MYC oncogene causes overexpression of DNA methyltransferase (DNMT) 1 and 3B, which contributes to tumor maintenance. By utilizing a tetracycline-regulated MYC transgene in a mouse T-ALL  and human Burkitt’s lymphoma (P493-6) model, we demonstrated that DNMT1 and DNMT3B expression depend on high MYC levels, and that their transcription decreased upon MYC-inactivation. Chromatin immunoprecipitation indicated that MYC binds to the DNMT1 and DNMT3B promoters, implicating a direct transcriptional regulation. Hence, shRNA-mediated knock-down of endogenous MYC in human T-ALL and Burkitt’s lymphoma cell lines downregulated DNMT3B expression. Knock-down and pharmacologic inhibition of DNMT3B in T-ALL reduced cell proliferation associated with genome-wide changes in DNA methylation, indicating a tumor promoter function during tumor maintenance. We provide novel evidence that MYC directly deregulates the expression of both de novo and maintenance DNMTs, showing that MYC controls DNA methylation in a genome-wide fashion. Our finding that a coordinated interplay between the components of the DNA methylating machinery contributes to MYC-driven tumor maintenance highlights the potential of specific DNMTs for targeted therapies.Aberrant DNA methylation is a hallmark of cancer. However, our understanding of how tumor cell-specific DNA methylation patterns are established and maintained is limited. Here, we report that in T-cell acute lymphoblastic leukemia (T-ALL) and Burkitt’s lymphoma the  The diiewed in ). While iewed in , 4). MYCiewed in ). The fiiewed in , holds eMYC is well known as a site-specific transcription factor regulating the expression of hundreds of target genes. MYC forms heterodimers with MAX, binding to a DNA-motif termed E-box (CACGTG) . MYC–MAXGCN5 [GCN5, MYC exerts broad effects on global chromatin structure establishing a tumor-cell specific pattern. In parallel, MYC suppresses chromatin regulators including SIN3B, HBP1, SUV420H1, and BTG1 through miR-17-92 [MYC oncogene establishes and maintains a genome-wide epigenetic state, while MYC inactivation triggers dramatic alterations in chromatin structure leading to cellular senescence as an important mechanism of tumor regression. However, how DNA methylation contributes to MYC-driven tumor maintenance, and how genome-wide aberrant DNA methylation patterns are established and maintained remains largely unknown.Hence, the classic model has recently been extended to incorporate MYC’s role as a global regulator of transcription. There is growing evidence that MYC induces genome-wide changes in the epigenetic landscape of the cell as an important part of its neoplastic features, including alterations of post-translational histone modifications (reviewed in ). N-MYC-GCN5 . By contiR-17-92 . CorrespiR-17-92 . These rde novo methyltransferases that establish new DNA methylation patterns [de novo methylation is a common event in human neoplasia and is known to contribute to tumorigenesis [DNA methylation represents an important feature that controls gene expression programs in mammalian development and disease , 17. DNApatterns . The curigenesis –21. TumoMYC, establish and maintain tumor-cell specific DNA methylation patterns on a genome-wide level through modulating individual components of the DNA methylating machinery. Here, we report that in T-ALL and Burkitt’s lymphoma, oncogenic MYC deregulates the expression of both DNMT1 and DNMT3B, which in turn establishes and maintains tumor cell-specific DNA methylation patterns in a genome-wide fashion with importance for tumor maintenanceTogether, these observations suggest that oncogenes, such as MYC-driven hematopoietic malignancies harbor aberrant DNA methylation patterns. However, how these tumor cell-specific marks are established and maintained through the interplay between the DNA methylating machinery is still elusive. To unravel the role of DNA methyltransferases (DNMTs) in MYC-driven hematopoietic malignancies, we performed expression profiling for DNMT1, DNMT3A and DNMT3B comparing MYC-driven T-ALL and Burkitt’s lymphoma cells to non-malignant control cells.First, we took advantage of EμSRα-tTA;tet-o-MYC transgenic mice, in which the ectopic expression of MYC in hematopoietic lineages gives rise to T-ALL . The malDNMT3B mRNA  and corresponding protein levels than non-malignant tissue. We also found DNMT1 mRNA to be overexpressed in all three T-ALL samples . In contrast, DNMT3A mRNA levels were lower in 2833 and 1329  than in non-malignant tissue; only 6780 expressed more DNMT3A . As expected, MYC expression levels were very low in non-malignant tissues compared to tumor cells.RT-qPCR and Western blot analysis of tumor cells derived from EμSRα-tTA;tet-o-MYC transgenic mice indicate that DNMT3B expression levels were elevated in MYC-driven T-ALL compared to normal spleen from wild-type C57BL/6J mice Figure . T-ALL cDNMT3B mRNA  and protein expression in comparison to non-malignant spleen and B-cells, respectively. Similarly, DNMT1 mRNA expression was increased in all human T-ALL and Burkitt’s lymphoma cell lines  compared to normal spleen and B-cells, respectively. In contrast, DNMT3A mRNA was increased in all human T-ALL , but not in all Burkitt’s lymphoma cell lines. While we detected 1.73-fold (P<0.001) more DNMT3A in CA46 cells, Raji cells showed a significantly decreased -27.91-fold (P<0.001) DNMT3A mRNA expression.Next, we compared a panel of human T-ALL and Burkitt’s lymphoma cell lines to non-malignant human spleen, peripheral blood mononuclear cells (PBMCs) and B-cells obtained from healthy donors Figure  and 1F. Based on our expression profiling, we conclude that DNMT1 and DNMT3B are consistently overexpressed in MYC-driven T-ALL and Burkitt’s lymphoma cell lines, while DNMT3A is overexpressed in human T-ALL cell lines only.DNMT1, DNMT3A and DNMT3B mRNA expression levels are elevated in clinical T-ALL compared to non-malignant cells , DNMT3A  and DNMT3B  compared to Peripheral Blood Mononuclear Cells (PBMCs).To evaluate whether clinical T-ALL and Burkitt’s lymphoma specimens resemble our data from mouse and human tumor cell lines, we took advantage of publically available clinical expression data sets. By analyzing Oncomine data derived from Haferlach et al.,  we deters Figure . In T-ALDNMT1  and DNMT3B  when compared to non-malignant B-cells (BC) obtained from healthy donors  than in normal B-cells, which is consistent with our cell line analysis. Taken together, clinical specimens resembled our data from mouse and human cell line models in regard to DNMT1 and DNMT3B overexpression in T-ALL and Burkitt’s lymphoma.Furthermore, by analyzing Oncomine data derived from Brune et al.,  we deters Figure . In contc-MYC allele in mouse T-ALL  as well as in a human Burkitt’s lymphoma-like cell line model (P493-6). P493-6 cells are derived from immortalized human peripheral blood B-lymphocytes engineered to harbor a tetracycline-repressible c-MYC transgene [To determine whether DNMT3B overexpression in T-ALL and Burkitt’s lymphoma cells is MYC-dependent, we took advantage of the tetracycline-regulated ransgene , 26. P49ransgene . Utiliziransgene , 27–29.DNMT3B mRNA and protein levels. DNMT1 mRNA levels also correlated with the time- and concentration-dependent depletion of MYC in both T-ALL or Burkitt’s lymphoma-like cells; however, while DNMT3A mRNA levels followed the same trend in T-ALL, DNMT3A levels were less consistent with the titration of MYC in Burkitt’s lymphoma-like cells. Taken together, DNMT1 and DNMT3B expression exhibited a correlation to MYC levels in both mouse T-ALL and human Burkitt’s lymphoma models.To determine whether MYC and DNMT expression correlate in a time dependent manner, we treated mouse T-ALL (2833) and human Burkitt’s lymphoma-like cells (P493-6) for 1 and 2 days with 20 ng/mL DOX. In parallel, we titrated MYC expression through increasing DOX concentrations (0.1-0.5ng/mL) for 2 days to measure the concentration-dependence. Subsequent RT-qPCR and Western blot analysis revealed a correlation between MYC and DNMT3B expression levels Figure . In bothDNMT1 and DNMT3B transcription in T-ALL and Burkitt’s lymphoma-like cells is directly regulated by oncogenic MYC, we performed chromatin immunoprecipitation (ChIP) analysis measuring MYC binding to the genomic loci of DNMT1, DNMT3A and DNMT3B.To determine whether DNMT1 locus directly upstream of exon 1 (relative enrichment: 33) and at the DNMT3B locus downstream of exon 1 (relative enrichment: 70). A high resolution view of the DNMT1, DNMT3A and DNMT3B loci in T-ALL including the location of the ChIP microarray probes is shown in DNMT1 and DNMT3B in T-ALL , and DNMT3B downstream of exon 1 (relative enrichment: 132) as well as at two more sites in the gene body, which was not covered by the promoter array analysis in mouse T-ALL. While the DNMT3A locus did not show any significant enrichment for MYC in T-ALL, we found multiple sites in the gene body to be bound by MYC in P493-6 cells.For MYC-driven T-ALL cells (2833) derived from EμSRα-tTA;tet-o-MYC transgenic mice we utilized ChIP followed by promoter microarray hybridization (ChIP-chip) Figure . We founin T-ALL . To detein T-ALL . ConsistDNMT1 and DNMT3B regulation by MYC, we retrieved the genomic DNA sequences of the MYC binding peaks including surrounding sequences from the UCSC genome browser and performed an E-box motif search using the transcription factor binding profile software, JASPAR [DNMT1 and DNMT3B in both T-ALL, and Burkitt’s lymphoma-like cells or a scrambled control shRNA (CTRL). Before and upon induction of the shRNA through addition of 100 ng/mL DOX to the culture medium for 2 days, the expression level of MYC, its canonical target gene ODC1, as well as DNMT1, DNMT3A and DNMT3B were determined by RT-qPCR. In parallel, we performed the same analysis on two additional T-ALL cell lines (6780 and 1329) derived from EμSRα-tTA;tet-o-MYC mice, before and upon suppression of transgenic MYC for 1 day via 20 ng/mL DOX  and 3B-sh2 -3.82-fold (P<0.001). MYC expression did not change significantly in 3B-sh1 but increased 1.5-fold in 3B-sh2 (P<0.05)  and -2.16-fold (P<0.001) for 3B-sh1, respectively, compared to the control cells , in 3B-sh1 and 3B-sh2 cells  [CDKN2B (p15INK4b) increased 1.60- and 1.65-fold (P<0.05 and P<0.001), CDKN2A (p16INK4a) increased 1.42- and 1.54-fold (P<0.01 and P<0.01), and CDKN2D (p19INK4d) increased 1.63- and 1.57-fold (P<0.001 and P<0.01). Flow cytometric cell viability and cell cycle analysis based on propidium iodide (PI) confirmed a decrease in cell proliferation, indicating a decrease in S phase cells from 40.3% for SCR to 32.1% (P<0.001) for 3B-sh1 and 37.6% for 3B-sh2 (P<0.05) . In summary, we conclude that loss of DNMT3B expression in T-ALL leads to increase of cycline-dependent kinase inhibitors and diminished cell proliferation through cell cycle arrest, rather