During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Signal Transduction and Targeted Therapy Open Access 19 June 2023
Biomarker Research Open Access 24 March 2022
Multi-objective optimization reveals time- and dose-dependent inflammatory cytokine-mediated regulation of human stem cell derived T-cell development
npj Regenerative Medicine Open Access 27 January 2022
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
NCBI Reference Sequence
Rothenberg, E.V. T cell lineage commitment: identity and renunciation. J. Immunol. 186, 6649–6655 (2011).
Yui, M.A. & Rothenberg, E.V. Developmental gene networks: a triathlon on the course to T cell identity. Nat. Rev. Immunol. 14, 529–545 (2014).
Avram, D. & Califano, D. The multifaceted roles of Bcl11b in thymic and peripheral T cells: impact on immune diseases. J. Immunol. 193, 2059–2065 (2014).
Ciofani, M. et al. A validated regulatory network for Th17 cell specification. Cell 151, 289–303 (2012).
Liu, P., Li, P. & Burke, S. Critical roles of Bcl11b in T-cell development and maintenance of T-cell identity. Immunol. Rev. 238, 138–149 (2010).
Li, L., Leid, M. & Rothenberg, E.V. An early T cell lineage commitment checkpoint dependent on the transcription factor Bcl11b. Science 329, 89–93 (2010).
Ikawa, T. et al. An essential developmental checkpoint for production of the T cell lineage. Science 329, 93–96 (2010).
Wakabayashi, Y. et al. Bcl11b is required for differentiation and survival of αβ T lymphocytes. Nat. Immunol. 4, 533–539 (2003).
Inoue, J. et al. Expression of TCRαβ partly rescues developmental arrest and apoptosis of αβ T cells in Bcl11b−/− mice. J. Immunol. 176, 5871–5879 (2006).
Li, P. et al. Reprogramming of T cells to natural killer–like cells upon Bcl11b deletion. Science 329, 85–89 (2010).
Yui, M.A., Feng, N. & Rothenberg, E.V. Fine-scale staging of T cell lineage commitment in adult mouse thymus. J. Immunol. 185, 284–293 (2010).
Naito, T., Tanaka, H., Naoe, Y. & Taniuchi, I. Transcriptional control of T-cell development. Int. Immunol. 23, 661–668 (2011).
Manesso, E., Chickarmane, V., Kueh, H.Y., Rothenberg, E.V. & Peterson, C. Computational modelling of T-cell formation kinetics: output regulated by initial proliferation-linked deferral of developmental competence. J. R. Soc. Interface 10, 20120774 (2013).
Guo, Y., Maillard, I., Chakraborti, S., Rothenberg, E.V. & Speck, N.A. Core binding factors are necessary for natural killer cell development and cooperate with Notch signaling during T-cell specification. Blood 112, 480–492 (2008).
Weber, B.N. et al. A critical role for TCF-1 in T-lineage specification and differentiation. Nature 476, 63–68 (2011).
García-Ojeda, M.E. et al. GATA-3 promotes T-cell specification by repressing B-cell potential in pro-T cells in mice. Blood 121, 1749–1759 (2013).
Scripture-Adams, D.D. et al. GATA-3 dose-dependent checkpoints in early T cell commitment. J. Immunol. 193, 3470–3491 (2014).
Franco, C.B. et al. Notch/Delta signaling constrains reengineering of pro-T cells by PU.1. Proc. Natl. Acad. Sci. USA 103, 11993–11998 (2006).
Germar, K. et al. T-cell factor 1 is a gatekeeper for T-cell specification in response to Notch signaling. Proc. Natl. Acad. Sci. USA 108, 20060–20065 (2011).
Del Real, M.M. & Rothenberg, E.V. Architecture of a lymphomyeloid developmental switch controlled by PU.1, Notch and Gata3. Development 140, 1207–1219 (2013).
Panne, D. The enhanceosome. Curr. Opin. Struct. Biol. 18, 236–242 (2008).
Spitz, F. & Furlong, E.E. Transcription factors: from enhancer binding to developmental control. Nat. Rev. Genet. 13, 613–626 (2012).
Iwafuchi-Doi, M. & Zaret, K.S. Pioneer transcription factors in cell reprogramming. Genes Dev. 28, 2679–2692 (2014).
Zhang, D.X. & Glass, C.K. Towards an understanding of cell-specific functions of signal-dependent transcription factors. J. Mol. Endocrinol. 51, T37–T50 (2013).
