This project is to exploit recent advances that can help break open the gene network circuitry that mediates a pivotal stage in T cell development. T cell development is a model for the mechanism of cell fate choice starting from a physiological pool of multipotent stem cells. Mammalian T-cell development permits highly refined dissection of the process of lineage commitment, because in this system one can isolate intermediate precursors with progressive degrees of developmental potential restriction in high purity. Excellent in vitro systems for T-lineage differentiation enable the whole lineage choice process to occur in an open, experimentally accessible way. Specific gene knockout experiments have also identified a group of transcription factors needed for T-cell specification, including GATA-3 and E proteins. Based on gene regulatory effects measured in short-term transcription factor perturbation experiments, we have published a provisional gene network model for T-cell development. However, to explain the properties of the commitment process, three functions were needed in the network for which the agents were not known. This proposal is based on two very recent advances which we predict will now make it possible to account for the crucial functions in the network. These enable us to see that the process consists of two separable phases, the first dominated by regulatory factors inherited from the stem-cell precursor, the second dominated by a T-cell associated factor set. First we found that Bcl11b is the long-sought T-cell specific negative regulatory component that is needed to shift cells from phase 1 to phase 2. Bcl11b is turned on only as phase 1 ends, then needed to allow commitment and to turn off the stem-cell associated regulatory genes for phase 2. Second, we have generated a major resource: a full survey of transcriptome and genome-wide epigenetic marking changes across five stages throughout the T-cell specification process. We can thus complete identification of all genes dynamically regulated during T-cell commitment, identify candidates for nearly all cis-regulatory elements where active regulatory change occurs, and correlate regulatory status marks with sites for specific transcription factor binding to predict function. We hypothesize that Bcl11b expression is a readout for successful activation of a first wave of T-cell regulators in phase 1, and that when transition to phase 2 occurs, previously expressed factors including GATA-3 and E proteins then refocus their functions to turn on new T-cell specific genes. Using Bcl11b as a wedge to split the T-cell commitment process, we will first identify the cis- and trans-elements that turn Bcl11b itself on at the completion of phase 1. Using ChIPseq mapping in normal and Bcl11b mutant cells, we will also determine how the target site binding and associated histone modifications of E proteins and GATA-3 may shift from phase 1 to phase 2. We will then use gain and loss of function to test how phase 1 or phase 2 interaction partners of E proteins and GATA-3 can cause specific shifts in their deployment.

Public Health Relevance

The T cell development process clearly reveals that when effector cells develop from stem cells, it is crucial for them to give up stem-cell like properties at a particular stage in the process, in order to acquire the proper regulation to go on to become normal differentiated cells. This project is to determine in detail how a trio of important T-cell gene regulatory factors coordinates this handover of control. The results are an important model for understanding the natural quality control checks that are built in to normal stem-cell based development, and to avoid the danger of improper biological quality control in future clinical use of stem-cell based models.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Cellular and Molecular Immunology - B Study Section (CMIB)
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Prabhudas, Mercy R
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California Institute of Technology
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Ungerbäck, Jonas; Hosokawa, Hiroyuki; Wang, Xun et al. (2018) Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1). Genome Res 28:1508-1519
Hosokawa, Hiroyuki; Ungerbäck, Jonas; Wang, Xun et al. (2018) Transcription Factor PU.1 Represses and Activates Gene Expression in Early T Cells by Redirecting Partner Transcription Factor Binding. Immunity 49:782
Hosokawa, Hiroyuki; Ungerbäck, Jonas; Wang, Xun et al. (2018) Transcription Factor PU.1 Represses and Activates Gene Expression in Early T Cells by Redirecting Partner Transcription Factor Binding. Immunity 48:1119-1134.e7
Ng, Kenneth Kh; Yui, Mary A; Mehta, Arnav et al. (2018) A stochastic epigenetic switch controls the dynamics of T-cell lineage commitment. Elife 7:
Hosokawa, Hiroyuki; Rothenberg, Ellen V (2018) Cytokines, Transcription Factors, and the Initiation of T-Cell Development. Cold Spring Harb Perspect Biol 10:
Rothenberg, Ellen V (2017) Fitting structure to function in gene regulatory networks. Hist Philos Life Sci 39:37
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
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; Ungerbäck, Jonas; Champhekar, Ameya (2016) Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control. Adv Immunol 129:109-74

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