than apoptosis.To determine whether loss of DNMT3B function affects tumor cell proliferation and viability we carried out shRNA-mediated knock-down as well as pharmacologic inhibition of DNMT3B in MYC-driven T-ALL cells. We compared T-ALL cells upon stable shRNA-mediated knock-down of ) Figure  and 7B. s Figure . In paras Figure . RT-qPCRctively) . Further) Figure  and 7G. MYC, DNMT3B, DNMT3A, and DNMT1 expression ), CDKN2D ) and CDKN2B mRNA levels )  upon 100nM NA treatment for 2 days  Figure . Flow cy) Figure  and 8F. s Figure  and 8H, s Figure . While tDNMT3B-specific shRNA (6780 3B-sh2) to cells harboring a scrambled shRNA (6780 SCR) as control . We compared the above described MYC-driven T-ALL cells  stably expressing a  Figures . We gene Figures , as well Figures . To idenSPRY1  and FOXA1 (forkhead box A1) -box 12) and GLIS2 (GLIS family zinc finger 2)  and FOXA1   and GLIS2   Figure  and 10C,inger 2) . For all) Figure  and 10D,P<0.001)  and 7D uIn summary, we conclude that DNMT3B contributes to tumor maintenance of MYC-driven T-ALL cells through its effects on DNA methylation, and that loss of DNMT3B causes the reactivation of gene transcription through reversing promoter/CpG island methylation.Aberrant DNA methylation is an important feature of tumor cells. However, how tumor cell-specific DNA methylation patterns are established and maintained through the coordinated action of DNA methylating enzymes remains less understood. Here, we report that in T-ALL and Burkitt’s lymphoma MYC directly caused the overexpression of both DNMT1 and DNMT3B. This suggests for the first time that the MYC oncoprotein controls DNA methylation patterns in a genome-wide fashion via DNMTs.microRNA-29b targets DNMT3A and DNMT3B directly and DNMT1 indirectly thereby inducing global DNA hypomethylation and tumor suppressor gene expression [To investigate the role of DNMTs in MYC-driven tumor maintenance, we performed expression profiling on T-ALL and Burkitt’s lymphoma compared to non-malignant tissue. We found that DNMT1 and DNMT3B to be consistently overexpressed in transgenic models, human tumor cell lines, as well as clinical specimens. In contrast, DNMT3A expression was not consistent in both MYC-driven tumor types. Burkitt’s lymphoma did not display increased DNMT3A levels, implicating a differential mechanism of regulation. Our results are consistent with reports that DNMT activity is often elevated in tumor cells and is subsequently implicated in disease progression. All three DNMT enzymes have been reported to be overexpressed in hematological malignancies such as acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), diffuse large B-cell lymphoma (DLBCL) as well as mantel cell lymphoma (MCL) –40. In Apression . Interespression –45. It rMYC transgene in a mouse T-ALL  and human Burkitt’s lymphoma (P493-6) model, allowed the time- and concentration-dependent inactivation of MYC, causing both DNMT1 and DNMT3B expression to decrease step-wise exhibiting a direct correlation with MYC levels. Subsequent ChIP analysis revealed that MYC occupies sites within the DNMT1 and DNMT3B regulatory regions, suggesting a direct transcriptional regulation in mouse T-ALL and human Burkitt’s lymphoma-like cells. Sequence analysis identified canonical E-box sites for the MYC enrichment peaks for both DNMT1 and DNMT3B loci, supporting the model of direct MYC binding. Extending our analysis to include the chromatin status of DNMT1, and DNMT3B, we found that MYC binding peaks are enriched for H3K27Ac and H3K4Me3 typically placed near active regulatory promoter elements. Furthermore, we found H3K4Me1 which linked to distal regulatory regions to co-localize, overall strengthening our model that MYC directly upregulates DNMT1 and DNMT3B transcription. Knock-down of endogenous MYC in human T-ALL and Burkitt’s lymphoma cell lines supports above findings, indicating that DNMT3B overexpression directly depends on high levels of MYC. Only few direct transcriptional regulators of DNMT3B such as SP1, SP3 or FOXO3A [DNMT3B promoter activity in lung cancer [DNMT3B in T-ALL and Burkitt’s lymphoma reveals a novel mechanism of deregulation in cancer. The notion that the effect of MYC on DNMT1 expression is less consistent than on DNMT3B might indicate that DNMT1 levels are the reflection of cell proliferation rather than MYC activity directly. Considering the role of DNMT1 as maintenance DNMT this fits the working model. However, since in T-ALL MYC levels directly affect proliferation, the two are difficult to dissect. In combination with the promoter binding assay, the expression analysis provides evidence that increased DNMT3B expression is directly dependent on a positive transcriptional regulation by MYC.Further examination of the underlying regulatory mechanism in T-ALL and Burkitt’s lymphoma revealed a direct correlation of DNMT1 and DNMT3B expression with high MYC levels, which led us to assess whether transcriptional regulation is MYC-dependent. Indeed, we found that DNMT1 and DNMT3B are overexpressed in a MYC-dependent manner. The tetracycline-regulated r FOXO3A , 47 haveCDKN2B (p15INK4b), CDKN2A (p16INK4a), CDKN2D (p19INK4d), and CDKN1A (p21CIP1) to increase upon DNMT3B knock-down. This indicates that the above genes are either directly or indirectly suppressed by DNMT3B during T-ALL maintenance, suggesting that DNMT3B acts as a tumor promoter in this context. Controversially, recent reports indicate that DNMT3B functions as tumor suppressor during tumor initiation. Knockout of DNMT3B accelerates the initiation of MYC-driven T- and B-cell lymphoma in mouse models  [DNMT1 has been shown to delay the onset of T-cell lymphoma by suppressing tumor cell proliferation [Above findings suggested that a complex interplay of DNMTs is required for MYC-driven tumor maintenance. Indeed, shRNA-mediated loss of DNMT3B expression impaired cell proliferation in T-ALL. While this was associated with a significant decrease of S phase cells, we did not see a drastic increase in apoptotic cells. Supporting the notion of a cell cycle arrest rather than cell death as mechanism, we found a number of tumor suppressor genes, including  Eμ-MYC) –50, whil Eμ-MYC) . Similar Eμ-MYC) . Conversferation . The divSPRY1, FOXA1, SOX12, and GLIS2, we show that loss of DNMT3B expression was associated with a marked decrease of DNA methylation in CpG islands that are located near the transcription start site, accompanied by reactivation of the corresponding genes. Hence, we conclude that DNMT3B contributes to tumor maintenance of MYC-driven T-ALL through its effects on DNA methylation, and that loss of DNMT3B causes the reactivation of gene transcription through reversing CpG island methylation.Gaining mechanistic insight into the role of DNMT3B in establishing and maintaining DNA methylation patterns in T-ALL cells, we found that loss of DNMT3B expression caused genome-wide changes associated with changes in gene expression. The approximately 30,000 differentially methylated regions that were hyper- or hypomethylated are distributed across CpG islands, CpG island shelves, CpG island shores and open sea, as well as gene regulatory elements like enhancers, exons, intergenic regions, introns and promoters. More than 6,200 genes were differentially methylated within their promoters and more than 8,600 CpG islands were differentially methylated. For four candidates, Taken together, we propose a working model in which MYC, in addition to its role as a site-specific transcription factor, controls DNA methylation in a global manner through overexpression of DNMT1 and DNMT3B during tumor maintenance Figure . In non-GCN5 which facilitates wide-spread acetylation of H3 and H4 [SIN3B, HBP1, SUV420H1 and BTG1 through miR-17-92 [Further expanding the list of chromatin modifying enzymes that MYC is known to regulate in the context of cancer, the direct control of the DNA methylating machinery reveals a novel facet of MYC’s global reach. It ties into the increasing number of reports that MYC acts as global regulator of chromatin structure (reviewed in ). The fi3 and H4 . More reiR-17-92 . The epiiR-17-92 . The latiR-17-92 , 55. TheFurther investigations are needed to unravel the complex interplay between the DNMT enzymes in establishing tumor-cell specific DNA methylation pattern. In the light of an elusive pharmacologic MYC inhibitor, our results highlight the importance of DNA methylation in MYC-driven tumor maintenance, and reveal the potential of specific components of the DNA methylating machinery for targeted therapeutic strategies.Investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines and has been approved by the authors’ institutional review board.Mouse T-ALL cell lines were derived from the transgenic T-ALL mouse model  . Human BCell lines were infected with lentiviral vectors (pTRIPZ or pLKO.1-puro) containing either scrambled control or specific shRNAs. Briefly, HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) with pTRIPZ/pLKO.1, pCMV-VSV-G and pCMV-dR8.2 dvpr plasmids. Virus particles were collected for spin infection. Upon selection of positive cells with 2-4 μg of puromycin, shRNA expression was induced with 100 ng/mL of DOX for the indicated times. The specific oligo sequences of shRNA are: MYC CCGGCCAAGGTAGTTATCCTTAAACTCGAGTTTAAGGATAACTACCTTGGTTTTTG; DNMT3B-sh1 TTGGCATTAGAATATCAGAGCCTCGAGGCTCTGATATTCTAATGCCAA; DNMT3B-sh2 AATTGCTGGGTACAACTTGGGCTCGAGCCCAAGTTGTACCCAGCAATT.Human spleen  obtained from a healthy donor, was purchased from Zyagen Inc. Human PBMCs  and B-cells  were obtained from the Augusta University Biorepository. 6-8 week old wild-type C57BL/6J mice were obtained from Jackson Laboratory. Mice were housed according to Augusta University’s animal care and IACUC guidelines.MYC F: CTGCGACGAGGAGGAGAA, R: GGCAGCAGCTCGAATTTCTT; Hs DNMT1 F: CC TAGCCCCAGGATTACAAGG, R: ACTCATCCGATT TGGCTCTTTC; Hs DNMT3A F: AGTACGACGA CGACGGCTA, R: CACACTCCACGCAAAAGCAC; Hs DNMT3B F: ACCTCGTGTGGGGAAAGATCA, R: CCA TCGCCAAACCACTGGA; Hs ODC1 F: TTTACTG CCAAGGACATTCTGG, R: GGAGAGCTTTTAACCAC CTCAG; Hs RPL13A F: CGGACCGTGCGAGGTAT, R: CACCATCCGCTTTTTCTTGTC; Mm MYC F: TCTCC ATCCTATGTTGCGGTC, R: TCCAAGTAACTCGGTC ATCATCT; Mm DNMT1 F: AAGAATGGTGTTGTC TACCGAC, R: CATCCAGGTTGCTCCCCTTG; Mm DNMT3A F: AAGGGGCCTTCAACGTG, R: ATTTATCC AGACTCGCGTGC; Mm DNMT3B F: GGCTT CAAGCCTACTGGGATCGAG, R: CCACAGGACAA ACAGCGGTCTTCC; Mm ODC1 F: GACGAGTTTGA CTGCCACATC, R: CGCAACATAGAACGCATCCTT; Mm SOX12 F: GGCTCCTCCCTAAGTCCATC, R: CC TAGCAACAACGTGCTTCA; Mm FOXA1 F: TGGA CTTCAAGGCATACGAGC, R:GCACGGGTCTGGAAT ACACA; Mm GLIS2 F: GACGAGCCCCTCGACCTAA, R: AGCTCTCGATGCAAAGCATGA; Mm SPRY1 F: GGTCATAGGTCAGATCGGGTC, R: GTCCCGTATTC CACCATGCT; Mm UBC F: AGCCCAGTGTTACC ACCAAG, R: ACCCAAGAACAAGCACAAGG.Total RNA was isolated using the NucleoSpin RNA Kit including DNase-I digest (Machery-Nagel Inc.) following the manufacturer’s protocol. 