Dynamic Nucleosome Reorganization during the Pioneer Factor GATA3-Mediated Cellular Reprogramming in Breast Cancer

Transcription factors (TFs) recognize short DNA sequences to regulate gene expression. They play crucial roles in cell development and reprogramming. Eukaryotic cells have chromatin structure to package huge DNAs inside of the tiny nuclei. Therefore, TFs need to overcome this physical barrier to initiate cell differentiation and reprogramming. A subset of transcription factors, called pioneer factors, are thought to be special as they are capable of overcoming the chromatin structure by using their ability to bind to nucleosomes and induce chromatin opening. Although various pioneer factors have been identified in multiple tissues and human diseases, the fundamental mechanisms underlying their gene-regulatory actions remain elusive. Previously, we demonstrated that GATA3, one of the most frequently mutated genes in breast cancer, acts as a pioneer factor to suppress tumor growth in MDA-MB-231 human breast cancer cells by inducing mesenchymal-to-epithelial transition (MET). Importantly, although GATA3 is capable of inducing chromatin opening, we found that GATA3 can only open ~50% of closed chromatin binding sites suggesting that GATA3 action is chromatin context-dependent. Context-dependent actions of other pioneer factors have been also reported in the other experimental systems. Here, we developed a high-resolution nucleosome mapping technique (Capture MNase-seq) in human cells to capture dynamic nucleosome remodeling during the MET cellular reprogramming. Capture MNase-seq revealed unique nucleosome positioning at the pioneer factor binding sites, associated with gene activation activities of GATA3. We also found that the nucleosome formation on the pioneer binding sites is regulated by chromatin remodeling factors. The nucleosome mapping data enabled us to determine the Cryo-EM structure of the GATA3-nucleosome complex. This structure revealed that GATA3 uses its two zinc finger domains to stably bind to nucleosomes. These data suggest that the engagement of the pioneer factor in a specific spatial location on the nucleosome may dictate productivity of new enhancer formation.