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: 10410677
• 负责人: Seth E Frietze
• 金额: $ 9.08万
• 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/10410677
• 关键词: ATPase Domain; Acetylation; Androgens; 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; Estrogens; 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; Proteins; RNA Interference; Relaxation; Renal carcinoma; Research; Resolution; 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; prognostic; protein function; structural biology; targeted biomarker; transcriptome sequencing; tumorigenesis

ATAD2 is an important co-activator of the estrogen and androgen receptors2,4. ATAD2 is known to be up- regulated in multiple different types of cancer including breast, lung, gastric, endometrial, renal, and prostate5,7,9-12. Up-regulation of ATAD2 is often correlated with poor patient outcomes, and can be used as prognostic marker9,12-14. 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 cells13,15,16. 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 bromodomain6. 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. The atomic resolution structures of the ATAD2/B bromodomains bound to their di-acetylated histone tail ligands will be solved by NMR 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 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2,4。众所周知ATAD2是 受到多种不同类型的癌症的调节，包括乳腺癌，肺，胃，子宫内膜，肾脏和 前列腺5,7,9-12。 ATAD2的上调通常与差的患者结局相关，可以用作 预后标记9,12-14。此外，通过RNA干扰沉默ATAD2的表达会抑制细胞 增殖和促进卵巢癌的凋亡，并抑制迁移和入侵 肝细胞癌和结直肠癌细胞13,15,16。 ATAD2B，是ATAD2的旁系同居很差的 基因，尽管ATAD2和ATAD2B是高度保守的，但对 ATAD2B或其在肿瘤发生中的作用。 ATAD2/B蛋白都包含两个保守域：AAA ATPase结构域和一个溴结构域6。拟议研究的总体目的是确定如何 ATAD2/B溴结构域的乙酰透析识别可调节这些蛋白质的细胞功能。这 建议的目的是：（1）表征组蛋白修饰之间的串扰如何调节乙酰透析 ATAD2/B溴化群的识别； （2）概述二乙酰化组蛋白的分子机制 ATAD2/B溴化群的识别； （3）确定二乙酰赖赛的功能意义 ATAD2/B溴化群的识别。体外生化，生物物理和 关于ATAD2/B的结构生物学研究将与体内功能基因组结合 使用乳腺癌进展模型的研究来表征ATAD2/B的生物学作用 溴化群。我们将评估相邻组蛋白修饰对组蛋白H4尾识别的影响 使用肽阵列测定法与等温滴定量热法（ITC）和核磁 共振（NMR）化学位移扰动技术。 ATAD2/B的原子分辨率结构 NMR或X射线晶体学将解决与其二乙酰化组蛋白尾配体结合的溴结构域。 为了表征ATAD2/B溴结构域与其组蛋白配体的结合模式，我们将执行 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和ITC相结合，将用于测量对配体结合的影响， 并确定ATAD2/B溴结构域的结合口袋的差异。我们将比较基因组 乳腺癌进展模型中ATAD2/B与组蛋白H4乙酰化模式的广泛关联 使用ChIP-Seq和RNA-Seq确定ATAD2/B对乳腺癌细胞表型的影响，然后是 细胞迁移和入侵测定。我们的多方面方法将与特定的组蛋白修饰相关联 使用ATAD2/B的结合和动作，这将使我们能够将组蛋白H4乙酰化标记连接到溴结构域 在癌细胞增殖中的功能。总体而言，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促进发现新型药物靶标，以帮助克服癌症。