Drosophila Wg-signalling, affecting pathway activity in the same manner, interacting genetically and physically, and co-localizing with β-catenin, the mediator of Wnt/Wg-signalling on microtubules. Following pathway activation, Kinesin-2/IFT-A mutant cells exhibit high cytoplasmic β-catenin levels, yet fail to activate Wg-targets. In mutant tissues in both, Drosophila and mouse/MEFs, nuclear localization of β-catenin is markedly reduced. We demonstrate a conserved, motor-domain dependent function of the Kinesin-2/IFT-A complex in promoting nuclear translocation of β-catenin. We show that this is mediated by protecting β-catenin from a conserved cytoplasmic retention process, thus identifying a mechanism for Kinesin-2/IFT-A in Wnt-signalling that is independent of their ciliary role.Wnt/Wg-signalling is critical signalling in all metazoans. Recent studies suggest that IFT-A proteins and Kinesin-2 modulate canonical Wnt/Wg-signalling independently of their ciliary role. Whether they function together in Wnt-signalling and their mechanistic role in the pathway remained unresolved. Here we demonstrate that Kinesin-2 and IFT-A proteins act as a complex during  Drosophila and mouse MEF Wnt signalling independent of its ciliary role.IFT-A proteins and Kinesin-2 modulate canonical Wnt/Wg-signalling independent of their ciliary role, but how is unclear. Here, the authors show that Kinesin-2 and IFT-A act as a complex to promote nuclear translocation of β-catenin in  Dysregulated expression of proteins in the Wnt/Wg pathway is often associated with diseases, including cancers7. Secreted Wnt proteins stabilize β-catenin (Armadillo/Arm in Drosophila), a multifunction protein that is critical in Wnt signaling as an essential transcriptional coactivator for Wg/Wnt-target gene expression4. Wnt/Wg proteins bind to Frizzled (Fz) receptors, resulting in alleviation of pathway inhibition caused by a “destruction complex”, composed of APC (Adenomatous Polyposis Coli), Axin, GSK3β, and Casein kinase I (CKI), targeting β-catenin for degradation. Pathway activation results in relocalization of Axin to the plasma membrane8 and stabilization of cytoplasmic β-catenin/Arm, which then translocates to the nucleus to act as coactivator of the Tcf/Lef transcription factors1. In the absence of Wg/Wnt, β-catenin/Arm is phosphorylated by the “destruction complex” and targeted to the proteasome10.Canonical Wnt/Wingless signalling is a highly conserved pathway with important roles in the regulation of a variety of developmental processes, including cell fate specification, proliferation, cell survival, and migrationDrosophila wing development serves as a paradigm for Wnt/Wg-signalling. In larval wing discs, Wg is expressed as a two-cell stripe at the dorso-ventral (D/V) boundary of the wing pouch, where it acts as a morphogen activating targets in a concentration-dependent manner13. Wg protein is detected in a gradient up to several cells away from the source14, patterning wing development and specifying future wing margin structures16. High threshold targets of Wg, in cells adjacent to D/V boundary, include senseless (sens), a gene responsible for the formation of margin sensory bristles of adult wings17, while lower threshold target genes include Distalless (Dll), required for wing growth and expressed in a graded fashion, decreasing towards the edges of the wing pouch18.Drosophila Kif3A, a KInesin-2 family member, and components of the IFT-A complex have recently been shown to be required for Wg signaling in non-ciliated Drosophila epithelial cells20. In absence of Klp64D function, Arm/β-catenin abnormally accumulates in cytoplasmic punctae, suggesting that Klp64D is involved in intracellular trafficking of Arm/β-catenin to enable Wnt/Wg signaling19. Klp64D/Kif3A is a subunit of the plus-end-directed microtubule-based motor Kinesin-2 21. Kinesin-2 is a heterotrimeric holoenzyme consisting of two motor subunits, Kif3A and Kif3B, and the nonmotor subunit Kap3. Kinesin-2 drives the anterograde transport of IFT particles along the cillium22. In Drosophila, localization of Arm/β-catenin to adherens junctions (AJs) is also regulated by Klp64D in developing photoreceptor cells23.Klp64D, 25. Despite their essential role in ciliogenesis and cilia-associated signaling pathways in vertebrates, such as Hedgehog signaling26, IFT-A proteins were also found to modulate canonical Wnt/Wg-signaling in non-ciliated cells in Drosophila imaginal discs20, suggesting that IFT-A must function in the Wnt-pathway in a non-ciliary context. In cilia, IFT-A complexes control retrograde protein transport, from the tip to the base of the cillium30. Among the five conserved IFT-A proteins, four  regulate Wnt/Wg-signaling in Drosophila through effects on β-catenin/Arm20. It remains unclear how this complex functions mechanistically in Wg/Wnt-signaling; whether IFT-A proteins associate with microtubular structures outside the cilium; and whether the non-ciliary Wnt-signaling-specific function of IFT-A is conserved in vertebrates.Intraflagellar Transport (IFT) is a movement of large protein complexes along axonemal microtubules, which is essential for the formation and maintenance of eukaryotic cilia and flagella. IFT particles are composed of about 20 proteins, organized into the IFT-A and IFT-B complexes31. There are many contradicting conclusions from analyses of Wnt signaling in the context of ciliary mutants, ranging from inactivation to hyperactivation of the pathway33. The barrier to understanding how ciliary proteins might function independently of the cilium stems from their crucial role in the biogenesis and maintenance of the cilium itself. It is thus difficult to distinguish cilia-dependent and independent effects in ciliated vertebrate cells. To overcome this problem we have used Drosophila, as all epithelial cells are non-ciliated in flies34. Consequently, phenotypes associated with loss of ciliary proteins must be due to cilia-independent function(s).The role of ciliary proteins in vertebrate Wnt-signaling has remained unresolved with existing data being confusing if not contradictory. Ciliary proteins have been suggested to limit response to Wnt signaling, affecting stability and localization of β-catenin/ArmHere we analyze the cilia-independent function of Kinesin-2 and IFT-A complexes in Wg/Wnt-signaling. Our data indicate that Kinesin-2 and IFT-A interact via Kap3 to form a large complex and together are critical for nuclear translocation of Arm/β-catenin, and thus its activity in Wnt/Wg-signaling. We demonstrate that the combined Kinesin-2/IFT-A complex interacts directly with β-catenin/Arm through IFT140 and that this interaction is critical for normal nuclear translocation of β-catenin/Arm. Subcellular localization and the observation that kinesin motor activity is critical for the process suggest that the Kinesin-2/IFT-A complex acts via transport along cytoplasmic microtubules. Moreover, IFT140 interacts with a 50 amino acid region in Arm/β-catenin that is deleted in the stable and fully active mutant isoform ArmS10, suggesting that an inhibitor of nuclear translocation of Arm/β-catenin competes with IFT140. We thus identify a mechanism as to how the Kinesin-2/IFT-A complexes serve a critical function during Wnt/Wg-signaling, independently of their ciliary role, by stabilizing and promoting Arm/β-catenin translocation to the nucleus.20, and so we asked whether they associate with each other during Wg-signaling. We first tested this hypothesis by examining genetic interactions between single IFT-A proteins and knockdown of Klp64D . C96-Gal4 was used to drive expression at/near the D/V-boundary36. klp64DRNAi under C96-Gal4 control (C96 > klp64DRNAi) caused wing margin notching  or Distal-less (Dll)38. Whereas C96 > klp64DRNAi alone caused a marked reduction/loss in expression of both, Sens and Dll, in the D/V boundary region  largely restored expression of Sens and Dll  caused notching of adult wings and partial loss of Sens and Dll expression along the wing D/V-boundary  affected wing development in a similar manner to Klp64D  and a nonmotor accessory subunit (KAP3). In ary Fig. . Coexpreion Fig. , suggest64D Fig. , knockdo64D Fig. , suggestC96 > axnRNAi) induced Wg-signaling gain-of-function phenotypes: ectopic margin bristles and Sens expression near the D/V boundary  or kap3  partially suppressed the single axn knockdown phenotype, suggesting that kap3 and klp64D are required downstream of Axin and might act on stable Arm/β-catenin released from the destruction complex . Similarly, double mutant clones for ift122179; klp64Dk1 , we analyzed target gene expression in double mutant clones of Klp64D and axin. Single axin mutant clones displayed increased Arm levels and ectopic Sens expression near the wing D/V boundary , although Arm/β-catenin levels were still increased as in axnE77 single clones . Their association was confirmed by direct binding in in vitro assays. IFT140 was sufficient to pull down Arm/β-catenin  that is not involved in Wg-signaling  from in vivo samples. Proteins extracted from third instar wing imaginal discs of nin Fig. . Interaccts Fig. . We testcts Fig. . As negaGal4 driver; see below in next section for assay description). Confocal microscopy of salivary glands revealed intracellular puncta costained for Klp64D-HA and IFT140-myc  Arm levels are high, indistinguishable from single axn− clones. However, loss of Sens expression in these double mutant clones suggested that the Kinesin-2/IFT-A complex is required for Arm/β-catenin function downstream of the destruction complex, possibly in the process of translocation to the nucleus.Our data suggest that the Kinesin-2/IFT-A complex is required for Wg-signaling downstream of the destruction complex and thus affect the function and/or localization of Arm/β-catenin. In double mutant C135-Gal4/or C805-Gal441 to activate the pathway and anti-Arm staining used to analyze its levels and localization . To facilitate detection of potential nuclear Arm/β-catenin, the salivary glands were also treated with Leptomycin B  before fixation and detection  on Arm/β-catenin localization.It is very difficult, if not impossible, to document nuclear Arm in imaginal disc tissues due to the small size of cells. We therefore turned to the salivary gland assay mentioned above Fig. , with itift140 and klp64D revealed a striking loss of nuclear Arm staining in this Wg-signaling assay , and Klp64DT114A, a Thr-to-Ala transition at T114 which blocks ATP binding19 . Strikingly, expression of either klp64DΔABD or klp64DT114A resulted in a loss of nuclear Arm/β-catenin localization, and abnormally high cytoplasmic Arm levels as expected by Wg-signaling activation  Arm/β-catenin level was detected, but it was constrained to the cytoplasm even in presence of LepB treatment  effect in wings with extra margin bristles and associated increase in cells expressing Sens within the Gal4-driver domain along the D/V boundary . The difference between ArmS10 and Arm* is that the phosphorylation region, targeting it for degradation, is deleted in ArmS10, while Arm* carries a non-phosphorylable mutation in the CK1 phospho-target site, T52A . Both are stable, because they cannot be phosphorylated by destruction complex kinases44. Expression of Arm* with C96-Gal4 has a similar effect on wing development as ArmS10 with ectopic margin bristles and increase in cells expressing Sens . We tested this hypothesis directly in in vitro pull-down experiments between IFT140 and Arm34-87 using purified recombinant GST-fusion proteins. Whereas Klp64D or IFT144, as negative controls, did not pull-down Arm34-87, IFT140 displayed strong binding to Arm34-87 Fig. , indistiC96 > IFT40-GFP and C96 > ArmS10; >IFT140-GFP were immunoprecipitated using anti-GFP and probed with anti-Arm  or ArmS10. Extracts from Arm Fig. , revealiArm Fig. .Together, these data indicate that association of the Kinesin-2/IFT-A