Importantly, PR5-LL-CM01 showed higher anti-tumor efficacy than the commercial PRMT5 inhibitor, EPZ015666, in both PDAC and CRC. This study clearly highlights the significant potential of PRMT5 as a therapeutic target in PDAC and CRC, and establishes PR5-LL-CM01 as a promising basis for new drug development in the future.Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are notoriously challenging for treatment. Hyperactive nuclear factor κB (NF-κB) is a common culprit in both cancers. Previously, we discovered that protein arginine methyltransferase 5 (PRMT5) methylated and activated NF-κB. Here, we show that PRMT5 is highly expressed in PDAC and CRC. Overexpression of PRMT5 promoted cancer progression, while shRNA knockdown showed an opposite effect. Using an innovative AlphaLISA high throughput screen, we discovered a lead compound, PR5-LL-CM01, which exhibited robust tumor inhibition effects in both cancers. An  Gastrointestinal cancer refers to malignant conditions of the gastrointestinal tract (GI tract) and accessory organs of digestion. Among them, pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are two of the most challenging cancer types. For instance, PDAC has a very poor prognosis with a median survival between 6-8 months , 2 and ~Equally devastating, CRC is the second leading cause of death in men and women combined in the United States . It resuetc., IκB kinase phosphorylates IκBα, leading to its degradation, the release of the p65:p50 complex, and the activation of NF-κB target genes [An important common feature of both PDAC –8 and CRet genes . A numbeet genes , 20. Thuvia dimethylating arginine 30 of its p65 subunit [Recently, we became the first to discover that the protein arginine methyltransferase 5 (PRMT5) serves as a potent activator of NF-κB  subunit . The nat subunit –24.in silico modeling, we proposed that PR5-LL-CM01 inhibits the activity of PRMT5 by direct binding to the glutamate 444 (Glu444) residue in the active pocket of PRMT5 [In these studies we provide the first evidence that PRMT5 is highly overexpressed in PDAC and CRC. Overexpression of PRMT5 promoted cancer progression through increased activation of NF-κB, cell growth, anchorage-independent growth and cell migration in both PDAC and CRC. Importantly, we developed a PRMT5-specific AlphaLISA HTS technique (Amplified luminescent proximity homogeneous assay-linked immunosorbent assay high throughput screen) by which we identified a novel small-molecule inhibitor of PRMT5, PR5-LL-CM01. We showed that PR5-LL-CM01 is much more potent than the only commercial PRMT5 inhibitor, EPZ015666 . Treatmeof PRMT5 .Here, we define for the first time a significant role of PRMT5 as a tumor promoter in PDAC and CRC. By discovering our leading compound PR5-LL-CM01 with the AlphaLISA HTS technique, we proved that PRMT5 is a promising therapeutic target in PDAC and CRC. Of extreme importance and significance, PR5-LL-CM01 could serve as an important basis for new drug development in PDAC and CRC.In order to determine whether PRMT5 is a tumor promoter in PDAC and CRC, we first examined PRMT5 expression in both cancers by Western blotting and immunohistochemistry (IHC) assay. As shown in Figure Since PRMT5 is highly expressed in PDAC and CRC, we wondered whether it serves as a tumor promoter in these cancers. In order to test this possibility, we examined the effect of PRMT5 on various characteristics of cancer cells including cell proliferation, anchorage-independent growth, and cell migration. Stable cells with either PRMT5 overexpression or shRNA knockdown were established in PANC1 (PDAC) and HT29 (CRC) cells. Western blotting was conducted to confirm the expected PRMT5 expression in these cells Figure . Using tMoreover, we conducted an anchorage-independent growth assay in soft agar. Data suggested that PRMT5 overexpression led to a dramatic increase in both the colony size and number in PANC1 and HT29 cells Figure , while sAnother well-known property of cancer cells is the strong migration ability, which is critical for tumor invasion and metastases . As obseCollectively, the above data provided strong evidence regarding the overall tumor promoting ability of PRMT5 in both PDAC and CRC.® Reader, which was proportional to the amount of dimethylated H4R3. Therefore, for any small-molecule inhibitor used to inhibit the activity of PRMT5, a reduced signal emission would be detected.Based on the above observations, we hypothesize that inhibition of PRMT5 with small-molecule inhibitors has potential therapeutic implications in PDAC and CRC. In order to successfully perform the HTS screen, we adapted the AlphaLISA technique (PerkinElmer) into an assay that could precisely quantify PRMT5 methylation of its substrate in a 384-well HTS platform. Compared to the conventional methods such as time resolved fluorescence energy transfer (TR-FRET) assay, the AlphaLISA technology is proved to be more sensitive, reliable, and adaptable for performing large-scale reactions. Thus it proves to be a more robust assay to be used for screening as compared to other methods used in the past , 33. To The PRMT5 enzyme used in our screen was purified by conducting co-immunoprecipitation with anti-Flag beads in a 293-PRMT5-Flag cell line established by us  . As showWe previously found that PRMT5 activated NF-κB through methylation of its p65 subunit , therefoin vitro models. Recently, Chan-Penebre et al. identified a PRMT5 inhibitor, EPZ015666 and showed its high efficacy in inhibiting PRMT5 in mantle cell lymphoma disease models [50 at 2-4 μM in PDAC cells    Figure , 4C, and) Figure , 4C. HowRC cells . Therefo50 observed in their cancer cell line counterparts  and interleukin 8 (IL8) in 293 cells. We therefore wondered whether an upregulation of these genes is observed in PDAC and CRC cells. Quantitative PCR showed that upon stimulation with IL-1β, there was a substantial increase in the expression of TNFα and IL8 in PRMT5 overexpressing cells, while a dramatic reduction was observed upon shPRMT5 knockdown, in both PANC1 and CRC cells Figure . Treatmein vitro, we wonder whether PR5-LL-CM01 exhibits tumor inhibition effect in vivo. Either PANC1 or HT29 cells were subcutaneously xenografted into NOD scid gamma (NSG) mice and then treated with PR5-LL-CM01 three times per week at 20mg/kg till the day the mice were sacrificed. Both body weight and tumor size were monitored during this process. As shown in Figure Since we have shown that PR5-LL-CM01 had great anti-cancer efficacy in PDAC and CRC cells In order to visualize the binding of PR5-LL-CM01 with PRMT5, we employed structural modeling approach using the existing information of PRMT5 crystal structure . PR5-LL-As described in Figure Taken together, we hypothesize that PRMT5 overexpression can promote the cancer phenotype through PRMT5-mediated NF-κB activation, whereas using PR5-LL-CM01 to block this activity could impede PDAC and CRC progression Figure . TherefoThe White House Cancer Moonshot's mission is to double the rate of progress in cancer research and treatment. Both PDAC and CRC are among the leading cause of cancer-related deaths in the United States. Despite important advances in recent years, many patients continue to experience disease recurrence following primary therapy. Moreover, there is currently “no cure” for a significant number of patients presenting with metastasis upon diagnosis. An estimated $20 billion will be spent on PDAC and CRC care in the United States in 2016, reflecting the urgent medical need for the discovery of newer PDAC and CRC treatment options.Post-translational modifications (PTMs) regulate protein function in eukaryotes and have been shown to play important roles in a variety of cancers . Methylaet al. reported that EPZ015666 is very effective in inhibiting mantle cell lymphoma [NF-κB is a critical transcription factor that is hyperactivated in various cancers. Since it also plays a crucial role in normal cellular functioning, direct targeting of NF-κB has not proven to be a successful therapeutic approach . These olymphoma . We showAdditionally, structural modeling experiments showed that PR5-LL-CM01 and EPZ015666 could interact with PRMT5 through quite different mechanisms. Particularly, PR5-LL-CM01 interacts with Glu444 on PRMT5, a critical residue for the catalytic activity of PRMT5 . InteresOf note, it is possible that PRMT5-mediated pathways are complicated. Though many mechanisms of the tumor-promoting tendencies of PRMT5 are still unclear, the insights provided by this study are extremely valuable in understanding the workings of PRMT5. They could also possibly be applied to other cancers beyond PDAC and CRC. PRMT5 is upregulated in various types of cancers, including but not limited to pancreas, colon, breast, prostate, and lung cancers, as well as lymphoma and melanoma –44. Due in silico model with purified PRMT5 crystal for the structure analysis. We will also determine the direct binding of PR5-LL-CM01 and its derivatives to PRMT5 with isothermal calorimetry (ITC) experiments, a cutting-edge technique that is able to monitor direct binding of a ligand to a protein.In summary, we have identified the significant role of PRMT5 as a tumor promoter in PDAC and CRC, as well as highlighted its potential to be exploited as an important therapeutic target. Building upon this exciting work, our immediate future plan would be to generate derivatives of PR5-LL-CM01 with the ultimate goal of moving this discovery to clinical application. In terms of how PR5-LL-CM01 inhibits PRMT5, one line of research we would like to pursue is to further verify our Overall, the discovery of PR5-LL-CM01 opens a door to promising new PDAC and CRC therapies and gateways to better understanding the workings behind PDAC and CRC as a whole. In the long run, we will move toward clinical trials with the few best-characterized PR5-LL-CM01 derivatives with the ultimate goal of improving PDAC and CRC patient survivorship and treatment.2 and used between passages 2 to 3. Cell lines were authenticated using 9 Marker STR Profile.Pancreatic control (HPNE) and cancer cell lines  were a kind gift from Dr. Murray Korc (Indiana University School of