Abstract
dc:description.abstractFOXA1 is a lineage-defining transcription factor called pioneer factor. Due to its role in recruitment of nuclear receptors, such as estrogen receptor α (ERα) to enhancer regions, it is highly implicated in ERα positive breast cancer. Structural predictions of FOXA1 suggest a highly disordered structure with no clear pockets for rational drug design, making small-molecule inhibition challenging. The identification of upstream regulatory circuitry of FOXA1 would provide potential new avenues for targeting drug-resistant breast cancer. To identify proteins influencing FOXA1 protein stability, we coupled whole-genome CRISPR screening with a reporter system expressing a FOXA1-fluorophore fusion. Using this approach, we identified a mild FOXA1 stabiliser – PIGS, a poorly characterised protein involved in GPI anchor biosynthesis in the endoplasmic reticulum. In breast cancer, PIGS is amplified in ~5% of patients and its upregulation correlates with decreased survival. Knockdown of PIGS leads to dramatic phenotypic and global transcriptional changes. Proteomic analysis revealed that PIGS primarily localises to the nucleus, where it interacts with chromatin-associated factors and modulates FOXA1 interactions with SWI/SNF complex members and other nuclear cofactors. PIGS depletion also impacts chromatin binding of both FOXA1 and ERα. To identify factors influencing FOXA1 protein levels, we conducted focused CRISPR screens coupled to FACS sorting based on endogenous FOXA1 staining. These screens identified several regulators, including NXF1, a nuclear RNA export factor. RNAscope analysis showed that NXF1 controls FOXA1 protein levels via regulation of FOXA1 mRNA nuclear export. Mechanistic experiments further revealed that NXF1 associates with nascent RNA co-transcriptionally on chromatin. Unbiased proteomics confirmed FOXA1 as one of the most affected proteins following short-term NXF1 modulation. As an alternative strategy to uncover upstream regulators of FOXA1, we employed FOXA1 mutants with impaired chromatin binding to identify interactors that change depending on FOXA1 chromatin association, highlighting candidates with potential roles in modulating FOXA1 activity. In parallel, we investigated the impact of naturally occurring FOXA1 isoforms as an additional regulatory layer that could be mediated by differential isoform expression. We identified a transcript variant arising from an alternative transcription start site that produces a FOXA1 isoform lacking the first 33 amino acids (ΔN33). This variant displayed altered chromatin binding and reduced interaction with key cofactors such as the androgen receptor, suggesting that the N-terminal region contributes to the stability of FOXA1 protein-protein and DNA interactions. Overall, by integrating functional genetic screens, proteomics, and mechanistic studies, we reveal new insights into upstream regulation of FOXA1. These findings may guide the development of future therapeutic strategies targeting FOXA1 and the downstream transcriptional events in breast cancer.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2025
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Sadzikowska, Krystyna
- Advisor dc:contributor.advisor
-
- Carroll, Jason
Subjects
dc:subject × 3Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.126659
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/397554