complex with Arm/β-catenin is required for efficient Wg-signaling by directly interacting with Arm/β-catenin and promoting its nuclear translocation to activate Wg target genes. As ArmS10 can translocate to the nucleus independent of Kinesin-2/IFT-A and Arm/β-catenin directly bound to IFT140 via the region deleted in ArmS10, these data indicate that Arm/β-catenin can interact with another factor/complex that inhibits its nuclear translocation. This means that the Kinesin-2/IFT-A complex is required to block the effects of an inhibitory factor for wild-type stable Arm to translocate to the nucleus. ArmS10 is missing the interacting region and thus does not require the protective function of the Kinesin-2/IFT-A complex in nuclear translocation, and ArmS10 can reach the nucleus passively or by other means (see Discussion).WT) MEFs revealed detectable levels of nuclear β-catenin upon Wnt3a treatment  had no effect on nuclear β-catenin localization following Wnt3a treatment  requirement for β-catenin nuclear translocation, and further demonstrates that presence of cilia is not required for Wnt-signaling and nuclear β-catenin localization, as ift88−/− MEFs completely lack cilia . We asked whether nuclear translocation of β-catenin depends on IFT140 in MEFs. Wild-type , β-catS10 and β-cat*  this function is specific to IFT-A and not observed with the IFT-B complex, and (iii) the requirement of amino acids deleted in the ArmS10/β-catS10 isoform for cytoplasmic retention is also conserved. In summary, these data support a non-ciliary mechanistic function of IFT-A complex components in Wnt-signaling that is evolutionarily conserved.These data support three conclusions: (i) the requirement of the Kinesin-2/IFT-A complex for nuclear translocation of β-catenin is conserved between 47, and it has been suggested to promote canonical Wnt/Wg-signaling independently of its ciliary role in Drosophila20. In the latter context it remained unclear whether (i) IFT-A also associates with microtubule structures via kinesin in the cytoplasm, (ii) how this complex mechanistically promotes Wg/Wnt–signaling, and importantly (iii) whether this IFT-A function is evolutionarily conserved in mammals. A function of Klp64D, the Drosophila Kif3A Kinesin-2 subunit, was also reported in the context of Wnt-signaling and Arm/β-Catenin trafficking in Drosophila19. This raised the possibility that IFT-A may interact with microtubules via Kinesin-2 and share a function in Wg/Wnt–signaling, possibly similar to Costal-2 (Cos2)/Kif7 in Hedgehog signaling, where Cos2 sequesters Ci/Gli proteins to microtubules49.The IFT-A complex is best known for its function in ciliary transportDrosophila and mammals to achieve high signaling levels. Note that while Sens expression is lost, low levels of signaling appear independent of this mechanism, as Dll, for example, is still expressed albeit at reduced levels. Kinesin-2 and IFT-A associate physically and act synergistically in vivo, and importantly the process requires the motor function of Klp64D, suggesting that movement along microtubules is essential. This is further supported by subcellular colocalization of IFT-A and Arm/β-Catenin punctae, which decorate filamentous structures, like “beads on a string” . Our data thus suggest that a Kinesin-2/IFT-A complex is required to transport Arm/β-catenin, independently of the cilium, and this function is conserved in mammals and promotes nuclear translocation of Arm/β-Catenin.Here we demonstrate the association between IFT-A, Kinesin-2, and Arm/β-Catenin and define a conserved mechanistic role of this complex in Wnt-signaling: the Kinesin-2/IFT-A complex is required to promote nuclear translocation of Arm/β-Catenin both in ng” Fig. . The non4. Although this pathway is highly conserved and fairly well dissected, how Arm/β-catenin is translocated to and retained in the nucleus remain poorly understood. It has been suggested that β-catenin is imported via a nuclear localization signal by interacting directly with nuclear pore proteins50. It has also been proposed that the β-catenin nuclear and cytoplasmic states are regulated by stimulation of both processes, Axin and APC retain β-catenin in the cytoplasm and TCF and BCL9 (a β-catenin co-activator) maintain β-catenin levels in the nucleus via multiple mechanisms51. It was further proposed that Rac1, JNK2, and β-catenin form a complex in the cytoplasm and JNK2 phosphorylates β-catenin to promote its nuclear translocation52, although it remains unclear how Rac1, normally linked to Wnt/Fz-PCP signaling54, is involved in canonical Wnt-signaling. We demonstrate a requirement of Kinesin-2/IFT-A for nuclear translocation of Arm/β-catenin. Strikingly, double mutant clones for either Kinesin-2 or IFT-A components with axin displayed high levels of stabilized nonjunctional Arm, same as in axin single mutant clones, but signaling (as detected in wing discs) and Arm/β-catenin nuclear translocation was markedly reduced, both in salivary gland Wg-signaling assay and mouse cells (MEFs). Given the function of Kinesin-2 as a motor protein, this suggested that the Kinesin-2/IFT-A complex promotes microtubule-based transport of Arm/β-catenin towards the nucleus. This hypothesis was corroborated with motor domain Kinesin-2 mutants, which displayed the same Wnt-signaling defects as Kinesin-2 or IFT-A null alleles. Importantly, this IFT-A function is conserved and independent of cilia.Wg/Wnt pathway activation induces relocation of Axin from the “destruction complex” to the plasma membrane, resulting in the release of Arm/β-catenin and its stabilization in the cytoplasm. As a consequence, Arm/β-Catenin enters the nucleus and stimulates transcription of Wnt/Wg target genes44). ArmS10 can enter the nucleus independently of the Kinesin-2/IFT-A function and, moreover, does not bind to the complex. In fact, the deleted 53 amino acid region of ArmS10 is sufficient to interact with IFT140 (IFT-A) on its own. This suggests that the small deletion in ArmS10 is critical for its differential behavior relative to wt Arm/β-catenin in terms of Kinesin-2/IFT-A requirements. The behavior of the equivalent mammalian β-catenin stable mutant isoforms is identical to Drosophila Arm/ArmS10, again supporting a fully conserved mechanism.Physical interaction studies indicate that the Kinesin-2/IFT-A complex directly interacts with Arm/β-catenin through IFT140, with IFT-A acting as a cargo adapter for Kinesin-2. The ArmS10 isoform, the stable “constitutively active” isoform of Arm/β-Catenin, is a small deletion within the N-terminal region, which is phosphorylated by the destruction complex to target it for degradation. Strikingly, ArmS10 behaves differently from other stable Arm/β-catenin proteins, which carry point mutations that block CK1 phosphorylation of Arm/β-catenin, but are otherwise full-length . ArmS10 and mammalian β-cateninS10 equivalent forms are indifferent to this mechanism and can translocate to the nucleus passively or through another mechanism, because the binding region for both factors, positive (IFT140) and antagonistic unknown protein, is eliminated.55 and our data in the Wg-signaling salivary gland assay are consistent with this, as almost all Arm/β-catenin is found in the nucleus in axnRNAi background, whereas more is retained in the cytoplasm upon pathway activation via Wg-expression  await further investigation.These observations raise two intriguing questions that will be the focus of future studies: (i) can the deleted residues of ArmS10/β-cateninS10 be developed as a therapeutic target to trap hyperactive β-catenin in the cytoplasm, and (ii) identification of the endogenous factor(s) that bind this region of Arm/β-catenin to inhibit nuclear translocation. Axin was suggested as a cytoplasmic anchor keeping Arm/β-catenin out of the nucleuscf. Fig. . HoweverThe Wnt canonical pathway is commonly activated by constitutive upregulation of wild-type or phosphorylation mutants of β-catenin that escape degradation via the destruction complex. With this study we have identified a process in the path that Arm/β-catenin requires on its way to the nucleus and one that will be of interest to biologists and clinical researchers alike. Our discovery that the IFT-A/Kinesin-2 complex plays a critical role in canonical Wnt-signaling via Arm/β-catenin identifies possible therapeutic targets for a wide spectrum of human malignancies, in which a hyperactive β-catenin is an oncogenic driver.GAL4 /UAS system was used for expression of RNAi and constructs, or in combination UAS-Dcr2 (where indicated). GAL4 driver for wing margin during wing development was C96-GAL4 (BL43343) or UAS-Dcr2; C96-Gal4 (BL25757) expressed around dorsal-ventral boundary of wing imaginal discs. Other GAL4 lines used were: tub-Gal4 and salivary gland-specific Gal4 line (PI)(2)C805C805 (BL6986) or (Pc135) (BL6978). All crosses were set up at 29 °C, unless otherwise indicated.The The following transgenic lines were used:UAS-IFT121GFP and UAS-IFT140GFP or oseg1179 (gifts from T. Avidor-Reiss)UAS-IFT121myc and UAS-IFT140myc UAS-Klp64DHA and UAS-Klp64DΔABD or UAS-Klp64DT114A (gifts from K.W. Choi)UAS-Arm* (gift from K. Cadigan)rempAl(2)21Ci-1 (gift from M. Kernan)tub-exdV5 (gift from R. Mann)UAS-Klp64D (BL32008)UAS-Klp64D RNAi (BL40945 and v103358)UAS-Kap3 RNAi (v4540 and v103548)UAS-IFT121 RNAi (oseg4) (v1098905)UAS-IFT122RNAi (oseg1) (v103598)UAS-IFT140 RNAi (rempA) (v31575)UAS-IFT 143 RNAi (v106366)UAS-Axin RNAi (BL31705)UAS-Axin GFP (BL7224)UAS-ArmS10 (BL4782)axine77 (BL17649)klp64Dk1 (BL5578)y w, hsflp; ubiGFP FRT2A, FRT82B/TM6b (BL5936)For tissue staining, primary antibodies used were:guinea pig anti-Sens ; rat anti-Dll (1:250 from Jun Wu); rabbit anti-IFT140 ; mouse anti-Arm ; rat anti-DE-cad ; rat anti-Dll (1:250); mouse anti-myc ; rabbit anti-myc ; chicken anti-GFP ; rabbit anti-RFP ; rabbit anti-Dlg ; mouse anti-HA ; rabbit-anti-β-catenin ; rabbit-anti-IFT88 ; rabbit anti-Arlb13 ; goat anti-γ-tubulin  and Hoechst 33342 .For immunoblotting, the following primary antibodies were used:mouse anti-Axin (1:500) (gift from Nusse); rabbit anti-Klp64D (1:500) (gift from Kwang Choi); mouse anti-Arm ; mouse anti-γ-tubulin ; mouse anti-GFP ; rabbit anti-IFT88 ; mouse anti-GST ; and rabbit anti-MBP ; rabbit anti-IFT140 ; rabbit anti β-catenin ; rabbit anti-laminb1 ; rabbit anti-Axin2 .All fluorescent secondary antibodies and HRP-coupled secondary antibodies were from Jackson Immuno Research Laboratories and used at 1:200.Imaginal wing discs were dissected at third instar larval stage in phosphate buffered saline (PBS) and fixed in PBS, 4% paraformaldehyde (PFA). Discs were washed two times in PBS 0.1% Triton-X100 (PBT), incubated in primary antibodies overnight at 4 °C. After washing in PBT, incubation with secondary antibodies was at room temperature for 2 h. Samples were mounted in Vectashield (Vector Laboratories). Wing disc images were acquired with a confocal microscope . Images were processed with ImageJ  and Photoshop .56, as implemented by the Microscopy Core facility.Salivary glands were dissected at the third instar larval stage in PBS and treated with 0.1% Leptomycin B (Sigma) in 10 min before fixation in PBS, 4% PFA. All subsequent steps were as described for wing imaginal discs. Colocalizations were performed using step size of 0.5× relative to the optical section thickness, according to the Nyquist theorem sampling criterionAnalyses of adult wings: wings were removed, incubated in PBS with Triton detergent, and mounted on a slide in 80% glycerol in PBS, and imaged using Zeiss Axioplan microscope. All adult images were acquired using Zeiss Axiocam color-type 412-312 camera and the Zeiss axiocam software.E. coli R2 cells (BL21 derivative) were transformed with plasmid constructs for fusion proteins MBP-Axin, MBP-Klp64D, MBP-Klp61F, MBP-Kap3 (Kap3cDNA (LD13052)), GST-IFT140, GST-Kap3, GST-Axin, and GST- Klp64D. Bacterial lysates were prepared