Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite.Examining essential biochemical pathways in Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2.Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2. Despite the efficacy of artemisinin-based combination therapy (ACT) in the treatment of malaria, an estimated 214 million cases and 438,000 malaria deaths occurred globally in 2015  was added to each well on the plate. The plate was shaken for 10 min to ensure mixing and the absorbance at 595 nm was obtained using a CLARIOstar plate reader. .PF3D7_0525900) was purchased (with codon optimization for Escherichia coli expression) from GeneArt™ Life Technologies. The codon optimized DNA sequence was as follows (the codon encoding V24 is highlighted in bold); ATGAGCAAACCGAAAATGATCGGTCCGTATGAAGTTGTGAAAAGCATTGGTCGTGGTAGCTTTGGTATTGTTACCGCAGTTAAAGATGAAAATGAAAAAATCTTTGTGATTAAAGAACTGGATATTAGCTGCATGAATAACAAAGAAAAAATGAATGTGGTGAATGAAATTCGTGCCCTGATTAAAATGAGCGTGCATCCGTTTATTGTGCGCTATAAAGAAGCCTTTGTTGAAGATTGCGTTCTGTATGTTGCGATGGATTATTGCATTAATGGTGATCTGGGCAAAGTGATCAAAAAACACAAAGAACTGGAAACCCCGATTCCTGAGAAAAAAATCAAACGTTGGCTGCTGCAGATTATTATGGCCATCAAATTCATTCATGATAAAAAACTGATCCATCGTGATCTGAAATGCAATAACATCTTCCTGGATGAAAAAGAACGTGCCAAAATTGGTGATTTTGGTCTGGCCAAATTTATCGAACAGACAGAACAGACCAATACCCTGTGTGGCACCATTGGTTATATGGCACCGGAAATTTGCAAAAATATCAATTACAGCTTCCCTGCCGATATTTGGAGCCTGGGTATTATTCTGTATGAACTGATTAGCCTGAAACCGCCTTTTAAAAGCAATAACAGCAATATGCTGAGCGTGGCCCAGAAAATTTGTGAAGATGAACCGGATCCGCTGCCGGATAGCTTTAGCAAAGATCTGATTAATCTGTGCTATTGGATGCTGAAAAAAGATTGGAAAGATCGTCCGACCATCTACGATATTATCAGCACCGATTATATCCAGGATGAACTGCAGCTGTTTAAACGTGAAATGCTGCAAGAACGTAACAGCCAGATT.The synthetic full-length coding sequence for Pfnek-2  and reverse (5′-tctttaactgcggtacaaataccaaagctaccacgaccaatgc-3′).E. coli BL21-Codon Plus(DE3)-RIPL strain (Agilent Technologies). Cells were treated with 1 mM IPTG when the optical density of the cell culture at 600 nm reached 0.6, and protein expression was induced overnight at 18 °C. For purification, cells were lysed in buffer  [1 tablet per 10 ml (cOmplete™ ULTRA Tablets by Roche)] by sonication for 10 cycles of 10 s bursts at 10 mA with a 30 s rest between each cycle. During sonication the cells were kept on ice. Lysates were then centrifuged at 20,000g for 30 min. The resulting supernatants were loaded onto a 0.5 ml pre-equilibrated Ni–NTA Superflow resin (Qiagen) column, washed with buffer  and proteins were eluted with the same buffer containing 400 mM Imidazole. Eluted proteins were dialysed against 50 mM bicine, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM DTT at 4 °C overnight. Dialysed Pfnek-2 protein was aliquoted and stored at −80 °C.Full-length Pfnek-2 as a 6-HIS-tagged protein was expressed in 2, 4 mM imidazole, 1 mM DTT) in the presence of 5 µg MBP. The reaction was started by the addition of 5 µl 200 µM ATP/32P-ATP (0.0185 MBq per reaction)—total volume 20 µl. Following incubation of 30 min at 37 °C, the reactions were stopped by adding 10 µl 2× Laemmli buffer and proteins were separated by SDS–PAGE on 12% gels and stained with Coomassie blue. Dried gels were exposed to X-ray film.Recombinant His-tagged WT or V24C mutant Pfnek-2 were assayed for protein kinase activity using myelin basic protein (MBP) as a substrate. 1.8 µg of either Pfnek-2 WT or Pfnek-2 V24C were pre-incubated at room temperature for 40 min with/without NCL-00016066  in Pfnek-2 buffer  was dissolved in DMSO to 10 mM and frozen in aliquots of 10 µl at −80 °C. These aliquots were kept in the dark and a fresh aliquot diluted prior to each kinase assay. All samples in each kinase assay were pre-incubated with NCL-00016066 for 40 min at room temperature and the reaction initiated by the addition of ATP/2, 1% glycerol, 4 mM imidazole, 1 mM DTT at room temperature for 30 min. 50 µl Ni–NTA Superflow resin (Qiagen) was added and the samples made up to 500 µl in buffer  and rotated for 1 h at room temperature. The beads were washed 3× in 500 µl buffer for 5 min rotation and supernatants discarded. The in vitro kinase assay protocol described above was followed and the enzyme immobilized on beads (washed and supernatant removed) is assumed to be 5 µl for the purpose of the kinase assay. During incubation at 37 °C the beads were gently re-suspended every 10 min.Recombinant Pfnek-2 wild type and Pfnek-2 V24C were purified as previously described and incubated (1:1) without/with 20 µM NCL-00016066 diluted in 10 mM bicine, pH 8.0, 40 mM NaCl, 10 mM MgCl2, 4 mM imidazole, 1 mM DTT) at room temperature prior to mass spectrometry analysis.Recombinant Pfnek-2 wild type and Pfnek-2 V24C were purified as previously described except glycerol was absent from all buffers. The absence of glycerol had no effect on the activity of the enzyme and was only added to previous preparations to stabilize the enzyme. Glycerol however interferes with the mass spectrometry and so was omitted here. Both wild type and mutant (V24C) Pfnek-2 were incubated (1:1) without/with 100 µM NCL-00016066 diluted in buffer  and molecular weight calculations made using ESIprot  for 1 h. The gel was stained with four parts colloidal blue to one part methanol and destained with 10% acetic acid in 25% (v/v). Protein bands were excised from the gel and cut into small pieces and washed three times with 200 μl of buffer B [20 ml 400 mM ammonium bicarbonate (3.16 g/100 ml) with 100% acetonitrile in ratio 1:1]. The gel pieces were then washed twice with 200 μl acetonitrile (100%) and gel pieces air dried.Recombinant Pfnek-2 (4 µl of 13 mg/ml stock solution of enzyme) was incubated with/without 1 mM ATP in Pfnek-2 buffer  followed by a further addition of acetonitrile for 10 min at room temperature followed by aspiration of the fluid and air drying. Trypsin solution (1 μg in 50 μl of 50 mM TEAB buffer per sample) was added and incubated at 37 °C overnight. The gel pieces were washed in 50 mM TEAB buffer for 5–10 min on a rotating platform. The digests and washes were combined and dried completely in a SpeedVac. The samples were then enriched for phosphopeptides as described below.g, and the flow-through (unbound peptides) collected and frozen. The IMAC beads were washed twice with 200 µl of IMAC load/wash buffer, once with 200 µl of HPLC grade water and eluted twice with 100 µl of solution containing 22 µl ammonia solution , 300 µl acetonitrile to a total volume of 1 ml with HPLC grade water. Eluates were concentrated to a small volume (15–20 µl) in a Speedvac centrifuge and submitted for mass spectrometry analysis.PHOS-Select iron affinity gel (Sigma) was equilibrated with 5 × 1 ml IMAC load/wash buffer . 500 µl IMAC load/wash buffer was added to completely dry samples and 50 µl of 50% PHOS-Select slurry was added followed by rotating at room temperature for 1 h. Following incubation with IMAC beads, the samples were transferred onto Mobicol ‘Classic’ spin columns (2B Scientific Ltd), centrifuged for 30 s at 1000×This was carried out as previously described  with one50 values of 23.95 ± 2.50 and 35.63 ± 3.03 µM as measured by SYBR Green I and LDH parasite death assays, respectively  were resolved by SDS PAGE and appeared as a single band at approximately 38 kDa  value, 6.30 ± 0.25 (n = 3)]  by NCL-00016066, the mutant kinase was pre-incubated with NCL-00016066 (20 µM) prior to immobilization on Ni–NTA beads, and unbound inhibitor were removed by extensive washing. As a control, Pfnek-2 was similarly incubated with high concentrations of NCL-00016066 (20 µM), which gave partial inhibition of kinase activity, which was reversed following washout Fig. a. In conIn an effort to further characterize the nature of the inhibition of the mutant Pfnek-2 with NCL-00016066, wild type Pfnek-2 or Pfnek-2(V24C) were pre-incubated with or without 100 µM NCL-00016066 and the intact protein subjected to analysis by mass spectrometry.m/z ratio of Pfnek-2 (red trace Fig. m/z ratio upon addition of NCL-00016066 (Fig. Pfnek-2 without inhibitor (black trace Fig. Table Mass spectrometry analysis of the intact protein as described above indicated that Pfnek-2 undergoes multiple autophosphorylation events. Analysis of tryptic phospho-peptides unequivocally identified three autophosphorylation sites, Ser20, Ser215 and Ser219 Fig. . InteresThe aim of the current study was to engineer Pfnek2 in a manner that would confer sensitivity of the kinase to a kinase inhibitor that otherwise would show low levels of activity to wild type Pfnek2. This was achieved by mutating Val24 in the wild type Pfnek-2 to cysteine. This mutation confers susceptibility to the kinase inhibitor NCL-00016066, and that the nature of this inhibition is likely through an irreversible covalent link of NCL-00016066 with the modified cysteine.Plasmodium berghei showed that parasites lacking the enzyme Pfnek-2 are able to undergo gametocytogenesis, gametogenesis and fertilization, but do not develop into ookinetes; this is likely due to a defect in meiosis, as pbnek-2− parasites appear to be unable to implement the pre-meiotic DNA replication that brings the DNA content to 4C in wild-type parasites, but remains 2C in the mutant parasites [Pfnek-2 is expressed predominantly in female gametocytes . Transmiarasites . The demarasites  suggestsStructural analysis of the Hsnek2 in complex with NCL-00016066 demonstrated that this inhibitor acts in an irreversible manner by covalent linkage to cysteine 22 in the glycine rich loop. Interestingly, Pfnek-2 does not contain this cysteine, or any other cysteine, in this loop. Demonstrated here is the weak inhibitor activity of NCL-00016066 against wild-type Pfnek-2 likely due to a lack of a cysteine in this position. Consistent with this notion is that NCL-00016066 showed potent inhibitory activity if a cysteine residue is introduced into the glycine-rich loop of Pfnek-2. Furthermore, this inhibition appeared to be irreversible, and mass spectrometry studies indicated that this is likely due to covalent modification of the variant kinase at the substituted cysteine.Recent studies have deployed a chemical genetic approach to the study of PfPKG where parasites expressing a mutant form of the kinase that is resistant to inhibition by a selective PfPKG inhibitor was used to identify cellular targets and physiological roles of the kinase . The engIn this way, the data presented here opens the door to investigating the essential role that Pfnek-2 plays in the transmission of malaria from the host to the mosquito vector and in doing so might inform on the mode of action of potential transmission-blocking drugs targeting this kinase.


