Molecular mechanism of chromatin targeting by BRPF1

描述(申请人提供)：人类BRPF1(溴域PhD Finger 1)是组蛋白乙酰转移酶(HAT)复合体MoZ/Morf的一个主要亚单位，在造血系统的发展中起关键作用，并与急性白血病有关。BRPF1是正常发育程序和转录调控所必需的，然而它在复合体中的作用仍然难以捉摸。BRPF1包含一簇多个锌指，称为PZP结构域。我们最近的研究表明，BRPF1的PZP模块识别组蛋白和DNA，揭示了MoZ/Morf介导的乙酰化和复杂组装之间的新联系。BRPF1这一新功能背后的分子机制尚不清楚，将在拟议的研究中阐明。我们假设BRPF1的PZP模块与组蛋白H3尾巴和DNA的结合可以招募和/或稳定染色质上的MoZ/Morf HAT复合体，而组蛋白H3的翻译后修饰(PTM)可以调节PZP的结合并微调HAT活性。我们试图阐明BRPF1 PZP与染色质相互作用的分子基础和功能意义。这项研究对于理解HAT刺激转录激活的表观遗传学机制具有重要意义。本项目的具体目标是：(1)阐明BRPF1锌指之间的功能和结构关系；(2)确定PZP在染色质上组装的分子基础和功能意义。为了确定染色质靶向的分子机制，将使用核磁共振光谱或X射线结晶学来确定组蛋白多肽和DNA络合物中PZP模块的原子分辨结构。组蛋白的PTM和DNA序列的特异性以及PZP在核小体上的组装将通过多肽文库筛选、凝胶迁移率改变、核磁共振和Forster共振能量转移来表征。组蛋白和DNA结合位点残基将被突变，突变的蛋白将在体外和体内进行测试，以确定PZP在染色质结合、BRPF1依赖的转录激活、组蛋白乙酰化的调节以及MoZ/Morf复合体的体内定位中的作用。在此背景下，我们将利用染色质免疫沉淀、聚合酶链式反应、荧光显微镜和HAT分析来评估BRPF1的PZP区域如何参与MoZ/Morf Hats的功能。这些研究将阐明BRPF1 PZP在MOZ/Morf复合体功能中的作用，使我们能够建立BRPF1/MoZ/Morf的信号模型，并将有助于更好地理解基因转录和染色质重塑的表观遗传学机制。

DESCRIPTION (provided by applicant): Human BRPF1 (bromodomain PHD finger 1) is a major subunit of the histone acetyltransferase (HAT) complexes MOZ/MORF, critical in the development of the hematopoietic system and implicated in acute leukemias. BRPF1 is required for normal developmental programs and transcriptional regulation, however its role in the complex remains elusive. BRPF1 contains a cluster of multiple zinc fingers, named a PZP domain. Our recent studies demonstrate that the PZP module of BRPF1 recognizes both histone and DNA, revealing a novel link between the MOZ/MORF-mediated acetylation and the complex assembly. The molecular mechanism underlying this novel function of BRPF1 is unclear and will be elucidated in the proposed studies. We hypothesize that concomitant binding of the PZP module of BRPF1 to histone H3 tail and DNA recruits and/or stabilize the MOZ/MORF HAT complexes at chromatin, and that posttranslational modifications (PTMs) of histone H3 modulate binding of PZP and fine-tune the HAT activity. We seek to elucidate the molecular basis and functional significance of interactions of BRPF1 PZP with chromatin. This study is of fundamental importance for understanding the epigenetic mechanisms of HAT-stimulated transcriptional activation. The specific aims of this project are: (1) to elucidate the functional and structural relationship between zinc fingers of BRPF1, and (2) to determine the molecular basis and functional significance of the PZP assembly at chromatin. To define the molecular mechanism of chromatin targeting, the atomic-resolution structures of the PZP module in complex with histone peptide and DNA will be determined using NMR spectroscopy or X-ray crystallography. Specificities for PTMs of histones and the DNA sequence, and the assembly of PZP on nucleosomes will be characterized by peptide library screening, electrophoretic mobility shift, NMR and Forster Resonance Energy Transfer. The histone- and DNA-binding site residues will be mutated and the mutant proteins will be tested in vitro and in vivo to determine the role of PZP in chromatin association, BRPF1-dependent transcriptional activation, and regulation of histone acetylation and in vivo localization of the MOZ/MORF complexes. We will utilize chromatin immunoprecipitation, PCR, fluorescence microscopy and HAT assays in this context to assess how the PZP region of BRPF1 contributes to the functions of MOZ/MORF HATs. These studies will shed light on the role of BRPF1 PZP in functioning of the MOZ/MORF complexes, allowing us to build a model of signaling by BRPF1/MOZ/MORF, and will lead to a better understanding of the epigenetic mechanisms for the regulation of gene transcription and chromatin remodeling.