小分子化合物2BROP对神经干细胞发育的影响及机制研究
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摘要
研究发现,作为胞内生命活动的主要执行者,许多蛋白质只有经历翻译后的加工才能具有生物活性。蛋白质的棕榈酰化是蛋白质翻译后修饰的重要方式之一。DHHC蛋白家族是最早在酿酒酵母中发现与棕榈酰化修饰作用相关的一类蛋白,它们大多具有蛋白质酰基转移酶(protein acyltransferase, PAT)活性。2004年随着Bredt DS研究小组完成人类和小鼠基因组23种DHHC蛋白基因的克隆与鉴定,人们对于这类蛋白功能的了解也逐渐深入。目前,有关棕榈酰化蛋白质组学和棕榈酰化转移酶DHHC家族的遗传学分析结果,暗示蛋白质的棕榈酰化在神经系统发育和相关神经疾病的病发过程中可能起到重要的调控作用。因此,阐明棕榈酰化修饰作用在神经系统中的作用及分子调控机制成为了近些年神经科学领域的研究热点。
在中枢神经系统中,神经干细胞(neural stem cells, NSCs)属于多潜能干细胞,具有自我更新能力,并且在胚胎发育及成年脑中均能分化产生神经元、星形胶质细胞和少突胶质细胞。NSCs的分化成熟,可以大致分为如下3个阶段：1)NSCs自我更新；2)NSCs被赋予具有分化潜能的神经前体细胞；3)退出周期,处于有丝分裂静止期,分化形成具有特殊形态和功能的神经细胞。其实,在NSCs分化过程中,这些细胞形态和功能的改变很大程度上是由细胞所处不同阶段的特定基因表达模式所决定的。其中,主要包括多潜能因子Sox2、原神经基因bHLH (basic Helix-Loop-Helix, bHLH)家族和细胞周期依赖激酶抑制因子(cyclin-dependent kinase inhibitors, CKIs)。 NSCs自我更新能力及干性维持的内部机制主要是通过多潜能因子的表达和分化命运决定因子的沉默而实现的。NSCs分化命运决定的内部机制也是通过时间和空间上高度精确的调控机制完成的,即分化命运决定因子的激活与表达以及多潜能因子的沉默。
目前,研究发现真核基因的表达在多个层次上受到精确的调控,而染色质的修饰是真核基因转录的第一调控靶点。其中,组蛋白的共价修饰对染色质的动态结构具有核心调节作用,它是表观遗传学重要的调节方式之一。在神经细胞命运决定过程中常常伴随着组蛋白乙酰化修饰水平的改变。组蛋白乙酰化转移酶(histone acetyltransferase, HAT) P300在神经系统中表达丰富,它参与了神经系统在形式和功能上的发生、可塑性和稳态的建立。P300通过自身的HAT活性,乙酰化组蛋白赖氨酸,染色质发生重塑,使转录因子易于与DNA结合,从而促进基因转录。其中,组蛋白H3和H4就是其乙酰化共价修饰的底物之一。
本研究中,我们通过建立视黄酸(retinoic acid, RA)诱导的P19细胞定向分化为神经样细胞的体外模型,利用棕榈酰转移酶抑制剂——小分子化合物2-bromopalmitate (2BROP),探讨棕榈酰化修饰在NSCs发育中的作用及相关表观遗传分子机制。
第一部分棕榈酰化修饰相关蛋白在神经干细胞发育过程中的表达
为了揭示棕榈酰化修饰作用是否参与NSCs发育过程,于NSCs发育的各阶段,包括P19细胞多潜能状态、NSCs增殖和NSCs分化等三个阶段,分别检测23个棕榈酰转移酶即DHHC家族成员的表达情况。结果显示,在NSCs发育阶段,共有20个DHHC家族成员的mRNA表达。其中,DHHC1、4、7、9、24于NSCs发育各阶段的mRNA表达无明显变化。DHHC5、13、14、16、17、20、21成员于NSCs增殖阶段mRNA的表达量获得增高,并且DHHC5、17、21在NSCs分化阶段也将持续性高表达。而DHHC2、6、8、11、15、18、19、23成员的mRNA仅于NSCs分化阶段高表达。
接下来,我们利用酰基-生物素交换法(Acyl-biotinyl exchange method, ABE)技术,检测NSCs增殖与分化两阶段,蛋白棕榈酰化修饰水平情况。结果显示,多种蛋白在NSCs增殖与分化阶段被棕榈酰化修饰,并在不同的NSCs发育阶段,呈现出一定的特异性,如NSCs增殖阶段,棕榈酰化蛋白条带分布于～10、25、45、50、70、170kDa；而NSCs分化阶段,蛋白分布于～25、55、60、70、100、125、150、290kDa。
本实验结果显示,大多数DHHC家族成员不同程度的表达于NSCs发育各阶段,并且在不同的NSCs发育阶段,棕榈酰化修饰的蛋白呈现出一定的特异性,暗示DHHC家族成员和它们所介导的棕榈酰化修饰参与NSCs发育过程的调控,棕榈酰化修饰可能通过特异性的影响NSCs发育各阶段中关键蛋白的生物活性,从而参与调控NSCs的命运决定。
第二部分小分子化合物2BROP对神经干细胞增殖的作用
为了研究棕榈酰化修饰对NSCs增殖的影响,我们首先利用MTT技术,检测不同剂量的2BROP对于细胞代谢活力的影响。结果显示,与对照组(0μ M)相比,1-15μM加药组中细胞活力表现出一定的增强；尤其是10uM加药组,但是统计学无明显差异。然而,与对照组相比,25μ M和50μM加药组中,细胞活力减弱,MTT值明显降低,并具有统计学显著性差异。与此同时,倒置相差显微镜,观察神经球的结构和形态变化。结果显示,随着RA诱导的进行,对照组中,细胞聚集体不断增大,最终形成一个结构致密、表面规整的细胞团。10μM终浓度加药组组中,细胞聚集体的发生和增长过程与对照组相比,未见异常。但是,25μM加药组中,细胞聚集体的结构发生明显改变,表现为细胞团形态不规则、内部有空隙。
接下来,我们利用TUNEL技术,在NSCs增殖阶段,检测小分子化学物2BROP对于细胞凋亡的影响。结果显示,2BROP处理48h后,与对照组相比,10μM加药组中,细胞团中TUNEL阳性细胞数没有明显差异；而25u M处理组中,TUNEL阳性细胞数明显增多。随后,我们利用、Vestern Blot和免疫荧光染色技术,检测了相关凋亡信号通路的变化。结果发现,25μM处理组中线粒体相关凋亡通路明显激活。与对照组相比,25μM处理组中抗凋亡Bcl-2的表达量减少,促凋亡的Bax和活化的Caspase3的表达量增多；而10μM加药组中Bcl-2、Bax与活化的Caspase3表达量没有明显变化
本实验结果,显示在一定的剂量范围内,小分子化合物2BROP对于NSCs增殖过程,并没有产生明显的影响；而高浓度2BROP将会对细胞产生毒性,引发细胞凋亡。
第三部分小分子化合物2BROP对神经干细胞分化的作用
为了探究蛋白棕榈酰化修饰在NSCs分化阶段中的相关作用及其分子机制,我们利用免疫荧光技术和Western Blot等技术,检测神经分化标记物MAP2的表达与分布情况。结果显示,与对照组相比,2BROP处理组中,MAP2阳性细胞数明显减少,统计学具有显著性差异；且神经分化各阶段,MAP2蛋白的表达均受到明显抑制。
接下来,利用Western Blot和qRT-PCR技术,我们检测了NSCs分化命运决定相关因子的表达情况,如多潜能因子Oct4; NSCs标记物Sox2、Nestin;神经前体细胞标记物β-tubulinⅢ和神经分化决定因子Neurogl、NeuroD1。结果显示,随着NSCs命运的获得,对照组与2BROP处理组中,Oct4的表达迅速下降直至消失,无明显差异。与对照组相比,2BROP组中神经前体细胞标记物β-tubulinIII、分化命运决定因子Neurog1和NeuroD1的表达于神经分化各阶段均呈现出明显地降低。与此相反,2BROP处理组中,NSCs标记物Sox2与Nestin却表现出一定的持续性高表达。同时,MTT实验结果显示,与对照组分化各阶段的MTT值无明显变化相比,2BROP处理组中,随着分化天数的延长,MTT值呈现出一定的增。这些结果提示我们,在神经分化阶段,2BROP可能通过增强NSCs的干细胞特性的维持,从而阻碍神经分化的发生。
为了验证NSCs分化阶段2BROP是否增强了细胞的增殖能力,我们首先利用BrdU掺入实验,检测分化阶段细胞的增殖情况。结果显示,在神经分化的第四天,与对照组相比,2BROP处理组中,BrdU阳性细胞数明显增多,并且统计学上具有显著性差异。然后,我们利用膜标记-FACS技术追踪细胞增殖的变化。分化阶段的细胞使用荧光染料标记。细胞每增殖一次,荧光强度便会减弱一半。结果显示,悬浮培养24h后,对照组中单个细胞的荧光强度并没有明显的变化；而2BROP处理后,单个细胞的荧光强度发生不同程度的减弱。