RIP (ribonucleoprotein immunoprecipitation) and RNA pull-down assays showed that Meg3 was co-immunoprecipitated with ATG3. In addition, Meg3 protected ATG3 mRNA from degradation following treatment with actinomycin D. Overall, our results suggest that the lncRNA Meg3 acts as a tumor suppressor in EOC by regulating ATG3 activity and inducing autophagy.Maternally expressed gene 3 (Meg3), a long non-coding RNA, has been reported to be associated with the pathogenesis of multiple malignancies. However, little is known regarding the role of Meg3 in epithelial ovarian cancer (EOC). In this study, we found that the expression of Meg3 was lower in epithelial ovarian carcinoma, and has potential to be considered as a biomarker for ovarian cancer. After transfecting the ovarian cancer cell lines OVCAR3 and A2780 with Meg3, phenotypic changes and autophagy-related molecules were examined. Upregulation of Meg3 inhibited cell proliferation, plate colony formation, induced cell cycle arrest in G2 phases, and promoted apoptosis. Observation of autophagosomes was performed by transmission electron microscopy. The expression levels of LC3-II, ATG3, LAMP1 were elevated, while SQSTM1/p62 expression declined. Upregulated expression of Meg3 also suppressed tumorigenesis  Epithelial ovarian carcinoma (EOC) remains one of the most common gynecologic malignancies, with 21,290 new cases diagnosed and 14,000 deaths reported in the United States in 2015 [Long non-coding RNAs (lncRNAs) are a family of non-protein-coding RNAs greater than 200 nucleotides in length . LncRNAsMeg3 mRNA expression was lower in EOC tissue as compared to normal ovarian tissues and benign ovarian carcinomas. In metastatic omentum tumors, expression was found to be significantly lower as compared to all above  assays were performed to confirm the functional interaction between MEG3 and autophagic protein ATG3. RNA from RIP assays with an antibody against ATG3 was used for qPCR analysis, which demonstrated an enrichment of the lncRNA Meg3 Figure . Next, wCells transfected with Meg3 were treated with actinomycin D to study the stability of ATG3 mRNA. Our results showed that ATG3 mRNA in cells transfected with Meg3 showed a slower rate of decay compared with control cells Figure .qRT-PCR and Western blot analysis were used to measure LC3, SQSTM1/P62, LAMP1 mRNA and protein expression after transfecting si-RNA to silence expression of ATG3 in OVCAR3 cells. Both mRNA and protein expression of LC3 Figure  and LAMPRecently, a series of studies have focused on the molecular mechanism of pathogenesis and progression of tumors, and the action of lncRNAs has attracted intense research interest. Studies have revealed that lncRNAs are involved in various processes including development, cell proliferation, metastasis, fate decision, invasion and migration –15, whicTo investigate the action of Meg3 in EOC, we analyzed the mRNA expression of Meg3 in normal epithelial ovarian tissues, benign tumor tissues, primary ovarian carcinomas, and metastatic omentum tumors. We found that the expression of Meg3 was reduced significantly in primary tumors as compared to normal ovarian tissue and benign tumors. Furthermore, the expression of Meg3 in metastatic omentum tumors was even lower than in primary ovarian carcinoma. These results demonstrated that there is a close relationship between Meg3 and tumorigenesis, as well as the development of ovarian cancer.in vivo. In conclusion, Meg3 plays a suppressive role in EOC, which is consistent with the role of Meg3 in gastric cancer, cervical cancer, lung cancer and hepatocellular carcinoma.Transfection of ovarian cancer cell lines with Meg3 resulted in inhibition of cell proliferation, colony formation, migration and metastasis, and promotion of G2 phase arrest, as well as cell apoptosis, while after given Meg3-transfected cells with autophagy inhibitor 3MA, the cell viability and colony formation rate was improved as compared with Meg3-transfected cells. Nude mice xenograft assays showed that overexpression of Meg3 could reduce tumorigenesis of ovarian cancer cells in vivo and in vitro. Therefore, we suggest that Meg3 acts as a tumor suppressor in EOC, which may be achieved by initiating autophagy and causing type II cell death.Furthermore, autophagosomes observed under transmission electron microscopy indicated that Meg3 induced autophagy in EOC. Autophagy, in general, is a survival process. Autophagic (type II) cell death usually represents a failed attempt to overcome lethal stress, and disruption of this process promotes rather than inhibits cell death in many cases , 25. SevRecent studies have provided a significant perspective on the important role of lncRNAs in regulation of gene expression. Reports have revealed that lncRNAs may act as either scaffolds that contain distinct protein-interacting domains to bring specific protein into proximity of each other, and form unique functional complexes, or as guides to recruit proteins –32. UpreIn conclusion, the present study has shown for the first time that upregulated expression of Meg3 triggers autophagic flux through an interaction with ATG3 to suppress tumorigenesis and progression of EOC. Meg3 may serve as a potential biomarker of EOC. Our findings may provide a novel insight into the early diagnosis and treatment of ovarian cancer. And further research should delineate the role and regulation of the crosstalk between Meg3 and autophagy in pathogenesis and progression of EOC.EOC tissues (Ca), metastatic omentum tissues (Om), borderline tumor (Bo) tissues, benign tumor tissues (Be) and normal ovarian specimens (No) were obtained from patients who underwent surgical resection at the Department of Gynecology, the First Affiliated Hospital of China Medical University  among 2005.01-2015.12. Tumor specimens were reviewed by microscopy by two independent pathologists and staged according to the FIGO staging system. Samples were frozen in liquid nitrogen immediately and stored at − 80°C until use. No patients underwent chemotherapy or radiotherapy treatment prior to surgery. Informed consent was obtained from all subjects. Ethics board approval was obtained from the China Medical University Ethics Committee, and all specimens were handled and anonymized in an ethical and legal manner.2. The medium was changed every one to two days based on the culture state. MEG3-specific pcDNA overexpression vector (pcDNA-MEG3), ATG3-specific siRNA (si-ATG3) and corresponding control including empty pcDNA, and si-scramble were completed by Shanghai Genechem Co., Ltd., China. These recombinants were transfected into OVCAR3 or A2780 cells using Lipofectamine 2000 reagent  according to the manufacturer's instructions. The sequence of Meg3 plasmid is CTCGAGCTAGCCCCTAGCGCAGACGGCGGAGAGCAGAGAGGGAGCGCGCCTTGGCTCGCTGGCCTTGGCGGCGGCTCCTCAGGAGAGCTGGGGCGCCCACGAGAGGATCCCTCACCCGGGTCTCTCCTCAGGGATGACATCATCCGTCCACCTCCTTGTCTTCAAGGACCACCTCCTCTCCATGCTGAGCTGCTGCCAAGGGGCCTGCTGCCCATCTACACCTCACGAGGGCACTAGGAGCACGGTTTCCTGGATCCCACCAACATACAAAGCAGCCACTCACTGACCCCCAGGACCAGGATGGCAAAGGATGAAGAGGACCGGAACTGACCAGCCAGCTGTCCCTCTTACCTAAAGACTTAAACCAATGCCCTAGTGAGGGGGCATTGGGCATTAAGCCCTGACCTTTGCTATGCTCATACTTTGACTCTATGAGTACTTTCCTATAAGTCTTTGCTTGTGTTCACCTGCTAGCAAACTGGAGTGTTTCCCTCCCCAAGGGGGTGTCAGTCTTTGTCGACTGACTCTGTCATCACCCTTATGATGTCCTGAATGGAAGGATCCCTTTGGGAAATTCTCAGGAGGGGGACCTGGGCCAAGGGCTTGGCCAGCATCCTGCTGGCAACTCCAAGGCCCTGGGTGGGCTTCTGGAATGAGCATGCTACTGAATCACCAAAGGCACGCCCGACCTCTCTGAAGATCTTCCTATCCTTTTCTGGGGGAATGGGGTCGATGAGAGCAACCTCCTAGGGTTGTTGTGAGAATTAAATGAGATAAAAGAGGCCTCAGGCAGGATCTGGCATAGAGGAGGTGATCAGCAAATGTTTGTTGAAAAGGTTTGACAGGTCAGTCCCTTCCCACCCCTCTTGCTTGTCTTACTTGTCTTATTTATTCTCCAACAGCACTCCAGGCAGCCCTTGTCCACGGGCTCTCCTTGCATCAGCCAAGCTTCTTGAAAGGCCTGTCTACACTTGCTGTCTTCCTTCCTCACCTCCAATTTCCTCTTCAACCCACTGCTTCCTGACTCGCTCTACTCCGTGGAAGCACGCTCACAAAGGCACGTGGGCCGTGGCCCGGCTGGGTCGGCTGAAGAACTGCGGATGGAAGCTGCGGAAGAGGCCCTGATGGGGCCCACCATCCCGGACCCAAGTCTTCTTCCTGGCGGGCCTCTCGTCTCCTTCCTGGTTTGGGCGGAAGCCATCACCTGGATGCCTACGTGGGAAGGGACCTCGAATGTGGGACCCCAGCCCCTCTCCAGCTCGAAATCCCTCCACAGCCACGGGGACACCCTGCACCTATTCCCACGGGACAGGCTGGACCCAGAGACTCTGGACCCGGGGCCTCCCCTTGAGTAGAGACCCGCCCTCTGACTGATGGACGCCGCTGACCTGGGGTCAGACCCGTGGGCTGGACCCCTGCCCACCCCGCAGGAACCCTGAGGCCTAGGGGAGCTGTTGAGCCTTCAGTGTCTGCATGTGGGAAGTGGGCTCCTTCACCTACCTCACAGGGCTGTTGTGAGGGGCGCTGTGATGCGGTTCCAAAGCACAGGGCTTGGCGCACCCCACTGTGCTCTCAATAAATGTGTTTCCTGTCTTAACAAAAAGGATCC. The sequence of the siRNA targeting ATG3 was as follows Sense: CAUUGAGACUGUUGCAGAAdtdt; Anti-sense:UUCUGCAACAGUCUCAAUGdtdt.The human ovarian carcinoma cell lines OVCAR3 and A2780 were obtained from the Tumor Cell Bank of the Chinese Academy of Medical Science . OVCAR3 cells were cultured in RPMI 1640  and A2780 cells were cultured in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum (FBS), and penicillin/streptomycin (100 U/mL). The cells were cultured in an incubator at 37°C and an atmosphere of 5% COThree thousand cells were seeded into each well of 96-well plate in 100 μL medium. At the time points of 0 h, 24 h, 48 h and 72 h, 20 μL of 5 mg/mL MTT reagent was added and plates incubated for 4 h. The medium was then removed and replaced with 150 μL DMSO. Absorbance at 490 nm was measured using a microplate spectrophotometer .3 cells in 100 μL medium were seeded into each well of the E-Plate. Following incubation at room temperature for 30 min, the E-Plates containing cells were placed on the RTCA SP/MP station positioned in a cell culture incubator. The CI values were measured automatically every 15 minutes to form a continuous proliferation curve.For real-time cell proliferation assays, 50 μL medium was added to each well of a 96-well E-Plate for background measurement. Subsequently, 5 × 10After washing with PBS, cells were treated with trypsin to be collected and washed, then fixed with 70% ice-cold ethanol at -20°C for at least 12 h. Cells were washed and re-suspended by centrifugation then stained with PI following the manufacturer's protocol  for cell cycle analysis by flow cytometry.Cells were trypsinized, counted, suspended and seeded into six-well plates at a density of 500 cells per well, in regular culture medium, then cultured at 5% CO2 and 37°C for 2 weeks until visible clones appeared. The medium was discarded and the cells were carefully washed twice with PBS. After being fixed with methanol for 15 min, the cells were stained with Giemsa's solution for 15 min before washing with tap water and air-drying. The clone formation rate was calculated with the formula: Plate clone formation efficiency = (number of clones/number of cells inoculated) × 100%. All the experiments were repeated 3 times and the average values were reported.Cells were collected and centrifuged for 5 min and washed twice with cold PBS. Cells were then resuspended in 100 μL 1×buffer with 5 μL 7AAD and 5 μL PE-labeled annexin V (KeyGen) per sample in the dark. An additional 400 μL of 1× buffer was added and the rate of apoptosis for each samples determined by flow cytometry within 1 h.TRIzol  was used to extract the total RNA from EOC cell lines and tissues. Isolated RNA was transcribed into cDNA using an avian myeloblastosis virus reverse transcriptase and random primers . Amplification of the cDNA was performed by real-time quantitative PCR using the SYBR Premix Ex Taq™ II kit . The expression level of each target gene was normalised to 18s mRNA. The data analysis was calculated according to the sample threshold cycle (Ct) value from three independent experiments, primers for RT-PCR could found in The complete proteome from ovarian cancer cells was extracted in RIPA buffer, preventing protease-mediated degradation of samples. Protein concentration was determined for each sample, and 40 μg of the denatured proteome was resolved by 15% SDS-polyacrylamide gel electrophoesis and then electro-transferred to Hybond membranes . After blocking the membranes with 5% fat-free milk at room temperature for 2 h, membranes were incubated with the primary antibodies primary antibodies targeting ATG3, LAMP1 , LC3 , and SQSTM1  at 4°C overnight. Membranes were washed three times with TBST then secondary antibodies (anti-rabbit), at dilutions of 1:5000 were added. Following incubation for 2 h at room temperature, the protein bands were visualized by enhanced chemiluminescence (ECL) following the manufacturer's instruction . GAPDH  served as the loading control.The interaction between LncRNA MEG3 and ATG3 protein was examined using Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo fisher) according to the manufacturer's protocols. Biotin-labeled MEG3 or antisense RNA was co-incubated with protein extract of OVCAR3 cells and magnetic bead. The generated bead-RNA-Protein compound was collected by low-speed centrifugation. After washed with Handee spin columns, bead compound was boiled in SDS buffer, and the retrieved protein was detected by western blot with expressional level of GAPDH as control.The Magna RIP RNA-Binding Protein Immunoprecipitation Kit  was used for RIP assays following the manufacturer's protocol. Briefly, OVCAR3 cells were collected and lysed using RIP lysis buffer. Whole-cell extracts were incubated with RIP buffer containing magnetic beads conjugated to human anti-ATG3 antibody or the control IgG. Proteinase K was added to the samples to digest the protein, and the immunoprecipitated RNA isolated. Purified RNA was used for qRT-PCR analysis. Expressional level of GAPDH was as control.Control and transfected cells were treated with actD (dissolved in 100% ethanol) at a final concentration of 1-5 μM. The actD was added to cells 0 h, 1 h, 2 h, 4 h, 6 h or 12 h prior to RNA extraction with TRIzol reagent. Subsequently, qRT-PCR was used to analyze changes in RNA levels.7  were injected into the right flanks of the mice. The tumour volume was directly measured following inoculation and weight calculated using the formula: (length × width2)/2.All animal studies were approved by the China Medical University Animal Care and Use Committee, and all experimentation on animals was performed in agreement with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Four-week-old female BALB/c nude mice with weights of approximately 20 g were obtained from Vital River Laboratories  and housed in a pathogen–free and temperature-controlled environment. Two hundred microliters of PBS containing approximately 1 × 102O2 to endogenous peroxidase activity. Next, the sections were heated in target retrieval solution (Dako) for 15 min in a microwave oven  to retrieve the antigen. Non-specific binding was blocked by incubation with 10% goat serum for 2 h at room temperature. Slides were then incubated overnight at 4°C with anti-ATG3 primary antibody. An appropriate secondary antibody was added and incubated for 20 min at 37°C, and binding was visualized with 3,39-diaminobenzidine tetrahydrochloride (DAB). After each treatment, slides were washed three times with TBST for 5 min. Three independent observers  randomly selected and counted 100 cells from five representative fields from each section. Any discrepancies were checked by three observers until a consensus was reached. Positive expression was graded as follows: 0 = negative; 1 = 1%-50%; 2 = 51%-74%; 3 ≥ 75%. The staining intensity was graded as follows: 1 = weak; 2 = intermediate; 3 = strong. The two grades were multiplied to obtain a final score: - = 0; + = 1-2; ++ = 3-4; +++ = 6-9).Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions and then incubated for 20 min in 3% HTransfected or control OVCAR3 cell slides were fixed with 4% formaldehyde, blocked using 5% skimmed milk in 0.01 M PBS, pH 7.2 (PBS-M). Primary antibodies targeting LC3, SQSTM1 (P62) or ATG3 , were diluted 1:500 in PBS and added to the slides. After incubation overnight at 4°C, slides were washed once with 0.2% Tween 20 in PBS and twice with PBS, and then incubated for 30 min at room temperature with anti-rabbit IgG–TRITC . The slides were washed three times with PBS, then fixed with glycerol/PBS , and observed and photographed using a laser confocal microscope .Data were analyzed using SPSS 17.0 software , and are presented as mean ± SD. Two groups were compared using an unpaired, two-tailed Student's t-test. Spearman rank correlation was used to test the association between Meg3 expression and FIGO stages. The ROC characteristics curve was used to calculate the sensitivity and specificity values of Meg3. and P < 0.05 was considered to be statistically significant.


