Drosophila melanogaster and Caenorhabditis elegans. According to the Rosetta-Stone hypothesis, proteins encoded as fusion proteins in one organism and as separate proteins in another organism may act in the same signalling pathway. Although a direct interaction of human nitrilase1 and Fhit has not been shown, our previous finding that Fhit interacts with β-catenin and represses its transcriptional activity in the canonical Wnt pathway suggested that human nitrilase1 also modulates Wnt signalling. In fact, human nitrilase1 forms a complex with β-catenin and LEF-1/TCF-4, represses β-catenin-mediated transcription and shows an additive effect together with Fhit. Knockdown of human nitrilase1 enhances Wnt target gene expression. Moreover, our experiments show that β-catenin competes away human nitrilase1 from LEF-1/TCF and thereby contributes to the activation of Wnt-target gene transcription. Inhibitory activity of human nitrilase1 on vertebrate Wnt signalling was confirmed by repression of Wnt-induced double axis formation in Xenopus embryogenesis. In line with this finding, the Drosophila fusion protein Drosophila NitFhit directly binds to Armadillo and represses the Wingless pathway in reporter gene assays. Genetic experiments confirmed the repressive activity of Drosophila NitFhit on Wingless signalling in the Drosophila wing imaginal disc. In addition, colorectal tumour microarray analysis revealed a significantly reduced expression of human nitrilase1 in poorly differentiated tumours. Taken together, repression of the canonical Wnt pathway represents a new mechanism for the human nitrilase1 tumour suppressor function.Nitrilase1 was classified as a tumour suppressor in association with the fragile histidine-triad protein Fhit. However, knowledge about nitrilase1 and its tumour suppressor function is still limited. Whereas nitrilase1 and Fhit are discrete proteins in mammals, they are merged in  D. melanogaster and C. elegans Fhit is expressed as a C-terminal fusion protein with the unrelated protein nitrilase (Nit). Sequence alignments identified hNit1 as the closest homologue of the Nit domain in the Drosophila and C. elegans NitFhit fusion proteins exhibiting about 50% identity at the amino acid sequence level within the Nit domain [C. elegans NitFhit fusion protein forms a tetrameric complex with four Nit domains building the central core and the C-terminal Fhit domains aligning as dimers at opposing sites of the Nit core [Human nitrilase1 (hNit1) was recognized during the characterization of the human fragile histidine triad (Fhit) tumour suppressor protein, when it was realized that in Nit core . A similNit core . MoreoveNit core . Based oD. melanogaster and C. elegans, Nit1 is a member of the 10th out of 13 branches of the Nit protein superfamily. Only branch 1 enzymes are known to have specific nitrilase activity performing nitrile hydrolysis, whereas most other branches have apparent or postulated amidase or amide condensation activities [In contrast to Fhit, little is known about the function of Nit1 and interaction partners have not been studied. Together with nitrilase2 and the NitFhit fusion proteins of tivities . Mammalitivities , 8. To d−/− mice developed normally and revealed similar phenotypes as reported for Fhit−/− mice such as accelerated cell proliferation, increased cyclin D1 expression and higher N-nitrosomethylbenzylamine-induced tumour incidence. Overexpression of Nit1 similar to Fhit overexpression resulted in enhanced apoptosis. Mutation of the Cys residue within the catalytic triad did not affect the tumour suppressive activity of Nit1 [−/− cells and vice versa, suggesting that both proteins can promote pro-apoptotic signalling independently [Nit1 of Nit1 . Moreove of Nit1 . Interesendently . In oesoendently . Howeverendently . Currentc-myc, cyclin D1, sp5, axin2, survivin and MMP-14 [β-Catenin acts as a multifunctional adaptor protein, for example, within the cadherin–catenin cell–cell adhesion complex and in the canonical Wnt signalling pathway, thereby regulating diverse cellular processes including morphogenesis, proliferation, differentiation, tumourigenesis and neurodegenerative disease . This red MMP-14 .Drosophila NitFhit (dNitFhit) has a modulatory role in the canonical Wnt/Wingless (Wg) pathway by both biochemical and genetic analyses.Previously, we have identified β-catenin as a direct Fhit interaction partner , 17. Bas6. As shown in 6 in cells co-transfected with both constructs but not in controls that were transfected with only a single plasmid. Moreover, using the monoclonal anti-Nit1 (1C3) antibody it was possible to precipitate endogenous hNit1/β-catenin complexes from lysates of HeLa and HEK-293 cells -β-catenin fusion protein pulled down MBP -hNit1 -hNit1 , lane 6,n)-hNit1 .6) and FLAG-TCF-4 together with hNit1. After immunoprecipitation with anti-FLAG-M2 antibody, hNit1 associates with TCF-4 in the absence of co-transfected β-catenin-S33A-myc6  under the control of the heterologous UAS promoter. To assess the repressive activity of UAS-dNitFhit we focused on the expression of the high threshold Wg/Armadillo target gene senseless. In the wild-type wing, imaginal disc of third larval instar senseless is expressed in two well-defined stripes along the dorsoventral boundary in close proximity to the Wg-secreting cells [engrailed-Gal4 driver in the posterior part of the wing discs dramatically reduced Senseless expression. The levels of senseless are decreased in the part where dNitFhit is overexpressed, in comparison with the anterior part that remains as wild type. This repressive effect was also apparent when the separated domains dNit and dFhit were overexpressed alone, but appeared more pronounced when Nit and Fhit were overexpressed together, either as dNitFhit fusion protein or co-expressed  revealed that in well-differentiated tumours hNit1 staining was significantly more intense compared with poorly differentiated tumours (P=0.036), consistent with its tumour suppressor function. Although a high number of pN1+ tumours showed reduced staining of hNit1, correlation with involvement of lymph nodes or patient survival was not statistically significant .In colon cancer, the canonical Wnt pathway is aberrantly activated at high frequency . Thus, dDrosphila Fhit is expressed as a fusion protein with Nit1 defining dNitFhit as a Rosetta-Stone protein [Xenopus embryos. Moreover, co-transfection of hFhit augmented the hNit1 repressive effect in reporter gene assays, suggesting that both proteins are able to cooperate in signalling pathways such as the canonical Wnt pathway. In line with this, an artificial hNit1–hFhit fusion protein exhibits similar repressive activity as co-transfected hNit1 and hFhit. Such a combined and additive repressive effect also was observed in Nit1−/−Fhit−/− double knockout mice [We have previously characterized the fragile histidine-triad protein hFhit as a new β-catenin interaction partner and repressor of β-catenin transcriptional activity , 17, pro protein . The Rosout mice .Drosophila dNitFhit Rosetta-Stone protein, where Fhit is C-terminally fused to the Nit part, similarly binds to the β-catenin homologue Armadillo and acts as a repressor of the Wg/Armadillo pathway. Interestingly, both subparts—dNit and dFhit—are able to independently repress Wg signalling and revealed an additive effect in Drosophila imaginal disc development, again supporting a common function as suggested by the Rosetta-Stone hypothesis. Furthermore, the analysis of the Drosophila fusion protein dNitFhit indicates that the Wnt/Wg-repressing activity of Nit and Fhit is evolutionary conserved.In addition, the In binding directly to the Arm-repeat region of β-catenin, hNit1 uses a different site compared with hFhit . Therefocyclin D1 promoter is in line with a previous report showing that Wnt target genes such as cyclin D1 [claudin-1 [−/− mice [cyclin D1, sp5 and MMP-14 [cyclin D1 expression at least in part appears to be mediated by β-catenin and probably is also involved in the regulation of cell survival. However, the amount of hNit1 within the nucleus appears to be limited and tightly regulated. The mechanisms of this regulation remain to be investigated.The association of hNit1 with the yclin D1  or claudlaudin-1  are upre−/− mice . Consistd MMP-14 , 49, 50.Analysis of colon carcinoma tissue microarrays revealed that hNit1 expression is significantly reduced in dedifferentiated tumour samples. Future studies are needed to unravel whether hNit1 mutations or loss of hNit1 expression occur in tumours at similar frequencies as reported for hFhit and how mutations of both hFhit and hNit1 affect outcome compared with mutations of a single gene. It will be interesting to see whether and how hNit1 expression is correlated with the β-catenin, APC and Ras status of tumours. Moreover, it currently is not clear how hNit1 expression itself is regulated.In conclusion, our studies identified hNit1 and the dNitFhit fusion protein as conserved negative regulators of the canonical Wnt/Wg signalling pathway. As postulated by the Rosetta-Stone hypothesis, hNit1 and hFhit share β-catenin as a common interaction partner and appear to have an additive repressive effect on β-catenin transcriptional activity. Our data further confirm the postulated tumour suppressor function of hNit1 and provide a molecular mechanism for this activity.HEK-293, HeLa and MCF-7 cells were grown as reported previously . MonocloBamHI site and the Nitrilase1C203A mutations were performed with the QuikChange site-directed mutagenesis kit . A human NitFhit fusion protein was generated by overlap extension PCR introducing the Drosophila linker sequence between hNit1 and hFhit. PCR products were ligated into pCS2+, pCS2+-myc6, pFLAG-CMV4, pGEX-4T1, pQlinkG [BamHI sites of pMal-C2X, pCS2+myc6, p3xFLAG-CMV10, pUAST-attB and pAc5.1-FLAG. Plasmids encoding Fhit [KpnI/HindIII.Oligonucleotides used for PCR are summarized in  pQlinkG  and pMALing Fhit , GST-Arming Fhit , GST-β-cing Fhit  and GST-ing Fhit  were des5 cells per well) or 24- (1×105 cells per well) well plates were transiently transfected with the indicated constructs. pGL4.26-BAR, -fuBAR, Siamois-luciferase (S5 and S0) [−) cells. Transfection efficiency was normalized by co-transfection of constitutive active Renilla luciferase (phRL-Null). Average values of at least three independent transfection experiments are presented for all reporter gene assays. Drosophila Kc (or S2R+) cells were transfected in 96-well plates by CellFectin  with wf-Luciferase reporter, tubulin α1-Renilla and empty vector or dNitFhit constructs. Cells were stimulated with Wg-conditioned or control medium 48 h after transfection for 24 h and luciferase activities were measured in triplicate and normalized to Renilla-luc activity.Reporter gene