We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy.Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the  in vivo environment. Most importantly, by combination of this NS-mouse model with mutant mice individually or simultaneously deficient for proteases KLK5 and KLK7, we elucidated the complex proteolytic networks that are dysregulated in the absence of LEKTI. We show that although the single ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency, simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality. Our results also provide novel insights into the roles of KLK5 and KLK7 in the inflammation and differentiation defects that are associated with NS. Based on these findings, we propose that both, KLK5 and KLK7 should become targets for NS therapy.Netherton syndrome (NS) is a genetic skin disorder caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. In this work, we aimed to explore the molecular mechanisms underlying this disease using a novel mutant mouse model for NS, which is based on mimicking a causative mutation known from human patients. This novel model reproduces the symptoms of NS and thus provides a useful tool to study the NS pathology in a complex  These conditions may improve with age and older patients often show less severe ichthyosis exhibiting erythematous plaques with double-edged scales at the periphery [Netherton syndrome (NS) is a life-threatening autosomal recessive disorder that affects approximately one in 200 000 newborn children ,2. Newboeriphery –3. The heriphery . NS may eriphery ,5.SPINK5 gene  that encodes LEKTI , an inhibitor of serine proteases expressed in the epidermis and other stratified epithelia [stratum corneum (SC) and disruption of the epidermal barrier in NS patients [NS is caused by mutations in pithelia . Full lepithelia ,8. LEKTIpithelia –11 as wepithelia –14. Unrepithelia , which cpithelia ,17. KLK5pithelia . Prematupatients . Recentlpatients  or by dipatients . In addipatients –24.in vivo roles of KLK5 and KLK7 using a set of mouse models that are simultaneously deficient for KLK5 and KLK7 on the genetic background of Netherton syndrome-like mouse model based on a mutation found in human patients. The close proximity of these genes (on the same locus) has so far prevented the generation of suitable animal models and therefore the in vivo roles of KLK5 and KLK7 could not be studied concurrently. Our study shows that individual functional ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of Spink5 mutation. In contrast, simultaneous deficiency of both KLK5 and KLK7 completely rescues the lethality allowing adult mice to survive to adulthood with a fully functional skin barrier.In this study, we revealed the in vivo, we generated a new mouse model mimicking a causative mutation of SPINK5 gene  previously described in human patients [SPINK5 nucleotide sequences, deletion of TG nucleotides at positions 402 and 403 of murine Spink5 (402delTG) produces the premature termination codon (PTC) at a similar position as described in human patients (p.A135X) (Spink5 in combination with a single stranded oligonucleotide (ssODN) carrying the desired mutation (XbaI restriction site as a marker (+/A135X) did not show any obvious phenotype and were used to obtain Sp5A135X/A135X mice, which showed dramatic downregulation of Spink5-RNA expression  . To intrmutation . Foundera marker . Heterozpression . The prequencing . Sp5A135ial area  and died-/-, Klk7-/-, Klk5-/-Klk7-/-), which were crossed with Spink5+/A135X line  when they exhibited reduced body-size and dry skin with severe scaling throughout the body surface  penetration assay and showed severe skin barrier disruption marked by penetration of TB through large areas of the body, mainly the abdomen, paws, and head. Ablation of KLK7 on the Sp5A135X/A135X background did not improve the barrier and Klk7-/-Sp5A135X/A135X newborns showed similar barrier disruption to Sp5A135X/A135X. In contrast, Klk5-/-Sp5A135X/A135X newborns developed less severe barrier phenotype characterised by multiple small stained patches and in Klk5-/-Klk7-/-Sp5A135X/A135X pups, the barrier integrity was almost completely recovered and the mice showed TB staining only in the area of nostrils  in P0 pups over time. Consistently with previous analyses of epidermal barrier properties, we found significantly impaired water retention in Sp55X/A135X .-/-Sp5A135X/A135X died at P5, mice were stained with TB at P5. Interestingly, Klk5-/-Sp5A135X/A135X mice showed clear penetration of the dye in the epidermis adjacent to hair shafts whereas this barrier defect was completely rescued in Klk5-/-Klk7-/-Sp5A135X/A135X  and markedly increased expression of keratin 6 (Krt6), suggesting hyperproliferation of keratinocytes in Sp5A135X/A135X embryos  are initiated by KLK7 activity which is independent of KLK5. Indeed, KLK7 was previously shown to induce inflammation and keratinocyte proliferation in the epidermis [in vitro [in vitro [Association of NS with abnormal epidermal differentiation accompanied by acanthosis, parakeratosis, and hyperproliferation of keratinocytes was previously reported in Spink5-deficient mouse models ,26,27 anytokines –24. In tpidermis ,39 and ain vitro . The mecin vitro  and thusin vitro , which cA135X/A135X. Only the concurrent ablation of both KLK5 and KLK7 can fully rescue the lethal phenotype of Sp5A135X/A135X mice, therefore both proteases should be investigated as clinical targets. We show that KLK7 plays an important role in the inflammation and defective differentiation in NS and its activity is not dependent on activation by KLK5. We also show that the pathological effects of unregulated KLK activities are remarkably age dependent. Altogether, this study expounds the complexity of the proteolytic network and its regulation, which are especially important to understand Netherton syndrome and its treatment.In summary, we show that the individual inactivation of KLK5 or KLK7 only partially rescues the defective skin barrier but not the lethal phenotype of Sp5All animal studies were ethically reviewed and performed in accordance with European directive 2010/63/EU and were approved by the Czech Central Commission for Animal Welfare.Klk5 was produced by introduction of targeting construct  via homologous recombination in embryonic stem cells (ESC). Positively targeted ESC were injected into developing wt embryos, to produce chimeric mice, which were used to establish Klk5-/- line.Knock-out first allele of Spink5 gene were designed using TAL Effector Nucleotide Targeter 2.0  [Spink5 gene (XbaI restriction enzyme (Thermo-Scientific).TALENs targeting exon5 of ll.edu/) ,44, assell.edu/) , and cloll.edu/) . DNA binnk5 gene  consistenk5 gene  and the Klk7 contained following repeats: NN-NG-NI-NI-NI-NN-NI-NI-NN-NN-HD-NG-HD-NN-HD (5´ TALEN-Klk7) and NN-NI-NG-NG-NN-HD-HD-NG-NG-NG-NN-NI-NN-HD-NI-NN (3´ TALEN-Klk7). TALEN mRNA with total RNA concentration of 40 ng/μl was microinjected into Klk5-/-oocytes. Genomic DNA isolated from tail biopsies of newborn mice was screened by PCR (primers F3: 5´- GGAGAAGGCCAGGGTCTGAA-3´ and R3: 5´- TGGTCAGAAACCCACGGAGA-3´) and subsequently analyzed by RFLP using NcoI restriction enzyme (Thermo-Scientific).TALENs were designed and synthesized as described above. DNA binding domains of TALENs specific for exon3 of murine Newborn pups from at least two independent litters were separated from mothers to prevent fluid intake. The rate of water loss was analyzed by measuring the reduction of initial body weight at 1h, 2h, 3h and 4 h.Newborn mice were euthanized and then dehydrated by incubation for 5 min in 25, 50, 75, and 100% methanol. After rehydration in PBS, mice were incubated for 4 hours in 0.1% toluidine blue (Sigma-Aldrich), washed in PBS and imaged.Newborn pups or skin tissues were fixed in 3.6% formaldehyde for 24 h and embedded in paraffin. 5-μm sections were prepared using microtome were stained by hematoxylin/eosin (H&E) or by 0.5% toluidine blue using standard protocols. Images were obtained using Zeiss Axioscan Z1 (Carl Zeiss AG).Dorsal skin of P5 pups was fixed in 3.6% formaldehyde for 24 hours and embedded in paraffin. 5-μm paraffin sections were used for antigen retrieval with Discovery Ultra automated IHC/ISH system (Ventana) and stained with antibodies against Dsg-1  and CDSN . After 1 hour incubation at room temperature, anti-rabbit peroxidase conjugated polymer (Zytomed GmBH) was applied for 30 min and the reaction was developed using DAB (DAKO) as a chromogen. Images were obtained using Zeiss Axioscan Z1 (Carl Zeiss AG).In order to stain the cryosections, dorsal skin of E18.5 dpc embryos or P5 pups was isolated, embedded in Tissue-Tek O.C.T (Sakura), and frozen at -80°C. 6 μm-sections were stained as described previously , using a2 (Sigma-Aldrich) and 0.25mM of each primer. Respective gene expression was normalized to the expression of TATA-binding protein (TBP). Normalized expression levels were then re-expressed relative to the mean expression level of the respective target in the wt mice. Primer sequences are detailed in Dorsal skin was obtained from newborn pups, crushed in liquid nitrogen and total RNA was isolated using TRIzol (Thermo-Scientific) according to the manufacturer's instructions. Residuals of genomic DNA were removed using 1 U of DNAse I (Roche) per 1 μg of RNA by 15 min incubation at 37°C. 1 μg of total RNA was used for reverse transcription by M-MLV Reverse Transcriptase (Promega) using oligo (dT) primers. RT-PCR was performed in a 20-μl reaction mixture containing SYBR Green JumpStart™ Taq ReadyMix with MgCl2 in a K 850 unit (Quorum Technologies Ltd). The dried samples were sputter-coated with 20 nm of gold in a Polaron Sputter-Coater (E5100) (Quorum Technologies Ltd). The final samples were examined in a FEI Nova NanoSem 450 scanning electron microscope (FEI) at 5 kV using secondary electron detector.The samples on cellulose filter paper strips were fixed with 3% glutaraldehyde in cacodylate buffer overnight at 4°C. After fixation, extensively washed samples were dehydrated through ascending alcohol concentrations followed by absolute acetone and critical point drying from liquid COThe levels of TNFα, IL-1β, IL-9, and IL-17 in mouse serum were analyzed using Bio-Plex Pro Mouse Cytokine Assay (Bio-Rad Laboratories) with high sensitivity range standard settings according to manufacturer’s instructions.S1 FigA135X/A135X pups, mutation present in Sp5A135X/A135X is underlined.Successful targeting was confirmed by sequencing of cDNA obtained from Sp5(TIF)Click here for additional data file.S2 Fig(A) Schematic of Klk5 knock-out allele, which is present in Klk5-/-, Klk5-/-Klk7-/-, Klk5-/-Sp5A135X/A135X and Klk5-/-Klk7-/-Sp5A135X/A135X mutant mice (B)Klk5 (loxP) allele was generated by breeding of Klk5-/-Klk7-/- mice with FLPe expressing strain. FLPe activity leads to excision of FRT flanked cassette present in the Klk5 knock-out allele, which leads to the restoration of Klk5 expression. Klk5 (loxP) allele is present in Klk7-/- and Klk7-/-Sp5A135X/A135X mutant mice.(TIF)Click here for additional data file.S3 Fig(A) Exon organization of Klk7 gene. TALEN-binding sequences are marked with red underline and NcoI restriction site that was used to analyse the targeting is marked with black underline. (B) Positively targeted founders  were analyzed by sequencing of exon 3 of Klk7, founder Klk7 ex3-A with targeted allele containing 20 bp deletion in exon 3 was selected for further breeding to establish Klk5-/-Klk7-/- line. (C) Klk7-targeting site was analyzed by sequencing; cDNA was obtained from Klk5-/-Klk7-/- animals. (D) KLK7 deficiency in Klk5-/-Klk7-/- was confirmed by western blot using anti-KLK7 antibody [antibody .