0.5 μg RNA were reverse transcribed into cDNA using the iScript cDNA Kit (BioRad). Quantitative PCR was performed using SYBR GREEN (BioRad) in an ABI StepOne Plus analyzer. The specific forward (F) and reverse (R) primer sequences are as follows: Hs Cells were fixed in 70% methanol and stained using a Propidium Iodide (Acros Organics) solution containing PBS+0.5% BSA, 50μg/mL PI, and 200μg/mL RNaseA. Cells were then analyzed immediately on a LSR II flow cytometer (Becton Dickinson). FACS data was analyzed using FlowJo software (Tree Star).Annexin V and propidium iodide staining was used for the study of cell cycle distribution and apoptosis using the Annexin V-FITC Early Apoptosis Detection Kit from Cell Signaling. Briefly, cells were resuspended in PBS and fixed with ice-cold ethanol. Cells were treated with RNase and propidium iodide and analyzed on a LSR II flow cytometer (Becton Dickinson). FACS data was analyzed using FlowJo software (Tree Star).t-test was used to calculate p-values; statistically significant values are specified in the figure legends.All experiments were performed on biological replicates unless otherwise specified. Sample size is reported in the respective figure legends. All quantitative PCR were run in triplicates and standard deviation is shown in the figures. Two-tailed Student’s https://imagej.nih.gov/ij/) [Total protein extracts were prepared using a lysis buffer , sonicated on ice using a Sonifier 250D (Branson Ultrasonics), and heated for 5 min at 99 °C. Protein concentrations were determined with the Bio-Rad DC Protein Assay Kit (BioRad) using bovine serum albumin as a standard. Protein extracts were separated in 10% SDS-PAGE and electrotransferred to PVDF membranes . Antibodies used were as follows: MYC #ab32  , #sc764 gov/ij/) .3). Chromatin was reverse-crosslinked by adding 0.2 M NaCl for 4 hours at 65°C. After RNase and proteinase K treatment, DNA was extracted using phenol-chloroform.For ChIP, cells were crosslinked with 1% formaldehyde followed by quenching with 0.2 M glycine. After washing with PBS, the cells were resuspended in lysis buffer . Chomatin was sheared by sonication for 10 min using a Branson 250 Sonifier, diluted in ChIP Dilution Buffer  and incubated with 5 μg of specific antibody overnight. The bound material was recovered after 2 hours incubation with 50 μl Dynabeads/Protein G (Invitrogen/Thermo-Fisher), rotating at 4°C. The beads were washed, once in Low Salt Buffer , twice in High Salt Buffer , twice in LiCl Buffer  and twice in TE buffer. ChIPed material was eluted by two 15 minute incubations at room temperature with 250 μl Elution Buffer (1% SDS and 0.1 M NaHCOhttps://genome.ucsc.edu/). E-box motif search was performed for the genomic DNA sequence surrounding MYC binding peaks using JASPAR (http://jaspar.genereg.net) [ChIP followed by promoter array analysis (ChIP-chip) was performed using the 244K Mouse Promoter Microarray Kit (Agilent Technologies) covering ~19,000 defined mouse genes following the manufacturer’s protocol. ChIP DNA was used for microarray hybridization following the manufacturer’s protocol. ChIP-chip data processing was performed using Agilent Technologies ChIP Analytics Platform v1.3.1 by mapping to data to NCBI36/mm8. ChIP figures were generated using the UCSC genome browser (reg.net) .DNMT1 ChIP-1  F: CATGGCCACAACATCTCACT, R: TTATCTCACCA GCCCCAGAC; Mm DNMT1 ChIP-2  F: GAATGACCGAGGACCAGAAA, R: TG TGTACATGTGCGTGGGTA; Mm DNMT1 ChIP-3  F: TCTTAGTAGCCAGG GCCAGA, R: TGGGTGCAGCTCTGAGTATG; Mm DNMT3B ChIP-1  F: TT GTGTTTCTCCAGTGGTTCAG, R: CATACATGTTCC CCCAACTACC; Mm DNMT3B ChIP-2  F: CTCAGTAGAGTGCTTCC GGACT, R: TACCTGAGGTTTCCAAGGTCTG; Mm DNMT3B ChIP-3  F: AGA TAGCGCTTGCTAAATCTGG, R: CACTACCTGGGGG TAAAGAACA;ChIP followed by quantitative PCR was performed using SYBR GREEN (BioRad) in an ABI StepOne Plus analyzer. The specific forward (F) and reverse (R) primer sequences and their genomic location are as follows: Mm https://www.ncbi.nlm.nih.gov/gds).RRBS was performed by Diagenode, Inc. as described before . Sequenc


P< 0.001, P< 0.001). We saw the similar correlation between D2, PFS and OS . Multivariate survival analyses showed that D-dimer was independently associated with OS and PFS in patients with endometrial cancer. The ΔD level was not related to the OS and PFS in endometrial cancer patients. The levels of D-dimer may be considered as an important predictor of PFS and OS in endometrial cancer patients treated with IMRT.Explain the important role of plasma D-dimer in cancers. Plasma D-dimer is increased in various tumors. However, the predictive value of plasma D-dimer is unclear. This study is aimed to evaluate the prognostic value of the D-dimer level in patients managed with intensity-modulated radiation for endometrial cancer. The D-dimer levels of forty patients with endometrial cancer were assessed before (D1) and after (D2) intensity-modulated radiation therapy (IMRT), respectively. The D-dimer level changes (ΔD) were defined as D2 minus D1. Cox regression and log-rank tests were used to evaluate the D-dimer levels in relation to progression free survival (PFS) and overall survival (OS). The OS and PFS of patients with a low D1 were significantly longer than those with a high D1 ( Endometrial cancer is a common gynaecological malignancies . AlthougTumor cells activate the coagulation pathway in endothelial cells, resulting in the secretion of procoagulants and a secondary increase of fibrinolysis and fibrin degradation product D-dimer , 5. D-diIn this study, the plasma D-dimer levels were measured in patients treated with hysterectomy and postoperational IMRT for endometrial cancer. The prediction value of D-dimer in relation to progression free survival (PFS) and overall survival (OS) was also investigated.Forty patients with complete clinical data were included in this study. The median age was 56 years . Clinicopathologic characteristics were shown in Table P< 0.001, P< 0.001), histologic grade , depth of myometrial invasion (MI) , lymph node metastasis , D1 , and D2  were significantly associated with OS and PFS. Multivariate analysis using the Cox proportional hazards model demonstrated that FIGO stage (hazard ratio (HR) = 33.338, 95 % confidence interval (CI): 0.259-49.805, P= 0.001; HR= 23.293, 95 % CI: 0.374-541.274, P< 0.001, respectively), D1  as well as D2  were independent prognostic indicator of OS and PFS . Similar correlation was found between OS and PFS and D-dimer on D2 . There was no differences in OS and PFS between patients with a higher ΔD and lower ΔD . As shown in Figure Kaplan–Meier analysis was performed to the levels of D-dimer on D1, D2 in relation to patients’ survivals. Based on the cut-off values described above, we divided patients into subgroups with lower level of D-dimer or higher level of D-dimer. The median OS and PFS times of all patients were 48 and 32 months, respectively. The Kaplan–Meier survival curves indicated that the OS and PFS of patients with a higher level of D-dimer on D1 were significantly shorter than those with a lower level on D1 , histologic grade , depth of MI , lymph node metastasis , PFS  and OS . However, there was no association between the levels of D-dimer on D1 and D2 and other prognostic factors such as age, BMI, ER, PR expression. ΔD levels had no correlation to the above factors.Moreover, to detect the prognostic value of D-dimer in endometrial cancer patients, the premonitory affection of the levels of D-dimer on D1 and D2 were analyzed in the subgroup stratified by the FIGO stage II–IV. The mean concentrations of D-dimer on D1 in patients with FIGO stage II, III, IV stages were 0.33±0.54mg/L, 1.76±1.90mg/L, 3.81±4.33mg/L, respectively. The mean concentrations of D-dimer on D2 in patients with FIGO stage II, III, IV stages were 1.89±2.02mg/L, 3.12±4.61mg/L, 6.71±6.04mg/L, respectively. Patients with a higher level of D-dimer on D1 or D2 had remarkably shorter OS and PFS than the patients with a lower level of D-dimer on D1 or D2 level in patients with the stage II and III subgroups . The progression disease was significantly related to the increase of D-dimer after IMRT. Our data exhibited that the D-dimer level may be considered as a predictor for IMRT response in endometrial cancer patients.We further assessed the relation between ΔD and IMRT response. The mean D2 level reduced by 0.74 mg/L compared to the D1 level in 14 patients with stable disease  were not collected because D-dimer can predict VTE in cancer patients , 22. It In summary, our data indicate that D-dimer levels before and after IMRT D-dimer is correlated with OS and PFS in endometrial cancer patients. Additionally, the D2 is associated with survival rate in patient with endometrial cancer. We demonstrated that D-dimer levels could be also used to assess prognosis and IMRT response in endometrial cancer patients. Further prospective research is needed to make sure these discoveries.The data of 40 patients with endometrial cancer patients in Shanghai Jiao Tong University Affiliated Sixth People's Hospital between 2006 and 2009 were collected in our study. The inclusion criteria of this study were the following: (1) patients with histological confirmed endometrial cancer based on the FIGO; (2) no previous treatment; (3) no previous others malignancies; and (4) none of the patients had taken in any anticoagulant drugs before enrollment. Ethical approval for the study protocols was obtained and informed consents were obtained from each patient. The mean follow-up period for these patients was 57 months (range: 9–84 months). All patients were followed up by determination of three monthly ultrasonography (US), computed tomography (CT) or magnetic resonance imaging (MRI) scan. The diagnosis of recurrence and metastases was based on two factors: histopathological findings of the tumor tissue in patients and on the characteristic appearance on US, CT and MRI.We collected clinical data including age, BMI, FIGO stage, histologic grade, depths of MI, lymph node metastasis, KPS score, OS and PFS time and so on. D-dimer levels were detected before and after IMRT.P value < 0.05 was considered statistically.Statistical analysis used the SPSS version 19.0 statistical software. Survival analysis was performed using the Kaplan–Meier method, and the log-rank test was used for analysis. Univariate and multivariate analyses were performed using Cox's proportional hazards model. The relationship between D-dimer levels and clinicopathological features were analyzed using χ2 test. The data are expressed as the mean±SEM, and


