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GATA-3 Dose-Dependent Checkpoints in Early T Cell Commitment

Scripture-Adams, Deirdre D. and Damle, Sagar S. and Li, Long and Elihu, Koorosh J. and Qin, Shuyang and Arias, Alexandra M. and Butler, Robert R., III and Champhekar, Ameya and Zhang, Jingli A. and Rothenberg, Ellen V. (2014) GATA-3 Dose-Dependent Checkpoints in Early T Cell Commitment. Journal of Immunology, 193 (7). pp. 3470-3491. ISSN 0022-1767. PMCID PMC4170028. doi:10.4049/jimmunol.1301663.

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GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4CD8 (double-negative [DN]) stages 1–3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3–deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3–deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro–T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment.

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Rothenberg, Ellen V.0000-0002-3901-347X
Additional Information:© 2014 by The American Association of Immunologists, Inc. Received for publication June 24, 2013. Accepted for publication August 4, 2014. Published online before print August 29, 2014. This work was supported by National Institutes of Health Grants R01CA98925, RC2CA148278, R01CA90233, and R01AI95943, the Louis A. Garfinkle Memorial Laboratory Fund, the Al Sherman Foundation, and the Albert Billings Ruddock Professorship to E.V.R. Initial support for D.D.S.-A. was also provided by the Elisabeth Ross Endowment at the California Institute of Technology and by National Institutes of Health Training Grant 5T32 HD07257. The sequences presented in this article have been submitted to the Gene Expression Omnibus ( under accession number GSE59215. We thank Gabriela Hernandez-Hoyos for the Gata3 shRNA construct, I-Cheng Ho and Sung-Yun Pai (Brigham and Women’s Hospital, Harvard Medical School) for GATA-3–floxed mice, Qiang Tu for advice and help with data deposition, and Barbara Kee (University of Chicago), James Di Santo and Roel Klein Wolterink (Institut Pasteur), and Marcos García- Ojeda (University of California Merced) for stimulating discussions and for sharing data before publication. We thank Christopher Franco and Kira Sarup-Lytzen for assistance with qPCR, Jerry Kwong and Benjamin Park for technical help, and Rochelle Diamond, Stephanie Adams, and Diana Perez for cell sorting. We also thank Frank Costantini (Columbia University), Mark Leid (Oregon State University), Pentao Liu (Cambridge University), and Stephen Nutt (Walter and Eliza Hall Institute) for ROSA26REYFP, two types of Bcl11bfl/fl, and Spi1fl/fl founder mice, respectively, Juan Carlos Zúñiga-Pflücker (University of Toronto) for OP9-DL4 cells, and John Rossi (City of Hope), Naoko Arai (DNAX), Tom Taghon (Ghent University), and laboratory members Mark Zarnegar, Sanket Acharya, and Elizabeth-Sharon David-Fung for providing and characterizing retroviral vectors. The authors have no financial conflicts of interest.
Funding AgencyGrant Number
Louis A. Garfinkle Memorial Laboratory FundUNSPECIFIED
Al Sherman FoundationUNSPECIFIED
Albert Billings Ruddock ProfessorshipUNSPECIFIED
Elisabeth Ross EndowmentUNSPECIFIED
NIH5T32 HD07257
Issue or Number:7
PubMed Central ID:PMC4170028
Record Number:CaltechAUTHORS:20140905-102102851
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Official Citation:GATA-3 Dose-Dependent Checkpoints in Early T Cell Commitment Deirdre D. Scripture-Adams, Sagar S. Damle, Long Li, Koorosh J. Elihu, Shuyang Qin, Alexandra M. Arias, Robert R. Butler III, Ameya Champhekar, Jingli A. Zhang, and Ellen V. Rothenberg J Immunol 1301663; published ahead of print August 29, 2014, doi:10.4049/jimmunol.1301663
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:49280
Deposited By: Ruth Sustaita
Deposited On:05 Sep 2014 18:12
Last Modified:10 Nov 2021 18:41

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