Profiling linkage-specific ubiquitin chain functions reveals a key role of K29-linked ubiquitylation in epigenome integrity

Linkage-specific ubiquitin (Ub) chains dictate the functional outcome of many critical Ub-dependent signaling processes. However, the functions and targets of several poly-Ub topologies remain poorly understood due to a lack of tools for their specific detection and manipulation. To remedy this knowledge gap, we applied a cell-based Ub replacement strategy enabling specific, conditional abrogation of each of the seven lysine-based Ub chain types in human cells to comprehensively profile global ubiquitylation changes resulting from disabling formation of individual Ub chain types. Focusing on K29-linked ubiquitylation, we found that this linkage type is enriched among chromatin-associated proteins, and that the H3K9me3 methyltransferase SUV39H1 is a prominent cellular target of this modification. We demonstrate that K29-linked ubiquitylation is essential for the proteasomal degradation of SUV39H1 despite its extensive modification by K48-linked ubiquitylation, and that K29-linked ubiquitylation of SUV39H1 is catalyzed and reversed by TRIP12 and TRABID, respectively. Importantly, preventing K29-linked ubiquitylation of SUV39H1 markedly increases H3K9me3 levels, but not other histone marks. Collectively, our Ub replacement cell line panel and datasets provide valuable resources for illuminating cellular functions of linkage-specific ubiquitylation processes and reveal a key role for K29-linked ubiquitylation in epigenome integrity.