Super-resolution measurements of hematopoietic stem cell chromatin using expansion microscopy

Abstract

Proper hematopoiesis, the formation of all blood and immune cell types (e.g., red blood cells, T cells, etc), is crucial for a healthy human being. Hematopoietic stem and progenitor cells (HSPCs) establish distinct lineage-specific gene programs to give rise to various blood and immune cell types. Gene expression is regulated by many different epigenetic mechanisms, such as changes in histone post-translational modifications (i.e., histone marks) and three-dimensional (3D) chromatin structure. Sequencing methods have identified a bivalent chromatin state, marked by two histone marks of opposing functions. Bivalent chromatin is characterized by the presence of H3K4me3, an active transcription mark, and H3K27me3, a histone mark of transcription repression. Developmental genes in particular are enriched in this bivalent chromatin state in stem cells. However, sequencing methods are unable to characterize the 3D organization of bivalent domains around in the native 3D conformation of a single-cell nucleus. In this dissertation, I sought to create new measurements to describe chromatin features using an imaging approach. Chapter 1 introduces concepts of gene regulatory mechanisms and their impact on hematopoiesis, as well as methodologies to study the aforementioned concepts. Chapter 2 introduces a quantitative imaging framework to characterize the spatial organization and formation of bivalent chromatin domains in single cells, establishing new metrics that link that attempt to link gene regulatory features associated with bivalency to their underlying three-dimensional chromatin structure. Chapter 3 contains supplementary information for Chapter 2.