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

ATAD 2是雌激素和雄激素受体的重要共激活剂2，4。ATAD 2已知是向上的- 在多种不同类型的癌症中调节，包括乳腺癌、肺癌、胃癌、子宫内膜癌、肾癌和 前列腺5，7，9-12。ATAD 2的上调通常与不良的患者结局相关，并且可以用作 预后标记物9，12-14.此外，通过RNA干扰沉默ATAD 2的表达抑制了细胞凋亡。 促进卵巢癌细胞的增殖和凋亡，并抑制卵巢癌细胞的迁移和侵袭。 肝细胞癌和结直肠癌细胞13，15，16. ATAD 2B是ATAD 2的一种研究较少的蛋白 虽然ATAD 2和ATAD 2B是高度保守的，但关于ATAD 2和ATAD 2B的功能知之甚少。 ATAD 2B或其在肿瘤发生中的作用。两种ATAD 2/B蛋白都含有两个保守结构域： ATP酶结构域和溴结构域6。这项研究的总体目标是确定如何进行- ATAD 2/B布罗莫结构域的乙酰赖氨酸识别调节这些蛋白质的细胞功能。这 该提案旨在：（1）表征组蛋白修饰之间的串扰如何调节乙酰赖氨酸 ATAD 2/B布罗莫结构域的识别;（2）概述了二乙酰化组蛋白的分子机制 ATAD 2/B溴结构域的识别;（3）确定二乙酰赖氨酸的功能意义 通过ATAD 2/B布罗莫结构域的识别。一种独特的体外生物化学、生物物理学和 对ATAD 2/B布罗莫结构域的结构生物学研究将与体内功能基因组研究相结合。 使用乳腺癌进展模型表征ATAD 2/B的生物学作用的研究 溴结构域。我们将评估相邻组蛋白修饰对组蛋白H4尾识别的影响 使用肽阵列测定结合等温滴定量热法（ITC）和核磁共振 共振（NMR）化学位移扰动技术。ATAD 2/B的原子分辨结构 结合到其二乙酰化组蛋白尾部配体的溴结构域将通过NMR或X射线晶体学解析。 为了表征ATAD 2/B溴结构域与其组蛋白配体的结合模式，我们将进行 分析超离心、尺寸排阻色谱、ITC和NMR T1/T2弛豫实验。 结合NMR和ITC的定点诱变将用于测量对配体结合的影响， 并鉴定ATAD 2/B布罗莫结构域结合口袋的差异。我们会比较基因组- 在乳腺癌进展模型中ATAD 2/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ATAD 2/B对乳腺癌细胞表型的影响， 细胞迁移和侵袭测定。我们多方面的方法将特定的组蛋白修饰 与ATAD 2/B的结合和作用，这将使我们能够连接组蛋白H4乙酰化标记溴结构域 在癌细胞增殖中起作用。总的来说，我们的综合生物化学，生物物理学，结构生物学和 功能基因组学方法将有助于发现新的药物靶点，以帮助克服癌症。

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.