Zhang, J.A., Mortazavi, A., Williams, B.A., Wold, B.J. & Rothenberg, E.V. Dynamic transformations of genome-wide epigenetic marking and transcriptional control establish T cell identity. Cell 149, 467–482 (2012).
Wang, H. et al. NOTCH1-RBPJ complexes drive target gene expression through dynamic interactions with superenhancers. Proc. Natl. Acad. Sci. USA 111, 705–710 (2014).
Yu, M. et al. Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors. Mol. Cell 45, 330–343 (2012).
Li, L. et al. A far downstream enhancer for murine Bcl11b controls its T-cell specific expression. Blood 122, 902–911 (2013).
Tydell, C.C. et al. Molecular dissection of prethymic progenitor entry into the T lymphocyte developmental pathway. J. Immunol. 179, 421–438 (2007).
Mingueneau, M. et al. Immunological Genome Consortium. The transcriptional landscape of αβ T cell differentiation. Nat. Immunol. 14, 619–632 (2013).
Schmitt, T.M. & Zúñiga-Pflücker, J.C. Induction of T cell development from hematopoietic progenitor cells by Delta-like-1 in vitro. Immunity 17, 749–756 (2002).
De Obaldia, M.E. & Bhandoola, A. Transcriptional regulation of innate and adaptive lymphocyte lineages. Annu. Rev. Immunol. 33, 607–642 (2015).
Weng, A.P. et al. c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma. Genes Dev. 20, 2096–2109 (2006).
Kueh, H.Y., Champhekar, A., Nutt, S.L., Elowitz, M.B. & Rothenberg, E.V. Positive feedback between PU.1 and the cell cycle controls myeloid differentiation. Science 341, 670–673 (2013).
Varnum-Finney, B. et al. Immobilization of Notch ligand, Delta-1, is required for induction of Notch signaling. J. Cell Sci. 113, 4313–4318 (2000).
Schmitt, T.M., Ciofani, M., Petrie, H.T. & Zúñiga-Pflücker, J.C. Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions. J. Exp. Med. 200, 469–479 (2004).
Taghon, T.N., David, E.S., Zúñiga-Pflücker, J.C. & Rothenberg, E.V. Delayed, asynchronous, and reversible T-lineage specification induced by Notch/Delta signaling. Genes Dev. 19, 965–978 (2005).
Hosokawa, H. et al. Gata3/Ruvbl2 complex regulates T helper 2 cell proliferation via repression of Cdkn2c expression. Proc. Natl. Acad. Sci. USA 110, 18626–18631 (2013).
Yu, S. et al. The TCF-1 and LEF-1 transcription factors have cooperative and opposing roles in T cell development and malignancy. Immunity 37, 813–826 (2012).
Kawazu, M. et al. Functional domains of Runx1 are differentially required for CD4 repression, TCRβ expression, and CD4/8 double-negative to CD4/8 double-positive transition in thymocyte development. J. Immunol. 174, 3526–3533 (2005).
Zarnegar, M.A., Chen, J. & Rothenberg, E.V. Cell-type-specific activation and repression of PU.1 by a complex of discrete, functionally specialized cis-regulatory elements. Mol. Cell. Biol. 30, 4922–4939 (2010).
Peter, I.S. & Davidson, E.H. Genomic Control Process: Development and Evolution (Academic Press, 2015).
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Ebina, W. & Rossi, D.J. Transcription factor-mediated reprogramming toward hematopoietic stem cells. EMBO J. 34, 694–709 (2015).
Lin, Y.C. et al. Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate. Nat. Immunol. 12, 1196–1204 (2012).
Liu, P., Jenkins, N.A. & Copeland, N.G. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484 (2003).
Hernández-Hoyos, G., Anderson, M.K., Wang, C., Rothenberg, E.V. & Alberola-Ila, J. GATA-3 expression is controlled by TCR signals and regulates CD4/CD8 differentiation. Immunity 19, 83–94 (2003).
Telfer, J.C., Hedblom, E.E., Anderson, M.K., Laurent, M.N. & Rothenberg, E.V. Localization of the domains in Runx transcription factors required for the repression of CD4 in thymocytes. J. Immunol. 172, 4359–4370 (2004).
Champhekar, A. et al. Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU.1. Genes Dev. 29, 832–848 (2015).
