CARM1在PC12细胞内与CLOCK的结合及功能性研究
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摘要
CARM1(Coactivator-Associated arginine(R)Methyltransferase 1,辅激活因子相关的精氨酸甲基转移酶1)是生物体内一种分布广泛,功能多样的辅调节因子,它具有蛋白精氨酸甲基转移酶活性,在类固醇激素刺激下,能够被SRC-3(Steroid ReceptorCoactivator-3,类固醇受体辅激活因子-3)募集,通过甲基化染色质内组蛋白上特异位点的精氨酸残基而参与调控靶基因的转录过程。而与SRC-3在结构上十分相似的CLOCK(Circadian Locomotor Output Cycles Kaput,昼夜节律运动输出周期故障)是细胞内进行生物节律调节的核心调控因子,它与BMAL1形成异二聚体结合于下游靶基因的E-box元件上,利用其C末端所带有的乙酰转移酶活性区域修饰启动子上的组蛋白赖氨酸残基,激活下游节律基因的表达,在此过程中是否需要其它辅调节因子的参与,目前的研究还较少。本研究旨在发现细胞内CARM1与CLOCK的相互作用,以及CARM1对CLOCK潜在的调节能力,进而寻找其参与生物节律调控的证据,为细胞内类固醇激素信号转导途径和生物节律调控体系间的交叉提供一定的理论支持。实验的具体内容及结果如下:
利用免疫共沉淀实验,在PC12细胞内,以CAMR1抗体进行免疫沉淀,CLOCK抗体进行免疫印迹,检测两者在内源条件下的结合情况,结果发现CARM1可以与CLOCK发生相互作用,且这一结合作用受血清刺激的影响,在存在血清或不存在血清的情况下,两者的结合有明显差异;另一方面在COS-7细胞内,外源转染HA-CARM1和Flag-CLOCK的真核表达质粒,模拟PC12细胞内实验,以HA抗体进行免疫沉淀,Flag抗体进行免疫印迹,检测两者的结合,结果也发现在外源转染条件下CARM1与CLOCK的结合。
为了检测CARM1和CLOCK在PC12细胞内的分布情况,利用CARM1和CLOCK的抗体进行免疫荧光实验,实验结果显示,正常培养的细胞内,CARM1及CLOCK在细胞核和细胞质内都有分布,且核内相对较多;而当用血清刺激之后,CARM1的分布没有变化,但CLOCK明显向核内聚集。
对于CARMl结合CLOCK的功能性研究,从转录活性和周期性两个方面着手。首先进行的是双荧光素酶报告基因实验检测CARM1对于CLOCK转录活性影响,结果显示正常的Ga14DBD-CLOCK能大大提高报告基因系统的表达,而当向其中同时转染如CARM1质粒后,报告基因系统的表达明显下降,说明CARM1能够抑制CLOCK的转录活性;随后进行的CARM1与CLOCK周期性结合情况分析,发现在血清刺激后,CARM1和CLOCK的结合随时间先增高后降低,在大约20小时处为峰值,存在一定周期性。
综上,本研究首次发现了CARM1与CLOCK这两个分属于类固醇受体信号转导途径与生物节律调节途径的蛋白因子间的相互作用,将细胞内相对独立的两种调控体系联系起来,并进一步证实两者的结合在血清刺激后会表现出一定的周期性,且CARM1会抑制CLOCK的转录活性,暗示其可能是一种潜在的生物节律调控因子。
CARM1 (Coactivator- Associated arginine(R) Methyltransferase 1) is a ubiquitous co-regulator with various functions like HMT activity. It can be recruited by SRC-3 (Steroid Receptor Coactivator-3) to the target gene to methylate special Arg of histone residue and then regulate gene transcription. While similar to SRC-3 in structure, CLOCK (Orcadian Locomotor Output Cycles Kaput) is a key regulator in biological rhythm, where it forms heterodimmer with BMAL1 to combine onto the E-box of target gene and activate transcription by acetylating lysines of histone through its C terminal HAT activity. However, it is still unknown whether there are still other co-regulators participating in this process. So our research is to find the interaction between CARM1 and CLOCK in vivo, and the potential regulation of CLOCK by CARM1 to prove the participant status of CARM1 in the biological rhythm regulation. These findings will definitely support the crossover theory between steroid signal transduction and biological rhythm regulation. And the methods and results are as follows:
The endogenous CARM1 complex is immunoprecipitated by antibody anti-CARM1 from lysates of PC12 cell in Immunoprecipitation, and proteins were blotted with antibody anti-CLOCK. We found that CARM1 interacts with CLOCK in vivo. Moreover, this interaction is dependent on serum and different in serum hungry and serum stimulation. Then plasmids expressing HA-CARM1 and Flag-CLOCK were transfected into COS-7 cell to detect the interaction of these two proteins in vitro with the same method, and identical result was found.
