imaging system (Millennium Science).Recql4K525A and Recql4G522Efs mice were generated using Cripsr/Cas9 methods by the Mouse Engineering at Garvan/ABR (MEGA) services . Lysine 525 was mutated to Alanine (AAG>GCA) in single cell C57Bl/6 embryos via sgRNA-directed gene targeting and homologous recombination with a single stranded DNA oligonucleotide substrate. Viable pups were screened by DNA sequencing and one C57Bl/6 male carrying the K525A mutation on one allele and a 2bp insertion (GA) after the T521 codon (G522Efs) on the other allele was identified as a founder. The chemically (ENU) induced Recql4M789K and Recql4R347X mutations were obtained from the Australian Phenomics Facility . Recql4fl/fl mice (C57BL/6-Recql4tm2272Arte) have been previously described [Rosa26-CreERT2 mice on a C57Bl/6 background were purchased from The Jackson Laboratory (B6.129-Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J, Stock Number: 008463) and have been previously described [escribed , 44. Rosescribed . All linGenotyping of the K525A mutants was performed by PCR using the following primers: mRecql4 K525A MO36-F9: 5’-TAGACCTTATGAAACCTCAAAGCC-3’ and mRecql4 K525A MO36-R3: 5’- AGAACATTGGGCATTCGGC-3’ to yield a 591bp product, which was then digested with MslI (NEB) restriction enzyme to generate two fragments of 416 and 175bp for the WT or three fragments of 361, 175 and 55bp for the K525A mutant. Primers for the M789K mutants are: mRecql4 M789K 1F: 5’- AATAGGTGGCAATGGGCAGG-3’ and: mRecql4 M789K 1R: 5’-GCACTCGGCGAAAGGATACA-3’ yielding a 420bp PCR product, uncut by MslI when M789K mutant, but cut in two (277 and 143bp) when WT. The presence of the G522Efs and R347X mutations was determined by KASP  technology (LGC) with custom designed (G522Efs) or facility provided  sequences according to manufacturer instructions.Thymocyte lysates were prepared in RIPA buffer  plus Complete protease inhibitor  and PhosStop  tablets. 25μg whole protein extracts were fractionated on pre-cast NuPAGE BOLT 8% Bis-Tris polyacrylamide gels (Invitrogen) and transferred onto Immobilon-P PVDF membranes (Merck Millipore). Membranes were blocked with 5% milk in TBST and incubated overnight with rat monoclonal anti-mouse Recql4 antibody  or mouse anti-β-Actin . Membranes were then probed with HRP-conjugated goat anti-rat  or anti-mouse  secondary antibodies and visualized using ECL Prime Substrate (Amersham).4Cl, 10mM KHCO3, 0.1mM Na2EDTA, pH7.3).Peripheral blood was analyzed on a hematological analyzer . For flow cytometric analysis, red blood cells were lysed using a red blood cell lysis buffer , CD41, CD105, CD150, either biotinylated or conjugated with phycoerythrin, phycoerythrin-Cy7, peridinin chlorophyll protein-Cy5.5, allophycocyanin, allophycocyanin eFluor780, eFluor660 or eFluor450 were all obtained from eBioscience, BioLegend or BD Pharmingen  20, 45,. Biotiny6 cells per well were cultured in 6-well plates for 4 days in RPMI supplemented with 10% FCS, 100U/ml penicillin, 100ng/ml streptomycin, 2mM L-glutamine, 1 x MEM nonessential amino acids, 1mM sodium pyruvate, 50μM ß-mercaptoethanol, 15μg/ml LPS  and 10ng/ml recombinant murine IL-4 , and stained with CellTrace Violet  and rat anti-mouse IgG1-APC  [B cells were purified from single cell spleen suspensions using a B Cell Isolation kit ; 10 550874) . StainedR26-CreERT2Recql4fl/+ (control) and R26-CreERT2Recql4fl/K525A cells were maintained in IMDM, 10% FBS (non-heat inactivated) and 1% GM-CSF containing media (BHK-HM5 cell conditioned media). The cells were treated for 4 days with 400nM 4-hydroxy tamoxifen (Merck Millipore) then genotyped to confirm complete recombination. Cells were then plated at 10,000 cells/well in 96 well plates  and incubated for 48 hours with the indicated concentration of drugs in triplicates per dose . Doxorubicin was obtained from St. Vincent’s Hospital Pharmacy. Hydroxyurea was purchased from Selleck. 4-Nitroquinoline and Topotecan were purchased from Sigma-Aldrich. Cell viability was measured using ATP-Lite (Perkin Elmer) as directed by the manufacturer and measured on an EnSpire plate reader (Perkin Elmer). Data were plotted and the IC50 value calculated using Prism 7 software. The dose-response curve was plotted as mean±SEM.Hoxb8 immortalized  R26-CreES1 FigGenomic PCR showing recombination of the floxed allele to produce the excised product of the expected size (left) and genotyping PCRs of the point mutants (right) of the following alleles: (A) M789K. (B) K525A. (C) G522Efs. (D) R347*.(TIF)Click here for additional data file.S2 FigData from COSMIC database. Image generated using Protein painter .(TIF)Click here for additional data file.S3 Fig50 values in R26-CreER Recql4Δ/K525A and R26-CreER Recql4Δ/+ with the non-tamoxifen treated isogenic controls. The X axis shows the individual drug doses of (A) Doxorubicin, (B) Hydroxyurea, (C) 4-Nitroquinoline, (D) Topotecan.Data showing the response to each individual drug dose used to calculate the IC(TIF)Click here for additional data file.S1 TableList of antibodies used for flow cytometry in this study.(PDF)Click here for additional data file.