Medicine). The normal colon cell line (FHC) and CRC  cell lines were purchased from the American Tissue Culture Collection. All pancreas-derived cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) , supplemented with 1% of penicillin/streptomycin, 10% fetal bovine serum (FBS). CRC cells were maintained in Roswell Park Memorial Institute Medium (RPMI 1640) , containing 1% penicillin/streptomycin and 10% FBS, while FHC cells were cultured under the same condition with further addition of 25mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005 mg/ml transferrin and 100 ng/ml hydrocortisone. All cell lines were cultured at 37°C under 5% COProtein samples were run on a 10% polyacrylamide gel and then transferred overnight to a polyvinylidene fluoride (PVDF) membrane (Fisher Scientific) at 4°C. Primary antibodies for anti-PRMT5 , anti-Flag , anti-dimethyl arginine motif (sdme-RG)  and β-actin  and their corresponding secondary antibodies were used. The enhanced chemiluminescent (ECL) detection method (PerkinElmer) was conducted to detect the protein signal.Pancreatic and colon cancer tissue microarrays with matched normal adjacent controls were acquired from US Biomax Inc. The tissue microarrays were blocked using protein-blocking solution (Dako Corp.) for 30 min. All subsequent staining steps were performed using the Dako FLEX SYSTEM and an automated Immunostainer. Incubations were carried out at room temperature and Tris buffered saline containing 0.05% Tween 20, pH 7.4 (Dako Corp.) was used for all the washes and diluents. Anti-PRMT5 primary antibody  was used to detect PRMT5 localization. Horseradish peroxidase-conjugated secondary antibody was then used, followed by addition of the chromogen, which formed a brown precipitate at the binding site of secondary antibody. Imaging was done using Aperio whole slide digital imaging system. The system imaged all slides at 20X magnification.The NF-κB luciferase construct p5XIP10 (containing five tandem copies of the NF-κB site from the IP10 gene)  was tran5 cells for each cell line were then mixed into top agar solution and layered on top of the bottom layer. The plates were incubated for 10-20 days at 37°C and 5% CO2. Images were captured using a Canon EOS Rebel T3i Digital SLR camera and quantification of colony size and number was performed using ImageJ.2.4% and 1.25% agar were used to prepare the bottom and top layers of the soft agar, respectively. The bottom agar was added to each well of a 6-well plate. 2×105 cells were suspended in serum-starved media in the upper chamber of the well. Corresponding serum rich media was added to the lower chamber. Migrated cells were fixed using 4% formaldehyde followed by crystal violet staining and counting using a microscope at 20X magnification. The images were captured using a Canon EOS Rebel T3i Digital SLR camera.Migration assay was conducted using Boyden chambers. 8μm pore size cell culture inserts (Corning) were placed in a 24-well plate. Each chamber was coated with gelatin on the side facing the lower chamber. 2×10® Reader.PRMT5 enzyme was purified with anti-Flag-M2 beads (Sigma-Aldrich) from 293 cells with the overexpression of Flag-PRMT5 protein, as described previously . The enz293 with stable overexpression of Flag-p65 protein, were treated with 20μM of PR5-LL-CM01 for 24 hrs. p65 was then pulled down with anti-Flag-M2 beads, using immunoprecipitation methods as described previously . The samCells were seeded at 90% confluence in 96-well plates and titrated with different dosages of PR5-LL-CM01 for 4 days. Media was then removed and 10μl of MTT (Sigma-Aldrich) was added per well. Cells were incubated for 2 hrs at 37°C before adding 100μl of DMSO to each well and quantified with the Synergy H1 Multi-Mode Reader (BioTek Instruments Inc).Cells were cultured to 90% confluence and total RNA was isolated using Trizol as described previously . cDNA waIn Vivo Therapeutics Core at Indiana University School of Medicine. After acclimation for 7 days, NSG mice (6-8 weeks old) were xenografted with Mycoplasma-free PANC1 or HT29 cells subcutaneously  (BD Biosciences). 5 mice were randomized in each group when tumor volumes reach ~100mm3. Mice were treated with either vehicle control or 20mg/kg of PR5-LL-CM01 (drug stock dissolved in 1:1 Cremophor:ethanol solution) intraperitoneally 3 times per week. Tumor volumes and body weights were measured twice a week. The study was performed in accordance with the guidelines and standards of the Institutional Animal Care and Use Committee (IACUC) and under the approved animal protocol # 11066 MD/R by Indiana University.Male NSG mice were obtained from the For docking, chain A of PRMT5 protein in 4×61.pdb from the Protein Databank was used. All docking experiments were performed by Glide program (version 6.8) of the Schrodinger suite 2015-3 in standard precision mode , 28. Thep value < 0.05 was considered statistically significant.Statistical analysis was performed using Prism 6 software . Results have been presented as mean ± SD or mean ± SEM, as specified. A two-tailed Student's t test was used while comparing two means to test for significant differences. All statistics were calculated on triplicate experiments and


ON state, Hippo signalling results in serine/threonine phosphorylation of Yes-associated protein (YAP1) and tafazzin (TAZ), promoting formation of the β-catenin destruction complex. In contrast, YAP1 or TAZ dephosphorylation (and YAP1 methylation) results in β-catenin destruction complex deactivation and β-catenin nuclear localization. In the Hippo OFF state, YAP1 and TAZ are engaged with the nuclear β-catenin and participate in the β-catenin-dependent transcription program. Interestingly, YAP1/TAZ are dispensable for intestinal homeostasis; however, upon Wnt pathway hyperactivation, the proteins together with TEA domain (TEAD) transcription factors drive the transcriptional program essential for intestinal cell transformation. In addition, in many CRC cells, YAP1 phosphorylation by YES proto-oncogene 1 tyrosine kinase (YES1) leads to the formation of a transcriptional complex that includes YAP1, β-catenin and T-box 5 (TBX5) DNA-binding protein. YAP1/β-catenin/T-box 5-mediated transcription is necessary for CRC cell proliferation and survival. Interestingly, dishevelled (DVL) appears to be an important mediator involved in both Wnt and Hippo (YAP1/TAZ) signalling and some of the DVL functions were assigned to the nuclear DVL pool. Wnt ligands can trigger alternative signalling that directly involves some of the Hippo pathway components such as YAP1, TAZ and TEADs. By upregulating Wnt pathway agonists, the alternative Wnt signalling can inhibit the canonical Wnt pathway activity.In this review, we address aspects of Wnt, R-Spondin (RSPO) and Hippo signalling, in both healthy and transformed intestinal epithelium. In intestinal stem cells (ISCs), the Wnt pathway is essential for intestinal crypt formation and renewal, whereas RSPO-mediated signalling mainly affects ISC numbers. In human colorectal cancer (CRC), aberrant Wnt signalling is the driving mechanism initiating this type of neoplasia. The signalling role of the RSPO-binding transmembrane proteins, the leucine-rich-repeat-containing G-protein-coupled receptors (LGRs), is possibly more pleiotropic and not only limited to the enhancement of Wnt signalling. There is growing evidence for multiple crosstalk between Hippo and Wnt/β-catenin signalling. In the  Ctnnb1 gene (the gene encoding β-catenin), TCF/β-catenin-dependent transcription is constitutively active [In this review, we describe the molecular mechanisms and possible crosstalk of the signalling pathways that directly influence intestinal homeostasis and tumorigenesis. The role of Wnt/β-catenin signalling in both processes was documented by several major discoveries published in the 1990s. In 1996, reports from three laboratories showed that β-catenin, the main mediator of canonical Wnt signalling, interacts with the T-cell factor (TCF)/lymphoid enhancer factor (LEF) and converts these DNA binding proteins to transcriptional activators ,3. In 19y active ,5,6. In y active . In agrey active ,9. In 20y active . These fy active ,12,13. Ay active . Moreovey active ,16. In sstemness and also determines stem cell numbers [The most distinct feature of stem cells is their self-renewal capacity and potency, i.e., the ability to differentiate into one or multiple types of differentiated cells. In contrast to embryonic stem cells that populate the early embryo and give rise to the entire organism, adult stem cells emerge during later developmental stages and their self-renewal and cell differentiation potential is limited to a particular organ or tissue . Virtual numbers .Intestinal epithelium—similarly to other tissues of the adult organism—is characterized by the presence of stem cells that ensure tissue homeostasis and regeneration upon tissue damage. With constituent cells renewing every 3–5 days, the intestinal epithelium represents the most dynamic cellular system in the mammalian body . In the + cells accompanied by their ability to differentiate into all cell lineages present in the intestinal epithelium. In the small intestine, these cell types include absorptive enterocytes, microfold (M) cells associated with the mucosal immune system and several populations of secretory cells. The latter are represented by mucus-producing goblet cells, various sorts of hormone-secreting enteroendocrine cells, tuft cells that secrete endogenous opioids but also contribute to immune type 2 responses against enteric parasite infections and Paneth cells characteristic by production of anti-bacterial enzymes and peptides [The idea that gene expression triggered by aberrant Wnt signalling in human intestinal cancer cells mirrors the Wnt-dependent transcription program in the crypt cells led to the discovery of several ISC-specific markers ,21. Althpeptides . Besidespeptides , SPARC-rpeptides  and Troypeptides . ISCs dipeptides . + CBC cells had been confirmed, a number of studies proposed that intestinal homeostasis is maintained by slowly dividing cells localized four cell positions from the bottom of the crypt [Before the stem cell identity of LGR5he crypt . The “+4he crypt . Howeverhe crypt ,30. Howehe crypt ,32. In the crypt .