Mutation in CBS enzyme causes homocysteine levels to rise, and gives rise to a condition called hyperhomocysteinuria. To date, numerous mouse knockout models for CBS enzyme has been generated, which show panoply of defects, reflecting the importance of this enzyme in development. In zebrafish, we and others have identified two orthologs of cbs, which we call cbsa and cbsb. Previous gene knockdown studies in zebrafish have reported a function for cbsb ortholog in maintaining ion homeostasis in developing embryos. However, its role in maintaining H2S homeostasis in embryos is unknown. Here, we have performed RNA analysis in whole zebrafish embryos that showed a wide expression pattern for cbsa and cbsb primarily along the embryonic axis of the developing embryo. Loss-of-function analysis using a combination of approaches which include splice morpholinos and CRISPR/Cas9 genomic engineering show evidence that cbsb ortholog is responsible for anterior-posterior axis development, and cbsa function is redundant. Cbsb loss of function fish embryos show shortened and bent axis, along with less H2S and more homocysteine, effects resulting from loss of Cbsb. Using a chemical biology approach, we rescued the axis defects with betaine, a compound known to reduce homocysteine levels in plasma, and GYY4137, a long term H2S donor. These results collectively argue that cells along the axis of a developing embryo are sensitive to changes in homocysteine and H2S levels, pathways that are controlled by Cbsb, and thus is essential for development.The cystathionine ß-synthase (CBS) is a critical enzyme in the transsulfuration pathway and is responsible for the synthesis of cystathionine from serine and homocysteine. Cystathionine is a precursor to amino acid cysteine. CBS is also responsible for generation of hydrogen sulfide (H In addition to H2S biosynthesis, CBS and CSE enzymes also participate in transsulfuration reactions involving cystathionine (precursor to cysteine) biosynthesis through a condensation reaction of serine and homocysteine  and cystathionine γ-lyase (CSE) are two key enzymes that are involved in the biosynthesis of hydrogen sulfide   and not cse showed a reduction in calcium influx in larval zebrafish  pattern data observed on zebrafish information network site. To investigate this hypothesis, we performed RNA analysis and whole mount ISH (WISH) across embryonic stages. We also performed loss-of-function analysis using MOs  and betaine (Trimethylglycine) chemicals were purchased from Sigma Aldrich. 1 M Stock solution of GYY4137 was prepared in dimethylsulfoxide (DMSO) and stored at 4°C. 50 mg/ml solution of betaine was prepared in water and stored at 4°C. 1 M stock solution of NaSH.xH2O was prepared in water and used immediately.GYY4137 (P-(4-Methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid), NaSH.xHcbsa and cbsb were generated using PCR-based methods. The primers for these probes include:Probes for sense and anti-sense Cbsa sense: Fwd 5′-CACCGAAATAATACGACTCACTATAGGGGATGGAGACAGACCCCCACA, Rev 5′-GGCACTTTTCCTTCAATTTCCGA and antisense primers: Fwd 5′- GATGGAGACAGACCCCCACA, Rev 5′ CACCGAAATTAACCCTCACTAAAGGGGGCACTTTTCCTTCAATTTCCGACbsb sense: Fwd 5′-CACCGAAATAATACGACTCACTATAGGGATCAACGGGGATGCTGACGAT, Rev 5′- CAATCTCAGCACCCAGAGCA and antisense primers: Fwd 5′- ATCAACGGGGATGCTGACGAT, Rev 5′- CACCGAAATTAACCCTCACTAAAGGGCAATCTCAGCACCCAGAGCAPrimer sequences used for amplification include:Cbsa, Fwd 5′- GATGGAGACAGACCCCCACA, Rev 5′- GGCACTTTTCCTTCAATTTCCGACbsb, Fwd 5′- ATCAACGGGGATGCTGACGAT, Rev 5′- CAATCTCAGCACCCAGAGCAactb1): Fwd 5′- GAAATTGTCCGTGACATCAA, Rev 5′- CACTGTGTTGGCATACAGGTActin  buffer containing protease inhibitors. Measurement of protein concentration was performed using BCA assay kit from Pierce, Grand Island, NY, USA. SDS-PAGE and immunoblotting was performed using standard protocol. The cell lysates were separated on 10% glycine SDS-PAGE gel and transferred to PVDF membrane. Membranes were blocked in 5% BSA in TBS with 0.1% TWEEN-20 (TBST) for 1 h at room temperature followed by incubation with indicated primary antibodies in TBST with 5% BSA. Antibodies were purchased from following vendors. CBS (D8F2P) purified rabbit antibody  was used at a 1:1000 dilution; anti-GAPDH antibody  was used at a 1:5,000 dilution at 4°C overnight. Horseradish peroxidase conjugated secondary antibodies were used and proteins were visualized by Clarity Western ECL Substrate (Bio-Rad). For the immunofluorescence experiment, CBSB-S1-injected embryos, ConMO-injected embryos and cbsb-CRISPR embryos were dechorionated at 48 hpf and fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at 4°C overnight. The following day, embryos were washed twice with PBST  Tween 20), permeabilized in ice-cold acetone for 10 min and washed in PBST. Embryos were blocked in 10% normal goat serum for 1 h and incubated with purified rabbit anti-homocysteine antibody  overnight at 4°C. Embryos were washed twice with PBST, incubated with the appropriate secondary antibody  for 2 h at RT, washed with PBST and imaged on a Zeiss AxioObserver Z1 fluorescence inverted microscope.For western blotting, 48 h post fertilization (hpf) CBSB-S1-injected, ConMO-injected and Figure 2C). Control MO sequence used in this study is 5′-CCTCTTACCTCAGTTACAATTTATA-3′. All MOs were each injected into Transgenic (Tg: flk:EGFP) , 200 μL of N, N-dimethyl-p-phenylenediamine sulfate (20 mM in 7.2 M HCl), and 200 μL of FeCl3 (30 mM in 1.2 M HCl). The mixture was incubated at room temperature in darkness for 15 min, and finally 600 μL of the mixture was added to a tube with 150 μL of trichloroacetic acid (10% wt/vol) to precipitate protein. The precipitated protein was removed by centrifugation at 10,000 g for 5 min, and absorbance at 670 nm of the resulting supernatant (200 μL) was determined. The H2S concentration of each sample was calculated against a calibration curve of NaHS.Hcbsb CRISPR embryos were treated with either 1 mM betaine or 1 mM GYY4137 or 1 mM NaHS at 6 hpf. All embryos were then developed to 30 or 52 hpf in the 28°C incubator. Pictures were taken as described earlier.CBSB-S1 injected or cas9 mRNA were synthesized by in-vitro transcription as previously described.  fish were fin-clipped at adulthood and screened for INDELs at the target location by Sanger sequencing. Two F0 founders containing the same 2 base pair deletion at the target site were bred to produce F1 mutants . F1 mutants were raised to adulthood and in-crossed for production of F2 embryos, for which phenotypes were observed and quantified. For rescue, CRISPR embryos were placed in 1 mM betaine at 6 hpf. CRISPR phenotypes were evaluated at 5 dpf. All CRISPR lines were generated in the background of (Tg: flk:EGFP)  stage, and the expression is constitutive in the remaining period of development  using gene-specific primers for s Figure . Both cbf Figure . Notablyt Figure . To dete Figures , an earln Figure  and in tn Figure ′. At 24 n Figure ′, cbsb es Figure , while ig Figure . At 48 h Figures ′. WISH fs Figure . Cbsa gen Figure , particucbsa and cbsb, we employed two strategies, morpholinos (MOs) and CRISPR/Cas9 genomic engineering methods. We designed MOs that targeted cbsa and cbsb ; cbsb: intron 2-exon3 (CBSB-S1), exon3-intron3 (CBSB-S2), and intron4-exon5 (CBSB-S3)]. MO sequences are provided -injected embryos. CBSB-S1 MO1 knockdown embryos showed reduction in H2S levels compared to their control counterparts .To investigate function of b Figure  genes atd Figure . Efficacd Figure  and protd Figure  level. Ct Figure . Of the d Figure . The bana Figure , but conf Figure . Becausecbsa and cbsb in embryonic development. CBSA-S1 alone injected embryos showed no phenotype (data not shown). CBSB-S1 when injected alone into 1-cell stage zebrafish embryos showed a shortened anterior-posterior axis and head edema , and correlated well with percentage mortality . At ~50 hpf, the axis defects were pronounced in CBSB-S1-injected embryos . We also investigated the vasculature grossly since we injected CBSB-S1 into transgenic embryos where VEGFR2 promoter or fms-like kinase (FLK) drives enhanced green fluorescent protein (EGFP) [Tg (flk:EGFP)] expression . Three distinct axis phenotypes were observed  was found to be statistically significant compared to the percentage of embryos with bent axis in the control flk:EGFP CASPER line. These results show that cbsb is mainly responsible for axis development in zebrafish.To complement MO loss-of-function analysis, we generated guide RNAs (gRNAs) that target exon 4 of e Figure . Guide Re Figure , and gRNe Figure . Indeed,e Figure . Also, ch Figure . The CBSh Figure  in cbsb d Figure , which wd Figure . The phed Figure  showed ~s Figure , the dif2S metabolism  is an important byproduct of cysteine metabolism, and was observed to be lower in CBSB-S1 knockdown embryos , & GYY4137  for the bent axis phenotype. Similarly, the differences between untreated morphant embryos and morphant embryos treated with GYY4137 were found to be statistically very significant (***P < 0.0001) for both the bent axis phenotype and head edema phenotype. We also treated cbsb CRISPR fish lines with betaine  were statistically very significant (***P < 0.001). The differences between the Controls Untreated and Controls + Betaine groups were not statistically significant.Based on s Figure , the dife Figure , which r2S homeostasis during embryonic zebrafish development. The salient features include, the characterization of the expression of cbsa and cbsb transcripts during embryonic development, the loss-of-function analysis using two complementary independent approaches, and finally the rescue of the axis phenotype with small molecules that are used for treatment of CBSB enzyme deficiency in the clinic.This manuscript reports the importance of Cbsb enzyme in maintaining Hcbsa and cbsb across developmental stages shows that cbsb is the dominant gene in terms of its levels, which later also is the case for function. The expression of cbsb is observed as early as bud stage (10 hpf) along the developing embryonic axis, and continues through 24 and 48 hpf along the anterior-posterior axis of the embryo. Cbsb expression is prominent in the brain and somite muscle, two tissues that are highly susceptible to changes in H2S levels during normal physiology.The RNA expression pattern for cbsb was to use complementary methods such as MOs and CRISPR-Cas9. Given the evidence that MOs induced phenotypes do not replicate fully in genetic mutants , and carbon monoxide (CO) collectively referred to as gasotransmitters interact leading to oxidative stress in the developing embryo (Olson et al., Previous work has shown that small molecule slow releasing Hcbsb in zebrafish using multiple approaches, and have demonstrated that cbsb is partly responsible for anterior-posterior axis development in zebrafish. A combination of homocysteine and H2S regulation is responsible for the axis development, and structures along the axis of the embryos are highly susceptible to this regulation.In summary, we have identified a novel phenotype for SP designed and performed experiments, interpreted data, wrote, and edited the manuscript; CK designed and performed experiments, and interpreted data, wrote, and edited the manuscript; AD designed and performed experiments, and interpreted data; SE-B designed and performed experiments, and interpreted data; NL performed experiments and interpreted data; RB provided intellectual input, direction in experimental design, and edited the manuscript; PM provided intellectual input, direction in experimental design, provided resources, and edited the manuscript; RR provided intellectual input, direction in experimental design, provided resources, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


