Chromatin Barriers Impacting Liver Development and Regeneration - Summary The goal of this proposal is to investigate how changes in transcription factor and heterochromatin states in embryos and the adult liver impact hepatocyte development, homeostasis, and regeneration. To do so we will use new in vivo imaging technologies and genetic perturbations of heterochromatin to elucidate the molecular interactions that drive cell fate specification. Previous work from the Zaret and Mir laboratories have revealed that the interplay between transcription factor kinetics and heterochromatin limits cell plasticity in model systems. We will use our new approaches to understand how ventral-lateral and -medial domains of the embryonic endoderm induce hepatic fates and the morphologic transitions that form the liver bud. We will similarly investigate, in wild type and heterochromatin mutant adult livers, how differential heterochromatin states and transcription factor kinetics in the periportal, midlobular, and pericentral liver zones impact homeostasis and regenerative responses. Although single-cell genomics have revealed heterogeneity of embryonic hepatocytes and of cells in each zone of the adult liver, the functional significance is unclear. We propose that quantifying regulatory protein and chromatin kinetics within living wild type and heterochromatin mutant embryos and adult liver will provide novel insight into the basis for two origins of the liver and the functional nature of zonal heterogeneity. The Zaret lab showed that heterochromatin promotes liver cell stability in mouse development and identified diverse heterochromatin proteins that repress liver genes in adult cells. Using single-molecule tracking in cultured liver cells they showed that the pioneer factors FOXA1/FOXA2 scan heterochromatin, whereas the liver differentiation factor HNF4 is restricted to open chromatin. The Mir lab engineered high- resolution light-sheet technologies to image transcription factor and chromatin kinetics within specific nuclear domains in live Drosophila and pre-implantation mouse embryos. Mir discovered how transient high- concentration hubs of transcription factors at target genes regulate zygotic gene expression in fly embryos. We will couple our laboratories’ expertise, as seen by our preliminary data showing that liver regulatory proteins and chromatin can be genetically perturbed and imaged at high resolution in live developing mouse embryos and in adult liver slices. We ask in Aim 1: How do changes in heterochromatin, and the molecular kinetics and sub- nuclear clustering of transcription factors and chromatin components that interact with it, dictate how fields of endoderm cells acquire liver fates? Aim 2: How do protein kinetics and heterochromatic features impart different homeostatic capacities in the three zones of the adult liver lobule and the abilities of the periportal and pericentral zones to respond to liver damage? By bringing together state-of-the-art live imaging of embryos and liver slices with genetic perturbation, we will reveal how heterochromatin states and chromatin and transcription factor kinetics impact development in two domains of endoderm, liver homeostasis, and regeneration. The work will inform new ways to make hepatic cells from pluripotent cells and paradigms for other gut-derived tissues.
