Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Schild-Poulter, Caroline

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.

Summary for Lay Audience

Our body contains trillions of cells that all contribute to a normal lifespan. Each cell contains the same DNA; however, not all cells perform the same tasks. By regulating gene expression – or how the DNA is read to produce mRNA that will eventually be translated to functional proteins – each cell is able to establish their own identity through unique transcriptional landscapes, regardless of their identical DNA sequences.

Regulating gene expression requires the coordinated function of many classes of proteins located in the nucleus that change how DNA is organized and how it is read. The resulting transcriptional landscape is the end product of these coordinated functions and leads to cellular identity. In order to maintain proper balance between all cell types and ensure healthy development, gene expression regulators themselves need to be regulated.

One process that regulates cellular proteins – or “substrates” in this case – is ubiquitination. This process involves “tagging” substrates with ubiquitin proteins that lead to substrate destruction or changes in substrate function. Throughout the ubiquitination pathway, it is the E3 ligase that selects which substrates to tag. E3 ligases can be single proteins or exist as a group of proteins known as a complex; an example of the latter is the C-terminal to LisH complex. Previous studies focussed on individual members of this complex suggest that it may regulate gene expression, however, it is unclear whether the complex – as a whole – plays a role in regulating gene expression.

In this thesis, I determined that there are multiple versions of the CTLH complex, one of which is predicted to function in the nucleus and regulate transcription. This complex contains WDR26: a protein that, when depleted, causes severe neurodevelopmental problems that occur when gene expression is dysregulated. I also identified gene expression regulators and chromatin-bound proteins as CTLH complex interacting proteins in the nucleus. In addition, I determined that altered chromatin structure leads to dysregulated gene expression in WDR26-depleted cells. Taken together, I have determined that a particular version of the CTLH complex is required to maintain normal DNA organization and subsequent gene expression.

Download

Available for download on Friday, May 08, 2026

Share

COinS