People who had ever smoked and those who were exposed to environmental tobacco smoke had significantly increased UC odds ratios (ORs) of 1.65 and 1.68, respectively. Participants who had smoked more than 18 pack-years had a significantly increased UC OR of 2.64. People who had ever smoked and who carried the A/A genotype of the TNF-α -308 G/A polymorphism had a significantly higher UC OR (10.25) compared to people who had never smoked and who carried the G/G or G/A genotype. In addition, people who had ever smoked and who carried the IL-8 -251 T/T genotype had a significantly increased UC OR (3.08) compared to people who had never smoked and who carried the T/A or A/A genotype. In a combined analysis of three major risk factors , subjects with any one, any two, and all three risk factors experienced significantly increased UC ORs of 1.55, 2.89, and 3.77, respectively, compared to individuals with none of the risk factors. Conclusions. Our results indicate that the combined effects of cumulative cigarette exposure and the TNF-α -308 A/A genotype and/or the IL-8 -251 T/T genotype on UC OR showed a significant dose-response relationship.Cigarette smoking and exposure to environmental tobacco smoke are well-known risk factors for urothelial carcinoma (UC). We conducted a hospital-based case-control study involving 287 UC cases and 574 cancer-free controls to investigate the joint effects of cigarette smoking and polymorphisms of inflammatory genes on UC risk. Tumor necrosis factor alpha (TNF- The bladder is the most common site of urothelial carcinoma (UC), a multifactorial malignancy influenced by exogenous exposure to environmental risk factors and molecular variations in metabolism-related genes . Cigaretα) and interleukin-8 (IL-8), which are involved in the development of various malignancies [α expression through upregulating the activator protein, AP-1 [α is involved in the immune response during the intravesical instillation of bacillus Calmette-Guerin [α and IL-8 [α initiates signaling pathways that activate proinflammatory gene expression via the transcription factor; nuclear factor-kappa B (NF-κB) is also produced by tumors and acts as an endogenous tumor promoter. Cytokines, like IL-6 and IL-8, are also involved in transformation and angiogenesis, respectively [Oxidative stress can induce proinflammatory cytokines such as tumor necrosis factor alpha  is located in promoter and has functional impact on the TNF-α expression [α, -308 G/A polymorphism was investigated and found to be related to several cancers, including breast, gastric, and bladder cancers [ IL-8 gene is located on chromosome 4q13-q21, and several polymorphisms were identified in this gene [ IL-8, and it was identified to be associated with various cancer risks in Asian group [ TNF-α and IL-8 genes may modify the risk of UC.The polymorphism ofpression –15. Comp cancers , 16–18. his gene . A commo TNF-α -308 G/A and IL-8 -251 T/A polymorphisms on the UC risk, we conducted a hospital-based case-control study. We also examined the combined effects of cumulative cigarette exposure, the TNF-α -308 A/A genotype, and the IL-8 -251 T/T genotype on UC risk.To investigate the effects of Declaration of Helsinki.This study involved 861 participants enrolled from the Departments of Urology and those undergoing a general health examination at National Taiwan University Hospital, Taipei Medical University Hospital, and Taipei Municipal Wan Fang Hospital between September 2002 and May 2009. In total, 287 UC cases were included (with a mean age of 62.95 ± 13.62 years). Each case was diagnosed with histopathological confirmation, which was performed using routine urological practices and verified by board-certified pathologists. In total, 574 age- and gender-matched cancer-free controls (with a mean age of 62.56 ± 13.50 years) were recruited from a hospital-based pool. All participants provided informed consent before the questionnaire interview and biospecimen collection. The Research Ethics Committee of National Taiwan University Hospital  approved this study, and this study complied with the World Medical AssociationWell-trained investigators interviewed each patient using a structured questionnaire. Information collected by the questionnaire included demographic characteristics and lifestyle habits such as cigarette smoking, secondhand smoke exposure, betel nut chewing, alcohol, tea, and coffee consumption, exposure to occupational and environmental carcinogens such as pesticides, and a family history of disease. Study subjects who had smoked more than 100 cigarettes during their lifetime were regarded as people who had ever smoked, while those who had smoked fewer than 100 cigarettes were defined as people who had never smoked. Environmental tobacco smoke (secondhand smoke exposure) was assessed through a questionnaire asking participants whether anyone had ever smoked around them. A 6~8-ml sample of venous blood was drawn from each participant for genotype determination. TNF-α -308 G/A polymorphism: 5′-TCCTCCCTGCTCCGATTCCG-3′ (sense) and 5′-AGG CAATAGGTTTTGAGGGCCAT-3′ (antisense). The thermal PCR conditions were as follows: one cycle at 95°C for 5 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 45 s; and a final extension at 72°C for 10 min. After complete digestion with the NcoI restriction enzyme, the resulting DNA fragments, which represented the TNF-α -308 G/A polymorphism, were determined . PCR-RFLP genotyping and sequencing of TNF-α -308 G/A are shown in  IL-8 -251 T/A polymorphism, the following primers were used: 5′-ATCTTGTTCTAACACCTGCCACTC-3′ (sense) and 5′-TAAAATACTGAAGCTCCACAATTTGG-3′ (antisense) [ IL-8 -251 T/A polymorphism . PCR-RFLP genotyping and sequencing of IL-8 -251 T/A are shown in Genomic DNA was extracted from the buffy coat of peripheral blood using standard methods and stored at −80°C. Genotyping was completed using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method modified from a study by Duarte et al. . Brieflytisense) . The PCR TNF-α -308 A/A genotype, and the IL-8 -251 T/T genotype on the UC risk were estimated by odds ratios (ORs) and associated 95% confidence intervals (CIs) using a multivariate-adjusted logistic regression. All data were analyzed using the Statistical Analysis Software for Windows, ver. 9.2 . p values of < 0.05 were considered statistically significant.A goodness-of-fit Chi-squared test was used to test for Hardy-Weinberg equilibrium (HWE) among the cancer-free controls. Study subjects who had smoked more than 100 cigarettes during their lifetime were regarded as people who had ever smoked, while those who had smoked fewer than 100 cigarettes were defined as people who had never smoked. The pack-years of cigarette smoking were calculated using the formula: pack-years = (cigarettes per day ÷ 20) × (number of years smoked). Because of dependent variable (UC) is binary, logistic regression is used to assess the association between UC and related risk factors. The combined effects of cumulative cigarette exposure, the TNF-α -308 A/A genotype and the IL-8 -251 T/T genotype among controls fit HWE . The genotype distribution of the control did not show significant difference from the Hardy-Weinberg equilibrium values. Subjects who carried the TNF-α-308 A/A genotype had a significantly increased OR for UC  compared to those with the G/G or G/A genotype. Subjects who carried the A/A genotype of the IL-8 -251 T/A polymorphism had a significantly lower OR for UC  than those carrying the T/T genotype (data not shown).No significant differences were found in age or gender between the UC cases and cancer-free controls. Occasional alcohol drinkers had a significantly lower OR for UC than people who had never consumed alcohol. After adjusting for age, gender, educational level, alcohol consumption, and other risk factors, people who had ever smoked had a significantly higher UC OR of 1.65 (95% CI: 1.19~2.27) compared to people who had never smoked. Individuals who were exposed to environmental tobacco smoke had a significantly higher UC OR  than those with no exposure. Frequencies of the TNF-α -308 G/A and IL-8 -251 T/A genotypes, individual characteristics, including age, gender, educational level, cigarette smoking, environmental tobacco smoke exposure, and alcohol consumption, displayed no significantly different distributions in either of the polymorphism strata  in a combined analysis (p for trend < 0.0001).Furthermore, we included three major risk factors  and found dose-response relationships for UC risk in study subjects with any one risk factor, any two risk factors, and all three risk factors.In this study, the risk of UC in smokers was 2~2.5-fold compared to nonsmokers. This result was consistent with a previous study . In our  TNF-α -308 G/A polymorphism contributes to carcinogenesis and that the variant A allele was associated with increased TNF-α expression [ TNF-α -308 G/A polymorphism was significantly associated with the tumor stage and grade of bladder cancer [ TNF-α -308 G/A polymorphism had a significantly higher OR for UC than those with the G/G or G/A genotype. These results suggest that the TNF-α -308 G/A polymorphism might regulate angiogenesis, which plays a role in the invasion and metastasis of various tumors. However, another study did not report the same findings in urinary stone diseases or bladder cancer in Taiwan [Functional polymorphisms of inflammatory genes can affect cytokine production. A previous study reported that thepression . A Korear cancer . In thisn Taiwan .