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• W2068201305 abstract "•Jade-2 is an E3 ubiquitin ligase that specifically targets LSD1 for degradation•Jade-2-mediated LSD1 elimination promotes neural commitment of ESCs•The Jade-2-LSD1 pathway regulates nervous system development•The Jade-2-LSD1 pathway is implicated in neuroblastoma differentiation Histone H3K4 demethylase LSD1 plays an important role in stem cell biology, especially in the maintenance of the silencing of differentiation genes. However, how the function of LSD1 is regulated and the differentiation genes are derepressed are not understood. Here, we report that elimination of LSD1 promotes embryonic stem cell (ESC) differentiation toward neural lineage. We showed that the destabilization of LSD1 occurs posttranscriptionally via the ubiquitin-proteasome pathway by an E3 ubiquitin ligase, Jade-2. We demonstrated that Jade-2 is a major LSD1 negative regulator during neurogenesis in vitro and in vivo in both mouse developing cerebral cortices and zebra fish embryos. Apparently, Jade-2-mediated degradation of LSD1 acts as an antibraking system and serves as a quick adaptive mechanism for re-establishing epigenetic landscape without more laborious transcriptional regulations. As a potential anticancer strategy, Jade-2-mediated LSD1 degradation could potentially be used in neuroblastoma cells to induce differentiation toward postmitotic neurons. Histone H3K4 demethylase LSD1 plays an important role in stem cell biology, especially in the maintenance of the silencing of differentiation genes. However, how the function of LSD1 is regulated and the differentiation genes are derepressed are not understood. Here, we report that elimination of LSD1 promotes embryonic stem cell (ESC) differentiation toward neural lineage. We showed that the destabilization of LSD1 occurs posttranscriptionally via the ubiquitin-proteasome pathway by an E3 ubiquitin ligase, Jade-2. We demonstrated that Jade-2 is a major LSD1 negative regulator during neurogenesis in vitro and in vivo in both mouse developing cerebral cortices and zebra fish embryos. Apparently, Jade-2-mediated degradation of LSD1 acts as an antibraking system and serves as a quick adaptive mechanism for re-establishing epigenetic landscape without more laborious transcriptional regulations. As a potential anticancer strategy, Jade-2-mediated LSD1 degradation could potentially be used in neuroblastoma cells to induce differentiation toward postmitotic neurons. LSD1 was the first histone demethylase identified to catalyze the removal of the mono- and dimethyl moieties from H3K4 (Shi et al., 2004Shi Y. Lan F. Matson C. Mulligan P. Whetstine J.R. Cole P.A. Casero R.A. Shi Y. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.Cell. 2004; 119: 941-953Abstract Full Text Full Text PDF PubMed Scopus (3156) Google Scholar). It is subsequently identified in a number of corepressor complexes, including REST/CoREST (Shi et al., 2005Shi Y.J. Matson C. Lan F. Iwase S. Baba T. Shi Y. Regulation of LSD1 histone demethylase activity by its associated factors.Mol. Cell. 2005; 19: 857-864Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar), Mi-2/NuRD (Wang et al., 2009Wang Y. Zhang H. Chen Y. Sun Y. Yang F. Yu W. Liang J. Sun L. Yang X. Shi L. et al.LSD1 is a subunit of the NuRD complex and targets the metastasis programs in breast cancer.Cell. 2009; 138: 660-672Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar), and SIRT1/HDAC (Mulligan et al., 2011Mulligan P. Yang F. Di Stefano L. Ji J.Y. Ouyang J. Nishikawa J.L. Toiber D. Kulkarni M. Wang Q. Najafi-Shoushtari S.H. et al.A SIRT1-LSD1 corepressor complex regulates Notch target gene expression and development.Mol. Cell. 2011; 42: 689-699Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) functioning in transcription repression. The LSD1-REST/NRSF complex has been described as a master regulator of neuronal gene expression (Ballas et al., 2005Ballas N. Grunseich C. Lu D.D. Speh J.C. Mandel G. REST and its corepressors mediate plasticity of neuronal gene chromatin throughout neurogenesis.Cell. 2005; 121: 645-657Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar, Lunyak et al., 2002Lunyak V.V. Burgess R. Prefontaine G.G. Nelson C. Sze S.H. Chenoweth J. Schwartz P. Pevzner P.A. Glass C. Mandel G. Rosenfeld M.G. Corepressor-dependent silencing of chromosomal regions encoding neuronal genes.Science. 