Project Abstract The overall theme of my research program is to understand the formation mechanism of chromatin structure and its role in gene regulation. We plan to address this problem at two different levels: 1) at the chromatin / nucleosome level, we will identify pioneer factors (PFs) and investigate the mechanism underlying nucleosome displacement and chromatin opening, and 2) at the chromosome level, we will study the mechanism of gene regulation by high-order 3D chromosome organization. In the past five years, supported by two NIGMS R01 grants, we have made significant progress in both directions. New observations and insights we gained from these studies, as well as several novel methods we developed, form the foundation of this proposal. Theme1: Pioneer factors (PFs) can invade nucleosome and increase chromatin accessibility near their binding sites and therefore play critical roles in gene regulation. Mis-regulation of PFs is highly linked to cancer and developmental disease. Despite their essential functions, only a handful of PFs have been identified and the mechanism underlying the pioneer activity is unclear. The long term goal of this theme is to systematically characterize PFs and their pioneer activities in health and disease cells, and to develop a full understanding of the mechanism of these activities. In our recent PF studies, we have innovated an Integrated Synthetic Oligo (ISO) assay to investigate PF function in a high-throughput manner. In the next five years, we plan to 1) adapt the ISO assay into human cell lines and pluripotent stem cells to dissect the genetic rules underlying PF binding and nucleosome displacement, and 2) further our understanding of the pioneering activity by investigating the co-factors of PFs, the sequence of events during nucleosome displacement, and the kinetic rates of these events. Theme2: Imaging and 3C-based methods have revealed 3D chromosome organization with extensive long- distance chromosomal interactions. The long-term goal of this theme is to understand the formation mechanism of these high-order chromosome structures and their roles in gene regulation. Our recent studies show that long- distance chromosomal interactions contribute to gene regulation in yeast. More specifically, some allelic or co- regulated genes cluster in the 3D space, and such clustering is correlated with higher gene activities. The former case is analogous to the “transvection” phenomenon in Drosophila. These novel observations revealed a new layer of gene regulation in yeast and opened the opportunity of using the powerful genetic system to investigate the 3D genome function. Currently we have little understanding of what mediates the cluster formation and how the clusters enhance gene expression. In the next five years, we plan to 1) use an unbiased genetic screen to identify factors that negatively regulate transvection, and investigate the underlying mechanism, and 2) test the hypothesis that some activators condense though liquid-liquid phase separation, leading to the clustering of co- regulated genes and enhanced expression.

Project Narrative Mis-regulation of gene expression are highly linked to various types of human diseases. Chromatin accessibility and 3D chromosome organization play essential roles in gene regulation. By studying these phenomena, we will contribute to our understanding of disease mechanisms, as well as providing potential treatment targets.