Over the course of development, regulation of gene transcription is the main mechanism by which pluripotent stem cells become restricted to the various distinct cell types found in the mature organism. Among the many different processes that regulate gene transcription, is the control of physical access to DNA and the genes for which it codes. DNA wound around histone proteins forms chromatin and the enzymes that modify the landscape of that chromatin control which regulatory elements, like promoters and enhancers, are active. This process confers different developmental competencies in cells, enabling them to respond uniquely to similar environmental and developmental signals, regulating gene transcription in turn. The study of these processes during in vitro differentiation of stem cells has enabled us and others to draw links between chromatin remodelers, transcription factors and cellular response to inductive cues during human development.
In Chapter 1, I explore the role of the lysine-specific demethylase (LSD1) during human pancreatic development using an in vitro system to differentiate human embryonic stem cells (hESCs) to the pancreatic endocrine lineage. Removal of LSD1 activity during a specific early time window of pancreatic development prevents endocrine formation. Investigation into enhancer regions occupied by LSD1 during this critical time window provided results that support a model in which LSD1-mediated decommissioning renders these enhancers insensitive to activation by external retinoic acid signaling.
In Chapter 2, I report my previous work dissecting the role of the transcription factor neurogenin-3 (NGN3) during human pancreatic development. Using the aforementioned hESC-based in vitro differentiation system, gain and loss-of-function studies showed that NGN3 is both necessary and sufficient to induce endocrine formation in human cells.
A final supplemental chapter provides an example of a hESC-based pancreatic differentiation protocol similar to the one employed for the studies outlined in Chapters 1 and 2 and discusses the importance of such model systems in dissecting the myriad mechanisms of human disease and development.