Structural studies of function and regulation of microtubules and transcriptional gene expression machinery

微管和转录基因表达机制的功能和调节的结构研究

• 批准号: 10623788
• 负责人: Eva Nogales
• 金额: $ 47.08万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623788
• 关键词: Acetylation; Area; Binding; Biochemical; Biological Assay; Biophysics; Cell Nucleus; Cells; Chromatin; Complement; Complex; Coupling; Cryoelectron Microscopy; DNA; Dedications; Development; Dinucleosome; Epigenetic Process; Eukaryotic Cell; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Growth; Guanosine Triphosphate; HTATIP gene; Health; Histone H3; Human; Hydrolysis; Knowledge; Lysine; Methylation; Microtubules; Molecular; Molecular Conformation; Nucleosomes; PRC1 Protein; Phase; Polycomb; Polymers; Preparation; Process; Property; RNA; RNA Polymerase II; Regulation; Role; SAGA; Structure; System; TAF1 gene; Techniques; Transcription Coactivator; Transcription Factor TFIIA; Transcription Factor TFIIB; Transcriptional Regulation; Tubulin; Visualization; Work; Yeasts; analog; chromatin remodeling; cofactor; dimer; histone modification; mutant; promoter; tool; transcription factor TFIIE; transcription factor TFIIF; transcription factor TFIIH

Project Abstract Our lab is dedicated to the mechanistic understanding of macromolecular function through the visualization of structure, dynamics, and regulatory interactions. Towards that goal, we use cryo-EM, together with biochemical and biophysical assays. Our areas of study are centered on the characterization of the regulatory molecular mechanisms governing the function of microtubules and of human transcription/epigenetic complexes. Microtubules (MTs) are essential polymers in eukaryotic cells s built of -tubulin dimers. Dynamic instability, the switching between growing and shrinking phases due to the coupling of the assembly process to the exchange and hydrolysis of GTP in -tubulin, is an essential property for MT function. Many MT cellular partners modulate MT dynamics or utilize it to carry out specific functions. In the past. In the last 5 years, we confirmed and complemented our past studies, which used non-hydrolyzable GTP analogs to characterize conformational changes in MTs that accompany GTP hydrolysis, now using of GTP-hydrolysis tubulin mutants. We also defined the mode of binding and action of cellular factors that regulate MT assembly, dynamics and organization in the cell and described the effect of tubulin acetylation on MT structure and function. We will continue this work with the central theme of adding complexity to our studies in order to bring us closer to the regulated function of microtubule cellular systems as we also add techniques complementary to our major tool, cryo-EM. Transcriptional regulation of gene expression is critical for growth and survival, and of obvious significance to human health. Its initiation involves RNA polymerase II (Pol II) together with TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Regulation is achieved by sequence-specific activators or repressors, co-factors, and chromatin remodeling/modifying complexes. During the last funding period we defined the structures of human TFIID[17] and TFIIH[18], as well as other large transcriptional coactivators like the human SAGA[19], and the yeast NuA4 (in preparation) and RSC complexes[20]. We will now characterize the human TIP60 complex, the binding of transcriptional co-activators to chromatin substrates, and pursue the dynamic visualization of TFIID engagement with promoter DNA to gain further knowledge of how these complexes work in the nucleus to regulate gene expression. Polycomb repressive complex 2 (PRC2) is an epigenetic gene silencer that methylates lysine 27 of histone H3 and is essential for cellular differentiation and development. After obtaining the structure of human PRC2 with its cofactors JARID2 and AEBP2 and its interaction with a di-nucleosome, we have now defined how PRC2 recognizes mono-ubiquitylated nucleosome, the substrate created by the other major Polycomb complex, PRC1. We will now further characterize the regulatory landscape of PRC2 looking into other histone modifications, different forms of PRC2 (i.e., other cofactors), the role of PRC2 auto-methylation, and binding of PRC2 to RNA.

项目摘要 我们的实验室致力于通过可视化来理解大分子的功能 结构、动态和监管互动。为了实现这一目标，我们使用低温EM，并结合生化 和生物物理化验。我们的研究领域集中在调控分子的特征上。 控制微管和人类转录/表观遗传复合体功能的机制。 微管是由-微管蛋白二聚体构建的真核细胞所必需的聚合物。动态不稳定性， 由于组装过程与交易所的耦合，在增长阶段和收缩阶段之间的切换 而-微管蛋白中GTP的水解性是MT功能的一个基本性质。许多MT蜂窝合作伙伴调制 MT动态或利用它来执行特定的功能。在过去。在过去的5年里，我们确认了 补充了我们过去的研究，这些研究使用非水解性GTP类似物来表征构象 伴随GTP水解的MTS的变化，现在使用GTP水解微管蛋白突变体。我们还定义了 调节MT组装、动力学和组织的细胞因子的结合和作用方式 并描述了微管蛋白乙酰化对MT结构和功能的影响。我们将继续这项工作， 增加我们研究的复杂性的中心主题是为了让我们更接近 微管细胞系统，因为我们还添加技术补充我们的主要工具，冷冻-EM。 基因表达的转录调控对生长和生存至关重要，对 人类健康。它与TFIIA、TFIIB、TFIID、TFIIE、TFIIF和 太好了。调节是通过序列特异性的激活或抑制因子、辅助因子和染色质来实现的 改造/改造综合体。在上一次资助期间，我们定义了人类TFIID的结构[17]和 TFIIH[18]，以及其他大型转录辅助激活因子，如人类SAGA[19]和酵母NuA4(in 制备)和RSC络合物[20]。我们现在将表征人类Tip60复合体，结合 转录共激活因子到染色质底物，并追求TFIID参与的动态可视化 利用启动子DNA进一步了解这些复合体如何在细胞核内调节基因 表情。 多梳抑制复合体2(PRC2)是组蛋白H3赖氨酸27甲基化的表观遗传基因沉默因子 对细胞分化和发育是必不可少的。在获得人类PrC2的结构后，通过其 辅因子JARID2和AEBP2及其与双核小体的相互作用，我们现在已经定义了PRC2 识别单一泛素化的核小体，这是由另一个主要的多梳复合体PRC1产生的底物。 我们现在将进一步描述PRC2的调控格局，寻找其他组蛋白修饰， 不同形式的PRC2(即其他辅因子)，PRC2自身甲基化的作用，以及PRC2与RNA的结合。

项目成果

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules.

• DOI: 10.1073/pnas.2114994119
• 发表时间: 2022-01-11
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: LaFrance BJ;Roostalu J;Henkin G;Greber BJ;Zhang R;Normanno D;McCollum CO;Surrey T;Nogales E
• 通讯作者: Nogales E

JARID2 and AEBP2 regulate PRC2 in the presence of H2AK119ub1 and other histone modifications.

• DOI: 10.1126/science.abc3393
• 发表时间: 2021-01-22
• 期刊: Science (New York, N.Y.)
• 作者: Kasinath V;Beck C;Sauer P;Poepsel S;Kosmatka J;Faini M;Toso D;Aebersold R;Nogales E
• 通讯作者: Nogales E

2.5 Å-resolution structure of human CDK-activating kinase bound to the clinical inhibitor ICEC0942.

• DOI: 10.1016/j.bpj.2020.12.030
• 发表时间: 2021-02-16
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Greber BJ;Remis J;Ali S;Nogales E
• 通讯作者: Nogales E