(TIF)Click here for additional data file.S4 FigA135X/A135X, Klk7-/-Sp5A135X/A135X, Klk5-/-Sp5A135X/A135X, and Klk5-/-Klk7-/-Sp5A135X/A135X muzzle area 12 hours after birth. Whiskers of Spink5-/- pups were sparse or completely missing in comparison to wt pups. Spink5-/-Klk5-/- pups did not show any absent hair, however hair shafts were thinner and twisted when compared to wt. Despite severe epidermal defects, vibrissae hair of Klk7-/-Sp5A135X/A135X showed almost normal appearance apart from a reduced length and uneven distribution. No hair defects were observed in Klk5-/-Klk7-/-Sp5A135X/A135X P0 animals.Magnification of wt, Sp5(TIF)Click here for additional data file.S5 Fig(A) and E18.5 dpc embryos (B) wt, Sp5A135X/A135X, Klk7-/-Sp5A135X/A135X, Klk5-/-Sp5A135X/A135X, and Klk5-/-Klk7-/-Sp5A135X/A135X lines. Although there were no significant differences in the weight of 18.5 dpc embryos between the individual lines, newborn (P0) Sp5A135X/A135X and Klk7-/-Sp5A135X/A135X pups showed significant reduction of body weight when compared to wt mice, as well as to Spink5+/+ littermates. The weight of Klk5-/-Sp5A135X/A135X and Klk5-/-Klk7-/-Sp5A135X/A135X newborn mice was comparable to wt animals and to Spink5+/+ littermates. Error bars represent standard deviations of mean, data were analysed by One-way ANOVA followed by Bonferroni post-hoc tests. n>5, ** p < 0.01.Body weight of 12 hours old newborn pups (TIF)Click here for additional data file.S6 FigA135X/A135X P0 pups showed occasional focal parakeratosis (white arrows) (A) and detachment of SC (double arrow) (B). SC detachment was found also in Klk7-/-Sp5A135X/A135X P0 pups. Sections were stained by hematoxylin and eosin. Scale bar, 100 μm.Apart from the differentiation defects described in (TIF)Click here for additional data file.S7 Fig-/-Sp5A135X/A135X mice. Scale bar, 100 μm.Skin sections obtained from P5 pups were stained with toluidine blue. Increased infiltration of mast cells (black arrowheads) was found in Klk5(TIF)Click here for additional data file.S8 Fig-/-Klk7-/-Sp5A135X/A135X. In contrast, only the inner root sheaths of non-hyperplastic hair follicles of Klk5-/-Sp5A135X/A135X P5 pups were CDSN-positive. No apparent staining was found at stratum granulosum/startum corneum interface or in the hyperkeratotic follicles. Scale bar, 100 μm.Skin sections obtained from P5 pups were stained with antibodies against DSG1 and CDSN. CDSN-positive staining was found in the inner root sheath (black arrowheads) and upper granular layer (asterisks) of wt and Klk5(TIF)Click here for additional data file.S9 Fig(A) Analysis of Klk14 expression in 18.5 dpc embryos. Klk14 was found to be expressed in the area of nostrils (black arrowhead) (B) Analysis of P5 skin sections revealed specific expression of Klk14 in hair follicles.Klk14 expression was monitored using a mutant strain carrying LacZ reporter under the control of Klk14 promoter. (TIF)Click here for additional data file.S1 TableSequences of primers used for qRT-PCR analysis.(DOCX)Click here for additional data file.


The JAK2V617F mutation is present in the majority of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The impact of this mutation on disease phenotype in ET and PMF is still a matter of discussion. This study aims to determine whether there are differences in clinical presentation and disease outcome between ET and PMF patients with and without the JAK2V617F mutation.In this single-center study, a total of 184 consecutive Philadelphia-negative chronic myeloproliferative neoplasms, 107 cases of ET and 77 cases of PMF, were genotyped for JAK2V617F mutation using the JAK2 Ipsogen MutaScreen assay, which involves allele-specific polymerase chain reaction.ET patients positive for JAK2V617F mutation had higher hemoglobin (Hb) and hematocrit (Hct) levels, lower platelet counts, and more prevalent splenomegaly at diagnosis compared to patients negative for the JAK2V617F mutation, but rates of major thrombotic events, arterial thrombosis, and venous thrombosis were comparable between the groups. At presentation, PMF patients with JAK2V617F mutation had significantly higher Hb and Hct levels and leukocyte counts than patients without the mutation. Similar to the findings of ET patients, thromboembolic rates were similar in PMF patients with and without theJAK2V617F mutation. For ET and PMF patients, no difference was observed in rates of death with respect to JAK2V617F mutational status. Moreover, leukemic transformation rate was not different in our PMF patients with and without JAK2V617F mutation.We conclude that JAK2V617F-mutated ET patients express a polycythemia vera-like phenotype and JAK2V617F mutation in PMF patients is associated with a more pronounced myeloproliferative phenotype. Philadelphia-negative chronic myeloproliferative neoplasms (Ph-negative MPNs) are a heterogeneous group including 3 major diseases: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Thrombotic events are the major cause of morbidity and mortality in ET. Other complications include hemorrhage and progression to myelofibrosis or acute myeloid leukemia ,2. PMF iA total of 184 consecutive Ph-negative MPN patients, 107 with ET and 77 with PMF, admitted to the Division of Hematology of the İstanbul University İstanbul Medical Faculty from 1995 to 2013 were included in the study. ET and PMF patients were diagnosed based on WHO criteria . InformeData were processed using SPSS 16 . Continuous variables were summarized as mean [standard deviation (SD)]. Chi-square statistics were used to compare categorical variables among the different patient groups categorized according to the JAK2V617F mutational status. Analysis of continuous variables among the groups was performed using the Mann-Whitney U test. A p-value of less than 0.050 was considered to indicate statistical significance; all tests were 2-tailed.A total of 184 patients (107 with ET and 77 with PMF) were included. Bone marrow fibrosis was detected in 90.7% (97 in 107) of ET and 100% of PMF patients. In ET patients, the grade of bone marrow fibrosis was scaled as follows: grade 0, 9.3%; grade 1, 62.7%; grade 2, 25.2%; and grade 3, 2.8%. All PMF patients had bone marrow fibrosis (grade 2 in 20.8% and grade 3 in 79.2%).JAK2V617F mutation was identified in 64 of 107 ET (59.8%) and 58 of 77 PMF (75.3%) patients (p=0.028). Clinical and laboratory correlates of ET patients according to JAK2V617F mutational status are summarized in JAK2V617F-positive and -negative ET patients showed no significant differences with respect to sex and age at diagnosis. ET patients with JAK2V617F mutation presented with higher hemoglobin (Hb) and hematocrit (Hct) levels and lower platelet count at diagnosis compared to patients without mutation . The leukocyte count and LDH levels were similar for the 2 groups.The 2 groups showed no significant difference with respect to mean spleen size. However, JAK2V617F-positive ET patients presented with more prevalent splenomegaly at diagnosis compared to patients without the mutation (p=0.044).ET patients with JAK2V617F mutation showed a higher, albeit not statistically significant, rate of bleeding events compared to the JAK2V617F-negative group .ET patients with and without JAK2V617F mutation showed no significant difference with respect to the degree of bone marrow fibrosis, prevalence of hydroxyurea use, and rate of splenectomy. In addition, no significant differences were observed in the use of other medical treatments in any of the categories (p>0.050). Duration of follow-up in patients with and without JAK2V617F mutation was 69.7 months (SD: 63.7) and 70.1 months (SD: 56.9), respectively (p=0.675). During follow-up, 3 of 64 (4.7%) JAK2V617F-positive ET and 2 of 43 (4.7%) JAK2V617F-negative ET patients succumbed to their disease (p=1.000).Clinical and laboratory parameters of PMF patients classified according to genotype are outlined in The rate of female patients was higher in the JAK2V617F-negative group compared to the JAK2V617F-positive group . PMF patients with and without JAK2V617F mutation showed no significant differences with respect to age at diagnosis. At initial diagnosis, PMF patients with the JAK2V617F mutation presented with significantly higher Hb and Hct levels and leukocyte counts compared to those without the mutation . Platelet count and LDH level did not differ between the 2 groups.The mean spleen size showed no significant difference among any of the categories, although PMF patients with JAK2V617F mutation showed a trend towards higher prevalence of massive splenomegaly at diagnosis compared to patients without mutation .JAK2V617F-positive PMF patients showed a trend towards a higher prevalence of bleeding events compared to JAK2V617F-negative PMF patients .There was no significant difference in the prevalence of total thrombotic events, arterial thrombosis, and venous thrombosis between JAK2V617F-positive and -negative PMF patients.The degree of reticulin fibrosis, prevalence of hydroxyurea use, rate of allogeneic hematopoietic stem cell transplantation (AHSCT), and history of splenectomy did not differ in any of the categories. In addition, the 2 groups showed no significant differences in the use of other medical treatments (p>0.050).No significant difference was observed in the distribution of karyotype categories and DIPSS-Plus risk stratification between JAK2V617F-positive and -negative PMF patients.Duration of follow-up in PMF patients with and without JAK2V617F mutation was 42 months (SD: 46.9) and 56.6 months (SD: 48.7), respectively (p=0.165). At the end of the data collection period, 11 of 58 (19%) PMF patients with JAK2V617F mutation succumbed to their disease, while the rate of death in patients without JAK2V617F mutation was 15.8% (p=1.000). During follow-up, rate of leukemic transformation was similar between the 2 categories.In our relatively large series of patients with Ph-negative MPNs, including 107 ET patients with a mean follow-up duration of more than 5 years and 77 PMF patients with a mean follow-up duration of more than 3 years, we documented that JAK2V617F mutation correlates with disease phenotype in adult Turkish patients with ET and PMF.Our results suggest that JAK2V617F positivity in ET induces a phenotype resembling PV. Confirming previous observations, we found that ET patients with JAK2V617F mutation presented with higher Hb and Hct levels and lower platelet counts compared to unmutated patients ,20,21,22In our series of 77 PMF patients, we found a significant association between JAK2V617F mutation and the expression of a more pronounced myeloproliferative phenotype. In PMF patients, JAK2V617F mutational status contributed to laboratory abnormalities, including higher Hb level and leukocyte count, but its association with platelet count is inconsistent . Our PMFCollectively, according to the results of our study, JAK2V617F mutation may identify distinct disease phenotypes of ET and PMF patients.Ethics Committee Approval: The study was approved by the Local Ethics Commitee of İstanbul University İstanbul Medical Faculty (file number: 2012/1571-1245), Informed Consent: Informed consent was obtained from all patients for being included in the study.


