Several factors control mESC pluripotency, including Wnt/β-catenin signaling pathway, which is essential for mESC differentiation and proliferation. Here we show that the activity of the Wnt/β-catenin signaling pathway safeguards normal DNA methylation of mESCs. The activity of the pathway is progressively silenced during passages in culture and this results into a loss of the DNA methylation at many imprinting control regions (ICRs), loss of recruitment of chromatin repressors, and activation of retrotransposons, resulting into impaired mESC differentiation. Accordingly, sustained Wnt/β-catenin signaling maintains normal ICR methylation and mESC homeostasis and is a key regulator of genome stability.Mouse embryonic stem cells (mESCs) are pluripotent and can differentiate into cells belonging to the three germ layers of the embryo. However, mESC pluripotency and genome stability can be compromised in prolonged  Mutations affecting the Wnt/β-catenin pathway often lead to disease, cancer progression and developmental defects.The evolutionarily conserved Wnt/β-catenin signaling pathway controls many cellular and developmental processes, including cell proliferation, cell fate determination and tissue homeostasis2. In the absence of Wnt ligands, cytoplasmic β-catenin is constantly degraded by the action of the AXIN/GSK3/APC/CKI destruction complex. On the contrary, the destruction complex is disassembled when Wnt ligands bind to the FZD receptors. As a consequence, β-catenin translocates to the nucleus where it associates with TCF/LEF (T-cell factor/lymphoid enhancing factor) nuclear complex and activates Wnt targeted gene expression3.The canonical Wnt/β-catenin-dependent pathway integrates membrane, cytoplasmic and nuclear components, such as Wnt ligands, Frizzled (FZD) receptors and co-receptors, AXIN/glycogen synthase kinase 3 (GKS3)/Adenomatosis polyposis coli (APC)/Casein Kinase I (CKI) destruction complex, β-catenin protein and several transcription factors5. Accordingly, Wnt/β-catenin represents a key pathway for mouse embryonic stem cell (mESC) identity and homeostasis.During embryogenesis Wnt/β-catenin signaling plays a fundamental role in the establishment of both dorso-ventral and anterior-posterior axis and its role is essential for normal gastrulation. Indeed, β-catenin knockout embryos are lethal since they fail to develop the mesodermal and endodermal germ layers7 are pluripotent stem cells, which are able to generate the three germ layers and can be expanded in vitro indefinitely. Their long-term self-renewal ability has been attributed to the protein regulatory network that includes several pluripotency factors, such as Nanog, Oct4 and Rex1, among others11. In this context, the role of β-catenin during mESC differentiation has been shown to be indispensable, since β-catenin null mESCs undergo apoptosis at the onset of the differentiation process14. However β-catenin function in ESC self-renewal has been largely debated20. The dual role of β-catenin can be attributed to its capacity to form complexes with many downstream factors, including key pluripotency genes, such as Oct422.Mouse ESCs, derived from the inner cell mass (ICM) of the blastocyst23. In particular, reversible changes on DNA methylation, followed by histone modifications, control both pluripotency and differentiation genes in mESCs, recapitulating the epigenetic profile of the pre-implantation embryo stage25. While developmental genes are characterized by flexible and reversible epigenetic regulation mechanisms to allow their dynamic expression, stable DNA methylation ensures silencing and protection of retrotransposons from moving around in the genome and causing potential mutations26. The same applies to imprinted genes, which reside in clusters27 and are regulated from one major cis-acting element called the imprinting control region, ICR. In mammals, DNA methylation is maintained stable and it can be propagated through cell division30 by a mechanism of DNA methylation maintenance coupled to DNA replication, which involves the action of different enzymes including DNA methyltransferase I (DNMT1). Along with DNA methylation, other epigenetic factors, such as ZFP57, KAP1, DNMT1, H3K9me3 and others, are involved in marking the ICRs and in protecting the methylated DNA. Indeed, loss of ZFP57, KAP1, DNMT1 or other repressors, leads to loss of imprinting and genomic instability in mESCs, and, thereby, to embryonic lethality34.In parallel to the core pluripotency factors, several epigenetic mechanisms control mESC identity through chromatin remodeling processes35 or prolonged in vitro culture of mESCs39. In particular, mESCs with global loss of methylation at the ICRs have been shown to contribute to chimeras, but mice developed several types of tumors by one year of age40. The mechanisms causing genomic aberrations and destabilization are still debated. However, downregulation of several epigenetic factors, such as DNMT1, KAP1, G9a, has been correlated with the epigenetic instability of the cells46.Epigenetic instability in imprinted genes and transposons has been observed in several mESC lines and can be attributed to culture conditions, sex of the cells, isolation proceduresin vitro the mechanisms that regulate embryo development. Therefore, it is important to fully understand the mechanisms that control cell identity, genomic stability and cell homeostasis. Wnt/β-catenin signaling has been investigated to be crucial for gene transcriptional regulation of mESCs, including pluripotency genes. Though, the connection between Wnt signaling and the epigenetic regulatory mechanisms has not been elucidated up to now. Here we investigated a novel role of Wnt/β-catenin signaling as a key player involved in epigenetic changes that preserve mESC identity and genome stability. We found that mESCs cultured in vitro for prolonged time showed loss of Wnt activity and downregulation of β-catenin protein, which correlated with a general loss of DNA methylation, affecting the ICRs, and leading to impaired mESC differentiation. On the contrary, sustained levels of Wnt/β-catenin ensure ICR methylation maintenance over time, suggesting a possible role for this signaling pathway in the protection of silent genomic regions and, therefore, in the maintenance of the genomic stability.Mouse embryonic stem cells represent an essential model to study The functional role of the Wnt/β-catenin pathway has been widely investigated in pluripotent stem cells. While the activation of Wnt pathway is indispensable for mouse embryonic stem cell (mESC) differentiation, its role in self-renewal and cell identity maintenance has been largely debated. Thus, we decided to analyze the activity of the Wnt/β-catenin pathway in mESCs cultured for a prolonged time, in particular its influence on pluripotency and homeostasis, including cell proliferation, differentiation potential and epigenetic stability.53.To this aim we cultured E14 mESCs for several passages in the Serum + LIF medium. We observed that E14 mESCs cultured for many passages, around seventy, , showed homogeneous morphology, characterized prevalently by flat clones, when compared to the same mESCs that were kept in culture for only fourteen passages  Fig. . SimilarAxin2, Lef1, Tcf1 and Sp5 were significantly downregulated, when compared to YP-mESCs  formation from both E14 and GS1 YP- and OP-mESCs , a technique that combines restriction enzymes and bisulfite sequencing to enrich for the genomic areas with high CpG content66, using both YP- and OP-mESCs. Only 3 genomic regions, localized close to Repin1, Nkx2-1 and Gm11846 genes, were hypermethylated in E14 OP-mESCs after prolonged culture, when compared to the YP-mESCs , while methylation was lost in several ICRs in OP-mESCs  coupled to pyrosequencing analysis. Methylation analysis by COBRA includes the use of methylation sensitive restriction enzymes that can digest DNA only when methylated. As expected, some of the ICRs were hypomethylated in OP-mESCs and not in YP-mESCs, such as ern Fig. . On the ion Fig. . In paraAirn, Rasgrf1, Peg10, Grb10, Ig-DMR and Gnas XL) that was comparable to the mouse genomic DNA (Ctrl gDNA)  and Inpp5fV2 ICRs in E14 OP- but not in YP-mESCs  at the analyzed ICRs (NA) Fig. . On the CRs Fig. . In addiSCs Fig. . The KvDSCs Fig.  left. Hoges Fig. , right.Overall these data indicate that downregulation of endogenous Wnt signaling and of β-catenin protein level occurs in prolonged mESC cultures and this correlates with loss of DNA methylation. In particular, we observed loss of methylation at several ICRs, including both maternally and paternally methylated loci that, in turn, control the expression of a variety of coding and non-coding genes within the imprinted clusters.70. We named the two E14-derived mESC clones, carrying the S33Y-mutant β-catenin protein, S33Y-β-cat #1 and S33Y-β-cat #2. To test if sustained Wnt/β-catenin signaling activity could preserve methylation at ICRs, we cultured the clones in the conditions that were used to derive E14 OP-mESCs  and we compared them with both E14 YP- and OP-mESCs. OP-S33Y-β-cat #1 and OP-S33Y-β-cat #2 retained high level of Wnt activity as indicated by the top-flash reporter -qPCR experiments for both ZFP57 and H3K9me3 on some known ICR target regions34. Both ZFP57 and H3K9me3 binding decreased in KvDMR, Rasgrf1, Airn and Inpp5fV2 ICRs, reflecting their methylation status. On the contrary, no reduction of ZFP57 and H3K9me3 binding occurs at the Gnas ICR, which did not show loss of DNA methylation in