2002; 298: 1747-1752Crossref PubMed Scopus (396) Google Scholar). Consistently, LSD1 is reported to maintain the silencing of several developmental genes in embryonic stem cells (ESCs) (Adamo et al., 2011Adamo A. Sesé B. Boue S. Castaño J. Paramonov I. Barrero M.J. Izpisua Belmonte J.C. LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells.Nat. Cell Biol. 2011; 13: 652-659Crossref PubMed Scopus (242) Google Scholar, Sun et al., 2010Sun G. Alzayady K. Stewart R. Ye P. Yang S. Li W. Shi Y. Histone demethylase LSD1 regulates neural stem cell proliferation.Mol. Cell. Biol. 2010; 30: 1997-2005Crossref PubMed Scopus (182) Google Scholar). Interestingly, it has been reported that neurospecific LSD1 (nLSD1) isoforms LSD1-8a and LSD1-2a/LSD1-8a exist and are highly expressed in the nervous system in order to promote neurite morphogenesis (Zibetti et al., 2010Zibetti C. Adamo A. Binda C. Forneris F. Toffolo E. Verpelli C. Ginelli E. Mattevi A. Sala C. Battaglioli E. Alternative splicing of the histone demethylase LSD1/KDM1 contributes to the modulation of neurite morphogenesis in the mammalian nervous system.J. Neurosci. 2010; 30: 2521-2532Crossref PubMed Scopus (117) Google Scholar). Moreover, recent studies found that the expression of LSD1 is elevated in pluripotent cancer cells (Wang et al., 2011Wang J. Lu F. Ren Q. Sun H. Xu Z. Lan R. Liu Y. Ward D. Quan J. Ye T. Zhang H. Novel histone demethylase LSD1 inhibitors selectively target cancer cells with pluripotent stem cell properties.Cancer Res. 2011; 71: 7238-7249Crossref PubMed Scopus (182) Google Scholar) and that LSD1 is also highly expressed in poorly differentiated neuroblastoma (Schulte et al., 2009Schulte J.H. Lim S. Schramm A. Friedrichs N. Koster J. Versteeg R. Ora I. Pajtler K. Klein-Hitpass L. Kuhfittig-Kulle S. et al.Lysine-specific demethylase 1 is strongly expressed in poorly differentiated neuroblastoma: implications for therapy.Cancer Res. 2009; 69: 2065-2071Crossref PubMed Scopus (364) Google Scholar). Collectively, current literatures point a critical role for LSD1 in stem cell biology particularly in the maintenance of the silencing of differentiation genes. However, how LSD1 is regulated and the brake is relieved in neural development is largely unknown. Gene for apoptosis and differentiation in epithelia (Jade) family proteins contain one canonical C4HC3 plant homology domain (PHD) followed by a noncanonical extended PHD domain. On the basis of expressed sequence tags, Jade-1, Jade-2, and Jade-3 proteins are homologous at N-terminal and PHD domains but variable at C-terminal portions (Tzouanacou et al., 2003Tzouanacou E. Tweedie S. Wilson V. Identification of Jade1, a gene encoding a PHD zinc finger protein, in a gene trap mutagenesis screen for genes involved in anteroposterior axis development.Mol. Cell. Biol. 2003; 23 (8553–2)Crossref PubMed Scopus (31) Google Scholar). Jade-1, a short-lived and kidney-enriched protein, was the first and only characterized protein in the Jade family and was physically associated with the von Hippel-Lindau tumor suppressor pVHL and the ING4/ING5/HBO1 histone acetyltransferase complex and functionally linked to apoptosis and DNA replication (Doyon et al., 2006Doyon Y. Cayrou C. Ullah M. Landry A.J. Côté V. Selleck W. Lane W.S. Tan S. Yang X.J. Côté J. ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation.Mol. Cell. 2006; 21: 51-64Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, Panchenko et al., 2004Panchenko M.V. Zhou M.I. Cohen H.T. von Hippel-Lindau partner Jade-1 is a transcriptional co-activator associated with histone acetyltransferase activity.J. Biol. Chem. 2004; 279: 56032-56041Crossref PubMed Scopus (36) Google Scholar, Tzouanacou et al., 2003Tzouanacou E. Tweedie S. Wilson V. Identification of Jade1, a gene encoding a PHD zinc finger protein, in a gene trap mutagenesis screen for genes involved in anteroposterior axis development.Mol. Cell. Biol. 2003; 