Drosophila melanogaster chromatin and a D. melanogaster-specific antibody into standard ChIP reactions. Specifically, the use of an antibody that exclusively recognizes the D. melanogaster histone variant H2Av enables precipitation of D. melanogaster chromatin as a minor fraction of the total ChIP DNA. The D. melanogaster ChIP-seq tags are used to normalize the human ChIP-seq data from DMSO and EZH2 inhibitor-treated samples. Employing this strategy, a substantial reduction in H3K27me3 signal is now observed in ChIP-seq data from EZH2 inhibitor treated samples.Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) has been instrumental in inferring the roles of histone post-translational modifications in the regulation of transcription, chromatin compaction and other cellular processes that require modulation of chromatin structure. However, analysis of ChIP-seq data is challenging when the manipulation of a chromatin-modifying enzyme significantly affects global levels of histone post-translational modifications. For example, small molecule inhibition of the methyltransferase EZH2 reduces global levels of histone H3 lysine 27 trimethylation (H3K27me3). However, standard ChIP-seq normalization and analysis methods fail to detect a decrease upon EZH2 inhibitor treatment. We overcome this challenge by employing an alternative normalization approach that is based on the addition of  ChIP-seq is a powerful and commonly used technique for the detection of transcription factor binding patterns and histone post-translational modification (PTM) occupancy profiles across the entire genome . ChIP-seIdentifying differences between data sets becomes more challenging when differences are not just occurring at specific sites across the genome, but involves global modification changes. An example would be a setting where an increase or decrease of a particular histone PTM occurs at all or most occupied sites across the genome, as is frequently the case when studying the effects of chromatin modifying enzyme inhibitors. Impairing the function of a histone methyltransferase (HMT) can result in a reduction in bulk methylation levels at the targeted histone residue, which in the case of H3K27 methylation affects a large part of the genome. In these instances, currently available bioinformatic-based normalization methods are not applicable since they assume invariance in the signal to noise ratio, the background signal level, or the height of shared peaks.Several recently described approaches alter the ChIP protocol by adding material that can be used to normalize the signal. For example, ChIP-Rx is based on the addition of a constant amount of reference cells from a different species, and allows for the genome-wide quantitative comparison of histone modifications across different biological samples . The metD. melanogaster-specific histone variant H2Av is added to all ChIP samples along with a constant amount of D. melanogaster chromatin. We employed this strategy effectively in two different cancer cell contexts using two different EZH2 inhibitors. Normalization using spike-in standards in these cell systems effectively uncover the genome-wide reduction in H3K27me3 occupancy upon inhibitor treatment, while other histone methylation marks not affected by EZH2 inhibitors were unaffected by the normalization. We suggest that this approach provides a broadly applicable solution for the normalization of ChIP-seq data sets.EZH2 is the major H3K27-specific HMT with an important role in transcriptional repression. Genomic and transcriptomic data have identified EZH2 as a candidate oncology target in a number of human malignancies, including prostate, breast and hematological malignancies , 10. Rec2 in RPMI-1640 (Invitrogen) supplemented with 10% FBS (HyClone) and antibiotics (Invitrogen). Cells were treated with 1 μM EZH2 inhibitor GSK126 in the same media with 5% FBS for 5 days. KARPAS-422, a human B cell lymphoma cell line containing a mono-allelic activating mutation in EZH2, was obtained from the Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures, and cultured at 37°C with 5% CO2 in RPMI-1640 medium (Invitrogen) supplemented with 10% FBS (HyClone) and antibiotics (Invitrogen). Cells were treated with 1.5 μM CPI-360 for 4 and 8 days. Schneider (S2), a D. melanogaster epithelial cell line, was obtained from ATCC and cultured at 28°C without CO2 in Schneider’s medium  containing 1% FBS. OSS, a D. melanogaster ovarian somatic sheath cell line, was obtained from the Drosophila Genomics Resource Center and cultured as described [PC9, a lung derived human adenocarcinoma cell line, was kindly provided by Dr. Kazuto Nishio  and cultured at 37°C with 5% CO2, 0.5% NP-40). KARPAS-422 cell pellet then was re-suspended in miccrococcal nuclease (MNase) reaction buffer  plus 20000 U of MNase (NEB M0247S) at 37°C for 15 min. Digestion was terminated by the addition of EGTA to 3 mM, SDS concentration was increased to 1%, and NaCl concentration was increased to 150 mM. Chromatin was sonicated at 15% output for 6 x 10 sec . Cell debris was removed by centrifugation at 20,000 x g for 10 min, and the resulting chromatin was diluted with IP dilution buffer  and pre-cleared with protein G-coupled magnetic beads (Invitrogen). PC9, OSS, and S2 cell pellets were treated the same as KARPAS-422 cells except the cells were sonicated at 15% output for 30 x 10 sec  in sonication buffer .PC9, KARPAS-422, OSS, and S2 cells were fixed with 1% formaldehyde (Sigma) for 15 min and quenched with 0.125 M glycine. Cells were washed twice in PBS and three times in cell lysis buffer  overnight at 4°C. For titration with D. melanogaster chromatin, 2.85 μg of S2 chromatin was incubated with 125 ng to 4 μg of H3K27me3 antibody , rabbit IgG (Jackson ImmunoResearch Lab 011-000-003), or 200 ng to 6.4 μg of H2Av antibody (Active Motif #39715) overnight at 4°C. Antibody-chromatin complex was captured by the addition of protein G magnetic beads and the beads were washed once with IP dilution buffer, twice with wash buffer 1 , once with wash buffer 2 , once with wash buffer 3 , and once with TE . DNA-protein complex was eluted twice with 100 μl elution buffer  at 65°C for 15 min with shaking at 1200 rpm (Eppendorf ThermoMixer C). Eluted chromatin was treated with 10 mg of RNAse A at 37°C for 1 h followed by proteinase K at 50°C for 2 h. Samples were then de-crosslinked at 65°C for 5 h. Reverse crosslinked DNA was purified by a PCR purification column , and eluted with 150 μl of buffer EB (10 mM Tris-HCl pH 8) for qPCR. Small aliquots of input chromatin samples were reverse crosslinked, RNase A, proteinase K digested and purified with PCR purification columns as described above. The concentration of the purified DNA was measured with Qubit Fluorometric Quantitation (Invitrogen) as a reference for the amount of chromatin used in each ChIP reaction. Quantitative polymerase chain reaction analysis of purified ChIP DNA (ChIP-qPCR) is described in the supporting information.For antibody titration Experiments in D. melanogaster chromatin, the samples were prepared and processed as above but with the addition of 0.4 μg of H2Av antibody  and 750 ng of D. melanogaster S2 cell chromatin for KARPAS-422 replicate #1 and 3.75 μg for replicate #2 and #3 (to increase the number of D. melanogaster tags) or 750 ng of D. melanogaster OSS cell chromatin for PC9 experiments.For ChIP-seq experiments, 30 μg of chromatin was pre-cleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 3 μg of H3K27me3 antibody , 4 μg anti-H3K4me3, a rabbit monoclonal antibody raised against a synthetic peptide corresponding to the N-terminus of histone H3 and containing a tri-methylated lysine 4  or 5 μl of anti-H3K9me3 serum from a rabbit immunized with a synthetic peptide corresponding to the N-terminus of histone H3 and containing a tri-methylated lysine 9 . ChIP reactions had a volume of 250 μl and were processed as described above. For ChIP-seq experiments with spike-in ChIP and input DNA libraries were prepared for Illumina sequencing by using one half of the total ChIP DNA from each reaction. The amount of ChIP DNA was not determined for all replicates but known quantities are presented in D. melanogaster melanogaster genome (dm3) using the BWA algorithm (default settings). Duplicate tags were removed and only uniquely mapped tags  were used for further analysis. Alignments were extended in silico at their 3’-ends to a length of 200 bp, which is the average genomic fragment length of the library molecules, and assigned to 32-nt bins along the genome. Peak locations were identified using the SICER algorithm (v1.1) with an E-value of 1 based on random background . The gap size parameter was set at 600 bp for H3K9me3 and H3K27me3, and 200 bp for H3K4me3. Peak regions were defined to be the union of peak intervals in control and treated samples, and average signal was reported for each region. Our standard normalization for the comparison of multiple samples was implemented by normalizing human tag numbers in each sample to the same number of tags present in the sample with the lowest number of uniquely mapped tags. Tag removal was performed by randomly removing excess tags from the larger files. The spike-in normalization was performed by counting D. melanogaster tags in each sample and using those tag counts to generate correction factors; calculated as DMSO tags/inhibitor tags. The primary method of mapping D. melanogaster tags used a partial dm3 reference genome containing only H2Av bound regions that we identified in H2Av ChIP-seq experiments using only D. melanogaster chromatin from S2 or OSS cells. The alternate method of mapping D. melanogaster tags used the entire dm3 reference genome. Correction factors were then applied to the human tag counts for all samples and scaling of human tags was performed by randomly removing tags as dictated by the scaling factor.Sequences  were separately aligned to either the human genome (hg19) or the https://www.broadinstitute.org/igv/) was used for visualization of ChIP-seq data sets.The Integrated Genomics Viewer (IGV) v2.3  Spike-in material is present in all samples in equal amounts. 2) Spike-in material is added early in the process as to be present during technical sample manipulations throughout the entire ChIP procedure and library generation. 3) Spike-in material is present in small amounts so that library generation is not affected, and so that the final sequencing data is not dominated by tags originating from the spike-in material. 