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

项目总结/摘要 适当的基因表达是细胞生物学的各个方面的基础，从病毒复制到多细胞 发展到癌症。适当的基因表达的一个关键方面是基因组的分裂 进入异染色质，基因组中更紧密压缩和转录沉默的部分， 常染色质，一个更开放，转录活性的部分。常染色质区域被认为是由以下定义的： 序列特异性转录的恒定活性，但定义和维持 异染色质是该领域中一个悬而未决的问题。异染色质蛋白1α（Heterochromatin Protein 1α，HP 1 α）是一种主要的 是组成性异染色质的结构组分，被认为介导染色质致密化。最近 研究表明，重组纯化的HP 1 α在加入DNA后发生液-液分层， 不同的富含蛋白质和缺乏蛋白质的阶段。类似地，N端磷酸化HP 1 α（nPhos HP 1 α） 甚至在没有DNA的情况下也会自发地解混。这种相分离能力提出了新的 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HP 1 α的分离能力表明，N端磷酸化可能使细胞 来调节它的异染色质区室。然而，在此之前，需要对nPhos HP 1 α生物化学进行更多的研究。 to make targeted目标models模型of in vivo体内function功能. 本提案的目的是确定nPhos HP 1 α可用于细胞保护的性质 和异染色质的隔离。我将测量nPhos之间的相互作用的强度和种类， 未修饰的HP 1 α，以及两种物质是否在体外使用光和荧光形成混溶相 显微镜我也将测量这些相内分子的粘弹性和扩散 通过相关的荧光波动和微观流变学来确定溶剂化分子是否可以 在nPhos /HP 1 α相之间自由交换。然后我将把纯金属的物理性质 和混合HP 1 α相的化学环境和异染色质相关的测量 使用已建立的酶测定以及荧光pH、盐 氧化还原和疏水性探针。最后，我将确定nPhos和HP 1 α液滴的中尺度结构 含有异染色质相关配体，使用软X射线断层扫描（SXT）。通过生成HP 1 α 液滴在各种异染色质背景下（染色质，RNAi，已知的蛋白质结合伴侣），我将能够 确定HP 1 α液滴结构是如何调节的，以及它与相应材料的关系， 化学性质这些研究将提供nPhos HP 1 α行为的全面表征， 并允许生成用于体内测试的nPhos HP 1 α功能的靶向模型。

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.