assays were performed as described previously , 55. HEK and S0)  or TopflCt) values of the target genes were normalized to the endogenous control glyceraldehyde-3-phosphate dehydrogenase. Differential expression was calculated according to the 2C−ΔΔt method.For hNit1 knockdown studies, MCF-7 cells were stably transfected with pGFP-V-RS short hairpin RNA Nit1 (GI344668) or pGFP-V-RS scrambled non-effective short hairpin RNA (TR30013) . Total RNA was isolated by using the NucleoSpin RNA II kit  and reverse transcription was performed with the High-Capacity cDNA Reverse Transcription Kit . Real-time PCR was done with UPL probes  using the primer/probe combinations given in 5 HEK-293 cells per six well were transiently transfected with the indicated constructs in different combinations. After 48 h, cells were lysed with ice-cold lysis buffer  Triton X-100 and Complete protease inhibitor mix (Roche)) for 20 min at 4 °C and centrifugated . Immunoprecipitation was performed with precleared lysate  and 2 μg of the appropriate antibody pre-bound to Protein A Sepharose  as described [−1 anti-FLAG M2, 1:1 000 anti-myc (9E10), 0.25 μg ml−1 anti-Fhit and 0.1 μg ml−1 anti-β-catenin).For immunoprecipitation, 8×10escribed . To deteescribed  using thEscherichia coli XL1 blue or BL21-RE4 grown in Luria broth media and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside. For expression of GST-Armadillo, bacteria were induced in M9 minimal medium. For affinity purification of GST fusion proteins on glutathione-agarose (Sigma), cells were washed and lysed in phosphate-buffered saline. MBP fusion proteins were purified in 40 mM Tris/HCl pH 8.0, 100 mM NaCl on amylose resin . GST and MBP fusion proteins were eluted with 20 mM glutathione, 0.1 M Tris/HCl pH 8.0 and 20 mM maltose, 40 mM Tris/HCl pH 8.0, 100 mM NaCl, respectively. For pull-down assays, 4 μg of GST or GST fusion proteins were incubated with 5 μg of MBP fusion proteins in pull-down buffer  Triton X-100) or  Triton X-100) for 20 min at 4 °C. Assays were performed as described before [All fusion proteins were expressed in d before .6) per six-well plate were seeded on a coverslide. After 48 h, PLA  was performed with monoclonal anti-Nit1 (1C3) and polyclonal anti-β-catenin (M14M) according to the manufacturer’s recommendations. Images were taken with an inverse fluorescence microscope  and quantified with Fiji Open Source software [For PLAs , MCF-7 ssoftware .Xenopus embryos. The embryos were then incubated at 19 °C and axis duplication was scored after 36 h. For luciferase experiments, 100 pg of the siamois-luciferase construct [Capped XWnt1 mRNA and hNit1 mRNA was synthesized from linearized plasmid template using the mMESSAGE mMACHINE kit . Four nanoliters of XWnt1 mRNA (5 pg) alone or in combination with hNit1 (500 pg) mRNA were injected into the equatorial regions of the two prospective ventral blastomeres of four-cell stage onstruct  were co-UAS promoter were generated using the ΦC31 integration system developed previously in our lab [ZP-attP-86Fb fly line harbouring a landing site on the third chromosome. To express the UAS-driven constructs the fly lines were crossed to yw; engrailed-Gal4,UAS-CD8-GFP line. Engrailed-Gal4 drives the expression in posterior part of wing imaginal discs as marked by co-expressed green fluorescent protein. Third instar larva wing imaginal discs were isolated, fixed and stained by standard techniques using guinea pig anti-Senseless antibody (provided by Hugo Bellen). Pictures were captured on a Zeiss LSM710 confocal microscope.Transgenic fly lines expressing dNitFhit or its sub-fragments dNit and dFhit under the control of  our lab . ConstruThe chromatin immunoprecipitation analysis was performed as described in Weiske and Huber  using 3


Wolbachia intracellular bacteria can manipulate the reproduction of their arthropod hosts, including inducing sterility between populations known as cytoplasmic incompatibility (CI). Certain strains have been identified that are unable to induce or rescue CI, including wAu from Drosophila. Genome sequencing and comparison with CI-inducing related strain wMel was undertaken in order to better understand the molecular basis of the phenotype.wAu were identified, including the absence of the wMel ortholog of a gene recently identified as a CI candidate in a proteomic study. The comparative analyses also focused on a family of transcriptional regulator genes implicated in CI in previous work, and revealed numerous differences between the strains, including those that would have major effects on predicted function.Although the genomes were broadly similar, several rearrangements were identified, particularly in the prophage regions. Many orthologous genes contained single nucleotide polymorphisms (SNPs) between the two strains, but a subset containing major differences that would likely cause inactivation in The study provides support for existing candidates and novel genes that may be involved in CI, and provides a basis for further functional studies to examine the molecular basis of the phenotype. Wolbachia pipientis is a maternally inherited intracellular bacterium that infects a very large number of arthropod and nematode species [Wolbachia-infected males, while Wolbachia-infected females produce viable offspring when mated with both infected and uninfected males. Bidirectional CI can also occur, in which Wolbachia-infected females are incompatible with males infected with a different Wolbachia strain. At present little is known about the molecular mechanisms of CI, and this represents a critical roadblock in our understanding of Wolbachia biology. Identification of CI genes would also be beneficial for disease control applications using Wolbachia. Some Wolbachia strains have been found to block or reduce transmission of human viruses [Plasmodium[Wolbachia for population replacement has shown considerable promise in field trials [ species  and can  species . The mos species . In the  viruses –7 and paasmodium, 8–11; ud trials .Wolbachia, comparing closely related incompatible strains with each other, or comparing CI-inducing strains with related strains that do not induce CI. In D. simulans the Wolbachia variant wAu expresses neither sperm modification in males, nor rescue of CI in females [wAu is ‘mod- resc-’, while CI-inducing strains are designated mod + resc + [wAu strain has, however, been shown to provide its Drosophila host with a degree of protection against the effects of pathogenic viruses [wMel strain, from D. melanogaster, is most closely related to wAu and does induce CI; thus genes differing between these strains are candidates for involvement in CI [wMel has already been reported [wAu genome sequencing in order to enable a comparative analysis.One approach to identifying genes involved in CI is genomic analysis of  females –17; in o+ resc + . The wAu viruses . The wMent in CI . The genreported , so hereWolbachia strains have been published [Wolbachia genomic DNA (gDNA). Wolbachia are obligate endosymbionts that cannot be cultured outside of their hosts, and are often present in relatively low abundance. Obtaining enough gDNA has thus required time-consuming amplification and purification protocols to minimize contamination with host gDNA. In addition, assembly has been complicated by numerous repeated sequences. Here we utilised the Pacific Biosciences (PacBio) RS II platform for sequencing; the long reads generated by this technology facilitate assembly through genomic repeats.Although the genomes of several ublished –26, acquWolbachia wPip sub-strains infecting Culex pipiens mosquitoes [wtrM identified in wPip from Cx. molestus (wPipMol) but absent in wPip from Cx. quinquefasciatus Pel and JHB (wPipPel and wPipJHB), which are bidirectionally incompatible with Cx. molestus[Cx. quinquefasciatus females with wtrM resulted in significant upregulation of CPIJ005623, a host gene implicated in CI based on knockdown studies [wMel . A specific comparison of these transcriptional regulator genes in wMel and their homologs in wAu was therefore conducted to further investigate the hypothesis that disruptions to these genes could be responsible for the different CI phenotypes of these strains.Previous comparative analysis of the genomes of mutual incompatibility-generating squitoes , 22 revewAu and host genes were performed to estimate the degree of contamination with host gDNA in wAu gDNA samples extracted from cultured cells and whole adult flies. The estimated purity of wAu gDNA was ~60% for the extract from cultured cells, and >90% for the extract from whole adult flies. The latter is comparable to the figure of up to 97% reported previously [Wolbachia gDNA extraction from cultured cells. One explanation for the lower purity could be that Wolbachia densities may be lower within cultured cells than in vivo.Approximate calculations based on quantitative PCR (qPCR) C(t) values for eviously  using thwAu genome sequencing was initially performed using the Illumina platform on gDNA extracted from whole adult files. However, the resulting assembly was fragmented in the regions of most interest, with scaffold positions uncertain. A second round of sequencing was therefore performed using the PacBio RS II system to obtain longer reads in an attempt to improve the assembly, using gDNA extracted from cultured cells rather whole adult files. The Illumina data was used to correct errors in the PacBio reads, which assembled into a single contig.Wolbachia, that cannot be cultured outside of host cells. The sequence generated was largely consistent with data produced using the Illumina platform, with only one single nucleotide polymorphism (SNP) between the two datasets. There were 88 indels relative to Illumina data; these were mostly single nucleotide insertions in the PacBio sequence, and were located in homopolymeric tracts, regions that are known to be prone to insertion errors in PacBio sequencing [The achievement of a single contig assembly shows that PacBio represents an extremely useful new sequencing platform for rapid generation of finished bacterial genome assemblies. Furthermore, the generation of this single contig from a very small amount of DNA (approximately 2 ng), containing a substantial amount of host DNA contamination (~40%), suggests that PacBio is well suited to use in cases where it is hard to obtain a large amount of gDNA, including obligate endosymbionts, like quencing , 29. Thequencing , 31.Wolbachia genomes have been sequenced using gDNA extracted directly from their native hosts. It is a time-consuming and often laborious process to rear sufficient numbers of the host insects for Wolbachia gDNA extraction, particularly for species with demanding rearing requirements. Transinfection of cells with Wolbachia is fairly easy to achieve, and