OP-mESCs   and we found 2631 common overlapping regions between β-catenin and KAP1 in BJ1 mESCs , as potential targets of β-catenin. Notably, the same two ICRs were also bound by ZPF5731, which specifically binds to methylated ICRs, as we previously observed in the overlapping regions between β-catenin and ZFP57. However, in this case, the total number of common binding sites was very low . This was also true when we overlapped the data with H3K9me3 peaks Fig. . Among t76. All mammalian LTRs derive from the vertebrate-specific endogenous retroviral elements (ERVs), which can be grouped into sub-classes (I–III) and subfamilies, such as murine retroviral-related sequences , ERVK, class II, ERVL and mammalian apparent LTR retrotransposons , and others77. The expression of retroelements has been previously detected in different developmental stages. However, in most tissues and during embryo development the transcription of ERVs is counteracted by several epigenetic mechanisms, including DNA methylation, chromatin repressors (i.e. KAP1) and heterochromatin-associated histone marks, such as H3K9me380.Long-terminal repeat (LTR) elements belong to the third repeat class and represent 10% of all mammalian transposable elementsAs expected most of the LTRs were found in the overlapping common regions between β-catenin and KAP1 corresponded to endogenous retroviral elements, with the ERVK family  and early transposons (ETn//MusD)) being the most represented one, followed by MaLR and ERV1 families Fig. . AccordiIAP and MusD subsets of ERVs were significantly upregulated in the E14 OP-mESCs  are located within repressed genomic regions and they have been associated with induction and spreading of heterochromatin marks, such as H3K9me381. Furthermore, they are controlled by KAP1-mediated repressive complexes, which prevent their activation and genomic spreading80. Therefore, to analyze whether the transcriptional up-regulation of the ERVs in OP-mESCs correlates with loss of heterochromatin marks, we performed ChIP- qPCR analysis for H3K9me3 in E14 and GS1 YP- and OP-mESCs. The recruitment of H3K9me3 decreased at IAP LTR in both E14 and GS1 OP-mESCs, and at MusD in E14 OP-mESCs  experiments in E14 YP-mESCs carrying either a short hairpin against a control sequence with no predicted genomic target (shCtrl) or against β-catenin (shβcat). In shCtrl-transduced mESCs, β-catenin was co-immunoprecipitated with KAP1 and DNMT1, suggesting that it can interact with each one of the components of the epigenetic repressive complex  were efficiently downregulated   did not almost change between shCtrl- and shβcat-transduced mESCs  of the blastocyst, such as the expression of pluripotency genes and the capacity to contribute to the formation of the three germ layers. Up to now, many studies have investigated embryonic developmental mechanisms by exploiting the capacity of mESCs to self-renew in vitro indefinitely. Mouse ESCs are usually cultured in vitro in the presence of LIF87, which directly controls the expression of the pluripotency gene-network that includes Oct4, Nanog and other factors88.Since mouse embryonic stem cells (mESCs) were isolated, in the early 80’sin vitro cultured mESCs are identical to the ICM has been a topic of debate of the last years. With the aim to obtain mESCs with features as much as possible similar to the ICM, the 2i-LIF culturing medium was designed to drive mESCs toward a naïve pluripotent state, characterized by high expression of several pluripotency genes, including Rex190. This has been attributed to the activity of two small drug inhibitors (2i) of MEK1/2 and GSK3β, the latter leading to Wnt/β-catenin signaling activation. However, the identity of mESCs is not only dependent on the pluripotency gene network but also on the maintenance of a correct epigenetic state. Indeed, many groups previously observed genomic instability of mouse and human ESCs when they were cultured in vitro for a long time36. Recently, it has been shown that the constant inhibition of MEK1/2 is detrimental for mESC homeostasis after prolonged in vitro culture, leading to chromosomal aberrations, severe global hypomethylation and impaired differentiation capacity39. However, this study did not addressed whether Wnt/β-catenin activity also had an epigenetic effect in prolonged mESC cultures.Nevertheless, whether the 14 or Wnt3a91 causes embryo cell death when gastrulation starts. Likewise, we observed that OP-mESCs, which showed decreased endogenous Wnt/β-catenin activity, could not generate beating embryoid bodies with the same efficiency as YP-mESCs. On the contrary, they expressed high levels of ectodermal markers, such as Otx2, Sox1, Pax6, Nestin and III β-Tubulin, during both EB and neural differentiation. E14 OP-mESCs also expressed high levels of Fgf5 during differentiation, but GS1 OP-mESCs did not, suggesting a possible epigenetic dysregulation of this gene in this strain. Since the differentiation potential of OP-mESCs was biased toward the ectodermal fate, these data might suggest that OP-mESCs could be more primed with respect to the YP-mESCs, and they could share some common features with epiblast stem cells (EpiSCs)93. It is important to note however that EpiSCs were shown as able to generate the three germ layers in vitro and maintain their genomic integrity93. In our study, the expression level of pluripotency genes was maintained high in OP-EBs, indicating that OP-mESCs could not properly exit from pluripotency state, in agreement with what was previously observed in β-catenin knockout mESCs85. Indeed, β-catenin knockout–derived teratomas displayed high level of pluripotency markers and features comparable to germ cell tumors.The essential role of Wnt/β-catenin signaling in mESC differentiation is widely accepted. Loss of β-catenin96. Accordingly, in our study, we observed changes in DNA methylation at several CpG enriched genomic regions, which were prevalently hypomethylated upon prolonged culturing, and consequently, impaired mESC differentiation potential. Moreover, the hypomethylated CpG regions appeared to control several biological processes as indicated by the gene ontology analysis, with the metabolic processes as the most represented. Additionally, some of the analyzed hypomethylated regions were found nearby genes that have been previously reported to interact with Wnt pathway and its downstream components in different cellular contexts101. Some of these genes have been described to inhibit Wnt/β-catenin signaling pathway, such as Grb10, Kdm2b, Tet3, Pcdhgc5, Mir148a101. On the contrary, other hypomethylated regions corresponded to genes that act as activators or inhibitors of Wnt signaling, depending on the molecular and cellular context, such as Runx3, En1, Nkx6-1, Epha2, Nr4a107.In addition to molecular pathways alterations, several epigenetic changes have been investigated in both mouse and human ESCs. These epigenetic changes can occur both at the level of DNA methylation and histone modifications and they affect the chromatin structure and ESC identity. In particular, DNA methylation is sensitive to external stimuli and cellular stress. DNA methylation pattern is maintained stable during cell replication through the action of DNA methyltransferases (DNMTs) and other repressors, since tuned methylation levels are indispensable for mESC stability and differentiation. Loss of DNMTs leads to severe global epigenetic deregulation, which causes developmental defects and embryonic lethality, as previously reportedIAP, when compared to YP-mESCs. Both imprinted genes and retrotransposons are tightly epigenetically controlled81 since they play essential roles during embryonic and extra-embryonic tissue formation. Loss of imprinting or transcriptional changes in retrotransposons, in particular in IAP, could indeed be detrimental to genomic stability and embryo development109. Accordingly, both E14 and GS1 OP-mESCs could not properly differentiate when compared to YP cells.In particular, the OP-mESCs showed high hypomethylation at the level of imprinted genes, which commonly show stable allele-specific methylation pattern in the pre-implantation embryo. It is important to take into consideration that the imprinted genes are organized in clusters with a common ICR. Methylation changes of one ICR can affect the expression of many imprinted genes within the cluster (around 4–5). In this study, we observed that the YP-mESCs show 50% of methylation at many ICRs, indicating that allele specific methylation was lost in prolonged cultures, extended to both maternally and paternally imprinted loci. Additionally, the OP-mESCs were characterized by higher retrotransposon expression, such as KvDMR ICR has been associated with lower expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1 C, (Cdkn1c)110. In addition, Wnt/β-catenin pathway has been reported to have an anti-proliferative effect, by directly regulating the expression of cell cycle repressor genes61, thus safeguarding mESC identity. In line with previous published studies, we observed that both E14 and GS1 mESCs downregulated the expression of Wnt downstream targets  and β-catenin protein at late passages. Wnt activity reduction was not associated with altered pluripotency gene expression, but it was translated into impaired differentiation potential, faster cell cycle progression and genomic instability, thus loss of mESC homeostasis. The factors causing Wnt/β-catenin pathway downregulation in prolonged cell cultures still remain unknown. Among possible