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

基本信息

批准号：
10005379
负责人：
Seth E Frietze
金额：
$ 48.15万
依托单位：
ALBANY COLLEGE OF PHARMACY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-19 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10005379
关键词：
ATP phosphohydrolase ATPase Domain Acetylation Affinity Amino Acid Sequence Androgen Receptor Apoptosis Automobile Driving Binding Biochemical Biological Biological Assay Biological Process Biophysics Breast Breast Cancer Cell Bromodomain Calorimetry Cell Proliferation Cell physiology Cells ChIP-seq Chemicals Chromatin Code Colorectal Cancer Complex Coupled DNA Data Development Disease Endometrial Endometrial Carcinoma Epigenetic Process Estrogen Receptors Family Fetal Development Gene Expression Gene Expression Profile Genes Genetic Code Genomic approach Goals Histone Acetylation Histone Code Histone H4 Histones In Vitro Investigation Journals Kidney Knowledge Ligand Binding Ligands Light Link Lung Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of prostate Measures Modeling Modification Molecular Molecular Sieve Chromatography Neoplasm Metastasis Nuclear Nuclear Magnetic Resonance Ovarian Carcinoma Patient-Focused Outcomes Pattern Peptides Phenotype Play Positioning Attribute Post-Translational Protein Processing Primary carcinoma of the liver cells Prognostic Marker Prostate Proteins RNA RNA Interference Reader Relaxation Renal carcinoma Research Role SECTM1 gene Signal Transduction Site-Directed Mutagenesis Stomach Structure Tail Techniques Testing Time Titrations Up-Regulation X-Ray Crystallography analytical ultracentrifugation base biophysical techniques breast cancer progression cancer cell cancer risk cancer therapy cancer type cell motility design experimental study functional genomics genome wide association study histone modification in vivo insight malignant breast neoplasm malignant stomach neoplasm migration new therapeutic target novel overexpression paralogous gene protein function recruit stem cells structural biology targeted biomarker transcriptome sequencing tumor progression tumorigenesis

项目摘要

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。 ATAD2 已知是 up- 在多种不同类型的癌症中受到调节，包括乳腺癌、肺癌、胃癌、子宫内膜癌、肾癌和前列腺癌。 ATAD2 的上调通常与患者预后不良相关，可作为预后标志物。此外，通过RNA干扰沉默ATAD2的表达可抑制细胞增殖和促进卵巢癌细胞凋亡，抑制肝细胞癌的迁移和侵袭，结直肠癌细胞。 ATAD2B 是 ATAD2 基因的一个旁系同源物，研究很少，尽管 ATAD2 和 ATAD2B 高度保守，人们对 ATAD2B 的功能或其在肿瘤发生中的作用知之甚少。 ATAD2/B 蛋白均包含两个保守结构域：AAA ATP 酶结构域和溴结构域。这拟议研究的总体目标是确定 ATAD2/B 如何识别二乙酰赖氨酸溴结构域调节这些蛋白质的细胞功能。该提案旨在：(1) 描述如何组蛋白修饰之间的串扰调节 ATAD2/B 溴结构域对乙酰赖氨酸的识别； (2) 概述 ATAD2/B 溴结构域识别二乙酰化组蛋白的分子机制； (3) 确定 ATAD2/B 溴结构域识别二乙酰赖氨酸的功能意义。一个独特的 ATAD2/B 溴结构域的体外生化、生物物理和结构生物学研究相结合将使用乳腺癌进展模型与体内功能基因组研究相结合描述 ATAD2/B 溴结构域的生物学作用。我们将评估邻近地区的影响使用肽阵列测定结合等温对组蛋白 H4 尾部识别进行组蛋白修饰滴定量热法 (ITC) 和核磁共振 (NMR) 化学位移微扰技术。我们将确定识别二乙酰化所需的 ATAD2/B 溴结构域的结构特征使用 NMR 和/或 X 射线晶体学分析组蛋白尾配体。表征 ATAD2/B 的绑定模式溴结构域及其组蛋白配体，我们将进行分析超速离心、尺寸排除色谱、ITC 和 NMR T1/T2 弛豫实验。与 NMR 结合的定点诱变 ITC 将用于测量对配体结合的影响，并识别配体结合口袋的差异 ATAD2/B 溴结构域。我们将比较 ATAD2/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 ATAD2/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。我们的多方面的方法将特定组蛋白修饰与 ATAD2/B 结合和作用关联起来，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溴结构域功能联系起来。全面的，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 ATAD2/B 的生物学作用并促进新药物靶点的发现，以帮助克服癌症。