amplification of cells to a suitable number is easier, quicker, and requires less space than whole organisms; this study used 24 flasks of cells, which were generated from a single flask in a few weeks. Concerns that the sequence of the wAu from cultured cells might have accumulated differences compared to the wAu genome found in flies, due to a relaxation in cell lines of the selective pressures that apply in its native host, were alleviated by the observation of only one SNP between the sequence obtained using wAu from cultured cells and that using wAu from its native host. It is possible that after a longer period of time more differences from Wolbachia in vivo would accumulate, so use of recently generated Wolbachia-infected cell lines, as employed here, is advisable.The use of cultured insect cell lines to obtain gDNA for genome sequencing represents a methodological departure from previous studies. All previous wAu genome is a single circular chromosome of 1,268,461 bp. It has 1266 predicted genes, corresponding to a coding content of 84%. The major features of the genome, along with those of the wMel genome, are shown in Table wAu and wMel genomes are similar, but with a significant amount of rearrangement  elements. For example, 27 putative IS5 elements were identified, although most are likely to be inactive due to mutations or frameshifts. Some elements appear to have been active since the divergence of wMel and wAu, as their locations in the genome differ between the two strains. In some cases their movement has resulted in disruption of genes in one strain. In many cases where there are structural differences between the genomes of the strains, it seems that mobile elements have provided a mechanism for the rearrangement.Like wAu genome, as for wMel, although the location and structure of these regions differs between the two strains  is inverted in wAu relative to wMel, and is further from the origin of replication (spanning WPWAU0631 – WPWAU0666). Several genes in this region differ in one strain relative to the other beyond SNPs , frameshift, nonsense mutation, or start codon mutation, two are disrupted in wMel relative to wAu, and two contain small in frame indels.There are three prophage regions in the  Figures , 4 and 5 Figures  in wMel wMel (spanning WD0582 – WD0644) is closer to the origin of replication in wAu (spanning WPWAU0282 – WPWAU0318). This region contains two segments, one closely related to P2 phage and the other to lambdoid phage [wAu the P2-like segment is inverted relative to its orientation in wMel. In addition, in wMel there are intervening genes between the lambdoid-like block and P2-like block, whereas in wAu the two blocks are contiguous. These differences mean that unlike wMel WO-B, the gene order of wAu WO-B is highly conserved with that of WO phage in wKue [wAu relative to wMel, due to truncation or frameshift; another five are disrupted in wMel relative to wAu, due to frameshift, IS element insertion or start codon mutation. Several of the genes between the lambdoid-like and P2-like blocks in wMel, which include three of the transcriptional regulator genes discussed below, also differ significantly between wMel and wAu  are absent in wAu, discussed further below.The region designated WO-B Figure  in wMel id phage . In wAu  in wKue , from wh in wKue . As in WWD0565 – WD0574 in wMel . One gene is disrupted in wAu relative to wMel, by a nonsense mutation, and four genes are disrupted in wMel relative to wAu, by frameshift, nonsense mutation or truncation. In addition, there is an insertion after the first gene in wAu relative to wMel, in which two additional genes are annotated. Overall the level of difference between wMel and wAu in these three prophage regions is much higher than elsewhere in the genome.The third prophage region, a small pyocin-like element comprising wMel genes were also identified with large differences potentially causing inactivation in wAu, or which are absent from wAu entirely, as listed in Table WD0506 to WD0518 is absent in wAu; no other indel of comparable size to the WD0506 to WD0518 segment was found to be absent in the wAu genome. However, of note was the absence in wAu of two genes, WD0631 and WD0632, that have recently been identified as CI candidates due to the detection in Cx. pipiens spermathecae of a protein corresponding to the WD0631 ortholog in the wPip strain [WD0631 and WD0632 orthologs are transcribed as an operon [wAu relative to wMel, by truncation, frameshift, mobile element insertion, nonsense mutation or start codon mutation, as shown in Table Various other p strain ; the WD0n operon . VariouswPip Wolbachia from Cx. pipiens mosquitoes [wtrM in CI, a comparison of the family of transcriptional regulator genes between wAu and wMel constituted a focus of this study. These transcriptional regulator genes were found to differ in both organisation in the genome and sequence. Figure wAu and wMel transcriptional regulator genes; also shown is a comparison between wMel and wRi, a CI-inducing strain found in D. simulans. Figure wMel transcriptional regulator genes and their corresponding sequences in wAu. Of particular note is that the ortholog of WD0622 is highly disrupted by a frameshift in wAu. The two identical sequences corresponding to WD0622 (WPWAU0256 and WPWAU0687) have a 1 bp insertion in wAu relative to wMel, after bp 212, which causes a frameshift that would lead to premature termination of translation after 85 amino acids. Genes corresponding to the last 82 amino acids of WD0622 (WPWAU0257 and WPWAU0686) are also predicted since a substitution has produced a new start codon, although these may be mis-predictions, as numerous SNPs and deletions have accumulated relative to WD0622. None of these genes include the DNA binding domains present in WD0622, suggesting that even if they are functional their activity is likely to differ significantly from that of their counterpart in wMel. Furthermore, there are IS5 elements inserted immediately downstream of the wAu genes corresponding to WD0622, so it may be that the regulation of the genes differs between the two strains. Genes such as this, which are conserved between multiple CI-inducing strains (WD0622 has two orthologs in the CI-inducing wRi strain) but disrupted in a non-CI strain, are prime candidates for involvement in CI.Following a prior comparative genomic study of sub-strains of squitoes , which iwMel genome, WD0254, WD0255, WD0622, WD0623 and WD0626 are in two regions that appear to be paralogous; WD0254 is a truncated paralog of WD0622 (with the truncation appearing to be due to a transposase insertion) and WD0255 a paralog of WD0623. In the wAu genome, there also appear to be two genome segments containing paralogous sequences at approximately the same genomic positions as in wMel. However, the segment at a similar position to WD0254 and WD0255 is inverted relative to its orientation in wMel and is adjacent to genes that match WD0628-WD0630 in wMel, suggesting that one or more translocations have occurred, encompassing WD0622 to WD0630; flanking IS elements provide a putative mechanism for its translocation and inversion. Unlike in wMel, in which there are sequence differences between the paralogs in the two paralogous transcriptional regulator regions, in wAu WPWAU0687, WPWAU0686 and WPWAU0688 are identical to WPWAU0256, WPWAU0257 and WPWAU0255 respectively, while WPWAU0691 is identical to WPWAU0252 after the first 84 bp; this suggests that replacement by intragenomic recombination has occurred. WD0296 appears to have an ortholog in wAu, WPWAU0324, in a similar genomic position, while there is no ortholog of WD0508 present in wAu, consistent with a previous study that found that the region spanning WD0506 to WD0518 in wMel is absent from wAu [Wolbachia strains [In the from wAu . This ge strains .wAu and wMel  both have three small insertions in the putative DNA binding domains that result in an extra four amino acids and one amino acid substitution; there are 37 further amino acid substitutions, 23 of which are located in the putative DNA binding domains. There are substitutions involving proline residues; due to the unique conformational rigidity of the proline side chain, this could have a large impact on the secondary structure of any protein produced, and thus probably also its function. There are also SNPs and a 6 bp insertion in 5′ upstream regions where promoter elements have been shown to occur in prokaryotes, centred at −45 and −52 [wAu homolog of WD0626, WPWAU0252, two small insertions in the putative DNA binding domains would result in an extra three amino acids and one amino acid substitution. In addition there are 48 amino acid substitutions, 27 of which are located in the putative DNA binding domains. There are also SNPs and a 16 bp deletion in the 5′ upstream region spanning the −45 and −52 positions. Another factor that may affect expression is their different genomic location; in wMel, WD0622, WD0623 and WD0626 are located within a prophage region, whereas in wAu this is not the case. The precise impact of all these differences described on protein function is hard to predict, but given in particular the changes in DNA binding domains it seems highly likely that their activity will be affected.Analysis of the other transcriptional regulator gene sequences indicates that protein products from these genes would be different between l Figure , particuWD0623 and WD0626 relative to their wAu orthologs are at the extreme end of the distributions of these measures over the genome  for e Figure . Comparie Figure . A KruskWolbachia transformation methodology. Expression vectors have been used for transfection and shown to change transcription levels of a host cell cycle regulator, but their tissue distribution is uneven, limiting their use for examining whether a CI-like phenotype can be induced [Wolbachia genes and assess whether a CI phenotype can be induced, as has been performed for ankyrin repeat-encoding genes previously [Wolbachia genes are required to produce the CI phenotype the use of individual genes in this way may not clearly reveal their role. As more Wolbachia genomes are sequenced, further comparison of these genes between different strains will also be useful.To confirm whether any of these transcriptional regulator genes are involved in CI, functional studies are required, although these are currently difficult to undertake in the absence of an effective  induced . The beseviously , althougWolbachia gDNA for genome sequencing using an infected cell line has been successfully employed, and the PacBio RS II sequencing platform has proved a very useful tool for achieving a complete bacterial assembly, particularly when combined with Illumina sequencing. Using this approach, a single contig