23 (8553–2)Crossref PubMed Scopus (31) Google Scholar, Zhou et al., 2002Zhou M.I. Wang H. Ross J.J. Kuzmin I. Xu C. Cohen H.T. The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1.J. Biol. Chem. 2002; 277: 39887-39898Crossref PubMed Scopus (67) Google Scholar, Zhou et al., 2005Zhou M.I. Foy R.L. Chitalia V.C. Zhao J. Panchenko M.V. Wang H. Cohen H.T. Jade-1, a candidate renal tumor suppressor that promotes apoptosis.Proc. Natl. Acad. Sci. USA. 2005; 102: 11035-11040Crossref PubMed Scopus (58) Google Scholar). More recently, Jade-1 was reported to inhibit Wnt signaling through its E3 ubiquitin ligase activity toward β-catenin (Chitalia et al., 2008Chitalia V.C. Foy R.L. Bachschmid M.M. Zeng L. Panchenko M.V. Zhou M.I. Bharti A. Seldin D.C. Lecker S.H. Dominguez I. Cohen H.T. Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL.Nat. Cell Biol. 2008; 10: 1208-1216Crossref PubMed Scopus (142) Google Scholar) and was therefore defined as a PHD-finger-type E3 ubiquitin ligase. However, the molecular function of other members of the Jade family is uncharacterized, and the gene encoding for Jade-2 is not cloned. Here, we report that elimination of H3K4 demethylase LSD1 promotes ESC differentiation toward neural lineage. We characterized Jade-2 as an E3 ubiquitin ligase specifically targeting LSD1 for degradation. We demonstrated that Jade-2-mediated LSD1 degradation promotes pluri- or multipotent stem cell differentiation toward the neural lineage in vitro as well as in vivo during mouse embryonic cerebral cortical development and neural induction in zebra fish embryos. We showed that the Jade-2-LSD1 pathway is implicated in neuroblastoma in order to induce differentiation of tumor cells into postmitotic neurons. To further explore the role of LSD1 in neural differentiation, R1 mouse ESCs were effectively induced for neural commitment in N2B27 medium (Ying and Smith, 2003Ying Q.L. Smith A.G. Defined conditions for neural commitment and differentiation.Methods Enzymol. 2003; 365: 327-341Crossref PubMed Scopus (251) Google Scholar, Ying et al., 2003Ying Q.L. Stavridis M. Griffiths D. Li M. Smith A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.Nat. Biotechnol. 2003; 21: 183-186Crossref PubMed Scopus (1154) Google Scholar) (Figure 1A ). Western blotting analysis and real-time quantitative RT-PCR (qRT-PCR) measurements indicate that the protein level of Lsd1 declined during this process, which was not a result of downregulation of Lsd1 mRNA (Figure 1B). In addition, analysis of mouse ESCs and cortical progenitors (NPCs) by western blotting indicated that the expression of Lsd1 is downregulated in NPCs (Figure 1C). These observations suggest that LSD1 is eliminated during neural differentiation. To further support this notion, ESCs were infected with lentiviruses carrying control (shCTR) or Lsd1 small hairpin RNA (shLsd1) and selected for stable clones (Figure S1A available online). These clones were subjected to alkaline phosphatase (AP) or immunofluorescent (IF) staining for neural progenitor marker Nestin in undifferentiated cultural environment. It was found that Lsd1 knockdown led to a loss of pluripotency of the ESCs, evidenced by the inability of the cells to grow in clones and by weaker staining for AP and to the induction of Nestin (Figure 1D). When the ESCs were induced for neural differentiation, knockdown of Lsd1 accelerated the emergence of neural progenitors and mature neurons, evidenced by the enhanced expression of Nestin and mature neuron markers βIII-tubulin, Gap43, and Map2 (Figure 1D). On the other hand, overexpression of LSD1 via doxycycline (DOX)-inducible lentiviral system (Brambrink et al., 2008Brambrink T. Foreman R. Welstead G.G. Lengner C.J. Wernig M. Suh H. Jaenisch R. