Transcription Factor Gene Fluorescent Reporters Track Lineage Fate in Lymphoid Commitment

The transition from multipotency to lineage commitment can be followed with particular clarity for T cell precursors. In this lineage, the role of environmental signals can be clearly separated from the role of intrinsic fate programming in individual cells and the cells' developmental responses to changing conditions and can be tracked in real time. T cell precursors are still multipotent when they first enter the thymus, and if they are removed from the thymic microenvironment at this stage they can give rise to non-T cells including dendritic cells and myeloid cells. For multiple cell divisions, they preserve this multipotency and are only kept in line to become T cells conditionally, by Notch signaling from the thymic stroma. Then at a specific point of no return, the cells become unable to give rise to anything except T cells regardless of environment, and this is the point of commitment. Commitment is clearly the readout of a change in internal transcriptional regulatory state. To determine how this is controlled, we and others have charted transcription factor expression changes across this interval and changes in chromatin modification and DNA accessibility that accompany the transition, and we have been able to use functional perturbation tests to narrow down the key regulators that catalyze and enforce this transition. A particularly important commitment factor is encoded by the Bcl11b gene, which is released from previously repressed chromatin and sharply activated at the transcriptional level just as the cells become committed. The Bcl11b gene product is required in all alpha beta and most gamma delta T cells to enable the commitment process to occur. These properties make it highly illuminating as an indicator of the regulatory state in individual differentiating T-cell precursors. We have generated a series of knock-in Bcl11b fluorescent reporter alleles to probe the correlation of Bcl11b expression with changes in specific target genes, to determine the transcription factor requirements for Bcl11b gene activation in the gene regulatory network controlling commitment in single cells, and to measure the role of epigenetic modification of the Bcl11b locus on the kinetics of transition from uncommitted to committed states. These results and their implications will be presented. Importantly, the use of live-cell reporters reveals a level of all-or-none, stochastic regulation in the responses of individual cells to combinatorial transcription factor action at this developmental watershed¹.

Reference:

1. Kueh HY, Yui MA, Ng KKH, et al. Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment. Nature Immunology. 2016.17, 956-965.