assembly has been generated for the genome of the wAu strain, which does not induce CI. Comparison of this genome to that of wMel, which does induce CI, revealed significant structural differences in the prophage regions and loss or potential inactivation of a number of genes. Transcriptional regulator genes in particular displayed considerable differences between wAu and wMel, both in terms of genomic location and sequence; of these the wMel gene WD0622 may be the most promising to examine as a CI candidate. Given that a transcriptional regulator gene has previously been implicated in CI, these represent important targets for further functional studies on the mechanism of CI. The wMel genome region containing the transcriptional regulator genes WD0622, WD0633, WD0626 and WD0627 is also of particular interest with respect to CI given the proximity of WD0631-2, absent in wAu, because a WD0631 ortholog protein was recently identified in mosquito spermathecae. The current study thus contributes to the important basic aim of gaining a better understanding of the molecular basis of CI.In this study, a methodology for conveniently extracting D. simulans flies infected with wAu from Coffs Harbour, Australia, were reared using standard techniques. Flies were maintained at 25°C with a 12:12 hour light: dark cycle, in plastic bottles containing 25 ml food, and transferred to fresh containers when necessary.Aedes albopictus Aα23 cells infected with wAu were maintained using standard cell culture techniques. Cells were maintained in an incubator at 28°C, in flasks containing 10 ml Schneider’s medium with 10% fetal bovine serum, penicillin and streptomycin, and passaged when required.et al. [2O, then further sterilised in 70% ethanol and rinsed again in filter-sterilised dH2O. The flies were then homogenised in cold SPG buffer  using a Polytron homogeniser . After homogenisation the sample was centrifuged at 3,200 g for 15 minutes. The supernatant was collected and the centrifugation repeated. The supernatant from the second centrifugation was sequentially filtered through 5 μm, 2.7 μm and 1.2 μm syringe filters. The filtrate was centrifuged at 18,000 g for 20 minutes to pellet Wolbachia, which were resuspended in cold SPG buffer. The suspension was then incubated with 600 ng of DNase I  at 37°C for 30 minutes and subsequently with 5 μl of RNase A  at 37°C for 15 minutes to remove host DNA and RNA contamination. Cells were then lysed by incubation with 200 μg of proteinase K  at 56°C. gDNA was purified using two phenol/chloroform/isoamyl alcohol extractions and one chloroform/isoamyl alcohol extraction.gDNA for genome sequencing was extracted using two different methods. To extract gDNA from whole flies, a slightly modified version of the protocol used by Iturbe-Ormaetxe et al.  was usedWolbachia were first purified from the cells. Cells were dislodged from flasks by pipetting and scraping, and lysed by vortexing with borosilicate beads. The lysate was centrifuged at 2,500 g for 10 minutes at 4°C, then filtered sequentially through 5 μm and 0.2 μm filters. Sucrose gradient centrifugation was performed at 18.500 g for 10–20 minutes at 4°C to pellet the Wolbachia. gDNA was purified using the method described by Livak [wAu-infected flies.To extract gDNA from cells, by Livak . To extrwAu gDNA was analysed for contamination with host gDNA using qPCR. Reactions were performed on five serial dilutions of the extracts, using primers specific for a wAu gene (wsp), a host nuclear gene  and a host mitochondrial sequence . The average relative C(t) values for each primer pair, corrected for differences in primer efficiencies, were calculated. Taking into account the different sizes of the genomes, these values were compared to give a ratio of the amount of wAu gDNA to host gDNA, and the figures in this ratio were converted into percentages.Extracted wAu gDNA extracted from whole files was sequenced using the Illumina HiSeq 2000 platform. A ~200-300 bp paired end library was constructed following the methods described by Quail et al. [wMel genome as a reference. The final assembly comprised 77 contigs, with a total length of 1,222,634 bp, an N50 of 29.5 kb and a mean coverage of 283×.l et al. , 41, usil et al. . The libl et al. , using twAu gDNA extracted from cells was sequenced using the PacBio RS II platform. A ~10 kbp library was constructed following standard protocols using a PacBio DNA Template Prep Kit. Three SMRT® cells from this library were sequenced, with a movie length of 2 hours. With filters set to exclude reads of quality <0.8, polymerase read length <500 bp and sub-read length <500 bp, 139 Mb of data was generated from 75,456 sub-reads , with a mean sub-read length of 1,847 bp. A de novo assembly was generated from these sub-reads using the Hierarchical Genome Assembly Process (HGAP) version 1.0 [sion 1.0 , with thErrors in the assembly were corrected using the data from the Illumina sequencing. The Illumina reads from the second sample were mapped to the assembly using SMALT , then thWolbachia sequences from RefSeq [The assembly was annotated using the Automated Annotation Pipeline at the Wellcome Trust Sanger Institute, with the software Prokka . Infernam RefSeq , using Cm RefSeq  to creatm RefSeq . Some anwMel and wAu genomes were compared using Artemis Comparison Tool (ACT) [wAu and wMel genomes was generated using Dotter [The ol (ACT) . Sequencol (ACT) . Predictol (ACT) . SNP anaol (ACT) , 59. Theol (ACT)  was usedg Dotter  with defwAu genome. Amplification was performed using standard PCR conditions. PCR products were run on an agarose gel to check their size, then purified using a Qiagen PCR purification kit and sequenced using GATC Biotech sequencing.The sequences of regions of interest were confirmed using PCR. Primers were designed to flank the regions of interest in the wAu genome sequence has been submitted to the EMBL/GenBank/DDBJ database with the accession number LK055284. The raw Illumina sequence reads have been submitted to the European Nucleotide Archive with the accession number ERS151014.The


We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IThe online version of this article (doi:10.1007/s13238-015-0241-6) contains supplementary material, which is available to authorized users. The IκB kinase β (IKKβ) is a key catalytic subunit of the IKK complex, involved in inflammatory responses. It is robustly activated by cytokines, bacterial and viral products and metabolic stresses. IKKβ activation leads to phosphorylation of inhibitor of κB (IκB), and subsequently, translocation of the nuclear factor κB (NF-κB) to nucleus. The nuclear NF-κB binds to the κB elements in gene promoters and enhancers to either activate or repress gene expression Perkins, . By reguIkkβ is knocked out in hepatocytes, the livers of the knockout mice have normal development, but exhibit elevated levels of reactive oxygen species (ROS). In addition, IKKβ-defective livers are susceptible to injuries by carcinogens, concanavalin A and bacterial infection  and -deficient  cells using the same strategy. Genes up-regulated in the IKKβ-competent cells were, as expected, enriched for pathways involved in immunity and inflammation, such as antigen processing and presentation, rheumatoid arthritis, and B cell receptor signaling pathway and allograft rejection, but intriguingly, genes down-regulated in the IKKβ-competent cells were enriched for focal adhesion, ECM-receptor interaction and, and the TGFβ signaling pathways .Infection of IkkβF/F/Ad-Cre cells lacked IKKβ, but surprisingly, they did not have detectable α-SMA expression immediately following Ad-Cre infection  labeling, was high in Ikkβ−/− but low in wild type cells . In addition, the expression of the oxidative stress-inducible biomarker gene Heme oxygenase 1 (Ho-1) was more abundant in Ikkβ−/− than wild type cells , encoding for a crucial redox scavenger. Correspondingly, compared to the IKKβ-competent, i.e. wild type and Ikkβ−/−/Ad-IKKβ, cells, the IKKβ-deficient Ikkβ−/−cells had decreased level of RNA pol II recruitment to the Sod2 promoter and reduced p65 bound at the gene enhancer  . As predicted by the hypothesis, BSO increased Tgfβ2 gene expression and promoter activity and NAC significantly attenuated them, and reduced SMAD activity and migration of the IKKβ-deficient cells , which could further augment ROS . Similarly, the HO-1 expression increased gradually after Ad-Cre infection of IkkβF/F cells, and by 180 days, it reached the levels similar to that in Ikkβ−/− cells and twice that in wild type or Ad-IKKβ-infected IkkβF/F/Ad-Cre cells . AP-1 is a stress responsive transcription factor; we tested its activation with a luciferase reporter bearing an AP-1 binding site and found that luciferase expression was induced by IKKβ ablation, but repressed by IKKβ expression and NAC treatment . Both AP-1 binding and H3K4me3 were potentiated by IKKβ ablation and BSO treatment, but reduced by IKKβ over-expression and NAC treatment  and -deficient (c-JunF/F/Ad-Cre) cells and showed that while c-Jun ablation did not affect HO-1 induction, it abolished Tgfβ2 induction under the oxidative stress conditions created by BSO treatment  in the ion Fig. D and 6E.IkkβF/F cells, but gradually increased following Ad-Cre infection; by 180 days after infection the activity reached approximately 50% of the level in Ikkβ−/−cells  (Cdkn1a), the extracellular matrix component Fibronectin (Fn1), and γH2AX, a histone modification associated with DNA double strand damage , Western blotting, measurement of ROS and GSH, and the RNA was extracted, labeled and hybridized to Affymetrix Mouse Genome 430 2.0 Arrays using standard protocol  from that of each sample in order to normalize ChIP assay  results. The ΔΔCT value was calculated by subtracting control ΔCT values from the corresponding experimental ΔCT values. The resulting values were converted to fold changes over control by raising 2 to the power of −ΔCT values.qRT-PCR was performed using a DNA Engine Opticon2 Real-Time PCR Detection System (MJ Research) and SYBR Green qPCR MasterMix (Applied Biosystems) and primers for the genes of interest as listed in Table S1. All experiments were performed at least in triplicates. The relative differences in qRT-PCR among samples were determined by the ΔCT value as described previously , using telomeric primers for the reference control gene (mouse 36B4 single copy gene). The telomere signal was normalized to the signal from the single-copy gene to generate a relative telomere to single copy gene (T/S) ratio indicative of relative telomere length. Equal amounts of DNA (300 pg) were used for each reaction with several repeats and average telomere length was calculated.Supplementary material 1 (PDF 772 kb)Below is the link to the electronic supplementary material.