α in the case and control groups of this study was 2.23% and 0.67%, respectively, and the TNF-αA/A genotype carried a 3.56-fold risk of UC. A meta-analysis study showed that the A/A genotype frequency of TNF-α in cervical cancer cases and controls was 0.97%~7.33% and 0%~6.78%, respectively. The TNF-α A/A genotype significantly elevating risks of cervical cancer was found in a Caucasian population  [α A/A genotype frequency was very low in Japanese (141 bladder cancer patients and 173 control subjects), the allelic frequency of patients (3.5%) was higher than that in controls (0.6%) [α expression in an animal model [α level was higher in people who had ever smoked compared to people who had never smoked [α -308 genotypes was associated with HCC risk in Taiwan males who were exposed to cigarette and alcohol [ TNF-α -308 polymorphism on the OR (10.25) for UC. This finding suggests that the TNF-α -308 G/A polymorphism can modify the OR for UC, especially in people who had ever smoked.Compared to previous studies, differences in genotype frequencies may have been due to ethnic variations. The A/A genotype frequency of TNF-34~3.25) . Althougs (0.6%) . A previ alcohol . In the  IL-8 promoter region can influence its expression and may increase one's susceptibility to bladder cancer [ IL-8 -251 T/A polymorphism and various cancer risks, but the findings were inconsistent [ IL-8 -251A allele has significantly higher promoter activity than the -251T allele [ IL-8 -251T allele had significantly higher transcriptional activity than the -251A allele of the IL-8 gene [IL-8 plays a critical role in inflammation. A common single-nucleotide polymorphism at the -251 position of ther cancer . In the nsistent , 30. SomT allele . HoweverL-8 gene . In addiL-8 gene , 11. IL-L-8 gene . A meta-L-8 gene . It is pα protein stability was impacted by amino acid residue substitutions of P84L (rs4645843) and A94T (rs1800620) [Single-nucleotide polymorphisms of genes can increase disease susceptibilities via affect gene expression. Silico analysis is a novel computational approach to identify potential SNPs causing transcription factor binding affinities to change and influence regulatory functions of genes . Since p1800620) . The eff1800620) . Althoug1800620) . TNF-α and IL-8 genes. More functional polymorphisms of the TNF-α and IL-8 genes should be included in future analyses with larger samples to validate our findings. Other proinflammatory cytokines such as NF-κB also need to be further investigated. In addition, interactions of cigarette smoking and other cytokines on the UC risk should be explored in the future.Our study has some limitations. We only investigated one polymorphism located in the promoter region, which might not entirely account for the functions of the TNF-α-308 A/A genotype and the IL-8 -251 T/T genotype, and who experience cumulative cigarette smoking have a significantly increased OR for UC in a dose-response manner.We found a dose-response relationship between the number of risk factors and the OR for UC. These findings suggest that individuals who carry high-risk genotypes, including the


Pichia pastoris is a widely used heterologous protein production workhorse. However, with its multiple genetic modifications to solve bottlenecks for heterologous protein productivity, P. pastoris lacks selectable markers. Existing selectable marker recycling plasmids have drawbacks . Here, zeocin-resistance marker recycling vectors were constructed using the Cre/loxP recombination system. The vectors were used to (i) knock in heterologous phytase, xylanase and lipase expression cassettes, (ii) increase the phytase, xylanase and lipase gene copy number to 13, 5, and 5, respectively, with vector introduction and (iii) engineer the secretion pathway by co-overexpressing secretion helper factors (Sly1p and Sec1p) without introducing selectable markers, giving a phytase field of 0.833 g/L. The vectors allow selectable marker recycling and would be a useful tool to engineer P. pastoris for high heterologous protein productivity. Pichia pastoris, currently reclassified as Komagataella phaffii, has become a workhorse to produce large quantities of medically and industrially important proteins1. P. pastoris is a valuable production system because of its ability to grow to very high cell densities using minimal media, its ability to produce gram amounts of recombinant protein per liter of culture both intracellularly and in a secretory fashion, and the availability of the strong and tightly regulated promoter AOX1 (PAOX1)2. Fermentation can be readily scaled to meet greater demand, and parameters influencing protein productivity and activity, such as the pH, aeration and carbon source feed rate, can be controlled3. Large quantities of heterologous proteins have been produced in P. pastoris for basic research as well as industrial applications5. Moreover, P. pastoris has been ruled as a generally recognized as safe (GRAS) strain for use in food industries by the Food and Drug Administration (FDA)7. To date, P. pastoris has been applied for the expression of many proteins used in the food and feed industry9.The budding methylotrophic yeast P. pastoris remains one of the early bottlenecks affecting heterologous protein productivity. Increasingly, arranged expression genes of target proteins show a trend of promoting their host cells to produce higher levels of proteins11. However, the achievable copy numbers are limited in the natural state: if the expression vector transformed into P. pastoris is a single copy, the probability of the emergence of multi-copy integration is approximately 10% and the copy number is not controllable12. To gain high copy numbers of heterologous genes, multi-copy integration, using several rounds of gradient antibiotic concentration screening of a large number of colonies, led to random copy numbers14, but required significant time and effort.The copy number of expression cassettes introduced into P. pastoris16 17. During protein secretion, at each trafficking step that requires soluble NSF  receptor (SNARE) complex formation involving the Sec1/Munc18 (SM) proteins, the cargo proteins are delivered by fusion of the membrane of transport vesicles and the target membrane18. Among the SM proteins, Sly1p regulates endoplasmic reticulum (ER)-Golgi trafficking and Sec1p interacts with the vesicle trafficking between the Golgi and cell membrane19. Engineering the secretion pathway may solve this bottleneck of poor secrection20. For pathway engineering, DNA transformation systems based on homologous recombination for integration into genomes are one of the most powerful genetic techniques and have been extensively used in research on P. pastoris. However, the multiple genetic modifications of P. pastoris have faced a shortage of selectable markers26. With limited selectable markers, it is hard to process further engineering to solve the bottlenecks in P. pastoris.Moreover, there are some other bottlenecks for heterologous protein productivity in 28. The first one used the uracil biosynthetic genes URA3 or URA5 as counter-selectable markers22. Unfortunately, uracil auxotroph host strains grow slowly, even in the presence of uracil23. The second, also based on counter-selection, used the Escherichia coli toxin gene mazF28 or the maize mitochondrial gene T-urf1327. However, a nearly 250 bp unwanted repeat sequence was left in the genome after each marker was rescued for knock-in of a gene of interest using mazF28, and the toxicity of the T-urf13 gene might cause conditional lethality for some gene deletions22.To solve this problem, some selectable marker recycling plasmids are currently in useloxP recombination system has been widely employed in various organisms31. When two loxP sites are placed flanking a marker gene, Cre recombinase can excise the marker gene and leave a loxP sequence behind. The use of mutant lox sequences, such as lox71 and lox66 can help avoid potential recombination between the newly introduced loxP site and the former loxP site left in the genome30.Recently, a Cre/P. pastoris based on the Cre/loxP system. We used the vectors (i) to recycle a zeocin-resistance marker, (ii) to increase target gene copy number by re-introducing vectors, and (iii) to engineer the secretion pathway by co-overexpressing the genes of SM proteins Sly1p and Sec1p to increase secretion of the heterologous protein phytase.In this study, we describe new marker recycling vectors for loxP recombination system for zeocin-resistance (ZeoR) marker excision, two novel expression vectors, pZACH and pGACH  containing AOX1 and GAP promoter, respectively, which are two of most commonly used promoters in P. pastoris32. First, the creG357C gene, a silent mutation of cre that avoids the BamHI site, was introduced into pPICZA. Second, the original ZeoR cassette and ori region were replaced with the Cre-ZeoR cassette with lox71 and lox66, resulting in pZAC and pGAC. Third, the gene HIS4, used for homologous fragment insertion, was introduced into pZAC and pGAC, resulting in pZACH and pGACH.Using the Cre/ACH Fig. , for P. creG357C was expressed with methanol induction under the control of the AOX1 promoter, a tightly regulated promoter2. During zeocin resistance selection, the AOX1 promoter was repressed on glucose-containing media. After shifting the cells to methanol-containing medium, creG357C was expressed, leading to the recombination of the lox71 and lox66 sites, resulting in replacement of the Cre-ZeoR cassette with lox72 34, XYN that encodes the xylanase A from Bacillus halodurans C-125 (Xyn)10 and ARL that encodes lipase from Acinetobacter radioresistens CMC-1 (Arl)35, which are three important kinds of enzymes applied in industry37, were used as reporter genes. Considering the advantages of the most commonly used promoter PAOX1 for expression of heterologous proteins in P. pastoris, e.g., strong transcription level and tightly regulated (recently reviewed by Ahmad38), and advantages of the secreted expression system, e.g., simple purification, reduced potential degradation of heterologous proteins and toxicity to hosts by accumulation of secreted heterologous proteins40, the vector pZACH was chosen for secreted heterologous proteins expression. Here, we used PHY as a detailed example for ZeoR marker excision. The plasmid, C-Phy, was verified to be integrated into the genome by PCR with the primer pair P1 and P2  carried the PHY gene as detected by PCR using the primer pair Phy-S and Phy-A. It typically took 3 or 4 days to complete the ZeoR marker excision 27 and lower than that used mazF as a counter-selectable marker (>90%)28.To test the Cre/ P2 Fig. . Three dloxP ZeoR marker recycling vectors could be used as the expression vector in P. pastoris hosts.The strains GS115/C-Phy, GS115/C-Xyn and GS115/C-Arl contained a single copy of the reporter gene, similar to the control strains Table . These r35. To test whether the Cre/loxP ZeoR marker recycling vectors can be used to increase gene copy number directly, the vectors were re-introduced into the hosts. Here, we used PHY as a detailed example. Plasmid P-6c, containing six copies of PHY, was constructed. Based on strain GS115/C-Phy, P-6c was transformed using a ZeoR selectable maker to form strain C-Phy/P-6c after Cre-ZeoR cassette excision. Similarly, another P-6c was transformed into C-Phy/P-6c to form C-Phy/P-6c/P-6c. Similarly, strains GS115/X-4c, C-Xyn/X-4c, GS115/A-4c and C-Arl/A-4c were constructed. These strains were confirmed to contain desired copies of reporter genes  of unwanted sequence remained after each marker was rescued for knock-in of a gene of