Syringomyelia (SM) is a common condition affecting brachycephalic toy breed dogs and is characterized by the development of fluid-filled cavities within the spinal cord. It is often concurrent with a complex developmental malformation of the skull and craniocervical vertebrae called Chiari-like malformation (CM) characterized by a conformational change and overcrowding of the brain and cervical spinal cord particularly at the craniocervical junction. CM and SM have a polygenic mode of inheritance with variable penetrance.PCDH17 on CFA22 and ZWINT on CFA26. PCDH17 codes for a cell adhesion molecule expressed specifically in the brain and spinal cord. ZWINT plays a role in chromosome segregation and its expression is increased with the onset of neuropathic pain. Targeted genomic sequencing of these regions identified respectively 37 and 339 SNPs with significantly associated P values. Genotyping of tagSNPs selected from these 2 candidate loci in an extended cohort of 461 CKCS  identified 2 SNPs on CFA22 that were significantly associated to SM strengthening the candidacy of this locus in SM development.We identified six cranial T1-weighted sagittal MRI measurements that were associated to maximum transverse diameter of the syrinx cavity. Increased syrinx transverse diameter has been correlated previously with increased likelihood of behavioral signs of pain. We next conducted a whole genome association study of these traits in 65 Cavalier King Charles Spaniel (CKCS) dogs . Two loci on CFA22 and CFA26 were found to be significantly associated to two traits associated with a reduced volume and altered orientation of the caudal cranial fossa. Their reconstructed haplotypes defined two associated regions that harbor only two genes: PCDH17 and ZWINT, significantly associated to two traits associated with syrinx transverse diameter. The locus on CFA22 was significantly associated to SM secondary to CM in the CKCS dog breed strengthening its candidacy for this disease. This study will provide an entry point for identification of the genetic factors predisposing to this condition and its underlying pathogenic mechanisms.We identified 2 loci on CFA22 and CFA26 that contained only 2 genes, The online version of this article (10.1186/s12863-018-0605-z) contains supplementary material, which is available to authorized users. Canine syringomyelia (SM) is a painful condition where fluid-containing cavities (syrinx or syringes) develop within the parenchyma of the spinal cord and which progress over time , 2. DepeIn the CKCS, risk of SM has been shown to be associated with increased brachycephaly with rostrocaudal doming i.e. a heightened cranium that slopes caudally  and reduBMP3 (BONE MORPHOGENETIC PROTEIN 3) variations to skull diversity GGAATGGGAAGTGAGATGAACAGCATAGGAGATTTGAAAGGTAGCAAACAATCCAGGAGACCTACAGGCCCGTGATCAATGACTTATAAGATGATATTAAGGAAACATTATATATGATACTATATCCTCCTTTGAGAGTCTGTATGCATT and TTAATCTTCAAAACGGCCCAATGAGGATATCCCCATTTTGCAGATGAAGAATGAAGGAACGTTGAAGTTCAGTGATTTGATCCAGAATAAGTGCAGTGAGACTTCAGATCCAGCTATGGGGTTTGCCAAATTCAATCTCTGCCCTTCCTC[A/C]CTATTCTTAACCGCCAAATATTATTTATATTTGTAAGAATGCAGTTTTAAAGGTTGAAATTTTCAACTTCTCACACAGAGCAGATAGCTGGAGACGAAGATGGTAGGACTGCTCTCTCATTGCCTGCATTGTGCTCTCTGAGTAGTGAAA respectively. Two SNPs were also amplified on CFA26 at position 32,757,080 (rs23302138) and 32,797,595 with the probes TGTCCTCCTGGCTTCTGAGGGGGTGGGTGCGGGGCCTGGAGGCCCAGAGGGGAACAGGATGTGGCCACAGGATGGAGAGCTGACTTGTGCACAGGGGCCTGTGTGGGTCAGTCTGTGTCCCCGGCACCCCTGAAGCTGCAGGTGTCTCAG[T/C]AGAGCCCCTCAGTGGGTAACTCTGCCCCCAATTCCCTCCTTGGAGACTGCATCTCCTCCTGTGCCTCCTGCAAGTCGCTGTCAGCTTCCCTCCCCTGAGGTCTGACGCCTCCTGCAGGAAGTTCTCTGGGATTGGATCTCAAAATGGTGC and CATGAGTTGGAAGGGCAGTTAAGGGCAGAAGGACTTAGAGGCGGAGAGCATAGAGAAGGAAAAGGCACGATGGTGTGTTTGATTATCTCCCCCTCTCCATTCTCATGGTGCCACCTATCCTAATTCCAGTTCGTATTATCATAGGTCTCA[C/T]TCCACCAATAACGTCTCAATCACACACACCATGTCCTGTCTTCTCGTTGGTCTGTGCTATGATCTGTGTGGTTCTTCTCTTTCCCAGGAGGCCAGATCTGTATTTTGCTGATTACAATCTACTCTTTAATTCTGGATTGAATTGCTAACT respectively. Genotyping was performed using the TaqMan assay (Applied Biosystems) on 393 CKCS dogs .Tagging SNPs were selected to maximize coverage of each locus. They were identified using the tagger routine in the Haploview V4.2 software with a maximum rP values obtained from the association were corrected together for multiple testing using a storey’s q value method [Initial GWAS analysis was done in 2 phases: association of the quantitative traits  to disease, followed by association of SNPs to these quantitative traits. Association of the quantitative traits to the disease was done using a linear regression with a type III sum of squares in R V3.0.1  and age e method . Haplotye method  and assoe method  with ageL4 + L7 all showed a significant association to STD size (Pbonferroni < 0.0019) and were therefore further investigated . This resulted in the identification of a group of 13 SNPs on CFA15  associated to ratio F-d/BC under a FDR of 0.05  and two SNPs on CFA26 , which were significantly associated to L4 + L7 under a FDR of 0.05 (both P = 0.03754)  and two on CFA26 (P = 0.01067 and 0.00231), that were significantly associated to ratio F-d/BC  surrounding the SNPs significantly associated to PCDH17 (PROTO-CADHERIN 17) on CFA22 and ZWINT (ZW10 INTERACTING KINETOCHORE PROTEIN) on CFA26. In order to identify potential SM -predisposing mutations, both CFA22 and CFA26 candidate regions were submitted to targeted next generation sequencing. The reads of the sequences obtained from the 65 CKCS were aligned on CanFam 3.1 resulting in 5608 SNPs on CFA22 and 10,814 SNPs on CFA26. Except for one SNP (rs2305483), that was identified in the coding region of PCDH17 and that was non-conserved and synonymous, all identified SNPs resided in intergenic and deep intronic regions. Using improved coverage of the region, linkage disequilibrium blocks were reevaluated based on the significant SNPs in the regions. A total of 37 and 339 SNPs were defined as significantly associated to F-d/BC and L4 + L7 respectively. Based on the hypothesis that causative SNPs would be significantly associated to their respective trait, these SNPs refined the regions of interest to 13,785,828-14,183,295 bp  on CFA22 and 32,721,057 bp to 33,094,292 bp  on CFA26.Each of the SM- associated regions harbored only one coding gene: P value = 0.7637 and 0.7614), the 2 selected TagSNPs on CFA22 at position 13,933,606 bp and 13,804,718 bp reached significance . Bonferroni corrected P value of the TagSNP at position 13,933,606 bp on CFA22, was still significant at a P value of 0.0104. Hence, we successfully identified a region on CFA22 associated to ratio F-d/BC and SM in the CKCS dogs.To investigate the potential association between the identified SNPs and SM, a cohort of 461 CKCS including 187 unaffected and 274 SM-affected  were genotyped with two TagSNPs from each of the associated regions on CFA22 and CFA26. While the 2 selected TagSNPs on CFA26 at position 32,797,595 bp and 32,757,080 bp did not show any significant association to SM  significantly associated to STD size. Genetic investigation of these traits identified significant association with SNPs on CFA15 with F-d/BC and on CFA26 with L4 + L7. Both these traits represented a combination that demonstrated a reduction in the overall size of the caudal cranial fossa and rearrangement of the neural parenchyma  identified a significant association of the candidate locus on CFA22 and SM. Hence, this candidate locus that was suggestive of association to the ratio F-d/BC in CKCS and to the line BC in a previous association study in GB dogs was found to be significantly associated to SM, strengthening its candidacy for SM. On the other hand, the tagSNPs on CFA26 that was significantly associated to PCDH17 and ZWINT (respectively). Targeted next generation sequencing of both CFA22 and CFA26 candidate loci identified a total of 37 and 339 significantly associated SNPs with ratio F-d/BC and L4 + L7 respectively. No mutation in the coding region of either gene was detected, except for one synonymous mutation in PCDH17. We hypothesize that predisposing mutations in these two regions are most likely regulatory that would affect RNA expression of either PCDH17 or ZWINT or other unannotated transcripts. Alternatively, these regulatory mutations could have a long-range expression effect on transcripts residing outside the 2 candidate regions. RNA-sequencing or quantitative RT-PCR studies in affected tissues from dogs carrying the associated haplotypes are needed to test this hypothesis.The associated loci on CFA22 and CFA26 harboured each only one gene: PCDH17 (PROTOCADHERIN 17) belongs to the family of protocadherins that are involved in the adhesion and sorting of cells during tissue morphogenesis. It is expressed specifically in several regions of the developing and adult brain and spinal cord [PCDH17 in primary cortical neurons is associated with significantly decreased dendritic spine density and abnormal dendritic morphology [ZWINT (ZW10-INTERACTOR) has an important role in kinetochore assembly and proper chromosome segregation [ZWINT in pain development and in SM associated with CM.nal cord –41. It rnal cord . Overexprphology  and it iregation . In ratsregation –46. We tCM with SM in the dog is very similar to a condition in humans called Chiari malformation I (CMI) with a reported frequency of 1 in 1280. As in dogs, the prevalence of SM secondary to CMI in humans is high reaching 65%–85% , 48. GenL4 + L7 respectively. Genotyping of a larger cohort of CKCS dogs confirmed association of the locus on CFA22 with SM in this breed. Each of these 2 haplotypes harbored only one gene: PCDH17 on CFA22 that codes for a cell adhesion molecule specifically expressed in the brain and spinal cord and ZWINT that plays a role in proper chromosome segregation and whose expression is increased with the onset of neuropathic pain. Additional molecular genetic studies in larger CKCS cohorts from various affected brachycephalic breeds and in cell and animal models are needed to further investigate the role of the 2 associated loci and the genes they harbor in the pathogenesis of SM secondary to CM. Our study represents an essential step towards a better understanding of the complex genetics of this devastating condition and development of breeding strategies that aim at eliminating it from the affected dog breeds. It also provides an important model for studying CMI/SM in humans.In this study, we have used a genome-wide association study to decipher the genetics of SM secondary to CM in the CKCS breed. We identified 6 cranial T1-weighed sagittal MRI measurements that were associated with the syrinx transverse diameter. We next identified 2 haplotypes on CFA22 and CFA26 that were significantly associated to ratio F-d/BC and Additional file 1:Table S1. Characteristics and measurements of 96 CKCS of the cohort. This table includes the gender, age, clinical status and all MRI cranial measurements taken on the 96 CKCS dogs included in this study. (XLSX 41 kb)Additional file 2:Table S2. SNPs suggestive of association to SM in the CKCS breed. This table enlists all SNPs suggestive of association with FDR corrected scores between 0.05 and 0.1. These SNPs were identified following a GWAS using a mixed linear model with age as a covariate on the previously identified traits . (DOCX 15 kb)Additional file 3:Figure S1.P value distribution inside the CFA15  associated region. This region spans 1.7 Mb surrounding SNPs associated to ratio F-d/BC and was identified using Haploview V4.2. (PDF 54 kb)


Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes.Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of  Repetitive DNA elements are widespread in genomes. They are located in centromeres, telomeres, rDNA genes, transposons, and intergenic regions . The newMSH2 and MLH1 causes microsatellite instability and a predisposition to colon and other types of cancer  and MutSβ  mutants exhibit instability of mono- and dinucleotide repeats and generate duplications of sequences flanked by repeats  complex has single-stranded 3′-exonuclease and endonuclease activities as well as structural functions in recombination processes . After 5nd MutSβ . S. cere repeats  and repaSchizosaccharomyces pombe encodes the MutS homologs Msh1, Msh2, Msh3, and Msh6, the MutL homologs Mlh1 and Pms1, and the exonuclease Exo1. Based on homology with S. cerevisiae Msh1, S. pombe Msh1 likely acts in MMR of mitochondrial DNA. S. pombe Msh2, Msh6, Mlh1, and Pms1 are indispensable for repair of base-base mismatches and small loops with one or two nucleotides , yeast extract agar (YEA), and yeast extract liquid (YEL), and general genetic methods were used as described ; OL937 − mlh1h::kanMX his3-D1 ura4-D18 (− exo1h::ura4 ura4-D18 (− rad2h::ura4 ade6-704 leu1-32 ura4-D18 (smt-0 rad50::kanMX ura4-D18 (+ rad51h::kanMX ade6 leu1-32 ura4-D18 (The − msh2h::hphMX ura4-D18 and KK37 − msh6h::hphMX ura4-D18 originated from transformations of OL2137 (− ura4-D18h), with DNA fragments obtained by fusion PCRs. pFA6a-hphMX (smt-0) was used to amplify either 450 bp of the 3′ UTR of the msh2 locus or 500 bp of the 5′ UTR of the msh6 locus. Primers for the msh2 disruption were msh2_For 5′-GAGGTTTTTTATTTATCCTTTTTGAGGACTTAACTGTGGCAAGGAGTTTCTTCTCCTGTTTTATACATTTCGCGTTCGCGCTTTAGAACATTCAATCAATCGGATCCCCGGGTTAATTAA; msh2_Rev 5′-TTTCCTCGTTTTAGTAAAAAATTATTTTATTCATAAAATGCGCTTCCAAAAAACATGTACCTTGGTTGAATTCTTTCAATTAGTACCTTGCTCACATTCTGAATTCGAGCTCGTTTAAAC; msh2_For3 5′-TTGAAAGAATTCAACCAAGG; and msh2_Rev3 5′-GCTAAAACAAAATTATGCCG. Primers for the msh6 disruption were msh6_For 5′-TATATATGTTATTTTGTGCTCTCATGTTAGCTTTGTTTACTATTAGAATGCTGCTTTTTGTAAATAACTGAACTTAGCCAAAACCAACACTTGTTCCAGTCGGATCCCCGGGTTAATTAA; msh6_Rev 5′-ATAACGTAAGTAAATGGTAAATAAAAGCAAGCTTCCGCTTGCCAGCAAACGAAAGATATTGCTTTGAATAGTCATAAAACTGATAGAGTGTTGACAGTTAGAATTCGAGCTCGTTTAAAC; msh6_For2 5′-CTCATCTTACCTAAACTCTC; and msh6_Rev2 5′-GAACAAGTGTTGGTTTTGGC.Strains KK42 DraIII-HindIII fragment of the ade6 gene containing a (GACC)7 repeat near the DraIII site . The fragment was obtained by PCR with primers ade6-GACC7 5′-TCCCACTTGGTGACCGACCGACCGACCGACCGACCGACCTTTATGTTGAAAAGTTCGTTC-3′ and ade6-H 5′-GGGCAAGCTTCAATGGTGTA-3′, and subsequent digestion with DraIII and HindIII. PCR was performed under standard conditions using pCG162 as template (3 run immediately 3′ to the (GACC)7 repeat was deleted. The 1.7-kb XhoI-EcoRI fragments of the plasmids containing either (GACC)7 or (GACC)7ΔT were transformed into the S. pombe strain AM1 7 his3-D1 leu1-32 ura4-D18h and – ade6-(GACC)7hΔT his3-D1 leu1-32 ura4-D18. A strain with a (GACC)8 repeat was isolated during a fluctuation test with an h+msh2::7his3 ade6-(GACC) mutant.In pAN-K, a pUC18 derivative, the kanamycin resistance gene was replaced by a 340-bp − ade6h::ura4 ura4-D18) was constructed by transformation of strain OL2137 (− ura4-D18h) with a PCR fragment obtained with primers ade6_d_ura4_F 5′-TCCTTTTGTACTGAAAAGTAAAACATTGGCTTACGACGGTCGTGGAAATTACGTTGTTCATCAACCATCTGAGATTCCTACTGCCATCAAAGCACTTGGTagcttagctacaaatcccac and ade6_d_ura4_R 5′-GAATGGTCTCAGTTGTAGGATAAGCATAAACTTTTCCGTCTAAACTGCGTACTACCATCACTGCAATTTCCATGGAGAAAGGAACGAACTTTTCAACATagcttgtgatattgacgaaac, and pAW1 as template  in ade6 were deleted and replaced by ura4 . After initial denaturation for 1 min at 94°, we applied two cycles with 30 sec at 94°, 30 sec at 45°, and 30 sec at 72°, followed by five cycles with 30 sec at 94°, 30 sec at 55°, and 30 sec at 72°. Reaction samples were transformed into strain DE1 using the method of 6ade6-(CTGCC). Primers Hepta-F 5′-ACATTGGCTTACGACGGTCGTGGAAATTACGTTGTTCATCAACCATCTGAGATTCCTACTGCCATCAAAGCACTTGGTGATCGTCCATCGTCCATCGTCCATCGTCC ATCGTCCTTTATG and Hepta-R 5′-AAGCATAAACTTTTCCGTCTAAACTGCGTACTACCATCACTGCAATTTCCATGGAGAAAGGAACGAACTTTTCAACATAAAGGACGATGGACGATGGACGATGGACGATGGACGATCACC were used for 5ade6-(ATCGTCC). Primers HeptadT-F 5′-AACATTGGCTTACGACGGTCGTGGAAATTACGTTGTTCATCAACCATCTGAGATTCCTACTGCCATCAAAGCACTTGGTGATCGTCCATCGTCCATCGTCCATCGTCCATCGTCCTTATG and HeptadT-R 5′-TAAGCATAAACTTTTCCGTCTAAACTGCGTACTACCATCACTGCAATTTCCATGGAGAAAGGAACGAACTTTTCAACATAAGGACGATGGACGATGGACGATGGACGATGGACGATCACC were used for 5ade6-(ATCGTCC)ΔT. Here, a single T immediately downstream of the repeats has been deleted ΔT, repeat tract changes were also analyzed by inspection of the color of Ade+ revertants 7ΔT, (CTGCC)6, and (ATCGTCC)7, insertions of one repeat unit and deletions of two repeat units are the major events detectable. The opposite is the case with (GACC)8 and (ATCGTCC)7ΔT, where deletions of one repeat unit and insertions of two repeat units are the principal events that can be detected, although in the case of (ATCGTCC)7ΔT, we identified exclusive deletions as described below.We have previously reported that in-frame nucleotide insertions and codon changes in a region around nucleotide 1397 of the  mutants . Inserti7of ade6-(GACC)ΔT, deletions and insertion can be distinguished by the color of revertants ΔT repeat in the various genetic backgrounds revealed deletions and sequencing of 23 7(GACC)ΔT revertants revealed insertions (7(GACC)ΔT repeat can be easily determined from a large number of revertants. Analyzing revertants of the other ade6 repeats did not allow a distinction by color, probably because Ade+ originating from deletions and insertions were both not fully functional. In these cases, PCR products of independent Ade+ revertants were subjected to sequencing to identify the number of repeats.In the case vertants . Deletiosertions . All revmsh2, msh3, msh6, mlh1, or exo1. In wild type, 8ade6-(GACC) and 7ade6-(GACC)ΔT reverted to Ade+ with 1.2 × 10−5 and 2.1 × 10−5 reversions per cell division, respectively (8ade6-(GACC) reversion rates increased 39–44-fold in msh2, msh6, and mlh1 mutants and slightly decreased in msh3 and exo1 mutants. Similarly, 7ade6-(GACC)ΔT reversion rates increased 17–22-fold in msh2, msh6, and mlh1 mutants and decreased 3–5-fold in msh3 and exo1 mutants. None of the differences between msh3, exo1, and wild type was statistically significant.We first analyzed stability of GACC tetra-nucleotide repeats in wild type, and in mutants deleted for either ectively . The ade7ade6-(GACC)ΔT can be determined by colony color as described above and in Materials and Methods, and as illustrated in msh3, and exo1 strains, reversions mainly occurred by deletions  revealed that in wild type, all 10 had two repeat insertions 6 repeat appeared to be more stable in the msh3 mutant. It reverted mostly through gain of one repeat unit in all strain backgrounds, with the possible exception of mlh1  to Ade+ . Rates w of mlh1 . We conc5ade6-(ATCGTCC)ΔT and 5ade6-(ATCGTCC) reverted to Ade+ at rates of 7.2 × 10−6 and 7.5 × 10−6, respectively ΔT (not significant) and 2.8–2.9-fold for 5ade6-(ATCGTCC) (significant) (5ade6-(ATCGTCC)ΔT revertants contained four hepta-nucleotide repeats and thus originated from deletion of one repeat (ade6 (+. 5ade6-(ATCGTCC) reverted mainly by insertion of one repeat and less frequently by deletion of two repeats without any significant differences between the spectra of wild type and any of the mutants  . All seqe repeat . The facat 5 repeat 5 repeat instability in msh3 and exo1 is not due to a defect in MMR. We therefore wanted to analyze the genetic context of msh3 and exo1 defects in microsatellite stability. To do this, we measured reversion rates of the hepta-nucleotide repeat in FEN1rad2, rad50, and rad51 mutants and in various double mutants 5 repeat when msh3 is mutated.The 5 repeat . Since d mutants . FEN1 anon rates . The msh mutants . Like wieat unit . In the S. cerevisiae, msh3 and msh6 mutants show little to moderate increases of mutation rates in mono- and dinucleotide repeats 7 and (GACC)8 tetra-, (CTGCC)6 penta-, and (ATCGTCC)5 hepta-nucleotide repeats 7ΔT, were the predominant reversion events in msh2 and msh6 mutants, slippage of one repeat can occur in the template and in the nascent strand during replication, and both types of events are corrected by MMR mediated by MutSα and MutLα.The assay with the (GACC)vertants . We founsertions . Thus, t6 in msh2, msh6, mlh1, and exo1 mutants were similar to that of wild type, but decreased in msh3 5ΔT and (ATCGTCC)5 (5ΔT and mainly by insertion of one repeat in (ATCGTCC)5. We conclude that loops in penta- and hepta-nucleotide repeats are not substrates of MutSα and MutLα in S. pombe.Mutation rates of the penta-nucleotide repeat (CTGCC) in msh3 . All stre repeat . InactivTCGTCC)5 . Like wimsh3 mutants had no significant defects in repair of base-base mismatches and of loops with one unpaired nucleotide in a T6 repeat and in nonrepetitive DNA (msh3 mutations caused some instability of a (GT)8 dinucleotide repeat, which was mostly evident by a reversion spectrum different to wild type (msh3 mainly reverted by two-nucleotide deletions in the (GT)8 repeat. However, this was clearly less frequent than in msh2, msh6, and pms1 mutants 8 repeat t strand . Rates wS. pombe Msh6, as part of MutSα, recognizes base-base mismatches and loops with one to four unpaired nucleotides, while Msh3 does not play a significant role in MMR, but rather maintains repeat stability independently of MMR. Consequently, S. pombe MMR cannot repair loops with five or more nucleotides, in contrast to human MMR  (S. pombe microsatellites. It is therefore critical for humans, but not for S. pombe, to have an MMR system that can deal with larger loops.We conclude from our studies that


