Investigating the Dynamic Composition, Subcellular Localization, and Functions of the Human C-terminal to LisH Complex
Abstract
The attachment of ubiquitin to protein substrates regulates cellular processes by controlling protein turnover and/or function. In this process, E3-ubiquitin ligases confer substrate specificity by coordinating the transfer of ubiquitin to – most commonly – lysine residues on bound substrates.
Recent studies demonstrate that the C-terminal to LisH (CTLH) Complex has E3 ligase activity; however, although its individual subunits have previously been implicated in various cytoplasmic and nuclear regulatory networks, a comprehensive assessment of CTLH complex function in these compartments has not been performed. This thesis investigates the composition, subcellular localization, and functions of this under-studied human E3 ligase.
I show that the CTLH complex forms different molecular weight complexes in nuclear and cytoplasmic fractions. Loss of the WDR26 subunit severely destabilizes nuclear CTLH complex subunits – including scaffolding protein RanBP9 – and impairs higher-order CTLH complex formation, revealing WDR26 as a critical determinant of CTLH complex nuclear stability. In addition, over 170 compartment-specific interactors involved in various conserved biological processes, such as ribonucleoprotein biogenesis and chromatin assembly, were identified by affinity-purification coupled to mass spectrometry of endogenous RanBP9.
I then demonstrate that CTLH complex activity is dictated by a dynamic exchange between WDR26 and the structurally similar subunit muskelin. The formation of separate WDR26 and muskelin complexes correlated with distinct proteomes in WDR26 and muskelin knockout cells. We then find that mTOR inhibition induces muskelin-associated CTLH complex-mediated degradation of HMGCS1 and autodegradation, likely as an autoregulatory feedback mechanism to regulate CTLH complex activity.
Finally, I show that the WDR26-containing CTLH complex has a protective effect on RanBP9 chromatin association and regulates chromatin accessibility and gene expression. The WDR26 KO transcriptome revealed over 2000 differentially expressed genes, many of which are involved in processes that CTLH subunits have been previously implicated in. Lastly, we find that the genes normally regulated by AP-1 and NF-1 transcription factor families in WDR26 KO cells are differentially expressed due to changes in their chromatin accessibility, implicating the CTLH complex in chromatin regulation.
Overall, this work demonstrates that CTLH composition is dynamic, leading to different functions in distinct cellular locations.