为了进一步分析2BROP在NSCs分化阶段对于细胞增殖和周期的影响,利用FACS技术,分析分化阶段细胞周期的分布。结果显示,神经分化第四天,对照组中细胞主要分布于G0/G1期；而2BROP处理组中,G0/G1期的细胞数量减少,S期的细胞数量明显增多。与此同时,我们利用qRT-PCR技术,检测了与神经分化相关的CKIs家族成员的表达情况,借以阐明2BROP阻碍周期退出的相关机制。结果显示,与对照组相比,2BRPP处理后,分化相关的CKIs成员的表达受到不同程度的降低。其中,P57的变化最为明显。作为对照,细胞周期增殖标记物Ki67的表达在2BROP处理组中获得了一定的持续性高表达。
本实验结果,显示在NSCs分化阶段,2BROP处理后,NSCs通过下调CKIs家族成员,尤其是P57的表达,从而阻碍细胞周期的退出与神经分化的发生,使分化状态下NSCs自我更新能力获得明显的提高。
第四部分小分子化合物2BROP对神经干细胞分化表观遗传机制的作用
已知,神经分化阶段,介导细胞周期调控的P57的表达依赖于组蛋白H3、H4的乙酰化水平。为了检测组蛋白乙酰化修饰是否参与2BROP介导的神经分化,我们利用免疫荧光、Western Blot等技术,检测NSCs分化阶段乙酰化组蛋白H3、H4的分布与表达。结果显示,当细胞进入分化阶段时,组蛋白H3与H4的乙酰化水平呈现出一定的上升,但是2BROP处理组中组蛋白H3与H4的乙酰化水平明显的低于对照组的水平。为了进一步验证组蛋白乙酰化修饰是否参与2BROP介导的神经分化,我们使用去组蛋白去乙酰化抑制剂曲古菌素A(Trichostatin A, TSA)预处理细胞,发现TSA处理后,2BROP抑制神经分化的现象得到一定的恢复。值得注意的是,TSA与2BROP共处理组中,β-tubulinⅢ的表达甚至高于正常分化组。
接下来,我们免疫共沉淀、ELISA等技术,检测组蛋白H3、H4乙酰化修饰酶P300乙酰转移酶活性。结果显示,与对照组相比,2BROP处理后,P300的乙酰转移酶活性受到明显的抑制。同时,我们利用Western Blot、免疫荧光等技术,检测了分化阶段P300的表达与分布。结果显示,虽然与对照组相比,2BROP处理组中P300的蛋白表达水平没有明显变化,但是P300在胞内的分布却发生明显的改变。对照组中,P300主要分布于细胞核中,而2BROP处理组中P300蛋白遍布于细胞质和细胞核中。
作为脂质化修饰方式之一,棕榈酰化修饰作用参与调节蛋白的胞内分布、物质运输、蛋白间相互作用等过程。接下来,我们探究2BROP是否通过调节P300的棕榈酰化修饰参与调控组蛋白乙酰化修饰与神经分化。首先,我们使用CSS-Palm4.0软件预测P300蛋白是否拥有典型的棕榈酰化位点。结果显示,P300蛋白拥有多个保守性较高的棕榈酰化潜在修饰位点。然后,我们使用ABE方法,检测NSCs分化阶段,对照组与2BROP处理组中P300的棕榈酰化修饰水平。结果显示,蛋白P300可以被棕榈酰化修饰,并且与对照组相比,2BROP处理组中P300的棕榈酰化修饰水平明显受到抑制,统计学上具有显著性差异。
本实验结果,显示在NSCs分化阶段,棕榈酰化修饰通过调节表观遗传关键因子P300的脂质化修饰水平,进而影响P300的核转位和HAT活性,最终调节组蛋白H3、H4的乙酰化水平和分化命运相关基因的转录激活。
结论
神经干细胞的分化、发育与成熟是现代神经生物学研究的热点之一。棕榈酰化修饰作用在神经发育过程中的作用及分子机制尚未阐明。我们建立P19细胞定向分化为神经样细胞的体外模型,利用棕榈酰转移酶抑制剂——小分子化合物2BROP,探讨棕榈酰化修饰在神经发育中的作用及分子机制。利用P19神经定向分化模型,研究发现,DHHC家族成员分别特异性地表达于NSCs的增殖和分化阶段。并且在这些阶段中,许多蛋白发生了明显的棕榈酰化修饰。2BROP (10μM)处理后,NSCs增殖的命运没有发生明显的改变。但是,在NSCs分化阶段,发现2BROP (10μM)处理后,NSCs的神经分化过程和细胞周期的退出受到阻断,而NSCs的数量却维持在一定的高水平状态。进一步的检测,表明细胞周期调节因子CKI家族中P57的表达明显降低。已知,神经分化阶段,P57的表达依赖于组蛋白H3/H4的乙酰化水平。当加入组蛋白去乙酰化抑制剂曲古菌素A(Trichostatin A, TSA)后,发现P57的表达和神经分化得到一定程度的恢复。最终,研究表明在神经分化阶段,2BROP处理后,由于特异性的抑制P300的棕榈酰化修饰水平,影响其核转移,进而阻碍组蛋白H3/H4的乙酰化修饰与神经分化。因此,本研究揭示了一个由基因表达、表观遗传修饰、蛋白翻译后修饰共同参与的神经分化命运决定调控网络。本研究不仅深化了棕榈酰化修饰在神经发育中的作用,拓展了其分子调控机制,同时为研究棕榈酰化修饰作用在组织发育过程中的作用开辟了一个全新的领域、提供了一个新的视野。
After mRNA translation, immature protein undergoes post-translational modifications to become the mature protein product. Among them, protein palmitoylation is an important lipid modification. Protein palmitoyltransferases were first identified and isolated in yeast and they share a domain referred to as the DHHC domain, a cysteine-rich domain with a conserved aspartate-histidine-histidine-cysteine signature motif. Evidence suggests that the DHHC domain is directly involved in the palmitoyl transfer reaction. In mammals twenty three members of this family have been identified and examined by by Bredt DS group at2004. Recently, the role of protein palmitoylation on neuronal development and function are just beginning to be appreciated by proteome and molecular genetic analysis. There are still many unsolved questions and much work is still needed to elucidate the specific functions and mechanisms of protein palmitoylation on neural development.
In mammals, neural stem cells (NSCs) have capability to self-renew and differentiate into neurons, astrocytes, and oligodendrocytes. During NSCs differentiation, NSCs proliferate (1), then begin to specify into distinct neural progenitor cell fates (2), and exit the cell cycle following a terminal mitosis and express markers of terminal differentiation to finally undergo neuronal or glial morphogenesis (3). In fact, these universal changes in cell types and function represent a major cellular reprogramming event, involving in activation or repression of Sox2, bHLH family and CKIs. NSCs have capability to self-renew through the activation of multipotency genes and repression of differentiation-related genes. Moreover, the precise temporal and spatial differentiation progression is associated with activation the specific transcriptional factors, up-regulation of differentiation-specific genes and repression the multipotency genes.
