Abstract Master transcription factors regulate cell-type-specific gene expression to define cellular identities. One such gene, GATA3, is a key regulator of multiple cellular programs, including T lymphocyte development, mammary luminal epithelial cell differentiation and trophoblast development. Recently, comprehensive genomic analysis has identified GATA3 as one of the most frequently mutated genes in breast cancer. It is also known that GATA3 expression levels directly correlate with favorable prognosis. These findings strongly suggest that GATA3 plays a critical role in tumorigenesis. However, the molecular mechanism(s) underlying GATA3-mediated gene regulation in breast cancer cells is not clearly defined. GATA3 participates in a complicated regulatory network with FOXA1 and ER-alpha, governing the transcriptional program in luminal tumors. Biochemical analyses indicate that: (1) GATA3 binds to chromatin in an estrogen-independent manner, (2) GATA3 acts upstream of FOXA1. These studies suggest GATA3 may act as a pioneer factor, which is capable of independently associating with closed chromatin and modulating chromatin structure to establish an active enhancer. In order to investigate GATA3 function as a pioneer transcription factor, we chose the MDA-MB-231 breast cancer cell line, which is GATA3, FOXA1 and ER-alpha negative, and established stable cell lines expressing wild-type GATA3 or GFP as a control. Consistent with previous results, GATA3-expressing cells represented an epithelial phenotype at the cellular and molecular level. To determine whether GATA3 can direct reprogramming of chromatin conformation, we performed genome-wide analyses of the chromatin binding activity of GATA3 and its impact on histone modifications and chromatin structure. We will present recent results describing how GATA3 licenses enhancer function to direct the luminal transcriptional program. Citation Format: Motoki Takaku, Sara A. Grimm, Takashi Shimbo, Lalith Perera, Shinichi Machida, Hitoshi Kurumizaka, Paul A. Wade. GATA3 modulates chromatin structure to establish active enhancers in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 964. doi:10.1158/1538-7445.AM2015-964