【作者】 齐天阳；

【导师】 曾宪录；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2011， 博士

【摘要】 肌动蛋白不仅作为细胞质骨架的主要成分存在于真核细胞内,而且还作为一种细胞核蛋白,对基因的转录进行调控。近期实验报道,在基因的转录过程中,细胞核肌动蛋白分别作为染色质改构复合物,转录前起始复合物和信使核糖核蛋白的组分参与了3种真核生物RNA聚合酶的转录调控。实验证明,肌动蛋白在3种真核生物RNA聚合酶早期的转录中是通用的,而且在转录过程中肌动蛋白与转录装置是直接结合的。此外体内实验证明,在RNA聚合酶Ⅱ依赖的转录过程中,肌动蛋白与特定系列的前信使核糖核蛋白结合形成复合物,并以这个复合物作为一个分子平台招募一些组蛋白修饰酶对转录进行调控。肌动蛋白已经被证明参与了真核生物RNA聚合酶的转录,但是在这一过程中,肌动蛋白是以什么结构形式参与其中的仍然不清楚。近期有报道称,单体细胞核肌动蛋白参与了信号转导,同时另一实验证明,聚合形式的肌动蛋白和肌球蛋白的一种异形体(肌球蛋白Ⅰ)相结合,参与了RNA聚合酶Ⅰ所介导的基因转录。此外,对脱氧核糖核酸酶I(DNaseⅠ)的沉淀复合物进行亲和色谱分析显示,肌动蛋白与核糖核蛋白U (hnRNP U),组蛋白乙酰化转移酶PCAF和RNA聚合酶Ⅱ形成复合物。DNaseⅠ与单体肌动蛋白有很高的亲和性这一众所周知的事实说明了单体肌动蛋白参与这一过程。以上结果提示我们,应该对肌动蛋白究竟是以单体还是聚合形式在细胞核内行使功能进行进一步的研究。在本文中,我们发现在转录延伸复合物中肌动蛋白与正性转录延伸因子P-TEFb的催化亚基,细胞周期依赖性激酶9(Cdk9)能够相互作用。免疫荧光和免疫沉淀实验证明,Cdk9通过T-loop上保守的186位苏氨酸与单体肌动蛋白相结合。过表达和体外激酶实验证明,单体肌动蛋白能够促进P-TEFb依赖的RNA聚合酶Ⅱ的C-末端结构域的磷酸化水平。体外转录实验表明,单体肌动蛋白与Cdk9之间的相互作用能够促进RNA聚合酶Ⅱ的转录延伸。我们利用染色质免疫沉淀实验和体外固定模板实验证明了肌动蛋白能够招募Cdk9到转录模板上。利用白细胞介素6(IL-6)诱导的p21基因表达体系进一步证实,肌动蛋白能够将Cdk9招募到内源基因上。而且我们发现肌动蛋白和Cdk9能够通过与组蛋白乙酰化转移酶p300之间的作用,促进组蛋白H3的乙酰化水平,并进而促进乙酰化的组蛋白H3结合到转录模板上。综上所述,我们的实验结果表明肌动蛋白通过招募Cdk9对RNA聚合酶Ⅱ的碳末端进行磷酸化参与转录延伸这一过程,并且在延伸的过程中促进染色质的改构,使得转录延伸能够顺利的进行。更多还原

【Abstract】 Actin is not only a major cytoskeletal component in all eukaryotic cells but also a nuclear protein that plays a role in gene transcription. Recent reports have shown that in gene transcription, nuclear actin plays a key role as a component of chromatin remodeling complexes, transcription pre-initiation complex and messenger RNP (mRNP) particles associated with all three eukaryotic RNA polymerases (pol). Recent studies have suggested that actin, in direct contact with the transcription apparatus, is required in an early step of transcription that is common to all three eukaryotic RNA polymerases. In addition, there is evidence from in vivo studies that actin is involved in the transcription elongation of classⅡgenes. In this case, actin is bound to a specific subset of premessenger RNA binding proteins, and the actin–messenger RNP complex may constitute a molecular platform for recruitment of histone-modifying enzymes.Actin has now been implicated in transcription by RNA polymerases, but the structural form it adopts in these processes remains unclear. Recently, a claim was made that monomeric nuclear actin plays a role in signal transduction; while a study of RNA polymeraseⅠtranscription has implicated polymeric actin, consorting with an isoform of its classical partner myosin. Moreover, actin in complex with hnRNP U, PCAF, and polⅡcan be coprecipitated by DNaseⅠaffinity chromatography, which, notoriously, has a high affinity for monomeric actin. It now seems reasonable to start thinking about functions for both monomeric and polymeric actin in the nucleus.In this study, actin was found to interact with Cdk9, a catalytic subunit of P-TEFb, in elongation complexes (ECs). Using immunofluorescence and immunoprecipitation assays, Cdk9 was found to bind to G-actin through the conserved Thr-186 in the T-loop. Overexpression and in vitro kinase assays showed that G-actin promotes P-TEFb-dependent phosphorylation of the PolⅡC-terminal domain (CTD). An in vitro transcription experiment revealed that the interaction between G-actin and Cdk9 stimulated PolⅡtranscription elongation. ChIP and immobilized template assays indicated that actin recruited Cdk9 to a transcriptional template in vivo and in vitro. Using cytokine interleukin-6 (IL-6)-inducible p21 gene expression system, we revealed that actin recruited Cdk9 to endogenous gene. Moreover, overexpression of actin and Cdk9 increased histone H3 acetylation and acetylized histone H3 binding to a transcriptional template through the interaction with histone acetyltransferase, p300. Taken together, our results suggested that actin participates in transcription elongation by recruiting Cdk9 for phosphorylation of the PolⅡCTD, and the actin-Cdk9 interaction promotes chromatin remodeling.更多还原