摘要
核小体核心组蛋白的尾部有多种共价修饰,包括乙酰化,磷酸化,甲基化和泛素化等。这些组蛋白的翻译后修饰可以改变染色质结构,进而影响非组蛋白转录因子与染色质的结合,在调节转录过程中发挥着重要的作用。与乙酰化不同,对组蛋白甲基化的作用知之甚少,尽管这种修饰已经被发现了近40年。当初由于不清楚具体发挥甲基化作用的酶类,很难探讨组蛋白甲基化与基因活化之间的直接联系。近些年一些具有精氨酸或赖氨酸甲基化作用的酶类陆续被鉴定出来。其中蛋白质精氨酸甲基转移酶家族(protein arginine methyltransferases, PRMTs)就是一类含有高度保守结构域的具有甲基转移酶活性的酶类。其作用底物非常广泛,涉及真核细胞DNA和RNA介导的许多过程。其修饰组蛋白,调节转录因子,调整mRNA的稳定性,参与DNA损伤修复及装配剪接复合体的功能是几乎所有真核细胞功能所必需的。
横纹肌肉瘤(Rhabdomyosarcoma, RMS)起源于肌肉前体细胞,是一种儿童时期的软组织肉瘤,其恶性程度很高且较常见。人类胚胎型横纹肌肉瘤衍生的RD细胞,尽管表达成肌调节因子MyoD和生肌蛋白(Myogenin),但未能完成分化。为了深入了解该肿瘤的发病机理,探索可能用于体内的治疗靶点,本文以RD细胞为模型,从该肿瘤终末分化阻滞机理出发,主要研究对基因转录具有重要调控作用的组蛋白修饰酶——蛋白质精氨酸甲基转移酶(PRMTs)在TPA (12-O-tetradecanoyl-phorbol-13-acetate)诱导RD细胞分化过程中的作用及其机制,并对RD细胞分化过程中早期分化标志——生肌蛋白基因启动子区染色质的重塑以及转录相关因子的结合进行研究,为横纹肌肉瘤的临床治疗提供一定的理论依据。
一、TPA诱导RD细胞分化过程中PRMT家族成员的表达情况
TPA处理RD细胞过程中,流式细胞术显示,G2／M期数值增加,细胞周期明显阻滞；RT-PCR、Western Blot方法检测PRMT家族基因在RD细胞中都有不同程度的表达,且随着RD细胞分化时间的延长,表达有所增加；组蛋白精氨酸甲基化水平增加；细胞免疫荧光染色发现诱导后细胞出现分化后的形态改变,如胞体伸长,细胞内颗粒物增多；PRMT家族酶主要定位在胞浆中,细胞分化后进入细胞核。
二、PRMT家族酶对TPA诱导的RD细胞分化过程中生肌蛋白基因表达的调控作用
1)构建并验证了PRMT家族各成员的有效siRNA质粒。构建并鉴定了PRMT5、PRMT6的过表达质粒pCDNA6-PRMT5(/PRMT6)-FLAG。
2)确定在RD细胞中,PRMT抑制剂AdOx的使用浓度为20μM。
3)流式细胞术结果显示,腺苷二醛AdOx处理的RD细胞退出细胞周期,处于G2／M期阻滞状态。Western Blot检测到分化标志生肌蛋白的表达受到了明显的抑制,说明细胞没有进入分化过程。免疫荧光实验结果也表明细胞形态上没有明显的分化趋势。结果还显示随着TPA的诱导PCAF的mRNA及蛋白的表达量均明显增加,且AdOx能明显抑制其表达。而SWI/SNF染色质重塑复合物核心亚单位Brgl不论是在TPA诱导的过程中,还是AdOx处理前后都没有明显的改变。启动子活性分析实验结果提示TPA通过某种机制激活生肌蛋白基因的表达,而可被AdOx抑制的精氨酸甲基化作用可能是其激活途径的必经环节。以上AdOx处理细胞的实验结果提示,精氨酸甲基化过程是细胞分化形态的改变、主要转录因子活化、以及成肌相关因子激活所必需的。
4)应用siRNA干扰技术,经Western Blot实验证明,TPA诱导增加的Myogenin蛋白在转染CARM1siRNA质粒后表达受到了明显的抑制,PCAF的表达也有所降低。且Myogenin和PCAF的入核量也都明显减少。过表达CARM1的实验结果显示Myogenin、PCAF和Brgl的核内表达都明显高于对照组。细胞免疫荧光实验结果显示,转染了CARM1siRNA的细胞,在TPA诱导过程中形态上没有明显的分化趋势。此外,在转染各个PRMT特异的siRNA后,myogenin基因启动子活性都被明显抑制。结合上述AdOx作用的相关实验,这些结果都显示PRMT家族酶在肌肉分化过程中发挥着重要的作用。
5)Myogenin基因启动子活性分析实验结果显示,在Brgl促进生肌蛋白表达的过程中,CARM1的作用是不可或缺的。CARM1也是PCAF增强生肌蛋白启动子活性所必需的调控因子。而p300可能与CARM1相互作用来协同增强myogenin启动子活性。
三、PRMT家族酶对TPA诱导的RD细胞分化过程中生肌蛋白基因染色质水平的调控作用
1)染色质免疫共沉淀(ChIP)实验结果表明,未经TPA诱导的对照RD细胞中,CARM1并不与生肌蛋白基因启动子区结合,TPA诱导3小时后,CARM1开始结合到启动子上。6小时后,结合更加明显,而SB的处理不改变CARM1的结合,说明CARM1被募集到myogenin启动子上是非p38依赖性的。