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells.Cell Stem Cell. 2008; 2: 151-159Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar) (Figure S1B) in differentiating ESCs decelerated the derivation of neural progenitors and mature neurons (Figure 1D). To investigate the role of Lsd1 in cortical progenitor differentiation, shCTR or shLsd1 vectors were electroporated along with GFP expression plasmids in utero into developing embryonic day (E) 13.5 mouse cortices (Figure S1C). The cortices were subsequently analyzed by IF staining for βIII-tubulin or Nestin at E15.5. In cortices electroporated with shCTRs, the expression of βIII-tubulin was absent in the ventricular zone (VZ) but was readily detected in the intermediate zone (IZ), whereas the expression of Nestin was mostly detected in the VZ (Figure 1E). However, a dramatic increase of βIII-tubulin expression and decrease of Nestin expression was detected in shLsd1-electroporated cells (and their progeny) in the VZ (Figure 1E). Image analysis of the whole cortex showed that the proportion of GFP+/βIII-tubulin+ cells was higher and the proportion of GFP+/Nestin+ cells was lower in shLsd1-electroporated cells in comparison to control cells (Figure 1E). Altogether, these data support a notion that Lsd1 inhibits neuronal differentiation of pluri- or multipotent stem cells, and thus the elimination of Lsd1 promotes neural differentiation. To answer the question of whether the ubiquitin-proteasome pathway is implemented in the downregulation of LSD1, we transfected U2OS cells with FLAG-LSD1 and treated with DMSO or proteasome inhibitor MG132. Western blotting analysis revealed a time-dependent increase in LSD1 level in the presence of MG132, which was not a result of increased LSD1 mRNA expression (Figure 1F), suggesting that LSD1 is a liable protein and subjected to regulation by the ubiquitin-proteasome-mediated process. To support this, we investigated whether LSD1 could be polyubiquitinated in vivo. U2OS cells were cotransfected with FLAG-LSD1 and HA-tagged ubiquitin. Immunoprecipitation (IP) of the cell lysates with anti-FLAG and immunoblotting (IB) with anti-HA revealed a robust increase of LSD1 polyubiquitination in the presence of MG132 (Figure 1F), supporting the argument that the steady-state level of LSD1 is controlled by the ubiquitin proteasome pathway. To further strengthen this argument, we treated ESCs or cortical progenitor cells with DMSO or MG132. Western blotting analysis showed that the decreased protein level of Lsd1 in cortical progenitor cells could be restored almost to the same level as ESCs when the activity of proteasome is inhibited by MG132 (Figure 1G). Consistently, in vivo ubiquitination assays with nickel bead precipitation for His-tagged ubiquitin showed an increase in Lsd1 polyubiquitination during neural differentiation (Figure 1H). Altogether, these results support the notion that LSD1 is downregulated via the ubiquitin-proteasome system during neural differentiation. To search for the E3 ubiquitin ligase for LSD1, we employed affinity purification and mass spectrometry in order to identify proteins that potentially interact with LSD1 in vivo. To this end, FLAG-tagged LSD1 was stably expressed in U2OS cells. Then, whole-cell extracts were prepared and subjected to affinity purification with anti-FLAG affinity gel. Mass spectrometry did not detect any known E3 ubiquitin ligase in the purified complex (Figure 2A ). However, interestingly, Jade-2, an uncharacterized member of Jade family protein whose gene has not been cloned yet, was identified in the LSD1-containing complex (Figure 2A). The presence of Jade-2 in the LSD1-containing complex was confirmed by western blotting with antibodies generated on the basis of the expressed sequence tag of Jade-2 (Figure 2B). To further support the observation that LSD1 is physically associated with Jade-2 in vivo, we extracted total proteins from HeLa cells, mouse ESCs, or mouse cortical neurons and performed co-IP experiments. IP with antibodies against LSD1 followed by IB with antibodies against Jade-2 showed that LSD1 was efficiently coimmunoprecipitated with Jade-2 (Figure 2C). Reciprocally, IP with anti-Jade-2 followed by IB with anti-LSD1 revealed that Jade-2 was also coimmunoprecipitated with LSD1 (Figure 2C). Co-IP experiments showed that Jade-2 is also associated with nLSD1 isoforms LSD1-8a and LSD1-2a/LSD1-8a in cortical neurons (Figure 2D). In addition, glutathione S-transferase (GST) pull-down assays indicated that LSD1 is capable of interacting with Jade-2 in vitro (Figure 2E) and the C