4) Spike-in material is uniquely identifiable in the final data set. 5) Spike-in material can be applied to ChIP reactions universally, independent of the nature of the experimental sample chromatin and independent of the experiment-specific antibody.D. melanogaster chromatin spike-in strategy that includes the addition of a D. melanogaster histone variant protein-specific antibody  as a control [D. melanogaster S2 cell line. Results showed a significant enrichment pattern at specific genomic loci and across the D. melanogaster genome  (In subsequent experiments tag counts were derived from ‘H2Av-only’ or tags) . The coror tags) , similaror tags) . Both coor tags) . ChIP-seor tags) , consistor tags)  but wereD. melanogaster sequence tags does not artificially introduce changes in ChIP-seq signals, we performed control experiments with antibodies against modifications that are not expected to change in response to EZH2 inhibitor treatment. H3K4me3 and H3K9me3 ChIP-seq were performed in PC9 cells treated with GSK126 and in KARPAS-422 cells treated with CPI-360 as compared to DMSO controls. The D. melanogaster tag counts in these control ChIP-seq reactions were more evenly distributed across samples than in the H3K27me3 ChIP reactions  into all samples and determine if the standard is differentially affected during processing. Recently, ChIP-seq spike-in strategies have been described , 25 thatD. melanogaster chromatin spike-in is simple. ChIP protocols remain unchanged except for addition of the D. melanogaster-specific H2Av antibody and a small amount of D. melanogaster chromatin from a common stock. Library generation and DNA sequencing are performed as usual as is the mapping of human sequence tags. Normalization of human tags is based on differences in D. melanogaster tag counts using an approach to map tags uniquely to the pre-defined H2Av binding sites or the alternate approach of using all tags that uniquely map across the entire D. melanogaster genome. In the case of the H3K27me3 antibody, it recognizes both human and D. melanogaster chromatin. Thus, it is possible to re-analyze the data according to a previously published method [D. melanogaster tag counts using only H3K27me3-specific regions of the D. melanogaster genome. Analysis of our data using this method resulted in a correction factor between DMSO and EZH2 inhibitor-treated samples that was similar to those obtained with our H2Av method  One can prepare a large amount of D. melanogaster chromatin and avoid batch to batch variations that may arise from repeatedly growing D. melanogaster cells along with culturing human cells for a given experiment. (2) D. melanogaster and human cells behave differently during chromatin processing. For instance, different sonication conditions are used to adequately fragment chromatin from both species. Even for human cells, sonication conditions may vary depending on cell type. If human and D. melanogaster cells are mixed, the human cell-specific conditions may be inadequate for D. melanogaster cells. Processing the cells separately will ensure that the quality of the reference standard is independent of human cell processing conditions.While Orlando et al. mix human and Ultimately, one of the major problems contributing to the inability of ChIP-seq to detect global changes in binding or histone PTM occupancy seemingly occurs at library generation. Our DNA quantitation data show that for inhibitor treated cells there is less DNA going into library generation protocols . HoweverD. melanogaster tags would be 1 million tags from a 50 million-tag experiment, assuming that the antibodies pull down a similar fraction of chromatin in each species. This number would be enough for reliable normalization but would not negatively impact the overall tag count from the experimental samples. The initial experiments contained 750 ng of D. melanogaster chromatin resulting in approximately 1.2 million D. melanogaster tags in untreated PC9 cells. However, in the first experiment in KARPAS-422 DMSO-treated cells, tag counts were only 102,000, well below the target of 1 million. In the second experiment we increased the amount of D. melanogaster spike-in chromatin to 3.7 μg in KARPAS-422 cells, resulting in D. melanogaster tag counts of 831,000. One of the reasons why more spike-in chromatin is required in KARPAS-422 cells than in PC9 cells to achieve adequate tag counts, is that KARPAS-422 cells harbor a mono-allelic mutation in EZH2 which leads to increased global H3K27me3 levels as compared to PC9. As a consequence, H3K27me3-enriched DNA from KARPAS-422 cells is more prominent in the final ChIP-DNA population, thus the ratio of ChIP DNA originating from the spike-in compared to ChIP DNA originating from H3K27me3 enriched human chromatin is lower than that from PC9 cells. Importantly, we observed that using antibodies against less abundant histone modifications or transcription factors in ChIP-seq reactions containing 750 ng of D. melanogaster chromatin may result in D. melanogaster tag counts exceeding 30% of the total tags in the final sequencing data set. Therefore, different contexts may require different amounts of D. melanogaster chromatin in order to achieve a reasonable number of D. melanogaster tags in the final data set. In summary, the amount of spike-in chromatin required to achieve 1 million D. melanogaster sequence tags varies depending on the cell type, the prevalence of the target and the quality of the experimental antibody.When designing the spike-in approach we rationalized that the ideal number of In conclusion, the ChIP-seq spike-in method described here was designed to be a broadly applicable solution for ChIP-seq normalization. This method overcomes a challenging and persistent barrier to our understanding of the mechanisms by which drugs targeting epigenetic modifying proteins may exert their clinical effect. EZH2 inhibitors have been available to the research community for several years, yet comparative H3K27me3 ChIP-seq profiles from DMSO and EZH2 inhibitor-treated disease-relevant cell models have not been broadly explored. Our spike-in system and normalization strategy used with EZH2 inhibitor treated samples revealed global reductions in H3K27me3 ChIP-seq signal in inhibitor treated samples. We expect that the adoption of this ChIP-seq spike-in normalization approach or other similar strategies , 8 will S1 Fig(A) Reduced global H3K27me3 levels were observed in PC9 cells treated with 1 μM of GSK126 for 5 days. Immunoprecipitation using anti-H3K27me3 followed by immunoblotting with anti-H3 shows reduced H3K27me3 levels in inhibitor-treated cells. (B) Mass spectrometry analysis of histone H3K27 and H3K9 methylation abundance in CPI-360-treated  KARPAS-422 cells compared to DMSO treated controls. (C) Mass spectrometry analysis of histone H3K27 and H3K4 methylation abundance in GSK126-treated (1 μM) PC9 cells compared to DMSO-treated controls. (D) The KARPAS DMSO samples from duplicate H3K27me3 ChIP-seq experiments were compared do demonstrate the consistency of the ChIP-seq protocol. The biological replicates display a correlation coefficient of 0.985. (E) The PC9 DMSO samples from duplicate H3K27me3 ChIP-seq experiments were compared do demonstrate the consistency of our ChIP-seq protocol. These biological replicates display a correlation coefficient of 0.998.(PDF)Click here for additional data file.S2 Fig(A) ChIP-qPCR was performed using chromatin from KARPAS-422 cells treated with the EZH2 inhibitor CPI-360. H3K9me3 occupancy did not change at the ZNF274 and ZNF829 positive control genes after treatment. (B) ChIP-qPCR was performed using chromatin from PC9 cells treated with the EZH2 inhibitor GSK126. H3K4me3 occupancy did not change at the ACTB and GAPDH promoters after treatment.(PDF)Click here for additional data file.S3 FigU6-5 gene was used as negative control for H3K27me3 occupancy. qPCR results showed that H3K27me3 occupancy is proportional to the amount of H3K27me3 antibody used in ChIP reactions within the tested range (250 ng to 8 μg antibody/ChIP).Different chromatin amounts (12 and 6 μg) were used in ChIP reactions with different anti-H3K27me3 antibody amounts (250–8000 ng). ChIP DNA was analyzed by qPCR at indicated promoters. Data is represented as mean enrichment from two independent experiments and qPCRs carried out in triplicate ±SD. A region in the (PDF)Click here for additional data file.S4 FigD. melanogaster(A) and human (B) chromatin. Antibody amounts used in ChIP reactions are indicated below the graphs. Chromatin amounts of 2.85 μg of D. melanogaster and 12 μg of human chromatin were used in each ChIP-reaction. ChIP DNA was analyzed by qPCR at indicated gene promoters. Data is represented as mean enrichment from two independent experiments and qPCRs carried out in triplicate ±SD. DUntr3L was used as negative control for H2Av occupancy. Results showed an antibody concentration-dependent increase of both H2Av and H3K27me3 occupancy at indicated D. melanogaster promoters. In ChIP reactions containing human chromatin, an antibody concentration-dependent increase in H3K27me3 at indicated human promoters was detected while H2Av ChIPs did not result in enrichment over background. (C) H2Av ChIP-seq data from reactions containing chromatin from only the D. melanogaster S2 cell line. The 13 million base pair region depicted shows hundreds of peaks detected on chromosome 2R. (D) H2Av ChIP-seq data from a reaction containing chromatin from only the human cell line PC9. Depicted are 95 million base pairs from human chromosome 5. The H2Av antibody does not cross react with human chromatin therefore no peaks are detected.Titration of H2Av and H3K27me3 antibodies with (PDF)Click here for additional data file.S5 FigD. melanogaster S2 chromatin for H3K27me3/H2Av ChIP. qPCR at different human and D. melanogaster genomic loci was performed to evaluate enrichment. U6-5 promoter was selected as the H3K27me3 negative locus in KARPAS-422 cells. CPI-360 treatment resulted in a significant decrease in H3K27me3 signal at human genes and slightly increased enrichment of D. melanogaster genes. Data represent the mean of three ChIP experiments with qPCR carried out in triplicates ±SEM. *Wilcoxon signed rank test.Chromatin from DMSO or CPI-360 treated KARPAS-422 cells were mixed with (PDF)Click here for additional data file.S6 FigD. melanogaster genome were determined using the H2Av antibody in ChIP-seq reactions containing D. melanogaster S2 or OSS chromatin. D. melanogaster tags from ChIP-seq spike-in reactions were mapped only to these pre-defined H2Av regions. The trends in this replicate are similar to those shown in (A) H3K27me3 ChIP-seq reactions using D. melanogaster spike-in in KARPAS-422 cells results in an increase in D. melanogaster tags mapping in CPI-360 treated cells both at 4 days and 8 days after treatment. (B) H3K27me3 ChIP-seq reactions using D. melanogaster spike-in in PC9 cells results in an increase in D. melanogaster tags mapping in GSK126 treated cells.H2Av bound regions of the (PDF)Click here for additional data file.S7 FigD. melanogaster tag counts are observed in EZH2 inhibitor treated samples in H3K27me3 ChIP-seq spike-in reactions when using tags mapped across the entire D. melanogaster genome. Data is similar to the strategy of mapping to only the pre-defined H2Av regions that is presented in (A) H3K27me3 ChIP-seq spike-in reactions using S2 D. melanogaster chromatin in KARPAS-422 cells. (B) H3K9me3 ChIP-seq spike-in reactions using S2 D. melanogaster chromatin in KARPAS-422 cells. (C) H3K27me3 ChIP-seq spike-in reactions using OSS D. melanogaster chromatin in PC9 cells. (D) H3K4me3 ChIP-seq spike-in reactions using OSS D. melanogaster chromatin in PC9.Elevated (PDF)Click here for additional data file.S8 Fig(A) Chromatin from DMSO or CPI-360-treated KARPAS-422 cells and (B) chromatin from DMSO or GSK126-treated PC9 cells were mixed with D. melanogaster S2 and OSS chromatin respectively for H3K27me3/H2Av and H3K4me3/H2Av ChIPs. ChIP-seq was carried out on a biological replicate distinct from the experiment shown in (PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.S1 TableChIP DNA was quantified using Qubit fluorometric quantitation. Total immunoprecipitated DNA mass and fold change after EZH2 inhibitor treatment are presented.(PDF)Click here for additional data file.S2 TableChIP-seq libraries were PCR amplified for 15 cycles. ChIP-seq library yields were measured using a NanoDrop spectrophotometer. Yields within groups of treated and untreated cells were similar.(PDF)Click here for additional data file.S3 TableD. melanogaster (bottom) reads in treated versus control samples.Normalization factors were calculated based on tags mapping to the entire dm3 genome , H2Av peak regions  or H3K27me3 peak regions . Normalization factors were calculated from two independent ChIP-seq spike-in campaigns . For KARPAS-422 cells, H3K27me3 ChIP-seq was carried out on cells that were treated with DMSO or 1.5 μM CPI-360 for 8 days and H3K9me3 ChIP-seq from cells treated for 4 and 8 days. For PC9 cells, H3K27me3 and H3K4me3 ChIP-seq was carried out on cells that were treated with DMSO or 1 μM GSK126 for 5 days. Numbers represent the ratio of human (top) and (PDF)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


