Mechanism and effects of communication between actin and gene regulatory complexes

摘要 尽管许多研究已经证明了细胞骨架动力学、基因组组织、 和基因表达，将它们联系在一起的潜在机制尚不清楚。最近，我们发现 Wiskott-Aldrich综合征蛋白家族异丙氨酸同源(WAVE)调节复合体之间的串扰 (WRC)，促进肌动蛋白聚合；SPT-AdA-Gcn5-乙酰基转移酶(SAGA)复合体，a 转录辅活化子。它们的关系是通过共享组成 佐贺脱泛素酶模块，包括脱泛素酶不间断。脱泛素酶模块突变--针对 例如，多聚谷氨酰胺在ataxin 7(ATXN7)中的扩展--导致一系列表型不能用来解释 佐贺的转录辅助激活功能，包括神经系统退化和失明。在这两个 在神经系统和眼睛中，WRC是肌动蛋白聚合的重要推动者，这是通过以下方式促进的 构成活性的酶亚基波动。波的活动性和局部化由 其余的WRC亚基，确保肌动蛋白聚合的空间和时间控制。肌动蛋白的错误调控 聚合复合体导致与SAGA突变体相似的表型谱，包括 神经系统退化和失明。这表明SAGA在神经系统和 眼睛，因为它需要控制这些组织中的WRC。我们发现佐贺脱泛素酶模块 离开传奇来捆绑WRC。在那里，不间断的去泛素化浪潮，增加了它在两个 细胞质和细胞核。因此，我们假设SAGA控制着WRC复合体的组成， 数量和位置；正是通过这些活动，佐贺完成了我们之前拥有的功能 单单归因于传奇。这一假设将从三个方面进行研究。首先，我们将确定和 亲和纯化和柱层析表征含核波的配合物 与质谱学相结合。由于波的活动在一定程度上受其局部化的调节，因此波的位置 这些复合体将被测定，以及Atxn7聚谷氨酰胺对复合体的影响 将测试其组成和本地化。第二，佐贺脱泛素酶模块之间的相互作用 WRC将在细胞和苍蝇中被破坏，以确定哪些SAGA/WRC功能需要它们。最后， SAGA脱泛素酶-WRC相互作用对失明和神经变性的影响将在#年进行研究 通过破坏它们并测量Atxn7聚谷氨酰胺扩展的表型特征。这些 除了这些联系外，研究还将为神经退行性变和失明的原因提供新的见解 转录调控复合体和细胞骨架调控复合体之间的关系。

Abstract Although many studies have demonstrated correlations between cytoskeletal dynamics, genome organization, and gene expression, the underlying mechanisms linking them remain unclear. Recently, we discovered crosstalk between the Wiskott-Aldrich syndrome protein family verprolin homolog (WAVE) regulatory complex (WRC), which promotes actin polymerization, and the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, a transcriptional coactivator. Their relationship is established through the sharing of subunits comprising the SAGA deubiquitinase module, including the deubiquitinase Non-stop. Deubiquitinase module mutations—for example, polyglutamine expansion in ataxin 7 (ATXN7)—lead to a spectrum of phenotypes not explained by SAGA’s transcriptional coactivator function, including nervous system degeneration and blindness. In both the nervous system and the eye, the WRC is an essential promoter of actin polymerization, which is facilitated by the constitutively active enzymatic subunit WAVE. WAVE activity and localization are regulated by the remaining WRC subunits, ensuring spatial and temporal control of actin polymerization. Misregulation of actin polymerizing complexes results in a similar spectrum of phenotypes as seen in SAGA mutants, including nervous system degeneration and blindness. This suggests that SAGA is important in the nervous system and eye because it is required to control the WRC in these tissues. We found that the SAGA deubiquitinase module leaves SAGA to bind the WRC. There, Non-stop deubiquitinates WAVE, increasing its level in both the cytoplasm and the nucleus. Therefore, we hypothesize that SAGA controls WRC complex composition, amount, and location; and it is through these activities that SAGA accomplishes functions we had previously attributed to SAGA alone. This hypotheses will be investigated in three aims. First, we will identify and characterize nuclear WAVE-containing complexes through affinity purification and column chromatography coupled to mass spectrometry. Because WAVE activity is regulated in part by its localization, the locations of these complexes will be determined, and the effects of Atxn7 polyglutamine expansion on complex composition and localization will be tested. Second, interactions between the SAGA deubiquitinase module and WRC will be disrupted in cells and flies to determine which SAGA/WRC functions require them. Lastly, the effects of SAGA deubiquitinase-WRC interactions on blindness and neurodegeneration will be investigated in flies, by disrupting them and measuring phenotypes characteristic of Atxn7 polyglutamine expansion. These studies will provide novel insight on the causes of neurodegeneration and blindness, in addition to the links between transcriptional and cytoskeletal regulatory complexes.