terminus of Jade-2, which is variable between Jade proteins (Figure S2A) and that the AOD/Tower domains of LSD1 are responsible for the molecular interaction between Jade-2 and LSD1 (Figure 2E). Importantly, Jade-2 is not associated with REST or CoREST (Figure 2F). To test the hypothesis that Jade-2 is an E3 ubiquitin ligase specific for LSD1, we performed experiments with gain- and loss-of-function of Jade-2 in U2OS cells. Western blotting analysis revealed that increased expression of Jade-2 was associated with a decreased level of LSD1 protein (Figure 3A ), an effect that requires the PHD domains and C terminus of Jade-2 (Figure 3B, left). In addition, the decreased LSD1 protein level under Jade-2 overexpression was not a result of downregulation of LSD1 mRNA (Figure 3B, right). Moreover, Jade-2-associated destabilization of LSD1 protein occurred only in the absence of MG132 (Figure 3A). Consistently, knockdown of Jade-2 expression (Figure S2B) resulted in an increased half-life time of LSD1 in U2OS, evidenced by cycloheximide (CHX) chase assays (Figure 3C, top), and the half-life of Lsd1 was extended in mouse ESCs (Figure 3C, bottom) when Jade-2 was depleted (Figure S1A). Furthermore, Jade-2 had no or only marginal effect on the expression of other histone modification enzymes or their cofactors, and LSD1 is not targeted by β-TRCP and Jade-1, the E3 ubiquitin ligases for REST and β-catenin, respectively (Chitalia et al., 2008Chitalia V.C. Foy R.L. Bachschmid M.M. Zeng L. Panchenko M.V. Zhou M.I. Bharti A. Seldin D.C. Lecker S.H. Dominguez I. Cohen H.T. Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL.Nat. Cell Biol. 2008; 10: 1208-1216Crossref PubMed Scopus (142) Google Scholar, Westbrook et al., 2008Westbrook T.F. Hu G. Ang X.L. Mulligan P. Pavlova N.N. Liang A. Leng Y. Maehr R. Shi Y. Harper J.W. Elledge S.J. SCFbeta-TRCP controls oncogenic transformation and neural differentiation through REST degradation.Nature. 2008; 452: 370-374Crossref PubMed Scopus (255) Google Scholar) (Figures 3D–3F). Altogether, these results support the notion that Jade-2 specifically targets LSD1 for destruction through the proteasome-mediated process. To further substantiate the argument, in vitro ubiquitination assays with baculovirally purified Jade-2, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme UbcH6 (E2), ubiquitin, and other reaction constituents showed that Jade-2 is able to polyubiquitinate itself (autopolyubiquitination) (Figure 4A ), a signature of proteins with E3 ubiquitin ligase activity (Pickart, 2001Pickart C.M. Mechanisms underlying ubiquitination.Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2909) Google Scholar). Consistently, incubation of bacterially expressed GST-LSD1 with bacterially expressed GST-Jade-2 resulted in the detection of LSD1 polyubiquitination but only when both E2 and ubiquitin were present, whereas incubation of GST-LSD1 with GST-Jade-2-ΔPHD resulted in no detectable LSD1 polyubiquitination, even when E2 and ubiquitin were included (Figure 4B). In vivo ubiquitination assays in ESCs showed that depletion of Jade-2 resulted in a dramatic decrease in the level of LSD1 polyubiquitination (Figure 4C) and that overexpression of Jade-2, but not Jade-1, led to a pronounced increase in the level of LSD1 polyubiquitination in SH-SY5Y cells (Figure 4D). Moreover, when wild-type (WT) ubiquitin used in in vivo ubiquitination assays was replaced with an ubiquitin mutant UbK48R, which is defective in the assembly of polyubiquitin chain that is recognized by proteasome for degradation (Chau et al., 1989Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1116) Google Scholar, Thrower et al., 2000Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. Recognition of the polyubiquitin proteolytic signal.EMBO J. 2000; 19: 94-102Crossref PubMed Scopus (1309) Google Scholar), high-molecular-weight LSD1 conjugates were no longer detected (Figure 4D). Detailed experiments with Jade-2 and LSD1 mutants indicated that the first PHD of Jade-2 is essential for its E3 ubiquitin ligase activity toward LSD1 (Figures 4E and 4F), although in vitro ubiquitination assays, for some reason, detected a slight decrease