Various types of stem cell lines have been derived from preimplantation or postimplantation mouse embryos: embryonic stem cell lines, epiblast stem cell lines, and trophoblast stem cell lines. It is not known if extraembryonic endoderm stem (XEN) cell lines can be derived from postimplantation mouse embryos. Here, we report the derivation of 77 XEN cell lines from 85 postimplantation embryos at embryonic day E5.5 or E6.5, in parallel to the derivation of 41 XEN lines from 69 preimplantation embryos at the blastocyst stage. We attain a success rate of 100% of XEN cell line derivation with our E5.5 whole-embryo and E6.5 disaggregated-embryo methods. Immunofluorescence and NanoString gene expression analyses indicate that the XEN cell lines that we derived from postimplantation embryos (post-XEN) are very similar to the XEN cell lines that we derived from preimplantation embryos (pre-XEN) using a conventional method. After injection into blastocysts, post-XEN cells contribute to extraembryonic endoderm in chimeras at E6.5 and E7.5. Mouse preimplantation embryonic development culminates in the blastocyst stage. A blastocyst consists of three cell lineages: epiblast, trophectoderm, and primitive endoderm (PrE). The epiblast develops into most of the embryo proper, the amnion, and the extraembryonic mesoderm of the yolk sac; the trophectoderm gives rise ultimately to the fetal portion of the placenta; and the primitive endoderm forms the two extraembryonic endoderm lineages – the visceral endoderm (VE) and the parietal endoderm (PE) of the yolk sac134in vitro model to identify patterning activities of the extraembryonic endoderm such as factors involved in cardiac induction17Stem cell lines have been derived from these three cell lineages91114Gata4 or Gata6 . We find that this and other pre-XEN cell lines are immunoreactive for XEN cell markers GATA4, GATA6, SOX7, SOX17, and DAB2, but negative for ES cell markers OCT4 and NANOG, and negative for TS cell marker CDX2.Xist locus on the X-chromosome; Sox17 and Gata6 are XEN-cell markers; and Cdx2 is a marker for trophoblast stem cells. We removed the ectoplacental cone of the embryos as much as possible, and transferred each embryo separately into a well of 4-well dish coated with 0.1% gelatin and covered with MEF in TS cell medium including 25 ng/ml FGF4 and 1 μg/ml heparin (referred to as F4H). One day later, the embryos had attached to the surface and started to form an outgrowth. The embryos had formed a large outgrowth after 5 days. We used TrypLE Express to disaggregate the outgrowths and passaged cells into a well of a 4-well dish. After cells reached 70–80% confluency, they were passaged into a well of a 12-well dish. After they reached 70–80% confluency again, cells were passaged into a well of a 6-well dish, and we then obtained stable post-XEN cell lines. The intrinsic red fluorescence of mTomato produced from the Gata6 promoter in the transgene was sufficiently high to detect it in the whole embryo and outgrowth, but not in the established post-XEN cell line at day 60  by immunofluorescence for GATA4 and counterstaining with DAPI. Sets of fluorescence images were captured for each line visualizing the intrinsic fluorescence of GFP, DAPI, and GATA4 immunoreactivity . The imaWe asked if post-XEN cells can differentiate into a VE identity by incubation with BMP438Dab2, Gata4, Gata6, Pdgfra, Sox7, and Sox17, versus low or no expression of ES cell-specific genes such as Nanog, Pou5f1/Oct4, Sox2, and Nr0b1. There is no expression of EpiSC-specific genes such as Cer1 and Fgf5 (data not shown). Thus, the NanoString gene expression analysis confirms and extends the immunofluorescence profiles.We applied the NanoString multiplex platform for gene expression4041in vivo. We injected into blastocysts  cells of two R26-tauGFP41+ pre-XEN cell lines (X-ICM-4 and X-ICM-5) and one PDGFRa-GFP+ pre-XEN cell line (X47) that we had derived from isolated ICMs or blastocysts, and cells from four R26-tauGFP41+ post-XEN cell lines  and one PDGFRa-GFP+ post-XEN cell line (X-E6.5-Z0663-9) that we had derived from E5.5 embryos or disaggregated E6.5 embryos  that are at the origin of the post-XEN cell lines.Sox17 locus, we cannot detect GFP expression in embryos and cell lines derived from them.) The Xist1loxGFP strainThe PDGFRa-GFP strainAdvanced RPMI-1640  was supplemented with 20% (vol/vol) FBS , 2 mM GlutaMAX Supplement (Gibco #35050), 1% penicillin/streptomycin , 0.1 mM β-mercaptoethanol (Gibco #21985-023), 1 mM sodium pyruvate (Gibco #11360-039), supplemented with 25 ng/ml FGF4 (Peprotech #100-31) and 1 μg/ml heparin (Sigma #H3149).ES cell lines were maintained on MEF-coated, pregelatinized tissue culture dishes  in DMEM  supplemented with 15% FBS (HyClone #SH30071.03), 2 mM GlutaMAX Supplement, 1% penicillin/streptomycin, 1% β-mercaptoethanol , 0.1 mM nonessential amino acids (Gibco #11140-035), 1 mM sodium pyruvate, and 1000 IU/ml leukemia inhibitory factor (LIF) (Millipore #ESG1107).Embryos were collected at the 2–8 cell stage by flushing oviducts using M2 medium (Sigma #M7167), and cultured in KSOM medium (Millipore #MR-106-D) to the blastocyst stage. The zona pellucida of blastocysts was then removed using acid Tyrode solution (Sigma #T1788). Blastocysts were transferred separately into a well of a 96-well dish  coated with 0.1% gelatin  and covered with MEF in ES medium supplemented with LIF and 1 μM PD0325901 (Axon #1408) and 3 μM CHIR99021 (Axon #1386), a combination of chemicals that is typically referred to as “2i”. After 4 days TrypLE Express (Gibco #12604-013) was used to disaggregate the embryonic outgrowths, and cells were passaged to a well of 24-well dish to derive ES cell lines.Embryos were collected at the 2–8 cell stage embryos and cultured in in KSOM medium to the blastocyst stage. The zona pellucida of blastocysts was removed using acid Tyrode solution. Blastocysts were transferred separately into a well of a 4 well-dish (Nunc #176740) coated with 0.1% gelatin and covered with MEF in ES medium supplemented with LIF. The XEN lines were derived as describedThe ectoplacental cone was removed with forceps or needles. A whole embryo was placed in a well of a 4-well dish (Nunc #176740) coated with 0.1% gelatin and covered with MEF in TS medium including F4H. After the embryos formed a large outgrowth, TrypLE Express was used to disaggregate the outgrowths and passage cells into a 4-well dish. When cells reached 70–80% confluency, they were passaged into a well of a 12-well dish until XEN cell lines were obtained, which were then passaged into a well of a 6-well dish. If the outgrowth of an E5.5 embryo grew well and XEN cells thrived, we continued to culture cells in TS medium including F4H. But if the outgrowth grew slowly and XEN cells were surrounded by trophoblast-derived cells, we cultured cells in ES medium supplemented with LIF, culture conditions that would inhibit trophoblast-derived cells; when XEN cells started to become abundant, we switched to TS medium including F4H. To prepare XEN cells for RNA extraction, cells were cultured in dishes coated with 0.1% gelatin but without MEF in TS medium including F4H.The ectoplacental cone was removed with forceps or needles. A whole embryo was treated with 0.1 mg/ml collagenase (Gibco #17104-019) and 0.01 mg/ml deoxyribonuclease (Gibco #D5025) for 20–30 min at room temperature, followed by 0.2 mg/ml TrypLE for 5 min at room temperature. The embryo was disaggregated into single cells using a glass pipette with a diameter of 50–60 μm. Cells were transferred into a well of a 4-well dish (Nunc #176740) coated with 0.1% gelatin and covered with MEF in TS medium including F4H. Three days later XEN colonies appeared. We picked these colonies, disaggregated them with a glass pipette or by TrypLE Express for 5 min at 37 °C, and passaged them into a well of a 4-well dish. When cells reached 70–80% confluency, they were passaged into a well of a 12-well dish until XEN cell lines were obtained, which were then passaged into a well of a 6-well dish.http://cellprofiler.org/ . The DAPI signal was used to segment individual cells by thresholding, de-clumping, and applying size and roundness filter in an effort to evaluated single cells that are not MEFs. After filtering, the GFP and GATA4 signals were evaluated in each cell, and the population characteristics for these two markers were quantified.Images representing Fields of View of post-XEN cell lines were imaged for intrinsic fluorescence of GFP, fluorescence of DAPI, and GATA4 immunoreactivity. Automated cell population characteristics were determined with CellProfiler Gelatin-coated plates were prepared by coating with 0.1% gelatin overnight at room temperature. XEN cells were cultured in TS medium with F4H and with or without 10 ng/mL BMP4  on gelatin-coated plates for four days.Cell lines X42, X44, X47 (PDGFRa-GFP × CAG::mRFP1); X35, X36 (B2D6F1 × D4/EGFP); X97, X107 (D4/EGFP × DBA2/N); X-E6.5-81346-8 (ROSA-STOP-taulacZ x Sox17-Cre); X-E6.5-Z0617-2, X-E6.5-Z0617-5, X-E6.5-Z0617-8 (R26-tauGFP41 x Sox17-Cre); X-E6.5-78097-4 (Xist1loxGFP × DBA/2 N); X-E6.5-82278-4 (Gata6-mTomato × Cdx2-GFP); X-E6.5-Z0663-12 (CD1 × PDGFRa-GFP); and X-E5.5-6, X-E5.5-8, X-E5.5-9, X-E5.5-10, X-E5.5-13 (R26-tauGFP41 × Sox17-Cre) were cultured in 4-well or 24-well dishes. Cells were fixed in 4% paraformaldehyde at 4 °C overnight or room temperature for 30 min, permeabilized with 0.1% Triton X-100 in 1× PBS (1× PBST) for 30 min and blocked with 5% normal donkey serum  diluted in 1× PBST (blocking solution) for 1 hr. Primary antibodies were diluted at 1:50–1:200 in blocking solution and samples incubated at 4 °C rotating overnight. After three 10-min washes in 1× PBST 10 min, samples were incubated for 1–1.5 hr at room temperature in a 1:500 dilution of secondary antibody in blocking solution, then washed and covered with 1× PBST containing DAPI. Images were taken with an AMG EVOS (Life Technologies), Zeiss LSM 710 confocal microscope, and Nikon SMZ25 stereofluorescence microscope.Primary antibodies from Santa Cruz Biotechnology were against GATA4 (#SC-1237), DAB2 (#SC-13982), OCT3-4 (#SC-5279), NANOG (#SC-376915), and CDX2 (#SC-166830). Primary antibodies from R&D Systems were against GATA6 (#AF1700), SOX7 (#AF2766), SOX17 (#AF1924), and PDGFRa (#AF1062). Primary antibodies against E-cadherin (ECCD2) were from Invitrogen (#13-1900). Secondary antibodies from Jackson ImmunoResearch Laboratories were Cy5™ AffiniPure Donkey anti-goat IgG (H+L) (#705-175-147), and Cy5™ AffiniPure Donkey anti-rabbit IgG (H+L) (#711-175-152). Secondary antibodies from Invitrogen were Donkey anti-rabbit IgG (H+L) with Alexa Fluor 546 (#A10040), Goat anti-rat IgG (H+L) with Alexa Fluor 546 (#A11081), Donkey anti-mouse IgG with Alexa Fluor 546 (#A10036), Donkey anti-mouse IgG with Alexa Fluor 488 (#A21202), and Goat anti-chicken IgY (H+L) with Alexa Fluor 488 (#A11039).Actb and Gapdh. Normalized counts are displayed in a heatmap generated by the nSolver Analysis Software v2.5, using agglomerative clustering.Cells were cultured in 12-well plates with TS medium including F4H or ES medium supplemented with LIF. Dissociated cells were collected by trypsinization and centrifugation. Cell pellets were dispensed directly in RNAlater Stabilization Solution (Qiagen) and stored in −80 °C for later use. Cell pellets were lysed in RLT Lysis Plus Buffer using a TissueLyser LT (Qiagen) at 40 Hz for 2 min. Total RNA extraction was performed using RNeasy Plus Micro kit (Qiagen) according to manufacturer’s protocol. The custom NanoString CodeSet “Extra” was used. 100 ng of total RNA samples were hybridized at 65 °C for 18 hr and processed with the nCounter Analysis System GEN1 (NanoString Technologies). The reporter counts were processed using nSolver Analysis Software v2.5 (NanoString). Two normalizations were performed to the counts, the first normalization to the generic positive controls, followed by normalization to the reference genes, Below are the nucleotide sequences of the NanoString probes; first line is the capture probe, second line the reporter probe.ActbACGATGGAGGGGCCGGACTCATCGTACTCCTGCTTGCGGGTGTAAAACGCAGCTCAGTAACAGTCCGCCTAGAAGCACTTGCGGTGCDab2GTCTCCTCGAGCATCAGGCACATCATCAATACCGATTAGCTTGGCCCAGCTGCTGCCATTCCCTTGAGTTTCATCATAGAATCCTGACTCATTTTDnmt3lGAGGCAGCGCATACTGCAGGATCCGGTGGAACTGGAACATGCCATGAATATCCAGAAGAAGGGCCGCTGACTCTCCTGGCDppa4CAAGTCTTTACAGTTGACTGCTGAACTGGTTATGACGCCCGTTGTGCTGGCACTACAACCCAGGGAAGAGGACATGCATGCGGAGGCTACAGGTATAAGCDppa5aCGCACGGCCCACAGCTCCAGGTTCAGGAAGTTTTAGTACCCTGCCAAGGAACCAGACTTCAGGGAAGACGAGATCAAGCTTATCCACCAEsrrbTTTCCAGAATGAACCGCTTCATCTTTAGGACATCCTGTCAACCCAAACCCGAGCAGGTAAAGCCGGAGGACTTGTCATGAAAGTGGCGTGTCCATFgf4ATTCTGGTAACAAAATTCCAAAGATACAGTCTTGTCCCTGGGCGCAGGAAACAGACCGACTCGGTAACAGTGGCAGATACAGAGCAGAAACATCAAACCCFoxa2CACAGACAGGTGAGACTGCTCCCTTGAGGCCTGAAGTCCCTTCCCTATTTAGAATGACAGATCACTGTGGCCCATCTATTTAGGGAFoxq1GCTGTCCTTACTCCGAGGTTTAGAGACTTTGAGCGGAAGACAAGCGTTTTGATTGTTGGGTGAAGTGAGGAGTGGAGTGATAGAAGTTGGTGCAGTFstAGGACTTTGTGATACACTTTCCCTCATAGGCTAATCCAATGGATCTGCCCCTTGGAATCCCATAGGCATTTTTTCCCGCCGCCACACTGGATATCTTCACFxyd3TGAGCCCGCCGACTCGGAGGCTGTACCAATCATAGTAGAAAGGATCATTTGCCACTCATAAGGACTATAATGCCCAGGGCACAGAGAATCCCTGCACAAAGapdhATACTTGGCAGGTTTCTCCAGGCGGCACGTCAGATCCACGACGCCTCAGATGCCTGCTTCACCACCTTCTTGATGTCATCGata4AGAGCCAGGTAACTGTCTGACTTAAGAGGGCTTGGCTTGGGCAAACAGTCTGTATTTTCTAGACAAAGGATCTGTGCTGGAGAAAGTCCCGata6ATCTGGACTGCTGGACAATATCAGACACAAGTGGTATGAGGCCTTCAGAGGGCACAGAAATCACGCATCGAAGGAATGTTATGTCTGCATTTTTGCTGCCKrt8TTCCCATCTCGGGTTTCAATCTTCTTCACAACCACAGCCTTGCAGTGGCCATTCACTTGGACACGACATCAGAAGACTCGGACACCAGCLama1CATTGGCTAAATCGGCATGGCGGTCATCCTTGATACAGACAGAACTCAGACATAACCTTTCCTACATGGACACTGACCTGGCCACTTTCLamb1CCAGGAAGGAATGCGGTCCTGAATGTACTGCCTTTCCACCACAATGACAAAAACTCCAAATAAGCCCCTTCAGGCACCCGGACGAACCCAGGTCCTGTNanogCATATTTCACCTGGTGGAGTCACAGAGTAGTTCAGGAATAATTCCAAGGCGAAGGAACCTGGCTTTGCCCTGACTTTAAGCCCAGATGTTGCGTAAGTCTNr0b1CACTTGAAAAAGAAACTCTTGATGGCCTGGACCGCAGCAGCTGGGAGCAACCTTTCAGATAGGCATACTCTTTGGTGTCAATGTTCAGACTCCAGPdgfraTATGGAGTAAGTCGCTCTCACACACTTACCACACCACCATGTTGGGAACAAAACATGAACAGGGGTATCTGGAAGCCATCTTGTATTGGAAGACCCTTCCPou5f1ACATGGTCTCCAGACTCCACCTCACACGGTTCTCAATGAGTGATCTGCTGTAGGGAGGGCTTCGGGCACTTCAGAAPth1rCCTGGGCACGGTGCAGCAGGAAAATCTGTTCCTCTTTGGTAAAGACATCGTGCTGTGTGCAGAACTTCCTTGAGCAGCTTGTCACATTGCGSox2CCCCGCCGCCCTCAGGTTTTCTCTGTACAAAAATAGTCCCCCAAAAAGAATGCGTAGTTTTTTTCCTCCAGATCTATACATGGTCCGATTCCCCCGCCCTSox7AGAAATCAGCACACCCCAACACTTTTGTGGACAGACGTTTTAGGTTTCTATCATCTTTTGCATCTGTAGATAAGAGTATGCTACAGCTCTGCTCTSox17GGCAGATACTGTTCGAATTCCGTGCGGTCCACCTCTGTCCCTGGTAGGGAAGACCCATCTCGGGCTTATACACAAAGTet3TTTTCAAAGAGCTGAATGAATGCACCAGGATTTTAGGATGGGCGTGTTTCGTCAGGCCCAACCTCTCAATGTCACAGAAATTAAAGCACCAACGTTCTCAAll animal studies were carried out in accordance with the German Animal Welfare Act, European Communities Council Directive 2010/63/EU, and institutional ethical and animal welfare guidelines of the Max Planck Institute of Biophysics and the Max Planck Research Unit for Neurogenetics. All experimental protocols were approved by the Regierungspräsidium Darmstadt and the Veterinäramt of the City of Frankfurt.How to cite this article: Lin, J. et al. Efficient derivation of extraembryonic endoderm stem cell lines from mouse postimplantation embryos. Sci. Rep.6, 39457; doi: 10.1038/srep39457 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm  β-globin gene locus can protect a gene promoter from DNA methylation. Protection from DNA methylation is separable from other insulator activities and is mapped to three transcription factor binding sites occupied by the zinc finger protein VEZF1, a novel chromatin barrier protein. VEZF1 is a candidate factor for the protection of promoters from DNA methylation. We found that VEZF1-specific binding sites are sufficient to mediate demethylation and protection of the APRT gene promoter from DNA methylation. We propose that barrier elements in vertebrates must be capable of preventing DNA methylation in addition to blocking the propagation of silencing histone modifications, as either process is sufficient to direct the establishment of an inactive chromatin state.DNA sequences known as chromatin insulator or barrier elements are considered key components of genome organization as they can establish boundaries between transcriptionally permissive and repressive chromatin domains. Here we address the hypothesis that barrier elements in vertebrates can protect genes from transcriptional silencing that is marked by DNA methylation. We have found that the HS4 insulator element from the  It has been proposed that genes and gene clusters are organized into chromatin domains that are maintained independent of their surroundings through the establishment of boundaries β-globin genes are clustered within a thirty kilobase domain of nuclease accessible chromatin, the 5′ boundary of which is marked by a constitutive DNaseI hypersensitive site called HS4  and adult chicken red blood cells (an abundant source of nuclear protein for purification purposes). Complexes of similar mobility and intensity are observed between the two nuclear extracts and each of the FI, FIII and FV sites  and determined whether it interacts with the G-rich footprinted sites of the HS4 insulator, as it was previously reported that human VEZF1  interacts with similar G-rich sites FV sites . The comA-globinβ promoter that contains a (dG-dC)16 string. A nuclear factor called Beta Globin Protein 1 (BGP1) was previously characterized as interacting with this site in vitro, but was considered to indirectly assist in activation by directing nucleosome placement A promoter site with an identical specificity to that of erythrocyte nuclear protein(s) (A promoter site A site is competed by unlabelled FI which contains a (dG-dC)9 string 6 string .The (dG-dC) strings present in the VEZF1 sites at HS4 are reminiscent of a site in the chicken s FV see , which mA sites  analyses were performed to analyze the binding of VEZF1 at the chicken analyses . VEZF1 bxpressed . None ofin vivo, as all three VEZF1 binding sites are required for protection from DNA methylation. We found that this was not the case, as VEZF1 remains tightly bound at HS4 when any one of its binding sites is deleted (in vitro (data not shown). We have attempted to disrupt VEZF1 function at HS4 following knockdown by RNAi. We strived to achieve substantial knockdown of VEZF1 to disrupt its binding to the high affinity sites at HS4. Prolonged knockdown was also required as we have previously found that the de novo DNA methylation of these transgenes is a gradual process that takes many days to establish de novo DNA methylation of the HS4 element and no change in HS4's ability to protect a transgene from silencing during this period (data not shown). The inadequacy of RNAi to strip constitutive transcription factor binding from high affinity sites has also been observed for CTCF Vezf1 null ES cells, as we recently found that they are defective for de novo DNA methylation due to the requirement of Vezf1 for full transcriptional activity of the Dnmt3b gene in these cells We tested whether VEZF1 requires all three of its sites for binding to HS4  deleted . VEZF1 aAPRT gene promoter and its effects on methylation. SP1-like binding elements have been shown to be required to prevent methylation of the mouse and hamster APRT CpG island elements: two earlier papers have shown that deletion of the SP1-like elements is sufficient to induce methylation in these islands APRT CGI that contain three SP1-like elements are subject to demethylation upon integration into mouse ES cells APRT gene remaining unmethylated and expressed normally in Sp1 null ES cells and embryos APRT CGI elements (APRT CGI stably integrated into mouse ES cells (in vitro (CCCCCCTTTCCCC) that is reminiscent of the VEZF1-specific bipartite footprint III site found at the HS4 insulator element (CCCCCCGCATCCCC).To address whether VEZF1 elements also protect CpG island (CGI) promoters from DNA methylation, we investigated VEZF1 binding to the elements . We perfES cells . We founin vitro . Site 3 APRT CGI from methylation, we replaced each of the three SP1-like elements with the VEZF1-specific FIII element from the HS4 insulator  a promoter can be protected from DNA methylation even when it lacks active histone modifications and transcriptional activity, and 6) short DNA elements bound by VEZF1 mediate the demethylation and protection of a CpG island from DNA methylation.Here we have studied the paradigm HS4 element to address our hypothesis that a barrier element in vertebrates must be capable of defending a gene from silencing by DNA methylation and have identified a novel CpG island factor. We have presented six findings: 1) a vertebrate barrier element protects a gene promoter from DNA methylation-mediated silencing, 2) the essential transcription factor VEZF1 is a barrier/anti-methylation factor, 3) there is a modular division of labor at the compound HS4 insulator as VEZF1-mediated protection from methylation is separable from CTCF-mediated enhancer blocking and USF-mediated recruitment of active histone modifications, 4) the de novo DNA methylation.We have previously demonstrated that the HS4 insulator acts as a barrier to the spread of histone methylation marks associated with repressive chromatin bona fide activity that is protective against DNA methylation.It was previously shown that the transgenes used in this study become marked by dense promoter DNA methylation upon chromosomal position effect silencing de novo DNA methylation is key to our understanding of how VEZF1 binding at