In the following step, we detect the distribution of CARM1 and CLOCK in PC12 cellwith Immunofiuorescence assay, in which the results showed that CARM1 and CLOCK colocates well in the whole cell with preparences in nuclear. When treated with serum, the distribution of CARM1 was unchanged but CLOCK translocated from cytoplasm to nucleas.
To investigate the functional effects of the interaction between CARM1 and CLOCK, Luciferase Assay was conducted, in which we found the luciferase activity was highly improved when Gal4DBD-CLOCK was overexpressed, but co-transfection of CARM1 plasmid simultaneously reduced the activity obviously. These results indicated that CARM1 can inhibit the transcriptional activity of CLOCK. Through the following cycle analysis of the interaction between CARM1 and CLOCK while treated with serum, we found the interaction strenthened first and then weakened, and the maximum combination happened at about 20hrs.
In conclusion, we found the interaction between CARM1 and CLOCK, which belongs to steroid receptor pathway and biological circadian pathway respectively. Our finding made a connection between these two independent regulation systems and proved that the interaction shows some sort of periodicity under the inducement of serum. Moreover, CARM1 was found to inhibit the transcriptional activity of CLOCK, which indicates CARM1 may be a potential circadian regulator.
利用免疫共沉淀实验,在PC12细胞内,以CAMR1抗体进行免疫沉淀,CLOCK抗体进行免疫印迹,检测两者在内源条件下的结合情况,结果发现CARM1可以与CLOCK发生相互作用,且这一结合作用受血清刺激的影响,在存在血清或不存在血清的情况下,两者的结合有明显差异;另一方面在COS-7细胞内,外源转染HA-CARM1和Flag-CLOCK的真核表达质粒,模拟PC12细胞内实验,以HA抗体进行免疫沉淀,Flag抗体进行免疫印迹,检测两者的结合,结果也发现在外源转染条件下CARM1与CLOCK的结合。
为了检测CARM1和CLOCK在PC12细胞内的分布情况,利用CARM1和CLOCK的抗体进行免疫荧光实验,实验结果显示,正常培养的细胞内,CARM1及CLOCK在细胞核和细胞质内都有分布,且核内相对较多;而当用血清刺激之后,CARM1的分布没有变化,但CLOCK明显向核内聚集。
对于CARMl结合CLOCK的功能性研究,从转录活性和周期性两个方面着手。首先进行的是双荧光素酶报告基因实验检测CARM1对于CLOCK转录活性影响,结果显示正常的Ga14DBD-CLOCK能大大提高报告基因系统的表达,而当向其中同时转染如CARM1质粒后,报告基因系统的表达明显下降,说明CARM1能够抑制CLOCK的转录活性;随后进行的CARM1与CLOCK周期性结合情况分析,发现在血清刺激后,CARM1和CLOCK的结合随时间先增高后降低,在大约20小时处为峰值,存在一定周期性。
综上,本研究首次发现了CARM1与CLOCK这两个分属于类固醇受体信号转导途径与生物节律调节途径的蛋白因子间的相互作用,将细胞内相对独立的两种调控体系联系起来,并进一步证实两者的结合在血清刺激后会表现出一定的周期性,且CARM1会抑制CLOCK的转录活性,暗示其可能是一种潜在的生物节律调控因子。
CARM1 (Coactivator- Associated arginine(R) Methyltransferase 1) is a ubiquitous co-regulator with various functions like HMT activity. It can be recruited by SRC-3 (Steroid Receptor Coactivator-3) to the target gene to methylate special Arg of histone residue and then regulate gene transcription. While similar to SRC-3 in structure, CLOCK (Orcadian Locomotor Output Cycles Kaput) is a key regulator in biological rhythm, where it forms heterodimmer with BMAL1 to combine onto the E-box of target gene and activate transcription by acetylating lysines of histone through its C terminal HAT activity. However, it is still unknown whether there are still other co-regulators participating in this process. So our research is to find the interaction between CARM1 and CLOCK in vivo, and the potential regulation of CLOCK by CARM1 to prove the participant status of CARM1 in the biological rhythm regulation. These findings will definitely support the crossover theory between steroid signal transduction and biological rhythm regulation. And the methods and results are as follows:
