Abstract
Developing thymocytes require pre-TCR signalling to differentiate from CD4-CD8- double negative to CD4+CD8+ double positive cell. Here we followed the transcriptional response to pre-TCR signalling in a synchronised population of differentiating double negative thymocytes. This time series analysis revealed a complex transcriptional response, in which thousands of genes were up and down-regulated before changes in cell surface phenotype were detected. Genome-wide measurement of RNA degradation of individual genes showed great heterogeneity in the rate of degradation between different genes. We therefore used time course expression and degradation data and a genome wide transcriptional modelling (GWTM) strategy to model the transcriptional response of genes up-regulated on pre-TCR signal transduction. This analysis revealed five major temporally distinct transcriptional activities that up regulate transcription through time, whereas down-regulation of expression occurred in three waves. Our model thus placed known regulators in a temporal perspective, and in addition identified novel candidate regulators of thymocyte differentiation.
Highlights
In this study we investigate the whole genome changes in gene transcription in response to a developmental stimulus through time, in a synchronized population of differentiating cells, in their physiological environment
Ragdeficient thymocytes arrested at DN3 can be induced to differentiate to double positive (DP) cell by anti-CD3 treatment, and this has been used extensively experimentally to investigate pre-TCR function and study the transition of developing cells in a synchronous manner [23]
Such cultures have been widely used to study this developmental transition, we first validated our experimental system, and tested if the changes in transcription measured by microarray through time, mirrored those observed in phenotypically defined differentiated thymocyte populations (DN3, DN4, DP)
Summary
In this study we investigate the whole genome changes in gene transcription in response to a developmental stimulus through time, in a synchronized population of differentiating cells, in their physiological environment. We followed the transcriptomes of developing thymocytes in response to pre-TCR signal transduction, under conditions where differentiation is synchronous. T lymphocytes develop in the thymus, which provides an essential environment for T cell fate specification, and for the differentiation of multipotent progenitor cells into functional T cells. This cellular developmental programme has been well defined in terms of stepwise changes in cell surface markers and rearrangement of TCR gene loci, as the developing thymocytes move through different thymic microenvironments. Notch signals maintain survival of early DN cells until the DN3 stage, but following pre-TCR signalling, they are abruptly down-regulated as Id3 protein rapidly rises and supresses E2A-mediated Notch [6]
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