Allosteric Regulation in the KDM5 Family of Histone Demethylases

组蛋白去甲基酶 KDM5 家族的变构调节

• 批准号: 9037534
• 负责人: Danica Galonic Fujimori
• 金额: $ 30.16万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 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/9037534
• 关键词: Affinity; Allosteric Regulation; Binding; Catalysis; Catalytic Domain; Chromatin; Complex; Coupled; Coupling; Development; Dinucleosome; Disease; Electron Microscopy; Enzymes; Eukaryota; Excision; Family; Feedback; Figs - dietary; Gene Expression; Gene Targeting; Genetic Transcription; Genome; Histone H3; Histones; Human; In Vitro; Intellectual functioning disability; Intervention; Ligand Binding; Ligands; Lysine; Malignant Neoplasms; Mediating; Methylation; Modeling; Molecular; Mono-S; Mutagenesis; Nucleosomes; Oranges; Peptides; Pharmaceutical Preparations; Physiology; Plants; Play; Positioning Attribute; Post-Translational Protein Processing; Proteins; Reader; Reading; Regulation; Role; Scanning; Signal Transduction; Staging; Structure; Substrate Specificity; Tail; Testing; Transcriptional Regulation; Up-Regulation; Work; Writing; base; demethylation; enzyme activity; histone demethylase; homeodomain; insight; member; novel; novel therapeutics; overexpression; protein function; public health relevance; reconstitution; research study; tumorigenesis

 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KDM 5家族的脱甲基酶拮抗组蛋白H3尾部赖氨酸4的三甲基化、二甲基化和单甲基化。在人类中，这个家族有四个成员，KDM 5A-D。虽然在生理学中这些酶作为发育和分化的调节剂，但它们通过过表达或诱变的错误调节是癌症（KDM 5A和B）和智力残疾（KDM 5C）的原因。尽管它们的重要性，这些酶的催化活性在染色质上的调节机制仍然是未知的。除了催化结构域之外，这些脱甲基酶还含有几个其他结构域，包括属于植物同源结构域（PHD）家族的两到三个染色质“阅读器”结构域。在我们的工作之前，脱甲基酶和脱甲基复合物中的PHD结构域已被证明作为染色质结合模块来调节脱甲基酶的募集和底物特异性。通过研究去甲基化酶KDM 5A的催化调控，我们发现了去甲基化酶中PHD结构域的一个新功能。具体而言，我们证明了PHD 1结构域的占用由配体肽变构刺激KDM 5A的去甲基化活性。PHD 1结构域的优选配体是未修饰的组蛋白H3肽（H3 K4 Me 0），并且该结构域对组蛋白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KDM 5去甲基化酶的催化活性，并定义了一种新的方式，其中阅读器和催化结构域的功能耦合来调节去甲基化。由于变构刺激利用了基于正反馈的机制，我们的研究结果提出了一种去甲基化可以在染色质上传播的模型。本申请的目的是确定变构调节在控制KDM 5脱甲基酶的催化活性中的作用。在目标1中，我们将确定KDM 5A的PHD 1染色质阅读器结构域识别组蛋白尾部的分子基础，并定量翻译后修饰对识别的影响。在目标2中，通过询问配体结合到PHD 1结构域如何影响催化活性构建体中的去甲基化，我们将确定PHD 1结构域占用对KDM 5去甲基化酶的催化活性的影响。在目标3中，通过使用体外重建的染色质底物，我们将评估PHD 1结构域和催化结构域之间的功能偶联是否可以允许染色质上的去甲基化扩散。通过提供对KDM 5去甲基化酶中阅读器和催化结构域之间的功能耦合的机械见解，我们的方法将扩大我们对染色质甲基化调控机制的理解，从而转录。