in Jade-2’s ubiquitination activity when the second PHD was deleted (Figures 4G and S2C) and that Jade-2-promoted polyubiquitination of LSD1 occurs on lysine 503 of LSD1 (Figures S2D and 4H). Altogether, these data support the arguments that Jade-2 targets LSD1 for degradation and that Jade-2 does so via its E3 ligase activity and through promoting LSD1 polyubiquitination mainly via its first PHD domain. In vivo ubiquitination assays showed that Jade-2 also promotes the polyubiquitination of nLSD1 doublets (Figure 4I). In order to explore the biological significance of Jade-2-promoted LSD1 degradation in neural differentiation, R1 mouse ESC clones stably expressing shCTR or shJade-2 were induced for neural differentiation in N2B27 medium. Western blotting analysis indicated that the decrease of Lsd1 protein level was significantly delayed upon Jade-2 depletion (Figure 5A ). Correspondingly, the emergence of neural progenitors and mature neurons from these cells was decelerated, as evidenced by decreased expression of Sox1, Nestin, and βIII-tubulin and the postponed elimination of pluripotency marker Oct4 (Figure 5A). Morphologically, Jade-2-depleted ESCs grew in aggregates with smoother-edged, rounder-shaped, and deeper-AP-stained cell clones (Figure 5B). Concurrent infection of lentiviruses carrying shLsd1 (Figure S1A) was able to rescue the phenotype of Jade-2-deficient cells (Figure 5B). Immunofluorescent staining showed that, during neural differentiation, although most (∼70%) of the control cells expressed neural markers and organized in rosettes with surrounding cells exhibiting neuronal morphology with extensive arborization, Jade-2-depleted cells grew in aggregates, failed to attach plates, and display decreased expression of neural markers (Figure 5B). However, simultaneous depletion of Lsd1 could restore the expression of the neural markers and rescue the morphological manifestations (Figure 5B). In addition, all-trans retinoic acid (RA)-induced neuronal differentiation of embryoid bodies (EBs) was suppressed upon Jade-2 depletion, evidenced by decreased numbers of βIII-tubulin-expressing neurites around EBs (Figure 5B). Accordingly, in vivo ubiquitination assays detected no increase in polyubiquitination of either LSD1K503R in control cells or LSD1 in Jade-2-depleted cells during neural differentiation (Figure 5C). In addition, IF staining showed that, although LSD1K503R counteracted Jade-2 overexpression-promoted neuronal differentiation, manifested by the repressed neural markers, LSD1 had limited effect (Figures S3A and 5D). Altogether, these data support a notion that the Jade-2-LSD1 pathway plays an important role in neural differentiation in which Jade-2 targets LSD1 for degradation, relieving the braking function of LSD1 on neural commitment. Real-time quantitative RT-PCR analysis showed that several LSD1 target genes known to be associated with neurogenesis, including Pax3, Ascl1, Zic1, Zic4, and Neurog1 (Adamo et al., 2011Adamo A. Sesé B. Boue S. Castaño J. Paramonov I. Barrero M.J. Izpisua Belmonte J.C. LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells.Nat. Cell Biol. 2011; 13: 652-659Crossref PubMed Scopus (242) Google Scholar, Whyte et al., 2012Whyte W.A. Bilodeau S. Orlando D.A. Hoke H.A. Frampton G.M. Foster C.T. Cowley S.M. Young R.A. Enhancer decommissioning by LSD1 during embryonic stem cell differentiation.Nature. 2012; 482: 221-225Crossref PubMed Scopus (422) Google Scholar), are regulated by Jade-2 (Figure 5E). Consistently, quantitative chromatin immunoprecipitation (qChIP) showed that neural differentiation of ESCs was associated with dramatic decreases in the recruitment of Lsd1 and increases in levels of H3K4me1 and H3K4me2 on the promoters of Pax3, Ascl1, Zic1, and Neurog1 genes, changes that were abrogated when Jade-2 was depleted (Figure 5F). Significantly, ectopic expression of Pax3 or Ascl1 (Figure S3B), which are both identified targets of the Jade-2-LSD1 pathway, in differentiating ESCs could, at least partially, rescue Jade-2 depletion-restrained derivation of neural progenitors and rosettes formation (Figure 5G), although Jade-2 depletion-associated repression of neuron maturation and neurite extension could be relieved by ectopic expression of Ascl1 but not Pax3 (Figure 5G). These results are consistent with the notion that Pax3 promotes early neural induction (Goulding et al., 1991Goulding M.D. Chalepakis G. Deutsch U. Erselius J.R. Gruss P. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis.EMBO J. 1991; 10: 1135-1147Crossref PubMed Scopus (750) Google Scholar, Li et al., 1998Li M. Pevny L. Lovell-Badge R. Smith A. Generation of purified neural precursors from embryonic stem cells by lineage selection.Curr. Biol. 1998; 8: 971-974Abstract Full Text Full Text PDF PubMed Google Scholar, Liem et al., 1995Liem Jr., K.F. Tremml G. Roelink H. Jessell T.M. Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm.Cell. 1995; 82: 969-979Abstract Full Text PDF PubMed Scopus (911) Google Scholar), although Ascl1 is involved in neuron maturation (Casarosa et al., 1999Casarosa S. Fode C. Guillemot F. Mash1 regulates neurogenesis in the ventral telencephalon.Development. 1999; 126: 525-534Crossref PubMed Google Scholar, Guillemot et al., 1993Guillemot F. Lo L.C. Johnson J.E. Auerbach A. Anderson D.J. Joyner A.L. Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons.Cell. 1993; 75: 463-476Abstract Full Text PDF PubMed Scopus (922) Google Scholar). Altogether, these data support the importance of the Jade-2-LSD1 pathway in neural differentiation. To explore a broad functionality for the Jade-2-LSD1 pathway in nervous system development during embryogenesis, Jade-2 or shJade-2, along with GFP, were electroporated in utero into E13.5 mouse cortices. Subsequent IF staining at E15.5 showed that Jade-2-overexpressing cells in the VZ exhibited a precocious expression of βIII-tubulin resembling the pattern with that of Lsd1-depleted cells, whereas Jade-2 knockdown led to a reduced βIII-tubulin expression of the electroporated cells in the IZ, which could be rescued by simultaneous electroporation of shLsd1 plasmid (Figure 6A ). Quantification analysis of the whole cortex showed that Jade-2 overexpression was associated with a higher proportion of GFP+/βIII-tubulin+ cells, whereas Jade-2 knockdown was accompanied by a lower proportion of GFP+/βIII-tubulin+ cells, which could be elevated by simultaneous depletion of Lsd1 (Figure 6A). In addition, Jade-2 overexpression led to a decrease, whereas Jade-2 knockdown resulted in an increase in the expression of Nestin in the electroporated cells, which could be offset when Lsd1 was coknocked down (Figure 6A). To investigate the functional importance of the Jade-2-LSD1 axis in neural development in vivo, we injected morpholino (MO) antisense oligonucleotides against phf15 (Jade-2 ortholog) and/or kdm1a (LSD1 ortholog) into one-cell-stage embryos of zebrafish (Figure S4), and the mRNA expression of a series of neuroectoderm markers was detected by in situ hybridization followed by statistical analysis in midgastrulation period (75% epiboly). Notably, the expressions of early neuroectoderm markers sox2, sox3, otx2, and hoxb1b decreased in phf15 morphants but increased in kdm1a morphants (Figure 6B). Coinjection of kdm1a MO offset the effect of phf15 MO (Figure 6B). These data indicate that the Jade-2-LSD1 pathway plays an important role in cortical progenitor differentiation and neuroectoderm induction. Next, we generated a series of SH-SY5Y cell lines stably expressing Jade-2 or its mutants and/or LSD1 by retroviral infection (Figure S5A) and verified Jade-2-mediated LSD1 degradation in these cells (Figure 7A ). qRT-PCR analysis showed that ZIC4, PAX3, and TPM1 (Schulte et al., 2009Schulte J.H. Lim S. Schramm A. Friedrichs N. Koster J. Versteeg R. Ora I. Pajtler K. Klein-Hitpass L. Kuhfittig-Kulle S. et al.Lysine-specific demethylase 1 is strongly expressed in poorly differentiated neuroblastoma: implications for therapy.Cancer Res. 2009; 69: 2065-2071Crossref PubMed Scopus (364) Google Scholar), but not ASCL1, are regulated by the Jade-2-LSD1 pathway in SH-SY5Y cells (Figure 7B). Then, SH-SY5Y cells were induced for differentiation by RA (Sidell, 1982Sidell N. Retinoic acid-induce" @default.
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