P16 (CDKN2Aink4a) gene is an endogenous CDK4/6 inhibitor. Palbociclib (PD0332991) is an anti-CDK4/6 chemical for cancer treatment. P16 is most frequently inactivated by copy number deletion and DNA methylation in cancers. It is well known that cancer cells with P16 deletion are more sensitive to palbociclib than those without. However, whether P16 methylation is related to palbociclib sensitivity is not known. By analyzing public pharmacogenomic datasets, we found that the IC50 of palbociclib in cancer cell lines (n = 522) was positively correlated with both the P16 expression level and P16 gene copy number. Our experimental results further showed that cancer cell lines with P16 methylation were more sensitive to palbociclib than those without. To determine whether P16 methylation directly increased the sensitivity of cancer cells to palbociclib, we induced P16 methylation in the lung cancer cell lines H661 and HCC827 and the gastric cancer cell line BGC823 via an engineered P16-specific DNA methyltransferase (P16-Dnmt) and found that the sensitivity of these cells to palbociclib was significantly increased. The survival rate of P16-Dnmt cells was significantly lower than that of vector control cells 48 hrs post treatment with palbociclib (10 μM). Notably, palbociclib treatment also selectively inhibited the proliferation of the P16-methylated subpopulation of P16-Dnmt cells, further indicating that P16 methylation can increase the sensitivity of cells to this CDK4/6 inhibitor. These results were confirmed in an animal experiment. In conclusion, inactivation of the P16 gene by DNA methylation can increase the sensitivity of cancer cells to palbociclib.The  The ability to sustain uncontrolled cell proliferation is one of the hallmarks of cancer cells . The norThe clinical implementation of first-generation nonselective CDK inhibitors was originally hampered by the high toxicity and low efficacy of these agents , 8. SecoCDKN2Aink4a gene, is an endogenous cellular CDK4/6 inhibitor that controls the G1-S phase transition of the cell cycle. This gene is one of the most frequently inactivated genes in cancer genomes; it is inactivated mainly by DNA methylation  [https://www.cancerrxgene.org/translation/Drug/1054#t_IC50] [rg/ccle] . The hal#t_IC50] .2.Human lung cancer cell lines H1975, H1395, H661, H596, H460, H358, H292 and HCC827, as well as human breast cancer cell lines HCC1937, MDA-MD-468 and MDA-MD-157, were purchased from the National Infrastructure of Cell Line Resource . Human lung cancer cell lines H1299 and A549; human breast cancer cell lines MDA-MB-231 and MCF7; human gastric cancer cell lines BGC823, MGC803 and AGS; human colon cancer cell lines HCT116, RKO and SW480; and human liver cancer cell line HepG2 were obtained from laboratories at Peking University Cancer Hospital and Institute. The H596 and MCF7 cell lines were cultured in DMEM  containing 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco), the AGS cell line was cultured in F12 medium (Gibco), and the remaining cell lines were cultured in RPMI 1640 medium (Gibco). All cells were maintained at 37°C in humidified air with 5% CO2O as a stock solution at concentration of 10 mM. Cells were seeded in 96-well plates at 4,000 cells per well (4 wells/group). Forty-eight hrs post treatment with various concentrations of palbociclib, cell confluence was evaluated and analyzed using the IncuCyte ZOOM system . The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 6 software.The CDK4/6 inhibitor palbociclib (PD0332991) was purchased from Selleck Chemicals  and dissolved in double-distilled HP16 transcription start site, an engineered P16 promoter-specific seven zinc finger protein (7ZFP) was fused with the catalytic domain of mouse Dnmt3a (approximately 608–908 aa) and integrated into the pTRIPZ vector, which contained a “Tet-On” switch . The lung cancer cell lines H661 and HCC827 and the gastric cancer cell line BGC823 were infected with lentiviral particles containing the P16-Dnmt or control vector and incubated for 48 hrs. Then, puromycin  was added to the medium  to kill nontransfected cells. The pooled cells treated with puromycin for two weeks were considered stably transfected cells. Then, these cells were treated with 0.25 μg/mL doxycycline  for 14 days to induce P16-Dnmt expression.The established P16-Dnmt expression vector and pTRIPZ empty control vector were prepared and used to transfect cells as previously described . BrieflyGenomic DNA was extracted from cells or tumor tissues and subjected to bisulfite treatment using an EZ DNA Methylation-Gold