as described in ref. 56. Equal amounts of blocked glutathione Sepharose 4B beads (Bioprogen) with GST, GST fusion protein, or beads alone were incubated in pull-down buffer , 10% glycerol, 0.1% Triton X100, 1 mM dithiothreitol (DTT) and protease inhibitor cocktail), 1× sample buffer was added, beads were boiled, and proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrophoretically transferred onto nitrocellulose, blocked in 5% skim milk (Bio-Rad) for 1 h, and incubated with primary mouse anti-GST (Santa Cruz) or rabbit anti-MBP antibody (Santa Cruz).For GST pull-downs, IPTG-inducible tub > AxinGFP, tub > IFT122GFP, tub > IFT140GFP  were precleared by incubating with protein A-sepharose beads (Thermo Scientific) for 1 h at 4 °C followed by centrifugation. A-sepharose beads were immunoprecipitated with specific antibodies at 4 °C for 1 h. Polyclonal anti-GFP antibody (Roche) was used. Immunoprecipitates were captured by protein A-sepharose at 4 °C in IP buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 100 mM NaCl, 0.05% Triton X100, 1 mM EDTA, 5 mM DTT, 10 mM NaVO4, 10% glycerol and protease inhibitor cocktail). Immunoprecipitates were resuspended in SDS sample buffer, boiled for 5 min, separated by SDS-PAGE, and transferred to nitrocellulose for immunoblotting. Protein was detected by enhanced chemiluminescence (Millipore).Lysates from 120 wing imaginal discs of Ift88 were obtained from mice containing the loxP and null Ift88 alleles57 crossed to the CAGG-creERTM mouse58 and maintained on a C57BL/6 background. MEFs were collected from E13.5 mouse embryos killed by cervical dislocation and dissected into cold Dulbecco's modified Eagle's medium (DMEM). Embryonic tissue not including the head and visceral organs was incubated in 0.25% trypsin for 10−15 min at 37 °C. Dissociated MEFs were grown in DMEM medium supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. Cells at 80−85% confluence were trypsinized, washed with PBS and plated for each experiment. To delete Ift88 in CreER; Ift88−/fx MEFs, 50 nM 4-hydroxytamoxifen (4OHT) solubilized in ethanol was added to the medium for 24 h. Ethanol-treated CreER; Ift88−/fx MEFs were used as control. Ift140−/− MEFs were a gift from Dr. Gregory Pazour (University of Massachusetts).MEFs carrying a tamoxifen-inducible knockout of 59. Cells were then fixed in cold methanol for 15 min at −20 °C and labeled with primary antibodies for β-catenin diluted 1:100 in 2% bovine serum albumin/PBS for 2 h, washed in PBS, incubated with fluorescent secondary antibodies  diluted 1:500 in PBS for 1 h and mounted with VECTASHIELD® mounting medium containing 4′,6-diamidino-2-phenylindole.For Wnt3A-induced β-catenin nuclear translocation, MEFs were grown at 70% confluence in DMEM medium supplemented with 10% fetal bovine serum and treated with Wnt3A-conditioned medium for 14−16 h as described in ref. Z-project on Image J), and a standardized region of interest at 4.705 μm × 4.843 μm avoiding cell membrane regions. Intensity ratios (nucleus/cytoplasm), standard deviations, and Student’s two-tailed test were performed using MicrosoftⓇ Excel.Confocal imaging was performed using a Zeiss LSM 880 Airyscan mounted on a Zeiss Axio-Observer inverted microscope. Z-stacks of 6 (1.083 µm) to 12 optical slices (4.362 µm) at 8-bit were captured and analyzed via ImageJ-Fiji. β-catenin translocation was evaluated through optical density assays of mean gray values between the nucleus and cytoplasm of individual cells. To maintain uniformity in all acquisitions, measurements were obtained under an image size of 1024 × 1024 pixels, FITC (green) channel, maximum projection  and gently homogenized for 1 min on ice using homogenizer. Samples were transferred to microfuge tube and kept on ice for 30 min at 4 °C. Supernatants were cleared by 20 min centrifugation at 4 °C and saved as the cytoplasmic fraction. The pellet or nuclei fraction was washed again by the lysis buffer above without protease inhibitor cocktail. Nuclear fraction was incubated for 30 min at 4 °C with nuclear extract buffer containing 20 mM HEPES pH 7.9, 15 mM MgCl2, 0.5 M NaCl, 1 M EDTA, 20% glycerol, 1% TritonX100, protease inhibitor cocktail and sonicated for 10 min after incubation. The supernatant was collected by 30 min centrifugation at 4 °C as the nuclear fraction. Protein extracts were boiled for 5 min at 95 °C in SDS-sample buffer, separated by 10% SDS-PAGE gel and transferred to nitrocellulose membrane. Protein levels were analyzed by immunoblotting with the corresponding antibodies.Wnt3A stimulated or unstimulated MEFs were gently washed with PBS buffer and cells were minced on ice by sharp scalpel and collected by centrifugation at 5000 rpm/4 °C. All samples were resuspended in 500 µl buffer comprising 250 mM sucrose, 50 mM Tris-Cl pH 7.4, 5 mM MgClTM Reagent (Invitrogen A12621). Cells were collected 12 h after transfection. The nuclear fraction assay was performed as mentioned above.For the β-catenin mutant isoform nuclear fraction experiments: The β-cateninFL-GFP, β-cateninS10-GFP, β-catenin*-GFP and β-cateninS33Y-GFP constructs were made from the original plasmid MSCV- β-catenin-IRES-GFP (Addgene Plasmid #14717) and pMXs- β-catenin-S33Y (Addgene Plasmid # 1371). All constructs were transfected into MEFs by Lipofectamine LTX & PlusAll the original western blot gels are provided in Supplementary Fig. Supplementary InformationPeer Review File


Because DHA has low toxicity and costs, it might be regarded as an important drug for fracture treatment and tissue engineering.Dihydroartemisinin (DHA), which is considered to be one of the active compounds within Artemisia annua, has extensively been used in recent years as the most effective drug against malaria, having many biological functions including anticancer, antifungal, and immunomodulatory activities. However, DHA plays a role in the regulation of the proliferation and human mesenchymal stem cells (hMSCs) osteogenic differentiation that remains unknown. We explored DHA's effect on hMSCs' proliferation as well as the osteogenic differentiation, together with its underlying mechanisms of action. We showed that DHA enhanced osteogenic differentiation but had no significant effect on hMSCs' proliferation. It probably exerted its functions through the signaling pathways of ERK1/2 as well as Wnt/ A great deal of fractures occurs every year due to trauma or pathology. Though a majority of fractures successfully heal in some months, approximately 5-10% of fractures show nonunion or delayed healing , resultiβ (TGF-β) [Recent findings have shown that certain cell types, such as human mesenchymal stem cells (hMSCs), can accelerate bone regeneration. MSCs possess multidirectional differentiation potential, including osteogenesis, adipogenesis, and chondrogenesis, making them hopeful seed cells to adopt within tissue engineering . MSCs po (TGF-β) , 6. The  (TGF-β) .Dihydroartemisinin (DHA), which is considered to be one of the active compounds within Artemisia annua, is classified into the family of sesquiterpene lactones. DHA shows important effectiveness against fibrosis, parasitic diseases, and malaria with minimal toxicity –10. The Recently, we characterized DHA's effect on hMSCs' proliferation as well as osteogenic differentiation and the underlying mechanism of action. Based on our findings and the low toxicity of DHA, it may have important applications in clinical settings.hMSCs had been gained from three healthy donors' femoral bone marrow  at the Second Affiliated Hospital of Zhejiang University. The informed consent was given by the three donors before assembling their bone marrow. The experimental protocol adhered to the ethical standards of the Declaration of Helsinki and to global and domestic guidelines and was permitted by the institutional review board of the writer. hMSCs were cleaned from the femoral fracture fixation's intramuscular nailing, following a formerly depicted strategy . The fraμM, 1 μM,100 nM, 10 nM or 0, and 1 nM DHA in the growth medium of hMSC for one day, two days, or three days and cultured within 96-well plates (103/well). The Cell Counting Kit-8 (CCK-8) assay had been adopted for measuring the proliferation of cells which follow the instructions of the manufacturers. In brief, the medium had been substituted with 10 μL of CCK-8 as well as 100 μL of hMSC growth medium at the appointed time points. After incubation for approximately four hours at room temperature, a microplate reader had been adopted for measuring the absorbance at 450 nm.hMSCs had been treated with 10 Before the experiment, dimethyl sulfoxide (DMSO) was used to dissolve DHA immediately before use, and the final concentration of DMSO did not exceed 0.1%.4/cm2 in 12-well culture plates and cultured with osteoinductive medium to induce osteogenic differentiation . The cells had been kept by adding fresh osteogenic induction medium every two to three days.The cells had been seeded at a density of 3 × 10μl of liquid was collected and transferred to 96-well plates. The absorbance was measured at 560 nm by using a microplate reader. The readings had been normalized to the overall concentrations of the proteins.ARS was used to evaluate mineral deposition. In brief, cells had been fixed for ten to twenty minutes under 4% paraformaldehyde at normal temperature, washed for three times by using the distilled water, treated for fifteen to thirty minutes by using ARS (0.5%) at normal temperature, and at last washed for three times by using distilled water. The stain was observed by incubating with 10% cetylpyridinium chloride  for 1 h. Approximately 200 β-catenin , p-ERK , runt-related transcription factor 2 , and glyceraldehyde 3-phosphate dehydrogenase . After washing within Tris-buffered saline including Tween 20 (TBST) three times (10 minutes each), the membranes had been incubated by using horseradish-peroxidase-conjugated goat anti-rabbit IgG  as a secondary antibody at normal temperature for an hour. After being washed with TBST for three times (ten minutes each), the immunoreactive bands had been noticed adopting a developed chemiluminescent detection reagent (Millipore) and further visualized through the explosion of the blot to X-ray film (Bio-Rad) for 0.2-2 minutes. The expression of proteins had been quantified through the determination of the ratio of the proteins' absorbance to that of the inner GAPDH control.Cells had been separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, lysed in RIPA buffer, and then transferred to a PVDF membrane. After being blocked for one to two hours at room temperature within 10% nonfat milk, the membranes had been incubated at 4°C overnight with antibodies particular to p-μM DHA as well as OIM for three to seven days after being implanted into 6-well plates (4 × 104 cells/well). First, RNAiso reagent  was used to extract the total cellular RNA, which was quantified through the measurement of the absorbance at 260 nm. Second, reverse transcription had been implemented by adopting PrimeScript RT Master Mix (Takara) in accordance with the instructions of the manufacturer. Every gene transcript had been quantified by qPCR adopting Power SYBR Green PCR Master Mix (Takara) on an ABI StepOnePlus System. Next, the target genes' mRNAs were quantified according to the following conditions: 95°C for half a minute followed by 45 cycles of 60°C for half a minute and 95°C for 5 seconds. The primer sequences adopted have been presented within hMSCs were cultured by using 1 μM DHA for 72 hours before being seeded within a 12-well plate. Fluorescence microscopy  was used to measure RUNX2, collagen α1 type I (COL1A1), p-ERK, and p-β-catenin. Briefly, cells had been settled at normal temperature within 4% paraformaldehyde for at least 15 min, washed with distilled water three times, blocked in 5% bovine serum albumin as well as 0.03% Triton X-100 for half an hour, washed with distilled water three times, and incubated overnight with anti-p-β-catenin , anti-p-ERK , anti-COL1A1 , or anti-RUNX2 . Cells had been incubated subsequently with a fluorescence-conjugated secondary antibody (Beyotime) for an hour. Cell nuclei had been stained with 4′, 6-diamidino-2-phenylindole for 5 minutes. Cell immunofluorescence was noticed adopting fluorescence microscopy (Leica).The cells had been cultured with OIM and 1 Statistical analysis had been implemented adopting SPSS statistical software for Windows . Statistical significance had been decided adopting the Wilcoxon nonparametric experiments as well as Mann–Whitney. The resources have been shown as the means ± standard deviation. A value of p ≤ 0.05 had been regarded to imply statistical significance.