HS4 could protect a promoter from epigenetic silencing. Previous studies using the same transgene system studied here found that non-insulated transgenes, regardless of integration site, are consistently subject to promoter methylation upon chromosomal silencing, and that flanking with HS4 elements can shield transgenes from this methylation β-globin locus, the spreading of repressive histone modifications is observed upon perturbation of active histone modification recruitment at the HS4 barrier Determining the source of de novo DNA methylation arise via spreading from the chromosomal integration site in our transgene system, we would expect to see high levels of methylation at compromised mutant insulators either coincident with, or prior to promoter methylation. However, we observe that promoters become methylated prior to the insulators, which remain unmethylated or become partially methylated. The observed independence of methylation states between insulator and promoter argue against spreading and clearly show that there can be no single mechanism that controls the methylation state of both the insulator and promoter. It remains possible that VEZF1 elements at HS4 are acting as a barrier to the spreading of a DNA methylation mechanism, but that additional processes prevent the accumulation of methylation at HS4 itself. An alternative possibility is that DNA methylation does not result from spreading, and that the insulator directly interacts with the promoter to deliver VEZF1 co-factors that prevent promoter methylation. In this model, the promoter itself would have its own program to recruit de novo DNA methylation, and VEZF1 would act as a factor that mediates inhibition of methylation. This would distinguish the activity of VEZF1 from those of USF1/USF2, which bind elsewhere in the insulator element and recruit a number of enzymes that deliver active histone modifications to the reporter gene Should Igf2/H19 domain de novo DNA methylation of HS4. It has previously been shown that DNA binding proteins can prevent the methylation of their binding sites simply by steric hindrance of de novo DNA methyltransferases (DNMTs) The 275 bp “core” HS4 element comprises a CpG island (CGI) that is free of DNA methylation regardless of neighboring gene expression de novo methylation during early development, and how they remain hypomethylated irrespective of transcriptional status. Recent epigenomic profiling studies have begun to reveal a significant portion of CGIs that are subject to varying degrees of tissue-specific methylation in human somatic tissues de novo methylation, which can be selectively inactivated during development and may become defective during cancer progression cis-regulatory elements and trans-acting factors that control CGI methylation status is key to unraveling these processes.All constitutively expressed genes and ∼40% of genes with tissue-restricted expression have CGI promoters APRT gene CGI promoter. It was previously shown that SP1-like binding elements are required to prevent CGI methylation APRT CGI. A promoter-less APRT CGI fragment containing only site 3 remains protected from DNA methylation de novo DNA methylation. Thus, VEZF1 binding elements can protect a CGI from DNA methylation. We attempted to definitively address the requirement for VEZF1, but discovered that global de novo DNA methylation mechanisms are defective in Vezf1 null ES cells DHFR gene  were cultured and assayed for IL-2R expression by FACS as described previously Chicken 6C2 erythroleukemia cells carrying Gel mobility shift assays were performed as described previously in vitro translation using rabbit reticulocyte lysate (Promega).FI- and FIII-binding proteins were purified from adult chicken red blood nuclear protein extracts by ion exchange chromatography. Throughout the purification, eluate fractions were analyzed for FI- and FIII-binding activity with gel mobility shift assays. The binding specificity of partially purified proteins was checked by competition analysis after each purification step. FI- and FIII-binding activities co-fractionated following ion exchange chromatography with SP XL and Heparin sepharose . Phosphocellulose, Q and Sephacryl S300 columns were used in early purification attempts to resolve FI- and FIII-binding activities but they co-fractionated in each case (data not shown). FI- and FIII-binding activities both eluted in two distinct fractions of approximately 200 and 400 kDa following gel filtration (data not shown). The active fractions eluted from heparin sepharose were pooled then split into two and fractionated in parallel by FI or FIII DNA affinity as described previously 5′CATGCCGCTCGAGCGGTTTTTTTTTTTTTTTTT was used in first strand cDNA synthesis with Superscript II reverse transcriptase (Invitrogen). The primers VEZF1_5′, 5′CCATGACCCATGGGCAGAGCCAAAGT and Adaptor 5′CATGCCGCTCGAGCGG were used to amplify a full length chicken VEZF1 cDNA by PCR which was TA cloned into pCRII (Invitrogen). VEZF1 cDNA was sub-cloned into pCITE4b (Novagen) to generate p4bVEZFfull for the purpose of in vitro transcription. cDNA encoding chicken ZF5 was isolated by RT-PCR from 6C2 cell total RNA using primers designed from the published sequence (U51640). We found that the bases CpG 1306-7 in the published sequence were GpC in our clone, causing codon 436 to translate as alanine instead of arginine. We obtained the vectors pCDNA3-ZF5 and pEVRFO-ZF5  and we also found the CpG to GpC conflict with the published sequence. Full length chicken SP1 and SP3 cDNAs cloned in the pBluescript-based vectors pH-SP1 and pH-SP3-3 were a kind gift from Marc Castellazzi .Chicken VEZF1/BGP1 cDNA was cloned following RT-PCR from 6C2 cell total RNA based on an assumption of conservation of 5′ cDNA sequence with human VEZF1/DB1. The oligonucleotide Adaptor-A E.coli . Peptides were produced in M15 [pREP4] E. coli followed by rapid lysis with B-PER reagent (Pierce). ZF5 peptide was soluble and purified on Ni-NTA agarose (Qiagen). VEZF1 peptide was insoluble and resulting inclusion bodies were prepared using B-PER reagent (Pierce), solubilized with 6M guanidium hydrochloride and immobilized on TALON Sepharose resin (Clontech) at pH 7. VEZF1 peptide was renatured in a stepwise manner with 6, 4, 3, 2, 1 and 0.5 M guanidium hydrochloride prior to elution. VEZF1 and ZF5 polyclonal IgG antibodies  were purified from rabbit serum using PROSEP-A media . Anti-full length chicken VEZF1 antibodies were raised previously Polyclonal antibodies were raised  against chicken VEZF1 (Ser376-Ala547) and chicken ZF5 peptides (Ser131-Lys248) produced in 7 cells/ml) and fixed with a final concentration of 0.25% formaldehyde at room temperature for 30 seconds. 6C2 cells were harvested in mid-exponential growth phase, divided into 30 ml aliquots containing 1×108 cells in fresh media and fixed with a final concentration of 0.8% formaldehyde at room temperature for 5 minutes. Reactions were stopped by adding glycine to a final concentration of 0.125 M. The cells were washed in PBS and resuspended in cell lysis buffer  to isolate nuclei. Chromatin was prepared following washing  and lysis  of nuclei. Crosslinked chromatin was fragmented by sonicaton (Misonix) for a total time of 10 minutes in regular 10 second pulses at 4°C. Debris was removed by centrifugation at 15000 g for 10 minutes and chromatin was diluted in 10 volumes of X-ChIP buffer . Agarose gel electrophoresis was used to confirm that chromatin fragments were ∼500 bp in length on average.ChIP analysis of transcription factor binding in chicken cells was performed using formaldehyde crosslinked chromatin prepared from chicken 10 day embryonic erythrocytes isolated from fertilized White Leghorn eggs  or cultured 6C2 erythroleukemia cells. 10 day erythrocytes were washed and resuspended in 25 mls of PBS . Incubation with antibodies was performed overnight at 4°C on a rotating wheel. Between 10 and 30 µg of specific antibodies or 10 µg of normal rabbit IgG (Santa Cruz) were used per ChIP. Chromatin was precipitated with protein A agarose  for 4 hours at 4°C with rotation. Immunoprecipitated chromatin was collected, washed, eluted and crosslinks reversed. DNA was then extracted by phenol-chloroform and ethanol precipitated in the presence of 10 µg glycogen.Chromatin was pre-cleared with 100 µl of protein A agarose  and 50 µg of normal rabbit IgG (Santa Cruz) for 3 hours at 4°C on a rotating wheel. Aliquots of chromatin were taken to generate input DNA and protein for western analysis. Individual ChIPs were performed using chromatin from 1×10β-globin locus as described previously Relative DNA enrichments were quantified by TaqMan real-time qPCR using the comparative Ct method relative to input DNA and normalized to the primer set 10.35 within the 16 kb condensed chromatin region upstream of the chicken GGAACAAGTTGGCAAGGTCCTAT10.35_For: TCTTCTGCCCTGCCCGTAT10.35_Rev: FAM-TGCAGTTCCCTGTTCATGTGCTTTTCG-TAMRA10.35_TM: TCCTGGAAGGTCCTGGAAG21.54_For: CGGGGGAGGGACGTAAT21.54_Rev: 6FAM-CCCAAAGCCCCCAGGGATGT-TAMRA21.54_TM: CTGTGGTCTCCTGCCTCACA39.8_For: AGGCTGGGTGCCCCTC39.8_Rev: FAM-CAATGCAGAGTGCTGTGGTTTGGAACTG-TAMRA39.8_TM: CACAGGAAACAGCTATGACATGATTPGI_5′_For TCTGCCTTCTCCCTGATAACGPGI_5′_Rev 6FAM-AATTCCTGCCCACACCCTCCTGC-TAMRAPGI_5′_TM 9 cells treated with 1% formaldehyde for 5 minutes. Chromatin was prepared as described above, where fragments sizes ranged from 500–700 bp. Semi-quantitative PCR was performed using the following primersChIP analysis of transcription factor binding in murine E14Tg2A.4 ES cell lines was performed using formaldehyde crosslinked chromatin prepared from 1×10AAAGGCGTGCGGGAGCCAGAAATAPRT1/2_For: CCTTGGTAGGTGGGGAPRT1/2_Rev: CCCTGTTCCTGGGCTCCAPRT3_For: TGACTGGCCAGGAGGAPRT3_Rev: ChIP analysis from human embryonic kidney 293-T cell line SD5 that contains a stably integrated copy of the 275 bp HS4 core chicken insulator was performed as described for cultured chicken cells above. SD5 cells were crosslinked with 1.6% formaldehyde for 5 minutes. The following primers were used in SYBR quantitative PCR analysis:CGGGATCGCTTTCCTCTGAHS4_21.726_F: CCGTATCCCCCAGGTGTCTHS4_21.726_R: TCGCCTGCACAAATAGGGACP_DHFR_F: AGAACGCGCGGTCAAGTTTP_DHFR_R: ControlGACAGCAGCCGAACTTCGTTVEZF1_CDS_F: TGGTGCCCGAGGAAGATGVEZF1_CDS_R: APRT elements. The following elements were amplified from Hamster liver genomic DNA:AvaII and HpaII restriction sites in bold.Sp1-like motifs in