This study is designed to analyze survival benefit of (neo-) adjuvant radiotherapy to patients with T2-3N0M0 stage esophageal adenocarcinoma (EAC).T2-3N0M0 stage EAC patients from 2004 to 2012 were searched from the Surveillance Epidemiology and End Results (SEER) data. Clinical factors including age, sex, race were summarized. Univariate, multivariate analysis, and stratified cox analysis based on different T stages were performed to explore the survival effect of (neo-)adjuvant radiotherapy to T2-3N0M0 stage EAC.T2-3N0M0 stage EAC patients with surgery were more likely to be white race, T3 stage. Univariate analysis showed sex, age, and T stage were the prognostic factors of survival (P<0.05). Multivariate analysis proved (neo-)adjuvant radiotherapy can prolong survival time of T2-3N0M0 stage EAC (P<0.05). Further analysis based on different T stages showed that both neoadjuvant radiotherapy  and adjuvant radiotherapy  significantly reduced the risk of death of T3N0M0 stage EAC, but neither of which significantly reduced death risk of T2N0M0 stage EAC (P>0.05).sex, age are the independent prognostic factors of T2-3N0M0 EAC. Significant survival benefit of (neo-)adjuvant radiotherapy is only observed in patients with T3N0M0 stage EAC, but not in those with T2N0M0 stage. Esophagus cancer is the world's eighth incidence and the sixth cause of death cancer. 455,800 new diagnostic esophageal cancers and 400,200 deaths were reported to occur worldwide in 2012  7]..7].The Surveillance, Epidemiology, and End Results (SEER) Program is a cancer related database founded by the National Cancer Institute (NCI) in the United States. It collects and reports cancer incidence and survival data from population-based cancer registries and covers approximately 28% of the US population. With large information of cancer, it is an important tool to analyze carcinoma.In view of above, we used SEER data for the analysis of EAC. Purpose to explore the efficacy of (neo-)adjuvant radiotherapy to the T2-3N0M0 stage EAC.A total of 918 patients were selected from the SEER database. In which, 338(36.8%) cases received surgery alone, 492(53.6%) cases received neoadjuvant radiotherapy with surgery (RT + Surg), and 88(9.6%) patients received surgery with adjuvant radiotherapy (Surg + RT). More than 95% patients were white race, so we combined black race in to other. Percent of male was 88.5%, much more than female. Independence test of the patients' treatment assignment and clinical characteristics indicated an obvious association of (neo-)adjuvant radiotherapy to age and T stage. Patients younger than 65 years and with the T3N0M0 stage were more likely to receive RT + Surg. A detailed listing of the patient characteristics and pathological features was presented in Table Univariate survival analysis of clinical characteristics was evaluated with log-rank test Table . Age Fi, T stageFinally, we performed stratified multivariate cox regression analysis to assess the efficacy of (neo-) adjuvant radiotherapy to survival time based on different T stages, by adjusting for sex, race, and age Table . The resEsophageal adenocarcinoma is one of the two major histological subtypes of esophageal cancer in the world, with a high and rapidly increased incidence in the western countries, including United States, Australia, France, and England , 8, 9. RIn this study, we summarized the clinical characteristics of surgical patients with T2-3N0M0 stage EAC, with information provided by the SEER database from 2004 to 2012. Characteristics analyzed in our study contained sex, race, T stage, age, and radiation therapy. We found that operative patients with T2-3N0M0 EAC were more likely to be T3 stage (60.0%), male (88.5%), and white (95.6%) race. More than half of these EAC patients received additional radiation therapy (63.2%). Of which, neoadjuvant radiotherapy was 84.8% (53.6%/63.2%), and adjuvant radiotherapy was 15.2% (9.6%/63.2%). Independence chi-square test between radiation therapy and other factors showed that radiation therapy was associated with age and T stage. Young (<65 years) and T3 stage patients were more likely to receive neoadjuvant radiotherapy. Univariate survival analyses showed that survival time was associated with sex, age, and T stage (P<0.05), but of no association with race and radiotherapy (P>0.1). While multivariate cox proportional hazards regression analysis displayed that T stage, age, (neo-)adjuvant radiation therapy were all significantly associated with survival (P<0.05). Death risk of patients in T2N0M0 stage was lower than those in T3N0M0 stage . Patients who younger than 65 years had a lower risk of death . Adjuvant radiotherapy could reduce nearly 35% of death hazards . And neoadjuvant radiotherapy could reduce nearly 30% of death hazards . Further multivariate analysis of (neo-)adjuvant radiotherapy based on different T stages showed that both neoadjuvant radiotherapy and adjuvant radiotherapy could reduce more than 40% risk of death in T3 stage (P<0.05). But neither of them could significantly reduce death risk in T2 stage (P>0.05).In conclusion, our study demonstrates that age and T stage are survival associated factors in T2-3N0M0 stage EAC. Compared with surgery alone, both neoadjuvant radiotherapy with surgery and surgery with adjuvant radiotherapy are of significant survival benefit to T3N0M0 stage EAC. But neither of them does significantly good to T2N0M0 stage of EAC.We chose SEER data between 1973 and 2012  for this study. The National Cancer Institute's SEER*Stat software (Version 8.2.1) was used for the identity of patients. The inclusion criteria contained: (1) primary esophageal cancer (C15.0-C15.9) with a confirmed diagnosis of microscopically, (2) entire adenocarcinoma histology (Histologic/Behavior codes: 8140/3) based on the International Classification of Diseases for Oncology, 3rd Edition (ICD-O-3), (3) being diagnosed between 2004 and 2012, (4) with the 6th AJCC stage of T2-3N0M0, and (5) received surgery. And the exclusion criteria contained: (1) unknown age, sex, race, T, N, M stage, (2) with a radiotherapy status of “radiation both before and after surgery”, “intraoperative radiation therapy”, “intraoperative radiation with other radiation given before or after surgery”, “surgery both before and after radiation (for cases diagnosed 1/1/2012 and later)”, or “sequence unknown, but both surgery and radiation were given”, (3) diagnosed solely on autopsy or death certificate. Survival data were extracted at 1-month intervals for a maximal follow-up of 60 months.This study based on public data from the SEER database. The reference number we obtained for the permission to access research data files was 10612-Nov2014. No human subjects or personal identifying information were used in this study. No informed consent was require in this study. This study was approved by the Review Board of Huai'an First People's Hospital, Huai'an, China.The enrolled population was divided into three groups based on different treatment: patients who were treated with surgery alone (Surg group), with surgery followed by radiotherapy (Surg+RT group), and with surgery following with radiotherapy (RT+Surg group). Chi-square test was used to analyze the differences of these three groups. Univariate analyses with log-rank test and multivariate analysis with cox proportional hazards regression model were performed to examine the clinical factors' association with survival respectively, with a statistically significant difference at the value of p<0.05. Finally, stratified cox regression survival analysis were performed based on different T stages. All analysis were performed with survival package  of R(ver


Km and Vmax for the NAs of A/PR8-S331G and A/PR8-S331R viruses were higher than for the NA of WT virus, and the corresponding values for the B/YAM-D342S virus were lower than for the NA of WT virus. Although there was initial variation in the kinetics of influenza A and B viruses’ replication in MDCK cells, their titers were comparable to each other and to WT viruses at later time points. All introduced substitutions were stable except for B/YAM-D342S and B/YAM-A395V which reverted to WT sequences after three passages. Our data suggest that inferring susceptibility to NAIs based on sequence information alone should be cautioned. The impact of NA substitution on NAI resistance, viral growth, and enzymatic properties is viral context dependent and should be empirically determined.Amino acid substitutions within or near the active site of the viral neuraminidase (NA) may affect influenza virus fitness. In influenza A(H3N2) and B viruses circulating in Thailand between 2010 and 2015, we identified several NA substitutions that were previously reported to be associated with reduced inhibition by NA inhibitors (NAIs). To study the effect of these substitutions on the enzymatic properties of NA and on virus characteristics, we generated recombinant influenza viruses possessing either a wild type (WT) NA or an NA with a single I222V, S331G, or S331R substitution [in influenza A(H3N2) viruses] or a single D342S, A395T, A395V, or A395D NA substitution (in influenza B viruses). We generated recombinant (7:1) influenza A and B viruses on the genetic background of A/Puerto Rico/8/1934  or B/Yamanashi/166/1998 (B/YAM) viruses, respectively. In contrast to the expected phenotypes, all the recombinant influenza A(H3N2) and B viruses carrying putative