triggering events, oxidative stress could be a putative inducing factor. Indeed, increased oxidative stress has been reported to antagonize Wnt signaling and has been previously described to induce genomic aberrations and loss of cell homeostasis in long-term in vitro cell culture114. In particular, increased oxidative stress can antagonize Wnt signaling by inducing expression of Forkhead box-O (FOXO) transcription factor115. FOXO competes with TCFs for its interaction with β-catenin, thus inhibiting TCF transcriptional activity and the canonical Wnt/β-catenin signaling pathway117. Additionally, among the hypomethylated regions analyzed by RRBS we found some inhibitors of Wnt/β-catenin pathway, including Kdm2b, which inhibits the stability of β-catenin protein98, thereby creating a feedback loop that could accelerate β-catenin degradation.OP-mESCs were characterized by significantly faster cell cycle progression with respect to YP-mESCs, suggesting that the epigenetic changes could also affect cell cycle check-points. Interestingly, loss of methylation at the in vitro culturing conditions or different timing of mESC isolation from the embryo62. YP-GS1 mESCs showed low levels of nuclear β-catenin protein, if compared to the YP-E14 mESCs. Interestingly, ICRs, such as Inpp5fV2 region were hypomethylated in GS1 mESCs already at early passages, suggesting that this cell line was less stable with respect to E14. The role of Wnt activity in homeostasis maintenance has been largely described in the adult stem cells. For instance, long-term hematopoietic stem cells show a decrease in the canonical Wnt/β-catenin signaling activity during ageing118. In parallel, other studies has reported that many epigenetic changes, including loss of imprinting, occur in hematopoietic and other adult stem cell compartments119. The observation that mESCs carrying gain of function Wnt mutants (S33Y-β-cat #1 and S33Y-β-cat #2) maintained normal or even higher level of methylation at the ICRs also after prolonged in vitro culturing, further strengthen the hypothesis of a possible “protective” role due to Wnt/β-catenin pathway, which can control epigenetic stability. Even after prolonged culturing, these clones retained Wnt/β-catenin activity and methylation at similar levels of YP-mESCs. However, it is important to take into account that additional epigenetic changes might occur in some of the clones, due to stochastic perturbations. Indeed, some of the ICRs, such as Peg10, Gpr1 were hypomethylated in the OP-S33Y-β-cat #2 mESC clone, whereas others showed an increase in DNA methylation level in both β-catenin mutant clones. However, in both β-catenin overexpressing clones the overall DNA methylation profile was similar to the YP-mESCs.It is important to note that different mESC lines can display disparate endogenous levels of Wnt/β-catenin activity, due to either the diverse 120 or with other chromatin factors in mESCs121. However, it is important to note that, when we silenced β-catenin in mESCs we did not detect drastic methylation changes at the ICRs, though shβcat- transduced cells upregulated the transcriptional levels of IAP and MusD endogenous retroviruses. These data suggest that either the endogenous levels of β-catenin are already limiting and its further decrease does not impair methylation maintenance, or, alternatively, that β-catenin does not directly act on the ICRs but it acts as a mediator for the repressive complexes. Up to date, the factors causing genomic instability in mESCs are not clear, even though many chromatin modifiers have been described to act on repressed genomic regions.Finally, we observed that β-catenin interacts with KAP1 and DNMT1 repressors, therefore it can be considered as a regulator of epigenetic stability maintenance. By analyzing published ChIP-sequencing datasets we observed that β-catenin and KAP1 share common target regions, located within intergenic regions and overlapping with endogenous retroviral elements. These data further strengthen the conclusion that β-catenin could mediate the action of repressors recruited by KAP1 on silent genomic regions, even though the potential mechanism still remains unknown. Interestingly, β-catenin protein has been previously described to interact with DNMT1 in colorectal cancer cellsin vitro culture. In conclusion, Wnt/β-catenin pathway, mediates a large number of molecular and biological processes including DNA methylation at ICRs to ensure correct cell and tissue homeostasis.All in all, our data suggest that Wnt/β-catenin activity need to be maintained constantly active in mESCs during passages to ensure correct cell identity and epigenetic stability. Loss of Wnt activity results in global hypomethylated DNA, loss of chromatin repressor recruitment and activation of silent genomic regions, resulting in impaired mESC differentiation and altered cell cycle progression Fig. . Sustain122 and purchased from ATCC, respectively. Both mESC cell lines were maintained in 0.1% gelatin (Millipore ES-006-B)-coated plates mESC medium, which consisted of DMEM supplemented with 15% fetal bovine serum (FBS), L– glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ ml), sodium pyruvate (1 mM), non-essential amino acid (NEAA) , 2-mercaptoethanol  and ESGRO mLif (1000 U/ml). E14 mESCs were thawed at passage 8 and expanded up to passage 14 for the analysis of young passage  E14 mESCs. GS1 were thawed from passage 18 and expanded up to passage 22 for the analysis of young passage  GS1 mESCs. To obtain the old passage  E14 and GS1 mESCs were kept in culture for ~70 and ~50 passages, respectively, in mESC medium. At each passage cells were detached by using trypsin  at 37 °C, centrifuged for 5 minutes and 300 rcf and plated with a dilution ratio of 1:15–1:20 at each passage.GS-1 (129 Sv) and E14 (129/Ola) mouse embryonic stem cells (mESCs) were obtained from Merrill’s laboratory2 bacterial dishes, where the cells grow in suspension. On day 5, the embryoid bodies were transferred onto gelatinized p100 dishes always in differentiation medium, which consisted of mESC culture medium without the LIF. The medium was changed every 2 days and the beating embryoid bodies were observed starting from day 8 of the differentiation process. For expression profile analysis the cells were harvested and pelleted at day 0 (ESC), 6 and 12 of the differentiation process.The differentiation medium for the production of embryoid bodies (EBs) consisted of mESC culture medium without LIF. The cells were harvested by trypsinisation, counted, and propagated in hanging drops  for 2 days, before being transferred to 10 cm4/cm2 in N2B27 medium [1:1mix of DMEM/F12 (GIBCO) supplemented with N2 (GIBCO) and Neurobasal medium (GIBCO) supplemented with B27 (GIBCO)], L–glutamine , 2-mercaptoethanol  and retinoic acid (1 μM). The medium was refreshed every other day65. For expression profile analysis cells were detached by using Accutase  and pelleted at 300 rcf for 5 minutes.For neural differentiation in monolayer, undifferentiated mESCs were gently dissociated using trypsin  at 37 °C and plated onto 0.1% gelatin-coated tissue culture plastic at a density of 0.5–1.5 × 10Cells were trypsinized  at 37 °C, pelleted at 300 rcf and washed twice with PBS. During each wash cells were pelleted at 300 rcf for 5 min 4 °C. Cell lysis was performed on ice for 25 min, in RIPA buffer  containing protease (SIGMA P8340) and phosphatase inhibitors (SIGMA P2850). Insoluble material was pelleted by centrifugation at 16,000 rcf for 30 min at 4 °C. Protein concentrations were determined using the Bradford assay (Bio-Rad 500-0006). Western blot was performed as specified in the apposite section with the antibodies indicated in Table 2 DTT 1 mM) containing protease inhibitors (SIGMA P8340) for 10 min at 4 °C. Cells were pelleted at 300 rcf for 5 min at 4 °C and plasma membrane lysis was performed in 0,25% NP-40 hypotonic buffer on ice for 15 min. Nuclei were pelleted at 300 rcf for 15 min at 4 °C and washed twice in hypotonic buffer. Isolated nuclei were incubated in RIPA buffer  containing protease (SIGMA P8340) and phosphatase inhibitors (SIGMA P2850). Insoluble material was pelleted by centrifugation at 16,000 rcf for 30 min at 4 °C. Protein concentrations were determined using the Bradford assay (Bio-Rad 500-0006). Western blot was performed as specified in the apposite section with the antibodies indicated in Table For nuclear protein extraction cells were trypsinized  at 37 °C, pelleted at 300 rcf and washed twice with cold PBS. During each wash cells were pelleted at 300rcf for 5 min at 4 °C. Cells were incubated in hypotonic buffer  were washed 3 times in 500 µl of cold CHAPS buffer  containing protease inhibitors (SIGMA P8340). To separate the beads from the wash solution the tubes were placed on the magnet. The isolated beads were re-suspended in 500 µl of antibody solution containing 8 µg of antibody  β-catenin  KAP1 ,) or IgG  in cold CHAPS buffer containing protease inhibitors) and incubated O/N at 4 °C on a rotating wheel.Cell fractionation and nuclei isolation was performed as described in the previous paragraph. For co-immunoprecipitation experiments, nuclei were lysed in CHAPS buffer  for 15 min at 4 °C and were immerged in liquid nitrogen for 2 min and successively thawed on ice to perform a freeze-thaw lysis. Insoluble material was pelleted by centrifugation at 16,000 rcf for 30 min at 4 °C and the supernatants (100 µg of the nuclear protein extract) were incubated with antibody-coupled dynabeads overnight O/N at 4 °C. Beads