+ ISCs in culture conditions. Upon embedding in Matrigel, the crypts (or single ISCs) generate three-dimensional structures called organoids or miniguts that resemble the organization, self-renewal and differentiation of the intestinal epithelium. The organoid medium has to be supplemented with the Wnt pathway agonist RSPO, the bone morphogenetic protein (BMP) pathway inhibitor noggin and epidermal growth factor (EGF). Essential factors, additional to RSPO, noggin and EGF, are supposedly produced by ISCs neighbouring Paneth cells. The Paneth cell-derived factors include Wnt3, the Notch ligand delta like 4 (Dll4) and tumour necrosis factor (TNF) [The ISC niche in the small intestine is composed of the extracellular matrix (ECM) and many cell types that include connective tissues cells (fibroblasts and myofibroblasts), endothelial and immune cells, cells of neuronal origin and smooth muscle and Paneth cells . Interesor (TNF) ,35. Intror (TNF) . The orgor (TNF) ,38.Fzd5 and Fzd7 mRNAs are highly enriched in the crypts and their expression declines towards the villus tip [Fzd7 gene in LGR5+ cells leads to stem cells loss and impaired regeneration capacity after damage [Fzd7 expression levels were linked to progression of human intestinal cancer [Wnt signalling represents one of the key signalling mechanisms that regulates intestinal epithelium homeostasis. Nineteen mammalian secreted Wnt ligands have been identified, which contribute to a multitude of cellular processes such as proliferation, differentiation, cell-fate determination, migration and polarization . Upon trllus tip . FZD7 apr damage . Additiol cancer . The Wntl cancer . Upon Wnl cancer ). Beta-cl cancer ,50. Tcf7l2 gene, encoding the β-catenin partner and Wnt signalling transcriptional regulator, caused complete loss of the intestinal crypt compartments accompanied by the absence of proliferating cells in the small intestine of new-born Tcf7l2−/− mice [Apc orthologue [Apc using the Cre/loxP system in LGR5+ cells initiates neoplastic growth in epithelia of the small intestine and colon [Ctnnb1 gene (encodes β-catenin) that stabilize the protein or mutations inactivating the additional component of the β-catenin destruction complex AXIN2 [The contribution of Wnt/β-catenin signalling to intestinal self-renewal and tumorigenesis was established by gene targeting studies performed in the mouse and by analysis of human cancer cells. For example, disruption of the −/− mice . In cont−/− mice ,53,54,55−/− mice . Two yeathologue . Correspnd colon . Anotherex AXIN2 . Wnt3 knock-in allele, brought valuables insights into the Wnt ligand movement in the intestine. The ligand is distributed in a single-cell distance from its source (Paneth cell) to the neighbouring ISC. Then, the Wnt-FZD complex is passively distributed to daughter cells through mitosis. Subsequent cell divisions in the ISC and TA compartments results in dilution of surface-bound Wnt leading to the gradient formation [The Wnt/β-catenin pathway activity in the intestine generates a descending gradient along the crypt-villus axis that controls epithelial cell division, differentiation and migration. As already indicated, Paneth cells produce Wnt3, which stimulates the neighbouring cells; however, how the Wnt gradient is established and controlled remained unclear ,44,60. Hormation . 2+) signalling. In Wnt/PCP signalling, the Wnt signal is transduced by a transmembrane receptor complex that involves the FZD receptor and the alternative co-receptors receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2), receptor-like tyrosine kinase (RYK) and protein tyrosine kinase 7 (PTK7). Wnt/PCP signalling activates the small GTPases RHO/RAS-related C3 botulinum toxin substrate 1 (RAC1) and results in cytoskeleton reorganization affecting cellular polarity and migration [2+ signalling, the Wnt FZD interaction leads to activation of phospholipase C (PLC) through heterotrimeric G proteins. Active PLC hydrolyses phospholipids to inositol 1,4,5-trisphosphate (IP3) and 1,2-diacyl glycerol. These molecules function as “second messengers” that induce multiple cellular responses such as Ca2+ release from the ER and protein kinase C (PKC) stimulation [The less-well characterized non-canonical Wnt pathways include Wnt/planar cell polarity (Wnt/PCP) and Wnt/calcium , a thrombospondin type 1 repeat domain and a basic amino acid-rich repeat domain . Based oPorcn gene in the intestinal epithelium showed no pathological changes but organoids derived from these mice failed to grow. Additionally, organoid viability was rescued by co-culture with cells producing RSPO1 (and Wnt3a). These findings support the idea that stromal production of RSPOs and Wnts is sufficient to support intestinal homeostasis [+ cells. As expected, Wnt pathway inhibition (infection with Ad-FZD-CRD/Ad-Dkk1) caused crypt demise that was not rescued by RSPOs. Similarly, crypts were damaged upon RSPO signalling inhibition (upon infection with Ad-RNF43/ZNRF3-ECDs and Ad-LGR5-ECD); however, in this particular case the crypt formation was rescued by (simultaneous) Wnt signalling activation. In conclusion, these studies underlined the role of Wnt ligands in priming epithelial cell renewal via establishment of the ISC fate. RSPO signalling can induce LGR5+ cell amplification; however, the signalling is insufficient for formation (or maintenance) of ISCs when the Wnt signal is absent [In the intestine, RSPO1/2/3 mRNAs are predominantly produced in the stroma, whereas their expression in the epithelium is negligible . The expeostasis . Very reeostasis , stimulas absent ,80.Lgr5 knockout mice develop normally but die shortly after birth because of ankyloglossia, a craniofacial defect that prevents new-borns from suckling. Otherwise, LGR5-deficient animals do not display any (remarkable) phenotypic abnormalities [Lgr6, is expressed in the embryonic hair placode and in the area interconnecting adult hair follicles with sebaceous glands. The LGR6+ cells were identified as adult stem cells with the potential to generate all skin cell lineages. Remarkably, Lgr6 expression is Wnt signalling independent and Lgr6 knockouts do not manifest any apparent defects [Lgr4 gene is embryonic/perinatal lethal [Lgr4 had damaging effects on ISC proliferation. Deficiency in both Lgr4 and Lgr5 genes worsened the phenotype and led to crypt demise [Lgr4 gene expression is activated by BMP2 in osteoblastic cells [Lgr4 null mice, which display delayed osteoblast differentiation [Lgr4 gene that is linked to low bone mineral density [Lgr4 stimulates white-to-brown fat transition and promotes energy expenditure [LGR5 and related LGR4 and LGR6 proteins represent the B subgroup of LGR transmembrane proteins, related to the G-protein-coupled receptors (GPCRs) of the hormone receptor class. All LGRs contain a large N-terminal extracellular domain; however, in contrast to LGR4/5/6, the other members of the LGR A and C subgroups bind hormones ,82,83. Lmalities . The oth defects . In contl lethal ,93. In tic cells  or by siic cells . The latntiation . It alsontiation . Interesntiation . In contntiation . In agre density . Interes density . Complemenditure . Currentenditure . Finallyenditure . In summRspo2 gene knockout is associated with facial skeletal defects, limb loss and lung hyperplasia resulting in perinatal respiratory failure [Rspo4 gene targeting are not available yet; however, in humans, mutations affecting RSPO4 function are associated with anonychia, the absence of finger- and toenails [Similar to LGRs, RSPO functions do not (completely) overlap. Nevertheless, all RSPOs have the capacity to induce crypt cell proliferation and β-catenin activation . RSPO1-d failure . RSPO3 d failure ,107. Restoenails .Apc, Ctnnb1 or Axin2 genes, genetic rearrangements in the Rspo2 or Rpso3 locus were identified. The detected alterations generate fusions between Rspo2 and the eukaryotic translation initiation factor 3, subunit E (Eif3e) gene or between Rspo3 and the protein tyrosine phosphatase, receptor type, K (Ptprk) gene. Both fusions possibly lead to increased production of functional RSPO proteins resulting in aberrant Wnt signalling [+ tumour cells in relapse after therapy [Aberrant RSPO/LGR signalling is frequently associated with CRC. Interestingly, in a fraction of CRCs harbouring intact gnalling ,110. Fur therapy ,112. No  therapy . Neverth therapy .Drosophila melanogaster as an organ size regulatory system; however, its core components are evolutionally conserved [The Hippo signalling pathway was recently added to the list of signalling mechanisms involved in intestinal tumorigenesis. Importantly, several of the Hippo and Wnt pathway components are either shared between both pathways, or physically interact. The Hippo pathway was originally discovered in onserved ,115. Theonserved ,117. Upoonserved ,119 and onserved . The phoonserved .OFF state, YAP1 and TAZ mediators are translocated into the nucleus, where they bind members of the TEA domain transcription factor (TEAD) family [In the Hippo signalling ) family  and prom) family , SMAD [1) family  and AP-1) family . YAP1/TA) family . The abs) family . Yap1 or Taz gene abolished intestinal adenoma formation in APC-Min mice [Apc gene is completely suppressed by simultaneous inactivation of Yap1 and Taz [12/13), RHO GTPases, and LATS1/2 promoting YAP1/TAZ activation and TEAD-mediated transcription expression in the mouse induced intestinal hyperplasia accompanied by the absence of mature Paneth and goblet cells. The observed phenotype, which was reversible and strictly dependent on YAP1 production, was not caused by cell dedifferentiation. Instead, differentiated cells were replaced by newly arising YAP1-expressing progenitors . In addiMin mice . In cont and Taz ,13. Inte and Taz . A diffe and Taz . Interes and Taz . Besides and Taz  (Figure cription . In prim


Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of β-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these β-catenin co-factors may coordinate Wnt and nuclear hormone responses.Wnt/β-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing β-catenin via the Legless/BCL9 adaptor. We used CRISPR/Cas9 genome engineering of DOI:http://dx.doi.org/10.7554/eLife.20882.001 In every animal, different cells must be able to communicate with each other to make sure that the body is correctly formed and maintained. Animal cells have many ways of communicating, but one important and well-studied mechanism involves a signaling molecule called Wnt that is released by some cells and received by others. The Wnt molecule and its effects are similar in all animals, and over-active Wnt signaling in humans contributes to a number of diseases including various cancers.The Wnt signal is carried from the surface of the receiving cell to the DNA in its nucleus via a protein called β-catenin. The β-catenin protein then helps to switch on a large number of genes. However, to do this β-catenin must interact with an assembly of other proteins collectively called the Wnt enhanceosome. There are still many unknowns about how exactly β-catenin cooperates with the enhanceosome.Now, van Tienen et al. investigated one component of the Wnt/β-catenin pathway called BCL9/B9L: a large protein that contains a number of flexible regions. First, a biochemical technique called BioID was used with human embryonic kidney cells to determine the proteins that BCL9/B9L encounters during a 12-hour period. This technique can detect when two proteins come close together, even if the interaction is weak or does not last very long.The BioID experiments showed that BCL9/B9L is close to two proteins in the Wnt enhanceosome in addition to β-catenin, and other techniques were used to confirm that one of these proteins contacts BCL9/B9L directly. The experiments also showed that BCL9/B9L acted as a tether to bring β-catenin close to the protein within the enhanceosome that binds to the DNA. Importantly, BCL9/B9L interacted with the enhanceosome both in the presence and absence of Wnt, indicating that the assembly is ready to switch genes on as soon as β-catenin reaches the DNA.Next, van Tienen et al. confirmed that the parts of BCL9/B9L that bind to the enhanceosome are important for its activity by using a gene-editing technology called CRISPR/Cas9 to essentially delete them both in the human cells and in fruit flies.Unexpectedly, the BioID experiments also revealed that BCL9/B9L binds to proteins that transmit signals from molecules other than Wnt, in particular from hormones such as estrogen and androgen. Future experiments could explore if, and how, BCL9/B9L integrates these signals from hormones with the signal from Wnt. A better understanding of this process might have important implications for the treatment of certain cancers, such as breast and prostate cancers that can be driven by over-active hormone signals.DOI:http://dx.doi.org/10.7554/eLife.20882.002 Drosophila), which is rapidly degraded in unstimulated cells. Absence of β-catenin thus defines the OFF state of these enhancers, which are silenced by Groucho/TLE co-repressors bound to TCF via their Q domain. This domain tetramerizes to promote transcriptional repression  histone acetyltransferase or its p300 paralog , resultiDrosophila where Pygo was discovered as an essential co-factor for activated Armadillo, its main function appears to be to assist Armadillo in overcoming Groucho-dependent repression , owing to a stop codon downstream of HD2  bind to Pygo and Armadillo/β-catenin via their conserved N-terminal homology domains 1 and 2 , respectively . Previourmadillo . Howeverrmadillo , which sm of HD2 , yet thim of HD2  which isDrosophila lgs (ΔHD3lgs) using the CRISPR/Cas9 system (2-8lgs) that deletes HD3 plus the entire C-terminus  show severely delayed development, and most die in their pupal cases, with <25% of them hatching as flies (Drosophila Wnt), as previously observed in flies bearing weak alleles of lgs, pygo, and dTCF  (Senseless (Sens) .To confirm this, we examined a Wg-responsive transcriptional enhancer from 8 discs) , demonstt clones . As expeengrailed, Distalless and H15 , we adopted a proximity-labeling approach called BioID, tagging B9L and BCL9 with BirA*  technique  to our cImportantly, the majority of the hits identified by B9L-BirA* or BCL9-BirA* hardly change after Wnt stimulation . This imAssociation of β-catenin with BCL9/B9L is undetectable prior to Wnt stimulation, as expected from its low abundance in the absence of signaling. This may also explain why the labeling of CBP and p300 by B9L-BirA* is stimulated ~4–6x upon Wnt stimulation, given that these histone acetyltransferases are known ligands of β-catenin. However, both proteins exhibit a significant level of labeling even without Wnt signaling, suggesting a constitutive association with the Wnt enhanceosome, consistent with the identification of CBP by RIME as a putative direct ligand of B9L. It thus appears that the binding of CBP and p300 to β-catenin upon its docking to the Wnt enhanceosome increases their proximity to the C-terminus of BCL9/B9L, which bears the BirA* tag.Drosophila, the association of dTCF with Wg-responsive enhancers appears to be strengthened by Wg signaling , and also with baits lacking HD1 (△HD1) expected to abrogate Pygo binding . We confThe lists of proteins associated with △C baits are similar to those found with the wt, but a few of them show reduced spectral counts, most notably in the case of BCL9ΔC which barely associates with TLE1 nor TLE3 . coIP asLikewise, a small number of proteins show reduced association with BCL9 or B9L baits lacking HD3, with the top hit being LDB1 whose association with B9L-BirA* is reduced by half compared to the wt control . Indeed,Next, we asked whether HD3 is a direct binding site for ChiLS. This short conserved element is predicted to form a single α-helix , with an15N-labelled 6xHis-Lipoyl (Lip)-tagged B9L509-537 (Lip-HD3) after bacterial expression, for binding assays with purified ChiLS complex, as previously described (15N-Lip-HD3 proved soluble and monomeric (as judged by size exclusion chromatography), and produces well-dispersed heteronuclear single-quantum correlation (HSQC) spectra. Incubation of 15N-Lip-HD3 with purified ChiLS complex, but not with purified SSDP alone, produces clear line broadening (‘bleaching’) of selective peaks  Armadillo/β-catenin via HD2 . LeglessDrosophila although, interestingly, this preceded transcriptional activation  are pivotal for initial assembly of the Wnt enhanceosome at Wnt-responsive enhancers during early embryonic development, or in uncommitted progenitor cells of specific cell lineages  RRID:AB_476744 ; α-BCL9 RRID:AB_2063609, α-BCL9-2 RRID:AB_2063747 ; α-β-actin (for fly lysates) RRID:AB_2305186, α-BirA RRID:AB_300830, α-Pygo2 RRID:AB_10863482 ; α-ABC RRID:AB_11127203 . Rat α-Lgs antiserum  was obtained from pre-bleed immunizations with gluthathione S-transferase-purified Lgs232-555. GFP-Trap resin RRID:AB_2631357  was used for coIP assays, and α-FLAG M2 affinity gel RRID:AB_10063035 (Sigma-Aldrich) was used for RIME.The following antibodies and resins were used: α-GFP RRID:crispr.mit.edu was used to design single-stranded oligomers for sgRNA targeting vectors. pCFD3-1S and pCFD3-2S were generated by hybridizing single-stranded oligomers with their complementary strands in 2 mM Tris-HCl (pH 7.4), 10 mM NaCl, 200 μM EDTA at 95°C for 5 min, and by subsequently ligating the double-stranded oligomers into a BbsI restriction site of pCFD3 . pCFD4-HD3 was generated by inserting a double-stranded oligomer coding for sgRNAs targeting upstream and downstream of HD3 (The CRISPR design tool at m of HD3  into pCFm of HD3 .vermilion strain), pCFD3-1S, pCFD3-2S and pCFD4-HD3 were purified using plasmid midi kit columns , and dissolved in injection buffer at 200 ng μl−1 containing 100 μM phosphate buffer and 5 mM KCl. Dechorionated embryos were injected by standard procedures, and sgRNA-expressing transgenic lines were identified on the basis of their +vermilion eye color, and subsequently crossed to a transgenic fly strain bearing act5c.cas9 (from Simon Bullock), essentially as described (lgs sequences were determined after PCR amplification using the following primers: 5’-CATCGGGAAGAACAGTTGGC-3’ and 5’-GGACTGGATGCAGCAAATCG-3’ (for PCR amplification), and 5’-TGAATCAATTTCTTTTTCCTG-3’ .Prior to injections into fly embryos  and BCL9/B9L DKO cells were generated essentially as described  for 20–25 days to isolate individual clones. These were screened by genotyping (see below) and Western Blot analysis .HEK293T cells were purchased from the European Collection of Cell Cultures (authenticated by STR DNA profiling). Upon receipt, cells were frozen, and individual aliquots were taken into culture, typically for analysis within <10 passages. Single KO  and thermocycled as specified by the manufacturers. 