Date of Award

December 2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Motoki Takaku

Abstract

In eukaryotic cells, chromatin acts as a physical barrier for many transcription factors, preventing them from efficiently binding to their recognition sequences within the nucleosome. Pioneer factors are a class of transcription factors that bind to nucleosomes and activate silent chromatin. Pioneer factors are frequently involved in various immune cell pathways, tissue development, and homeostasis, thereby underscoring their important roles in development and disease. Previous research found that pioneer factors have at least three fundamental properties: 1) they can interact with their cognate recognition sequences before transcription activation, 2) they can increase local chromatin accessibility, and 3) they have integral roles in lineage establishment. However, the molecular mechanisms of pioneer factor-mediated cellular reprogramming are largely unknown. For this study, I use GATA3-mediated MET (mesenchymal to epithelial transition) to study pioneer factor-mediated cellular reprogramming. GATA3 has been implicated in various cellular processes such as proliferation, migration, and invasion and is a critical regulator in breast cancer, including triple-negative breast cancer (TNBC). In TNBC, GATA3 expression is often downregulated, correlating with poor prognosis. Our lab and others have shown that GATA3 functions as a pioneer factor that actively changes the chromatin state from closed to open. In luminal breast cancer cells, ER-alpha and FOXA1 are well-known GATA3 co-factors. In mesenchymal breast cancer cells, exogenous expression of GATA3 can suppress tumor metastasis by inducing MET in the absence of well-known GATA3 co-factors FOXA1 and ER-alpha [114]. We have been studying how GATA3 activates silent chromatin. More than 7 million GATA3 motifs exist in the human genome, yet the experimental data from GATA3 ChIP-seq analysis indicates less than 1% of the motifs are occupied by GATA3 [80]. Based on the previous genomics data, GATA3 action on chromatin is site-specific (context-dependent) and only induces chromatin opening and enhancer formation at a subset of binding sites, suggesting that GATA3 might work with other co-factors. To identify a novel co-factor that is involved in GATA3-mediated MET, we performed a Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) assay [86], which identified the chromatin-modification enzyme, PARP1, as a potential GATA3 co-factor. Previous studies have shown PARP1 as a co-factor of the pioneer factor, SOX2 [45]. Our overall goal was to uncover the roles of GATA3-PARP1 interaction in breast cancer with our central hypothesis being that PARP1 is required for successful GATA3-induced MET. We break this down into three sections: 1) Determine the function of PARP1 in the selective nucleosome binding of GATA3. 2) Determine the function of the GATA3-PARP1 complex on chromatin opening. 3) Determine the role(s) of the PARP1-GATA3 interaction in gene activation (Figure 1).

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