A unique double PHD finger and bromodomain in epigenetic signaling

描述(由申请人提供)：单核细胞白血病锌指(MOZ)组蛋白乙酰转移酶(HAT)融合蛋白中表观遗传阅读器结构域的保留被推测为指导HAT异常活性，并可能与致癌转化有关。然而，将MozHat复合体与其组蛋白底物连接起来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读取域如何将Moz Hat复合体与组蛋白连接起来。溴域-PhD指蛋白1(BRPF1)在白血病易位中与MoZ结合，并将MoZ催化亚基与生长抑制因子5(ING5)和Moz HAT复合体中的hEaf6亚基连接起来。BRPF1包含多个表观遗传阅读器结构域，包括一个独特的双植物同源域(PHD)和锌指(ZF)组装(PZP)，一个溴域(BRD)和一个PWWP结构域。这项建议的总体目标是阐明PZP和BRD区域在招募BRPF1到组蛋白中的作用。中心假说是，BRD和PZP区域独立地将BRPF1连接到组蛋白尾部，相邻的PhD和锌指基序调控PhD1的组蛋白靶标，使其功能多样化。本研究的目的是：(1)通过BRPF1独特的PhD指状区建立识别组蛋白H3的分子基础；(2)确定BRPF1 BRD的组蛋白配体(S)，并从结构上表征针对BRPF1组蛋白尾部的特异性决定因素。将使用一种独特的生化、生物物理和结构生物学技术组合来表征BRPF1中PZP和BRD区域的结构和功能作用。组蛋白尾部配体将使用多肽阵列分析结合 核磁共振化学位移微扰技术。色氨酸荧光、等温滴定热法(ITC)和/或核磁共振滴定实验将用于研究翻译后修饰对结合的影响。与组蛋白尾部配体结合的BRPF1、PZP和BRD结构域的原子分辨结构将通过核磁共振或X射线结晶学来解决。结构数据将被用来模拟BRPF1、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是重要的。这些结果将建立一个新的PhD指状区识别染色质的唯一机制，并增加我们对结构不同的BRD识别其组蛋白配体的分子机制的理解。这些信息将阐明针对染色质底物的Moz HAT复合体的原理，并为多个组蛋白读取器结构域在BRPF1亚基中如何协同发挥作用提供深入的见解。这些数据将为BRPF1靶向组蛋白尾部的机制提供关键的结构见解，并将使人们更好地理解这些染色质结合效应器如何将表观遗传信号与正常和病理基因表达的调控联系起来。

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.