N-terminal phosphorylation as a potentially tunable dial to modify the properties of HP1alpha-mediated heterochromatin

N-末端磷酸化作为潜在的可调节旋钮来改变 HP1α 介导的异染色质的特性

• 批准号: 10007584
• 负责人: Emily Wong
• 金额: $ 6.53万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10007584
• 关键词: Affinity; Behavior; Binding; Binding Proteins; Biochemistry; Biological Assay; Cells; Cellular biology; Chemical Structure; Chemicals; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Collaborations; Cytoprotection; DNA; DNA Binding; Development; Diffusion; Environment; Enzymes; Euchromatin; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Gene Expression; Generations; Genes; Genetic Transcription; Genome; Goals; Growth; Heritability; Heterochromatin; Histones; Human; Hydrophobicity; In Vitro; Label; Laboratories; Ligands; Liquid substance; Maintenance; Malignant Neoplasms; Measurement; Measures; Mediating; Methyltransferase; Microscopy; Modeling; Molecular Conformation; N-terminal; Nucleosomes; Oxidation-Reduction; Phase; Phosphorylation; Property; Proteins; RNA Interference; Recombinants; Reporting; Rheology; Site; Sodium Chloride; Structure; Testing; Tissues; Virus Replication; Work; X-Ray Tomography; chemical property; chemical reaction; experimental study; heterochromatin-specific nonhistone chromosomal protein HP-1; in vivo; in vivo Model; in vivo evaluation; light microscopy; organizational structure; public health relevance; viscoelasticity

Project Summary / Abstract Appropriate gene expression underlies every aspect of cellular biology from viral replication to multicellular organismal development to cancer. A key aspect of appropriate gene expression is the division of the genome into heterochromatin, a more tightly compacted and transcriptionally silent portion of the genome, and euchromatin, a more open, transcriptionally active portion. Euchromatic regions are thought to be defined by the constant activity of sequence-specific transcription, but the interactions that define and maintain heterochromatin are still an outstanding question in the field. Heterochromatin Protein 1α (HP1α) is a major structural component of constitutive heterochromatin and is thought to mediate chromatin compaction. Recent studies show that recombinantly purified HP1α undergoes liquid-liquid demixing on the addition of DNA to form distinct protein-rich and protein-poor phases. Similarly, N-terminally phosphorylated HP1α (nPhos HP1α) spontaneously demixes even in the absence of DNA. This phase separating ability has suggested new potential mechanisms for heterochromatin function. In particular, the differential chromatin affinities and phase separating abilities of nPhos and unmodified HP1α suggest that N-terminal phosphorylation may allow the cell to tune its heterochromatin compartment. However, more work on nPhos HP1α biochemistry is required prior to make targeted models of in vivo function. The goal of this proposal is to determine the properties of nPhos HP1α available to the cell for the protection and sequestration of heterochromatin. I will measure the strength and kinds of interaction between nPhos and unmodified HP1α, and whether the two species form miscible phases in vitro using light and fluorescence microscopy. I will also measure the viscoelasticity of and the diffusion of molecules within these phases through correlated fluorescence fluctuations and micro-rheology to determine whether solvated molecules can freely exchange between nPhos / HP1α phases. I will then correlate the physical material properties of pure and mixed HP1α phases with measurements of chemical environment and heterochromatin-associated enzymatic activity within HP1α droplets using established enzymatic assays as well as fluorescent pH, salt, redox, and hydrophobicity probes. Lastly, I will determine the mesoscale structure of nPhos and HP1α droplets containing heterochromatin-associated ligands using Soft X-ray Tomography (SXT). By generating HP1α droplets in various heterochromatin contexts (chromatin, RNAi, known protein binding partners) I will be able to determine how HP1α droplet structure is regulated, and how it relates to the corresponding material and chemical properties. These studies will provide a comprehensive characterization of nPhos HP1α behavior, and permit generation of targeted models for nPhos HP1α function for in vivo testing.

项目摘要/摘要 从病毒复制到多细胞，适当的基因表达奠定了细胞生物学的方方面面 从生物体发育到癌症。适当的基因表达的一个关键方面是基因组的分裂 转化为异染色质，这是基因组中一种更紧密且转录沉默的部分，以及 常染色质，一种更开放、转录活性更高的部分。常色区域被认为是由 序列特异性转录的持续活性，但定义和维持 异染色质仍然是该领域一个悬而未决的问题。异染色质蛋白1α(hp1α)是一种主要的 构成异染色质的结构成分，被认为是调节染色质压缩的物质。近期 研究表明，重组纯化的hp1DNA在加入α后发生液-液分离形成 不同的富含蛋白质和缺乏蛋白质的阶段。同样，N-末端磷酸化的Hp1α(nPhos Hp1α) 即使在没有DNA的情况下，也会自发地分离。这种相分离能力表明了新的 异染色质功能的潜在机制。特别是，不同的染色质亲和力和相位 NPhos和未修饰的hp1α的分离能力表明N末端的磷酸化可能允许细胞 来调整它的异染色质隔间。然而，需要在nPhos hp1α生物化学方面做更多的工作 制作体内功能的靶向模型。 这项建议的目标是确定细胞可用于保护的nPhos HP1α的属性 异染色质的隔离。我将测量nPhos和nPhos之间的强度和交互类型 未修饰的Hp1α，以及这两种物种在体外是否形成可混溶的相 显微镜。我还将测量分子在这些相中的粘弹性和扩散。 通过相关的荧光波动和微流变学来确定溶剂化分子是否可以 在nPhos/hp1α相间自由交换。然后我将把纯净的物质的物理性质关联起来 和混合hp1α相的化学环境和异染色质相关的测量 使用已建立的酶分析方法以及荧光pH，盐， 氧化还原和疏水性探针。最后，我将确定nPhos和hp1α液滴的中尺度结构 含有异染色质相关配体的软X射线断层扫描(SXT)。通过生成Hp1α 各种异染色质环境中的液滴(染色质、RNAi、已知的蛋白质结合伙伴)我将能够 确定hp1α液滴结构是如何调节的，以及它与相应的材料和 化学性质。这些研究将提供nPhos HP1α行为的全面表征， 并允许生成用于体内测试的nPhos HP1α功能的靶向模型。