2-bromopalmitate Regulates Neural Stem Cell Differentiation By Modulation Of EID1 Activity And Its Mechanisms

In mammals,neural stem cells(NSCs)have the capability to self-renew and differentiate into neurons,astrocytes,and oligodendrocytes.In response to appropriate signals,multipotent NSCs exit the cell cycle and initiate terminal differentiation.This precise temporal and spatial differentiation progression is associated with activation of specific transcriptional factors,upregulation of differentiation-specific genes,and repression of multipotency genes.However,regulation of the fate switch between self-renewal and differentiation of NSCs has not been well characterized.It is becoming increasingly apparent that chromatin accessibility plays a key role in the transcriptional regulation of cell-type-specific gene expression.Modification of histone N-terminal tails can alter the interaction between histones and DNA,serving as a mechanism to regulate gene expression.Transcriptional co-activators,such as the CBP(CREB-binding protein)/p300 family,have been shown to possess histone acetyltransferase(HAT)activity.CBP/p300 can acetylate core histones and regulate transcription by linking transcriptional machinery with its acetylation-dependent regulation.CBP/p300 is essential for the formation of the mammalian nervous system,since mice nullizygous for p300 die between gestation days E9 and E11.5 and exhibit strong defects in neurulation and cell proliferation.2-Bromopalmitate(2-BRO),a non-metabolisablepalmitate analogue,has been identified as a palmitoylation inhibitor and is often referenced as the only pharmacological tool to block protein palmitoylation.We recently reported that 2-bromopalmitateresulted in decreased acetylation of histones and interfered with cell cycle withdrawal during retinoic acid-induced P19 differentiation.However,it is still unknown specifically how protein palmitoylation modulates this process,as well as whether palmitoylation is involved in the regulation mouse NSC self-renewal and differentiation.Here,the mouse neural stem cells were used in this study as a model system to elucidate the functional importance of protein palmitoylation during neuronal development.Part ? Effects of 2-bromopalmitate on proliferation and differentiation of mouse NSCsThe effects of palmitoyl acyl transferase(PAT)inhibitor,2-bromopalmitate,on the proliferation of NSCs were assessed first.After 2-bromopalmitate treatment,NSCs grew at rates comparable to control cells,and 2-bromopalmitate did not have obvious effect on expression of the sternness-related genes Sox2 and Nestin.All the results suggested that 2-bromopalmitate did not have obvious effect on the proliferation of mouse neural stem cells.For cell differentiation analysis,we examined the neuronal marker MAP2,astrocytic marker GFAP;and oligodendrocytic marker NG2,after plating with immunofluorescentstaining.2-bromopalmitate treatment blocked the differentiation of NSCs into both neurons and glia compared to control cells.A decrease in MAP2,GFAP,and NG2 protein expressions was also detected with the western analysis,indicating that 2-bromopalmitate has a functional role in both neuronal and glial differentiation.To examine the quantitative effects of 2-bromopalmitate on the differentiation process,real-time PCR was applied to the stemnness-related gene markers,Sox2 and Nestin,and the differentiation-specific gene markers,NeuroDl(neuronal marker),GFAP(astrocytic marker),and Olig2(oligodendrocytic marker).The NSCs exposed to 2-bromopalmitate expressed higher levels of Sox2 and Nestin notably during the differentiation stage.This apparent increase in differentiated NSCs was in agreement with the results of the immunofluorescent staining with Sox2 and Nestin antibodies.Once differentiated,however,NSCs treated with 2-bromopalmitate expressed reduced levels of the differentiation-specific gene markers,NeuroD1,GFAP,and Olig2,when compared to controls.These results suggested that,while cells exposed to 2-bromopalmitate produced increased numbers of NSCs,their differentiation was less efficient.Part ? 