2)未经处理的RD细胞生肌蛋白启动子区组蛋白H3有一定水平的乙酰化,而组蛋白H4的精氨酸甲基化水平较低,TPA诱导后组蛋白乙酰化和精氨酸甲基化水平都明显升高,且经AdOx或SB处理后都受到了抑制。
3) SWI/SNF染色质重塑复合物亚基BAF60和Brg1处理前不结合在生肌蛋白启动子上,TPA诱导后结合明显增加,这种结合依赖于p38和精氨酸甲基化作用。
4)乙酰基转移酶p300在生理状态下就结合在生肌蛋白的启动子上,TPA诱导和SB处理后都没有改变,但AdOx处理明显抑制其结合。p300／CBP协同因子—-PCAF只在TPA诱导后结合到启动子上,但这种结合并不依赖于p38,而依赖于可被AdOx抑制的精氨酸甲基化作用。
5)肌细胞分化调节因子MyoD不论是在TPA诱导前后,还是SB或AdOx处理过程中,都结合在启动子上且没有明显的改变。PRMT5生理条件下不结合到启动子上,TPA诱导后明显结合,这种结合依赖于p38和精氨酸甲基化作用。
6)RNA聚合酶polⅡ生理状态下在生肌蛋白启动子上有一定的结合,TPA诱导后结合增加,而SB或AdOx处理都可对其结合有一定的抑制作用,但抑制不完全。
综合以上结果,我们认为PRMT家族酶在RD细胞的诱导分化过程中具有正调节作用。通过该类酶促的蛋白质精氨酸甲基化,以及其他调节因子对组蛋白的修饰或转录因子的活化,可有效地促进生肌蛋白基因表达,并导致RD细胞的分化。本研究显示深入研究精氨酸甲基转移酶的调控机制有助于对肌肉细胞诱导分化机制的认识,并将为横纹肌肉瘤的临床治疗提供一定的理论依据。
The N-terminal tails of core histones in a nucleosome are covalently modified in vivo in a variety ways, such as acetylation, phosphorylation, methylation, and ubiquitination, etc. Histone modification plays a pivotal role in the regulation of eukaryotic gene transcription mainly by changing the interactions between nuclear proteins and DNA sequences in a nucleosome. Despite that the histone methylation has been known for over 35 years, its role in gene transcription remains unclear. In the past decade, several families of enzymes that act to methylate either arginine or lysine residues in the histones have been identified. Protein arginine methyltransferases (PRMTs) are a family of enzymes that share a highly conserved domain encompassing methyltransferase activity. PRMTs are capable of methylating a broad spectrum of substrates including histones, transcription factors and other proteins functions RNA processing, nucleo-cytoplasmic transport and DNA damage repair.
Rhabdomyosarcoma, the most common pediatric soft tissue sarcoma, arises from skeletal muscle progenitor cell with high malignancy. RD is an embryonic rhabdomyosarcoma cell line that is taken as a model in this study, in which, despite the expression of MyoD and myogenin, is not efficient in terminal differentiation. TPA (12-O-tetradecanoyl-phorbol-13-acetate) is used here to induce RD cell differentiation and to investigate the functions of PRMTs therein.
