Role of nucleosome architecture in cellular reprogramming

PROJECT SUMMARY Enhancer formation at precise chromatin loci by transcription factors enables appropriate gene activation to biological demand. This essential process is challenged by the millions of matches to transcription factor binding motifs present in the human genome. Chromatin structure is also known to act as a physical barrier for many transcription factors. How do transcription factors overcome chromatin barriers and selectively activate appropriate genomic loci while they are competing against chromatin barriers? The answer to this fundamental question is still largely unknown. Recently, a subset of transcription factors, so-called pioneer factors, have been identified as essential proteins required to modify chromatin accessibility during cellular reprogramming. Unlike other transcription factors, pioneer factors are capable of binding to nucleosomes at closed (inactive) chromatin sites and inducing chromatin opening. Since nucleosome formation was thought to act as a physical barrier, identification of pioneer factors’ action on chromatin is fundamental to understanding the cellular reprogramming processes. Misregulation of pioneer factors is associated with various human diseases including cancer. Therefore, there is a critical need to identify the molecular mechanisms underlying pioneer factor-induced cellular reprogramming. Our long-term goal is to elucidate the molecular mechanisms of transcription factor-induced cellular reprogramming. We previously demonstrated that GATA3 acts as a pioneer factor that can directly bind closed chromatin and activate epithelial marker genes during the mesenchymal-to-epithelial transition (MET) in triple-negative breast cancer cells. Using this cellular reprogramming model system, we further identified the unique nucleosome positioning enriched at GATA3 target loci. Our Cryo-EM structure analysis of the GATA3-nucleosome complex revealed that the motif recognition by GATA3 on the nucleosome differs from histone-free DNAs. Chromatin structural analysis during MET also identified that chromatin opening and de novo enhancer formation by GATA3 is site-specific and only observed at a subset of GATA3 binding sites. Based on this evidence, we hypothesize that nucleosome positioning and structure at the pioneer factor GATA3 binding site dictate the readout (chromatin opening and gene activation) of GATA3 binding and is directly involved in MET. The primary goals of this application are to: (1) identify specific nucleosome positioning and conformation that are essential for successful GATA3- induced cellular reprogramming; (2) identify the roles of chromatin remodeling enzymes during GATA3- induced MET, and; (3) discover minimum chromatin components for GATA3-induced enhancer formation. We will use several novel approaches including high-resolution mapping methods for nucleosome positioning and transcription factors’ footprint. These results will provide a basis for understanding cellular reprogramming processes induced by transcription factors.