were washed three times with CHAPS buffer containing protease inhibitors and elution was performed by boiling beads in Laemmli buffer (1x) at 95 °C for 10 min. Western blot was performed as specified in the apposite section with the antibodies indicated in Table Either total protein extract or nuclear protein extract was mixed with 4 × Laemmli buffer  and denatured at 99 °C for 10 minutes. Either total protein extract or nuclear protein extract, or co-immunoprecipitation eluate was separated by SDS-PAGE, and transferred to poly vinylidene difluoride membrane (BIO-RAD 162-0177). The membranes were blocked with 5% non-fat dry milk (SIGMA 70166) in TBS-Tween 20  (SIGMA P1379) for 60 min, incubated with primary antibodies , NANOG , OCT-4 , β-tubulin , DNMT1 , KAP1 ) overnight at 4 °C. The working dilution of each antibody is listed in Table 124 to the Mus musculus genome (assembly NCBI37/mm9). CpG methylation calls were extracted using the Bismark methylation extractor v0.7.9. The methylation level of a DNA region was defined using SeqMonk v0.32.1 pipeline  considering at least 2 CpGs covered by at least 10 reads. Hypomethylated DNA regions were identified by searching for sequences with common symmetric CpGs (at least 10 CpGs covered by at least 10 reads that were less than 2 kb apart) with a decrease in methylation of >25%. Clustering and correlation analysis were performed using R package methylKit125. The RRBS data are available under the GSE109417 accession number.Reads were processed by adaptor trimming (Illumina Pipeline Casava v1.8.2), filtered for low quality reads  and subjected to quality control (FastQC). Reads were aligned using Bismark v0.7.9126 to overlap ChIP-seq peak data coordinates. The annotatePeaks tools from HOMER suite of programs127 was used to annotate the resulting peak overlaps (using mm9 version of Mus musculus genome assembly).The CpG islands data was downloaded from the UCSC genome annotation data-base for the July 2007 assembly of the mouse genome . The repeat types and coordinates were extracted from the RepeatMasker file (UCSC table browser). We used bedtools (v2.25.0)128, reshape2129 VennDiagram130 and UpSetR131 packages.Relevant figures were produced in the R environment using mainly ggplot2Supplementary Information and figuresMovie S1 YP-E14Movie S2 YP-GS1Movie S3 OP-E14Movie S4 OP-GS1Table S1Table S2Table S3Table S4


Cx3cr1-deficient microglia in young mice display a gene expression profile similar to microglia in aged mice, suggesting premature microglial aging.Gyoneva et al use RNA-seq to show that  Cx3cr1 signaling are not well understood. Here, we analyzed transcriptomes of Cx3cr1-deficient microglia under varying conditions by RNA-sequencing (RNA-seq). In 2-mo-old mice, Cx3cr1 deletion resulted in the down-regulation of a subset of immune-related genes, without substantial epigenetic changes in markers of active chromatin. Surprisingly, Cx3cr1-deficient microglia from young mice exhibited a transcriptome consistent with that of aged Cx3cr1-sufficient animals, suggesting a premature aging transcriptomic signature. Immunohistochemical analysis of microglia in young and aged mice revealed that loss of Cx3cr1 modulates microglial morphology in a comparable fashion. Our results suggest that CX3CR1 may regulate microglial function in part by modulating the expression levels of a subset of inflammatory genes during chronological aging, making Cx3cr1-deficient mice useful for studying aged microglia.CX3CR1, one of the highest expressed genes in microglia in mice and humans, is implicated in numerous microglial functions. However, the molecular mechanisms underlying  Microglia are the parenchymal tissue-resident macrophages of the central nervous system (CNS). They support brain development and adult CNS homeostasis, functions which are often dysregulated in acute or chronic neurological conditions . DecipheThe factors that regulate the microglial transcriptome under homeostatic conditions are starting to emerge. Microglial identity is conferred by their yolk-sac origin and the brain environment . The traCx3cr1) is highly expressed in microglia. Genetic studies with deletion of one or both copies of Cx3cr1, or its monogamous ligand Cx3cl1, have shown pleiotropic roles for the receptor. Mice lacking Cx3cr1 expression show altered brain development during embryogenesis, dysregulated neuronal firing in the first few weeks after birth, and abnormal results in tests designed to mimic sociopsychological behaviors  model of Parkinson’s disease and in tauopathies such as frontotemporal dementia, but promotes amyloid clearance in models of amyloid β deposition  motif receptor 1  to define how transcriptional responses to diverse biological processes were affected by loss of Cx3cr1. Our results indicate that CX3CR1 signaling contributes to the regulation of microglial morphology and a subset of inflammatory genes. Importantly, loss of CX3CR1 signaling in young mice resulted in a microglial transcriptome reminiscent of aged mice.Despite the importance of CX3CR1 for microglial function in the dynamic contexts of development and disease, there is relatively little understanding of how CX3CR1 regulates microglial function at the molecular level. We performed a transcriptomic analysis of Cx3cr1 expression: Cx3cr1+/+ (WT), Cx3cr1+/eGFP (Het), and Cx3cr1eGFP/eGFP (KO). In WT mice, the microglial expression profile obtained here is consistent with the published microglial transcriptomes (Table S1) . The samples separated well by Cx3cr1 genotype on the first principal component  on isolated brain microglia from young adult (2 mo) mice with variable able S1) . To examomponent , showingiability .Comparison between highest expressed genes in current data set and selected published data sets.Table S1 Cx3cr1 resulted in 165 DEGs. Of these, 100 genes showed lower expression in Cx3cr1-KO microglia, whereas 65 genes showed higher expression (Table S2). Similarly, when comparing Cx3cr1-Het to WT microglia, there were 55 genes with lower expression in Het animals and only four with higher expression (Table S3). Importantly, most of the genes differentially expressed between Het and WT microglia were also differentially expressed between KO and WT microglia  by performing pairwise comparisons between the genotypes. Using the cutoffs described in the Methods, loss of icroglia , indicatsee also .Quantification results in 2-mo KO versus WT microglia.Table S2 Quantification results in 2-mo Het versus WT microglia.Table S3 P-value) showed a striking resemblance between the transcriptomes of Cx3cr1-KO and -Het microglia , chemokine receptors and ligands , proteases , and other genes with immune function . Although fewer genes had higher expression in Cx3cr1-deficient microglia, some of these included genes with pleiotropic functions such as Tnf, the transcription factors Egr1 and Klf2, and the chemokine-like receptor Ccr1l1. We confirmed the altered expression of selected genes by Cx3cr1 genotype using quantitative reverse transcriptase PCR (qRT-PCR) of microglia sorted from a separate cohort of mice  differentiates microglia from other myeloid cells . Cx3cr1 re genes , and onlCx3cr1-WT and -KO microglia. Unexpectedly, only 19 genes displayed differential binding between WT and KO animals (We performed chromatin immunoprecipitation for RNA polymerase II (Rbp2) followed by sequencing (ChIP-seq) to determine whether changes in transcriptional activity indicated by Rbp2 binding explain the differential gene expression between  animals . These fQuantification results for Rbp2 ChIP-seq.Table S4 Quantification results for H3K27Ac ChIP-seq.Table S5 Klhl42 and Auh  injection. The LPS treatment  induced a strong transcriptional response in both peritoneal cells and in microglia, resulting in 5,362 and 5,084 DEGs, respectively. This was visible by PCA, where LPS and saline samples formed distinct clusters . Yet, some of the genes affected by Cx3cr1 deficiency in microglia from LPS-injected mice included up-regulation of Adora1 and Klf2 and down-regulation of Ninj1 , middle-aged (1 yr), and aged (2 yr) Cx3cr1+/+, Cx3cr1+/eGFP, and Cx3cr1eGFP/eGFP mice. Fewer live microglia were recovered from aged mice, independent of genotype , chemokine receptors (Ccr3 and Ccr5), Clu, Mmp9, and Lyz2. Thus, the absence of a single copy of Cx3cr1 at young age results in a transcriptome comparable with that of aged microglia. Additional clusters of genes were up-regulated with aging but showed highest expression in aged KO microglia. Noteworthy genes in these clusters included Adora1, Cd34, H2-Dma, and Tnf. The widespread dysregulation of immune genes by Cx3cr1 deletion was also confirmed by gene ontology analysis  or old (18–24 mo) mice by quantitative morphometry, using immunohistochemistry for Iba1, a cytosolic marker, to delineate the cells. Microglia in the cortex (Ctx)  , hippocaex (Ctx) , and strex (Ctx)  displayeex (Ctx) .Cx3cr1-deficient microglia had significantly decreased total Iba1 area, Iba1 area in processes directly connected to cell bodies, and process perimeter compared with those found in WT mice  is down-regulated in Cx3cr1-deficient microglia, and Klotho protein has been linked to aging and homeostatic support of brain function . The changes we detected in young Cx3cr1-KO versus Cx3cr1-WT mice were strikingly similar to those seen in Cx3cr1-WT aged versus young mice, suggesting that loss of Cx3cr1 accelerated microglial aging.Only by examining the effect of Cx3cr1-deficient mice, in which both copies of Cx3cr1 are disrupted