2 μl supernatant was used for PCR amplification, and the resulting PCR products were purified using the QIAquick purification kit  and sequenced. Sequence chromatograms were analyzed using MacVector software . The following primers were used: 5’- CGAGATTTTCCTCTGGCAGC-3’ and 5’-AAGGAGTCGGCGGAAATACT-3’ (amplification of BCL9); 5’-GGATCCTGGCTAACAAGACAAG-3’ and 5’-AGAAGTCCGACCACTCTGTG-3’ (amplification of B9L); 5’-AGTCCAGAAAAGAAGCGAAGG-3’ and 5’-CAGAAGCTTCAGTGGTCAGC-3’ (amplification of PYGO2); 5’-ACCCACTTCCACAGCAGAG-3’ (sequencing of BCL9); 5’- TGTCTGAGGAAGCCATGGAG-3’ (sequencing of B9L); 5’-CTCGATCTCCTGACCTCGTG-3’ (sequencing of PYGO2).For genotyping, 2–5 × 10Drosophila strains were used: 20Flgs were provided by Simon Bullock  or chicken α-β-galactosidase RRID:AB_2313752  as described (Wing and leg discs dissected from late third instar larvae (or pupating larvae if specified) were fixed with paraformaldehyde, and stained with α-Sens , α-Wg RRescribed . All dis8 cells were grown adherent to full confluence, washed once with phosphate-buffered saline (PBS), flash-frozen in liquid nitrogen, and stored at −80°C for 1–20 days  before lysis and boiling in sample buffer. All samples were resolved on 4–12% Bis-Tris polyacrylamide gels , and gels were stained with Imperial Protein Stain . Gel slices (2–3 mm) were prepared for mass spectrometric analysis by manual in situ digestion with trypsin, and digests were analyzed by nano-scale capillary LC-MS/MS using an Ultimate U3000 HPLC . The analytical column outlet was directly interfaced via a nano-flow electrospray ionization source, with a hybrid dual pressure linear ion trap mass spectrometer . LC-MS/MS data were searched against a protein database (UniProt KB) using the Mascot search engine program . MS/MS data were validated using the Scaffold program , and processed with R (RRID:SCR_001905).To generate BioID plasmids, BirA*(R118G) was amplified from pcDNA3.1 mycBioID  by PCR a3VO4, 0.2% v/v Triton-X-100) supplemented with protease inhibitors , and sonicated twice for 10 s with an amplitude of 15 μm using a Soniprep 150 plus . Samples were cleared by centrifugation at 16,100 x g for 10 min, and supernatants were incubated with resin for 2 hr at 4°C. All inputs shown are equivalent to 10% of IPs.HEK293T cells were cultured and transfected essentially as described . For coIFor luciferase reporter assays, SuperTOP  was co-tRNA extractions, cDNA synthesis and RT-qPCR reactions were conducted as described above for fly wing discs. The following TaqMan probes were used: Hs00610344_m1, Hs01370227_mH and Hs00427620_m1 .56-285, 6xHis-Lip-SSDP1-92 and 6xHis-Lip-HD3509-537 (from human B9L) as well as the acquisition and analysis of [1H-15N]fast-HSQC spectra were done as described . Backbone resonance assignments were obtained for [13C-15N]-double-labelled Lip-HD3 using standard procedures  The authors do not discuss some inconsistencies of their data with current models of Wnt signaling. They need to provide a more comprehensive interpretation of their data and how it might be reconciled with their previous studies. If the C-terminal region of Bcl9 proteins functions by binding the repressor TLE, one would expect deleting this region to enhance basal expression of Wnt target genes, rather than reducing their activation by Wnt. Analysis of Wg target gene expression in the arm and lgs, as well as the negatively acting component gro.It is also not clear why the Chip heterozygous phenotype, which looks like a loss of wg function, should be suppressed by removing positively acting components of the complex such as The absence of any Runx protein in the BCL9 and B9L biotinylation assays argues against such a protein being a central part of the Wnt enhanceosome as proposed by Fiedler et al.The observation that Wnt signaling is necessary for the interaction of Lgs with β-catenin and TCF proteins and does not reduce its association with TLE proteins does not support a model in which the enhanceosome is present at TCF-bound enhancers in the absence of Wnt signaling.2) The Authors employed BioID (using BirA*) followed by mass spectrometry to identify BCL9 (and the related B9L) binding partners . They went one step further and combined BioID and spectral counting as a (semi) quantitative measurement for determination of protein interactions with and without Wnt. It is not clear to me whether these methods offer reliable quantification. For example, spectral counts for LDB1 and SSDP in 3) The NMR results showed that HD3 directly interacts with LDB1/SSDP, and HD3 deletion and W472A mutants reduced BCL9-LDB1 coIP. But HD1 appeared to have a more critical role as its deletion completely abolished BCL9-LDB1 coIP. The authors suggested, reasonably, that HD1 binding to Pygo, which interacts with LDB1, has a critical role in this multivalent interaction. The authors should be able to demonstrate this point directly using Pygo knockdown or knockout (via CRISPR). Summary:This paper presents interesting data that has the potential to help to resolve the mechanism of transcriptional activation of Wnt target genes. The manuscript addresses an important mechanistic detail of the TCF/b-catenin complex, concerning the addition of the ChiLS complex and how the Wnt "enhanceosome" may be assembled. The dominant model on TCF/b-catenin mediated transcription is the "chain of co-activator" model, which posits that TCF recruits β-catenin that in turn recruits the BCL9 and Pygo complex and other co-activators. The authors have challenged this model through a series studies, including the eLife 2015 paper, and they have argued that the BCL9, Pygo and the new ChiLS complex is preassembled on Wnt enhanceosome to recruit β-catenin. Understanding the mechanism concerning TCF/β-catenin is of general interest and important so the paper in principle is worthy of publication in eLife. However, there are a number of aspects of the work which need to be strengthened to make a compelling case. A few additional experiments should allow the authors to provide the supporting evidence to solidify this model and the authors need to modify the text and discussion around their model where the data are inconsistent. We realize it may take time to conduct the two main experiments suggested but we are willing to allow that time to strengthen this story.lgs truncation allele, (2) comparative BioID for PYGO2 as well as PYGO2 knock-out by CRISPR/Cas9, and (3) coIP assays to complement our evidence from BioID. We believe that our new results provide compelling support for the enhanceosome model. They also strengthen our insights into the general components of the Wnt enhanceosome that are not dedicated to Wnt signaling, such as Groucho/TLE, CBP/p300 and the subunits of the BAF complex (in particular Osa/ARID1), which broadens the interest of our study.We were pleased that the referees found our study of interest, and stimulated by their challenging of our Wnt enhanceosome model and their generally constructive critique. We thus decided to design and conduct additional experiments to further test our conclusions and proposals, taking up the editors’ offer of a substantial extension of our revision deadline. In a nutshell, our additional work includes (1) in-depth analysis of transcriptional Wg responses in imaginal discs from larvae bearing our Inevitably though, our additional work  nearly doubled the data described in our manuscript, and we were thus compelled to recast it as a Research Article. This also allowed us more space to explain more clearly the rationales underlying our approaches, and to outline how our results advance and revise current models of Wnt-dependent transcription switches . Neither of this was possible in our original submission as a Research Update, because of length restrictions. We hope the referees will agree with us that the new format is appropriate for our revised manuscript.The specific points to be addressed are:2-8 mutant might help to clarify this issue.1) The authors do not discuss some inconsistencies of their data with current models of Wnt signaling. They need to provide a more comprehensive interpretation of their data and how it might be reconciled with their previous studies. If the C-terminal region of Bcl9 proteins functions by binding the repressor TLE, one would expect deleting this region to enhance basal expression of Wnt target genes, rather than reducing their activation by Wnt. Analysis of Wg target gene expression in the lgsIn our revised manuscript, we discuss our data in the context of current views of Wnt-dependent transcription switches, adding several references to refer to results published by others on this topic, much of which is consistent with our new evidence (or might be re-interpreted in the light of it). For example, we explain that Groucho/TLE is a bimodal regulator, not only conferring silence on the enhanceosome in the absence of signaling, but also earmarking it (together with other core components) for subsequent Wnt induction : owing to its multivalent interactions with TCF, ChiLS and the Legless/BCL9 C- terminus, Groucho/TLE assumes the role of a cornerstone within the enhanceosome  that the CBP/p300 co-activator associates with the Wnt enhanceosome prior to Wnt signaling , possibly via direct interaction with Legless/BCL9, as indicated by RIME new .Although we have not mapped this interaction, the best candidate for the CBP/p300 binding site in Legless/BCL9 is its C-terminus . If so, this would further explain the positive role of this C-terminus, given that CBP/p300 is a well-established co-activator of transcription.Interestingly, like Groucho/TLE, CBP/p300 is a bimodal regulator, acting activating or repressive depending on context, as shown by the Cadigan group and ourselves . Furthermore, our BioID data indicated that both bimodal regulators are constitutively associated with the enhanceosome . Indeed,lgs2-8 mutant larvae and flies (new dpp (dpp.lacZ) . This supports our conclusion that the C-terminal portion of the protein, downstream of its Pygo-Armadillo adaptor sequences, is essential for promoting transcriptional Wg responses during fly development.Importantly, we have extended our phenotypic analysis of It is also not clear why the Chip heterozygous phenotype, which looks like a loss of wg function, should be suppressed by removing positively acting components of the complex such as arm and lgs, as well as the negatively acting component gro.Drosophila) are notoriously difficult to interpret. This gets worse if the proteins in question are components of a multiprotein complex, because it is hard to predict how reducing the expression levels of individual components will affect complex assembly, especially if the structure of this complex and its stoichiometry are poorly defined.Genetic interactions caused by heterozygosity of genes .Nevertheless, in the revised figure legendof chip/+ should therefore be exacerbated by lowering expression of its obligatory partner SSDP. It might be ameliorated by lowering the dose of enhanceosome components that, at their normal expression level, potentially sequester the limiting amounts of Chip by direct binding .This haploinsufficiency phenotype caused by The absence of any Runx protein in the BCL9 and B9L biotinylation assays argues against such a protein being a central part of the Wnt enhanceosome as proposed by Fiedler et al.Drosophila embryo  is reminiscent of its pioneering functions in other uncommitted cell lineages, e.g. in the hematopoietic system. However, HEK293 cells are epithelial cells, and our data from BioID and Wnt reporter assays clearly show that