NA resistance substitutions were susceptible to NAIs. The  Influenza A and B viruses are important human respiratory pathogens that cause annual epidemics worldwide. Anti-influenza prophylaxis and treatment with antiviral drugs are used to prevent and control influenza virus infections, particularly when vaccines are ineffective or unavailable . The Cenin vitro and in vivo [50s to oseltamivir when compared to the wild-type NAs [50 values for these compounds) [The influenza neuraminidase (NA) is a tetrameric type II integral membrane protein with sialidase activity that is responsible for releasing newly produced viral particles from virus-infected cells , 4. Base in vivo –12. The type NAs . Antivirmpounds) –17. Thesmpounds) , 15. A rmpounds) .Previously, we reported substitutions at residues 222 and 331 in the NA of influenza A(H3N2) viruses and at residues 342 and 395 in that of influenza B viruses circulating in Thailand . HoweverTable 1) [The novel NA substitutions were identified in influenza A(H3N2) and B viruses in polymerase chain reaction (PCR)-positive respiratory samples from patients in Thailand (Table 1) . SubstitInfluenza B/Brisbane/60/2008 (B/BR/60/08) virus was obtained from the repository at St. Jude Children’s Research Hospital. Madin-Darby canine kidney (MDCK) cells  were maintained in Eagle’s Minimum Essential Medium supplemented with 5% fetal bovine serum and a mixture of antibiotics and antimycotics . Human embryonic kidney 293T cells  were maintained in Opti-MEM reduced-serum medium with GlutaMAX supplement at a final concentration of 2 mM  and no antibiotics.BsaI [BsmBI [BsmBI, and the NA segments were ligated with T4 DNA ligase  into pHW2000 (for influenza A NAs) or pAD3000 (for influenza B NAs).Influenza viral RNA was extracted using a RNeasy Kit . The full-length NA genes  and B/Yamanashi/166/1998  and B 22 virusesBsaI  or BsmBIPlasmids were transformed into One Shot chemically competent Escherichia coli cells , and positive clones were inoculated into Terrific Broth medium containing ampicillin. The positive clones were purified using HiSpeed Plasmid Maxi Kits , and the plasmid DNA concentrations were determined by both UV spectrophotometry at 260 nm and analysis on agarose gels. The constructed plasmids were sequenced to ensure their identity with the field strains.50) of each recombinant influenza virus was determined in MDCK cells incubated for 72 h at 35°C (for influenza A viruses) or 33°C (for influenza B viruses). The TCID50 values were calculated by the Reed-Muench method [Recombinant (7:1) influenza viruses carrying NAs of influenza A(H3N2) or B viruses circulating in Thailand  were genh method . The HA 50) values [50s of viruses were determined using the curve-fitting model in GraphPad Prism 5 software . The phenotypic NAI susceptibility of influenza A and B viruses was evaluated based on the fold change in their IC50 values when compared to those of susceptible viruses from the same NA subtype/lineage, as previously described by the WHO Influenza Antiviral Working Group [The NAIs  were dissolved in sterile distilled water, and aliquots were stored at −20°C until use. A fluorescence-based NA inhibition assay with the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid  was used to determine the 50% inhibitory concentration (IC) values , 26. Bring Group . The inh10TCID50 of the virus. The thermostability of the NA was determined by incubating viruses in a gradient thermocycler at 33, 37, 45, or 55°C for 15 or 30 min, then all viruses were brought to room temperature, and NA activity was subsequently measured [The NA activity of recombinant viruses was measured by a fluorometric assay with MUNANA substrate as described above , 26. Themeasured , 26. TheVmax and Km) of the NAs of recombinant influenza A and B viruses with a single NA amino acid substitution were determined as previously described [Km) and velocity of catalysis (Vmax) values were calculated by fitting appropriate Michaelis-Menten equations to the data by using nonlinear regression in GraphPad Prism 5 software.The enzyme kinetics  and at 24, 48, and 72 h (for an MOI of 0.001 TCID50/cell) after virus inoculation and stored at −70°C until use. Virus titers were determined by TCID50 assays in MDCK cells.Multi-step growth curves were determined for each recombinant influenza virus in MDCK cells. Confluent cell monolayers were infected with recombinant virus at a multiplicity of infection (MOI) of 1 or 0.001 TCID50 values, enzyme kinetic parameters (Km and Vmax), and virus titers were compared by two-tailed Student’s t-tests. The results were considered significantly different if the P-value was less than 0.05.The NA activity, ICTable 2). When compared to the IC50 value of the A/PR8-WT virus, the fold changes in the IC50 values of recombinant influenza A viruses were > 10-fold. These results demonstrated that A/PR8 viruses carrying NA from A(H3N2) virus with single I222V, S331R, or S331G NA substitutions were susceptible to NAIs. For the recombinant B/YAM viruses, the fold changes in the IC50 values were > 5-fold when compared to the IC50 of the B/BR/60/08 virus. These results revealed that B/YAM viruses carrying NAs from currently circulating influenza B viruses with single D342S, A395T, A395V, or A395D NA substitutions were susceptible to NAIs.We used NA inhibition assays to determine the NAI susceptibility profiles of 7:1 recombinant influenza A and B viruses carrying single NA amino acid substitutions (10TCID50) and expressed as the quantity of 4-MU (in μg) (Table 3). The NA substitutions at residue 331 (S331G and S331R) significantly decreased the NA activity of recombinant A/PR8 viruses as compared to that of A/PR8-WT NA , whereas the I222V NA substitution slightly increased the NA activity (Table 3). In contrast, the B/YAM-D342S and B/YAM-A395T viruses had significantly higher NA activity when compared to the reference B/BR/60/08 virus (P < 0.01). The A395D substitution decreased the NA activity of recombinant B/YAM virus, whereas the A395V NA substitution increased the NA activity relative to that of the B/BR/60/08 virus (Table 3). Thus, the S331G substitution had the greatest impact on the NA activity of recombinant A/PR8 viruses, followed by the S331R substitution, and the D342S and A395T substitutions had the greatest effect on the NA activity of recombinant B/YAM viruses.We investigated the effect of NA amino acid substitutions on the NA activity of 7:1 recombinant influenza A and B viruses by using a modified fluorescence-based assay. The NA enzyme sialidase activity was standardized according to the amount of infectious virus (per 1 logKm and Vmax of NAs from recombinant influenza A and B viruses by using the fluorogenic MUNANA substrate (Table 3). The Km of A/PR8 virus NAs carrying S331G and S331R NA substitutions was significantly increased relative to that of WT NA (P < 0.01), whereas the D342S and A395T NA substitutions decreased the Km of recombinant B/YAM viruses . The S331G and S331R NA substitutions increased the Vmax of recombinant influenza A virus NAs  (Table 3).To investigate the effect of NA amino acid substitutions on NA enzymatic kinetics, we measured the Vmax (P < 0.05). The higher Vmax of the influenza A virus NA may facilitate the release and spread of A/PR8-S331G and A/PR8-S331R viruses, but the higher Km indicates that the NA has a low affinity for its substrate. These results suggest that the NA substitutions contribute to the reduction in NA enzymatic activity.In contrast, the NAs of B/YAM viruses carrying D342S or A395T NA substitutions had a reduced Fig 1A and 1B). The NA activity of recombinant B/YAM viruses carrying T/V/D changes at residue 395 and the B/BR/60/08 virus was undetectable after incubation at 55°C. In contrast, virus carrying the D342S NA substitution possessed high NA activity that was thermostable .NA amino acid substitutions can contribute to NA protein stability . Therefoin vitro of recombinant influenza A and B viruses carrying NA amino acid substitutions, we studied their replication capacity in MDCK cells. At 6 and 12 h post-infection (hpi), the growth of A/PR8-I222V virus was similar to that of A/PR8-WT, but the A/PR8-S331G and A/PR8-S331R virus yields were significantly lower (P < 0.05) . At 48 hpi, the yields of all recombinant influenza A viruses were comparable  . At 6 and 12 hpi, the recombinant influenza B virus yields were significantly lower than that of B/BR/60/08 virus (P < 0.05) . At 48 hpi, all influenza B viruses replicated to significantly higher virus titers in MDCK cells when compared to B/BR/60/08 virus (P < 0.05) . At 72 hpi, the viral titers of the recombinant influenza B viruses were 4.2 and 5.2 log10TCID50/mL. These results demonstrated that the NA substitutions S331G and S331R (on an A/PR8 background) and D342S, A395T, A395V, and A395D (on a B/YAM background) reduced the enzyme activity and thermostability of the NA. Although replication ability of recombinant influenza A and B viruses was heterogeneous at early time points, they showed at least equivalent yields as A/PR8-WT and B/BR/60/08 viruses at later time points. However, using B/BR/60/08 instead of the recombinant B/YAM virus with the wild-type NA is a caveat of this comparison.To evaluate the fitness in vitro, the NA genes were amplified from viruses before and after three passages in MDCK cells, and the NA sequences were determined. The I222V, S331G, and S331R NA substitutions were genetically stable in NA proteins on the genetic background of the A/PR8 virus. A diverse pattern was observed for influenza B viruses. The A395T and A395D substitutions were genetically stable and were maintained unchanged during three passages, whereas the D342S and A395V substitutions were unstable and the NAs reverted to WT after three passages (Table 4). These findings indicate that genetically unstable D342S and A395V NA substitutions are unlikely to emerge at a high frequency in influenza B viruses.To determine whether NA substitutions introduced into the A/PR8 and B/YAM genetic backgrounds could be stably maintained in vitro. We generated recombinant (7:1) influenza A and B viruses containing the NA glycoprotein from viruses currently circulating in Thailand on the homogeneous genetic background of A/PR8 and B/YAM viruses for the recombinant influenza A and B viruses, respectively. The conserved I222 framework residue in the NA of influenza virus provides support to the catalytic residues [In this study, we investigated the effect of novel NA amino acid substitutions on the NAI susceptibility, NA enzymatic properties, and replication capacity of influenza viruses ombinant :1 influeresidues . Our datresidues .Km) for MUNANA substrate. The Vmax of the A/PR8-S331G and A/PR8-S331R virus NAs was nearly double that of the A/PR8-WT NA, demonstrating the compensatory effect as reflected in the lower replication capacity of viruses carrying S331G and S331R substitutions when compared to viruses with WT or I222V NAs. The significant decrease in NA activity and the increased Km value demonstrated that S331G and S331R NA substitutions