Nakano, T., Kodama, H. & Honjo, T. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265, 1098–1101 (1994).
Schroeder, T. Long-term single-cell imaging of mammalian stem cells. Nat. Methods 8 (suppl.), S30–S35 (2011).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
We thank M. Lerica Gutierrez Quiloan for assistance with mouse genotyping and maintenance; N. Verduzco and I. Soto for animal husbandry; J. Longmate for help with statistical analysis of alternate-lineage-potential experiments; R.A. Diamond, K. Beadle, J. Grimm, D. Perez and J. Verceles for cell sorting; N. Feng for initial flow cytometric analysis; J. Hahn for advice on BAC recombineering; S. Qin for assistance with qPCR experiments; X.Wang for performing pilot studies with microwell arrays; and J. Ungerbäck for assistance with visualizing genome track data. We also thank A. Bhandoola, L. Xu and W. Pear (University of Pennsylvania); J. Telfer (University of Massachusetts) and N. Masuyama (University of Tokyo) for constructs. This work was funded by a CRI/Irvington Postdoctoral Fellowship and a US National Institutes of Health (NIH) K99/R00 Award (K99HL119638A) to H.Y.K.; a California Institute for Regenerative Medicine Bridges to Stem-Cell Research award to K.K.H.N. (TB1-01176); NIH grants to E.V.R. (R01 AI083514, R01 AI095943, RC2 CA148278, R33 HL089123, R01 CA90233 and R01 HL119102) and M.A.Y. (R01 AI064590); NIH/HHS grant U01HL100395 (I.D.B.); the Albert Billings Ruddock Professorship to E.V.R.; and the Al Sherman Foundation and the Louis A. Garfinkle Memorial Laboratory Fund to E.V.R.'s lab.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Binding profiles of key transcription factors to the Bcl11b gene and enhancer.
A)-C) UCSC genome browser tracks (mm9 coordinates) showing published ChIP-Seq binding data of the following transcription factors to the Bcl11b gene: Notch11, CSL1, Gata32, PU.12, TCF-13, Runx14 (both WT and Runx1-/- control) and Ikaros5. The H3K4me2 histone mark is also included2. The primary cell types on which ChIP-Seq was performed are also indicated. A) Global view of Bcl11b locus, showing gene body in relation to distal enhancer and downstream gene desert. Shaded yellow bars indicate gene and enhancer locations. B) Bcl11b gene body, and C) Bcl11b enhancer domain. Gray shaded bars indicate regions containing clusters of transcription factor binding sites and H3K4me marks. Asterisk indicates binding sites tested in enhancer transfection assays6. Note that a high resolution version of this figure is also provided separately.
A) Schematic showing targeted insertion of fluorescent protein reporter into 3’-untranslated region (UTR) of Bcl11b locus (top), along with excision of the loxP-flanked PGK-neomycin cassette using Cre recombinase. Cut sites for restriction enzymes PacI, CspCI, used for Southern Blot validation of targeted insertion in B) are shown. Diagram is not drawn to scale. B) Correct insertion of fluorescent protein cassette was validated by Southern Blot analysis on the targeted ES cell clone (left), which was later used for knock-in mouse generation. Bacterial Artificial Chromosomes (BAC) containing either the wildtype Bcl11b locus (center), or a Bcl11bYFP (+neo) locus (right) were used as controls.
Supplementary Figure 3 Bcl11b-YFP levels are not affected by presence of the neomycin drug resistance cassette.
Flow cytometry analysis of Bcl11bYFP/+ mice either with or without the neomycin drug selection cassette [Bcl11b‑YFP(+neo), Bcl11b-YFP(-neo)], together with non-fluorescent controls (B6), using cells from the whole thymus (A), DN thymus (B), or the spleen (C). Bcl11b-YFP reporter expression is identical with or without the drug resistance cassette. ISP – immature single positive, DP – double positive.
Supplementary Figure 4 Experimental and analysis workflow for time-lapse imaging of Bcl11b-YFP levels in progenitor cells.
A) Experimental setup, microscope configuration and illumination settings used for timelapse image acquisition. To confine cells to a single imaging field-of-view, progenitors were seeded into hollow PDMS microwells (Microsurfaces; Flemington, Australia) adhered to a glass-bottomed 24 well dish (Mattek; Ashland, MA). B) Workflow for image analysis showing (I) raw differential interference contrast (DIC), mCherry and YFP images; (II) representative images overlaid with automatically segmented cell boundaries; and (III) heat maps showing cytometric analysis of segmented cells, showing OFF and ON populations.
