Molecular mechanism of chromatin targeting by BRPF1

描述（由申请人提供）：人BRPF 1（溴结构域PHD指1）是组蛋白乙酰转移酶（HAT）复合物MOZ/MORF的主要亚基，在造血系统发育中至关重要，并与急性白血病有关。BRPF 1是正常发育程序和转录调节所需的，但它在复合体中的作用仍然难以捉摸。BRPF 1含有一个多个锌指簇，称为PZP结构域。我们最近的研究表明，BRPF 1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的装配之间的新联系。BRPF 1这种新功能的分子机制尚不清楚，将在拟议的研究中阐明。我们假设BRPF 1的PZP模块与组蛋白H3尾部和DNA的伴随结合招募和/或稳定染色质处的MOZ/MORF HAT复合物，并且组蛋白H3的翻译后修饰（PTM）调节PZP的结合并微调HAT活性。我们试图阐明BRPF 1 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刺激的转录激活的表观遗传机制具有重要意义。本项目的具体目标是：（1）阐明BRPF 1锌指之间的功能和结构关系;（2）确定PZP在染色质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NMR光谱学或X射线晶体学确定与组蛋白肽和DNA复合的PZP模块的原子分辨率结构。组蛋白和DNA序列的PTM的特异性，以及PZP在核小体上的组装将通过肽库筛选、电泳迁移率变化、NMR和Forster共振能量转移来表征。将突变组蛋白和DNA结合位点残基，并在体外和体内测试突变蛋白，以确定PZP在染色质缔合、BRPF 1依赖性转录激活、组蛋白乙酰化调节和MOZ/MORF复合物体内定位中的作用。我们将利用染色质免疫沉淀，PCR，荧光显微镜和HAT测定在这种情况下，以评估如何BRPF 1的PZP区域有助于MOZ/MORF HAT的功能。这些研究将揭示BRPF 1 PZP在MOZ/MORF复合物功能中的作用，使我们能够建立BRPF 1/MOZ/MORF信号转导模型，并将更好地理解基因转录和染色质重塑调控的表观遗传机制。

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.