Research ArticleIMMUNOLOGY

Functional definition of a transcription factor hierarchy regulating T cell lineage commitment

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Science Advances  31 Jul 2020:
Vol. 6, no. 31, eaaw7313
DOI: 10.1126/sciadv.aaw7313
  • Fig. 1 Tcf1-deficient DN3b cells show promiscuous gene expression compared to WT littermate controls.

    (A) Heat map of the top 100 differentially expressed gene as determined by RNA-seq of sorted DN3b cells from WT and Tcf1-deficient thymi. GSEA of the differentially expressed genes (Tcf1−/− KO over Tcf1 WT for DN3b) is enriched for DN2 genes (DN2a and DN2b with NES +1.23 and + 1.53, respectively). (B) qPCR validation of RNA-seq data for selected T cell–specific genes, genes expressed in non-T cells, and legacy genes whose expression is inherited from stem cells/multipotent progenitors. The levels of expression are normalized by ABL-2 expression as housekeeping gene. (Mann-Whitney U test; *P < 0.05, **P < 0.01, and ***P < 0.001. Error bars represent the SD of three pooled mice and from two independent experiments.)

  • Fig. 2 Chromatin accessibility analysis in Tcf1-deficient versus WT DN thymocytes.

    (A) Chromatin immunoprecipitation with an antibody specific for Tcf1 revealed that the Gata3 promoter and the Bcl11b enhancer are occupied by Tcf1 in vivo, whereas in Tcf1 KO DN thymocytes, no binding can be detected. Negative controls with IgG instead of anti-Tcf1 showed no enrichment. (Multiple t test. Error bars represent the SD of at least three pooled mice and from two independent experiments.) (B) Heat map of DESeq2 normalized read counts of ATAC-seq shows differentially accessible regions between WT and Tcf1−/− in DN3a and DN3b. Motif analysis was performed in the differentially accessible regions using HOMER showing the three highest scores and Tcf1 score. (C) ATAC-seq data mined for the Bcl11b, Gata3, and Trbj (T cell Receptor Beta) genomic regions. Per locus, the relative abundance of transposase accessible regions is indicated. The individual ATAC-seq profile from each genotype is shown. Data are shown as normalized read density.

  • Fig. 3 Tcf1-deficient DN3 cells dedifferentiate into DN1/2-like cells with multipotent lineage capacity.

    (A) WT DN3 cells sorted and seeded on OP9 WT/OP9-DL1 (10:1) cells develop largely further into DN4 or remain DN3 after 7 days in culture, while Tcf1-deficient cells develop into DN1 and DN2 cells (pregated Thy1+ Lin cells) with prominent B cell (B220+ CD19+) and myeloid cell (CD11b+ Gr1+) development. (B) Quantification of the developmental plasticity and dedifferentiation effects of DN3 Tcf1-deficient thymocytes into DN1, DN2, myeloid, and B cells. (C) Intracellular TCRβ staining reveals the dedifferentiated DN1 and DN2 cells to be derived from DN3 cells. (Mann-Whitney U test. Error bars represent the SD of three samples from three independent experiments.)

  • Fig. 4 Reexpression of Gata3 suppresses B and myeloid development in Tcf1 deficiency.

    (A) Layout of retroviral complementation experiments with GFP control and/or Gata3. (B) Gata3 expression partially overcomes the DN1 thymocyte block and (C) suppressed the enhanced non-T cell lineages (B and myeloid cell development) after 7 days in the OP9-DL1 culture system. FACS shows representative plots and graphs quantitative data from replicate measurements. (Multiple t test analysis. Error bars represent the SD from two independent experiments.) (D) In vivo complementation [Ly5.2 Tcf1 (WT or KO)–transduced stem cells transplanted into Ly5.1 recipient mice] reveals suppression of B cell development also in the thymus (right) 8 weeks after transplantation but minimal and partial rescue of T cell development in the thymus (left and middle). (Middle: Multiple t test analysis. Right: Paired t test. Error bars represent the SD from three individual mice per group.)

  • Fig. 5 Reexpression of Bcl11b rescues T cell development in Tcf1-deficient stem cells.

    (A) Layout of retroviral complementation experiments with Bcl11b. (B) Thy1 expression is rescued by Tcf1 deficiency by expression of Bcl11b after 14 days in OP9-DL1 culture (pregated LinGFP+ cells). (C) Bcl11b fully rescues T cell development from Tcf1−/− stem cells that otherwise are arrested in DN1 (pregated Thy1+LinGFP+). (D) Intracellular TCRβ expression can be restored in Tcf1-deficient cells by expression of Bcl11b (pregated Thy1+LinGFP+ DN subset). (E) Bcl11b overexpression does not affect myeloid and B cell development. (Two-way ANOVA. Error bars represent the SD from three independent experiments.)

  • Fig. 6 Hierarchy of the core transcription factors in immature T cell development.

    On the basis of the proven functional interactions shown in Figs. 4 and 5. Notch signaling (indicated by the open arrow symbols) induces Tcf1 expression that subsequently has two target genes: Gata3 and Bcl11b. Gata3 has a minor role in supporting development along the T cell linage but mainly acts to suppress the myeloid and B cell fates. In contrast, Bcl11b induces a T cell–specific program but has minor roles in suppressing alternative lineages with exception of NK cell development that is suppressed by Bcl11b. Collectively, there is a clear functional hierarchy of transcription factors. Potential additional roles for Runx1 and E2A are not shown here.

Supplementary Materials

  • Supplementary Materials

    Functional definition of a transcription factor hierarchy regulating T cell lineage commitment

    Laura Garcia-Perez, Farbod Famili, Martijn Cordes, Martijn Brugman, Marja van Eggermond, Haoyu Wu, Jihed Chouaref, David San León Granado, Machteld M. Tiemessen, Karin Pike-Overzet, Lucia Daxinger, Frank J. T. Staal

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