A unique double PHD finger and bromodomain in epigenetic signaling

描述（由申请人提供）：据推测，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解读域的保留可指导异常的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。进展受到阻碍，因为我们不知道染色质解读域如何将MOZ HAT复合体连接到组蛋白。bromodomain-PHD手指蛋白1 （BRPF1）在白血病易位中与MOZ相关，并将MOZ催化亚基与MOZ HAT复合物促进活性的生长抑制剂5 （ING5）和hEaf6亚基连接起来。BRPF1包含多个表观遗传解读域，包括一个独特的双植物同源结构域（PHD）和锌指（ZnF）组装（PZP），一个溴结构域（BRD）和一个PWWP结构域。本提案的总体目标是阐明PZP和BRD区域在将BRPF1招募到组蛋白中的作用。核心假设是BRD和PZP区域独立地将BRPF1连接到组蛋白尾部，相邻的PHD和锌指基序调节PHD1的组蛋白靶点，使其功能多样化。本课题旨在：(1)通过BRPF1独特的PHD指区建立组蛋白H3识别的分子基础；(2)鉴定BRPF1 BRD的组蛋白配体，并对BRPF1靶向组蛋白尾部的特异性决定因子进行结构表征。生物化学、生物物理和结构生物学技术的独特组合将用于表征PZP和BRD区域在BRPF1中的结构和功能作用。组蛋白尾部配体将使用肽阵列分析结合

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.