Tight regulation of these gene expressions may be at some levels, including transcriptional level, posttranscriptional level, as well as posttranslational level. Indeed, the chromatin modification firstly regulates the gene transcription. The acetylation status of histones is thought to act as a general regulator of chromatin structure that mediates the removal of epigenetically controlled repression and enhances transcriptional activity. Recently, studies provide evidence for general alterations in histone modification, such as the increased histone H3and H4acetylation occurring during neuronal differentiation. P300, a histone acetyltransferase (HAT), has the capacity to acetylate histone H3and H4both in vitro and in vivo, and plays an important role in neural development and function. Mechanistically, P300acetylates lysine residues within the histone tails, resulting in promoting transcription factors access to DNA in chromatin by neutralising the positive charge associated with the lysine e-amino group.
Here, Retinoic acid (RA) induced P19cells were used in this study as a model system to elucidate the functional importance of protein palmitoylation during neuronal differentiation.
Part I Protein palmitoylation is occurred during neural stem cells development
To investigate whether protein palmitoylation was involved in NSCs development, we first analyzed the expression of the23potential DHHCs in RA induced P19cells at different stages of neural development by RT-PCR. It was found that20out of23DHHCs can be detected. More interestingly, DHHC5,13,14,16,17,20and21were up-regulated in proliferation of NSCs compared to non-treated cells. Among them, DHHC5,17and21also up-regulated during differentiation of NSCs. On the other hand, DHHC2,6,8,11,15,18,19and23were only up-regulated during differentiation of NSCs, while DHHC1,4,7,9and24showed no alteration at the mRNA levels.
Next, we used ABE method to confirm that protein palmitoylation occurred during development of NSCs. During proliferation of NSCs, P19cells clearly showed several palmitoylation bands and the most prominent ones were at the MW of～10,25,45,50,70,170kDa. Moreover, the palmitoylation bands were at the MW of～25,55,60,70,100,125,150,290kDa.
Taken together, these results indicate that cells express many DHHCs and some proteins are palmitoylated during neural development. During different stages of neural development, different cell types may have specific sets of palmitoylated proteins and these specific palmitoylated proteins specifically regulated the different process of neural development.
Patr II Effects of2BROP on proliferation of neural stem cells
To examine the effect of2BROP on proliferation of NSCs, different concentrations of2BROP were added to the RA induced P19cells. The results of MTT assay showed that2BROP treatment improved the cell viability of P19cells in a dose-dependent manner (1μM,5μM,10μM and15μM) compared to the negative control. Among them,10μM2BROP favored the optimal effect on cell viability, but the result was statistically insignificant. However, higher level of2BROP (25μM and50μM) resulted in a clear reduction of cell viability. Next, we observed the morphological characteristics of neurospheres under different concentrations of2BROP. Both the control neurospheres and neurospheres treated with lower level of2BROP (10μM) showed the morphology of densely packed and regular colonies, while neurospheres treated with higher level of2BROP (25μM) showed loose and irregular colonies.
