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

染色质的特定组成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丰富的非组蛋白的关联。虽然我们对某些单个细胞状态下染色质组成的了解已经相当先进，但染色质组成在细胞状态之间的逐渐转变过程中如何变化仍然是未知的。目前的蛋白质组学方法是跟踪这些变化的主要障碍，因为它们太有限，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翻译后修饰的复杂性。在从年轻细胞到年老细胞的转变过程中，本构性压力导致逐渐累积的损伤，最终导致不可逆转的生长停滞，即细胞衰老。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改变的程度，也不知道它们是如何逐渐建立的，以及它们如何决定衰老的开始及其稳定表现。本提案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的组成，从而填补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染色质组成的变化和不同的衰老模型。具体来说，我将开发蛋白质组学工作流程，允许精确量化I)复杂的组合组蛋白修饰，ii)蛋白质与染色质或核质的差异结合，以及iii)染色质上的蛋白质-蛋白质相互作用。进一步的目标是测试资源的可用性，以识别和验证调节细胞衰老的发生或维持的染色质因子。细胞衰老与组织损伤后的再生、机体老化、肿瘤进展以及化疗后的复发有关，因此，确定调节这一过程的机制将具有强烈而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.