Gene amplification demonstrated the presence of viral sequences in 82.4% of cases and we detected expression of T-Antigen in 64.6% of cases by immunohistochemistry. Further, we found that T-Antigen and β-catenin co-localized in the nuclei of tumor cells and we confirmed the physical binding between these two proteins in vitro. The nuclear presence of T-Antigen and β-catenin resulted in the significant enhancement of TCF-dependent promoter activity and activation of the β-catenin downstream targets, c-Myc and Cyclin D1. These observations provide further evidence for a role of JCV T-Antigen in the dysregulation of the Wnt signaling pathway and in the pathogenesis of colon cancer.During the last decade, mounting evidence has implicated the human neurotropic virus JC virus in the pathology of colon cancer. However, the mechanisms of JC virus-mediated oncogenesis are still not fully determined. One candidate to mediate these effects is the viral early transcriptional product T-Antigen, which has the ability to inactivate cell cycle regulatory proteins such as p53. In medulloblastomas, T-Antigen has been shown to bind the Wnt signaling pathway protein β-catenin; however, the effects of this interaction on downstream cell cycle regulatory proteins remain unknown. In light of these observations, we investigated the association of T-Antigen and nuclear β-catenin in colon cancer cases and the effects of this complex in the activation of the transcription and cell cycle regulators c-Myc and Cyclin D1  Polyomaviridiae family, which also includes SV-40, BK virus (BKV) and the newly discovered Merkel Cell Polyomavirus (MCPyV), found to be clonally integrated in a high percentage of Merkel cell carcinomas. JCV has been established as the causative agent of Progressive Multifocal Leukoencephalopathy (PML), a demyelinating disease affecting HIV patients Colorectal carcinoma (CRC) remains a serious health problem around the world despite advances in diagnosis and treatment. It ranks third in overall cancer incidence and is the second most common cause of cancer mortality in the United States where there are an estimated 140,000 new cases and 50,000 CRC-related deaths each year et al showed that viral particles isolated from sewage remained intact after treatment at a pH of 1 for 30 minutes, indicating that ingestion of contaminated water or food may be sufficient for JCV infection of the gastrointestinal tract JCV is a ubiquitous virus that infects a large proportion of the general population. According to sero-epidemiological studies, primary infection with JCV occurs in early childhood and over 70–90% of adult individuals are positive for anti-JCV antibodies et al showed that the JCV viral load was 10- to 50- fold higher in colon adenomas and adenocarcinomas compared to matched normal tissue et al recently demonstrated that CRC tissue has a significantly higher relative frequency of JCV genomic sequences than adjacent normal mucosa and distant normal mucosa The JCV genome is approximately 5.2 kb in size and can be divided into three regions: the early viral genomic region (EVGR), the late viral genomic region (LVGR), and the non-coding control region (NCCR), which contains the origin of replication, and is located between early and late genomic regions. The EVGR encodes a powerful oncogenic protein, large T-Antigen, while the LVGR encodes the viral capsid proteins VP1, VP2 and VP3, as well as the Agnoprotein, a small accessory product in vitro are well known In vivo T-Antigen causes a variety of Central Nervous System tumors when injected into the brain of rodents and non-human primates, ranging from medulloblastomas to glial tumors The transforming abilities of JCV T-Antigen β-catenin is often dysregulated in colon cancer In the current study, we measure the prevalence of T-Antigen genomic sequences and protein expression in human colon cancer tissues using a collection of well-characterized biopsy samples. Importantly, we demonstrate an association of T-Antigen and nuclear β-catenin in human colon cancer samples and elaborate on this association by elucidating the molecular effects of this interaction in the dysregulation of the Wnt signaling cascade and the activation of β-catenin targets using colon cancer cell lines.A total of 113 de-identified paraffin-embedded samples of colorectal carcinoma and pre-neoplastic polyps were collected from the pathology archives of the following institutions: LSUHSC-New Orleans (34 samples) and the Ochsner Clinic (79 samples). The tumors were were histologically graded and immunohistochemically characterized according to the WHO Classification of Tumors of the Gastrointestinal Tract.AGTCTTTAGGGTCTTCTACC; PEP2 sequence: AGGTGCCAACCTATGGAACA). For amplification of the Control Region (CR), we used the NCRR1 and NCRR2 primers, corresponding to nucleotides 4993–5004 and 258–279, respectively . Amplification was carried out on 250 ng of template DNA with the FailSafe PCR system (Epicentre) in a total volume of 25 µl. PCR was run at the following conditions: denaturation at 95°C for 10 min, followed by 30 cycles of denaturation at 95°C for 15 s, annealing at 62°C for 30 s, and extension at 72°C for 30 s. As a termination step, the extension time of the last cycle was increased to 7 min. Samples amplified in the absence of template DNA served as a negative control, whereas DNA extracted from T-Antigen positive BSB8 cells was used as a positive control for each procedure. Southern blot was performed by resolving 10 µl of each PCR reaction on a 2% agarose gel. The gel was then treated for 15 min each with 0.2 M HCl for depurination, 1.5 M NaCl/0.5 M NaOH for denaturation, and 1.5 M NaCl/0.5 M Tris-HCl (pH 7.4) for neutralization, followed by transfer of the amplified fragments from the gel to nylon membranes . The membranes were then pre-hybridized for 1 h in EasyHyb solution (Roche), followed by hybridization in the same solution containing 2 µg DIG-labeled oligonucleotide specific for JCV T-Antigen (nucleotides 4304–4405) overnight. Primers used for synthesis of DIG labeled probe are PEP INT 1 (TTGGGATCCTGTGTTTTCATC) and PEP INT 2 (AATGGGAATCCTGGTGGAAT). For the CR probe we utilized primers from nucleotides 62–81. Probes were synthesized using the DIG DNA Labeling and Detection Kit (Roche). Membranes were hybridized overnight, washed, and developed using the DIG Wash and Block Buffer Set following the manufacturer’s instructions (Roche).A dedicated microtome was used to section the paraffin blocks in order to avoid contamination. Furthermore, the block and blade holder were periodically autoclaved, and a new, disposable blade was used for each specimen. DNA was extracted from approximately 10 sections of 10 µm in thickness from each of the tissue samples by using the QIAamp Tissue Kit, according to the manufacturer’s instructions (Qiagen). PCR amplification of the T-Antigen gene was performed using PEP1 and PEP2 primers , which amplify sequences in the N-terminal region of JCV T-Antigen  at 97°C in a vacuum oven for 35 min. After a cooling period of 25 min, sections were rinsed with PBS, and endogenous peroxidase was quenched by incubating the slides in methanol/3% H2O2 for 30 min at room temperature. Sections were blocked in 2% horse serum and incubated overnight with primary antibodies at room temperature in a humidified chamber. Antibodies used to detect viral proteins included a mouse monoclonal antibody against SV40 large T-Antigen that cross-reacts with JCV T-Antigen  and a mouse monoclonal anti-β-catenin antibody . After rinsing the sections in PBS, the slides were incubated for 1 hour at room temperature with biotinylated anti-mouse secondary antibodies and then were rinsed in PBS. The tissue was subsequently incubated with avidin-biotin-peroxidase complexes for 1 hour at room temperature according to the manufacturer’s instructions (Vector Laboratories), and finally, the sections were developed with a 3,3′-diaminobenzidine substrate (Sigma), counterstained with Hematoxylin, and coverslipped with Permount (Fisher). To assess the fraction of immunolabeled cells in specimens from each patient case, the labeling index defined as the percentage of positive cells of the total number of tumor cells counted was determined.Paraffin-embedded tissue previously fixed in 10% buffered formalin was sectioned at 4-For immunofluorescence and double labeling of paraffin embedded sections, deparaffinization, antigen retrieval, endogenous peroxidase quenching and blocking were performed as described above. For immunofluorescent double labeling of HCT116 cells in culture, the cells were washed with PBS, fixed in cold acetone, and blocked in PBS containing 5% horse serum and 0.1% BSA for 2 h. Sections or cells were then incubated with mouse anti-T-Antigen antibody  and rabbit anti-β-catenin antibody  for 16 hours followed by washing in PBS and incubation in anti-mouse Rhodamine antibody and anti-rabbit Fluorescein antibody . Finally, sections were washed in PBS and mounted in aqueous mounting media with DAPI (Vector Laboratories), and visualized in a confocal microscope (Olympus FV1000).HCT116 cells were kindly provided by Dr. Hamid Boulares. Cells were transfected using Xtreme Gene-HP (Roche). The following constructs were used for luciferase assays: M72 Super 16x TOPflash reporter construct: Addgene plasmid 17165; M51 Super 8x FOPflash: Addgene plasmid 12457; c-Myc promoter: Addgene plasmid 16595; Cyclin D1 promoter: Addgene plasmid 32727. These reporter constructs were transfected alone or together with pcDNA-T-Antigen and/or pcDNA-β-catenin and assayed for luciferase activity using a Luciferase Assay System (Promega) at 24 hours post-transfection.Cytoplasmic and nuclear protein extracts from HCT116 cells were collected in using cytoplasmic lysis buffer and nuclear lysis buffer as previously described This study was performed in strict accordance to Louisiana State University’s Institutional Review Board guidelines and approval. All 113 paraffin-embedded human samples of colorectal carcinomas and pre-neoplastic polyps, which were collected from the pathology archives of LSUHSC New Orleans (34 samples) and the Ochsner Clinic (79 samples). The Louisiana State University Institutional Review Board (IRB) waived the need for written consent, as the samples are archival, de-identified and follow under the NIH guidelines for exemption as tissue that would be discarded and cannot be traced back to patients (IRB Protocol #8077).in situ and invasive carcinoma. We found expression of T-Antigen by immunohistochemistry in the nuclei of tumor cells in 73 of the samples (64.6%). Interestingly, T-Antigen was also present in the nuclei of epithelial cells from pre-neoplastic polyps, but completely absent in the normal colonic epithelium  and found that in the normal colon, where T-Antigen is negative, β-catenin is expressed in the cytoplasm; however, in polyps and neoplastic cells, that express T-Antigen, β-catenin is co-localizing with the JCV oncoprotein in the nuclear compartment. To confirm the presence of the T-Antigen gene, we performed PCR amplification followed by Southern blot using a highly specific and sensitive probe for JCV T-Antigen in a selected number of 34 colon cancer cases that contained high-quality DNA. A schematic representation of the JCV genome is shown, containing the location of primers for Southern blot amplification and probe synthesis . The regWe amplified genomic sequences from the T-Antigen coding region in 28 samples of the 34 samples (82.4%), 21 of which demonstrated T-Antigen protein expression by immunohistochemistry. In only six cases we found the presence of viral DNA but no protein expression, and none of the cases had T-Antigen expression in the absence of viral genomic sequences. We tested the amplified sequences with specific probes for BKV and SV40, resulting negative in all cases. High quality genomic DNA was confirmed in each sample by PCR for GAPDH. . FinallyNext, we performed PCR experiments with primers and probes specific for the regulatory region of JCV. We amplified viral sequences in 14 of the 34 cases. in vitro and to determine whether T-Antigen and β-catenin are present in the same cellular compartment in colon cancer cells, we performed dual labeling immunofluorescence for these two proteins in the HCT116 colon cancer cell line transiently transfected with T-Antigen. We found that cells expressing the oncoprotein show a strong nuclear expression of β-catenin  driving expression of the luciferase gene. HCT116 cells were transfected with TOPflash and either T-Antigen, β-catenin, or both T-Antigen and β-catenin. Luminescence was measured at 24 hours post-transfection. All groups were separately transfected with FOPflash to measure baseline luminescence. T-Antigen or β-catenin alone significantly activated TOPflash by 2-fold and 5-fold over empty vector control, respectively. Strikingly, cells expressing T-Antigen and exogenous β-catenin showed a 113-fold increase in TOPflash activity over baseline , p≤0.001To determine activation of specific β-catenin target genes that control cellular transcription and cell cycle regulation by T-Antigen by T-Antigen, we chose to study two known proto-oncogene targets of β-catenin mediated transcription, c-Myc and Cyclin D1. We co-transfected HCT116 cells with luciferase constructs containing either the c-Myc promoter or the Cyclin D1 promoter along with either T-Antigen, β-catenin or both, and performed promoter reporter activation assays. Our findings demonstrate that T-Antigen and β-catenin individually increased c-Myc , p≤0.01 in vitro are well recognized, despite the fact that its role in the development of human tumors was debated for some time. However, during the last decade, an increasing number of studies provide clear evidence for the role of JCV in brain and gastrointestinal tract tumorigenesis. More than 6 independent laboratories have detected JCV and studied the mechanisms behind its oncogenic role in colon cancer in vitro on the activation of the β-catenin target genes c-Myc and Cyclin D1.The human neurotropic virus JCV is the established opportunistic agent of the fatal demyelinating disease Progressive Multifocal Leukoencephalopathy, frequently seen in patients with AIDS in vitro and interact to enhance the activation of the β-catenin target genes c-Myc and Cyclin D1. This discovery suggests that T-Antigen mediates β-catenin translocation to the nucleus and transcription of downstream target genes.We show that T-Antigen and β-catenin co-localize in the nucleus of colon cancer cells et al showed that JCV infection causes chromosomal instability in vitroet al showed that JCV-positive colon cancer cases are more likely to metastasize to the liver; this effect was shown to be T-Antigen-dependent in vitroThere have been many different, but not mutually exclusive hypotheses about the downstream effects of the interaction between β-catenin and T-Antigen. Ricciardiello Increased β-catenin target gene activation by T-Antigen in the absence of Wnt activation may proceed by a number of different mechanisms. T-Antigen can block or sterically hinder the phosphorylation of β-catenin JCV and its oncoprotein T-Antigen have long been a focus of investigation to elucidate its transformative properties and to determine its role in the development of cancer. Many groups have demonstrated the presence of T-Antigen in tumors of the GI tract and CNS, with a few explanatory mechanisms of transformation. Here we provide evidence for an association between T-Antigen and β-catenin in colon cancer and an enhanced transcriptional activation of Wnt pathway target genes by β-catenin in the presence of T-Antigen. These data suggest that the mechanism underlying the proliferation of cells expressing JCV T-Antigen involves the activation of Wnt pathway target genes and therefore may provide important therapeutic or prognostic value.


