Allosteric Regulation in the KDM5 Family of Histone Demethylases

 描述(申请人提供)：组蛋白去甲基酶是一类染色质修饰酶，通过调节染色质中组蛋白的甲基化状态来调节基因表达。属于KDM5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，KDM5A-D。虽然在生理学上，这些酶起着发育和分化的调节作用，但它们通过过度表达或突变而产生的错误调控会导致癌症(KDM5A和B)和智力残疾(KDM5C)。尽管它们很重要，但这些酶的催化活性在染色质上的调节机制仍不清楚。除催化结构域外，这些去甲基酶还含有其他几个结构域，包括两到三个属于植物同源结构域(PhD)家族的染色质“阅读器”结构域。在我们的工作之前，已经证明去甲基酶和去甲基化复合体中的PHD结构域作为染色质结合模块来调节去甲基酶的募集和底物专一性。通过研究去甲基酶KDM5A的催化调节，我们发现了去甲基酶中PHD结构域的一个新功能。具体地说，我们证明了配体多肽占据PhD1结构域可以变构刺激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多肽(H3K4Me0)，随着Lys 4甲基化程度的增加，该结构域与组蛋白H3多肽的结合亲和力逐渐降低。我们的工作表明，PhD结构域可以积极地调节KDM5去甲基酶的催化活性，并定义了一种新的途径，即阅读器功能和催化结构域的偶联来调节去甲基化。由于变构刺激使用了基于正反馈的机制，我们的发现提出了一种模型，通过该模型，去甲基化可以在染色质上传播。本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KDM5A的PhD1染色质读取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，通过询问配体与PhD1结构域的结合如何影响催化活性构建物中的去甲基化，我们将确定PhD1结构域占据对KDM5去甲基酶催化活性的影响。在目标3中，通过使用体外重组的染色质底物，我们将评估PhD1结构域和催化结构域之间的功能偶联是否允许染色质上的去甲基化扩散。通过提供对KDM5去甲基酶中阅读器和催化域之间的功能耦合的机械性见解，我们的方法将扩大我们对染色质甲基化调控以及随后的转录调控的机制的理解。

 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.