2-bromopalmitate modulates the cell cycle profile in NSCs during the differentiation stageDuring NSC differentiation,cell fate determination is coordinated with cell cycle regulation.We found that 2-bromopalmitate at the concentration used had little effect on cell survival,but obviously elevated the cell viability of NSCs.Therefore,we turned our attention to estimating the cell cycle distribution of NSCs after 2-bromopalmitate treatment during the differentiation stage.The result of BrdU incorporation assay showed that the BrdU-positive cells in 2-bromopalmitate-treated cells were much more than the control cells after 5 days of plating.Next,we examined the cell cycle distribution with fluorescence-activated cell sorting(FACS).The results showed that the percentage of S phase cells increased significantly while the percentage of Gl/GO-phase cells was reduced in 2-bromopalmitate-treated cells when compared to control cells,indicating their inability to arrest cell division and to induce differentiation.Cell cycle regulators,such as cyclin-dependent kinase(CDK)inhibitors,are important for the anti-proliferative effects.We noted that,concomitant with these results,there was reduced expression of CDK inhibitors p21,p27,and p57,in NSCs after 2-bromopalmitatetreatment.Taken together,these results suggest that the NSC differentiation mediated by 2-bromopalmitate involves a downregulation of both differentiation-specific genes and CDK inhibitors to promote cell proliferation and impair both neuronal and glial fate commitment.Part ? 2-bromopalmitate blocks the acetylation of histone H3 to suppress expression of differentiation genesAs reported,chromatin packaging determines accessibility and availability of DNA for transcription.Therefore,we assessed the effect of 2-bromopalmitate onchromatin organization in nuclei of NSCs by transmission electron microscopy(TEM).During differentiation,heterochromatin clumps increased at the nuclear periphery of NSCs exposed to 2-bromopalmitate,similar to that of proliferative NSCs,indicating that 2-bromopalmitate impeded the reorganization of chromatin in NSCs.Histone modifications play vital roles in the regulation of chromatin packaging and organization.We analyzed the status of histone H3 lysine 9 trimethylation and lysine 9/14 acetylation,because lysine 9 trimethylation in histone H3 typically indicates transcriptional silence regions in chromatin and lysine 9/14 acetylation is associated with transcriptional activation.The status of histone methylation was analyzed by an H3K9me3 antibody and found to be mostly unaffected in 2-bromopalmitate-treated NSCs when compared to that in control cells.However,we found that K9/14 acetylation of histone H3 decreased in NSCs exposed to 2-bromopalmitate during the differentiation stage.ChIP analysis using H3K9/14ac antibody showed that 2-bromopalmitate resulted in impaired acetylation in the promoters of differentiation-related genes,but not the stemness-related genes,indicating that 2-bromopalmitate maintains NSC self-renew through suppression of differentiation-related gene expression.K9/14 lysine residue of histone H3 was identified as the substrates of HATs CBP/p300.Our western analysis showed that 2-bromopalmitate did not clearly change the protein level of CBP or p300.Therefore,we decided to assess the effect of 2-bromopalmitate on the regulation of CBP/p300 HAT activity with an in vitro HAT assay.We found that 2-bromopalmitate remarkably reduced CBP/p300 HAT activity.Part ? 2-bromopalmitate prevents EID1 degradation to maintain self-renewal of NSCsWe attempted to understand the basis for the 2-bromopalmitate-associated effects on CBP/p300 HAT activity.EID1 has been identified as an inhibitor of CBP/p300 by suppression of the acetyltransferase activity,and reported to be degraded upon cell cycle exit in skeletal muscle during myogenesis;therefore,we expected similar behaviors during NSC differentiation.We first wanted to assess protein levels of EID1 undergoing NSCs differentiation.Surprisingly,we observed that EID1 was degraded during NSC exposure to differentiated medium.In contrast,the EID1 degradation was attenuated in NSCs after 2-bromopalmitate treatment.The results of co-immuoprecipitation showed that both EID1-CBP and EID1-p300 complex formation dramatically improved in the 2-bromopalmitate treated cells during NSC differentiation,suggesting that it was the interaction of EID1 with CBP/p300 that was responsible for the deficit in transcription regulation and differentiation observed in NSCs exposed to 2-bromopalmitate.As expected,transfection of EID1-shRNA impaired 2-bromopalmitate-induced transcriptional suppression of