μM). DHA treatment at 1 nM to 1 μM did not affect greatly the proliferation of hMSCs . Basal culture medium had been adopted for treating hMSCs for a day, two days, or three days with different concentrations of DHA  exhibited less calcium deposition at day 14 after osteogenic differentiation , or U0126 alone for 3 days. An important increase was found in the expression of p-ERK as well as RUNX2 in the 1 μM DHA group, and treatment with U0126 alone inhibited RUNX2 and p-ERK expression  exhibited less calcium deposition at day 14 after osteogenic differentiation  [μM, with no cytotoxic effects on bone marrow-derived macrophages. Hence, the effects of DHA on hMSCs may be dose dependent. When considering the inhibition of proliferation at high concentrations of DHA and the acceleration of osteogenic differentiation, 1 μM DHA was selected for the later tests of osteogenic differentiation  . FurtherμM)  showed t00 μM . Fβ, ERK1/2, and BMP-Smad pathways [β and ERK1/2 had some relationships with DHA's capacity for inducing MSC osteogenic differentiation. The pathway of ERK is involved in the protein kinase pathway which is activated by mitogen. The adipose-derived stem cells' osteogenic reaction would be decreased by ERK1/2 signaling pathway inhibition [There is a relationship between MSC osteogenic differentiation and numerous signaling pathways, containing the phosphatidylinositide-3 kinase (PI3K)/Akt, Wnt/pathways –24. In thibition . Neverthhibition  pointed β-catenin pathway. Being a member of a family of secreted proteins, Wnt may activate β-catenin at a downstream location and bind to seven-pass transmembrane Frizzled receptors [β-catenin is considered to be rather significant in cell proliferation, differentiation, growth, regeneration, self-renewal, and the determination of cell fate [β-catenin had been induced essentially during fracture healing and has thus been considered to be osteoinductive. The signaling pathway of Wnt is included in promoting the impacts of osteoblast as well as osteoblastogenesis, leading to improvements in adult bone mass [β-catenin and RUNX2 expression. Using DKK-1 to control β-catenin activation, calcium deposition and the expression of RUNX2 were slightly inhibited. Therefore, the pathway of Wnt/β-Catenin has been partly involved in the hMSCs' osteogenic differentiation induced by DHA.Another significant pathway that is included in osteogenesis is the Wnt/eceptors . The sigell fate . Krishnaone mass . In our μM DHA. An increasing number of researches have been warranted for illustrate the impacts of diversified concentrations of DHA. Second, we indicated that both the signaling pathways of Wnt/β-catenin and ERK had been involved in the DHA-induced osteogenic differentiation of hMSCs, but it is still not clear if there is crosstalk between Wnt/β and ERK signaling pathways existing. Third, we did not conduct an in vivo experiment to prove that DHA promoted fracture healing.There are some limitations about the research. First, we assessed hMSCs' potential mechanisms as well as their osteogenic differentiation just by using a concentration of 1 β as well as ERK1/2 (In conclusion, our study indicates that DHA has no significant effect on the proliferation of hMSCs but enhances osteogenic differentiation via the signaling pathways of Wnt/s ERK1/2 .


As acetylation and deacetylation regulate β-catenin stability, we searched for histone acetyltransferases (HATs) or histone deacetylases (HDACs) affecting δ-catenin acetylation status and protein levels. We showed that p300/CBP-associated factor (PCAF) directly bound to and acetylated δ-catenin, whereas several class I and class II HDACs reversed this effect. Unlike β-catenin, δ-catenin was downregulated by PCAF-mediated acetylation and upregulated by HDAC-mediated deacetylation. The HDAC inhibitor trichostatin A attenuated HDAC1-mediated δ-catenin upregulation, whereas HAT or autophagy inhibitors, but not proteasome inhibitors, abolished PCAF-mediated δ-catenin downregulation. The results suggested that PCAF-mediated δ-catenin acetylation promotes its autophagic degradation in an Atg5/LC3-dependent manner. Deletions or point mutations identified several lysine residues in different δ-catenin domains involved in PCAF-mediated δ-catenin downregulation. PCAF overexpression in prostate cancer cells markedly reduced δ-catenin levels and suppressed cell growth and motility. PCAF-mediated δ-catenin downregulation inhibited E-cadherin processing and decreased the nuclear distribution of β-catenin, resulting in the suppression of β-catenin/LEF-1-mediated downstream effectors. These data demonstrate that PCAF downregulates δ-catenin by promoting its autophagic degradation and suppresses δ-catenin-mediated oncogenic signals. Structurally, it contains 10 Armadillo (ARM) repeat domains, whereas β-catenin has 13 ARM repeat domains. Moreover, β- and δ-catenin conduce the adhesive potential of cadherin-based cell-cell contacts and share similar binding partners in signaling pathways including E-cadherin4. δ-Catenin promotes the fragmentation of E-cadherin , leading to increased total β-catenin protein levels and nuclear distribution, and resulting in the activation of β-catenin/LEF-1-mediated transcription5. These findings suggest that δ- and β-catenin are closely related and share similar signaling functions.The catenins  are cytoplasmic proteins which are related to the Drosophila Armadillo protein. β-Catenins are components of adherens junctional cadherin complex by bind to the cytoplasmic tail of E-cadherin and can transduce intracellular signal to the nucleus in the Wnt signaling pathway. The p120-catenin family  is homologous to both β- and γ-catenin and is a substrate of tyrosine kinases with cadherin/catenin complex at adherens junctions7. δ-Catenin is also overexpressed in various human malignancies, including prostate8, brain9, breast10, lung11, ovary12, esophagus13, and colorectal cancer14. In prostate cancer, δ-catenin accumulation promotes cancer cell growth and tumorigenesis by altering the cell cycle and the expression profiles of survival-related genes8. Furthermore, δ-catenin promotes prostate tumor growth by increasing angiogenesis through the upregulation of HIF-1α and VEGF15. Human prostate cancer cells overexpressing δ-catenin show an increase in multi-layer growth and substantial processing of plasma membranous E-cadherin, suggesting that δ-catenin plays a role in prostate cancer progression by inducing E-cadherin processing and thereby the release of β-catenin and increased oncogenic signaling5. Increased β-catenin translocates to the nucleus, where it functions in transcriptional regulation through interactions with transcription factors of the LEF-1/TCF family16.δ-Catenin is abundantly expressed in the developing neurons, which suggests the involvement of it in neuronal progenitor cell migration and dendrite development17. Post-translational modifications such as phosphorylation, acetylation, methylation, and ubiquitination modulate the activity or stability of proteins19. The cellular protein degradation machinery includes the ubiquitin-proteasome pathway and the endosome-lysosome pathway, which control the degradation of the majority of eukaryotic proteins. We previously showed that δ-catenin is ubiquitinated and targeted for degradation by the ubiquitin-proteasome pathway4. However, the molecular mechanism of δ-catenin degradation mediated by the lysosomal pathway remains unknown.Transcription is the first step in gene expression leading to the generation of a functional protein product21 and regulatory T cells22. The acetyltransferase p300/CBP-associated factor (PCAF) catalyzes β-catenin acetylation and promotes its stability in cells21. PCAF is a transcription cofactor that possesses intrinsic histone acetyltransferase (HAT) activity23. PCAF-mediated acetylation affects different biological functions, such as transcriptional activity, stability, and subcellular localization. PCAF regulates p21 transcription by catalyzing the stress-induced acetylation of histone H3, and acetylates the tumor suppressor p53 in response to DNA damage25. In the present study, we show that PCAF acetylates and significantly downregulates δ-catenin by promoting its degradation via the autophagosomal pathway. Our results suggest that targeting acetylation may be an effective strategy in δ-catenin-associated carcinomas.To clarify the mechanisms underlying the regulation of δ-catenin and the maintenance of adequate δ-catenin protein levels in cells, we investigated δ-catenin stabilization through acetylation. Acetylation mostly results in protein stabilization, which is the case for β-catenin21. To determine whether acetylation is involved in the regulation of δ-catenin, several HAT enzymes including p300, PCAF, and MOZ were co-transfected with δ-catenin, and changes in the level of δ-catenin were analyzed by immunoblotting. Among the tested HAT enzymes, PCAF significantly downregulated δ-catenin, whereas p120-catenin remained unchanged  in δ-catenin that are acetylated by PCAF. Several deletion mutants of δ-catenin including ΔN85–325, ΔC207, ΔN&ΔC, 1–690, and 691–1040 were generated in previous studies. 4 Fig. . In the . 4 Fig. . However. 4 Fig. . Deletio. 4 Fig. . The pre. 4 Fig. . Also, t. 4 Fig. .Figure 4To determine whether PCAF affects the expression and thus the oncogenic activity of endogenous δ-catenin in prostate cancer cells, several prostate cancer cell lines with different metastatic potential, including CWR22Rv-1 (Rv), DU145, PC3, and C42, were analyzed. As shown in Fig. 5. In the present study, PCAF overexpression dose-dependently decreased E-cadherin processing in Rv/δ cells  PCAF and HDACs regulate the acetylation status of δ-catenin; (2) acetylated δ-catenin is degraded through the Atg5/LC3-mediated autophagosomal pathway; (3) multiple Lys residues in the N-terminus of δ-catenin are involved in PCAF-mediated δ-catenin acetylation; (4) PCAF-mediated δ-catenin degradation decreases the growth and motility of prostate cancer cells; and (5) PCAF-mediated δ-catenin degradation inhibits E-cadherin processing and inactivates β-catenin-mediated signaling. The present results indicate that acetylation-dependent autophagic δ-catenin degradation by PCAF reduces oncogenic δ-catenin signaling, as indicated by E-cadherin processing and β-catenin-mediated signals, thereby inhibiting the progression of prostate cancer Fig. .30. Studies show that PCAF acetylates and stabilizes β-catenin, upregulating the expression of Wnt-responsive genes31. The present study is the first to provide evidence that PCAF acetylates δ-catenin leading to its downregulation, whereas HDACs deacetylate and upregulate δ-catenin. Acetylation of Lys residues protects them from ubiquitination, which stabilizes proteins in most cases32. There are few examples of acetylation resulting in protein destabilization. Acetylation destabilizes fatty acid synthase (FASN), which inhibits de novo lipogenesis and tumor cell growth, suggesting that targeting FASN acetylation could be an anticancer strategy33. ARD1-mediated acetylation destabilizes hypoxia-inducible factor 1 (HIF-1α)34. These studies illustrate the effect of acetylation on promoting ubiquitination-dependent proteasomal degradation of proteins, and suggest that acetylation-dependent destabilization plays an important role in tumorigenicity. The results of the present study support that acetylation can mediate protein destabilization. However, in contrast to previous reports, acetylation of δ-catenin promoted autophagic degradation and decreased oncogenic δ-catenin signaling.