HMAX is a well-known computational model of visual recognition in cortex consisting of just two computational operations – a “template match” and non-linear pooling – alternating in a feedforward hierarchy in which receptive fields exhibit increasing specificity and invariance . InteresTo test HMAX on word spotting, recorded speech samples from the TIMIT corpus  were firThese results are novel in that they provide support for the hypothesis that the simple computational framework implemented in HMAX – consisting of a feedforward hierarchy of only two alternating computational operations – may generalize beyond vision to support auditory recognition as well. It is possible that such a representation could give rise to stable neural encodings that are invariant to behaviorally irrelevant characteristics as seen in higher order visual and auditory cortices ,6. While


Consistently, we observed that subsequent increase in β-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the β-catenin-E-cadherin association at the plasma membrane was disrupted during acute cytokine exposure. Additionally, it was demonstrated that disrupting cell-cell interactions led to increased migration of LNCaP cells in real-time migration assay. Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of β-catenin by inhibiting Akt(S473) phosphorylation, therefore, partially rescued the disrupted β-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of β-catenin at the cell membrane as well as increased Akt(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression. Thus, the data indicate that the β-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered β-catenin signaling upon TNFα exposure, and relation to loss of function of the tumor suppressor NKX3.1 was examined in prostate cancer cells. We used an  Due to its high occurrence in the western world, prostate cancer constitutes a major health problem (S473) results in inhibitory phosphorylation of GSK3β(S9). The inhibition of GSK3β suppresses the phosphorylation of β-catenin at S33, ultimately resulting in its cytoplasmic accumulation. Eventually, a part of free β-catenin translocates to nucleus β-catenin is a dual-function protein that is an important component of the plasma membrane and plays a central role in cell-cell adhesion. Accordingly, solid tumors, including those of the prostate, frequently display considerable β-catenin accumulation (S552) by Akt kinase During inflammation, mutations in components of the Wnt/β-catenin signaling pathway or induced secretion of Wnt glycoproteins from activated macrophages may thus result in stabilization of β-catenin. Regardless of Wnt signaling, inflammation-mediated increase in the transcriptional activity of β-catenin may also be enhanced via phosphorylation of β-catenin(Y654) and (Y142) influence interactions with E-cadherin and α-catenin, respectively. Further, the loss of the interaction between β-catenin and E-cadherin enhances the transcriptional activity of β-catenin and promotes the epithelial-to-mesenchymal transition During carcinogenesis, increased transcriptional activity of β-catenin correlates with the loss of E-cadherin-mediated cell adhesion NKX3.1 is an androgen-regulated tumor suppressor in prostate cells In this study, using the prostate cancer cell line (LNCaP), the mechanism underlying inflammation-induced tumorigenesis, β-catenin localization change and E-cadherin association, and the role of NKX3.1 in these processes were investigated. First, a tightly controlled cytokine exposure that partially mimics prostatic inflammatory atrophy 2. To achieve macrophage differentiation and cytokine production, cells (8×105) were seeded into 75 cm2 culture flasks for 2 h prior to the treatments. Next, PMA was added at final concentrations of 2, 4, 8 and 16 nM for 16 h, and the adherent clusters were followed. The cells were washed twice, and 20 ml of fresh medium was added. After allowing the cells to rest for 3 h, lipopolysaccharide (LPS) was added at a final concentration of 10 ng/ml, and the cells were incubated for 24 h. Finally, the supernatant (conditioned medium - CM) was collected and filtered (using a 0.22 µm filter) for further use.The U937 monocyte cell line was cultured in RPMI 1640 medium including 10% FBS  at 37°C in the presence of 5% COBefore feeding the LNCaP cells with the collected CM, TNFα , interleukin-6 (IL6) and interleukin-1beta (IL1β)  levels were examined using an ELISA according to the manufacturer's instructions. Because exposure is a major component in our inflammation model, the time  and the dose  for the courses of CM treatments were optimized separately. Ultimately, TNFα was chosen as a measure of CM exposure.2. The acute inflammation treatments were performed with CM (500 pg/ml TNFα) at appropriate periods of 6 or 24 h. In chronic inflammation model, cells were treated with 62 or 125 pg/ml TNFα-containing CM for 4 weeks. TNFα concentrations were adjusted by diluting the CM using RPMI 1640 medium as described previously LNCaP cells were obtained from the American Type Culture Collection  and were propagated as recommended using RPMI 1640 medium supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C in the presence of 5% CONKX3.1 open reading frame was amplified (using the primers F: GGATCCATGCTCAGGGTTCCGGAGCCG and R: GAATTCGGTTGTCACCTGAGCTGGCATTA) and cloned into the pcDNA4/HisMax-TOPO vector  according to the manufacturer's instructions. The transfections were performed using the Fugene HD transfection reagent  for 24 h. Briefly; 4×105 cells were seeded into 6 cm plates. Next day, medium was changed to serum-free medium (SFM), and the transfection mix was prepared by adding 3 µl of Fugene HD into 100 µl of SFM. Then, the mix was incubated for 5 min at RT, 1 µg of DNA was added, and 15 min later, the transfection mix was added onto cells dropwise. After 6 h of incubation, pre-warmed RPMI medium including 2× FBS was added, and the cells were incubated for an additional 18 to 42 h.The (S33), p-Akt(S473), p-Akt(S308), p-GSK3β(S9), c-myc, cyclin D1, E-cadherin, NKX3.1 and poly-ubiquitin , p-β-catenin(S552), Matrix metalloproteinase 2 (MMP2)  and Glucose 6 phosphate dehydrogenase (GAPDH) . The NKX3.1 custom antibody was a gift from Dr. F. Saatcioglu (University of Oslo). The HRP-conjugated anti-mouse and anti-rabbit  and Alexa Fluor 488- and 594-conjugated  secondary antibodies were purchased and used as recommended.The following antibodies were purchased from manufacturers: β-catenin, p-β-cateninFor protein extraction, cells were grown in 6 cm culture dishes  and washed once with PBS prior to cell lysis. Cells were resuspended in 250 µl of modified RIPA buffer (10 mM Tris.Cl (pH: 8.0), 1% Triton X-100, 0.1% SDS, 0.1% Na deoxycholate, 1 mM EDTA, 1 mM EGTA, 140 mM NaCl) containing protease and phosphatase inhibitors. Cells were collected from culture plates using a cell scraper and transferred to eppendorf tubes. Lysates were sonicated for 20 seconds , centrifuged at 12000 g for 10 minutes, and cleared supernatants were collected into new tubes. Protein concentrations were determined using BCA assay . SDS-PAGE and western blots were performed under standard conditions using 50 µg of protein lysate per lane; proteins were separated on a 10–12% gel and transferred to PVDF membrane  using a wet transfer blotter. The PVDF membrane was blocked with 5% dry milk in TBS-T . Primary and secondary antibody incubations were performed using TBS-T containing 0.5% dry milk or 5% BSA at RT for 1 h or at 4°C for o/n. Membranes were developed using ECL plus reagent  for 5 min and were photographed using Kodak X-Ray films in a dark room.7) were washed with PBS and pelleted for 5 min at 300 g. The cell pellet was resuspended in 500 µl buffer A  and cell lysis was performed by sonication on ice . The suspension was centrifuged at 800 g for 15 min and the pellet A was saved to isolate nuclei. The supernatant A was centrifuged again at 1000 g for 15 min. The supernatant B was saved to isolate the cytosolic proteins. The pellet A, saved for isolation of nuclei, was dissolved in 500 µl buffer A, and centrifuged at 1000 g for 15 min. The supernatant C was added to the supernatant B for isolating cytosolic proteins and stored on ice until then. The pellet C was resuspended in 500 µl buffer B1  and layered onto a 1.5 ml cushion of buffer B2 . Then, centrifugation at 2100 g for 1 h was done. The pellet D was taken up in 250 µl buffer D  and incubated for 1 h by rotating at 30 rpm. The suspension was sonicated on ice again  and centrifuged at 9000 g for 30 min. nuclear protein lysate was collected as supernatant. Furthermore, the pooled supernatants B and C were centrifuged for 150 min at 16000 g to isolate cytosolic protein lysate from supernatant. The pellet was dissolved in 250 µl buffer C , incubated 1 h by rotating at 30 rpm and centrifuged at 9000 g for 30 min, to isolate membrane components from supernatant. All incubations and centrifugations were carried out at 4°C, buffers were supplemented with 1× protease inhibitor cocktail  and 1 mM DTT directly before use. Fifty µg of each fraction was subjected to SDS-PAGE.LNCaP cells  were added and incubated in a humidified chamber for 1 h, and cells were washed twice with PBS. Secondary antibody incubations were performed at RT for 20 min using Alexafluor 488 (anti-rabbit) and/or Alexafluor 594 (anti-mouse) antibodies. Finally, cells were washed twice with PBS and mounted on coverslips with 30% glycerol in PBS including 0.5 µg/ml DAPI, and analyzed immediately using Leica DM4000B LED fluorescent microscope . Images were captured using Leica imaging software.3) were untransfected or transfected with the HM control vector and HM-NKX3.1 (24 h), seeded into 96-well plates  and grown for 24 h. CM treatments were performed, and the growth rate and morphological changes were followed. The data  were collected every 10 min for an additional 48 h. For the migration assay, 8×103 cells were seeded into 16-well plates . FBS was used as a chemo-attractant, and the migration rate was followed. The data were collected every 10 min for an additional 136 h.The Xcelligence migration and proliferation assay system was used for real time measurements. Briefly, the LNCaP cells (8×105) cells were seeded and grown in 6-well plates for different time points . At the end of treatments, WST formazan dye assay was performed for cells grown in cultures.LNCaP (2×10The protein lysate (500 µg) was subjected to immunoprecipitation using 2 µg of the β-catenin antibody at 4°C overnight. After the incubation, the antigen-antibody complexes were collected using 20 µl of Protein A/G plus agarose beads  for 1 h at 4°C. After centrifugation at 4000×g for 5 min, the pellets were washed with 500 µl of modified RIPA buffer 5 times. The collected beads were resuspended in 20 µl of RIPA buffer, and half of each sample was used for the SDS-PAGE analysis.The tissue blocks containing the most representative areas from 14 radical prostatectomy specimens  were chosen based on H&E staining, and 5 µm sections were cut and mounted on poly-L-lysine-coated slides for immunohistochemical staining. A standard streptavidin-biotin immunoperoxidase method was used to stain the tissue sections with ß-catenin and NKX3.1 antibodies . Briefly, the tissue sections were treated in xylene, rehydrated in an alcohol series and immersed in distilled water. Endogenous peroxidase activity was blocked using a 0.3% solution of hydrogen peroxide in phosphate-buffered saline at room temperature (RT) for 10 min, and the sections were then rinsed with washing buffer . The primary antibodies were applied for 1 h at RT, the sections were washed, the streptavidin-labeled peroxidase-conjugated antibody  was added at RT for 10 min, and the sections were washed again. The peroxidase activity was visualized with 0.03% 3,3-diaminobenzidine tetrahydrochloride (DAB)  for 5 min. The sections were then washed in deionized water, counterstained with Mayer's hematoxylin and mounted.Dokuz Eylul University, Medical Faculty's institutional ethics committee approved this study . The same committee waived the need for consent, as the same samples were previously used in another study square. Mean intensity (nucleus/cytop.)  =  mean intensity (nucleus/cytop.) – mean background. Ratio  =  Mean intensity (nucleus)/Mean intensity (cytop.). The data are presented as the mean ± SEM . The differences in the mean values between the groups were analyzed by a two-tailed Student's t-test, and P<0.05 was considered statistically significant.The images were analyzed using image J software. Briefly, expression intensity measurements were performed individually for each nuclear and cytoplasmic area in pre-defined boundaries, which were selected in equal areaTo understand the role of the inflammatory microenvironment in prostate cells, optimized conditioned media (CM) including TNFα was added onto the LNCaP cells (S473) upstream of β-catenin. As this phosphorylation event subsequently inhibited the GSK3β kinase by increasing the serine 9 phosphorylation, eventually resulted in a decrease in β-catenin(S33) phosphorylation ((S552) phosphorylation, which is a direct target of Akt kinase.To investigate the consequences of these changes in morphology of LNCaP cells, the components of β-catenin signaling pathway was studied after CM treatment. Specifically, CM-treated cells for growth, altered cell morphology and increased β-catenin stability at time points up to 24 h treatments were examined. Additionally, the molecular changes leading to β-catenin accumulation and phospho-specific changes that control the stability and transcriptional activity of β-catenin were also examined. As a result, we observed a significant increase in pAktrylation . Since, rylation . Therefo(S552) was increased at cytoplasm at 3 h of CM treatment  phosphorylation and consistently restored GSK3β(S9) and β-catenin(S33) but not β-catenin(S552) phosphorylation. Subsequently, the β-catenin transcriptional targets c-myc, cyclin D1 and MMP2 were downregulated both in the control and in the 6 h CM-treated samples  phosphorylation increased following low dose but prolonged exposure to CM. As the p-β-catenin(S33) was decreased and the total β-catenin accumulated, target genes c-myc and cyclin D1 were significantly upregulated  of CM including TNFα for longer periods of time (4 weeks). Consistent with our findings in the acute model, Aktegulated . Thus, tTo study the migration of LNCaP cells real-time Boyden chamber based migration assay was used (Xcelligence system). In this assay, FBS was used as a chemo-attractant, and 10% normal serum (N10) was used for positive control. We then used 2% FBS (N2) in the upper chamber of the CIM plate. As a negative control, 2% FBS (N2) was placed into both chambers. When the LNCaP cells were treated with the CM (including 250 or 500 pg/ml TNFα for 3 h and split onto the CIM-plate) the migration ability of the cells were correlated with increasing CM doses significantly (p<0.001) . AdditioHuman radical prostatectomy specimens from 14 patients with prostatic inflammatory disease and prostate cancer were examined. The sample sizes were  for normal, proliferative inflammatory atrophy (PIA), H-PIN and cancer samples respectively. We examined these tissue sections for β-catenin and NKX3.1 expression using immunohistochemistry (IHC) staining. β-catenin was found uniformly expressed in the normal prostate epithelium with a remarkably low cytoplasmic expression with apparent membrane localization in the histological sections. However, the sections from different stages of prostate pathology exhibited a substantial increase  in cytoplasmic β-catenin level when compared to normal epithelium . As, theβ-catenin, one of the major components of cell-cell adhesion plays a crucial role in many aspects of cell function and development (S473), which consequently inhibits GSK3β kinase activity by increasing the phosphorylation from (S9) residue ((S33) phosphorylation implies that either proteosomal degradation is activated and β-catenin(S33) is depleted by ubiquitination dependent proteosomal machinery, or there can be an increase in the stabilizing phosphorylation of β-catenin(S552), which suppresses the S33 phosphorylation  phosphorylation and contributes to the disassembly of β-catenin from membrane, we studied the ectopic expression of androgen-regulated NKX3.1 with CM treatments. As the NKX3.1 is an intracellular Akt kinase regulator in prostate cell As adherent junction components, E-cadherin and β-catenin have been extensively studied in models of tumor invasion and development, these molecules are phosphorylated by many kinases such as CK2, Src, Abl, Fer, and Fyn, which subsequently affect the adherent association of the cell membrane in vivo study has indicated that some of the atrophic glands, which inherit PIN lesions, are in close proximity to adenocarcinomas, exhibit extensive E-cad expression and increased Akt(S308) priming phosphorylations (data not shown). Consistent with the previous report Moreover, the observations obtained from cell line studies suggested that the β-catenin localization increased substantially in cytoplasm after 6 h of CM treatment and this was confirmed in human prostate tissues from patients with prostatic inflammatory disease. Further, the As the effects of β-catenin on tumorigenesis, invasion and metastasis of prostate cells have been reported previously, increased translocation of β-catenin upon Wnt signaling has also been detected in 25–38% of castration-resistant metastatic prostate cancers Furthermore, the interaction between the adhesion and transcriptional activity of β-catenin has been intensely investigated in a recent study Figure S1This figure demonstrates the increased E-cad and p-Akt(S308) levels in human samples especially in PIA (red rectangles) and PIN (blue rectangles) regions. The block arrows indicate that the cells with increased p-Akt(S308) level in PIA regions, suggesting that the growth and the expression heterogeneity is higher in PIA glands than normal glands.(TIF)Click here for additional data file.


Wnt signaling and cadherin-mediated adhesion have been implicated in both processes of embryonic development and the progression of carcinomas. Recent experimental studies revealed that Wnt signaling and cadherin-mediated cell adhesion have close crosstalk with each other. A comprehensive model that investigates the dynamic balance of β-catenins in Wnt signaling and cell adhesion will improve our understanding to embryonic development and carcinomas. We constructed a network model to evaluate the dynamic interplay between adhesion and Wnt signaling. The network is decomposed into three interdependent modules: the cell adhesion, the degradation circle and the transcriptional regulation. In the cell adhesion module, we consider the effect of cadherin’s lateral clustering. We found adhesion negatively contributes to Wnt signaling through competition for cytoplasmic β-catenins. In the network of degradation circle, we incorporated features from various existing models. Our simulations reproduced the most recent experimental phenomena with semi-quantitative accuracy. Finally, in the transcriptional regulation module, we developed a function selection strategy to analyze the outcomes of genetic feedback loops in modulating the gene expression of Wnt targets. The specific cellular phenomena such as cadherin switch and Axin oscillation were archived and their biological insights were discussed. Our model provides the theoretical basis of how spatial organization regulates the dynamics of cellular signaling pathways. We suggest that cell adhesion affects Wnt signaling in both negative and positive ways. Cadherins can inhibit Wnt signaling not only in a way as a stoichiometric binding partner of β-catenins that sequesters them from signaling, but also in a way through their clustering to impacts the rate at which β-catenins are involved in the destruction loop. Additionally, cadherin clustering increases the phosphorylation rate of β-catenins and promotes its signaling in nucleus. The Wnt signaling pathway has crucial roles in the normal processes of embryonic development The gene expression of the Wnt signaling targets is regulated by the cytoplasmic distributions of β-catenins, a central component in the Wnt signaling network Recent progresses in experimental studies of Wnt signaling pathway begin to throw light on how the cytoplasmic pool of β-catenins is maintained and regulated, and how the destructive, signaling or adhesive pathway of β-catenin can interplay with each other. The study on endogenous destruction complex indicated that level of phosphorylated beta-catenin remains unchanged in ‘Wnt on’ scene Comparing with experimental studies, computational modeling and theoretical analysis possess of unique advantages. They allow one to test conditions that may currently be difficult to attain or even unapproachable in the laboratory. During the past decade, a variety of mathematical or computational models have been applied to study the Wnt signaling pathway at different levels Here we constructed a network model to reconsider the dynamics of Wnt signaling pathway by integrating the most updated experimental information. The network is decomposed into three interdependent modules: the cell adhesion, the degradation circle and the transcriptional regulation. In the degradation circle module, we applied the “Axin inactivation” model to describe the degradation pathway of β-catenins and the interactions between destruction complexes and Wnt receptors. In the cell adhesion module, we also consider the effect of cadherin’s lateral clustering, which leads to the formation of cellular junctions and modulates their cytoplasmic interactions with β-catenins. In the transcriptional regulation module, we developed a function selection strategy to analyze the outcomes of genetic feedback loops in regulating the gene expression of Wnt targets such as cadherin and Axin. In these modules, the spatial information of protein interactions and the effects of phosphorylation are taken into account. These new features allow us to investigate the interplay between cell adhesion and Wnt signaling in both molecule scale  and sub-cellular scale (like transcription feedback). We explored if cadherin clustering play other regulatory roles, in addition to simply compete β-catenins with Wnt signaling pathway. The effects of mutations in network components have also been estimated. Overall, this work will help us form a comprehensive understanding towards the molecular mechanism of Wnt signaling pathway.There are three modules in the network Fig. 1. The module of cadherin-mediated cell adhesion describes the dynamic interactions between cadherins and β-catenins during the formation of adherens junctions. The classical cadherins consist of five extracellular domains (EC1-EC5); a single trans-membrane segment; and a highly conserved cytoplasmic domain that contains binding sites for P120-catenin, β-catenin and α-catenin The concentration of Cad/Cat complexes on cell surfaces is modulated by three processes Fig. 2a.trans-interactions. The clustering of cadherins through the cis-interactions triggers the final formation of adherens junctions trans” Cad/Cat complexes that form trans-dimers with cadherins on the neighboring cells; and “cis” Cad/Cat complexes that form cis clusters