Along with the above, we examined the incidence of apoptosis of neurospheres used by TUNEL assay. The results of TUNEL have showed that neurospheres treated with lower level2BROP did not appear any difference compared with untreated neurospheres, but the higher level of2BROP induced apoptosis of neurospheres. Consistent with this, higher level of2BROP (25μM) could lead to mitochondrial apoptosis pathways, as shown by a decrease in the expression of Bcl-2, and a clear up-regulation in the expression of Bax and cleaved Caspase-3.
Taken together, these results showed that lower concentration of2BROP did not clearly change the characteristics of neurospheres, during self-renewal of NSCs, but higher level of2BROP showed toxicity to neurospheres and led to cell apoptosis.
PartⅢ Effects of2BROP on differentiation of neural stem cells
To investigate the effects of palmitoylation on neuronal differentiation, we examined the neuronal marker, MAP2, after plating with immunofluorescent staining and Western blot analysis. Compared to the negative control, a significant reduction in the percentage of MAP2-positive cells and MAP2protein expression was observed in2BROP-treated cells.
Next, we turned to define whether the early steps of the differentiation process were equally affected in the2BROP-treated cell model, the Oct4, Nestin, Sox2, β-tubulin III Neurogl and NeuroDl gene markers were tested with qRT-PCR and Western Blot analysis. After RA induction, the expression of Oct-4was lost, and there were no obvious differences between2BROP-treated and untreated cells. Once attached, cells that were treated with2BROP expressed reduced levels of β-tubulin Ⅲ, Neurogl and NeuroDl. However,2BROP treated cells expressed higher levels of Sox2and Nestin, notably during NSCs differentiation. Consistent with these, the results of MTT also showed that, during neuronal differentiation,2BROP improved the capacity for neural stem/progenitor cell renewal and hampered the passage of neural stem/progenitor cells to form differentiated neurons.
In order to test whether cells that were treated with2BROP had an enhanced capacity for cell division, we used a series of BrdU incorporation assays to assess cell proliferation during NSCs differentiation. The results showed that the percentage of BrdU-positive cells was higher in2BROP-treated cells compared to control after4days of plating. The enhanced capacity for cell division was also detected by membrane-labelling experiments at day4of differentiation in suspension culture. A fluorescence-activated cell sorting (FACS) analysis revealed that cells that were treated with2BROP lost fluorescence more rapidly during the initial24h of culture after labelling than control cells did, indicating that they had undergone more cellular divisions than control cells or that they contained more dividing cells.
Next, we tested the hypothesis that the increase in the neural stem/progenitor cell numbers of2BROP-treated cells was associated with changes in cell cycle distribution with FACS. As expected, the percentage of S-phase cells was increased significantly, while the percentage of G1/GO-phase cells was reduced in2BROP-treated cells compared to control cells, indicating their inability to arrest cell division and to induce neuronal differentiation. We further examined the expression of cell cycle regulators during NSCs differentiation between control and2BROP-treated cells with qRT-PCR. Among them, the levels of Ki67, which is a proliferative marker, in2BROP-treated cells showed obviously increased, while the expression of P57, which promotes cell cycle exit and post-mitotic neuron differentiation, was markedly decreased.
Taken together, these results suggested that the reduction in the differentiation capacity of2BROP-treated cells was most likely due to a reduction of P57expression and increased neural stem/progenitor cell renewal, in which the neural stem/progenitor cells were delayed in cell cycle progression and unable to exit the cell cycle on time.
PartIV Effects of2BROP on the epigenetic mechanisms during differentiation of neural stem cells
As reported, the P57gene, which is epigenetically regulated by the acetylation level of histones H3/4. In order to detect changes in histone acetylation, protein extracts were harvested from2BROP-treated and2BROP-untreated cells and analysed by a Western Blot analysis with antibodies that were specific for the acetylated histones H3and H4. There appeared to be less histone H3and H4acetylation in the2BROP-treated cell model compared to control cells. The immunofluorescent staining analysis supported this finding, and, in2BROP-treated cells, a significant reduction in the percentage of acetylation H3or H4-positive cells was detected compared to control cells. Next, in order to test whether increased HAT activity could rescue the2BROP-induced phenotype during neuronal differentiation, we treated cells with TSA, a histone deacetylase, for24h. TSA appeared to increase neuronal differentiation as shown by the expression of β-tubulin III. Interestingly, after TSA treatment, the β-tubulin III levels of the cells that were treated with2BROP were even higher than those of untreated cells.
We attempted to understand the basis for the2BROP-associated effects on HAT. Thus, we turned to assess the effect of2BROP on the regulation of P300HAT activity by an in vitro HAT assay. We found that2BROP remarkably reduced P300HAT activity. Next, we detected the sub-cellular localization and expression level of P300during NSCs differentiation. The Western Blot analysis showed that2BROP did not clearly change the protein level of P300. Interestingly, we found a change in the sub-cellular localization of P300in the immunofluorescent staining analysis. We detected relatively high levels of P300in the nucleus of control cells. However, there appeared to be an abundance of P300in the cytoplasm of2BROP-treated cells, indicating that2BROP might impair the nuclear import of P300after translation.
The post-translational modifications of proteins with lipids provide an important mechanism for regulating protein sub-cellular localization, stability, trafficking, and other aspects of protein function. Because2BROP is widely used as an inhibitor of protein palmitoylation, one possibility is that2BROP impairs the nuclear import of P300by inhibiting its palmitoylation status. As predicted by the CSS-Palm4.0software, P300has numerous potential palmitoylation sites, which are conserved in P300from different species. In order to detect changes in the palmitoylation of P300, protein extracts were immunoprecipitated with P300and analysed by the ABE method. A significant reduction in the palmitoylation level of P300was observed in2BROP-treated cells compared to control cells during different stages of neural development.
Taken together, these results suggested that2BROP inhibited P300palmitoylation and hampered the nuclear import of P300, resulting in a change in the histone H3and H4acetylation status in differentiation cells.
