Revealing the contribution of phase separation for establishing heterochromatin domains

揭示相分离对建立异染色质域的贡献

• 批准号: 419067076
• 负责人: Professor Dr. Axel Imhof
• 金额: --
• 依托单位: Forschungsbereich Physiologische Chemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/419067076?language=en
• 关键词: Revealing; contribution; phase; separation; establishing

Pericentric repeat sequences in Drosophila and mouse self-organize in the cell nucleus into distinct µm-size heterochromatin subcompartments called chromocenters. Chromocenters accumulate protein factors like heterochromatin protein 1 (HP1) and are marked by histone H3 lysine 9 trimethylation. Intact chromocenters are essential for silencing repeat transcription and genome stability. In the first funding period we studied liquid-liquid phase separation (LLPS) mechanisms for maintaining chromocenters. The results obtained with mouse fibroblasts argue against LLPS of HP1 being involved this process, in line with other studies that report the lack of an HP1 phenotype on mesoscale chromatin structure in differentiated cells. However, LLPS could be relevant for formation/disassembly of chromocenters during cellular differentiation and the cell cycle. Accordingly, we now focus on chromocenter transitions between the condensed heterochromatin state and the decondensed transcriptionally active state as well as loss of clustering of pericentric repeat sequences. This will allow us to distinguish between factors that govern chromocenter formation versus its maintenance. Furthermore, we will also broaden our analysis to include protein factors other than HP1 that are involved in this process. Since our super-resolution microscopy analysis of chromocenters points to a multi-layered granular structure with smaller subdomains we will characterize their functional properties. To reveal intermediates that form during chromocenter state transitions we will apply and further advance dCas9 based approaches that we have developed. A focus will be on the histone acetylase p300 that we found to spatially segregate into liquid-like droplets during chromocenter activation. Furthermore, we will assess if mechanisms inferred from studies of ectopically perturbed chromocenters also apply to endogenous transitions during Drosophila embryonic development and the cell cycle. Our findings from the last funding period suggest that components of the chromocenter that regulate its formation have differentially evolved in Drosophila species. We will now continue to study the influence of phase separation on hybrid viability. By comparing different chromocenter states with a combination of advanced fluorescence microscopy methods and mass spectrometry-based analysis of their proteome we will be able to address the question if the establishment/disassembly of chromocenters involves phase separated intermediates. Thus, our project with reveal how the dynamic molecular organization of chromocenters is linked to their function and shed light on the role of phase separation in this process.

果蝇和小鼠的着丝粒周重复序列在细胞核中自我组织成不同的μ m大小的异染色质亚区室，称为染色中心。染色中心积累蛋白质因子，如异染色质蛋白1（HP 1），并通过组蛋白H3赖氨酸9三甲基化标记。完整的染色中心对于沉默重复转录和基因组稳定性是必不可少的。在第一个资助期内，我们研究了液-液相分离（LLPS）维持色心的机制。用小鼠成纤维细胞获得的结果反对HP 1的LLPS参与该过程，这与其他报告在分化细胞中的中尺度染色质结构上缺乏HP 1表型的研究一致。然而，LLPS可能与细胞分化和细胞周期过程中染色中心的形成/分解有关。因此，我们现在专注于染色中心之间的凝聚异染色质状态和去凝聚的转录活性状态，以及损失的聚簇的臂间重复序列的转换。这将使我们能够区分控制色心形成和维持的因素。此外，我们还将扩大我们的分析，包括蛋白质因子以外的HP 1参与这一过程。由于我们的超分辨率显微镜分析的色心点的多层颗粒结构，较小的子域，我们将其功能特性的特点。为了揭示在色心状态转变过程中形成的中间体，我们将应用并进一步推进我们开发的基于dCas 9的方法。一个重点将是组蛋白乙酰化酶p300，我们发现空间分离成液体样液滴在染色中心激活。此外，我们将评估，如果机制推断异位扰动chromocenters的研究也适用于果蝇胚胎发育和细胞周期的内源性转换。我们在上一个资助期的研究结果表明，调节其形成的染色中心的组成部分在果蝇物种中有差异地进化。我们现在将继续研究相分离对杂交体生存力的影响。通过比较不同的色中心状态与先进的荧光显微镜方法和质谱分析的蛋白质组的组合，我们将能够解决的问题，如果建立/拆卸的色中心涉及相分离的中间体。因此，我们的项目揭示了色心的动态分子组织如何与它们的功能联系在一起，并阐明了相分离在这一过程中的作用。