A unique double PHD finger and bromodomain in epigenetic signaling

描述（由申请方提供）：推测单核细胞白血病锌指（MOZ）组蛋白乙酰转移酶（HAT）白血病融合蛋白中表观遗传阅读器结构域的保留可指导异常HAT活性，并可能导致致癌转化。然而，连接MOZ HAT复合物与其组蛋白底物的分子机制尚不清楚。进展受阻，因为我们不知道染色质阅读器结构域如何将MOZ HAT复合物连接到组蛋白。溴结构域-PHD指蛋白1（BRPF 1）在白血病易位中与MOZ相关联，并在MOZ HAT复合物中将MOZ催化亚基与生长抑制剂5（ING 5）和hEaf 6亚基连接，从而促进活性。BRPF 1含有多个表观遗传阅读器结构域，包括独特的双植物同源结构域（PHD）和锌指（ZnF）组装体（PZP）、溴结构域（BRD）和PWWP结构域。该提案的总体目标是阐明PZP和BRD区域在招募BRPF 1到组蛋白中的作用。中心假设是BRD和PZP区域独立地将BRPF 1桥接到组蛋白尾部，并且相邻的PHD和锌指基序调节PHD 1的组蛋白靶标，使其功能多样化。该提案旨在：（1）通过BRPF 1的独特PHD指区建立组蛋白H3识别的分子基础，和（2）鉴定BRPF 1 BRD的组蛋白配体，并在结构上表征靶向BRPF 1至组蛋白尾部的特异性决定簇。生物化学，生物物理学和结构生物学技术的独特组合将用于表征BRPF 1中PZP和BRD区域的结构和功能作用。组蛋白尾配体将使用肽阵列测定结合 核磁共振（NMR）化学位移微扰技术。色氨酸荧光、等温滴定量热法（ITC）和/或NMR滴定实验将用于研究翻译后修饰对结合的影响。BRPF 1 PZP和BRD结构域与其组蛋白尾部配体结合的原子分辨率结构将通过NMR或X射线晶体学解析。结构数据将用于模拟BRPF 1 PZP或BRD与组蛋白配体的相互作用，并设计突变，测试哪些残基对与组蛋白尾部的相互作用很重要。这一结果将建立一种新的PHD指状结构域识别染色质的独特机制，并增加我们对结构多样的BRD识别其组蛋白配体的分子机制的理解。这些信息将阐明MOZ HAT复合物靶向染色质底物的原理，并深入了解多个组蛋白阅读器结构域如何在BRPF 1亚基内协同发挥作用。这些数据将为BRPF 1靶向组蛋白尾部的机制提供关键的结构见解，并将更好地了解这些染色质结合效应物如何将表观遗传信号与正常和病理基因表达的调节联系起来。

DESCRIPTION (provided by applicant): Retention of epigenetic reader domains in the monocytic leukemic zinc-finger (MOZ) histone acetyltransferase (HAT) leukemic fusion protein is speculated to direct aberrant HAT activity and may be responsible for oncogenic transformations. However, the molecular mechanisms connecting the MOZ HAT complex to its histone substrates are unknown. Progress is hampered because we do not know how chromatin reader domains bridge the MOZ HAT complex to histones. The bromodomain-PHD finger protein 1 (BRPF1) associates with MOZ in leukemic translocations and links the MOZ catalytic subunit with the inhibitor of growth 5 (ING5) and hEaf6 subunits in the MOZ HAT complex promoting activity. BRPF1 contains multiple epigenetic reader domains including a unique double plant homeodomain (PHD) and zinc finger (ZnF) assembly (PZP), a bromodomain (BRD) and a PWWP domain. The overall objective of this proposal is to elucidate the role of the PZP and BRD regions in recruiting BRPF1 to histones. The central hypothesis is that the BRD and PZP regions independently bridge BRPF1 to the histone tail, and the adjacent PHD and zinc finger motifs modulate the histone target of PHD1 diversifying its functionality. This proposal aims to: (1) establish the molecular basis of histone H3 recognition by the unique PHD finger region of BRPF1 and (2) identify the histone ligand(s) of the BRPF1 BRD and structurally characterize the specificity determinants targeting BRPF1 to the histone tail. A unique combination of biochemical, biophysical and structural biology techniques will be used to characterize the structural and functional role of the PZP and BRD regions in BRPF1. The histone tail ligands will be identified and verified using peptide array assays in combination with nuclear magnetic resonance (NMR) chemical shift perturbation techniques. Tryptophan fluorescence, isothermal titration calorimetry (ITC) and/or NMR titration experiments will be used to investigate the effects of post-translational modifications on binding. The atomic resolution structures of the BRPF1 PZP and BRD domains bound to their histone tail ligands will be solved by NMR or X-ray crystallography. The structural data will be used to model the BRPF1 PZP or BRD interaction with histone ligands and to design mutations testing which residues are important for the interaction with the histone tail. The results will establish a uniqe mechanism of chromatin recognition by a novel PHD finger domain and increase our understanding of the molecular mechanism utilized by structurally diverse BRDs to recognize their histone ligands. This information will elucidate the principles targeting the MOZ HAT complex to chromatin substrates and provide insight into how multiple histone reader domains function cooperatively within the BRPF1 subunit. These data will provide critical structural insights into the mechanism used by BRPF1 to target the histone tail, and will impart a greater understanding on how these chromatin binding effectors link epigenetic signals to the regulation of normal and pathological gene expression.