Function of GATA3-PARP1 complex in breast cancer during GATA3-mediated cellular reprogramming

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Project Summary GATA3 is a transcription factor that acts as a pioneer factor in breast cancer cells. However, the function of pioneer factors isn’t well understood. Specifically, how they activate silent chromatin is largely unknown. The long-term goal is to provide novel insights into the molecular mechanisms of pioneer factor- mediated cellular reprogramming at a chromatin level. More than 7 million GATA3 motifs exist in the human genome, but the experimental data from GATA3 ChIP- seq analysis indicates less than 1% of the motifs are occupied by the pioneer factor, GATA3. In addition to binding selectivity, pioneer factor action, including GATA3, is known to be site-specific (context-dependent), and they can only induce chromatin opening and enhancer formation at a subset of binding sites. The overall objective of this proposal is to identify the binding specificity of the pioneer factor, GATA3, in the genome and elucidate the impact of the interaction between GATA3 and chromatin-modification enzyme, Poly ADP-ribose polymerase 1 (PARP1), on gene activation. Our preliminary data strongly suggest that PARP1 is involved in such context-dependent action of GATA3. The central hypothesis is that GATA3-mediated cellular reprogramming is dependent on the PARP1 activity. The rationale for this study is that the identification of the roles of GATA3 and PARP1 interaction will provide novel molecular mechanisms underlying the site- specific expression of pioneer factor activities during cellular reprogramming. The central hypothesis will be tested by (1) investigating the function of PARP1 in the binding selectivity of GATA3 and (2) identifying the role(s) of GATA3-PARP1 interaction in gene activation. To test this hypothesis, I will use our unique DOX- inducible system in MDA-MB-231 basal breast cancer cells. In this system, exogenous GATA3 expression initiates mesenchymal-to-epithelial transition (MET). This system recapitulates GATA3 function in patient breast tumors since GATA3 expression is known to be higher in the luminal/epithelial breast tumors, and is essential for the maintenance of epithelial phenotypes. Under the first aim, we will investigate changes in GATA3 binding across the genome in the presence and absence of PARP1. The second aim will utilize next- generation sequencing to evaluate the effects that GATA3 and PARP1 have on the steps in pioneer factor- mediated cellular reprogramming. The proposed research is innovative because we are addressing the novel concept of context-dependent enhancer formation by a pioneer factor during cellular reprogramming. This research is significant because it will fill a critical gap in our knowledge about how pioneer factors mediate cell fate transition.