the Wnt enhanceosome is fully functional in these cells without RUNX . A corollary is that RUNX functions in a hit-and-run fashion, to help set up the Wnt enhanceosome on Wnt-responsive enhancers in uncommitted cell lineages; however, once assembled , RUNX is no longer required for enhanceosome- mediated ON/OFF switching. Hit-and-run is a well-established concept in other contexts of transcriptional control during development, e.g. in setting up Polycomb-repressive complexes in the early Drosophila embryo.Absolutely, and we now state this explicitly . We offer the following (in our view plausible) explanation for this finding: namely, the role of RUNX in promoting Wnt enhanceosome function in the The observation that Wnt signaling is necessary for the interaction of Lgs with β-catenin and TCF proteins and does not reduce its association with TLE proteins does not support a model in which the enhanceosome is present at TCF-bound enhancers in the absence of Wnt signaling.This was our most intriguing BioID result – namely the Wnt-inducible labeling of TCF factors by both BCL9-BirA* and B9L-BirA*. We proposed in our initial submission that this reflects a β-catenin-dependent increase in proximity between TCF and the BCL9/B9L C- terminus . We now provide two new strands of evidence to support this notion: firstly, we use coIP assays to show that the association of BCL9/B9L with TCF is similarly stable before and after Wnt stimulation, like its association with Pygo, while its association with β-catenin depends on Wnt stimulation new . SecondlAs in the case of the BCL9/B9L baits, the list of constitutively labeled components by PYGO2-BirA* includes TLE3 and TLE1, underscoring our conclusion that these co- repressors remain associated with the Wnt enhanceosome even during Wnt signaling. Although this is against the prevailing view of Groucho/TLE detaching from TCF factors as a result of their binding to β-catenin, but to our knowledge, this has never been explicitly tested (e.g. by ChIP). Unfortunately, the Groucho antiserum previously used for ChIP (e.g. by the Jennings group) and initially produced by Kharchenko et al. (2011) is no longer available. However, this notion of competition between Groucho/TLE and Armadillo/β-catenin was seriously challenged recently by the discovery of Weis and Waterman and co-workers that TLE and β-catenin can bind to TCF simultaneously , as we point out in our Introduction.PYGO2-BirA* also labels the nuclear co-receptor components, like BCL9-BirA* and B9L- BirA*. This strengthens the link between the Wnt enhanceosome and nuclear hormone receptor signaling, as discussed in the last paragraph of the Discussion. In this paragraph, we also cite work by the Carroll group who found a strong association of TLE1 with estrogen receptor-occupied loci in breast cancer cells, and a positive role of TLE1 in promoting the function of this nuclear hormone receptor . As mentioned above, this is reminiscent of its role in promoting Wnt enhanceosome function and Wnt responses.Drosophila, the association of dTCF with target enhancers is increased by Wg signaling. This is not inconsistent with out BioID data from the BCL9/B9L baits which show slightly increased labeling of most Wnt enhanceosome components, although this increase is not seen with the PYGO2 bait. In any case, TCF factors clearly associate with target enhancers in the absence of Wnt signaling in HEK293 cells, as shown by Frietze et al. (2012) (cited in the aforementioned paragraph). Whether or not the binding of β-catenin to Legless/BCL9 and/or TCF results in a stabilization of these interactions will require future testing.Finally, in the second paragraph of the subsection “β-catenin triggers a Wnt-dependent apposition of the BCL9/B9L C-terminus to TCF”, we refer to work by Parker et al. (2002) who provided ChIP-based evidence that, in 2) The Authors employed BioID (using BirA*) followed by mass spectrometry to identify BCL9 (and the related B9L) binding partners . They went one step further and combined BioID and spectral counting as a (semi) quantitative measurement for determination of protein interactions with and without Wnt. It is not clear to me whether these methods offer reliable quantification. For example, spectral counts for LDB1 and SSDP in In the revised manuscript, we state more clearly that coIP assays assess (relatively stable) associations between proteins, while BioID assesses primarily their proximity (even if this reflects weak binding affinities). We have added an introductory paragraph on BioID , explaining how the spectral counts obtained by this approach can be taken as (semi)quantitative measurements of proximity, which can provide insight into the spatial organization of large protein complexes. This was demonstrated in the original Roux et al. (2012) paper by comparing biotin labeling efficiencies of interactors of Laminin A tagged with N-terminal or C-terminal BirA*. Proximity-dependent labeling efficiencies have since been documented by others, and we cite a compelling example of BioID data obtained by the Stearns group on components of the human centrosomal complex . In the same paragraph, we also mention other factors that can affect the efficiency of biotin labeling by the bait, but these are less relevant for the comparison of hits obtained under different conditions (with or without Wnt), or of hits labeled by different baits under the same conditions (BCL9/B9L versus PYGO2).Finally, as mentioned above, we have complemented our evidence from BioID with coIP assays, which revealed a relatively stable association between TCF4 and B9L in unstimulated cells, which is only slightly increased upon stimulation  available. Instead, we have conducted coIP experiments, and also comparative BioID labeling by PYGO2-BirA*, to complement and consolidate our conclusions based on the BCL9/B9L BioID baits (as detailed above).3) The NMR results showed that HD3 directly interacts with LDB1/SSDP, and HD3 deletion and W472A mutants reduced BCL9-LDB1 coIP. But HD1 appeared to have a more critical role as its deletion completely abolished BCL9-LDB1 coIP. The authors suggested, reasonably, that HD1 binding to Pygo, which interacts with LDB1, has a critical role in this multivalent interaction. The authors should be able to demonstrate this point directly using Pygo knockdown or knockout (via CRISPR).We have done this, by knocking out PYGO2 by CRISPR/Cas9 new . The res


Studies disrupting the chemokine pathway CX3CL1 / CX3CR1 have shown decreased atherosclerosis in animal models but the techniques used to interrupt the pathway have not been easily translatable into human trials. DNA vaccination potentially overcomes the translational difficulties. We evaluated the effect of a DNA vaccine, targeted to CX3CR1, on atherosclerosis in a murine model and examined possible mechanisms of action. DNA vaccination against CX3CR1, enhanced by dendritic cell targeting using DEC-205 single chain variable region fragment (scFv), was performed in 8 week old ApoE-/- mice, fed a normal chow diet. High levels of anti-CX3CR1 antibodies were induced in vaccinated mice. There were no apparent adverse reactions to the vaccine. Arterial vessels of 34 week old mice were examined histologically for atherosclerotic plaque size, macrophage infiltration, smooth muscle cell infiltration and lipid deposition. Vaccinated mice had significantly reduced atherosclerotic plaque in the brachiocephalic artery. There was less macrophage infiltration but no significant change to the macrophage phenotype in the plaques. There was less lipid deposition in the lesions, but there was no effect on smooth muscle cell migration. Targeted DNA vaccination to CX3CR1 was well tolerated, induced a strong immune response and resulted in attenuated atherosclerotic lesions with reduced macrophage infiltration. DNA vaccination against chemokine pathways potentially offers a potential therapeutic option for the treatment of atherosclerosis. Atherosclerosis is a chronic inflammatory disease characterized by progressive infiltration of monocytes into the endothelium with the formation of plaques containing lipids, leukocytes, smooth muscle cells (SMCs) and inflammatory mediators. These plaques can become unstable and prone to rupture, triggering acute thrombotic vascular events resulting in myocardial infarction, stroke, and sudden cardiac death.hi) monocytes have high levels of CCR2 and low levels of CX3CR1, whereas non-classical (Ly-6Clo) monocytes are CCR2 low and CX3CR1 high. Early in atherogenesis CCL2 plays an important role in attracting inflammatory monocytes but crucially these monocytes also require CX3CR1 to enter into the plaque [Monocytes play a major role in atherosclerotic plaque development. There are two major subsets of circulating murine monocytes based on the chemokine receptors expressed. Classical , 95°C for 45 s, 60°C for 45 s, and 72°C for 1 min (35 cycles) with the final cycle at 72°C for 7 min. The CX3CR1 PCR products were ligated separately by T4 DNA ligase (Promega) into DEC205 (DEC-CX3CR1) and control vector (Con-CX3CR1) plasmids to make the two sets of DNA vaccines. The ligated CX3CR1 constructs were cloned into JM109 competent E.coli cells (Promega) and verified by DNA sequencing after cloning using specific primers, 5’-GCGAATGAATTGGGACCT-3’ and 5’-CTTCTGAGATGAGTTTTTGTTCG. Plasmid DNA was prepared using Qiagen Plasmid Max Kit (Qiagen).Vectors DEC205 (pSC-DEC-OLLA) and control vector (pSC-GL117-OLLA) were kindly provided by Dr Godwin Nchinda from The Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10065–6399, USA. The pSC4 /ml of serum free Gibco® Dulbecco's Modified Eagle's medium . DNA plasmids were diluted in serum free Opti-MEM® I Medium  and mixed with pre-diluted Lipofectamine® LTX Reagent for 30mins at RT before adding to the CHO cells and incubating at 37°C for 24–48 hours. Total cell supernatant were collected for the incoming western blot analysis on CX3CR1 expressing NIH 3T3 cell lysate.The fusion DNA constructs (DEC-CX3CR1 and Con-CX3CR1) were transiently transfected into CHO cells (ATCC) using Lipofectamine® LTX Reagent  for immunoblot analysis to verify their expression. One day prior to