by the gene for eGFP . Generally, microglia isolated from individual mice were treated as individual samples. For ChIP-seq analysis, microglia from three mice (gender-matched) were pooled to obtain individual samples.All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Biogen. for eGFP , were inEscherichia coli strain O111:B4 and euthanized 24 h later for peritoneal cell and microglia isolation. Control mice received saline injections.For some experiments, mice were injected with 1 mg/kg LPS derived from 2 asphyxiation, peritoneal cells were isolated by flushing the peritoneal cavity with cold PBS, and then perfused. In all cases, the brain was then isolated and stored in PBS on ice until processing. Brains were processed in sets of 5–7 per day; each day contained a mixture of genotypes and experimental conditions . Sequencing results showed no batch effects based on processing days (data not shown).Except for mice used for the LPS study, mice were deeply anesthetized with 10 mg/kg ketamine, 30 mg/kg xylazine until they failed to respond to toe pinch, and transcardially perfused with cold PBS. Mice for the LPS study were euthanized by COg for 5 min. The cell pellet was resuspended in FBS, and the volume was adjusted to 10 ml with isotonic 33% Percoll  and centrifuged at 800g at room temperature for 15 min with no brake. The myelin layer was first aspirated from the top, and then the isolated cells collected from the bottom of the tube. The cells were washed in FACS buffer  and stained for microglial markers.All subsequent steps were performed on ice. After isolation, whole brains were finely minced with a razor blade and transferred to 15-ml polypropylene conical tubes containing 3 ml accutase enzyme . The samples were incubated for 30 min at 4°C to allow tissue dissociation. This was followed by manual pipetting consecutively with 5-ml serological pipette and a 1-ml pipette tip to complete the homogenization. The supernatant containing single-cell suspension was passed through a 250-μm filter in a clean 15-ml conical tube, and the tubes were filled with HBSS supplemented with 25 mM Hepes and centrifuged at 600+CD45lo events. Post-sort purity analysis included DRAQ5  for evaluating nucleated events; microglial purity was generally over 90% of nucleated events. The cells were maintained, chilled during the sort, and stored on ice. After sorting, the samples were spun down at 800g for 5 min to pellet the cells. The cell pellet was either processed right away for RNA isolation or frozen at −70°C.Once single cell suspensions were obtained, the samples were blocked with anti-mouse CD16/CD32  for 10 min on ice and stained with anti-mouse CD11b-PECy7  and anti-mouse CD45-BV605  for 30 min on ice. Staining antibodies were washed away with FACS buffer. Right before sorting, Draq7  was added to the cells to exclude dead cells. Sorting was performed on a BD Aria Fusion cell sorter equipped with four lasers: 405 nm violet (85 mW), 488 nm blue (50 mW), 561 nm yellow-green (50 mW), and 640 nm red (100 mW). Detection filters were 780/60 (LP735) for PE-CY7, 610/20(595LP) for BV605, and 780/60 (735LP) for DRAQ7. The cell suspension was sorted with an 85-μm nozzle at 45 PSI pressure. Cells were sequentially gated on FSC-A/FSC-H and SSC-A/SSC-H to select singlet events, followed by dead cell exclusion with DRAQ7. The final sort gate selected CD11bFor samples used for ChIP-seq, cells from three gender-matched animals were pooled together in one sample after staining and fixed in 1% paraformaldehyde for 10 min at room temperature, as described before . The celPeritoneal cells obtained from LPS-injected mice were not sorted. After washing with HBSS containing 25 mM Hepes, the cell pellets were processed for RNA isolation.The RNA (containing microRNAs) was isolated with the QIAGEN Universal AllPrep kit  according to the manufacturer’s instruction. The RNA samples were treated on the column with DNAse I  to remove potential DNA contamination. The RNA concentration was quantified with the Quant-iT RiboGreen reagent . The RNA quality was determined on a bioanalyzer with the RNA Pico 6000 kit , according to the manufacturer’s instructions.Because of the low RNA content of microglia and our desire to use each animal as an individual sample, we used low RNA input protocols for RNA-seq library preparation. The cDNA for RNA-seq libraries was prepared using 10 ng of RNA using the SmartSeq v4 Ultra Low RNA Input Library preparation protocol  according to the manufacturer’s instruction. The cDNA  was then tagged and fragmented with the NexteraXT DNA Library Preparation kit  according to the manufacturer’s instructions. Quality of amplified cDNA and fragmented libraries was assessed on a bioanalyzer with the High Sensitivity DNA chips . Tagged libraries were pooled  and sequenced on an Illumina HiSeq2500 sequencer, with a target depth of 20 million of 50 bp paired end reads. Quality control was performed using an Illumina’s BaseSpace run summary tool.Although microglial RNA samples for the aging study were obtained at different times, all libraries were prepared together to reduce batch effect in sequencing. It should be noted that the samples from 2-mo-old mice were used twice for sequencing—as a standalone set and as part of the aging study. The results from the two sequencing runs correlated very well with each other.For LPS-injected mice, RNA from peritoneal cells  was also subjected to sequencing, to confirm the peripheral effect of the LPS injections. The RNA-seq libraries were prepared with 8 ng RNA as the starting material for both peritoneal cells and matched microglia from the same animals to allow direct comparison of the cell types.Ccr1l1 (Mm00432606_s1), H2-Aa (Mm00439211_m1), Klf2 (Mm00500486_g1), S100a8 (Mm00496696_g1), Egr1 (Mm00656724_m1), Trem2 (Mm04209424_g1), and Gapdh (Mm99999915_g1). Gene expression within each sample was normalized to Gapdh and then to group average expression in WT mice. Samples are plotted individually and with their mean ± SEM.To validate the expression of selected genes by qRT-PCR, we sorted microglia from a separate cohort of mice and isolated RNA as described above. Fifty nanograms of RNA was reverse-transcribed to cDNA using the High-capacity cDNA Reverse Transcription kit , which was then amplified with TaqMan Fast Advanced Master Mix  on an Applied Biosystems QuantStudio 12K Flex thermocycler. The probes used were The raw sequencing data were first analyzed using an in-house data-processing pipeline consisting of STAR  and RSEMCx3cr1-KO and -WT mice. Binding/peaks were considered significantly different between comparisons if they had at least twofold difference in fold change and IDR less than 0.05. Two independent samples, each consisting of pooled microglia from three animals, were analyzed for each genotype.ChIP-seq libraries were prepared from recovered DNA by blunting, A-tailing, and adapter ligation using barcoded adapters  as previously described . LibrariCx3cr1+/+ or Cx3cr1eGFP/eGFP mice were euthanized by CO2 asphyxiation, transcardially perfused with ice-cold PBS, and the brains were dissected out and cut into left and right hemisphere. One brain hemisphere was flash-frozen in liquid nitrogen, and the other was fixed in 10% neutral buffered formalin. After 48–72 h, formalin-fixed hemispheres were embedded in paraffin. Blocks were trimmed, and sections discarded until a level of 1.2 mm lateral of midline was reached. At that level, one section was collected for dual immunohistochemistry with Pu.1/Iba1 . Slides were scanned on a 3D-Histech Pannoramic-250 whole slide scanner and imported into Viosiopharm image analysis software. Tissue regions were manually annotated by a blinded analyst. A custom image analysis algorithm was designed to identify Pu.1-immunoreactive nuclei, their associated Iba1 immunoreactive microglial processes, and all other Iba1 immunoreactivity present in each tissue region. The cell soma were identified as Pu.1-positive when pixel intensity surpassed a minimum threshold. All contiguous Iba1 immunoreactivity within a 2-μm radius of a Pu.1-positive nucleus was defined as its microglial processes. The Iba1 immunoreactive area not directly attributed to a Pu.1-positive nucleus was defined as non-process Iba1. The computer-based algorithm quantified the areas, count, and perimeters of all identified cell features, for each brain region. Here, we report the number of Iba1+/Pu.1+ objects as the number of microglia per imaged area, the total Iba1-immunoreactive area per Pu.1+ nucleus, the area of Iba1+ processes attached to a Pu.1+ nucleus as microglial process area, and the perimeter of Iba1+ processed attached to a Pu.1+ nucleus as the microglial process perimeter. In general, in young WT mice, a lower process area is associated with a more homeostatic phenotype, whereas an increased process area is associated with microglial activation.Young (2 mo) or aged (18–24 mo) post hoc test. Statistical significance on Tukey’s test is indicated in the figures as *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001.The data were graphed by grouping animals by age and genotype; each point in the dot plots represents an individual animal. Statistical significance was determined by two-way ANOVA for the main effect of age or genotype (or their interaction), followed by pairwise comparisons between all groups with Tukey’s GSE131869 and GSE130960). Additional quality control plots and interactive data exploration tools built by QuickRNASeq pipeline  to take the full advantage of this shared resource including gene matrix tables in count, reads per kilobase of transcript per million mapped reads, and TPM measures and associated visualization widgets.Raw sequencing files for the RNA-seq and ChIP-seq data sets are available at Gene Expression Omnibus (pipeline  are avai


