Typas, D. | bioRxiv Science

bioRxiv2024

Targeted disruption of linkage-specific ubiquitylation reveals a key role of K29-linked ubiquitylation in epigenome integrity

Linkage-specific ubiquitin chains dictate the functional outcome of numerous critical ubiquitin-dependent signaling processes. However, the functions and targets of several poly-ubiquitin topologies remain poorly defined due to a paucity of tools for their specific detection and manipulation. To remedy this knowledge gap, we applied a cell-based ubiquitin replacement strategy enabling targeted conditional abrogation of each of the seven lysine-based ubiquitin chain types in human cells to profile system-wide impacts of disabling formation of individual chain types. Focusing on K29-linked ubiquitylation, we reveal a strong association of this linkage type with chromatin-associated proteins and show that the H3K9me3 methyltransferase SUV39H1 is a prominent cellular target of this modification. We demonstrate that K29-linked ubiquitylation is essential for 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. Preventing K29-linked ubiquitylation-mediated control of SUV39H1 stability deregulates the H3K9me3 landscape, but not other histone marks. Collectively, our ubiquitin replacement cell line panel and datasets provide valuable resources for illuminating cellular functions of linkage-specific ubiquitin chains and establish a key role of K29-linked ubiquitylation in epigenome integrity.

cell biology↗

bioRxiv2023

VCF1 is an unconventional p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates

The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discovered that FAM104A, a protein of unknown function that we named VCF1 (VCP/p97 Cofactor FAM104 1), acts as a novel p97 cofactor in human cells. Detailed structure-function studies revealed that VCF1 directly binds p97 via a conserved novel -helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.

molecular biology↗