【作者】 宋朝霞；

【导师】 郝水； 曾宪录；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2004， 博士

【摘要】 肌动蛋白是一类高度保守的蛋白质，它不仅广泛存在于真核细胞的细胞质中，参与许多重要的生理活动，而且普遍存在于各类细胞的细胞核中。相关的结构和功能方面的研究已经表明，肌动蛋白在细胞核中可能参与了mRNA加工和运输、染色质集缩和染色体构建、染色质结构调整、基因转录调控、细胞的凋亡与衰老等许多重要生理功能。其中关于肌动蛋白可能参与了基因转录的报道大都是通过肌动蛋白抗体或肌动蛋白结合蛋白的作用间接证明的，肌动蛋白直接参与基因转录调控活动的报道还很少见。 近年来，肌动蛋白存在于依赖于ATP的染色质改构复合物(BAF)的发现引起了相关科学工作者的普遍关注。依赖于ATP的染色质改构复合物对核小体结构的调整是基因转录的必要前提。肌动蛋白在BAF复合物中与其核心亚基BRG1直接作用，是BRG1发挥最大ATP酶活性所必需的。鉴于肌动蛋白与BRG1的这种作用，细胞核中肌动蛋白很可能在转录调控方面具有尚不为人们所知的作用。 本文通过瞬时向细胞核中转染肌动蛋白，检测CSF-1和c-fos基因的转录情况，研究细胞核肌动蛋白是否直接参与了基因转录的调控活动。荧光素双报告系统检测及RT-PCR实验结果证实，细胞核肌动蛋白直接调控了内源和外源的CSF-1和c-fos基因的转录。肌动蛋白促进CSF-1基因转录，而抑制c-fos的基因转录，表明细胞核肌动蛋白调控基因转录具有复杂的机制。实验证明肌动蛋白对CSF-1和c-fos基因转录的调控活动需要基因的启动子序列形成染色质结构和Z-DNA结构。肌动蛋白分子的羧基端、氨基端及ATP结合区域合并羧基端的缺失突变可以明显降低对基因转录的调控作用，其中缺失ATP结合区域合并羧基端的突变体几乎丧失了转录调控作用。实验结果还发现过表达细胞核肌动蛋白能够提高细胞核裂解液的ATP酶活性，而肌动蛋白缺失突变体则没有此作用。肌动蛋白及其缺失突变体对细胞核裂解液的ATP酶活性的影响与它们对基因转录的调控作用趋势相一致。这些实验结果提示肌动蛋白参与基因转录的调控很可能与细胞核的ATP酶活性密切相关。 本论文明确了细胞核肌动蛋白在基因转录调控中的作用，为进一步深入探讨其参与基因转录调控活动的机制奠定了基础。 关键词:细胞核肌动蛋白;依赖ATP的染色质改构复合物;基因转录调控;CSF一1;c一:;ATP酶更多还原

【Abstract】 Actin is a highly conserved protein which is known to act on many crucial evens not only in the cytoplasm but also in the nuclei of eukaryotic cells. The studies on the structure and function have indicated that nuclear actin may take part in the process and transport of mRNA, the chromotin contraction and chromosome formation, the chromatin remodeling, the regulation of gene transcription and the cell senescence. Nuclear actin was tentatively implicated in transcription by earlier experiments showing the injection of anti-actin antibodies or actin-binding proteins into amphibian oocyte nuclei. But until today, there is no report for the nuclear actin directly participate the regulation of gene transcription.Recently, that actin was found to be a component of the human chromatin remodeling complex (BAF) interests the researchers. It is necessary for facilitating transcription to disrupt nucleosome structure by the ATP-dependent nucleosome remodeling complexes. Biochemical analysis indicates that actin is tightly bound to BRG1 during gene activation processes in the BAF complex, and the actin subunit is required for optimal ATPase activity of BRG1. So nuclear actin is likely to play a role in transcription, but the precise mechanisms are still to be revealedIn this study, we overexpressed actin in nuclei using a transient transfection method to investigate whether it directly takes part in the transcription of CSF-1 and c-fos. The dual-luciferase assay and RT-PCR results showed that nuclear actin augments CSF-1 while represses c-fos gene transcription, implying that nuclear actin plays an complex role on the regulation of gene transcription. And both of the regulations require the formation of chromatin and Z-DNA structure. We built three actin mutants, (the deletion of ammo- terminus, the deletion of carboxy- terminus and the deletion of carboxy- terminus with ATPase binding site), and found that themutants have lower capacity for regulating and the deletion of carboxy-terminus with ATPase binding site has nearly lost the capacity of regulation. Furthermore, the overexpressed actin can increase the nuclear ATPase activity while the mutants can not. The reduction of increasing nuclear ATPase activity is correspond to that of regulating gene transcrption, indicating that the activity of ATPase is important for actin in the regulation of gene transcription.Results presented in this paper has prove that nuclear actin takes part in regulating gene transcription and these original results will be helpful for understanding the mechanism of nuclear actin playing roles in gene transcripition.更多还原