on cell surfaces. A “free” Cad/Cat complex can transit to “trans” state  formation of destruction complex and retention of β-catenins by APC and Axin; (2) destruction complex cycle; (3) Wnt stimulation and (4) Wnt signal transduction. They are labeled with numbers in After the formation of destruction complex, we extended the simplified destruction complex cycle in Lee’s model based on recent new experimental evidences. The new destruction complex cycle consists of two parts: destruction complex mediated phosphorylation and degradation of β-catenin after phosphorylation. The phosphorylation of β-catenin has been found to have sequential steps: kinase CK1 first phosphorylates β-catenins at residue S45  could trigger Wnt target gene expressions The module of gene expression describes the transcriptional regulations of Wnt target genes. After an active β-catenin binds to Bcl9, the dimer  increases. The amount of ABCs further decreases when cis-clstering is taken into account, represented by the value of Y/X that increases from 0 to 10. In another word, after forming adherens junctions, more ABCs are recruited to cell surfaces and Wnt signaling pathway is weaken. Therefore, our model illustrates that cell adhesion negatively contributes to Wnt signaling through competition for cytoplasmic ABCs. More importantly, our simulations gave a quantitative estimation of how the amount of ABCs in cytoplasm is related to the strength of trans and cis interactions.In details, multiple values of X and Y/X were chosen to investigate the effects of competition within a wide range of adhesive conditions. The ratios of 1:X:Y are ranged from 1∶0∶0 to 1∶10∶100. For each ratio, the average amount of free ABCs left in cytoplasm after 0.5 hr of Wnt treatment was calculated from 50 simulations trajectories. The simulation results for different ratios of X and Y are plotted as a two-dimensional profile Fig. 6. Competition for β-catenins is not the only connection between cell adhesion and Wnt signaling. Maher et al demonstrated that cadherin-based cell adhesion can enhance the kinetics of β-catenin degradation in membrane-associated destruction complexes 1 (α1>1) is a coefficient to describe the increase of rates, including the acceleration of protein-protein association and phosphorylation, while α2 (α2<1) is a coefficient to describe the decrease of rates, including the deceleration of protein-protein dissociation and structural transformation of destruction complexes in crowding environments. Assuming that corresponding proteins and complexes are uniformly distributed on cell surfaces, the average effects caused by adhesion can be derived by calculating the proportion of Cad/Cat complexes under “cis” state (Pcis), where 0 in the original rate in “non-interface” regions. Similarly, the average rates of reactions that become slower are We hypothesized that membrane surfaces are divided into “interface” and “non-interface” regions. Cellular junctions are formed at “interface” regions. Therefore, only the reactions at “interface” regions are changed due to spatial organizations. In additional to the slower structural transformation of destruction complex as explained in the method, it was found that protein association is accelerated and dissociation is decelerated in a crowded environment trans-dimers due to the diffusion trap mechanism. At cellular interface, majority of trans-dimers further form cis-clusters. As a result, the ratio X and Y were set to be 10 and 100, respectively. This gives. We first investigated the adhesion-induced changes of β-catenin distributions when there is no Wnt stimulation. Maher et al have found that without Wnt stimulations, the cytoplasmic level of ABC decreases under strong adhesive condition, but the level of GSK3-phosphorylated β-catenins increases. We assume that majority of cadherins form ytoplasm Fig. 7b We further explored how adhesion changes β-catenin distributions under Wnt stimulations. The values α2 = 0.4 and α1 = 1.5 were chosen to quantitatively reflect the experimental data in Maher’s study. In order to minimize the complexity, we decomposed the multiple factors caused by adhesion and added them step by step. Firstly, the red squares in Cell adhesion also affects Wnt signaling by regulating the phosphorylation rate of β-catenins. Brembeck et al have found that the functional switch of β-catenin between adhesion and Wnt signaling is modulated by its phosphorylation status at residue Y142. The kinases related to this phosphorylation are membrane associated receptors which locations are close to cellular junctions. Therefore, we assume that cell adhesion facilitates the phosphorylation of β-catenin at Y142 and the phosphorylation accelerates the association kinetics between ABC and Bcl9. Consequently, the simulation results show that although the acceleration of Y142 phosphorylation does not change the kinetic response of ABC Fig. 8c.In summary, our computational studies are consistent with various recently experimental data about interplay between cell adhesion and Wnt signaling. In additional to the simple competition for β-catenins, we provide the theoretical basis of how spatial organization of cell adhesion actively regulates the degradation pathway and phosphorylation rate of β-catenins. We suggest that slow releasing of ABCs from destruction complexes is the major factor cause by cell adhesion, which gives potential insights to modulate the Wnt signaling pathway under strong cell adhesion.Activation of Wnt target genes changes the transcription rates of proteins that participate in cell adhesion and β-catenin degradation. This provides an additional interplay between cell adhesion and Wnt signaling pathway. For examples, after Wnt stimulations, the transcription rates of N-cadherin increase, while the transcription rates of E-cadherin decrease. Wnt target genes such as Snail and Slug have been identified to inhibit transcription of E-cadherins, while others such as Twist are able to activate transcription of N-cadherins. The expression of E-cadherins is also influenced by GSK3, because GSK3 was found to inhibit the activities of Snail. The transcriptional regulations of N- and E-cadherins lead to the process called “cadherin switch”, in which cells are switched from expression of E-cadherins to expression of N-cadherins. The cadherin switch is one of the most important hallmarks of EMT.The process of cadherin switch is numerically archived after adding the module of gene expression. As listed in It was found that transcription rates of Axin increase after Wnt activation. The transcriptional feedbacks result in the periodic oscillations of cellular concentrations of Axin under continuous Wnt stimulations Consequently, our simulation results have not only demonstrated the oscillation of Axin, but also demonstrated the oscillations in destruction complex cycle Fig. 9b.Overall, our computational model demonstrated the functional roles of transcriptional feedback in regulating the interplay between Wnt signaling and cadherin-mediated adhesion. Specific dynamic properties were archived, such as cadherin switch and Axin oscillation. The decayed oscillation in cadherin switch brings further insights to understand the mechanism of EMT.In this article we constructed a network model to evaluate the dynamic interplay between cell adhesion and Wnt signaling pathway. The model integrates different functional modules of cadherin clustering, β-catenin degradation circle and transcriptional regulation. Our calibrated model reproduced the most recent experimental phenomena with semi-quantitative accuracy. We have computationally illustrated that Wnt signaling and cadherin-mediated cell adhesion crosstalk with each other through multiple pathways. Except the simple competition for β-catenins between these two systems, the slow releasing of ABCs from membrane associated destruction complexes caused by spatial organization of cell adhesion plays an additional role to inhibit Wnt signaling. Cell adhesion also affects Wnt signaling by regulating the phosphorylation rate of β-catenins. Adding transcriptional feedback loops into the model, we have further revealed a decayed oscillation during cadherin switch. Finally, our results demonstrated the importance of spatial information in regulating the dynamics of cellular signaling pathways.trans” state to “cis” state. Although it was shown theoretically that receptor clustering is a phase transition from a diluted state to a condensed state, the details of many kinetic features, such as the cooperativity between cadherin’s trans and cis interactions, cannot be quantitatively taken into account by current model. More sophisticated spatial simulation techniques are expected to resolve this issue. The interactions, aggregations and organizations of proteins in specific cellular environments are well approached by particle-based simulation methods In order to incorporate the spatial information, our model divided the signaling network into multiple layers or compartments according to the subcellular locations of different network components, including membrane surface, cytoplasm, Golgi and nucleus. The same type of molecules will be denoted by different states and possess of different properties if they translocate from one layer to another. For instance, only the active β-catenin in cell nucleus can trigger the target gene expression. Similarly, the destruction complexes change the properties of phosphorylation after they bind to the Wnt receptors. However, the protein-protein interactions within each layer or compartment are still assumed to take place in a well-mixed, homogeneous medium according to our simulation design. This assumption is limited by the fact that proteins tend to be spatially organized into specific patterns at subcellular locations. The cadherin clustering in adherens junctions is one of the best examples. In our cell adhesion module, we have introduced a network model to partially compensate the heterogeneity of cadherin clustering and its interaction with β-catenins. In this adhesion network model, clustering is simplified by a transition process. Junctions form if a larger portion of cadherin-catenin complexes transit from “Several aspects of the Wnt pathway are still poorly understood. Firstly, the molecular mechanism from Wnt receptor activation to the engagement of intracellular partners remains elusive. The “initiation-amplification” model suggests that there is cooperation between LPR6 receptor phosphorylation and Axin recruitment It has been demonstrated that the concentrations of Wnt signaling proteins in different cell types is significantly different The Wnt signaling pathway is critical to induce the epithelial-mesenchymal transition, in which the cadherin-based cell adhesion is significantly repressed. Recent experimental studies revealed that Wnt signaling and cadherin-mediated cell adhesion interplay with each other through multiple pathways. The simulation results from our computational model gave supportive evidences that cell adhesion affects Wnt signaling in both negative and positive ways. Cadherins can inhibit Wnt signaling not only in a way as a stoichiometric binding partner of β-catenins that sequesters them from signaling, but also in a way through their clustering to impacts the rate at which β-catenins are involved in the destruction loop. In contrast, cadherin clustering also increases the phosphorylation rate of β-catenins and promotes its signaling in nucleus. Finally, adding transcriptional regulation into the model helps us to understand the mechanism of EMT.Document S1The full mathematical description of the model.(DOCX)Click here for additional data file.