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.

ATAD2是雌激素和雄激素受体的重要共激活剂。众所周知ATAD2是 在多种不同类型的癌症中调节，包括乳腺癌，肺，胃，子宫内膜，肾脏和前列腺。 ATAD2的上调通常与差的患者结局相关，并且可以用作预后标记。 此外，通过RNA干扰沉默ATAD2的表达会抑制细胞的增殖和 促进卵巢癌的凋亡，并抑制肝细胞癌的迁移和侵袭 结直肠癌细胞。 ATAD2B，是ATAD2基因的旁系同源物，尽管ATAD2和 ATAD2B是高度保守的，对ATAD2B的功能或其在肿瘤发生中的作用鲜为人知。 ATAD2/B蛋白都包含两个保守域：AAA ATPase域和溴结构域。这 拟议的研究的总体目标是确定ATAD2/B的二乙酰透明苷识别 溴结构域调节这些蛋白质的细胞功能。该建议的目的是：（1）表征 组蛋白修饰之间的横向词通过ATAD2/B溴化群调节乙酰透析剂的识别； （2） 概述了ATAD2/B溴结构域的二乙酰化组蛋白识别的分子机制； （3） 确定ATAD2/B溴结构域识别二乙酰透明苷的功能意义。一个独特的 关于ATAD2/B溴结构域的体外生化，生物物理和结构生物学研究的结合 将使用乳腺癌进展模型与体内功能基因组研究结合 表征ATAD2/B溴结构域的生物学作用。我们将评估邻近的影响 组蛋白对组蛋白H4尾识别的修饰，使用肽阵列分析与等温组合 滴定量热法（ITC）和核磁共振（NMR）化学位移扰动技术。我们 将确定识别二乙酰化所需的ATAD2/B溴结构域的结构特征 组蛋白尾配体使用NMR和/或X射线晶体学。表征ATAD2/B的结合模式 溴化域及其组蛋白配体我们将进行分析性超速离心，尺寸排斥 色谱，ITC和NMR T1/T2松弛实验。定点诱变与NMR ITC将用于测量对配体结合的影响，并确定在结合口袋的差异 ATAD2/B溴结构域。我们将比较ATAD2/B的全基因组关联与组蛋白H4 乳腺癌进展模型中的乙酰化模式，以确定ATAD2/B对乳房的影响 使用CHIP-SEQ和RNA-SEQ进行癌细胞表型，然后进行细胞迁移和侵袭测定。我们的 多面方法将将特定的组蛋白修饰与ATAD2/B结合和动作相关联，这将 允许我们将组蛋白H4乙酰化标记连接到癌细胞增殖中的溴结构域功能。全面的， 我们综合的生化，生物物理，结构生物学和功能基因组学方法将揭示 ATAD2/B的生物学作用，并促进了新的药物靶标有助于克服癌症。