Molecular mechanism of chromatin targeting by BRPF1

BRPF1 染色质靶向的分子机制

基本信息

批准号：
8996687
负责人：
TATIANA G KUTATELADZE
金额：
$ 32.5万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8996687
关键词：
Acetylation Acute leukemia Affinity Binding Binding Sites Biochemical Biological Biological Assay Bromodomain Calorimetry Chromatin Chromatin Structure Complex DNA DNA Binding DNA Sequence Data Development EMSA Energy Transfer Epigenetic Process Fingers Fluorescence Fluorescence Microscopy Genetic Transcription Health Hematopoietic System Histone Acetylation Histone H3 Histones Human Lead Length Light Link Measures Mediating Modeling Molecular Mutate NMR Spectroscopy Names Nucleosome Core Particle Nucleosomes PHD Finger Peptide Library Peptides Plants Play Post-Translational Protein Processing Proteins Recruitment Activity Regulation Resolution Role Signal Pathway Signal Transduction Specificity Structure Tail Titrations Transcriptional Activation Transcriptional Regulation Western Blotting X-Ray Crystallography Zinc Zinc Fingers base chromatin immunoprecipitation chromatin remodeling combinatorial design histone acetyltransferase homeodomain in vitro testing in vivo insight leukemia loss of function mutant novel peptide structure prevent programs screening

项目摘要

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.

描述（由申请人提供）：人 BRPF1（溴结构域 PHD Finger 1）是组蛋白乙酰转移酶 (HAT) 复合物 MOZ/MORF 的主要亚基，对于造血系统的发育至关重要，并与急性白血病有关。 BRPF1 是正常发育程序和转录调节所必需的，但其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 模块可识别组蛋白和 DNA，揭示了 MOZ/MORF 介导的乙酰化与复杂组装之间的新联系。 BRPF1 这一新功能的分子机制尚不清楚，将在拟议的研究中得到阐明。我们假设 BRPF1 的 PZP 模块与组蛋白 H3 尾部和 DNA 的同时结合会募集和/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 的结合并微调帽子活动。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机制至关重要。该项目的具体目标是：(1) 阐明 BRPF1 锌指之间的功能和结构关系，(2) 确定染色质 PZP 组装的分子基础和功能意义。为了定义染色质靶向的分子机制，将使用 NMR 光谱或 X 射线晶体学来确定与组蛋白肽和 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 和福斯特共振能量转移来表征。组蛋白和 DNA 结合位点残基将发生突变，突变蛋白将在体外和体内进行测试，以确定 PZP 在染色质关联、BRPF1 依赖性转录激活、组蛋白乙酰化调节和体内定位中的作用。 MOZ/MORF 复合物。在这种情况下，我们将利用染色质免疫沉淀、PCR、荧光显微镜和 HAT 测定来评估 BRPF1 的 PZP 区域如何促进 MOZ/MORF HAT 的功能。这些研究将阐明 BRPF1 PZP 在 MOZ/MORF 复合物功能中的作用，使我们能够建立 BRPF1/MOZ/MORF 信号传导模型，并将导致更好地了解调节基因转录和染色质重塑。