δ-Catenin shares many characteristics with β-catenin, and the two proteins have common binding partners4. Phosphorylation of δ-catenin by GSK-3β at Thr1078 enables β-TrCP-mediated ubiquitination of δ-catenin, targeting δ-catenin for degradation by the ubiquitin-proteasome pathway. In a previous study, the degradation of δ-catenin by the lysosomal pathway was suggested by the increased level of δ-catenin in response to CQ treatment4; however, the exact mechanisms underlying lysosomal δ-catenin degradation remained elusive. In the present study, treatment with the proteasome inhibitor MG132 did not restore PCAF-mediated δ-catenin downregulation. However, treatment with autophagy inhibitors with various mechanisms of action, such as blocking the early step of autophagosome formation (3-MA), the maturation (BafA1), and the autophagic process (CQ and E-64)26, restored PCAF-mediated δ-catenin downregulation, suggesting that δ-catenin acetylation is required for the initial step of the autophagosomal degradation process. Consistently, conditional knockdown of Atg5 suppressed PCAF-mediated δ-catenin downregulation, and this effect was reversed when Atg5 expression was restored. Overexpression of LC3 further decreased δ-catenin levels, and PCAF increased the colocalization of LC3 with δ-catenin. These results indicate that acetylated δ-catenin by PCAF can undergo degradation via the autophagosomal pathway, which is related to the expression of the autophagosomal marker Atg5/LC3. Autophagosomal targeting of non-histone proteins by acetylation was reported previously35. Acetylation of mutant Huntingtin (Htt) at Lys444 facilitates trafficking of mutant Htt into autophagosomes, increasing the clearance of Htt by autophagy. The identification of the acetylation site on δ-catenin is important. However, despite a series of experiments with deletion and arginine substitution mutants, we were only able to show that multiple Lys residues in the N-terminus and ARM repeat domain of δ-catenin are responsible for PCAF-mediated δ-catenin downregulation.Studies indicate that the degradation of δ-catenin is mediated by the ubiquitin-proteasome pathway37. Our previous study demonstrated that prostate cancer cell lines overexpressing δ-catenin display noticeable multi-layer growth, E-cadherin processing, and nuclear β-catenin localization, with the subsequent activation of β-catenin downstream target genes5. PCAF-mediated δ-catenin acetylation/destabilization is involved in prostate cancer progression by suppressing E-cadherin processing and β-catenin-mediated oncogenic signals, thereby inhibiting prostate cancer cell motility and tumorigenesis. The present investigation of the mechanism of δ-catenin acetylation provides a new perspective on the process of δ-catenin degradation and improves our understanding of the effects of δ-catenin on E-cadherin or β-catenin, which is implicated in cancer progression.δ-Catenin is overexpressed in several carcinomas, and its ability to interact with E-cadherin underlies the oncogenic activity of δ-catenin in epithelial-derived carcinoma cells38. These findings highlight the complexity of nucleosome modification associated with proto-oncogene induction, and identify HATs as key factors in this process. PCAF is also involved in virus-induced tumorigenesis. The oncogenic effect of human papillomavirus E7 (zinc-finger mutation) is dependent on PCAF and its deregulated HAT function in tumor development39. However, PCAF also functions as a tumor-suppressor gene, and its loss of function causes tumorigenesis. PCAF can induce cell apoptosis by modulating the GLI1/BCL-2/BAX axis or by acetylating histone H4 and inactivating AKT signaling, which in turn suppresses hepatocellular carcinoma progression41. PCAF-mediated acetylation of the transcriptional factor HOXB9 suppresses lung adenocarcinoma progression42. Studies suggest that PCAF modulates the activity or stability of several oncogenes or tumor suppressors through the acetylation of histones or transcription factors, consequently impacting cancer progression. The present study demonstrated that PCAF suppresses δ-catenin-mediated oncogenic signals by promoting δ-catenin degradation through the autophagy pathway, thereby suppressing prostate cancer cell growth and motility.The results of the present study indicate that PCAF inhibits the progression of carcinoma by promoting the acetylation-mediated degradation of δ-catenin and the suppression of its oncogenic signaling. However, PCAF affects non-histone proteins as an acetyltransferase involved in cancer, and acts as both oncogene and tumor suppressor. PCAF can function as a cofactor for several proto-oncogenes. PCAF possesses intrinsic ubiquitination activity that is critical for the control of Hdm2 expression levels, and thus p53 function43. The deletion mutants of δ-catenin  and the point mutants of δ-catenin, in which lysine (Lys) residue(s) are substituted for arginine, were generated using a PCR-based exchange site-directed mutagenesis kit . All constructs were confirmed by sequencing. The p300, PCAF, and monocytic leukemic zinc finger (MOZ) expression plasmids, and shPCAF, were kindly provided by Prof. Nacksung Kim , and histone deacetylase (HDAC) 1–9 expression plasmids were kindly provided by Prof. Hyun Kook . GFP-tagged LC3 expression plasmids were kindly provided by Prof. Kwang Youl Lee .The construction of N-terminal GFP-tagged δ-catenin full length (FL), ∆N85–325, ∆C207, ∆N85–325&∆C207, 1–690, and 691–1040 in pEGFP-C1 and RFP-tagged δ-catenin was previously describedThe following antibodies were used in the present study: anti-δ-catenin , anti-GFP , anti-tubulin , anti-lamin B , anti-ß-actin (Santa Cruz Biotechnology), anti-flag (Sigma-Aldrich), anti-PCAF , anti-E-cadherin (BD Bioscience), anti-acetylated-Lys , anti-p120-δ-catenin (BD Bioscience), anti-autophagy-related protein 5 (Atg5) , anti-myc (Santa Cruz Biotechnology), and anti-cyclin-D1 .27 and cultured in DMEM containing 10% (v/v) FBS with or without 10 ng/ml doxycycline . Cells were cultured in 5% CO2 in a humidified atmosphere at 37 °C. HEK293T cells were transfected with X-tremeGENE 9 DNA Transfection Reagent , and M6A and PC3 cells were transfected with Lipofectamine Plus reagent  according to the manufacturer’s instructions. For protein stability assay, cycloheximide (Sigma-Aldrich) dissolved in DMSO was used.Human embryonic kidney HEK293T cells were cultured in DMEM  supplemented with 10% fetal bovine serum (Gen Depot) and 1% penicillin-streptomycin solution. The human prostate cancer cells CWR22Rv-1 (RV1), DU145, C42, and PC3 were cultured in RPMI1640 culture medium (Gen Depot) supplemented with 10% fetal bovine serum (Gen Depot) and 1% penicillin-streptomycin solution. Tet-Off Atg5 conditional knockout m5–7 mouse embryonic fibroblasts (MEFs) were kindly provided by Dr. N. Mizushima 4. Immunoprecipitation was performed using lysates from transfected cells that were incubated with primary antibodies overnight at 4 °C and pulled down with Protein A/G Sepharose  for 3 h. The immunoprecipitated proteins were washed twice with the same buffer, and bound proteins were resolved by SDS-PAGE followed by immunoblotting.Immunoblotting was performed as previously described3 human prostate cancer  cells were plated at least in triplicate in 96-well plates and transfected with PCAF-expressing plasmid or empty vector. After incubating 72 h, 0.5 mg/ml MTT was added to each well and incubated for an additional 4 h. Then, the blue MTT formazan precipitate was dissolved in 100 μl of DMSO. The absorbance at 540 nm was measured on a multi-well plate reader.The MTT  assay was used to measure cell viability. In total, 2 × 1044. After transfection, the cells were seeded in 6-well plates at a density of 3000 cells/well and cultured for 9–14 days until visible colonies appeared. Then, the cells were stained with crystal violet, and colonies containing more than 50 cells were counted. The pixel intensity of the colony area was measured by the IMT iSolution software  in randomly selected microscope fields in each plate. To measure the percent area of colonies, the pixel amount of each colony area was normalized to pixel × pixel square. Data represent the mean of three experiments.The clonogenic assay was performed as previously described5 cells/well in RPMI1640/DMEM containing 0.2% bovine serum albumin (BSA) in the upper compartment of the chamber. RPMI1640/DMEM containing 10 μg/ml fibronectin was then added to the lower chamber as a chemotactic agent. After 24 h of incubation, upper chamber cells were fixed with a Diff Quik kit . The cells inside the chamber were removed from the membrane with a cotton swab, and the cells adhering to the underside of the membrane were stained and counted under a light microscope (five fields per chamber). Quantification of migrated and invaded cells was performed as previously described45. Each assay was repeated in three independent experiments. The results are expressed as the mean number of cells migrating per high-power field.Migration and invasion assays were performed in Transwell chambers  with 8 μm pore size polycarbonate membranes. For invasion assays, the upper chamber was coated with 1% gelatin. Cells transfected with flag-PCAF or empty vector were plated at a density of 2–2.5 × 1046. Extracts of cytoplasm and nuclear were separated using the NE-PER nuclear and cytoplasmic extraction kit . Briefly, harvested cells were resuspended in ice-cold CER I, followed by ice-cold CER II. Then, lysates were homogenized and centrifuged at 16,000 g for 5 min at 4 °C, and the supernatant was collected as the cytoplasmic fraction. The remaining pelleted lysates were resuspended in ice-cold NER within 40 min and centrifuged at maximum speed, and the supernatant was collected as the nuclear fraction. All extracts were stored at −80 °C until use.Cell fractionation was performed as previously describedBriefly, total RNA was isolated from human prostate cancer cells transfected with flag-PCAF or empty vector using RNAiso Plus  according to the manufacturer’s instructions. 1 μg of total RNA from each group of transfected cells was synthesized to cDNA using a M-MLV Reverse Transcriptase Kit (Invitrogen) and SYBR green . The qRT-PCR reaction and data analysis were performed using CFX .Data are expressed as the mean ± SD. Results comparing three or more samples were analyzed using analysis of variance and Bonferroni’s tests; comparisons between two samples were analyzed using Student’s t-tests. Probabilities of p < 0.05 were considered statistically significant.Dataset 1