Conclusion
The 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 P19cells to investigate the connection between palmitoylation and cell fate specification of NSCs.20out of23DHHCs can be detected and they specifically expressed at the different stage of NSCs development. Consistent with these, different cell types may have specific sets of palmitoylated proteins. During NSCs induction and proliferation, lower concentration of2BROP did not clearly change the characteristics of neurospheres. However, their differentiation to neurons and cell cycle exit were less efficient when cells were cultured in the presence of2BROP. These effects were associated with an extensive change in the transcriptional profile of neural stem/precursor cells and a decrease in acetylated histone H3and H4. These results suggested that2BROP inhibited P300palmitoylation and hampered the nuclear import of P300, resulting in a change in the histone H3and H4acetylation status 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 vertebrate development.
在中枢神经系统中,神经干细胞(neural stem cells, NSCs)属于多潜能干细胞,具有自我更新能力,并且在胚胎发育及成年脑中均能分化产生神经元、星形胶质细胞和少突胶质细胞。NSCs的分化成熟,可以大致分为如下3个阶段：1)NSCs自我更新；2)NSCs被赋予具有分化潜能的神经前体细胞；3)退出周期,处于有丝分裂静止期,分化形成具有特殊形态和功能的神经细胞。其实,在NSCs分化过程中,这些细胞形态和功能的改变很大程度上是由细胞所处不同阶段的特定基因表达模式所决定的。其中,主要包括多潜能因子Sox2、原神经基因bHLH (basic Helix-Loop-Helix, bHLH)家族和细胞周期依赖激酶抑制因子(cyclin-dependent kinase inhibitors, CKIs)。 NSCs自我更新能力及干性维持的内部机制主要是通过多潜能因子的表达和分化命运决定因子的沉默而实现的。NSCs分化命运决定的内部机制也是通过时间和空间上高度精确的调控机制完成的,即分化命运决定因子的激活与表达以及多潜能因子的沉默。
目前,研究发现真核基因的表达在多个层次上受到精确的调控,而染色质的修饰是真核基因转录的第一调控靶点。其中,组蛋白的共价修饰对染色质的动态结构具有核心调节作用,它是表观遗传学重要的调节方式之一。在神经细胞命运决定过程中常常伴随着组蛋白乙酰化修饰水平的改变。组蛋白乙酰化转移酶(histone acetyltransferase, HAT) P300在神经系统中表达丰富,它参与了神经系统在形式和功能上的发生、可塑性和稳态的建立。P300通过自身的HAT活性,乙酰化组蛋白赖氨酸,染色质发生重塑,使转录因子易于与DNA结合,从而促进基因转录。其中,组蛋白H3和H4就是其乙酰化共价修饰的底物之一。
本研究中,我们通过建立视黄酸(retinoic acid, RA)诱导的P19细胞定向分化为神经样细胞的体外模型,利用棕榈酰转移酶抑制剂——小分子化合物2-bromopalmitate (2BROP),探讨棕榈酰化修饰在NSCs发育中的作用及相关表观遗传分子机制。
第一部分棕榈酰化修饰相关蛋白在神经干细胞发育过程中的表达
为了揭示棕榈酰化修饰作用是否参与NSCs发育过程,于NSCs发育的各阶段,包括P19细胞多潜能状态、NSCs增殖和NSCs分化等三个阶段,分别检测23个棕榈酰转移酶即DHHC家族成员的表达情况。结果显示,在NSCs发育阶段,共有20个DHHC家族成员的mRNA表达。其中,DHHC1、4、7、9、24于NSCs发育各阶段的mRNA表达无明显变化。DHHC5、13、14、16、17、20、21成员于NSCs增殖阶段mRNA的表达量获得增高,并且DHHC5、17、21在NSCs分化阶段也将持续性高表达。而DHHC2、6、8、11、15、18、19、23成员的mRNA仅于NSCs分化阶段高表达。
接下来,我们利用酰基-生物素交换法(Acyl-biotinyl exchange method, ABE)技术,检测NSCs增殖与分化两阶段,蛋白棕榈酰化修饰水平情况。结果显示,多种蛋白在NSCs增殖与分化阶段被棕榈酰化修饰,并在不同的NSCs发育阶段,呈现出一定的特异性,如NSCs增殖阶段,棕榈酰化蛋白条带分布于～10、25、45、50、70、170kDa；而NSCs分化阶段,蛋白分布于～25、55、60、70、100、125、150、290kDa。
本实验结果显示,大多数DHHC家族成员不同程度的表达于NSCs发育各阶段,并且在不同的NSCs发育阶段,棕榈酰化修饰的蛋白呈现出一定的特异性,暗示DHHC家族成员和它们所介导的棕榈酰化修饰参与NSCs发育过程的调控,棕榈酰化修饰可能通过特异性的影响NSCs发育各阶段中关键蛋白的生物活性,从而参与调控NSCs的命运决定。
第二部分小分子化合物2BROP对神经干细胞增殖的作用
为了研究棕榈酰化修饰对NSCs增殖的影响,我们首先利用MTT技术,检测不同剂量的2BROP对于细胞代谢活力的影响。结果显示,与对照组(0μ M)相比,1-15μM加药组中细胞活力表现出一定的增强；尤其是10uM加药组,但是统计学无明显差异。然而,与对照组相比,25μ M和50μM加药组中,细胞活力减弱,MTT值明显降低,并具有统计学显著性差异。与此同时,倒置相差显微镜,观察神经球的结构和形态变化。结果显示,随着RA诱导的进行,对照组中,细胞聚集体不断增大,最终形成一个结构致密、表面规整的细胞团。10μM终浓度加药组组中,细胞聚集体的发生和增长过程与对照组相比,未见异常。但是,25μM加药组中,细胞聚集体的结构发生明显改变,表现为细胞团形态不规则、内部有空隙。
接下来,我们利用TUNEL技术,在NSCs增殖阶段,检测小分子化学物2BROP对于细胞凋亡的影响。结果显示,2BROP处理48h后,与对照组相比,10μM加药组中,细胞团中TUNEL阳性细胞数没有明显差异；而25u M处理组中,TUNEL阳性细胞数明显增多。随后,我们利用、Vestern Blot和免疫荧光染色技术,检测了相关凋亡信号通路的变化。结果发现,25μM处理组中线粒体相关凋亡通路明显激活。与对照组相比,25μM处理组中抗凋亡Bcl-2的表达量减少,促凋亡的Bax和活化的Caspase3的表达量增多；而10μM加药组中Bcl-2、Bax与活化的Caspase3表达量没有明显变化
本实验结果,显示在一定的剂量范围内,小分子化合物2BROP对于NSCs增殖过程,并没有产生明显的影响；而高浓度2BROP将会对细胞产生毒性,引发细胞凋亡。
第三部分小分子化合物2BROP对神经干细胞分化的作用
为了探究蛋白棕榈酰化修饰在NSCs分化阶段中的相关作用及其分子机制,我们利用免疫荧光技术和Western Blot等技术,检测神经分化标记物MAP2的表达与分布情况。结果显示,与对照组相比,2BROP处理组中,MAP2阳性细胞数明显减少,统计学具有显著性差异；且神经分化各阶段,MAP2蛋白的表达均受到明显抑制。
接下来,利用Western Blot和qRT-PCR技术,我们检测了NSCs分化命运决定相关因子的表达情况,如多潜能因子Oct4; NSCs标记物Sox2、Nestin;神经前体细胞标记物β-tubulinⅢ和神经分化决定因子Neurogl、NeuroD1。结果显示,随着NSCs命运的获得,对照组与2BROP处理组中,Oct4的表达迅速下降直至消失,无明显差异。与对照组相比,2BROP组中神经前体细胞标记物β-tubulinIII、分化命运决定因子Neurog1和NeuroD1的表达于神经分化各阶段均呈现出明显地降低。与此相反,2BROP处理组中,NSCs标记物Sox2与Nestin却表现出一定的持续性高表达。同时,MTT实验结果显示,与对照组分化各阶段的MTT值无明显变化相比,2BROP处理组中,随着分化天数的延长,MTT值呈现出一定的增。这些结果提示我们,在神经分化阶段,2BROP可能通过增强NSCs的干细胞特性的维持,从而阻碍神经分化的发生。