are deleterious to NA functions. The NA activity of the A/PR8-S331G and A/PR8-S331R viruses was decreased, and their viral titers in MDCK cells were lower than those of the WT virus at 6 and 12 hpi. The A/PR8-S331G and A/PR8-S331R viruses were characterized by a loss of NA activity at higher temperature (55°C), whereas PR8-WT and A/PR8-I222V viruses did not lose their NA activity. These data indicate that the viral replication capacity in vitro is dependent on the NA activity and the thermostability of the viruses [Km and Vmax values decreased. These data suggest that there is an inverse relationship between NA activity and Km and Vmax values and that these amino acid substitutions are beneficial to NA functions. Moreover, B/YAM-D342S, B/YAM-A395T, and B/YAM-A395V viruses gave significantly higher viral yields at 48 hpi in MDCK cells, as compared to B/BR/60/08 virus. At higher temperature (55°C), the NA activity of recombinant influenza A viruses carrying NA substitutions was more stable than that of influenza B viruses. The A395V and D342S NA substitutions on the B/YAM background were unstable, and the NAs reverted to WT after three passages in vitro.Regarding the S331G/R, D342S, and A395T/D/V NA amino acid substitutions, these are found outside the NA active site, and viruses with these substitutions showed normal inhibition by NAIs. The S331G and S331R substitutions affected the kinetic properties of the NA enzyme, changing its binding affinity  PCR-positive samples with I222V, S331G, and S331R NA substitutions were generated in the A/PR8 (H1N1) backbone. The replication efficiency of these viruses in MDCK cells may differ from that of natural isolates because of the effect of the HA-NA balance [50 does not account for defective/non-infectious virions in the virus preparations, which may be enzymatically active. NA content can be reliably standardized in purified NA proteins; however, protein production on this scale, and when evaluating numerous viruses/proteins, is not readily achievable.There were a few limitations in this study. As we could not generate a recombinant B/YAM virus with WT NA protein by using reverse genetics, we used the B/BR/60/08 virus, which is genetically similar to viruses circulating in Thailand, and compared the NA enzymatic properties and viral replication efficiency  balance , this apin vitro. Continuous influenza antiviral surveillance is important for the identification of molecular markers of antiviral resistance and the characterization of NA enzyme functions and viral fitness.In conclusion, the recombinant influenza A and B viruses containing different NA substitutions located outside the NA active site exhibited normal inhibition by NAIs but differed in their NA enzymatic properties and their replication capacity


Our results showed that the expression level of ZFP3 was highly suppressed by NaCl, mannitol and sucrose. Constitutive expression of ZFP3 enhanced tolerance of plants to salt and osmotic stress while the zfp3 mutant plants displays reduced tolerance in Arabidopsis. Gain- and Loss-of-function studies of ZFP3 showed that ZFP3 significantly changes proline accumulation and chlorophyll content. Furthermore, over-expression of ZFP3 induced the expressions of stress-related gene KIN1, RD22, RD29B and AtP5CS1. These results suggest that ZFP3 is involved in salt and osmotic stress response.Plants are continuously challenged by various abiotic and biotic stresses. To tide over these adversities, plants evolved intricate regulatory networks to adapt these unfavorable environments. So far, many researchers have clarified the molecular and genetic pathways involved in regulation of stress responses. However, the mechanism through which these regulatory networks operate is largely unknown. In this study, we cloned a C Plants are constantly challenged by a variety of stresses such as high salinity, extreme temperature and drought ,2. EnvirSOS) genes. Mutations in SOS genes make plants more sensitive to Na+ stress [+ / H+ antiporter [High salt stress can cause osmotic and ionic stress ,13. Salt+ stress . SOS3, atiporter ,15. Hightiporter .Zat10 or knockout and RNAi plants of Zat10 can both increase the tolerance of osmotic and salinity stresses which suggest that it plays an important role as a negative and positive regulator [ThZFP1 in Arabidopsis showed more tolerance to salt and osmotic stress by increasing POD and SOD activities, proline content and their relative genes such as delta-pyrroline-5-carboxylate synthetase (P5CS), superoxide dismutase (SOD) and peroxidase (POD) [Arabidopsis, four ZPT2-related C2H2-type zinc finger proteins  are all involved in salinity and abscisic acid stress by interaction with the EP2 sequence [O.sativa subspecies indica stress-associated gene (OSISAP1) in tobacco revealed strong tolerance to cold, dehydration and salt stress [Arabidopsis, also play a role in salt tolerance by its EAR-motif. A deletion or a mutation of the EAR-motif abolishes this gene’s function [Zinc Finger Proteins (ZFPs) play significant roles in physiological and biochemical processes of plants, such as hormone signal transduction, DNA/RNA combination, morphogenesis, transcription regulation, trichome and root hair development, biotic and abiotic responses –23. Seveegulator . Overexpse (POD) . In Arabsequence . A zinc sequence . Overexpfunction .2-4CX12HX3-5H , which includes about 30 amino acids consisting of two antiparallel β hairpin and one α helix. In this structure, two pairs of conserved cysteine residues and histidine residues combine with a zinc ion tetrahedrally [ZPT2-1 (EPF1), was found in petunia. It can integrate with the promoter of EPSPS [On the basis of the number and order of cysteine residues and histidine residues in finger secondary structure, ZFPs are divided into nine types, including C2H2, C8, C6, C3HC4, C2HC, C2HC5, C4, C4HC3 and CCCH type . Cys2/Hihedrally . It is rhedrally . ZFPs exhedrally ,30. The of EPSPS ,18–23,29ZFP3 (AT5G25160), was previously shown to regulate light and ABA signaling in seeds germination and plant development in Arabidopsis [ZFP3 enhances tolerance of plants to salt and osmotic stress while the zfp3 mutant plants displays reduced tolerance in Arabidopsis. Our results further demonstrated that overexpression of ZFP3 could increase proline accumulation, chlorophyll content and the gene expressions of stress-related genes  in regulating salt and osmotic stress tolerance in Arabidopsis.C2H2 transcription factor, bidopsis . In thisArabidopsis thaliana ecotype Columbia Col-0 was used as the control in all experiments. The plants were grown in an environmentally controlled growth chamber programmed for cycles of 16 hours of light , 8 hours dark (18°C) and relative humidity of 70–80% as we previously described [Arabidopsis seeds were surface-sterilized with 5% NaClO: water (v/v) for 8 min and washed five times with distilled water. Seeds were plated on Murashige and Skoog (MS) medium and vernalize at 4°C for 3 days in dark before being transferred to the growth chamber with growth condition described previously [escribed ,21. Arabeviously .zfp3 mutant was identified from the Nottingham Arabidopsis Stock Centre . Homozygous mutant was confirmed by screening on MS medium containing 5.25 mg/L sulfadiazine.A SalI and NotI restriction sites. Then, the fragment was purified with a gel extraction kit (Axygen) before restriction digestion and cloning. The primers, which used for ZFP3 overexpression, were as follows: FP (5'-GCGCGTCGACAATCATCTCCTGAAAAAATCTATC-3'), RP (5'-TGGCGGCCGCACTAACAATCACATGAAAAAACAG-3'). The construct in binary vector was transferred into Agrobacterium tumefaciens Stain GV3101, which was then used to transform Arabidopsis thaliana ecotype Columbia Col-0 by the floral-dip method as described by Clough and Bent (1998) [For overexpression construct, sequences were first inserted into the pENTR-1A vector (Invitrogen) and then recombined into the destination vector using the Gateway LR reaction (Invitrogen) . The dest (1998) . TransgeFor phenotypical analyses, seeds were sown on the 1/2 MS medium containing different concentrations of NaCl, mannitol and sucrose. Emergence of radicles from the seed coat was used as the standard of seed germination. The germination rate was calculated as the percentage of number of germinated seeds/number of total seeds, which was recorded daily for seven days.For the root length measurement, seeds were cultured on 1/2 MS medium and grown on vertically placed plates for 3 days under normal condition and uniform seedlings were then transferred to 1/2 MS mediums with or without different concentrations of NaCl, mannitol and sucrose. After 7 days treatment, photos were taken and the root lengths of different plants were measured by Image-Pro plus 6.0.For gene expression analysis, 10 days seedlings were transferred onto filter paper saturated with 0, 200mM NaCl, 250mM sucrose and 300mM mannitol in MS liquid media. The samples were harvested at different time points.Arabidopsis plants using Trizol (Takara). First-strand cDNA was synthesized from 2 μg of total RNA by M-MLV transcriptase (Promega) and oligo (dT18) primer according to the manufacturer’s instructions [UBQ10 (At4g05320) and TUB2 (At5g62690) were used as the endogenous control gene for qRT-PCR analyses. Relative expression of target genes was calculated as previously described [Total RNA was extracted from ructions ,23. For escribed . The priThe chlorophyll was extracted in 95% ethyl alcohol from the fresh leaves at darkness. Absorbance of solution was measured at wavelength of 470, 649 and 665 nm with spectrophotometer  to analysis the content of chlorophyll(a) and chlorophyll(b), respectively.Arabidopsis seedlings was determined according to the procedure of Bates et al. (1973) [Proline content of 10 days . (1973) .All data displayed in figures were analyzed by means of ANOVA for significance using SPSS, version 14.0 software . Student’s t-test was calculated to evaluate the difference at the probability of either 5%  or 1%  .ZFP3 in abiotic stress responses, a zfp3 mutant  was ordered and screened from the Nottingham Arabidopsis Stock Center. We also constructed 35S:ZFP3 transgenic lines and selected two representative lines, which showed higher level of ZFP3 expression in comparison to the wild type  with 0 mM, 100 mM NaCl, 200 mM mannitol and 150 mM sucrose treatments. As shown in zfp3 mutant was significantly less than wild type when treated with NaCl, mannitol and sucrose , RD22 (At5g25610), RD29B (At5g52300) and osmotic substance synthesis gene AtP5CS1 (At2g39800) that encodes pyrroline-5-carboxylate synthetase [zfp3 mutant and ZFP3 overexpression line (35S:ZFP3-6) were treated with 200 mM NaCl, 300 mM mannitol and 250 mM sucrose for 5h and 10h respectively. As shown in RD29B. However, the expressions of these genes in wild type, zfp3 mutant and overexpression line were all significantly induced when treated by NaCl, mannitol and sucrose. The expressions of these genes in overexpression line were significantly higher than wild type while the expressions of these genes in zfp3 mutant were significantly lower than wild type  and G5SA is converted to proline by the Δ1-pyrroline-carboxylate reductase (P5CR). Among them, the γ-glutamyl kinase activity of P5CS is the key rate-limiting factor [AtP5CS1. Our results show that the expression level of AtP5CS1 was significantly higher in overexpression line while the expression of this gene was significantly lower in the zfp3 mutant in comparison to wild type (Chlorophyll content is an important parameter to determine the capacity of plant photosynthesis. Analysis of chlorophyll content revealed that the content of l stress . Furtherl stress ,39. In tc stress . Prolineg factor –38,48,49ild type .KIN1, RD22 and RD29B (ZFP3 affects plants’ tolerance to salt and osmotic stress by regulating the expressions of these genes. However, the mechanism of ZFP3 interacts with these genes controls salt and osmotic stress should be further investigated.To further test the change of expression level in stress pathway genes, we analyzed several stress marker genes including nd RD29B . The res