red. CTAGAGGATCCGGACAACACCCACACCGGCCCCTCCAGGTCCAGAAAGCTGGCCCTGCGAGAAGCGGGACTGAAAAGGCGTGCGGGAGCCAGAAATCCAAAAGGGTGCCAAGGCATGCGTCCTTTTTCCACCCAGAAATAACCCCAGGCTTTCAATTTGAGGTTATTTCAATATCCAGCAAATGCGTTACTTCCTGCCAAAAGCCAGCCTCCCCGCAACCCACTCTCCCAGAGGCCCCGCCCCGTCCCGCCCCCTCCCGGCCTCTCCTCGTGCTGGATCGCTCCCTAAGGACGCCCCGCTCCAGAAGCCCCACCTACCAAGGACGCCCCACCCTTGTTCCCGGACTGGTATGACCCCAGCCTGCTGACATCCCTCCGCCCTTTCTCGTGCACGCGGCTATGGCGGAATCTGAGTTGCAGCTGGTGGCGCAGCGATCCGCAGTTTCCCCGACTTCCCCATCCCCGGCGTGCTGTTTAGGTGAGATCACGAGCCAGCAAGGCGTTGGAGCCCTGTTCCTGGGCTCCCGGCGAGGCGCATGGGCAGTCTCGGGGATCTTGTGGGGTCTCCGCCCCCCTTTCCCCGGCCACCAGCCTCTCCTTGTTCCCAGGGATATCTCGCCCCTCCTGAAGGACCCCGCCTCCTTCCGAGCTTCCATCCGCCTCCTGGCCAGTCACCTTAAGTCCACGCATGGCGGCAAGATCGACTACATCGCAGGTCTAAvaII and HpaII restriction sites in bold.Mutated bases in blue were introduced by site directed mutagenesis. Overall CpG content increased from 46 to 48 following these mutations. CTAGAGGATCCGGACAACACCCACACCGGCCCCTCCAGGTCCAGAAAGCTGGCCCTGCGAGAAGCGGGACTGAAAAGGCGTGCGGGAGCCAGAAATCCAAAAGGGTGCCAAGGCATGCGTCCTTTTTCCACCCAGAAATAACCCCAGGCTTTCAATTTGAGGTTATTTCAATATCCAGCAAATGCGTTACTTCCTGCCAAAAGCCAGCCTCCCCGCAACCCACTCTCCCAGAGACCCCCCGCATCCCCGACGCTACCCCCCGCATCCCCGATCTCCTCGTGCTGGATCGCTCCCTAAGGACGCCCCGCTCCAGAAGCCCCACCTACCAAGGACGCCCCACCCTTGTTCCCGGACTGGTATGACCCCAGCCTGCTGACATCCCTCCGCCCTTTCTCGTGCACGCGGCTATGGCGGAATCTGAGTTGCAGCTGGTGGCGCAGCGATCCGCAGTTTCCCCGACTTCCCCATCCCCGGCGTGCTGTTTAGGTGAGATCACGAGCCAGCAAGGCGTTGGAGCCCTGTTCCTGGGCTCCCGGCGAGGCGCATGGGCAGTCTCGGGGATCTTGTGGGGTCCGCTCCCCCCGCATCCCCGACACCAGCCTCTCCTTGTTCCCAGGGATATCTCGCCCCTCCTGAAGGACCCCGCCTCCTTCCGAGCTTCCATCCGCCTCCTGGCCAGTCACCTTAAGTCCACGCATGGCGGCAAGATCGACTACATCGCAGGTCTAHpaII site in bold.TCTAGATTGCTAGGAGTAGCACCTAAGATGAACTAGATGCTAAAAAATGCTGTATCTTTGGGGCACACGAGGGCATGCCTGGGCAGGCTTAGAGCCTGGTAGTCTCAGGGGCTGCACCAAAGTGTAATTCTTGTGCTAAATAACTTTCACTTACCAGTGCCAAGCACGGGCTTCAGAAACACCCTAGGGTCGCTGAATGTCCACCAGGGGAGTCAGACATGTCCAGAGGGTGAGAACCCCAGAGAATTCGGTAGCCCTGACATGTGCTACAATTACTGATGCCCACTTCCTACTGGTTCCTCCTGGCCATACCTCAGGAATTAGGGCATGCTTTCTGCCTGCTACAGTAGCTCATCCTCCCTGGAAGTGACCCCAGACATATACCCTGAACTGTAACCGATAAAGTGCGCCTGGGCAGATGTATTTGAGAGGTGGCAAAAGTAAACCATAGGTGTCCCCGAGCTAGATACAGAAGGCAGATAACATCCCCAAGGCTAAGCTGCTGCCCCAATAGCCATCAGCCTTCTAGTTATAGCTAGTAAGACCTAGTATTCCTGGTCAATACTATTCACTCAATCCTTACACCTCAGCCCTAACACGCCCCCTCTCTCATCCTAACAGGCCTAGACTCCAGGGGATTCTTGTTTGGCCCCTCCCTAGCTCAGGAGCTGGGCCTGGGCTGTGTGCTCATCCGGAAGCGAGGGAAGCTGCCAGGCCCCACAGTGTCAGCCTCCTATGCTCTCGAGTATGGCAAGGTAAGCAGGCAGTGGGTAGCTGTCTAGGAGTAAATGTGGGGGCTCAGAGAGGTTAAGTCATCAGGCCAGGTTTATACCACCAGGAAACATGGAGAAGCTAGGGGTGGTGGTTCTAGAAPRT CpG islands and an 870 bp non-island region of the APRT gene were cloned into pUC19. Each element was PCR amplified, half of which was subject to in vitro CpG methylation by M.SssI methyltransferase(New England Biolabs). In vitro methylation was validated by HpaII and MspI digestion. 1 µg of each APRT element was transfected into murine E14Tg2A.4 ES cells (BayGenomics) with 1 µg of the XbaI fragment of pREP7 (Invitrogen). Hygromycin resistant clones were grown in LIF without feeder co-culture. Genomic DNA was extracted after two weeks of culture. Probes for Southern blotting were generated using Ready-to-go dCTP beads .∼720 bp wild type and mutant Oligonucleotides were generated on an ABI 394 DNA synthesizer.The top strand sequences for each of the duplexes used for gel mobility shift analyses were:GACAGCCCCCCCCCAAAGCCCCCAGGGA5′ GGAGCTCACGGG,FI wt CGCTCCCCCCGCATCCCCGAGCCGGggcgcgcct5′ aggcgcgccCCGGTCCGG,FIII wt CCTGGGGGGATACGGGGAAAAAGCTTTAGG5′ CCTGCAGACA,FV wt 5′ ATTCGATCGGGGCGGGGCGAGC,Sp1 5′ AATTGCAGAGCTGGGAATCGGGGGGGGGGGGGGGGCGGGTGGTGGTGTGG,glo wt 5′ AATTGCAGAGCTGGGAATCGGGGGGGCGGGTGGTGGTGTGG,glo 7G 5′ AATTGCAGAGCTGGGAATCGGGGGGCGGGTGGTGGTGTGG.glo 6G Asc I restriction sites used for cloning FIII sites in an earlier study are shown as lower case. All FI and FIII oligonucleotides were identical to the wt sequences above except for mutations indicated in DNA affinity columns were prepared using the following oligonucleotides5′ gatcTCACGGGGACAGCCCCCCCCCAAAGCCCCCAFI-DA TOP 5′ gatcTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAFI-DA BOTTOM 5′ gatcGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAFIII-DA TOP 5′ gatcTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCFIII-DA BOTTOM PCR primers used in bisulfite sequencing were5′ GGTATTAGAGTAGATTGTATTGAGAGTGTAHS4 5′ double forward 5′ CATAACTATTTCCTATATAAATCCCCHS4 5′ double reverse 5′ AGAGTAGATTGTATTGAGAGTGTATTATAHS4 5′ single forward 5′ ACATCCCTAAAAACTTTAAAAAAAAHS4 5′ single reverse 5′ GTTAAGGTTGGGGGTTTTTTIL-2R forward 5′ AAAACTCTACCTAACAACCAAACACIL-2R reverse PCR primers used in RT-PCR gene expression analysis were5′CAGTGCACGTTTGGCATTTGAAGGgVEZF805anti 5′GAAAAGGCTTCTCGAGGCCTGATCGgVEZF716sense 5′-6FAM-TCCAGGAGCGTGACCCCAGCA-TAMRAGgGAPDH-247T 5′ ATGGGCACGCCATCACTATCGgGAPDH-226F 5′ AACATACTCAGCACCTGCATCTGGgGAPDH-302R 5′ TGCTGCGCTCGTTGTTGAGgB-ACTIN_F 5′ CATCGTCCCCGGCGAGgB-ACTIN_R 5′-6FAM-TGGCTCCGGTATGTGCAAGGCC-TAMRAGgB-ACTIN_T APRT non-island element were:The primers used for methylation specific PCR analysis of the 5′ TCTAGATTGCTAGGAGTAGCAPRTNI_3′ TCTAGAACCACCCCTAGCAPRTNI_Figure S1SacI - HindIII fragment originally defined as the “250 bp core” HS4 element in vitro DNaseI footprints AscI cloning sites present in all functional assays of the HS4 core are shown in italics.Core HS4 sequences. (A) The 244 bp (0.87 MB TIF)Click here for additional data file.Figure S2Identification of FI- and FIII-binding proteins. (A) Peptide sequences obtained from tandem MS sequencing of proteins isolated by FI- and FIII-DNA affinity. (B) Alignment of the amino acid sequences of chicken VEZF1/BGP1, human VEZF1/DB1, and mouse Vezf1 . Peptides obtained from tandem MS sequencing that match VEZF1 are indicated by lines above the alignment. Six C2H2 zinc fingers motifs are boxed. (C) Schematic representation of the domain structure of VEZF1. Only the zinc finger motifs share homology with factors other than VEZF1 orthologs, the nearest relative being the MAZ transcription factor.(4.96 MB TIF)Click here for additional data file.Figure S3A-globinβ promoter. Gel mobility supershift assays using 32P-labelled FI (A), FIII (B), FV (C), and glo wt (D) oligonucleotide duplexes. Adult chicken erythrocyte nuclear extract (ery) and recombinant chicken VEZF1, SP1, or SP3 used in the reactions are indicated by brackets above the lanes. Proteins were pre-incubated with antibodies (indicated above each lane) prior to incubation with DNA. Supershifts are evidenced by abrogation of specific complexes (asterisks) and/or formation of low mobility ternary complexes (SS). Antibodies alone do not give rise to complexes with any of the duplexes used (not shown).Supershift analysis of VEZF1, SP1, SP3, and ZF5 interactions with HS4 footprints and the (3.49 MB TIF)Click here for additional data file.Figure S432P-labelled FI and FIII oligonucleotide duplexes. Unlabelled competitor duplexes (indicated above each lane) were added at 50 fold molar excess. Recombinant chicken SP1, SP3, and ZF5 used in the reactions are indicated by brackets above the lanes. The competition profile of these proteins does not match that of red blood cell nuclear extract Click here for additional data file.Figure S5in vivo. (A) ChIP analysis of transcription factor interactions at the β-globin locus in 6C2 erythroid progenitor cells. DNA enrichments at the {lower case beta}A promoter or the HS4 and 3′HS insulators were normalized to a negative control located in the 16 kb condensed chromatin region upstream of the β-globin locus. (B) ChIP analysis of transcription factor interactions with a stably integrated HS4 element in transgenic human 293 cells. Interactions with the DHFR CpG island promoter are shown as a positive control for SP1 and SP3 binding. DNA enrichments are normalized to an AT-rich negative control locus.SP1, SP3, and ZF5 do not interact with HS4 (0.49 MB TIF)Click here for additional data file.Figure S6IL-2R transgene showing location of QPCR primer sets used to analyze the interaction of VEZF1 with the transgenic HS4 and promoter elements. (B–D) ChIP analysis of (B) VEZF1, (C) CTCF, or (D) USF1 interactions at the endogenous HS4 insulator (eHS4) and the transgenic HS4 (tHS4) and promoter (β-IL2Rpro) elements in the same 6C2 cell lines used for DNA methylation  Schematic representation of the hylation  and hist(1.09 MB TIF)Click here for additional data file.Figure S7β-globin locus.VEZF1 binding to HS4 is not significantly affected following VEZF1 RNAi. (A) Quantitative RT-PCR analysis following 48 hours of doxycycline-induction of two chicken 6C2 cell lines harboring lentiviral vectors that express VEZF1-specific miRNA. Expression levels are normalized to those of β-actin (ACTB) and untreated cells. VEZF1 mRNA levels are reproducibly knocked down by 70%. (B) Western blot analysis of chicken VEZF1 protein levels in one of the above lines with and without 14 days of doxycycline induction of VEZF1-specific miRNA. TBP levels were monitored as a loading control. VEZF1 and TBP band intensities visualized on a FUJI LAS3000 imager were quantified using AIDA software (shown below). Following normalization to TBP levels, VEZF1 protein levels were quantified to be knocked down by ∼97%. VEZF1-specific miRNA may cause translational inhibition in addition to the mRNA degradation observed by RT-PCR (C) ChIP analysis of VEZF1 interaction with the endogenous HS4 element following 14 days of induced VEZF1 knockdown. DNA enrichments were normalized to a negative control located in the 16 kb condensed chromatin region upstream of the (1.64 MB TIF)Click here for additional data file.Figure S8APRT CpG island elements. VEZF1 and SP1 interact with the wild type APRT CpG island, but the FIII mutant APRT element is only bound by VEZF1. Gel mobility supershift assays using 32P-labelled oligonucleotide duplexes containing either the wild type APRT SP1 sites 1&2 (lanes 1–4) and site 3 (lanes 5–8) or the mutant sites 1&2 (lanes 9–12) and site 3 (lanes 13–16). The core sequences of the duplexes are as shown in Transcription factor interactions with (1.78 MB TIF)Click here for additional data file.Table S1VEZF1 sites in the HS4 barrier protect a promoter from DNA methylation. CpG methylation of transgene promoters flanked by wild type or mutant HS4 insulators after 30 or 90 days of culture. The scoring of individual CpG bases from each clone subject to bisulfite sequencing is shown. Methylated bases are marked as ‘1’ and shaded blue. Average CpG methylation values are shown in (0.02 MB PDF)Click here for additional data file.Table S2de novo methylation of HS4. CpG methylation of wild type (WT) or mutant (ΔI - ΔV) HS4 insulators after 30 or 90 days of culture. Both copies of HS4 were sequenced, except for ΔII and ΔV, where only the outermost copy was sequenced (see Mutations of insulator protein binding sites result in the nced see . The sco(0.03 MB PDF)Click here for additional data file.Text S1DNA binding activities of candidate HS4-binding proteins; RNA interference methods; western blotting methods.(0.04 MB DOC)Click here for additional data file.