transfection, CHO cells were grown and maintained at 80% confluence and cells were then diluted to 5X10Six weeks old male C57/BL6 ApoE-/- mice (stock number 002052) were purchased from the Animal Resources Centre in Perth, Australia and maintained under standard sterile conditions in the Department of Animal Care at Westmead Hospital, NSW. These mice were generated by backcrossing the Apoetm1Unc mutation 10 times to C57BL/6J mice. This study was specifically approved by the Animal Ethics Committee of Sydney West Area Health Service. All mice were fed with normal diet. Mice were divided into four groups: DEC-CX3CR1 vaccinated (n = 6), Con-CX3CR1 vaccinated (n = 6), adjuvant only control (n = 6), and non-vaccinated control (n = 6). Six weeks old male wildtype C57/BL6 mice (n = 6) were also obtained from the Animal Resources Centre in Perth, Australia, they were fed with normal chow and maintained under standard sterile conditions in the Department of Animal Care at Westmead Hospital, NSW. The Health and well-being of mice were monitored 3 times every week.All mice except the non-vaccinated controls were pretreated with 0.5% bupivacaine  by intramuscular injection into the tibialis anterior muscle two weeks before plasmid DNA vaccination. Plasmid DNA (50 μg) was injected at the same site as bupivacaine three times at two weekly intervals, into the mice from the vaccinated groups. The mice were anaesthetised with Isoflurane inhalation (Sigma), the isoflurane vaporizer were adjusted to 3.5% for induction and approximately 1.5% for maintenance, the oxygen flowmeter was adjusted to approximately 0.9 L/min. The efficiency of DNA vaccination was enhanced by electroporation system (BTX Harvard Apparatus). One week after the third DNA vaccination, mice from the DEC-CX3CR1 and Con-CX3CR1 vaccinated groups were immunized with a prime boost of CX3CR1 peptide mixture  and Poly IC (50 μg/mouse). Mice from the non-vaccinated and adjuvant only control groups were injected with saline only. Blood was collected by tail bleeding from all mice at 2, 4, and 8 weeks after peptide boost or saline injection, for ELISA. All mice were sacrificed at 34 weeks, by inhalation of CO2 gas at which point, the whole blood was collected by cardiac puncture for ELISA and cholesterol measurement. The aortas and brachiocephalic arteries were excised, fixed in OCT tissue freezing medium (Leica systems) and frozen for histology and immunostaining analysis.in vitro Technology, Australia) was coated with CX3CR1 peptide (Genetex) at a concentration of 1 μg/well in 100 μl coating buffer and incubated overnight. Sample mouse sera were diluted 50 and 500 times and added to the coated ELISA plate. Wild type C57/BL6 mouse serum was used as a negative control. Goat anti-mouse IgG alkaline phosphatase conjugated Ab (Sigma) and p-Nitro-phenol phosphate (Sigma) as substrate were added sequentially for the Ab titer analysis. All samples and controls were added in duplicate to the plates. Absorbance was read at 450 nm on an ELISA plate reader .Serum anti-CX3CR1 antibody titers were evaluated by ELISA assay for all groups of mice. A 96 well Immuno ELISA microtiter plate  stained samples were used to visualize plaque size. Frozen sections of brachiocephalic arteries were fixed in ice-cold acetone (10 min) and washed with running tap water for 1 minute. Sections were re-hydrated in 2 changes of absolute alcohol, 5 minutes each, followed by 2 mins in 95% alcohol and another 2 mins in 70% alcohol. Sections were washed briefly in distilled water and stain in Harris hematoxylin solution for 10 minutes. Sections were washed in running tap water for 5 minutes and differentiated in 1% acid alcohol for 30 seconds. Sections were washed in running tap water for 1 minute and then Blued in 0.2% ammonia water for 1 minute. Sections were then washed in running tap water for 5 minutes and rinsed in 95% alcohol for 10 dips. Sections were counterstained in eosin solution for 1 minute. Sections were dehydrated in graded ethyl ethanol  and two changes of xylene, 5 minutes each and then permanently mounted with VectaMount mounting medium (Vector Laboratories).OCT-embedded frozen brachiocephalic artery samples were sectioned proximal to distal at 5 μm intervals and lipids were detected with Oil Red O. Frozen sections of brachiocephalic arteries were fixed in ice-cold acetone (10 min) and washed with running tap water for 1 minute. Sections were rinsed with 60% isopropanol and stained with freshly prepared Oil Red O working solution 15 mins and then rinsed with 60% isopropanol. Sections were stained for nuclei with alum haematoxylin in 5 dips and rinsed with distilled water. Sections were mounted in glycerine jelly. At least 3 sections per mouse each 100 μm apart were inspected for each histostaining.Aperio Imagescope software, images of the stained sections were scanned at original size and 20X of magnification for analysis.Plaque size and lipid content were quantified using Buffered Saline and Tween 20 (TBST). Peroxidases were quenched by Peroxidase-Blocking Solution (Dako) for 10 min. Sections were washed and blocked with Protein Block Serum-Free solution (Dako) for 10 min. Sections were incubated with the primary antibodies for 60 min , Santa Cruz final concentration: 0.01mg/ml). Sections were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min . After washing, sections were developed with incubation with 3, 3’-Diaminobenzidine tetra-hydrochloride  and sections were counterstained with hematoxylin for 1 min followed by bluing within continuous water for 20 minutes. Sections were dehydrated in graded ethyl ethanol  and two dips of histolene, and then permanently mounted with VectaMount mounting medium (Vector Laboratories). The macrophage, smooth muscle cell and M1 and M2 contents were quantified in three equidistant stained sections (100 μm apart) using Aperio Imagescope software. Images of the stained sections were scanned at original size and 20X of magnification for analysis.Frozen sections of brachiocephalic arteries were fixed in ice-cold acetone (10 min) and washed with Tris-At the time of euthanasia, whole blood was drawn by cardiac puncture. Serum was obtained by centrifugation for 10 minutes at 2,000 x g 4°C. Serum cholesterol levels were measured with a commercial cholesterol kit (Wako Diagnostics) as per the manufacturer’s instructions. Serum was also obtained from Wildtype C57/BL6 mice (n = 6) and analysed as control.The BD Cytometric Bead Array Mouse Inflammatory Kit (BD Bioscience) was used to measure IL-6, IL-12, CCL2, TNF-α, TFN-γ and IL-10 serum proteins, following the manufacturer’s instructions (BD Bioscience). In brief, all serum samples were pre-diluted to the manufacturer’s recommended concentration with distil water and mixed with the prepared capture beads at a 1:1 ratio (50ul each) before incubation at 37°C for 2 hours without exposure to light. Samples were washed with wash buffer and centrifuged at 300g and then resuspended before measurement by FACS Calibur flow cytometry. The data were analyzed by FCAP Array software (BD).t-test, with a p value (two-tailed) less than 0.05 considered statistically significant.Results are expressed as the group mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) for multiple comparisons using GraphPad Prism software. Two group differences were analyzed by Student’s We generated fusion DNA constructs with CX3CR1 genes fused in frame to the C-terminus of scDEC as shown in To determine whether DNA vaccines could induce a strong humoral response, we measured serum anti-CX3CR1 antibody level using ELISA. Both the anti-CX3CR1 vaccines stimulated a marked response compared to controls (p<0.001 for both vaccines) but on direct comparison this was greater in the DEC-CX3CR1 vaccinated mice beyond 2 weeks post CX3CR1 prime boost (p<0.001) . There wTo test the effect of vaccination on atherosclerosis we examined the brachiocephalic arteries and aortic arches from 34 week-old ApoE-/- mice  as theseAtherosclerotic plaques are made up mainly of lipid laden macrophages and inflammatory infiltrates. To examine the effect of the vaccination on the lipid content of the plaque, we stained sections with oil red O, . DEC-CX3Since CX3CL1 is a known chemoattractant for monocytes and macrophages we next examined whether DNA vaccination against CX3CR1 resulted in lower numbers of macrophages in the plaque. We found significantly less macrophage accumulation in the brachiocephalic artery lesions of the DEC-CX3CR1 vaccinated mice compared with control groups . DEC-CX3Macrophages can be further subtyped based on surface markers and biological function, with the M1 inflammatory phenotype predominating in atherosclerosis. We therefore examined if DNA vaccination against CX3CR1 resulted in altered macrophage phenotype within the atheromatous plaques. Although both DEC-CX3CR1 and Con-CX3CR1 vaccinated mice exhibited a minor increase in M2 macrophage accumulation in brachiocephalic artery lesions, the difference (133% and 118% compared to Adjuvant only) was not significant . It has In addition to being a chemoattractant for leucocytes, CX3CL1 is also involved in the migration of smooth muscle cells into atherosclerotic plaques . We therDNA vaccination did not alter the total serum cholesterol levels in ApoE-/- mice in our study . All athAtherosclerosis is a multifactorial disease process with interactions between various immune and inflammatory pathways. To test if anti-CX3CR1 vaccination had a direct impact on other inflammatory molecules we measured various serum cytokines. CX3CR1 DNA vaccination led to a non-significant reduction in IL-12 and IL-6 , and it This study demonstrates that DNA vaccination against CX3CR1 results in decreased atherosclerotic plaque size in a murine model of atherogenesis. This was associated with decreased numbers of macrophages within the plaques and may offer a potential therapeutic alternative or supplemental treatment to those currently available for atherosclerosis.The role of chemokines and chemokine receptors in atherogenesis is well established however many of the models investigating these pathways have used mice where one or more chemokine or receptor has been