为了验证NSCs分化阶段2BROP是否增强了细胞的增殖能力,我们首先利用BrdU掺入实验,检测分化阶段细胞的增殖情况。结果显示,在神经分化的第四天,与对照组相比,2BROP处理组中,BrdU阳性细胞数明显增多,并且统计学上具有显著性差异。然后,我们利用膜标记-FACS技术追踪细胞增殖的变化。分化阶段的细胞使用荧光染料标记。细胞每增殖一次,荧光强度便会减弱一半。结果显示,悬浮培养24h后,对照组中单个细胞的荧光强度并没有明显的变化；而2BROP处理后,单个细胞的荧光强度发生不同程度的减弱。
为了进一步分析2BROP在NSCs分化阶段对于细胞增殖和周期的影响,利用FACS技术,分析分化阶段细胞周期的分布。结果显示,神经分化第四天,对照组中细胞主要分布于G0/G1期；而2BROP处理组中,G0/G1期的细胞数量减少,S期的细胞数量明显增多。与此同时,我们利用qRT-PCR技术,检测了与神经分化相关的CKIs家族成员的表达情况,借以阐明2BROP阻碍周期退出的相关机制。结果显示,与对照组相比,2BRPP处理后,分化相关的CKIs成员的表达受到不同程度的降低。其中,P57的变化最为明显。作为对照,细胞周期增殖标记物Ki67的表达在2BROP处理组中获得了一定的持续性高表达。
本实验结果,显示在NSCs分化阶段,2BROP处理后,NSCs通过下调CKIs家族成员,尤其是P57的表达,从而阻碍细胞周期的退出与神经分化的发生,使分化状态下NSCs自我更新能力获得明显的提高。
第四部分小分子化合物2BROP对神经干细胞分化表观遗传机制的作用
已知,神经分化阶段,介导细胞周期调控的P57的表达依赖于组蛋白H3、H4的乙酰化水平。为了检测组蛋白乙酰化修饰是否参与2BROP介导的神经分化,我们利用免疫荧光、Western Blot等技术,检测NSCs分化阶段乙酰化组蛋白H3、H4的分布与表达。结果显示,当细胞进入分化阶段时,组蛋白H3与H4的乙酰化水平呈现出一定的上升,但是2BROP处理组中组蛋白H3与H4的乙酰化水平明显的低于对照组的水平。为了进一步验证组蛋白乙酰化修饰是否参与2BROP介导的神经分化,我们使用去组蛋白去乙酰化抑制剂曲古菌素A(Trichostatin A, TSA)预处理细胞,发现TSA处理后,2BROP抑制神经分化的现象得到一定的恢复。值得注意的是,TSA与2BROP共处理组中,β-tubulinⅢ的表达甚至高于正常分化组。
接下来,我们免疫共沉淀、ELISA等技术,检测组蛋白H3、H4乙酰化修饰酶P300乙酰转移酶活性。结果显示,与对照组相比,2BROP处理后,P300的乙酰转移酶活性受到明显的抑制。同时,我们利用Western Blot、免疫荧光等技术,检测了分化阶段P300的表达与分布。结果显示,虽然与对照组相比,2BROP处理组中P300的蛋白表达水平没有明显变化,但是P300在胞内的分布却发生明显的改变。对照组中,P300主要分布于细胞核中,而2BROP处理组中P300蛋白遍布于细胞质和细胞核中。
作为脂质化修饰方式之一,棕榈酰化修饰作用参与调节蛋白的胞内分布、物质运输、蛋白间相互作用等过程。接下来,我们探究2BROP是否通过调节P300的棕榈酰化修饰参与调控组蛋白乙酰化修饰与神经分化。首先,我们使用CSS-Palm4.0软件预测P300蛋白是否拥有典型的棕榈酰化位点。结果显示,P300蛋白拥有多个保守性较高的棕榈酰化潜在修饰位点。然后,我们使用ABE方法,检测NSCs分化阶段,对照组与2BROP处理组中P300的棕榈酰化修饰水平。结果显示,蛋白P300可以被棕榈酰化修饰,并且与对照组相比,2BROP处理组中P300的棕榈酰化修饰水平明显受到抑制,统计学上具有显著性差异。
本实验结果,显示在NSCs分化阶段,棕榈酰化修饰通过调节表观遗传关键因子P300的脂质化修饰水平,进而影响P300的核转位和HAT活性,最终调节组蛋白H3、H4的乙酰化水平和分化命运相关基因的转录激活。
结论
神经干细胞的分化、发育与成熟是现代神经生物学研究的热点之一。棕榈酰化修饰作用在神经发育过程中的作用及分子机制尚未阐明。我们建立P19细胞定向分化为神经样细胞的体外模型,利用棕榈酰转移酶抑制剂——小分子化合物2BROP,探讨棕榈酰化修饰在神经发育中的作用及分子机制。利用P19神经定向分化模型,研究发现,DHHC家族成员分别特异性地表达于NSCs的增殖和分化阶段。并且在这些阶段中,许多蛋白发生了明显的棕榈酰化修饰。2BROP (10μM)处理后,NSCs增殖的命运没有发生明显的改变。但是,在NSCs分化阶段,发现2BROP (10μM)处理后,NSCs的神经分化过程和细胞周期的退出受到阻断,而NSCs的数量却维持在一定的高水平状态。进一步的检测,表明细胞周期调节因子CKI家族中P57的表达明显降低。已知,神经分化阶段,P57的表达依赖于组蛋白H3/H4的乙酰化水平。当加入组蛋白去乙酰化抑制剂曲古菌素A(Trichostatin A, TSA)后,发现P57的表达和神经分化得到一定程度的恢复。最终,研究表明在神经分化阶段,2BROP处理后,由于特异性的抑制P300的棕榈酰化修饰水平,影响其核转移,进而阻碍组蛋白H3/H4的乙酰化修饰与神经分化。因此,本研究揭示了一个由基因表达、表观遗传修饰、蛋白翻译后修饰共同参与的神经分化命运决定调控网络。本研究不仅深化了棕榈酰化修饰在神经发育中的作用,拓展了其分子调控机制,同时为研究棕榈酰化修饰作用在组织发育过程中的作用开辟了一个全新的领域、提供了一个新的视野。
After mRNA translation, immature protein undergoes post-translational modifications to become the mature protein product. Among them, protein palmitoylation is an important lipid modification. Protein palmitoyltransferases were first identified and isolated in yeast and they share a domain referred to as the DHHC domain, a cysteine-rich domain with a conserved aspartate-histidine-histidine-cysteine signature motif. Evidence suggests that the DHHC domain is directly involved in the palmitoyl transfer reaction. In mammals twenty three members of this family have been identified and examined by by Bredt DS group at2004. Recently, the role of protein palmitoylation on neuronal development and function are just beginning to be appreciated by proteome and molecular genetic analysis. There are still many unsolved questions and much work is still needed to elucidate the specific functions and mechanisms of protein palmitoylation on neural development.
In mammals, neural stem cells (NSCs) have capability to self-renew and differentiate into neurons, astrocytes, and oligodendrocytes. During NSCs differentiation, NSCs proliferate (1), then begin to specify into distinct neural progenitor cell fates (2), and exit the cell cycle following a terminal mitosis and express markers of terminal differentiation to finally undergo neuronal or glial morphogenesis (3). In fact, these universal changes in cell types and function represent a major cellular reprogramming event, involving in activation or repression of Sox2, bHLH family and CKIs. NSCs have capability to self-renew through the activation of multipotency genes and repression of differentiation-related genes. Moreover, the precise temporal and spatial differentiation progression is associated with activation the specific transcriptional factors, up-regulation of differentiation-specific genes and repression the multipotency genes.
