Identifying the substrates and mechanisms of ubiquitin E3 ligases that shape the heterochromatin landscape in the fission yeast S. pombe

真核细胞的遗传信息被组织成一个称为染色质的高阶结构，染色质被划分为不同的功能域，称为常染色质和异染色质。我以前的研究揭示了染色质结构域如何通过染色质相关因子的选择性泛素依赖性降解（“染色质雕刻”）而受到限制的新范式。这一机制描述了一个因子如何最初被募集到染色质上，随后被一个保守的泛素连接酶以一种空间受限的方式降解，从而导致其特定的染色体分布，从而有助于染色质结构域的身份。其他泛素连接酶在染色质形成中也有作用，这表明存在类似的机制。然而，这些连接酶的具体功能和相应的底物尚不清楚，并且这些调控途径如何促进异染色质动力学的时空网络尚不清楚。识别这些底物，阐明它们的功能，并将这些途径映射到时空调节网络，对于理解染色质结构域如何形成以及它们的三维组织如何控制基因表达至关重要。过去，由于现有方法的局限性，解决这些问题一直受到阻碍。我们建议结合遗传筛选和先进的蛋白质组学来克服这些挑战，通过(1)剖析决定染色质相关泛素依赖性降解空间控制的信号和潜在机制；(2)鉴定与异染色质形成有关的其他泛素连接酶的底物，并研究它们对染色质结构域形成的贡献。解决这些目标将阐明泛素化如何有助于染色质结构域的形成以及三维组织如何控制基因表达的调节。

The genetic information of eukaryotic cells is organized into a higher-order structure called chromatin that is partitioned into distinct functional domains referred to as euchromatin and heterochromatin. My previous studies revealed a new paradigm of how chromatin domains are confined through selective ubiquitin-dependent degradation of a chromatin-associated factor ('chromatin sculpting'). This mechanism describes how a factor is initially recruited to chromatin and subsequently degraded in a spatially confined manner by a conserved ubiquitin ligase, resulting in its specific chromosomal distribution that contributes to the identity of the chromatin domain. Other ubiquitin ligases have been previously identified with roles in chromatin formation, suggesting the existence of similar mechanisms. However, the specific functions and the corresponding substrates of these ligases are unknown, and how these regulatory pathways contribute to the spatiotemporal network of heterochromatin dynamics is poorly understood. Identifying these substrates, elucidating their functions and mapping these pathways to a spatiotemporal regulatory network will be critical to understand how chromatin domains are formed and how their three-dimensional organization controls gene expression. In the past, addressing these questions has been hampered by the limitations of the available methods. We propose to overcome these challenges with a combination of genetic screens and advanced proteomics, by (1) dissecting the signals and underlying mechanisms that determine the spatial control of chromatin-associated ubiquitin-dependent degradation; (2) identifying the substrates of other ubiquitin ligases implicated in heterochromatin formation and investigating their contribution to the shaping of chromatin domains.Addressing these aims will shed light on how ubiquitylation contributes to the shaping of chromatin domains and how the three-dimensional organization controls the regulation of gene expression.