genetically deleted. Whilst providing confirmatory information on etiology they are not viable treatment options in humans and we therefore sought to develop a method of blocking the CX3CR1-CX3CL1 pathway that could be further developed for human therapy.in vivo within host cells in order to induce a desired host immune response[DNA vaccination delivers genes that encode target antigens and allows their expression  response. This te response. Further response. Selecti response–22. Othe response. Various response, 28–30.A further advantage of DNA vaccinations is the generation of specific antibodies that appear to block the function of encoded molecules on targeted tissues only. This was demonstrated clearly in our study as there was no depletion of other pathways of the immune system as shown by the cytokine profiling, and by others in previous studies, which is crucial for future clinical studies , 31, 32.Given the potential advantages of DNA vaccination there has been recent interest in its use against a variety of immune targets including co-stimulatory pathways, cytokines and chemokines , 33, 34.Our finding of a 35% reduction in atherosclerotic plaque size following DEC-CX3CR1 vaccination is in keeping with interruption of the CX3CL1/CX3CR1 pathway using genetic manipulation or pharmacological blockade , 40, 41.et al argued that CX3CR1 is essential in monocyte/macrophage survival  since CX3CR1 deficiency results in reduction of Ly6Clow cells [The primary mode of action of CX3CL1/CX3CR1 in atherosclerosis seems to be recruitment of monocytes and accordingly in this model there was a marked reduction in the macrophage content of the plaques. Other models of interruption of CX3CL1/CX3CR1 also reported decreases in macrophage/monocyte content of between 40% and 80%, consistent with our findings , 40, 41.ow cells . Howeverow cells , the autet al demonstrated that CX3CR1 antagonist (a novel drug AZ1220 targeting CX3CR1) reduced SMC infiltration at porcine model [et al suggested that CX3CL1/CX3CR1 plays a pivotal role on SMC proliferation within plaque neovascularization at murine model [CX3CL1/CX3CR1 can contribute to other features of the atherogenesis process, including the chemoattraction, patrolling of monocytes , survivane model , while Kne model .In atherosclerosis other groups have targeted DNA vaccines to vascular endothelial growth factor receptor 2 (VEGFR2) , 50, TIEIn conclusion, this study shows that modified DEC205-CX3CR1 DNA vaccine shows greater protection in atherosclerotic mice. Vaccination results in the attenuation of macrophage infiltration and an overall decrease in atheromatous plaque size. Therefore blocking the CX3CR1 pathway using DNA vaccination may potentially complement current therapies such as lipid lowering agents and anti-platelet drugs.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.


Cupriavidus necator H16. The production of the N-PEW is an alternative to oxidized polyethylene wax (O-PEW) (that has been used as a carbon source previously) as it is less time consuming to manufacture and offers fewer industrial applications. A range of molecular structural analytical techniques were performed on the PHAs obtained; which included nuclear magnetic resonance (NMR) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS). Our study showed that the PHA formed from N-PEW contained 3-hydroxybutyrate (HB) with 11 mol% of 3-hydroxyvalerate (HV) units.There is an increasing demand for bio-based polymers that are developed from recycled materials. The production of biodegradable polymers can include bio-technological (utilizing microorganisms or enzymes) or chemical synthesis procedures. This report demonstrates the corroboration of the molecular structure of polyhydroxyalkanoates (PHAs) obtained by the conversion of waste polyethylene (PE) via non-oxygenated PE wax (N-PEW) as an additional carbon source for a bacterial species. The N-PEW, obtained from a PE pyrolysis reaction, has been found to be a beneficial carbon source for PHA production with  Polyhydroxyalkanoates (PHAs) are a family of polyhydroxyesters with 3-, 4-, 5-, and 6-hydroxyalkanoic acids that are biodegradable, non-toxic, biocompatible organic polyesters synthesized by some species of bacteria . CurrentWaste polyethylene (PE) is a potential carbon source that could be utilized to make value-added biopolymers, particularly as it is the most commonly-produced plastic, making up over 29% of worldwide plastic manufacture, while only 10% of it is recycled ,8,9,10. Cupriavidus necator H16. The pyrolysis of PE samples in anaerobic conditions yields a complex mixture of relatively low molecular weight hydrocarbons with a molecular mass of 200–1000 + at m/z at 1155 m/z is presented. This parent ion produces fragment ions of subsequent oligomers according to the theoretical fragmentation pathway diagram. The loss of 86 Da equates to the loss of crotonic acid , and the loss of 100 Da equates to an HV unit. The fragmentation spectrum of these ions confirms that the most intensive ions in the clusters correspond to sodium adducts of 3-hydroxybutyrate oligomers. Therefore, the fragmentation spectrum for the precursor ion at m/z 1155 confirms the presence of 3-hydroxybutyrate and randomly-distributed 3-hydroxyvalerate co-monomer units in polyester chains produced from N-PEW and TSB.In −1 ) when it was added to BSM fermentations. The cell growth analysis also showed that N-PEWs do not have an adverse effect upon the growth rate of C. necator H16 in either TSB (not significant) or BSM. This was also reported by Radecka et al. [β-oxidation. However, the GPC data was not able to fully support this theory as any reduction in Mn or Mw occurred in the controls without bacteria. Those smaller hydrocarbon chains that were not successfully recovered after the 48 h fermentation could have either been metabolized (in the inoculated flask) or they were fully dissolved in the medium. Another possibility is that some of the wax attached to the sides of the flasks as a thin film. For these reasons it was not possible to give a definitive value of the wax metabolized by the bacteria; a possible answer to this would be use a labelled variant of the wax. The presence of accessible carbon sources, such as fatty and carboxylic acids from O-PEW means that more carbon is present in the wax-supplemented cultures in a form that promotes the synthesis of PHAs [The FTIR spectra showed that the N-PEWs received were non-polar, saturated hydrocarbons . Howevera et al.  it was s of PHAs ,16. C. necator with N-PEW in BSM (shown by cell counts), however, no PHAs were recovered after 48 h. This is an indication that the addition of N-PEW into TSB has an effect upon the PHA production and the structure, with a Mn of 64,000 g/mol with a molecular distribution of 9.7 (Mw/Mn). The Mn results for PHAs produced with TSB only were lower, in the range of 52,000 g/mol with a distribution of 5.7 (Mw/Mn). These dispersity values indicate that N-PEW with TSB produces a more varied range of polyesters, and it is further evidence that N-PEWs are being metabolized.The PHA yield of TSB cultures supplemented with N-PEW was 32% with a cell dry weight 1.42 g/L compared to shake flask experiments using TSB exclusively, which produced a lower yield of 20% and a dry weight of 0.98 g/L. There was growth of 1H-NMR was used to determine the structure of the copolymers produced. It was found that PHB was produced in the TSB only fermentation (data not shown) [2CH3) and δ = 5.17 (-CH) correspond to HV repeated units that were observed. These signals are typical for PHAs with longer aliphatic groups in the β-position [t shown) . In the position ,33. Afte3  determined copolymers were present. This method is known to provide more detail than GC and NMR when used for PHBV characterization. This is because it can corroborate whether a copolymer is a co-block polymer or randomly distributed. Therefore, it is feasible to find the precise sequence distribution of the oligomers generated and construct their sequences . The oli3 . This prC. necator. The N-PEWs did not mix with growth media, such as TSB, due to their hydrophobic nature; however, with ultrasound sonication, these waxes formed an emulsion with reduced-length hydrocarbon chains that did provide an increased cell count, clearly acting as a viable carbon source. The structural and thermal characterization of the biopolymers produced showed that by adjusting the carbon source, the types of polymer synthesized can be changed. This variation affects the molecular masses and properties of the PHB or PHBV biopolmyers. N-PEW does not limit the growth rate of bacterial cells; in fact the cultures with N-PEW as an additional carbon source produced a higher yield of PHAs. BSM supplemented with N-PEW produced no PHAs, while TSB and N-PEW can produce yields of up to 40% (w/w), TSB only experiments gave a yield of 20% (w/w).N-PEW, a substance produced from waste PE materials is a potential carbon source for PHA production with 1H-NMR and ESI-MS/MS analysis indicated that in the presence of TSB/N-PEW the chains of the resulting biopolyester contained 3-HB repeating units with 11 mol% of 3-HV units randomly distributed along the copolymer chain. This is different to the PHAs reported from O-PEW/TSB fermentations, that produced PHBV and 3HH repeating units. The N-PEW/TSB media also yielded polymers with greater polymer diversity. Ultimately this analytical technique has enabled the characterization of PHAs synthesized from N-PEW/TSB media using C. necator H16, at the molecular level even when the contents of 3HB is high and the levels of other PHA oligomers (such as HV) are limited. This study demonstrates that plastic manufacturing companies could recycle more and utilize a wider range of materials for the production of value-added bioplastics and possibly direct the type of polyesters formed by selecting the level of wax oxidation. By using N-PEWs, food-based materials previously used for biopolymer synthesis, such as corn seed or sugar cane, could be reserved. These N-PEWs are a viable carbon source that can yield truly biodegradable, bio-compatible, non-toxic bioplastics for diverse applications.The