Tight regulation of these gene expressions may be at some levels, including transcriptional level, posttranscriptional level, as well as posttranslational level. Indeed, the chromatin modification firstly regulates the gene transcription. The acetylation status of histones is thought to act as a general regulator of chromatin structure that mediates the removal of epigenetically controlled repression and enhances transcriptional activity. Recently, studies provide evidence for general alterations in histone modification, such as the increased histone H3and H4acetylation occurring during neuronal differentiation. P300, a histone acetyltransferase (HAT), has the capacity to acetylate histone H3and H4both in vitro and in vivo, and plays an important role in neural development and function. Mechanistically, P300acetylates lysine residues within the histone tails, resulting in promoting transcription factors access to DNA in chromatin by neutralising the positive charge associated with the lysine e-amino group.
Here, Retinoic acid (RA) induced P19cells were used in this study as a model system to elucidate the functional importance of protein palmitoylation during neuronal differentiation.
Part I Protein palmitoylation is occurred during neural stem cells development
To investigate whether protein palmitoylation was involved in NSCs development, we first analyzed the expression of the23potential DHHCs in RA induced P19cells at different stages of neural development by RT-PCR. It was found that20out of23DHHCs can be detected. More interestingly, DHHC5,13,14,16,17,20and21were up-regulated in proliferation of NSCs compared to non-treated cells. Among them, DHHC5,17and21also up-regulated during differentiation of NSCs. On the other hand, DHHC2,6,8,11,15,18,19and23were only up-regulated during differentiation of NSCs, while DHHC1,4,7,9and24showed no alteration at the mRNA levels.
Next, we used ABE method to confirm that protein palmitoylation occurred during development of NSCs. During proliferation of NSCs, P19cells clearly showed several palmitoylation bands and the most prominent ones were at the MW of～10,25,45,50,70,170kDa. Moreover, the palmitoylation bands were at the MW of～25,55,60,70,100,125,150,290kDa.
Taken together, these results indicate that cells express many DHHCs and some proteins are palmitoylated during neural development. During different stages of neural development, different cell types may have specific sets of palmitoylated proteins and these specific palmitoylated proteins specifically regulated the different process of neural development.
Patr II Effects of2BROP on proliferation of neural stem cells
To examine the effect of2BROP on proliferation of NSCs, different concentrations of2BROP were added to the RA induced P19cells. The results of MTT assay showed that2BROP treatment improved the cell viability of P19cells in a dose-dependent manner (1μM,5μM,10μM and15μM) compared to the negative control. Among them,10μM2BROP favored the optimal effect on cell viability, but the result was statistically insignificant. However, higher level of2BROP (25μM and50μM) resulted in a clear reduction of cell viability. Next, we observed the morphological characteristics of neurospheres under different concentrations of2BROP. Both the control neurospheres and neurospheres treated with lower level of2BROP (10μM) showed the morphology of densely packed and regular colonies, while neurospheres treated with higher level of2BROP (25μM) showed loose and irregular colonies.
Along with the above, we examined the incidence of apoptosis of neurospheres used by TUNEL assay. The results of TUNEL have showed that neurospheres treated with lower level2BROP did not appear any difference compared with untreated neurospheres, but the higher level of2BROP induced apoptosis of neurospheres. Consistent with this, higher level of2BROP (25μM) could lead to mitochondrial apoptosis pathways, as shown by a decrease in the expression of Bcl-2, and a clear up-regulation in the expression of Bax and cleaved Caspase-3.
