New proteomic methods to quantify chromatin changes to identify regulators of cellular senescence

染色质的特定成分是细胞状态的重要决定因素。染色质的组成由多种因素决定。其中包括组蛋白的翻译后修饰和大量非组蛋白的关联。虽然我们对某些单个细胞状态的染色质组成的了解已经相当深入，但在细胞状态之间的逐渐转变过程中，染色质的组成是如何变化的仍是未知的。目前的蛋白质组学方法是跟踪这些随时间变化的主要障碍，因为它们太有限了，无法可靠地量化蛋白质及其在染色质上的相互作用，以及组蛋白和非组蛋白蛋白翻译后修饰的复杂性。在从年轻细胞到老年细胞的过渡过程中，结构性应力导致逐渐积累的损伤，最终导致不可逆转的生长停滞，称为细胞衰老。最近的研究首次揭示了衰老细胞中染色质组成的重组。然而，目前我们既不知道人类原代细胞衰老过程中染色质变化的程度，也不知道它们是如何逐渐建立的，以及它们如何有助于定义衰老的开始及其稳定的表现。这一建议旨在通过开发定量蛋白质组学方法来可靠地监测不同条件下染色质及其相关蛋白质的组成，以填补这一技术和概念上的空白。虽然这些方法将广泛适用，但我将应用它来生成一个资源，定量描述细胞衰老过程中和不同衰老模型中染色质组成的变化。具体地说，我将开发蛋白质组学工作流程，允许精确量化i)复杂的组蛋白组合修饰，ii)蛋白质与染色质或核质的不同联系，以及iii)染色质上的蛋白质-蛋白质相互作用。另一个目标是测试资源的可用性，以确定和验证调节细胞衰老开始或维持的染色质因素。细胞衰老与组织损伤、组织老化、肿瘤进展以及化疗后复发有关，因此，识别调节这一过程的机制将具有强烈的和直接的生物医学意义。

The specific composition of chromatin is an important determinant for the cell state. Multiple factors define the composition of chromatin. Among them are post-translational modifications at histones and the association of a wealth of non-histone proteins. While our knowledge of the chromatin composition in some individual cell states is already quite advanced, how the composition of chromatin changes during the gradual transitions between cell states remains mostly unknown. Current proteomic methods present a major obstacle to follow the changes over time, because they are too limited to reliably quantify proteins and their interactions at chromatin as well as the complexity of post-translational modifications at histones and non-histone proteins. During the transition from young to old cells, constitutive stress leads to progressively accumulating damage, which ultimately causes an irreversible growth arrest, known as cellular senescence. Recent studies provided a first glimpse in the reorganization of the composition of chromatin in senescent cells. However, currently we neither know the extent of chromatin alterations during cellular aging in primary human cells nor do we know how they are progressively established and how they contribute to define the onset of senescence and its stable manifestation. This proposal aims to fill the technological and conceptual gap by developing quantitative proteomic methods to reliably monitor the composition of chromatin and its associated proteins between different conditions. While these methods will be broadly applicable, I will apply it to generate a resource that quantitatively describes the changes to the composition of chromatin during the course of cellular aging and in different senescence models. Specifically, I will develop proteomic workflows that allow the precise quantification of i) complex combinatorial histone modifications, ii) differential association of proteins to chromatin or the nucleoplasm and iii) protein-protein interactions at chromatin. A further goal is to test the usability of the resource to identify and validate chromatin factors that modulate the onset or maintenance of cellular senescence. Cellular senescence is implicated in regeneration following tissue damage, organismal aging, tumour progression as well as relapse after chemotherapy, hence, identifying mechanisms that modulate this process will have strong and immediate biomedical relevance.