1. Expression of PRMTs during cell differentiation
After treated with 100nM TPA, flowcytometry analyses show G2/M is increased and the cell cycle is arrested. Real-time RT-PCR, Western Blot experiments indicate PRMTs are expressed in RD cells and the expression of PRMTs increases during cell differentiation. The level of histone arginine methylation increases. Immunofluorescence analyses show RD cells acquire typical myogenic modality gradually after TPA inducing, such as a more elongated shape, more granule materials. The subcellular distributions of PRMTs are cytoplasm, then they enter nucleus after differentiation.
2. The effect of PRMTs on the expression of myogenin during cell differentiation
1) We construct PRMT-siRNA plasmids, PRMT5, PRMT6 expression plasmids [pCDNA6-PRMT5 (/PRMT6)-FLAG], and verify the availability of them.
2) We use 20μM as the concentration of PRMT inhibitor AdOx in RD cells.
3) Flowcytometry analyses show after AdOx treatment, the cells arrest at G2/M phase. The expression of myogenin is inhibited shows the cells fail to complete differentiation. Immunofluorescence analyses show RD cells did not acquire typical myogenic modality. The expression of PCAF mRNA and protein increases during cell differentiation and is restrained by AdOx. But the expression of Brgl, the core unit of SWI/SNF chromatin remodeling complex, does not change during differentiation or AdOx treatment. The analysis of promoter activity shows that TPA can activate the promoter activity of myogenin and AdoMet-mediated arginine methylation is indispensable for the full expression of myogenin. All the experiments of adenosine dialdehyde suggest that arginine methylation is necessary for (i) morphological differentiation, (ii) activation of essential transcription factors, and (iii) activation of myogenic relative factors.
4) Transfecting RD cells with CARM1siRNA, we find (i) the expression and nuclear translocation of myogenin and PCAF protein is inhibited. (ii) RD cells do not acquire typical myogenic modality. (iii) The promoter activity of myogenin gene is obviously restrained. Overexpressing CARM1 makes the expression and nuclear translocation of myogenin, PCAF and Brgl obviously increase. In summary, protein arginine methyltransferase activity is necessary for the activation of critical transcription factors and cofactors involved in RD cells terminal differentiation.
5) The assay of promoter activity of myogenin illuminates that Brgl can effectively increase myogenin promoter activity during RD cell differentiation and CARM1 is indispensable for this effect of Brg1. CARM1 may also cooperate with PCAF and p300 to regulate the expression of myogenin.
3. The effect of PRMTs on the binding of chromatin modifiers on the promoter of myogenin during RD cell differetiation
1) Chromatin Immunoprecipitation (ChIP) shows CARM1 can not bind the promoter of myogenin in RD cells. After induced by TPA for 3h, it begins to bind the myogenin promoter. The p38 inhibitor SB203580 can not restrain the binding of CARM1, which indicates the recruitment of CARM1 to the promoter is not p38-dependent.
2) Histone H3 is acetylated at a low degree in RD cells, but the arginine methylation of histone H4 is very little. After induced by TPA, both the acetylation of H3 and arginine methylation of H4 is increased. They can be inhibited by SB or AdOx treatment.
3) BAF60 and Brg1 can be recruited to the promoter of myogenin during RD cell differentiation, while they did not bind in normal RD cells. The binding is dependent on p38 and AdoMet-mediated methylation.
4) P300 can bind the promoter of myogenin in RD cells. TPA inducement or SB treatment can not influence the binding, but AdOx can obviously inhibit it. PCAF can be recruited to the promoter during RD cell differentiation, whereas it can not bind the promoter in normal RD cells. The binding is p38-independent and depends on AdoMet-mediated methylation.
5) MyoD can bind the promoter of myogenin in normal RD cells, and no matter TPA, SB or AdOx treatment can not influence the binding. PRMT5 can bind the myogenin promoter only when RD cells are induced by TPA. The binding is dependent on p38 and AdoMet-mediated methylation.
6) RNA polymerization enzyme polⅡcan bind the promoter of myogenin at a low degree in RD cells, and the binding can be increased after TPA inducement. Both SB and AdOx treatment can partially influence the binding.
In summary, PRMTs play an enhancing role in regulating the RD cells in TPA induced differentiation. Histone arginine methylated by PRMTs along with other histone modifications and the activation of transcription factors that jointly exert an increased expression of myogenin gene and lead the RD cells to differentiation. The regulatory functions of PRMTs in RD cell differentiation shed lights on novel target in drug development and its application to make the rhabdomyosarcoma reversal in the clinic.
引文
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