N-glycosylated proteins is upregulated in a wide range of malignancies including ovarian cancer. Prior studies have shown that ST6Gal-I-mediated sialylation of select surface receptors remodels intracellular signaling to impart cancer stem cell (CSC) characteristics. However, the mechanisms that contribute to ST6Gal-I expression in stem-like cancer cells are poorly understood.The ST6Gal-I glycosyltransferase, which adds α2–6-linked sialic acids to SOX2 and ST6GAL1 are located within the same tumor-associated amplicon, 3q26, and these two genes exhibit coordinate gains in copy number across multiple cancers including ~ 25% of ovarian serious adenocarcinomas. In conjunction with genetic co-amplification, our studies suggest that Sox2 directly binds the ST6GAL1 promoter to drive transcription. ST6Gal-I expression is directed by at least four distinct promoters, and we identified the P3 promoter as the predominant promoter utilized by ovarian cancer cells. Chromatin Immunoprecipitation (ChIP) assays revealed that Sox2 binds regions proximal to the P3 promoter. To confirm that Sox2 regulates ST6Gal-I expression, Sox2 was either overexpressed or knocked-down in various ovarian cancer cell lines. Sox2 overexpression induced an increase in ST6Gal-I mRNA and protein, as well as surface α2–6 sialylation, whereas Sox2 knock-down suppressed levels of ST6Gal-I mRNA, protein and surface α2–6 sialylation.Herein, we identify the master stem cell transcription factor, Sox2, as a novel regulator of ST6Gal-I expression. Interestingly, SOX2 and ST6GAL1 are coordinately amplified in cancer cells, with the Sox2 protein then binding the ST6GAL1 promoter to further augment ST6Gal-I expression. Our collective results provide new insight into mechanisms that upregulate ST6Gal-I expression in ovarian cancer cells, and also point to the possibility that some of the CSC characteristics commonly attributed to Sox2 may, in part, be mediated through the sialyltransferase activity of ST6Gal-I.These data suggest a process whereby  Ovarian cancer is the deadliest gynecological malignancy due primarily to late detection and development of drug resistance , 2. WhilN-glycosylated proteins destined for the cell surface or secretion [One of the predominant glycosyltransferases upregulated in ovarian and other cancers is ST6Gal-I, a sialyltransferase that adds α2–6-linked sialic acids to ecretion –8. High ecretion , 10, theecretion –17. We aecretion , invasivecretion , 19, tumecretion , and resecretion , 20–25.In addition to CSCs, ST6Gal-I expression is enriched in select stem/progenitor niches within normal tissues, such as the basal layer of epidermis and fallopian tube fimbriae , 18. In SOX2 and ST6GAL1 genes lie within the same amplicon, referred to as “3q26”, which spans from 3q26-3q29 [SOX2, PI3KCA and ECT2 [ST6Gal-I is dramatically upregulated upon transduction of somatic cells with the four Yamanaka factors to generate induced pluripotent stem cells (iPSCs) , 36, andq26-3q29 –50. The q26-3q29 . Amplifiq26-3q29 , 51. Whiand ECT2 , the potSOX2 and ST6GAL1 and showed that these two genes are coordinately amplified in patient specimens across a wide range of cancer types, including ovarian cancer. Furthermore, protein levels of Sox2 and ST6Gal-I were found to strongly correlate in established ovarian cancer cell lines. We next interrogated a possible direct interaction between Sox2 and ST6Gal-I by performing Chromatin Immunoprecipitation (ChIP) assays, which revealed that Sox2 binds to sequences proximal to the ST6GAL1 P3 promoter. To confirm that Sox2 regulates ST6Gal-I expression, Sox2 was knocked-down in Pa-1 ovarian cancer cells, which have high endogenous ST6Gal-I, or overexpressed in Skov3 ovarian cancer cells, which have relatively low ST6Gal-I expression. Sox2 knock-down reduced ST6Gal-I mRNA and protein expression, and correspondingly diminished surface α2–6 sialylation, whereas Sox2 overexpression increased ST6Gal-I mRNA and protein, and enhanced surface sialylation. These data suggest that Sox2 is a key transcription factor responsible for upregulating ST6Gal-I expression in ovarian cancer cells.In the current study we investigated a novel function for Sox2 in regulating the expression of ST6Gal-I. We first analyzed The Cancer Genome Atlas (TCGA) databases for copy number alterations in Skov-3, Pa-1, OVCAR3, OVCAR4, and OVCAR5 cell lines were obtained from ATCC. A2780 parental cells (IP2) and cisplatin resistant cells (CP20) were generously donated by Dr. Charles Landen (University of Virginia). Cells were grown in RPMI  or DMEM  media containing 10% fetal bovine serum  and antibiotic/antimycotic supplements (Invitrogen). OVCAR3 cells were grown in RPMI with 20% FBS and 0.01 mg/mL of bovine insulin (Sigma). Normal human astrocytes  were cultured in AGM media, and immortalized neural progenitor cells  were propagated in DMEM/F12 supplemented with EGF, FGF and Gem21 (Gemini Bio-Products). Stable polyclonal cell lines with either forced expression of Sox2 (GeneCopoeia), or shRNA against Sox2 (Sigma), were created by lentiviral transduction followed by puromycin selection. Cells with inducible Sox2 expression were generated using lentivirus harboring a tetracycline-inducible Sox2 construct (GeneCopoeia) followed by selection with blasticidin. Sox2 expression was induced in this latter cell line with 1 μg/ml doxycycline. In a pilot experiment, dox-induced Sox2 expression was measured at multiple time points, and based on these data, all further dox treatments were conducted at 96 h. Modulation of Sox2 expression in these various cell models was confirmed by immunoblotting.Cells were lysed in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (Sigma). Protein quantification was performed by BCA assay (Pierce). Lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Sigma). Blots were blocked in 5% non-fat dried milk and then incubated with primary antibodies against Sox2 , ST6Gal-I , or β-tubulin (AbCam). Blots were subsequently incubated with the appropriate secondary antibody , and then developed using either Clarity (Bio-Rad) or SuperSignal West Femto (Pierce) enhanced chemiluminescence (ECL) substrates.H isoform: forward: GTCTCTTATTTTTTGCCTTTGCAG, reverse: CCACACACAGATGACTGCAAYZ isoform: forward: AGTCCAGGGAGAAGTGGTGA, reverse: CCACACACAGATGACTGCAAX isoform: forward: CTTCTCCCATACCTTGCTCTACA, reverse: GAAGATGTGTTCAGGGAAGTCACCoding region: forward: TATCGTAAGCTGCACCCCAATC, reverse: TTAGCAGTGAATGGTCCGGAAG.GAPDH: forward: TGGTATCGTGGAAGGACTCA; reverse: AGTGGGTGTCGCTGTTGAAG.RNA was isolated from cells using the Ambion RNA extraction kit (Life Technologies). cDNA was then synthesized according to the vendor protocol (Promega), and PCR was used to amplify specific ST6Gal-I isoforms. Primers with the following sequences were obtained from Integrated DNA Technologies:Isoform-specific PCR products were visualized on a 1.2% agarose gel with ethidium bromide.p < 0.05 based on a Student’s T Test.For analyses of ST6Gal-I and Sox2 mRNA expression, TaqMan Fast Master Mix (Thermo Fisher Scientific) was used and primers were purchased from Applied Biosystems: ST6Gal-I (Assay ID: Hs00949382_m1) and Sox2 (AssayID: Hs00602736_m1); mRNA expression was normalized to GAPDH (Assay ID: HS02786624_g1). At least 3 independent experiments were conducted, with each individual experiment performed with 3 technical replicates. Statistical significance was defined as 8 cells per 150 cm2 dish, and allowed to adhere overnight. Cells were fixed with 3.7% paraformaldehyde (Thermo Fisher Scientific) for 10 min, and then fixation was stopped by adding 2.5 M glycine. Cells were washed with phosphate-buffered saline (PBS) containing protease inhibitors, removed from the plate using a cell scraper, and centrifuged at 1000 x g for 5 min. Cell pellets were then resuspended in hypotonic buffer  with fresh protease inhibitors, and incubated on ice for 5 min. NP-40 (Sigma Aldrich) was added to the solution at a final concentration of 0.5%. Samples were incubated on ice for 5 min. Cells were centrifuged and the pellets resuspended in TE buffer (Thermo Fisher Scientific) with fresh protease inhibitors. Samples were then sonicated using a BioRupter for 20 cycles of 30 s on; 30 s off; with temperature remaining at 4 °C. Following sonication, samples were centrifuged at 4 °C for 15 min and cell pellet debris was removed. 5% of the sample was removed for the input control. A 1:1 volume of 2x RIPA buffer was then added to each sample along with either a ChIP-validated anti-Sox2 antibody  or a nonspecific IgG control antibody . Chromatin samples were incubated with the antibodies for 2 h at 4 °C with rotation. Samples were then incubated with Protein G dynabeads (Invitrogen) overnight at 4 °C with rotation. The antibody/chromatin complexes were collected by placing the samples in a magnet , and complexes were washed with the following series of buffers at 4 °C with rotation for 5 min each: Low salt buffer ; High salt buffer , and LiCl Buffer . Complexes were subsequently washed twice with TE buffer. Following the washes, samples were incubated with fresh elution buffer  at room temperature for 15 min; this step was repeated once. Eluates were collected, and crosslinking reversed by adding 0.2 M NaCl and 10 mg/mL RNase A (Thermo Fisher Scientific) and incubating at 65 °C for 4 h. Protein was then degraded by adding proteinase K (20 mg/mL) to a final concentration of 50 μg/ml at 60 °C for 1 h. DNA was purified using the SimpleChIP DNA purification kit  according to the manufacturer’s instructions. PCR was conducted using the SYBR®Green system (Thermo Fisher Scientific). Primer sequences for PCR are as follows:1A: forward: TTGTGGCTGTGATCCTTTCA, reverse: CTGCACAGATGGGCTGATAA;1B: forward: TGCCCCCACTCTGCTTTATC, reverse: TTTAAGCACACAGGGATGGCT;2A: forward: GGTTACTCCAGGCTGAGTCG, reverse: CTCCAGGAGGTGAAGGTGAG;2B: forward: CCGCTTGGGCATCAGACTAA, reverse: CGACTCAGCCTGGAGTAACC;3A: forward: GAGGAGGGATTGGGTTTGTT, reverse: TCCTTAAACAGCACAGTTCCA;3B: forward: ACTGTGCTGTTTAAGGATCAACTG, reverse: AACTGGGCTCCACAATGCAA.Cells were plated at a density of 1 × 10Data were normalized to the percent input, and then values for the nonspecific IgG ChIP were normalized to 1.0.Cells were detached from tissue culture plates with Accutase (Corning) and washed with cold PBS. Cells were blocked in 1% bovine serum albumin (BSA) in PBS for 30 min on ice. Cells were stained with Dylight 649-conjugated SNA (EY labs), a lectin that specifically binds α2–6 linked sialic acids. Cells were stained with SNA-649 at dilutions of 1:200 or 1:400, depending on cell type, in 0.1% BSA in PBS for 40 min on ice. Binding of SNA-649 was quantified using the LSRII flow cytometer courtesy of the UAB Flow Cytometry Core.ST6GAL1 and SOX2 in human tumor tissues was evaluated in TCGA datasets using cBioportal [The presence of copy number alterations for ioportal , 53. cBiSOX2 and ST6GAL1 copy number. There were 74 cohorts with available data for copy number alterations, spanning a broad range of cancer types. The 74 cohorts were aligned from left to right based on the prevalence of SOX2 copy number variations . For example, the first two bars on the left of the graph represent lung squamous cell carcinoma (SCC) cohorts (tan circles), whereas the third bar, marked with an asterisk (*), depicts an ovarian serous carcinoma cohort (light blue circle). Results in Fig. SOX2 and ST6GAL1 genes are extensively amplified, but rarely deleted, across multiple cancer types. Secondly, in almost every case, SOX2 and ST6GAL1 are amplified together. Among the 50 out of 74 cohorts that display copy number alterations in either SOX2 or ST6GAL1, 48 cohorts had amplification of both genes. A third important finding is that there is a striking similarity between the frequency of SOX2 and ST6GAL1 copy number gains (CNGs) across the cohorts. For instance, the rates of SOX2 and ST6GAL1 CNG within the ovarian serous carcinoma cohort (*) are 27 and 24%, respectively.TCGA databases were screened for changes in SOX2 or ST6GAL1 . In fact, significant co-occurrence is found for all of the 48 TCGA cohorts with dual SOX2 and ST6GAL1 amplification.The ovarian serous carcinoma cohort (*) was subsequently examined in more detail. This cohort includes specimens from 579 patients. Each individual tumor specimen is represented by a single bar, with red bars indicating patients with CNGs in SOX2 and ST6GAL1 in human tumor tissues, we next examined the copy number status of these genes in the NCI-60 panel of established human cancer cell lines. Again, we noted a strong association between cells with high copy numbers of SOX2 and ST6GAL1  in the Pa-1 line, given that these cells have high Sox2 expression, and overexpressed (OE) in Skov3 cells, which have relatively low Sox2 expression. Sox2 mRNA levels were evaluated by qRT-PCR to verify successful KD