interest, compared to nearly 250 bp using the mazF counter-selection vector28. Second, the excised ZeoR marker and remaining lox72 did not influence the growth of P. pastoris when the strains grew slowly using URA3 or URA5 as counter-selectable markers23. Third, only 3 or 4 days were required to complete selectable marker excision compared to 8 days or longer using URA markers23.The vectors have several advantages compared to other selectable marker recycling vectors in P. pastoris cells was performed for antibiotic-resistance marker excision, and the recombinase gene creG357C was excised when the antibiotic-resistance marker was excised. Compared with the method for recycling antibiotic-resistance marker using two plasmids45, our method saves time and effort for another transformation of P. pastoris cells and minimizes the risk of leaked expression of the remaining recombinase gene cre, which could lead to genetic instability of engineered P. pastoris.Using the vector in this study, only one transformation of PHY expression cassette (~18 kb) was difficult to construct in vitro33. Using different selectable markers can also increase the target gene copy number46. However, the achieved copy number was limited, and to gain multiple integrated strains of target genes, we had to screen many antibiotic-marker-resistant colonies at gradient antibiotic concentrations33. These methods were not always successful47. Even after significant screening for higher copy gene integration strains at gradient antibiotic concentrations, excessive copy number can be detrimental for recombinant protein productivity in some cases33. Using the selectable marker recycling vector in this study, we could increase target gene copy number in vivo by re-introducing the same expression vectors as many times as desired, and obtained higher copy gene integration strains than by constructing expression cassette tandem-repeats in plasmids10 or by using different selectable marker combinations46. We can evaluate whether there is a trend in which increasing recombinant protein gene copy number promoted protein productivity. When increasingly recombinant protein gene copy number becomes detrimental for recombinant protein productivity, re-introducing vectors can be stopped to save effort compared with screening large numbers of resistant colonies14.In our previous study, a plasmid containing more than six tandem-repeats of 17. In some cases, secretory proteins are retained intracellularly with incomplete secretion15. Because of the limited selectable markers available, it is difficult to further engineer in P. pastoris. Here, enhancement of the secretion of Phy by co-overexpression of Sly1p and Sec1p was achieved using the zeocin-resistance marker recycling vector, suggesting that vesicle trafficking between the ER to Golgi and Golgi to cell membrane are the bottlenecks for Phy expression in P. pastoris. Furthermore, high recombinant protein expression may cause increasing accumulation of misfolded proteins, which causes ER stress and activates the unfolded protein response (UPR)49. With the vector in this study, further engineering to improve the folding of recombinant proteins by overexpressing disulfide isomerase (Pdi1p)50 and/or endoplasmic reticulum oxidoreductin 1 (Ero1p)51 can be performed without introducing selectable markers. Enhancing the expression of one sole chaperone or transcription factor might shift the bottleneck to the next step52. Further pathway engineering can be performed to solve the bottlenecks of heterologous protein production using the vectors introduced in this study. These vectors provide a useful tool for pathway engineering to improve recombinant protein expression in P. pastoris.By changing transgene copy number, protein secretion could become a bottleneck for heterologous protein expressionloxP recombination system for zeocin-resistance selectable marker recycling, can be used to integrate a gene of interest and increase gene copy numbers. These will be useful tools for engineering P. pastoris for enhancing heterologous protein productivity.Thus, the vectors in this study, using a Cre/E. coli TOP10  cells were grown at 37 °C in LB or low-salt LB  medium. Standard cloning procedures were performed in E. coli TOP10, as described by Sambrook & Fritsch53. Plasmid selection and maintenance was performed using 25 mg/L zeocin (Invitrogen) or 100 mg/L kanamycin (Invitrogen).P. pastoris strain GS115 (Invitrogen) was used to construct yeast strains. P. pastoris GS115 was cultured at 30 °C and 250 revolutions per minute (rpm) in YPD or YPM medium . BMGY or BMMY medium  and 1% glycerol or 1% methanol) was used for P. pastoris fermentation. Transformants were selected on YPDSZ plates .The Strains, vectors and primers used in this study are summarized in Supplementary Table BamHI site in cre, the cre silent mutation creG357C was amplified by PCR from the plasmid pSH4754 and cloned into pPICZA (Invitrogen) as follows: 5′ arms of creG357C were amplified by PCR using the primer pair Cre-F and Cre-G357C-R and the 3′ arms using Cre-G357C-F and Cre-R. The creG357C was generated using Cre-F and Cre-R, resulting in the vector pPICZA-creG357C. To avoid the BamHI site in the Cre-ZeoR cassette of pPICZA-creG357C, the 5′ arms of the Cre-ZeoR cassette were amplified from pPICZA-creG357C using AOX1-lox71-F and AOXTT-A and the 3′ arms using AOXTT-F and Zeo_lox66_A. These two arms were assembled with fragments amplified from pPICZA or pGAPZA using AOX1-G-F and AOXTT-G-A or GAP-G-F and AOXTT-G-A using a Gibson Assembly Cloning kit , resulting in the vectors pZAC and pGAC. To create the vectors pZACH and pGACH, HIS4 was amplified from pPIC9k (Invitrogen) using HIS4-F and HIS4-R and was assembled with fragments amplified from pZAC or pGAC using 3AOX-F and 3AOX-R using the Gibson Assembly Cloning kit (NEB).To avoid the d1+201-αE10-phy-HKA (αE10)33 was ligated into the pZACH plasmid using BglII and BamHI sites to create the vector pZACH-phy (C-Phy). Similarly, pZACH-xyn (C-Xyn) and pZACH-arl (C-Arl) were constructed based on pPICHKA-xynA10 and pPICHKA-epARL35. A similar method was used to obtain the six-copy plasmid pZACH-(phy)6 (P-6c), four-copy plasmid pZACH-(xyn)4 (X-4c) and pZACH-(arl)4 (A-4c) using the plasmid pPICZA-αE10-HKA/(Phy)6 (6c)33, pPICZA-(xynA)410 and pPICHKA-(epARL)435.A phytase expression cassette from the plasmid pAOX1SLY1 [GenBank ID: CAY71482.1] and SEC1 [GenBank ID: CAY71361.1] were obtained from P. pastoris strain GS115 genomic DNA using the appropriate primer pairs. All PCR products were ligated into the pGACH plasmid using PmlI-SacII sites to create the vectors pGACH-SLY1 and pGACH-SEC1.The genes Restriction enzyme digestion and DNA sequencing assured that all plasmids matched their design.Eam1105I  and transformed into P. pastoris GS115-competent cells using the electroporation method described by Cregg47. The transformed cells were selected on YPDSZ plates. The integration of these plasmids into the GS115 genome was verified by PCR using the appropriate primer pairs.Plasmids pZACH, C-Phy and P-6c were linearized with R transformants were shifted from YPDSZ plates to 5 mL YPM medium and grown for 20 h. Then, 20 μL of the YPM cultures was streaked onto YPD plates and incubated at 30 °C until colonies were visible (nearly 36 h). These colonies were picked, added to 10 μL sterilized water and spotted onto YPD and YPDZ (YPD plus 100 mg/L zeocin) plates. Colonies that grew on YPD plates but not on YPDZ plates indicated that the Cre-ZeoR cassette might have been excised and that the selectable marker could be reused. The result was verified by PCR using appropriate primer pairs. After Cre-ZeoR cassette excision, these strains formed GS115/pZACH, GS115/C-Phy and GS115/P-6c. Similarly, GS115/C-Xyn, GS115/C-Arl, GS115/X-4c, GS115/A-4c, C-Xyn/X-4c, C-Arl/A-4c, C-Phy/P-6c and C-Phy/P-6c/P-6c were constructed. A similar method was used to construct P-6c/GH, P-6c/SLY1, P-6c/SEC1, P-6c/SLY1/SEC1 and P-6c/SLY1/GH.As shown in Supplementary Fig. 33. The standard plasmids pPICZαA-phy-G33, pPICZα-G-xynA10 and pPICHKA-AG35 consisted of a portion of the GAPDH gene sequence, which was used as the reference sequence because there is only a single copy in the P. pastoris genome55. Total RNA were extracted by the hot acidic phenol method56. Next, the cDNA synthesis was carried out using the PrimeScript™ Kit .The quantitative PCR (qPCR) assay protocol was developed as described previouslyGAPDH gene  as an endogenous control. The PHY, XYN or ARL copy number in each transformant was calculated using the Ct value of the genomic DNA and a standard curve.The qPCR and real-time PCR (RT-PCR) assays were repeated three times per sample. RT-PCR data were normalized using the P. pastoris transformant was transferred into 5 mL of BMGY at 30 °C and 250 rpm for 20 h. The main cultures inoculated from precultures in BMGY to an initial optical density of 0.5 were collected by centrifugation and transferred into 20 mL of BMMY at 30 °C and 250 rpm. OD600 and phytase, xylanase or lipase activity were monitored and 1% (v/v) fresh methanol was added to BMMY every 24 h.A single 33. The enzyme reaction mixture was preheated at 37 °C for 5 min. Next, 5.0 mM sodium phytate (pH 5.5) was added and the mixture was incubated at 37 °C for 30 min. The reaction was stopped by adding coloration solution. The absorbance of mixture was quantified at 415 nm.Phytase activity was determined as described previously10. An appropriately diluted enzyme source was added to 1% (w/v) beechwood xylan and incubated at 70 °C for 30 min. Then, the reaction was stopped by adding dinitrosalicylic acid reagent. The absorbance of the mixture was quantified at 540 nm.Xylanase activity was determined as described previously35. The enzyme reaction mixture was preheated at 55 °C for 5 min. Next, 0.05 M p-nitrophenyl caprylate was added and the mixture was incubated at 55 °C for 5 min. The produced p-nitrophenol in the reaction mixture was quantified at 405 nm.Lipase activity was determined as described previouslyAll results were repeated thrice and used GS115/pZACH as background samples.Samples (culture supernatants) were heated at 100 °C for 5 min in loading buffer. The mixture was subjected to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a Mini-gel system . The proteins in the gel were stained with Coomassie Brilliant Blue R-250 (Invitrogen).The phytase protein concentration in the supernatants was analyzed by SDS-PAGE with bovine serum albumin  as a standard. The phytase, xylanase or lipase and BSA bands in the gel were quantified by Quantity One (Bio-Rad). All results were reproduced three times.Proteins in the SDS-PAGE gels were transferred to nitrocellulose (NC) membranes. The membranes were incubated with a mouse anti-FLAG (1:1000) or anti-HA monoclonal antibody (1:2000) and then exposed to the HRP-conjugated goat anti-mouse IgG monoclonal antibody. The protein bands were visualized by exposure using FluorChem M .P < 0.05.All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Differences between groups were tested for statistical significance by using a two tailed by unpaired T-test in Microsoft Excel 2010 . Differences were considered significant at Supplementary information


