Mammalian stem and progenitor cells activate the expression of specific regulatory genes to establish and stabilize different cell fates, but the mechanisms and general principles controlling this process are not well understood. Cells utilize fate-specifying regulatory genes to establish and maintain distinct fate identities during development, but it is not clear how they activate and maintain the expression of these genes to establish fate identity. Here, I propose to study this question in the context of two systems: T-cell fate commitment, which is driven by the activation of the T-cell specific transcription factor Bcl11b (Aims 1 and 2);and macrophage development, which we recently found is driven by the cell-cycle length dependent accumulation of the myeloid transcription factor PU.1 (Kueh et al., 2013)1 (Aim 3). Using these two systems, I will test a number of widely debated ideas in the field of developmental gene regulation. First, I will test the idea that developmental signals directly activate the expression of regulatory genes to instruct cell fate (Aim 1). Next, I will tet two proposed classes of mechanisms for stabilizing regulatory gene expression and fate identity: cis-acting mechanisms involving stable and heritable epigenetic modifications at regulatory gene loci (Aim 2);and trans- acting mechanisms involving self-reinforcing positive feedback loops on regulatory gene expression (Aim 3). My main approach will be to use timelapse live-cell imaging to track the expression dynamics of Bcl11b during T-cell development, and PU.1 during macrophage development. As cell differentiation is a dynamic and intrinsically heterogeneous process, single-cell tracking by timelapse imaging will reveal insights that are difficult to obtain with conventional discrete time-point population measurements. To gain mechanistic insights, I will perturb the mechanisms under investigation, and measure their resultant effects using timelapse imaging. These perturbations will involve over-expression or knockdown of genes;for studies of cis-epigenetic mechanisms, I will also develop a CRISPr-based system for perturbing chromatin marks at specific sites in the genome. To better understand this experimental data, and generate predictions for future experiments, I will then use mathematical modeling to analyze the behavior and dynamics of the different regulatory mechanisms studied. Finally, I will complement these approaches with genome-wide measurements of gene expression states in developing cells using high throughput sequencing, which will provide a more global picture of developmental changes, and potentially yield new directions for future work. Through these studies, I hope to uncover fundamental insights into how mammalian cells establish and maintain their distinct fate identities. These insights will potentially help us develop new therapies for leukemia and other cancers, and help us better manipulate stem cells for regenerative medicine.
The goal of this study is to understand how mammalian stem and progenitor cells make and stabilize cell fate decisions, using immune cell development as a model system. Insights gained from study will potentially help us manipulate stem cells for regenerative medicine, and develop more effective treatments for leukemia, which arises when blood cells fail to stabilize differentiated cell identities.
|Longabaugh, William J R; Zeng, Weihua; Zhang, Jingli A et al. (2017) Bcl11b and combinatorial resolution of cell fate in the T-cell gene regulatory network. Proc Natl Acad Sci U S A 114:5800-5807|
|Manesso, Erica; Kueh, Hao Yuan; Freedman, George et al. (2016) Irreversibility of T-Cell Specification: Insights from Computational Modelling of a Minimal Network Architecture. PLoS One 11:e0161260|
|Kueh, Hao Yuan; Yui, Mary A; Ng, Kenneth K H et al. (2016) Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment. Nat Immunol 17:956-65|
|Rothenberg, Ellen V; Kueh, Hao Yuan; Yui, Mary A et al. (2016) Hematopoiesis and T-cell specification as a model developmental system. Immunol Rev 271:72-97|