Mechanism of chromatin accessibility, 3D chromosome organization, and their functions in gene regulation

Project Summary / Abstract The overall theme of my research program is to unravel the mechanisms underlying chromatin structure and understand its role in gene regulation. Our approach tackles this challenge on two interconnected levels: at the chromatin / nucleosome level, we investigate the mechanisms driving nucleosome depletion and chromatin accessibility, while at the higher-order chromosome level, we explore the structure and dynamics of 3D genome organization and its functions in various nuclear processes, particularly transcriptional regulation. Over the past five years, primarily supported by the previous R35, we have made significant progress in both areas, yielding valuable new insights and innovative methodologies. We have also expanded our research from budding yeast to mammalian cells. These advances serve as the foundation for this proposal. Theme1: Pioneer factors (PFs) can invade into nucleosomes and increase chromatin accessibility near their binding sites and often serve as master regulators for gene expression during differentiation. Despite their essential functions, only a handful of PFs have been identified, and the mechanism underlying the pioneer activity remains poorly understood. The long-term goal of this theme is to systematically identify PFs and characterize their pioneer activities in health and disease cells, and to develop a full mechanistic understanding of how they access their nucleosomal binding sites, generate accessible chromatin, and regulate gene expression. Our recent studies have shed light on the mechanisms of PF invasion, their coordination with chromatin remodelers, and their targeting specificity. In the next five years, we plan to 1) Identify PFs responsible for generating constitutive open regions in human cells using integrated synthetic oligo (ISO) technology, 2) investigate the mechanism of PF binding cooperativity, 3) further our understanding of PF/TF specificity towards chromatin remodelers, and 4) explore intriguing connections between cohesin and PF/TF nucleosome invasion. Theme2: Imaging and 3C-based methods have revealed intricate chromosome folding patterns with extensive long-range intra- and inter-chromosomal interactions, yet the functional significance of these 3D chromosomal contacts remains elusive. The long-term goal of this theme is to understand the formation mechanism of high- order chromosome structures and dissect their functions in gene regulation and other nuclei processes. Recently, we developed innovative methods for detecting and manipulating chromosomal interactions in yeast, and uncovered connections between TF condensates, 3D genome organization, and gene regulation. Building on these advancements, we plan to extend one of our methods, chemically induced chromosomal interactions (CICI), from budding yeast to mammalian cells. This approach will allow us to study chromosome dynamics across various 3D structures, and explore the relationship between heterochromatin condensates, genome organization, and gene repression.