The endogenous CARM1 complex is immunoprecipitated by antibody anti-CARM1 from lysates of PC12 cell in Immunoprecipitation, and proteins were blotted with antibody anti-CLOCK. We found that CARM1 interacts with CLOCK in vivo. Moreover, this interaction is dependent on serum and different in serum hungry and serum stimulation. Then plasmids expressing HA-CARM1 and Flag-CLOCK were transfected into COS-7 cell to detect the interaction of these two proteins in vitro with the same method, and identical result was found.
In the following step, we detect the distribution of CARM1 and CLOCK in PC12 cellwith Immunofiuorescence assay, in which the results showed that CARM1 and CLOCK colocates well in the whole cell with preparences in nuclear. When treated with serum, the distribution of CARM1 was unchanged but CLOCK translocated from cytoplasm to nucleas.
To investigate the functional effects of the interaction between CARM1 and CLOCK, Luciferase Assay was conducted, in which we found the luciferase activity was highly improved when Gal4DBD-CLOCK was overexpressed, but co-transfection of CARM1 plasmid simultaneously reduced the activity obviously. These results indicated that CARM1 can inhibit the transcriptional activity of CLOCK. Through the following cycle analysis of the interaction between CARM1 and CLOCK while treated with serum, we found the interaction strenthened first and then weakened, and the maximum combination happened at about 20hrs.
In conclusion, we found the interaction between CARM1 and CLOCK, which belongs to steroid receptor pathway and biological circadian pathway respectively. Our finding made a connection between these two independent regulation systems and proved that the interaction shows some sort of periodicity under the inducement of serum. Moreover, CARM1 was found to inhibit the transcriptional activity of CLOCK, which indicates CARM1 may be a potential circadian regulator.
引文
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[2] KING D,ZHAO Y L.SANGORAM A, et al. Positional Cloning of the Mouse Circadian Clock Gene [J].Cell, 1997,89:641-653.
[3] HIRAYAMA J,SAAAONE-CORSI P. Structural and functional features of transcription factors controlling the circadian clock [J].Current Opinion in Genetics & Development, 2005,15:548-556.
[4] MASSARI M E,MURRE C. Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms [J]. Molecular and Cellular Biology, 2000, 20:429-440.
[5] CHAUDHARY J, SKINNER M K. Basic helix-loop-helix proteins can act at the E~box within the serum response element of the c-fos promoter to influence hormone-induced promoter activation in Sertoli cells [J].Molecular Endocrinology, 1999,13:774-786.
[6] HEPTI M H.FRANCOIJS K J, DE VRIES S C, et al.The PAS fold. A redefinition of the PAS domain based upon structural prediction [J]. European Journal of Biochemistry, 2004,271:1198-1208.
[7] MOGLICH A.AYERS R A,MOFFAT K. Structure and Signaling Mechanism of Per-ARNT-Sim Domains [J]. Structure, 2009,17:1282-1294.
[8] NAKAHATA Y.GRIMALDI B.SAHAR S, et al.Signaling to the circadian clock: plasticity by chromatin remodeling [J]. Current Opinion in Cell Biology, 2007,19:230-237.
[9] CHEN C, AGNES F.GELINAS C. Mapping of a serine-rich domain essential for the transcriptional, antiapoptotic, and transforming activities of the v-Rel oncoprotein [J].Molecular and Cellular Biology, 1999,19:307-316.
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