Aberrant activation of EGFR represents a common event in non-small cell lung carcinoma (NSCLC) and activates various downstream signaling pathways. While EGFR activation of β-catenin signaling was previously reported, the mediating mechanism remains unclear. Our current study found that EGFR activation in NSCLC cells releases SHC-binging protein 1 (SHCBP1) from SHC adaptor protein 1 (SHC1), which subsequently translocates into the nucleus and directly promotes the transactivating activity of β-catenin, consequently resulting in development of NSCLC cell stemness and malignant progression. Furthermore, SHCBP1 promotes β-catenin activity through enhancing the CBP/β-catenin interaction, and most interestingly, a candidate drug that blocks the CBP/β-catenin binding effectively abrogates the aforementioned biological effects of SHCBP1. Clinically, SHCBP1 level in NSCLC tumors was found to inversely correlate with patient survival. Together, our study establishes a novel convergence between EGFR and β-catenin pathways and highlights a potential significance of SHCBP1 as a prognostic biomarker and a therapeutic target. Lung cancer is the most commonly diagnosed cancer type and a leading cause of cancer death globally. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. Despite the availability of surgical therapy, radiotherapy, and chemotherapy, prognosis of NSCLC is still poor with overall five-year survival rate being as low as 15%, mainly due to development of resistance to chemo- and radiotherapy, postoperative recurrence and early metastasis . Even thAPC, resulting in the loss of ability to binding β-catenin [AXIN, which also lead to disruption of the destruction complex, have been identified likewise. In addition, mutations of β-catenin phosphorylation sites and consequent abrogation of β-catenin phosphorylation have been found in melanoma, which leads to β-catenin accumulation in the nucleus and transcription activation of its target genes [Cancer stem cells (CSCs) are subpopulations of malignant cells that possess the abilities to self-renew and differentiate within a tumor . The bio-catenin . Mutatioet genes , 17. In et genes  and hypeet genes , the afoet genes . Hence, Of note, activating mutations of EGFR are common in NSCLC. Previous reports have shown a positive correlation between the presence of activating EGFR mutations and activation of β-catenin signaling in NSCLC , and theIn our present study, we show for the first time that SHC-binging protein 1 (SHCBP1), a unique protein specifically bound to the SHC1 SH2 domain and previously reported to disassociate from SHC adaptor protein 1 (SHC1) in response to EGF stimulation, mediates EGF-induced activation of β-catenin signaling in NSCLC cells. In response to EGF stimulation, SHCBP1 translocates to the nucleus, promotes interaction between β-catenin and CBP, activates β-catenin driven transcription, and enhances development of stem cell-like properties of NSCLC. These results indicate a novel convergence of the EGFR and β-catenin signaling pathways in the nucleus through nuclear SHCBP1. We also have identified that SHCBP1 may be indispensable for the stem cell-like phenotype driven by EGF-β-catenin signaling and is up-regulated in NSCLC and other cancers in a manner correlated with unfavorable clinical prognosis of the disease.The previous finding that EGFR signaling could activate canonical Wnt/β-catenin pathways in cancer prompted us to further investigate the mechanism underlying such a crosstalk , 25, 30.http://cancergenome.nih.gov/)  Fig. . Interesv/) Fig. , suggestv/) Fig. , stronglv/) Fig.  nor the v/) Fig. , we postv/) Fig. , our datv/) Fig. , indicatTo explore how SHCBP1 mediates EGF activation of β-catenin signaling, the effects of EGFR activity on SHCBP1/β-catenin binding were examined, and we found that EGF stimulation of EGFR in 293T or A549 cells increased SHCBP1-binding β-catenin in the nuclear extraction, while TKI treatment in HCC827 or HCC4006 NSCLC cells revealed the opposite effects Fig. , indicatThe previously well-established understanding that EGF-stimulated EGFR signaling can induce and/or sustain cellular stemness under both physiological and cancer settings , 40, togFurther, our above-mentioned finding that EGFR activation enhances the affinity between CBP and β-catenin as well as activation of the β-catenin pathway via SHCBP1 translocation prompted us to ask whether the binding between CBP and β-catenin is also important for the stem cell properties mediated by EGF. Our results demonstrated that the ICG-001 compound effectively suppressed EGF-induced formation of tumor cell spheres, expression of stemness markers as well as Survivin, drug resistance against Cisplatin Fig. , suggestInterestingly, we observed that upregulation of SHCBP1 per se led to nuclear redistribution and β-catenin activation as well Fig. , and fur2 A549 cells with ectopic EGF expression led to growth of tumors, whereas at least 5 × 104 control NSCLC cells were required to form a tumor  and T-, N-, and M-classification   cells Fig. . In addiConsidering that nuclear SHCBP1 mediated EGF-induced β-catenin activation and cellular stemness, we further examined whether it was increased in NSCLC tissue. By using nuclear extraction and western blot assay, we detected far more nuclear SHCBP1 in NSCLC tumor tissues as compared with that in the corresponding adjacent non-cancerous lung tissues Fig. . MeanwhiIt is well established that in response to EGF stimulation, EGFR recruits scaffold protein SHC1 and consequently activates downstream signals, such as those mediated by Ras/Erk and PI3K/Akt pathways. This notion suggests a model in which SHC1 serves as a hub for its binding partners to trigger and/or transduce subsequent EGF–EGFR signaling and the downstream biological events. Along this context, Yong Zheng et al.  analyzedNotably, previous reports have shown that cytoplasmic activation of β-catenin can be regulated by EGFR signaling, leading to increased nuclear translocation of β-catenin through various extranuclear mechanisms, which either promotes release of β-catenin from the cytoplasmic membrane or accumulation of free β-catenin in the cytoplasm, or disrupts the β-catenin degradation complex so that more β-catenin enters the nucleus , 44–48. Interestingly, our data showed that disruption of CBP/β-catenin interaction with the ICG-001 compound could abrogate the EGFR enhancement of β-catenin transactivating activity. ICG-001 is an inhibitor that specifically blocks CBP/β-catenin binding. Moreover, it was found to eliminate drug-resistant tumor-initiating cells and promote differentiation of chemotherapy-insensitive CSCs by inhibiting β-catenin signaling , 49–52. Our data presented in this report highlight a functional significance of SHCBP1 in mediating the crosstalk between EGFR signaling and β-catenin transactivation activity. The SHCBP1 gene, located in chromosome 16q11.2, is evolutionarily conserved across species spanning from insects to mammals . PreviouIn summary, this work has identified SHCBP1 as a new mediator for the crosstalk between EGFR and β-catenin signaling pathways and a new mechanism mediating the development and maintenance of NSCLC stem cells. Our data also support that SHCBP1 is a potential therapeutic target and prognostic biomarker for the disease. Future opportunity of developing anti-NSCLC strategies targeting SHCBP1/β-catenin/CBP interaction under different EGFR activity statuses is under investigation in this laboratory.All tumor cell lines were obtained from the Cell Bank of Shanghai Institutes of Biological Sciences , Fu Erbo Biotechnology Co., Ltd , or ATCC, and cultured as previously described . PrimaryClinical tissue specimens were histopathologically diagnosed at the Sun Yat-Sen University Cancer Center from 2000 to 2004. The histological characterization and clinicopathologic staging of the cases were determined by following the standard provided in the current Union for International Cancer Control (UICC) Tumor-Node-Metastasis (TNM) classification. Each tumor and adjacent non-cancerous lung tissue pair was obtained according to our previous reports , 56. PriThe SHCBP1 expression plasmid was generated by PCR subcloning the human SHCBP1 coding sequence into the lentiviral transfer plasmid pSin-puro  to generate plasmid pSin-SHCBP1. To deplete SHCBP1 expression, two human shRNA sequences  were cloned into the pSuper-retro-puro plasmid to generate pSuper-retro-SHCBP1-sh1 and pSuper-retro-SHCBP1-sh2. Human EGF-coding sequence was subcloned into the lentiviral transfer plasmid to generate plasmid pSin-EGF. Retroviral and lentiviral production and infection were performed according to the manufacturer’s instructions.