Taken together, these results showed that lower concentration of2BROP did not clearly change the characteristics of neurospheres, during self-renewal of NSCs, but higher level of2BROP showed toxicity to neurospheres and led to cell apoptosis.
PartⅢ Effects of2BROP on differentiation of neural stem cells
To investigate the effects of palmitoylation on neuronal differentiation, we examined the neuronal marker, MAP2, after plating with immunofluorescent staining and Western blot analysis. Compared to the negative control, a significant reduction in the percentage of MAP2-positive cells and MAP2protein expression was observed in2BROP-treated cells.
Next, we turned to define whether the early steps of the differentiation process were equally affected in the2BROP-treated cell model, the Oct4, Nestin, Sox2, β-tubulin III Neurogl and NeuroDl gene markers were tested with qRT-PCR and Western Blot analysis. After RA induction, the expression of Oct-4was lost, and there were no obvious differences between2BROP-treated and untreated cells. Once attached, cells that were treated with2BROP expressed reduced levels of β-tubulin Ⅲ, Neurogl and NeuroDl. However,2BROP treated cells expressed higher levels of Sox2and Nestin, notably during NSCs differentiation. Consistent with these, the results of MTT also showed that, during neuronal differentiation,2BROP improved the capacity for neural stem/progenitor cell renewal and hampered the passage of neural stem/progenitor cells to form differentiated neurons.
In order to test whether cells that were treated with2BROP had an enhanced capacity for cell division, we used a series of BrdU incorporation assays to assess cell proliferation during NSCs differentiation. The results showed that the percentage of BrdU-positive cells was higher in2BROP-treated cells compared to control after4days of plating. The enhanced capacity for cell division was also detected by membrane-labelling experiments at day4of differentiation in suspension culture. A fluorescence-activated cell sorting (FACS) analysis revealed that cells that were treated with2BROP lost fluorescence more rapidly during the initial24h of culture after labelling than control cells did, indicating that they had undergone more cellular divisions than control cells or that they contained more dividing cells.
Next, we tested the hypothesis that the increase in the neural stem/progenitor cell numbers of2BROP-treated cells was associated with changes in cell cycle distribution with FACS. As expected, the percentage of S-phase cells was increased significantly, while the percentage of G1/GO-phase cells was reduced in2BROP-treated cells compared to control cells, indicating their inability to arrest cell division and to induce neuronal differentiation. We further examined the expression of cell cycle regulators during NSCs differentiation between control and2BROP-treated cells with qRT-PCR. Among them, the levels of Ki67, which is a proliferative marker, in2BROP-treated cells showed obviously increased, while the expression of P57, which promotes cell cycle exit and post-mitotic neuron differentiation, was markedly decreased.
Taken together, these results suggested that the reduction in the differentiation capacity of2BROP-treated cells was most likely due to a reduction of P57expression and increased neural stem/progenitor cell renewal, in which the neural stem/progenitor cells were delayed in cell cycle progression and unable to exit the cell cycle on time.
PartIV Effects of2BROP on the epigenetic mechanisms during differentiation of neural stem cells
As reported, the P57gene, which is epigenetically regulated by the acetylation level of histones H3/4. In order to detect changes in histone acetylation, protein extracts were harvested from2BROP-treated and2BROP-untreated cells and analysed by a Western Blot analysis with antibodies that were specific for the acetylated histones H3and H4. There appeared to be less histone H3and H4acetylation in the2BROP-treated cell model compared to control cells. The immunofluorescent staining analysis supported this finding, and, in2BROP-treated cells, a significant reduction in the percentage of acetylation H3or H4-positive cells was detected compared to control cells. Next, in order to test whether increased HAT activity could rescue the2BROP-induced phenotype during neuronal differentiation, we treated cells with TSA, a histone deacetylase, for24h. TSA appeared to increase neuronal differentiation as shown by the expression of β-tubulin III. Interestingly, after TSA treatment, the β-tubulin III levels of the cells that were treated with2BROP were even higher than those of untreated cells.
We attempted to understand the basis for the2BROP-associated effects on HAT. Thus, we turned to assess the effect of2BROP on the regulation of P300HAT activity by an in vitro HAT assay. We found that2BROP remarkably reduced P300HAT activity. Next, we detected the sub-cellular localization and expression level of P300during NSCs differentiation. The Western Blot analysis showed that2BROP did not clearly change the protein level of P300. Interestingly, we found a change in the sub-cellular localization of P300in the immunofluorescent staining analysis. We detected relatively high levels of P300in the nucleus of control cells. However, there appeared to be an abundance of P300in the cytoplasm of2BROP-treated cells, indicating that2BROP might impair the nuclear import of P300after translation.
The post-translational modifications of proteins with lipids provide an important mechanism for regulating protein sub-cellular localization, stability, trafficking, and other aspects of protein function. Because2BROP is widely used as an inhibitor of protein palmitoylation, one possibility is that2BROP impairs the nuclear import of P300by inhibiting its palmitoylation status. As predicted by the CSS-Palm4.0software, P300has numerous potential palmitoylation sites, which are conserved in P300from different species. In order to detect changes in the palmitoylation of P300, protein extracts were immunoprecipitated with P300and analysed by the ABE method. A significant reduction in the palmitoylation level of P300was observed in2BROP-treated cells compared to control cells during different stages of neural development.
Taken together, these results suggested that2BROP inhibited P300palmitoylation and hampered the nuclear import of P300, resulting in a change in the histone H3and H4acetylation status in differentiation cells.
Conclusion
The 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 P19cells to investigate the connection between palmitoylation and cell fate specification of NSCs.20out of23DHHCs can be detected and they specifically expressed at the different stage of NSCs development. Consistent with these, different cell types may have specific sets of palmitoylated proteins. During NSCs induction and proliferation, lower concentration of2BROP did not clearly change the characteristics of neurospheres. However, their differentiation to neurons and cell cycle exit were less efficient when cells were cultured in the presence of2BROP. These effects were associated with an extensive change in the transcriptional profile of neural stem/precursor cells and a decrease in acetylated histone H3and H4. These results suggested that2BROP inhibited P300palmitoylation and hampered the nuclear import of P300, resulting in a change in the histone H3and H4acetylation status 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 vertebrate development.
引文
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