Kit  according to the manufacturer’s instructions. The modified DNA was stored at -20°C until use.P16 CpG islands was assessed by a 150/151-bp methylation-specific PCR (MSP) or 115-bp quantitative MethyLight assay as previously described .The methylation status of escribed –34. Brieht assay . All PCRP16 mRNA was performed using primer sets shown in GAPDH was used as the reference gene. The amplification was performed with FastStart Universal SYBR Green Master (ROX)  in the Applied Biosystems 7500 Real-Time PCR System, as previously described  via oral gavage (i.g.) daily for 3 weeks. Then, mice were sacrificed by spine dislocation, and the tumors were weighed. Tumor samples were processed for formalin-fixed and paraffin-embedded (FFPE) sections or Western blot analysis.HCC827 cells  sections were incubated with primary antibody against Ki-67 (1:300) overnight at 4°C. The remaining IHC steps were the same as previously described .P16 (CDKN2Aink4a) mRNA level or copy number in various cancer cell lines was assessed using the nonparametric Spearman correlation test and a linear regression model. The Mann–Whitney test was used for nonnormally distributed data. Student’s t-test was used for normally distributed data. Statistical significance was accepted at p<0.05.Statistical analyses were performed using SPSS 23 software or GraphPad Prism 6 software. The Kolmogorov–Smirnov test and Shapiro–Wilk test were used to estimate the normality of distributions. The relationship between the IC50 value of palbociclib and the relative P16 methylation and the sensitivity of cancer cells to palbociclib, we first analyzed the correlation between the IC50 levels of palbociclib in 522 cell lines and the transcription level and copy number of the CDKN2A/P16 gene using public pharmacogenomics datasets [CDKN2A mRNA level , P16-methylated (P16-M), and P16-deleted (P16-D) groups according to the results of MSP (P16-U cell lines (n = 12) were consistently higher than those in the P16-M cell lines (n = 5) and P16-D cell lines (n = 5) [18.88 vs. 4.61 and 3.99 (μM), p<0.01; P16-M and P16-D cell lines. These results indicate that similar to copy number deletion, P16 methylation is positively associated with high sensitivity of cancer cells to palbociclib.Then, we validated the correlation in 22 cancer cell lines, which were divided into s of MSP . We founP16 methylation directly increases the sensitivity of cancer cells to palbociclib, we stably transfected three P16-U cell lines  with the P16-Dnmt vector and induced P16-Dnmt expression in these cells by doxycycline treatment for 2 weeks. P16 methylation was successfully induced in these P16-Dnmt cells, as evidenced by MSP  cells is selectively inhibited by palbociclib treatment  or control buffer for three weeks. No adverse event was observed in these mice. Compared with control buffer, palbociclib significantly inhibited the growth of P16-Dnmt tumors  did not improve outcomes of the administration of palbociclib plus letrozole in HR-positive breast cancer patients [CCND1 amplification vs. CDKN2A and CDKN2B codeletion trended toward a longer vs. shorter treatment duration [P16 methylation could increase the resistance of lung (and stomach) cancers to paclitaxel treatment in a clinical trial [P16 methylation can increase the sensitivity of lung and stomach cancers to palbociclib. It is worth performing clinical trials to assess whether P16 methylation could be used to predict the therapeutic efficacy of CDK4/6 inhibitors such as palbociclib in patients with lung and stomach cancers.In the phase II clinical study PALOMA-1, it was found that patient selection based on patients . In the patients , 40. In duration . We receal trial . In contP16 methylation can increase the sensitivity of cancer cells to the CDK4/6 inhibitor palbociclib. P16 methylation may be used as a predictive marker for sensitivity to CDK4/6 inhibitors.This study demonstrates that S1 FigThe results of immunofluorescence and confocal microscopy analyses to directly detect alterations in the subpopulation of cells staining positive for P16 protein within HCC827 lung cancer cells stably transfected with P16-Dnmt. Scale bar, 400 μm. Student’s t-test, **p<0.01.(TIF)Click here for additional data file.S2 Figp<0.01.The results of immunofluorescence and confocal microscopy analysis to directly detect alterations of the subpopulation of cells staining positive for P16 protein within the BGC823 gastric cancer cells stably transfected with P16-Dnmt. Scale bar, 400 μm. Student’s t-test, **(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.