−△△CT formula.Total RNA extraction, reverse transcription, and real-time PCR were performed as described previously . Primers7 293T cells transduced with Flag-tagged SHCBP1 or vector using lysis buffer. Lysates were then incubated with FLAG affinity agarose  overnight at 4 °C. Beads containing affinity-bound proteins were washed six times with immunoprecipitation wash buffer. Proteins were separated on SDS polyacrylamide gels stained with Coomassie blue, and all bands were subjected to mass spectrometry analysis. SHCBP1 and β-catenin protein purification was acquired by using immunoprecipitation and FLAG/HA competing peptides  . Forty-eight hours after transfection, Dual-Luciferase reporter assays were performed according to the manufacturer’s protocol of Dual Luciferase Reporter Assay Kit . The reporter plasmids containing wild-type  or mutated  TCF/LEF DNA-binding sites were purchased from Upstate Biotechnology .NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit  was used and the assays were performed according to the manufacturer’s instructions.Tumor spheres were cultured according to a previous report . Cells wIHC assays in 207 NSCLC tissues were performed by using primary SHCBP1 antibody  and quantified according to our previous report , 60. TheFemale BALB/c-nu mice  were purchased and housed in specific pathogen-free facilities on a 12-h light/dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University. At least five nude mice per group were used to ensure the adequate power and each mouse with different weight was randomly allocated. ICG-001 or vehicle has begun to be used 7 days after tumor cell injections in animals. ICG-001 50 mg/kg) or vehicle (PBS) were intraperitoneally injected every 3 days for 3 weeks as described previously  mg/kg or. For bioP < 0.05 and all sample sizes were appropriate for assumption of normal distribution. Variance within each group of data was estimated and was similar between compared groups. Data analysis was performed by two independent investigators who were blinded to the sample groups. All statistical analyses were performed using the SPSS 13.0 (IBM) statistical software package. The Kaplan–Meier method was used to establish survival curves. The statistical significance of various variables for survival was analyzed using the Cox proportional hazards model in the multivariate analysis. Correlation between SHCBP1 expression and T-, N-, M-classification was analyzed using chi-square test. Comparisons between groups were performed with a two-tailed paired Student’s t-test. In all cases, P < 0.05 was defined as statistically significant.Sample size was determined by power analysis to achieve a minimum effect size of 0.5 with Supplementary figure1Supplementary figure2Supplementary figure3Supplementary figure4Supplementary figure5Supplementary figure6Supplementary figure7Supplementary figure legendsSupplementary tablesSupplementary materials and methods


The Wnt pathway has been shown to regulate bone homeostasis and to influence some bone disease states. We utilized a zebrafish model system to study the effects of a synthetic, orally bioavailable glycogen synthase kinase-3β (GSK3β) inhibitor LSN 2105786, which activates Wnt signaling during bone healing and embryogenesis. GSK3β inhibitor treatment was used to phenocopy GSK3β morpholino oligonucleotide (MO) knockdown in zebrafish embryos. Human and zebrafish synthetic mRNA injection were similarly effective at rescue of GSK3β MO knockdown. During caudal fin regeneration, bony rays are the first structure to differentiate in zebrafish fins, providing a useful model to study bone healing. Caudal fin regeneration experiments were conducted using various concentrations of a GSK3β inhibitor, examining duration and concentration dependence on regenerative outgrowth. Experiments revealed continuous low concentration (4–5 nM) treatment to be more effective at increasing regeneration than intermittent dosing. Higher concentrations inhibited fin growth, perhaps by excessive stimulation of differentiation programs. Increased Wnt responsive gene expression and differentiation were observed in response to GSK3b inhibitor treatment. Activating Wnt signaling also increased cell proliferation and osteoblast differentiation in fin regenerates. Together, these data indicate that bone healing in zebrafish fin regeneration was improved by activating Wnt signaling using GSK3b inhibitor treatment. In addition, caudal fin regeneration is useful to evaluate dose-dependent pharmacological efficacy in bone healing, various dosing regimens and possible toxicological effects of compounds. The Wnt signaling pathway controls bone formation, homeostasis, and repair . Wnt ligLoss of function mutations in the LRP 5/6 co-receptor produce osteoporosis-pseudoglioma syndrome, where patients have significantly lower bone mass and an increased risk of fracture ,8. AdditThe canonical Wnt pathway is important in the bone healing process . Wnt patlef1 is expressed in the wound epidermis as early as 12 hpa in zebrafish [lef1, and β-catenin signaling is sufficient to repress expression of FGF target genes [Wnt/β-catenin signaling is the earliest genetically identified step in fin regeneration and is required during all stages of regeneration . The Wntebrafish . Previouet genes . These ret genes ,22.axin2 and sp8) expression is activated during blastema formation [In addition to the regenerative response, β-catenin signaling is also needed to establish the blastema. Using a transgenic zebrafish TOPdGFP, β-catenin signaling is detected in the blastema by 48 hpa. Additionally, Wnt signaling target genes  transgenic zebrafish were used to block β-catenin signaling at various times post amputation and showed that β-catenin signaling is needed during each stage of regeneration. Inhibition of β-catenin signaling at 0 or 1 day post-amputation (dpa) does not affect epidermal wound healing, but prevents blastema formation and subsequent regenerative outgrowth. Disrupting β-catenin signaling after the blastema has already formed allows only partial fin regeneration. Canonical Wnt-induced β-catenin signaling also regulates blastema maintenance during later outgrowth phases. Increasing β-catenin signaling enhanced fin regeneration rate. Fish with a mutation in one copy of the Wnt/β-catenin pathway inhibitor, at 7 dpa .Interestingly, β-catenin independent, non-canonical Wnt signaling regulates fin regeneration. Blocking the non-canonical Wnt signaling pathway increased fin regenerative outgrowth, and over activation of non-canonical Wnt signaling completely blocks fin regeneration , showingSynthetic orally bioavailable GSK3β inhibitors, which also activate the canonical Wnt signaling pathway have been shown to enhance bone growth in rats . In thisDanio rerio) AB strain and Tg(TOP:GFP) [Zebrafish (TOP:GFP)  transgenTOP:GFP) . IndianaTOP:GFP) .Zebrafish, 6–12 months of age, were used for caudal fin regeneration experiments. Fish were anesthetized in tricaine (Ethyl 3-aminobenzoate methanesulfonate), and approximately 50% of the caudal fin was amputated using a razor blade. Fish were placed in 2 L of water with various concentrations of GSK3β inhibitor compound (LSN 2105786) or dimethyl sulfoxide  vehicle control and kept at 31 °C to promote rapid regeneration. Tank water was replaced daily, including fresh compound. Tanks and fish were rinsed between treatments to remove any residual compound.http://rsb.info.nih.gov/ij/), and the average length of the regenerate was calculated for each fin.At 4 and 7 dpa, fish were anesthetized and images of regenerating fins were collected using a Leica MZ12 microscope equipped with Leica DFC290 camera . The length of the regenerate  at the third, fourth and fifth dorsal and ventral fin rays were measured using ImageJ software  in embryo medium. Various concentrations of the drug were used to treat embryos from 6 to 28 h post fertilization (hpf), in Petri dishes wrapped with parafilm and maintained at 28.5 °C. Embryos were imaged using a Leica MZ12 microscope equipped with Leica DFC290 camera.shh, lef1, bmp2b, bmp4 and sox9b were synthesized using DIG RNA Labeling Kit  according to manufacturer’s instructions. Images were collected using a Leica MZ12 microscope equipped with Leica DFC290 camera.Fins collected at various time points after amputation were fixed overnight at 4 °C in freshly made 4% paraformaldehyde in phosphate-buffered saline (PBS). Fins were then washed two times in PBS, dehydrated in methanol and stored at 20 °C overnight. Fins were rehydrated stepwise in methanol in PBS containing 0.1% Tween 20 (PBST). Next, fins were treated for 30 min in proteinase K (10 μg/mL) in PBST. Then, fins were washed two times in PBST, and post-fixed in 4% paraformaldehyde in PBS for 20 min. Fins were washed five times in PBST, and then prehybridized for 2 h at 70 °C in hybridization buffer . Following prehybridization, fins were hybridized overnight in hybridization buffer containing 0.5 μg/mL digoxigenin-labeled RNA probe at 70 °C. Then, fins were washed at 70 °C for 10 min each in 75% hybridization buffer/25% 2× SSC, 50% hybridization buffer/50% 2× SSC, 25% hybridization buffer/75% 2× SSC, and 2× SSC. Next, fins were washed two times in 0.05× SSC for 30 min each at 70 °C, followed by washes at room temperature for 5 min each in 75% 0.05× SSC/25% PBT, 50% 0.05× SSC/50% PBT, 25% 0.05× SSC/75% PBST, and PBST. Tissues were blocked for 2 h at room temperature in block buffer . Fins were then incubated overnight in a 1:5000 dilution of anti-digoxigenin antibody  coupled to alkaline phosphatase in block buffer. The following day, fins were washed with PBST five times for 15 min each. Fins were then washed with alkaline phosphatase buffer (NTMT) three times for 15 min each, and then, fins were incubated with NBT/BCIP substrate in NTMT to allow color development. Development of the staining reaction was monitored carefully, and compound-treated and control fins were stopped at the same time to allow accurate comparison. Following color development, fins were washed five times in