SERPINE1) by methylating H3K9. These results suggest that the ubiquitination of SETDB1 at lysine 867 controls the expression of its target gene by activating its H3K9 methyltransferase activity.Posttranslational modifications (PTMs) of proteins play a crucial role in regulating protein-protein interactions, enzyme activity, subcellular localization, and stability of the protein. SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that regulates the methylation of histone H3 on lysine 9 (H3K9), gene silencing, and transcriptional repression. The C-terminal region of SETDB1 is a key site for PTMs, and is essential for its enzyme activity in mammalian and insect cells. In this study, we aimed to evaluate more precisely the effect of PTMs on the H3K9 methyltransferase activity of SETDB1. Using mass spectrometry analysis, we show that the C-terminal region of human SETDB1 purified from insect cells is ubiquitinated. We also demonstrate that the ubiquitination of lysine 867 of the human SETDB1 is necessary for full H3K9 methyltransferase activity in mammalian cells. Finally, we show that SETDB1 ubiquitination regulates the expression of its target gene, serpin peptidase inhibitor, clade E, member 1 ( N-acetylglucosamine transferase , and the cells were lysed by sonication. GST-SETDB1 proteins from cell extracts were purified using glutathione-sepharose 4B.Samples were sent to the Center for Medical Research and Education, Osaka University Graduate School where they were analyzed by mass spectrometry as a custom contract service. To prepare the sample for mass spectrometry analysis, GST affinity-purified DEL570  was cleTo prepare whole cell extracts, HeLa cells were lysed in 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Sigma).Each cell extract was resolved on SDS-PAGE and electroblotted onto the PVDF membrane. Western blot analysis was performed using anti-SETDB1 antibody (Bethyl Laboratories), anti-GST antibody (Santa Cruz Biotechnology), anti-mono- and polyubiquitin (FK2) antibody (Enzo Life Sciences), and anti-lamin B2 antibody (Abcam). The signals were visualized with the SuperSignal West Dura extended duration chemiluminescent substrate (Pierce).The GST-SETDB1 proteins were purified by affinity chromatography on glutathione-sepharose 4B. Measurement of the H3K9 methyltransferase activity was carried out using the EpiQuik Histone Methyltransferase Activity/Inhibition Kit (H3K9), as recommended by the manufacturer (EPIGENTEK). Absorbance at 450 nm was measured by a Microplate reader Model 550 (Bio-Rad).5 cells on 6-well plates. The cells were transfected with 2.8 μg of the expression vector. Forty-eight hours after the first transfection, the cells were transfected with 2.8 μg of the expression vector again. Twenty-four hours after the second transfection, the cells were cultured in DMEM supplemented with 10% FBS and 2% P/S. The cells were collected 120 h after the first transfection. Total RNA extraction and cDNA synthesis were performed as described previously [DNA transfection of HeLa cells was performed with Lipofectamine 2000 (Invitrogen), as described previously . The expeviously . PCRs weChIP assays were performed as described previously . DNA traAll data are presented as mean±standard errors (SEM). Statistical analyses were performed using the unpaired Dunnett's test with Statcel version 3.0 (OMS Publishing Inc.).Shultz et al. reported that SETDB1 purified from the cell extracts was observed as two bands by SDS-PAGE analysis and Coomassie blue staining . We hypoFirst, to reveal the amino acid site and type of PTMs in SETDB1, we prepared the affinity-purified GST fusion SETDB1 full-length protein (WT) from transfected HeLa cell extracts . We alsoNext, we investigated the H3K9 methyltransferase activity using the four kinds of purified SETDB1 proteins from HeLa cells . WT, DELWe also expressed and affinity-purified SETDB1 WT and the three mutants from baculovirus-infected Sf9 cell extracts, and performed the immunoblot analysis and H3K9 methyltransferase activity assay. The results obtained were similar to that with affinity-purified SETDB1 from HeLa cell extracts . These rThe pattern of PTMs of SETDB1 (570–1291) purified from HeLa cells did not differ from that of the protein purified from Sf9 cells. To identify the type of PTMs in SETDB1 using mass spectrometry, we performed a large-scale preparation and purification of GST-SETDB1 (570–1291) using Sf9 cells. GST-SETDB1 (570–1291) from Sf9 cell extracts was affinity-purified by using glutathione-sepharose 4B, and GST was removed from GST-SETDB1 (570–1291) by enzymatic cleavage . SubsequTo ascertain whether ubiquitin is attached to SETDB1, we performed immunoblot analysis with an anti-ubiquitin antibody using the four kinds of affinity-purified SETDB1 proteins from HeLa cell extracts. We were able to detect a single band of ubiquitinated protein from WT, DEL417, and DEL570 , althougNext, we examined the site of ubiquitin-modified lysine residue on SETDB1. Since DEL681 does not show evidence of ubiquitination in To identify the site of ubiquitination of SETDB1 between amino acids 681 and 1291, we reanalyzed the LC-MS/MS analysis results . SETDB1 Next, we affinity-purified DEL570, K597R, and K867R from HeLa cell extracts, and performed immunoblot analysis with an anti-ubiquitin antibody . In DEL5et al. reported that the H1224K, C1226A, and C1279Y mutants of SETDB1 showed impaired H3K9 methyltransferase activity [We examined the effects of SETDB1 ubiquitination on the H3K9 methyltransferase activity in affinity-purified SETDB1 from HeLa cell extracts. In K597R, which is modified by ubiquitin, methyltransferase activity at the same level as DEL570 was observed . On the SETDB1 suppresses the expression of its target genes by methylating H3K9 , 8, 23. SERPINE1 gene, a SETDB1 target gene, after transfection with SETDB1 WT, whereas it did not change in the cells transfected with SETDB1 K867R  is modified by ubiquitination in HeLa and Sf9 cells . We also1 is modiEscherichia coli that did not undergo PTMs failed to show the enzyme activity [We revealed that the ubiquitination of K867 of the human SETDB1 protein upregulates its H3K9 methyltransferase activity. SETDB1 protein expressed in Macaca mulatta, Mus musculus, and Rattus norvegicus), amphibia (Xenopus laevis), fishes (Danio rerio), and insects (Drosophila melanogaster). In addition, this lysine site is located within an insert region (SET-I) where the SET domain is bifurcated. SET-I is approximately 30 amino acids in length and is a region of considerable sequence variability within the SET family. Although the lengths of the SET-I region vary across family members, the structural architecture of SET-I is conserved and it is structurally static [We compared the lysine residue at amino acid position 867 of human SETDB1 with that of other species . The K86y static , 35. In y static . In casey static . Based oIn this study, we showed that the region between amino acids 570 and 680 is essential for the ubiquitination of K867 in human SETDB1 . An MBD In conclusion, we have demonstrated for the first time that ubiquitination of human SETDB1 at lysine 867 is required for its H3K9 methyltransferase activity and regulation of the expression of its target gene. Since, SETDB1 controls a variety of intracellular physiologic processes through its H3K9 methyltransferase activity, this finding will be useful in planning future studies to elucidate its functions.S1 Fig(A) GST affinity-purified SETDB1 proteins in Sf9 cells were resolved on 7% SDS-PAGE. Western blot analysis was performed using anti-GST antibody. Asterisks indicate degradation products from SETDB1. (B) H3K9 methyltransferase activity of GST affinity-purified SETDB1 proteins in Sf9 cells was measured. The values represent means±SEM (n = 3). N. D.: not detected.(TIF)Click here for additional data file.S2 FigThe red text denotes the amino acid residues that were detected by LC-MS/MS analysis; the black box denotes the region between amino acids 845 and 886; the white text on a black background denotes invariant residues.(TIF)Click here for additional data file.S3 FigSETDB1 proteins were expressed as GST fusion proteins in HeLa cells and purified on glutathione-sepharose beads. The H3K9 methyltransferase activity of the GST affinity-purified SETDB1 proteins in HeLa cells was measured. The values represent mean ± SEM (n = 3).(TIF)Click here for additional data file.S4 Fig(A) Schematic view of the mutation map for GST-SETDB1 (570–1291). (B and C) SETDB1 proteins were expressed as GST fusion proteins in HeLa cells and purified on glutathione-sepharose beads. The purified SETDB1 proteins were resolved on 5% SDS-PAGE and electroblotted onto PVDF membranes. Western blots of GST-SETDB1 proteins were probed with anti-SETDB1 antibody or anti-ubiquitin antibody.(TIF)Click here for additional data file.S5 FigHeLa cells were transfected with pcDNA3-hSETDB1 (SETDB1 WT), pcDNA3-hSETDB1 K867R (SETDB1 K867R), or pcDNA3 (Empty). (A) Purified genomic DNA after ChIP was analyzed by real-time PCR. Data were normalized to the input DNA. Fold change was calculated relative to Empty. All values are expressed as the mean ± SEM (n = 3). (B) mRNA levels were normalized to those of cyclophilin A mRNA. All values are expressed as the mean ± SEM (n = 3).(TIF)Click here for additional data file.S6 FigHomo sapiens, Macaca mulatta, Mus musculus, Rattus norvegicus, Xenopus laevis, Danio rerio, and Drosophila melanogaster is shown. The white text on a black background denotes invariant residues; the black text on a gray background indicates conserved residues.An alignment of the SETDB1 protein from (TIF)Click here for additional data file.S7 Fig(A) Schematic view of the mutation map for GST-SETDB1 (570–1291). (B) Twenty-four hours after transfection, cell extracts were prepared, and the extracts were subjected to SDS-PAGE. Western blot analyses were probed with anti-SETDB1 antibody or anti-lamin B2 antibody.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.