Supplementary Figure 5 Notch signaling promotes all-or-none Bcl11b activation in a dose-dependent manner.
Flow cytometry analysis of bone marrow derived Bcl11b-YFP- DN2 cells cultured under the indicated conditions with 5 ng/mL IL-7 and Flt3L for four days. CD25 versus Bcl11b-YFP levels (top), and histograms of Bcl11b-YFP and CellTrace Violet levels (bottom) are shown. Initial levels (Day 0) are shown as gray dotted lines. GSI: γ-secretase inhibitor (Calbiochem/EMD Millipore, Billerica, MA), a small molecule inhibitor of Notch signaling.
Quantitative real time (RT)-PCR analysis of Tcf7 transcript levels in DN progenitors from E14.5 fetal livers infected with the indicated shRNA constructs for 2 days and sorted. RNA was extracted and processed as previously described7. Forward and reverse primers used for Tcf7 detection are CAAGGCAGAGAAGGAGGCTAAG and GGCAGCGCTCTCCTTGAG respectively, as previously described8.
A) Flow cytometry analysis of bone marrow DN progenitors transduced with either shRNA to Tcf7 (shTcf7) or a non-targeting control (shRandom), then cultured on plates coated with 6 μg/mL DL1 with 5 ng/mL SCF, IL-7 and Flt3L for four days. Levels of the dendritic cell marker CD11c against FSC (top), as well as CD25 levels versus Bcl11b-YFP levels (middle) or Bcl11b-YFP level distributions (bottom) for CD11c- populations are shown. Knockdown of Tcf7 promotes DC trans-differentiation in DN2 cells, but does not impede Bcl11b activation. B) Flow cytometry analysis of Bcl11b-YFP expression levels in bone marrow DN progenitors transduced with a control or VEX-GFP retroviral construct for over-expression of TCF-1 (encoded by Tcf7), and cultured on OP9- or OP9-DL1 monolayers in 5 ng/mL IL-7 and Flt3L for three days. TCF-1 over-expression does not affect Bcl11b activation from either ETP or DN2 progenitors.
Supplementary Figure 8 Runx1, but not TCF-1 or GATA-3, controls Bcl11b expression amplitude in mature T cells.
A) Published transcriptomic data on Bcl11b expression in wildtype or Tcf7-/- DN3 thymocytes9 (left), or in wildtype or Gata3-/- Th2 cells10 (right). Levels of Bcl11b expression are similar in the absence or presence of either factor. B) Flow cytometry analysis of CD4 and CD8 splenic T-cells activated with anti-TCRb and anti-CD28 for 1 day, transduced with shRNA constructs, and cultured for 3 days. Plots show CD8 versus CD3 for cells transduced with retrovirus (top), along with Bcl11b level distributions for gated non-CD8 T-cell populations (bottom). Corresponding data for CD8 T-cells are shown in Fig. 8. Solid black lines indicate cells transduced with random shRNA, and dotted lines indicate background levels from non-fluorescent T-cells. Data from (A) represent mean and S.D. of two independent experiments.
Supplementary Figures 1–8 and Supplementary Tables 3 and 4 (PDF 2633 kb)
Transcript levels for genes differentially expressed in Bcl11b-YFP– versus Bcl11b-YFP+ cells (XLSX 49 kb)
Transcript levels for transcriptional regulators expressed in developing T cells (XLSX 243 kb)
About this article
Cite this article
Kueh, H., Yui, M., Ng, K. et al. Asynchronous combinatorial action of four regulatory factors activates Bcl11b for T cell commitment. Nat Immunol 17, 956–965 (2016). https://doi.org/10.1038/ni.3514
This article is cited by
Signal Transduction and Targeted Therapy (2023)
Runx factors launch T cell and innate lymphoid programs via direct and gene network-based mechanisms
Nature Immunology (2023)
TCF-1 promotes chromatin interactions across topologically associating domains in T cell progenitors
Nature Immunology (2022)
Multi-objective optimization reveals time- and dose-dependent inflammatory cytokine-mediated regulation of human stem cell derived T-cell development
npj Regenerative Medicine (2022)
Nature Reviews Immunology (2022)