PBST and stored in 80% glycerol in PBST. Digoxigenin-labeled riboprobes for Fins were collected at various time points post-amputation and fixed overnight at 4 °C in 4% paraformaldehyde in PBS. Following fixation, fins were briefly washed several times with PBS. Fins were incubated overnight at room temperature in block solution, and then, fins were incubated with primary antibody in block solution for 1 to 3 days. Primary antibodies used were: mouse anti-Zns5  at 1:200; and rabbit anti-phospho-histone H3  at 1:500. After incubation in primary antibody, fins were washed with PBST six times for 10 min each, and then incubated overnight with secondary antibody in block solution. Goat anti-mouse Texas Red (Invitrogen), and goat anti-rabbit Alexa 488 (Invitrogen) were used. Nuclei were visualized by incubating fins in 1 µM To-PRO3 iodide for 1 h at room temperature. Confocal images were acquired using a Zeiss Observer Z1 LSM 700 confocal microscope . To measure cell proliferation phospho-histone 3 (PH3)-positive cells were counted from at least three fins per group. The number of PH3-positive cells per unit area was calculated for each fin using Volocity software .DNA sequence alignment was done by using Clustal Omega program, The European Bioinformatics Institute (EMBL-EBI), Hinxton, Cambridgeshire, UK. 3D-protein structure prediction was done by Phyre2 web portal for protein modeling, prediction and analysis  .gsk3β (this morpholino targets both gsk3βa and gsk3βb). The previously published sequence of the MO is GTTCTGGGCCGACCGGACATTTTTC [Morpholino oligonucleotide (MO) was synthesized by Gene Tools, LLC  to block the translation of CATTTTTC .MO concentration was determined spectrophotometrically. Different concentrations (2–6 ng/nL) of the MO were injected into 2–4 cell stage embryos to determine optimal effective concentrations. Injection of 1 nL of 3.5 ng/nL MO consistently produced defective embryos.gsk3β resistant to MO and human GSK3β genes were synthesized in vitro at Lilly Research Laboratories and cloned in pAN104b and pcDNA3.1 vectors, respectively. Messenger RNAs were synthesized using T7 polymerase. Different concentrations of mRNA ranging from 100 to 500 ng/nL were injected along with the MO into 2–4 cell stage embryos. Co-injection of 200 ng/nL of either zebrafish or human synthetic GSK3β mRNA reduced the severity with MO (3.5 ng/nL) induced defects.Zebrafish t-test .Analyses on fin regenerate length and cell proliferation were performed using unpaired two-tailed Student’s The human GSK3Β protein consisting of 420 amino acids is 95% identical and 98% similar to the zebrafish GSK3β protein consisting of 421 amino acids ; n, no. of embryos). The lower concentrations used (350–500 nM) resulted in variable phenotypes, ranging from reduced eye size to eyeless phenotypes. Treatment with 300 nM LSN 2105786 produced nearly normal appearance to eyes   A–T. To dlicates) U–Y. GFP licates) Y, perhapTo test the effects of LSN 2105786 on the rate of zebrafish fin regeneration, half of the caudal fin was amputated from adult zebrafish (6 to 12 months old) and the fish were treated either with compound or with vehicle DMSO immediately following amputation. To ascertain the optimal treatment regimen for maximal regenerative outgrowth, fish were either treated intermittently  with higher concentrations (50–300 nM) or continuously with lower concentrations (1–50 nM) of LSN 2105786. Continuous exposure with high concentration of LSN 2105786 killed the fish. At 4 and 7 dpa, fins were imaged, and regenerative outgrowth was measured. Continuous exposures with low concentrations of LSN 2105786 were more effective at stimulating regeneration than intermittent exposure, as detailed below.The optimal dosing regimen for maximal regenerative outgrowth was ascertained when caudal fin was amputated in fish that were treated continuously with various concentrations of LSN 2105786 . Treatments using 4 or 5 nM LSN 2105786 were most effective at stimulating regeneration. In both the 4 and 5 nM continuous treatment groups, fin regenerates were significantly longer than control fins at 4 and 7 dpa A–D. Contlef1, and downstream genes shh, bmp2b, and bmp4 were analyzed by using in situ hybridization. Previous studies showed that lef1 controls blastema formation [lef1 is expressed in the distal blastema in two sets of cells on each side of the fin ray [lef1 expression at 1 and 2 dpa compared to vehicle treated control fins (lef1 was restricted to the area immediately surrounding the blastema in DMSO treated fins; whereas, LSN 2105786 treated fins showed an expansion of lef1 expression in to the wound epidermis and inter ray tissue. At 3 dpa, lef1 staining in two groups was similar (To determine the spatial and temporal effects of GSK3β inhibition on Wnt responsive gene expression in fin regenerates, the Wnt/β-catenin signaling target gene, ormation ,32, and  fin ray . LSN 210rol fins A–D. At 1 similar E,F.bmp2b and bmp4 are expressed in the blastema [bmp2b is expressed in differentiating scleroblasts and in the basal layer of the epidermis [bmp4 is expressed in the distal blastema [bmp2b in the LSN 2105786 treated group was nearly indistinguishable from DMSO treated control group at 3 and 7 dpa (bmp4 expression at the blastema region in 3 and 4 dpa fins showed weak bmp4 expression at the blastema in controls, but LSN 2105786 treated group displayed more intense expression of bmp4 (bmp4 expression was similarly expressed in the blastema, comparing in LSN 2105786 and DMSO treated control groups (shh, which is involved in patterning of various structures during vertebrate development, was previously shown to be expressed in the basal layer of wound epidermis in the regenerative outgrowth [lef1, shh expression is typically restricted to two sets of cells on each side of the distal tip of the fin ray during the regenerative outgrowth phase. In the LSN 2105786 treated fins, there was a slight expansion of the shh expression domain at 3 dpa, with shh transcripts being detected in the tissue proximal to the blastema and extending to near the amputation plane (shh staining remained high in the LSN 2105786 treated group as compared to the DMSO treated control group. By 7 dpa, shh expression was similar in LSN 2105786 and DMSO treated control groups (Previous fin regeneration studies showed that blastema ,34. Duripidermis , and bmpnd 7 dpa G–J. In c of bmp4 K,L. At 4l groups M,N, but utgrowth ,35. Simion plane O,P. At 4l groups Q,R.The Wnt signaling pathway controls cell proliferation throughout development . ContinuTo test the effect of GSK3β inhibitor LSN 2105786 on osteoblasts, fins were immunostained with the monoclonal antibody Zns-5, which labels differentiated osteoblasts and nuclear stain To-PRO3 iodide. Confocal microscopy images of fins treated with 5 nM LSN 2105786 showed larger Zns-5 positive area in the blastema compared to DMSO treated fins at 2 dpa A–F. At 7Previously, Flemming et al.  and otheaxin1+/- fish had increased in regenerative outgrowth, as compared to wild type fish at the same time point [wnt8 transgene expression was sufficient to increase cell proliferation, but did not affect the length of fin regenerates [Our studies are in agreement with Stoick-Cooper et al., showing that activating Wnt/β-catenin signaling improves zebrafish caudal fin regenerative outgrowth . LSN 210me point . Interesenerates . A possiThe inhibitory effects on regeneration observed following continuous LSN 2105786 treatment at higher concentrations above 5 mM may indicate that over-activation of the Wnt/β-catenin pathway excessively stimulates differentiation programs, perhaps depleting the progenitor cell population and preventing outgrowth. Additional studies aimed at determining effects of higher concentrations of GSK3β inhibitor on Wnt/β-catenin signaling in fin regeneration are needed, and these studies may provide insight into modulation of the regeneration process.lef1 provides strong evidence that LSN 2105786 activates canonical Wnt signaling during fin regeneration. Experiments using human and zebrafish GSK3β synthetic mRNA to rescue Gsk3β morpholino oligonucleotide knockdown phenotypes also strongly supports the conclusion that the human and zebrafish enzyme are interchangeable in the zebrafish embryo. Together, these results indicate that the zebrafish is a useful model to study GSK3β effects on Wnt/β-catenin signaling pathways.GSK3β inhibitor effects recapitulated previous studies using ectopic Wnt signaling phenotypes in embryos, showing LSN 2105786 acts specifically. For example, upregulation of the direct Wnt target, shh was expanded at 3 dpa, which induces proliferation and alignment of osteoblasts in the regenerating fin [LSN 2105786 treatment was sufficient to activate cell proliferation and expression of downstream genes in Wnt/β-catenin signaling. LSN 2105786 treatment, which induces blastema cell proliferation and outgrowth, may help induce and maintain cell proliferation and regenerative outgrowth. Expression of ting fin .In summary, our analysis of the effects of GSK3β inhibition on zebrafish fin regeneration showed that enhanced Wnt/β-catenin signaling produced Wnt/β-catenin target gene expression; cell proliferation; and more rapid fin regeneration. Taken together, the results indicate that chemical biology of zebrafish fin regeneration will help us understand Wnt-mediated bone growth and repair. In particular, mechanistic features can be studied and applied to clinically important questions for potential bone healing therapies.