Fig.  a or OE Cells were then evaluated for ST6Gal-I mRNA expression. Sox2 KD in Pa-1 cells decreased the expression of ST6Gal-I mRNA Fig.  e, whileA plethora of literature has established Sox2 as a master regulator of stemness. Sox2 plays a seminal part in balancing the transcription of genes that regulate a cell’s differentiation state , 69, 70.SOX2 and ST6GAL1 genes lie within the same amplicon, 3q26, and are coordinately amplified in the vast majority of tumor specimens. Likewise, there is a strong correspondence between SOX2 and ST6GAL1 CNGs in the NCI-60 panel of established cancer cell lines. The 3q26 amplicon is one of the most pervasively amplified chromosomal regions in cancer, and is known for promoting stem-like cancer cell characteristics [SOX2, PRKCI, ECT2, and PIK3CA, and these genes work together to promote carcinogenesis [SOX2, the presence of ST6GAL1 within the 3q26 amplicon has escaped attention. We postulate that some of the tumor-promoting activity of the 3q26 amplicon may be mediated by ST6GAL1.While an association between Sox2 and ST6Gal-I mRNA levels has been noted previously –40, resueristics . This paeristics , with ameristics , and beteristics , 76, 77.eristics , 78–80. ogenesis , 80, 81.ST6GAL1 P3 promoter, one at ~ 4500 bp upstream, and another ~ 1000 downstream, of the TSS. A third site was identified at ~ 68,000 bp downstream of the P3 TSS, an area that lies within an intron between exons Z and I. Using ChIP assays, we confirmed Sox2 binding to all three of these sites in ovarian cancer cells. These data are noteworthy because prior ChIPSeq experiments were conducted primarily in stem cell models, or other types of cancers. More specifically, ChIPSeq databases show that Sox2 binds to the ~ 4500 bp site upstream of the TSS in ESC-derived neural progenitor cells (NPCs) [In conjunction with genetic co-amplification, Sox2 may directly regulate the expression of ST6Gal-I. Our analyses of publically available ChIPSeq databases uncovered two Sox2 binding elements proximal to the s (NPCs) , whereass (NPCs) –64, 82. s (NPCs) , 82–84. We further established that Sox2 promotes ST6Gal-I expression by modulating Sox2 levels and examining ST6Gal-I mRNA and protein. While Sox2-induced ST6Gal-I protein expression has not been previously reported, other groups have described Sox2-mediated changes in ST6Gal-I mRNA. In a glioblastoma (GBM) model focused on the regulation of cell plasticity, Berezovsky et al. found that knock-down of Sox2 resulted in a 3.9 fold decrease in ST6Gal-I mRNA; this study also revealed that Sox2 was essential for the maintenance of GBM CSCs . Zhu et The dearth of information regarding mechanisms that mediate differential ST6Gal-I expression in diverse cell types represents a major gap in the field. In particular, very little is known about ST6Gal-I regulation in stem and progenitor cells. On the other hand, there is some insight into pathways that induce ST6Gal-I expression in cancer cells. Several groups, including ours, have shown that ST6Gal-I is upregulated in response to signaling by oncogenic ras , 85, 86.ST6GAL1 promoter to further enhance ST6Gal-I expression. These collective events may contribute to the remodeling of cancer cells into a more stem-like cell phenotype.Studies herein describe a complex relationship between Sox2 and ST6Gal-I. Both genes are commonly amplified in cancer cells due to their shared presence within the 3q26 amplicon, and then the transcribed Sox2 protein binds the


Pparg2 gene is tightly regulated by many adipogenic and also anti-adipogenic factors. Four hours after the treatment of adipogenic hormones, more than fifteen TFs including glucocorticoid receptor (GR), C/EBPβ and AP-1 cooperatively bind the promoter of Pparg2 gene covering 400 bps, termed “hotspot”. In this study, we show that TEA domain family transcription factor (TEAD)4 reinforces occupancy of both GR and C/EBPβ on the hotspot of Pparg2 during early adipogenesis. Our findings that TEAD4 requires GR for its expression and for the ability to bind its own promoter and the hotspot region of Pparg2 gene indicate that GR is a common component of two positive circuits, which regulates the expression of both Tead4 and Pparg2. Wnt3a disrupts these mutually related positive circuits by limiting the nuclear location of GR in a β-catenin dependent manner. The antagonistic effects of β-catenin extend to cytoskeletal remodeling during the early phase of adipogenesis. GR is necessary for the rearrangements of both cytoskeleton and chromatin of Pparg2, whereas Wnt3a inhibits both processes in a β-catenin-dependent manner. Our results suggest that hotspot formation during early adipogenesis is related to cytoskeletal remodeling, which is regulated by the antagonistic action of GR and β-catenin, and that Wnt3a reinforces β-catenin function.Adipogenesis is a process which induces or represses many genes in a way to drive irreversible changes of cell phenotypes; lipid accumulation, round cell-shape, secreting many adipokines. As a master transcription factor (TF), PPARγ2 induces several target genes to orchestrate these adipogenic changes. Thus induction of  The 3T3-L1 cells can be differentiated into mature adipocytes by two-day exposure to 3-isobutyl-1-methylxanthine (IBMX/M), dexamethasone (Dex/D), and insulin (I)4. Recently, genome wide analyses of DNase I hypersensitive regions revealed that after 4 h MDI treatment, approximately three times more DNA regions became accessible5. ChIP-seq analyses revealed that during such dynamic chromatin remodeling (within 4 h after MDI treatment), hundreds of DNA sequences covering 400 bps, termed “hotspots”, including the promoter of Pparg2, are cooperatively occupied with at least five TFs including C/EBPβ/δ, GR , STAT5A, and other TFs. These early TFs recruit coregulators to induce chromatin remodeling and form early enhanceosomes at the hotspots6. Hotspots are enriched with enhancer histone marks, namely, H3K4me1, H3K4me2, and H3K27ac, suggesting that hotspots are key enhancers9. Similarly, intracellular and extracellular structures were also remodeled during adipogenesis when preadipocytes changed to round and lipid-laden adipocytes12. Within 24 h, MDI treatment rearranged the actin cytoskeleton from stress fibers to cortical structures in preadipocytes13. Thus, MDI dramatically rearranged both chromatin and cytoskeleton in this short period.The mouse 3T3-L1 cells have been widely used as an in vitro model system to investigate molecular mechanisms of adipogenesis15. 3T3-L1 preadipocytes secrete Wnt10b, which maintains preadipocytes in undifferentiated state via autocrine and paracrine signaling19. After binding to the frizzled receptor and coreceptor LRP5/6, the canonical Wnt ligand stabilizes β-catenin  by protecting it from the APC/AXIN/GSK-3β destruction complex. Stabilized β-catenin localizes in the nuclei and is recruited on Wnt target genes via protein-protein interactions with DNA-bound T-cell factor/lymphoid enhancer factor (TCF/LEF)23. As a coactivator, β-catenin increases the expression of TCF/LEF target genes such as Axin2 and Ccnd125. In addition, β-catenin maintains cytoskeletal integrity by interacting with membrane bound cadherins and α-catenin, which are connected to actin fibers28.Canonical Wnt signaling represses adipogenesis, but enhances osteogenesis of mesenchymal stem cells34. However, how Wnt signaling prevents adipogenic hormones from de-repressing and activating the transcription of Pparg2 during early adipogenesis (within 48 h after MDI treatment) remains unclear. Here, we showed for the first time that canonical Wnt signaling inhibits not only hotspot formation of Pparg2, but also cytoskeletal rearrangement in a β-catenin-dependent manner. These two events are regulated by the antagonistic actions of GR and β-catenin, and Wnt3a reinforces β-catenin function.Previous studies on the anti-adipogenic effects of Wnt/β-catenin signaling focused on the inhibition of PPARγ activity by β-catenin35. Anti-PPARγ (E-8), C/EBPα (14AA), C/EBPβ (H-7), GR , p300 (C-20), CBP (A-22), KLF4 (H-180), c-Jun (H-79), and β-catenin (H-102) antibodies were purchased from Santa Cruz Biotechnology . Anti-TAZ (M2-616) antibody was purchased from BD Biosciences , anti-lamin A/C and anti-STAT5 antibodies from Cell Signaling Technology , anti-FLAG (5A8E5), anti-Myc (2G8D5), and anti-HA (5E11D8) antibodies from GenScript , anti-TEAD4 and anti-H3 antibodies from Abcam , anti-14-3-3γ (CG31-2B6) from Millipore , and anti-β-actin (AC-15) from Sigma-Aldrich, fluorescent phalloidin conjugate from Invitrogen . cDNAs of HA-GR or Myc-Tead4 was subcloned into the pLenti-vector. pBABE-3× FLAG-C/EBPβ and pCMV-3× FLAG vector have been described previously36. Primer sequences for qRT-PCR and ChIP-qPCR are summarized in Supplementary Table Insulin, dexamethasone, 3-isobutyl-1-methylxanthine, Hoechst33258, Oil Red-O and RU486 were purchased from Sigma-Aldrich . Recombinant mouse Wnt3a was purchased from R&D Systems . Wnt3a-conditioned medium was obtained from confluent Wnt3a-expressing L929 cells35. AmphoPackTM 293 cells , HEK-293T cells, C3H10T1/2 cells  and Wnt3a-L929 cells were maintained in DMEM containing 10% (v/v) FBS. For adipogenesis, post-confluent 3T3-L1 cells and C3H10T1/2 cells were exposed to the adipogenic cocktail (MDI) containing 2 μM D, 0.5 mM M, and 5 μg/ml I in DMEM supplemented with 10% FBS as described previously35. Lipid droplets were stained using Oil Red-O as described previously37.3T3-L1 mouse preadipocytes  and mouse NIH-3T3 cells were maintained in DMEM containing 10% (v/v) bovine serum as describedTM 293 cell line)35. GR-L1, Cβ-L1, or Tead4-L1 cells were generated by infecting mouse 3T3-L1 preadipocytes with lentiviruses expressing HA-tagged GR, FLAG-tagged C/EBPβ, or Myc-tagged TEAD4, respectively. GR/Cβ-L1 or EV/EV-L1 cells were generated by infecting Cβ-L1 and EV-L1 cells with lentiviruses encoding HA-tagged GR or empty vector, respectively, using the pWZL retroviral vector system. shTead4-L1, shβ-cat-L1, shGR-L1, and shCtrl-L1 cells were generated by infecting mouse 3T3-L1 cells with lentivirus encoding shRNA against mouse Tead4 (5′-GCTGAAACACTTACCCGAGAA-3′), mouse Ctnnb1 (5′-CCCAAGCCTTAGTAAACATAA-3′), mouse Nr3c1 (encoding GR) (5′-TGAGATTCGAATGACTTATAT-3′) and control (5′-CCTAAGGTTAAGTCGCCCTCG-3′), respectively, using the pLKO.1 lentiviral vector system .C/β-NIH cells and EV-NIH cells were generated by infecting mouse NIH-3T3 cells with retroviruses encoding FLAG-tagged C/EBPβ and empty vector, respectively, using the pBABE retroviral vector system and HEK293-based packaging cells  as described previously39. Briefly, sonicated chromatin lysates were phase-separated by two rounds of phenol/chloroform extraction. Nucleosome-free DNA in the upper aqueous phase was obtained using ethanol precipitation. DNA was further treated with 10 μg RNase A and 20 μg proteinase K, and extracted using the QIAquick PCR purification kit . The isolated genomic DNAs were used for FAIRE-qPCR. The Ct value of a target gene in the isolated DNA sample of a ChIP or FAIRE experiment was normalized to the Ct value of the target gene in the input DNA (ΔCt = Ct sample−Ct input). The percentage of input indicates the value of 100 × 2ΔCt.ChIP analyses were performed as described previously35. To obtain nuclear extracts, the cells were washed twice with ice-cold phosphate buffered saline (PBS), harvested, and then lysed with hypotonic buffer (20 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP-40, 10 mM β-glycerophosphate, 10 mM NaF, 1 mM Na3VO4, and protease inhibitors) and incubated for 10 min on ice. The supernatant (cytosol extracts) was removed and the nuclear pellet was washed with hypotonic buffer and lysed with NETN buffer (20 mM Tris-HCl (pH 8.0), 140 mM NaCl, 0.5% NP-40, 10 mM β-glycerophosphate, 10 mM NaF, 1 mM Na3VO4, and protease inhibitors), followed by 10 cycles of sonication . Nuclear extracts (supernatant) were obtained by centrifugation at 13,000 × g for 10 min at 4 °C. The reporter plasmids, C/EBP-Luc and GRE-Luc, contained the luciferase gene under the regulation of three copies of C/EBP binding sequences (GTTGCGCAAG) and one copy of glucocorticoid responsive element (GRE) (AGAACACTGTGTTCT), respectively. Reporter assays were performed using Lipofectamine reagent as described previously40. The pRL-TK plasmid encoding Renilla luciferase was cotransfected for normalizing transfection efficiency.Western blot analyses were performed as described previouslyThe cellular F-actin was stained with fluorescent phalloidin conjugates (25 μΜ) for 40 min at room temperature prior to Hoechst staining. The stained cells were observed under a Zeiss LSM510 inverted confocal microscope according to the manufacturer’s instructions. F-actin structures in individual cells were categorized into three groups; S (stress) fiber, where F-actin stress fibers were observed both in the nucleus and cytoplasm; T (transition state) fiber, where F-actin stress fibers were observed in the cytoplasm but not in the nucleus; C , where F-actin stress fibers were observed neither in the nucleus nor in the cytoplasm, but were observed as cortical structures near the cellular membrane. Cells (13–48) in each treatment were observed and categorized into three groups.t-tests were used to compare the data between controls and indicated experimental groups. *p-values < 0.05; **p-values < 0.01; ***p-values < 0.001 were considered statistically significant.All quantitative measurements were performed in at least three independent experiments. Two-tailed unpaired Student’s Axin2, a Wnt target gene, it did not effectively reduce the mRNA and protein levels of Pparg2 and Cebpa  in the presence of recombinant Wnt3a Fig. . MDI indbpa Fig. . These rPparg2 transcription start site (TSS) , the promoters (−5 to +1 kb from TSS) of which contain GR binding peaks, as identified in the ChIP-seq analyses5. We found that Wnt3a reduced GR occupancy in the promoter of all seven genes, which are also occupied by C/EBPβ; interestingly, Wnt3a also reduced C/EBPβ binding to these promoters  Fig. . Althougrg2 Fig. . We seleers Fig. . These rPparg2 promoter, we generated C/β-NIH cells that ectopically express FLAG-tagged C/EBPβ  reinforced GR binding to a subset of GR target genes as a heterodimer binding partner of GRner Fig.  to S3C. ad4 Fig. . These rad4 Fig. . ChIP reent Fig. . Interesad4 Fig. , and thaad4 Fig. . These rsis Fig. .Fig. 3GRPparg2 promoter during early adipogenesis. We found that Tead4 knockdown reduced Pparg2 induction and adipogenesis . Unlike the ectopic expression of only C/EBPβ (Cβ-L1 cells), overexpression of both GR and C/EBPβ resulted in reinitiation of adipogenesis in 3T3-L1 cells even in the presence of Wnt3a , revealed that MDI reduced F-actin stress fibers in 50% cells from 4 h, whereas they disappeared in 86% cells within 48 h; instead, F-actin reorganized only at the cortical region. The amount of β-catenin protein decreased during the rearrangement of F-actin from stress fibers to cortical structures. Wnt3a prevented MDI from reducing β-catenin protein and disrupting F-actin stress fibers  isolated from GR (Nr3c1) knockout and GR dimerization-defective mutant mice was impaired45. Knockdown of GR or omission of Dex from MDI did not induce Pparg2 in 3T3-L1 cells46. These previous studies indicated that GR is essential for Pparg2 induction.The findings that treatment of Dex followed by IBMX was sufficient for adipogenesis, but that IBMX treatment followed by Dex treatment did not recapitulate this effect suggested that Dex primed preadipocytes to a novel commitment state for adipogenesis1. Furthermore, enhancers with large numbers of TFs are more sensitive to small changes in TF concentration compared to those with smaller numbers of TFs49. This is consistent with our findings that reduction in nuclear GR level by β-catenin sensitively changes super-enhancer formation on Pparg2 and other genes  is involved in commitment for adipogenesis. Primary preadipocytes embedded in stiffer matrices showed reduced rates of adipogenesis10. ECM stiffness increases tissue tension, which leads to increased actin and myosin fiber and cell stretching57. Interestingly, mesenchymal stem cells exposed to mechanical strain show increase in β-catenin level and could not differentiate into adipocytes, suggesting that Wnt/β-catenin conveys signals from intracellular tension to the nuclei58. In agreement with this result, we found that Wnt signal could not prevent MDI from changing F-actin stress fibers to cortical F-actin structures during adipogenesis in the absence of β-catenin.Unlike lipogenesis in other cells such as hepatocytes and myocytes, adipogenesis requires dramatic cytoskeletal remodeling of F-actin stress fibers to cortical actin structures, which is required to hold a large lipid vacuole in the center, relocate the nucleus and other organelles, and attain a round shape. Cytoskeletal remodeling starts within 24 h after adipogenic hormone treatments59. Furthermore, GR is connected to actin filaments through HSP90, a main component of GR-chaperone complex suggesting that the nuclear transport of GR and cytoskeletal rearrangement are closely related60. Upon ligand binding, the GR-chaperone complex recruits a motor protein dynein to form the liganded GR-HSP90-FKBP52-dynein complex, which is able to move along the microtubules through the nuclear pore complex (NPC) to the nucleus62. However, when the cytoskeleton is disrupted like adipogenesis, liganded GR simply diffuses in and out of the nucleus mainly via importins, exportins, and the RanGTPase system63. The mechanism via which the liganded-GR translocates into the nucleus during adipogenic cytoskeletal rearrangement remains unknown. In contrast, β-catenin harbors armadillo repeats, which are similar to the importin-β HEAT repeats66. Therefore, β-catenin can pass through NPC like importin and equilibrates between the nucleus and the cytoplasm by passive diffusion. Therefore subcellular distribution of β-catenin in the nuclei, cytoplasm, and the membrane can be determined from its location and the levels of diverse interacting complexes, namely, the TCF/LEF transcription factors in the nucleus, APC and AXIN in the cytoplasm, and cadherin complex in the membrane67. It remains to be investigated whether GR and β-catenin compete with each other for nuclear translocation during adipogenic cytoskeletal rearrangement.Interestingly, Wnt3a reduced GR level in the nuclei during the early phase of adipogenesis, but not in Dex-treated preadipocytes, suggesting that the antagonistic effect of Wnt/β-catenin on GR is specific for adipogenesis Figs. ,  6h. ExIn conclusion, this study provides insights into an intriguing question regarding chromatin remodeling and actin rearrangement, which are interdependently regulated during adipogenesis. Our findings that GR is necessary for the rearrangement of both cytoskeleton and chromatin, and that canonical Wnt3a inhibited both processes in a β-catenin-dependent manner, suggest that rearrangements of both chromatin and cytoskeleton are related in ways that involve the antagonistic activities of GR and β-catenin, and that Wnt3a reinforced β-catenin function.Clean_Supplementary_FiguresSupplementary_Tables


Understanding the brain is one of the most challenging scientific problems faced by humanity. In addition to offering an improved understanding of who we are, deciphering how our brains operate will provide payoffs for the treatment of mental and neurological disease as well as the creation of intelligent systems. Consequently, developed countries are pouring ever-increasing research funds into brain science. Prominent examples include the BRAIN initiative in the United States and the Human Brain Project in Europe. Although Africa may not yet possess financial and technological resources on this scale, Africans represent a vast and growing human resource poised to make important contributions to neuroscience. As the third yearly International Brain Research Organisation (IBRO)-Simons Computational Neuroscience Imbizo recently wrapped up in South Africa, we call for greater efforts to empower Africans to join the global neuroscience community and argue that the subfield of computational neuroscience is the ideal vehicle to do so.Computational and theoretical neuroscience, also known as neurotheory, or just “theory” within neurobiology, is a young, rapidly expanding field within neuroscience; its goal is to determine and articulate the general principles by which nervous systems perform their computations. We think it is a good example of how theory, as a subfield, can serve as a catalyst to accelerate the development of science in Africa.Due to the diversity of methods required for understanding the brain at multiple levels , skills There is growing momentum for experimentalists to make their data available for public access and for increased collaboration between groups, a movement lead by flagship projects that curate, systematize, and make freely available large-scale neuronal data –8. A parThe limiting factor for computational neuroscience development in Africa (and anywhere in the world) is quantitative training. Although Africa does not currently contribute a considerable fraction of global neuroscience output see , educatiSeveral promising educational initiatives have recently emerged for Africa. These include annual schools in computational neuroscience  and machine learning , which both started in 2017. These initiatives bring young, predominantly African scientists together with leading international experts in their field. It is the relationships between students, and particularly between students and faculty, that will support the careers of these fledgling, African computational neuroscientists and machine learning practitioners.Entering the field of computational neuroscience can be a daunting prospect; there are a proliferation of online courses, numerous textbooks, diverse conferences, and several summer schools. In addition, mailing lists and social media have become an integral component for engaging with current discussions in the field, connecting with authors, and sharing opportunities and information. Although the type of quantitative training background can inform a focus in the field, there are valuable general principles to be gained from the resources in Thus far, the major strides in computational neuroscience development in Africa have come from foreign sources. To retain these initiatives and benefit from these pilot projects, African governments and academic institutions must also be willing to support projects, both financially and logistically. In addition to increasing focus on the field for African scientists, we provide several recommendations for the global computational neuroscience community to enhance the field in Africa:Make funding available for Africans to attend international computational neuroscience conferences, workshops, and summer schools by providing cheaper (or free) registration and travel grants.Support or organise computational neuroscience schools and workshops in Africa.Provide exchange opportunities for students to visit world-class research labs, and, on the other side of the exchange, bring foreign researchers to experience and enhance an African research environment.Make available and accessible teaching material and computational resources.Collaborate with African theoretical and experimental researchers.Incorporate computational neuroscience research groups into African academic institutions.In the century of the brain, African scientists and educators are poised to make important contributions to global neuroscience research. We believe that theoretical sciences, and specifically computational and theoretical neuroscience are the ideal discipline for the African continent.