Thermus thermophilus shows DNA-guided DNA interfering activity at high temperatures, complicating its application in mammalian cells. A recent work reported that the argonaute protein from Natronobacterium gregoryi (NgAgo) had DNA-guided genome editing activity in mammalian cells. We compared the genome editing activities of NgAgo and Staphylococcus aureus Cas9 (SaCas9) in human HEK293T cells side by side. EGFP reporter assays and DNA sequencing consistently revealed high genome editing activity from SaCas9. However, these assays did not demonstrate genome editing activity by NgAgo. We confirmed that the conditions allowed simultaneous transfection of the NgAgo expressing plasmid DNA and DNA guides, as well as heterologous expression of NgAgo in the HEK293T cells. Our data show that NgAgo is not a robust genome editing tool, although it may have such activity under other conditions.The argonaute protein from the thermophilic bacterium  CCR5 has been tested in a clinical trial [The availability of nucleases that recognize long specific target sequences greatly enhances our ability to edit the mammalian genomes. Zinc finger nucleases ,2 were tal trial . Howeveral trial  and the al trial , a TALE al trial . Althougclustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system was shown to have genome editing activity in bacteria [Streptococcus pyogenes (SpCas9) was most widely used, while Cas9 from Staphylococcus aureus (SaCas9) is increasingly used due to its smaller protein size enabling Adeno-Associated Viral vector delivery [Recently, a bacteria . Subsequbacteria –12. Sincdelivery –15. Cas9Thermus thermophilus, Pyrococcus furiosus, and Methanocaldococcus jannaschii [Natronobacterium gregoryi  had higher genome editing activity than Cas9 in mammalian cells [Recently short single stranded DNA-guided sequence-specific DNA endonuclease activity was reported in argonaute proteins from nnaschii –18, as annaschii . These aan cells . Since iHere we compared the genome editing activities of NgAgo and SaCas9. We consistently observed genome editing activity from SaCas9, but not NgAgo.MDYKDDDDKAPKKKRKVGTGG . The N-terminal additional sequence for HA-tagged NgAgo is MAYPYDVPDYAPKKKRKVGGGSKRPAATKKAGQAKKKKGTGG . The NgAgo peptide is C-terminal to the added tags and nuclear localization signals. The cDNA and protein sequences for the two NgAgos are listed in Figures A to D in Three NgAgo expressing constructs were used in this study. nls-NgAgo-GK was a gift from Chunyu Han (Addgene plasmid # 78253). The othBsaI sites of pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA  [HBB) gene causing sickle cell disease: AGTAACGGCAGACTTCTCCACAGGAGT . Another target sequence was from the human interleukin 2 receptor gamma (IL2RG) gene whose mutation causes X-linked severe combined immunodeficiency (SCID-X1): TAGTCTGTCTGTGTCAGGAACCTGGGT. The Cas9/sgRNA expression constructs were named pSaCas9-HBB-sgRNA1 and pSaCas9-IL2RG-sgRNA1 respectively.Two SaCAS9 based constructs were made by inserting the guide sequences between the two # 61591) . Two comIL2RG-HBB-GFP, for making a GFP reporter cell line) was made by inserting the synthesized DNA between the EcoRI and BamHI sites of pSin-EF2-Sox2-Pur . The synthesized DNA sequence is: TGA-GGCCACC-ATG-GAACCCAGGTTCCTGACACAGACAGACTACACCCAGGGAATGAAGAGCAAGCGCCAT-ACTCCTGTGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATTGGCTAGC-(EGFP Codon 2–239)-GGATCCGAATTC- . A stop codon (in bold) is included at the 5’ end to prevent possible unexpected translation through into EGFP cDNA. The target sequences for IL2RG  and HBB  were inserted between the start codon (underlined) and the second codon of EGFP cDNA. Insertion of the 119 bp target sequences disrupted the reading frame of EGFP. Deletions of 3N+2 or insertions of 3N+1 base pairs by genome editing will restore EGFP expression.A lentiviral vector (pSin-EF2-659AA fusion protein (exclusively cytoplasmic) expression plasmid was used, as described previously [An mCherry-MEX3Ceviously . All DNAeviously .6 HEK293T cells were seeded in 10-cm dishes in DMEM medium 24 hours before transfection. Then pSin-EF2-IL2RG-HBB-GFP Plasmid DNA was co-transfected with psPAX2 and pMD2G DNA  into the cells mediated by 54 μl Fugene 6 according to the instructions of the manufacturer . Lentiviral vector production and transduction were performed according to published protocol [An EGFP reporter cell line was made to facilitate detection of genome editing events by observing restored EGFP expression. To make the lentiviral vector for reporter cell line construction, 5x10protocol . The virIL2RG-sgRNA1 and pSaCas9-HBB-sgRNA1 at the University of North Carolina at Chapel Hill Vector Core). NgAgo expression was achieved by plasmid DNA transfection. For plasmid DNA transfection, HEK293, HEK293T, and HEK293T-derived reporter cells were cultured in DMEM with 10% FBS, and 100 U/ml penicillin and 100 μg/ml streptomycin (ThermoFisher Scientific). The cells were seeded into 24-well plates at a density sufficient to reach 70–80% confluence 18 hours after being seeded: 1.25x105 cells/well for HEK293T cells and 6x104 cells/well for HEK293 cells. When only plasmid DNA was transfected, Fugene 6 (Roche) was used. When guide DNA was transfected, Lipofectamine 2000 (ThermoFisher Scientific) was used. For cells in one well of a 24-well plate, 0.25–0.5 μg plasmid DNA, 100 ng guide DNA and 1.25–2 μl Lipofectamine 2000 were used respectively. DNA and Lipofectamine 2000 were each re-suspended in 50 μl of OPTI-MEM (ThermoFisher Scientific) before mixing. If two guide DNA oligos were used, 100 ng of each guide DNA was used. When co-transfecting the EGFP reporter plasmid (pSin-EF2-IL2RG-HBB-GFP) and the NgAgo expressing plasmid, the amounts of reporter DNA and NgAgo DNA were 100 ng and 400 ng respectively. For NgAgo and guide DNA transfection, protocols in Gao’s paper [Cas9/sgRNA expression was achieved by direct plasmid DNA transfection or AAV transduction . In some experiments, the guide DNA oligos were further treated by T4 polynucleotide kinase (New England Biolabs) according to the manufacturer’s instructions to ensure 5’ phosphorylation. The sequences of the guide DNA oligos are listed in Table A in 4 cells/well in 24-well plate) were transfected with NgAgo expressing plasmid DNA with Fugene 6 or Lipofectamine 2000 (similar transfection efficiency in our hands). Twenty-four hours after transfection, the cells were fixed in 4% paraformaldehyde/PBS, pH7.4 at room temperature for 30 min, and permeablized with 0.1% Triton X-100/PBS at room temperature for 30 min. The cells were then blocked with 5% fat-free milk and incubated with anti-Flag antibody  or anti-HA antibody  at room temperature for 1 hour. After washing three times with PBS, the cells were stained with Alexa594 conjugated anti-mouse secondary antibody (for anti-Flag primary antibody) and anti-Rabbit secondary antibody (for anti-HA primary antibodies). Published immunostaining protocol was followed for immunostaining [HEK293 cells grown on coverslips  and phosphatase inhibitors . The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) before transferring to nitrocellulose membranes. SDS-PAGE was performed according to published protocol . The folCultures were tested for presence of mycoplasma using the PCR Mycoplasma Detection Kit from Applied Biological Materials Inc. . Culture medium was collected 48 hours after incubation and used as template for PCR. The presence of PCR products between 370–550 bp were examined by electrophoresis and ethidium bromide staining. PCR mycoplasma detection was performed according to the instructions of the manufacturer.http://www.ebi.ac.uk/Tools/msa/clustalo/) to visualize insertions, deletions and mutations. To detect insertions and deletions (INDELs), a program was written in SAS software to calculate the distance between two designated sequences located at the 5’ and the 3’ end of each read. The percentages of the 20 most appearing lengths were then calculated.Genomic DNA was isolated from cells treated with Cas9 or NgAgo with the QIAamp DNA Mini Kit  according to the manufacturer’s instructions. Then the target region was amplified from the genomic DNA by PCR amplification  expression was disrupted by insertion of a 119-base pairs (bp) sequence (missing 1 bp to make 40 inframe codons) between the start codon ATG and the second codon of EGFP cDNA, disrupting the EGFP reading frame  and pSaCas9-HBB-sgRNA1 (expressing SaCas9 and sgRNA for HBB) DNA was transfected into the reporter cells. 24 hours after transfection, increasing numbers of cells became EGFP-positive , deletions of 3N+2 or insertions of 3N+1 base pairs will restore the HBB-sgRNA1 transfected reporter cells 24 hours post-transfection  expressIL2RG and HBB genes as the SaCas9 system  [659AA-mCherry indicates successful plasmid DNA entrance into the endosome, escape from the endosome, and subsequent expression of the MEX3C659AA-mCherry protein. The bright green fluorescence signals also indicated the presence of Alexa488-labeled oligos in the endosomes. It was difficult to observe oligos already escaped from the endosomes due to their diffuse distribution. However, the escape of the co-transfected plasmid DNA from the endosome suggests the escape of some oligos as well. Similar co-transfection experiments were performed in a recent publication to demonstrate successful transfection of the guide DNA into the cells [To test whether the transfection conditions we used (the same as reported in the Gao paper ) could dprotein)  into theprotein) , 25. Thuhe cells . Thus, iBecause it was reported that guide DNA could be loaded into NgAgo at 55°C but not 37°C , we thusThermus thermophilus) were reported to cleave plasmid DNA in in vitro cleavage assays [IL2RG-HBB-GFP reporter plasmid DNA were co-transfected into HEK293T cells. Again, pSaCas9-IL2RG-sgRNA1 and pSaCas9-HBB-sgRNA1 DNA were used as positive controls. Whereas co-transfecting pSaCas9-IL2RG-sgRNA1 and pSaCas9-HBB-sgRNA1 with the reporter DNA  produced many bright EGFP-positive cells 24 hours after transfection, co-transfecting NgAgo DNA expressing the HA- or Flag-tagged NgAgo, guide oligos and the reporter DNA produced no EGFP-positive cells, even after 72 hours  that NgAgo was sensitive to mycoplasma contamination. We tested our HEK293T cells and reporter cells, and both were negative for mycoplasma (data not shown).The authors of the original NgAgo paper indicated in a protocol posted on the Addgene website Click here for additional data file.