differentiation genes,to some degree.2-bromopalmitate did not change EID1 mRNA cytoplasm/nuclei distribution,indicating that 2-bromopalmitate regulates EID1 stability through its own protein level.We found that endogenous EID1 was indeed ubiquitinated in NSCs during the differentiation stage.2-bromopalmitate changed the ubiquitination level of EID1 in NSCs,but 2-bromopalmitate did not obviously affect the happening of EID1 ubiquitination.The result was verified via an in vitro ubiquitination reconstitution assay,suggesting that 2-bromopalmitate may hamper EID1 proteolysis.Notably,EID1 degradation was blocked by the proteasome inhibitor MG132,indicating that its degradation is dependent on proteasomes.Under normal conditions,EID1 could be detected in proteasomes but was less localized in the nuclei.In contrast,EID1 was well distributed in both the cytoplasm and nuclei after 2-bromopalmitate treatment,but the cytoplasmic protein showed a weak overlap with the proteasome marker,PSM?1.These results suggest that 2-bromopalmitate changed the distribution of EID1 in the cytoplasm,hampered the passage of ubiquitinated EID1 to degrade,and improved the nuclear translocation of EID1 during NSC differentiation.The posttranslational modification of proteins encompasses diverse functions,including sorting proteins into membrane lipid microdomains and regulating intracellular protein trafficking,protein-protein interactions and degradation.Because 2-bromopalmitate has been identified as an inhibitor of protein palmitoylation,it is possible that 2-bromopalmitate impairs the sub-cellular localization of EID1 by inhibiting its palmitoylation status.As predicted by the CSS-Palm 4.0 software,EID1 has numerous potential palmitoylation sites,which are conserved in EID1 across different species.We next examined the palmitoylation levels using an acyl-biotin exchange(ABE)assay after immunoprecipitation of EID1,and found that the EID1 was palmitoylated.A significant reduction in the palmitoylation level of EID1 was observed in 2-bromopalmitate-treated cells compared to control cells.Taken together,these results suggest that 2-bromopalmitate inhibited EID1 palmitoylation and hampered the proteasome localization of EID1,resulting in a change in the CBP/p300 HAT activity during NSC differentiation.CONCLUSIONThe mechanisms of proliferation,differentiation and maturation of NSCs are of great importance in the study of neuroscience.Recently,more and more studies have suggested that palmitoylation,a post-translational lipid modifications plays a key role in neural development,but the molecular mechanisms that control these processes are not yet fully understood.In this series of experiments,we used a well-established model system of mouse stem cells to investigate the connection between palmitoylation and cell fate specification of NSCs.During NSCs proliferation,lower concentration of 2-bromopalmitate did not clearly change the characteristics of neurospheres.However,their differentiation to neurons and cell cycle exit were less efficient when NSCs were cultured in the presence of 2-bromopalmitate.These effects were associated with an extensive change in the transcriptional profile of neural stem/precursor cells and a decrease in acetylated histone H3.These results suggested that 2-bromopalmitate inhibited the degradation of EID1,then,hampered the activity of CBP/P300,resulting in a change in the histone H3 acetylation and a downregulation of differentiation-related genes in differentiation cells.Thus,our findings established a role of palmitoylation in the epigenetic regulation of genetic expression during neuronal differentiation.This study will contribute to understand the mechanism of NSCs differentiation and provided a novel insight into the function of palmitoylation during neurodevelopment.Novelty:1.We found that 2-bromopalmitate impeded the differentiation of both neurons and glias.2.Mechanistically,we found that 2-bromopalmitate suppressed the palmitoylation level of EID1 and leaded to an inhibition of EID1 degradation.We further found that EID1-CBP/p300 complex was involved in the regulation of the balance between the maintenance of multipotency and differentiation in NSCs.3.We found that EID1 was the direct target of 2-bromopalmitate at the fiest time,which make it possible to remodel the function of NSCs with small molecular compound in the future.