Allosteric Regulation in the KDM5 Family of Histone Demethylases

组蛋白去甲基酶 KDM5 家族的变构调节

基本信息

批准号：
9330881
负责人：
Danica Galonic Fujimori
金额：
$ 30.05万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
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：
2015-09-15 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9330881
关键词：
Affinity Allosteric Regulation Binding Cancer Etiology Catalysis Catalytic Domain Chromatin Complex Coupled Coupling Development Dinucleosome Disease Drug resistance Electron Microscopy Enzymes Eukaryota Excision Family Feedback Gene Expression Gene Targeting Genetic Transcription Genome Histone H3 Histones Human In Vitro Intellectual functioning disability Intervention KDM5B gene Ligand Binding Ligands Lysine Malignant Neoplasms Mediating Methylation Modeling Molecular Mono-S Mutagenesis Nucleosomes Oranges Peptides Pharmacology Physiology Plants Play Positioning Attribute Post-Translational Protein Processing Proteins Reader Recruitment Activity Regulation Role Scanning Signal Transduction Structure Substrate Specificity Tail Testing Transcriptional Regulation Up-Regulation Work Writing base demethylation enzyme activity experimental study histone demethylase homeodomain insight member novel novel therapeutics overexpression peptide B polypeptide C protein function public health relevance reconstitution tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Histone demethylases, a class of chromatin-modifying enzymes, regulate gene expression by modulating the methylation status of histone proteins within chromatin. Demethylases that belong to the KDM5 family antagonize tri-, di-, and monomethylation of lysine 4 in the tail of histone H3. In humans, this family has four members, KDM5A-D. While in physiology these enzymes act as regulators of development and differentiation, their misregulation via overexpression or mutagenesis is causative of cancer (KDM5A and B) and intellectual disability (KDM5C). Despite their importance, the mechanisms by which the catalytic activities of these enzymes are regulated on chromatin are still unknown. In addition to the catalytic domain, these demethylases contain several other domains, including two to three chromatin "reader" domains belonging to the plant homeodomain (PHD) family. Prior to our work, PHD domains in demethylases and demethylation complexes had been shown to act as chromatin binding modules to regulate recruitment and substrate specificity of demethylases. By investigating regulation of catalysis in demethylase KDM5A, we uncovered a novel function of PHD domains in demethylases. Specifically, we demonstrate that occupancy of the PHD1 domain by a ligand peptide allosterically stimulates demethylation activity of KDM5A. The preferred ligand for the PHD1 domain is unmodified histone H3 peptide (H3K4Me0), and the binding affinity of this domain for histone H3 peptide progressively decreases with the increase in methylation of Lys 4. Our work demonstrates that PHD domains can actively regulate catalytic activity of KDM5 demethylases and defines a new way in which the function of reader and catalytic domains are coupled to regulate demethylation. As the allosteric stimulation exploits a positive feedback-based mechanism, our findings suggest a model by which demethylation could spread on chromatin. The objective of this application is to define the role of allosteric regulation in controlling the catalytic activities of KDM5 demethylases. In Aim 1, we will determine the molecular basis of histone tail recognition by the PHD1 chromatin reader domain of KDM5A and quantitate the impact of post-translational modifications on recognition. In Aim 2, by interrogating how ligand binding to the PHD1 domain influences demethylation in the context of catalysis-competent constructs, we will determine the impact of the PHD1 domain occupancy on the catalytic activity of KDM5 demethylases. In Aim 3, by using in vitro reconstituted chromatin substrates, we will evaluate whether the functional coupling between the PHD1 domain and the catalytic domain can allow for spreading of demethylation on chromatin. By providing mechanistic insight into the functional coupling between the reader and catalytic domains in KDM5 demethylases, our approach will expand our understanding of the mechanisms that underlie regulation of chromatin methylation and consequently transcription.

描述（由申请人提供）：组蛋白去甲基化酶是一类染色质修饰酶，通过调节染色质内组蛋白的甲基化状态来调节基因表达。属于 KDM5 家族的去甲基化酶可拮抗赖氨酸的三甲基化、二甲基化和单甲基化。 4 位于组蛋白 H3 的尾部。在人类中，该家族有四个成员，KDM5A-D。酶作为发育和分化的调节因子，它们通过过度表达或诱变而产生的错误调节会导致癌症（KDM5A 和 B）和智力障碍（KDM5C），尽管它们很重要，但这些酶在染色质上的催化活性的调节机制尚不清楚。除了催化结构域外，这些去甲基化酶还包含几个其他结构域，包括属于植物同源结构域的两到三个染色质“读取器”结构域。在我们的工作之前，去甲基化酶和去甲基化复合物中的 PHD 结构域已被证明可以作为染色质结合模块来调节去甲基化酶和底物特异性。通过研究去甲基化酶 KDM5A 的催化调节，我们发现了 PHD 的新功能。具体来说，我们证明配体肽对 PHD1 结构域的占据会变构地刺激 KDM5A 的去甲基化活性。 PHD1 结构域的首选配体是未修饰的组蛋白 H3 肽 (H3K4Me0)，该结构域与组蛋白 H3 肽的结合亲和力随着 Lys 4 甲基化的增加而逐渐降低。我们的工作表明，PHD 结构域可以主动调节 KDM5 的催化活性去甲基化酶并定义了一种新的方式，其中读取器和催化结构域的功能耦合以调节去甲基化，因为变构刺激利用了积极的作用。基于反馈的机制，我们的研究结果提出了一种可以在染色质上传播去甲基化的模型。该应用的目的是确定变构调节在控制 KDM5 去甲基酶催化活性中的作用。在目标 1 中，我们将确定分子基础。在目标 2 中，通过探究配体结合方式，检测 KDM5A 的 PHD1 染色质读取器结构域对组蛋白尾部的识别，并定量翻译后修饰对识别的影响。鉴于 PHD1 结构域在具有催化能力的构建体中影响去甲基化，我们将确定 PHD1 结构域占用对 KDM5 去甲基化酶催化活性的影响。在目标 3 中，通过使用体外重构的染色质底物，我们将评估是否PHD1 结构域和催化结构域之间的功能耦合可以通过提供对染色质的机制了解来实现去甲基化的扩散。通过阅读器和 KDM5 去甲基化酶催化结构域之间的功能耦合，我们的方法将扩大我们对染色质甲基化及其转录调控机制的理解。