Deciphering the molecular mechanisms of histone code recognition by ATAD2/B

破译 ATAD2/B 组蛋白密码识别的分子机制

• 批准号: 10592912
• 负责人: Seth E Frietze
• 金额: $ 1.01万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-19 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10592912
• 关键词: ATPase Domain; Acetylation; Androgen Receptor; Apoptosis; Binding; Biochemical; Biological; Biological Assay; Biophysics; Breast; Breast Cancer Cell; Bromodomain; Calorimetry; Cell Proliferation; Cell physiology; ChIP-seq; Chemicals; Colorectal Cancer; Coupled; Development; Disease; Endometrial; Endometrial Carcinoma; Estrogen Receptors; Genes; Genomic approach; Histone Code; Histone H4; Histones; In Vitro; Investigation; Kidney; Ligand Binding; Ligands; Lung; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Measures; Modeling; Molecular; Molecular Sieve Chromatography; Nuclear Magnetic Resonance; Ovarian Carcinoma; Patient-Focused Outcomes; Pattern; Peptides; Phenotype; Play; Primary carcinoma of the liver cells; Prognostic Marker; Prostate; Proteins; RNA Interference; Relaxation; Renal carcinoma; Research; Role; Site-Directed Mutagenesis; Stomach; Structure; Tail; Techniques; Titrations; Up-Regulation; X-Ray Crystallography; analytical ultracentrifugation; breast cancer progression; cancer cell; cancer type; cell motility; design; experimental study; functional genomics; genome wide association study; histone modification; in vivo; malignant breast neoplasm; malignant stomach neoplasm; migration; new therapeutic target; paralogous gene; protein function; structural biology; targeted biomarker; transcriptome sequencing; tumorigenesis

ATAD2 is an important co-activator of the estrogen and androgen receptors. ATAD2 is known to be up- regulated in multiple different types of cancer including breast, lung, gastric, endometrial, renal, and prostate. Up-regulation of ATAD2 is often correlated with poor patient outcomes, and can be used as prognostic marker. Furthermore, silencing the expression of ATAD2 through RNA interference inhibits cell proliferation and promotes apoptosis in ovarian carcinoma, and inhibits migration and invasion of hepatocellular carcinoma and colorectal cancer cells. ATAD2B, is a poorly studied paralog of the ATAD2 gene, and although ATAD2 and ATAD2B are highly conserved, there is little known about the function of ATAD2B or its role in oncogenesis. Both the ATAD2/B proteins contain two conserved domains: an AAA ATPase domain and a bromodomain. The overall objective of the proposed research is to determine how di-acetyllysine recognition by the ATAD2/B bromodomains regulates the cellular function of these proteins. This proposal aims to: (1) characterize how cross-talk between histone modifications modulate acetyllysine recognition by the ATAD2/B bromodomains; (2) outline the molecular mechanism(s) of di-acetylated histone recognition by the ATAD2/B bromodomains; (3) determine the functional significance of di-acetyllysine recognition by the ATAD2/B bromodomains. A unique combination of in vitro biochemical, biophysical, and structural biology studies on the ATAD2/B bromodomains will be coupled with in vivo functional genomic investigations using a breast cancer progression model to characterize the biological roles of the ATAD2/B bromodomains. We will evaluate the impact of neighboring histone modifications on histone H4 tail recognition using peptide array assays in combination with isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) chemical shift perturbation techniques. We will determine the structural features of ATAD2/B bromodomains required for recognition of di-acetylated histone tail ligands using NMR and/or X-ray crystallography. To characterize the binding mode of the ATAD2/B bromodomains with their histone ligands we will carry out analytical ultracentrifugation, size-exclusion chromatography, ITC, and NMR T1/T2 relaxation experiments. Site-directed mutagenesis coupled with NMR and ITC will be used to measure the effects on ligand binding, and identify differences in the binding pockets of the ATAD2/B bromodomains. We will compare the genome-wide associations of ATAD2/B with histone H4 acetylation patterns in a breast cancer progression model to determine the impact of ATAD2/B on breast cancer cell phenotypes using ChIP-seq and RNA-seq, followed by cellular migration and invasion assays. Our multi-faceted approach will correlate specific histone modifications with ATAD2/B binding and action, which will allow us to connect histone H4 acetylation marks to bromodomain function in cancer cell proliferation. Overall, our integrated biochemical, biophysical, structural biology and functional genomics approach will reveal the biological roles of ATAD2/B and facilitate the discovery of novel drug targets to help overcome cancer.

ATAD 2是雌激素和雄激素受体的重要共激活剂。ATAD 2已知是向上的- 在包括乳腺癌、肺癌、胃癌、子宫内膜癌、肾癌和前列腺癌在内的多种不同类型的癌症中调节。 ATAD 2的上调通常与不良患者结局相关，并且可用作预后标志物。 此外，通过RNA干扰沉默ATAD 2的表达抑制了细胞增殖， 促进卵巢癌细胞凋亡，抑制肝细胞癌的迁移和侵袭， 大肠癌细胞ATAD 2B是ATAD 2基因的一个研究较少的基因，尽管ATAD 2和 ATAD 2B基因高度保守，其功能及其在肿瘤发生中的作用尚不清楚。 两种ATAD 2/B蛋白都含有两个保守结构域：AAA ATP酶结构域和布罗莫结构域。的 本研究的总体目标是确定ATAD 2/B如何识别二乙酰赖氨酸 布罗莫结构域调节这些蛋白质的细胞功能。本建议旨在：（1）描述如何 组蛋白修饰之间的串扰调节ATAD 2/B溴结构域对乙酰赖氨酸的识别;（2） 概述ATAD 2/B溴结构域识别二乙酰化组蛋白的分子机制;（3） 确定ATAD 2/B溴结构域识别二乙酰赖氨酸的功能意义。一个独特 对ATAD 2/B布罗莫结构域的体外生物化学、生物物理学和结构生物学研究的组合 将结合使用乳腺癌进展模型的体内功能基因组研究， 表征ATAD 2/B溴结构域的生物学作用。我们将评估邻国的影响， 肽阵列结合等温分析法研究组蛋白修饰对组蛋白H4尾识别的影响 滴定量热法（ITC）和核磁共振（NMR）化学位移微扰技术。我们 将确定ATAD 2/B布罗莫结构域的结构特征，所述布罗莫结构域是识别二乙酰化的 使用NMR和/或X射线晶体学对组蛋白尾配体进行分析。表征ATAD 2/B的结合模式 溴结构域与其组蛋白配体，我们将进行分析超离心，尺寸排阻 色谱法、ITC和NMR T1/T2弛豫实验。定点突变与NMR偶联 和ITC将被用来测量对配体结合的影响，并确定在结合口袋的差异， ATAD 2/B布罗莫结构域。我们将比较ATAD 2/B与组蛋白H4的全基因组关联 乳腺癌进展模型中的乙酰化模式，以确定ATAD 2/B对乳腺癌的影响。 使用ChIP-seq和RNA-seq分析癌细胞表型，然后进行细胞迁移和侵袭测定。我们 多方面的方法将使特定的组蛋白修饰与ATAD 2/B结合和作用相关，这将 使我们能够将组蛋白H4乙酰化标记与癌细胞增殖中的布罗莫结构域功能联系起来。总的来说， 我们的综合生物化学，生物物理，结构生物学和功能基因组学方法将揭示 ATAD 2/B的生物学作用，并促进发现新的药物靶点，以帮助克服癌症。