A gene regulatory network armature for T lymphocyte specification

Abstract

Choice of a T lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification, tests of the short-term Notch dependence of these gene expression changes, and analyses of the effects of overexpression of two essential transcription factors, namely PU.1 and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through which T cell precursors progress from primitive multipotency to T lineage commitment. Our analyses reveal separate contributions of Notch signaling, GATA-3 activity, and down-regulation of PU.1. Using BioTapestry (www.BioTapestry.org), the results have been assembled into a draft gene regulatory network for the specification of T cell precursors and the choice of T as opposed to myeloid/dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfi1 against Egr-2 and of TCF-1 against PU.1 as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose dependence of GATA-3 effects, the gene-specific modulation of PU.1 activity based on Notch activity, the lack of direct opposition between PU.1 and GATA-3, and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.

Fig. 1.

Gene expression changes during normal T lineage specification and regulatory perturbation: depiction in principal component space. (A) Coordinates of key genes (black or magenta stars) and gene expression signatures of DN1, DN2, DN3a, DN3b, and DN4 stages, projected on axes representing the first two principal components of gene expression change (full data in Fig. S1 and Tables S2–S4). (B) Targets of GATA-3 overexpression in fetal thymocytes, positioned relative to first two principal component axes shown in A (14). The coordinates of the normal, adult DN1–DN4 phenotypes are shown for orientation by blue vectors. Genes positively affected by GATA-3 overexpression are targets of green arrows, with negatively affected genes indicated by red arrows. (C) Targets of PU.1 overexpression in fetal thymocytes (13), depicted as in B. (D) Effects of short-term exposure to Notch–Delta signaling in fetal thymocytes. These effects, compiled from refs. and , are calculated independently of effects of PU.1 or GATA-3 in the experiments. (E) Supraadditive modulation of PU.1 effects by Notch–Delta signaling (P < 0.05; Tables S5 and S6). Genes that are protected from repression supraadditively by Notch–Delta signaling are shown in green. A gene that is prevented from up-regulation by Notch–Delta signaling is shown in red.

Fig. 2.

Direct Notch regulation of early T cell genes compared with developmental regulation during the DN1 to DN3 transitions. Line graphs show the log₁₀ of the ratio of expression of indicated genes in adult DN3a cells relative to DN1 cells in two independent studies (7, 8) (E.-S.D.-F. and M.A.Y., unpublished results); breaks indicate genes not included. Bars show the effect of 24-h Notch–Delta signaling on gene expression in Thy-1⁺ embryonic day 15.5 fetal thymocytes after a 16-h preculture without Delta. Secondary y axis gives log₁₀ of the expression ratio with and without restored Notch–Delta signaling. For additional results see Fig. S3. Data are from empty vector controls in ref. .

Fig. 3.

Gene regulatory network model for T cell specification. (A) View from all nuclei: comprehensive map of relationships included in the network, integrating over all stages. For sources of each link, see Table S7. For expanded size figure, see Fig. S4A. For predicted differential activity of different network links at different stages, see Fig. S4 G–K. (B) Close-ups of one region of the network with background highlighting indicating differential gene expression levels at five different developmental states. For full network versions, see Fig. S4 B–F.