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

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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.