title: GATA-3 dose-dependent checkpoints in early T cell commitment.
abstract: GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4(-)CD8(-) (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.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Deirdre D
authors.initials: DD
authors.lastname: Scripture-Adams
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Sagar S
authors.initials: SS
authors.lastname: Damle
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Long
authors.initials: L
authors.lastname: Li
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Koorosh J
authors.initials: KJ
authors.lastname: Elihu
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Shuyang
authors.initials: S
authors.lastname: Qin
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Arias
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Robert R
authors.initials: RR
authors.lastname: Butler
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Ameya
authors.initials: A
authors.lastname: Champhekar
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Jingli A
authors.initials: JA
authors.lastname: Zhang
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125 evroth@its.caltech.edu.
authors.forename: Ellen V
authors.initials: EV
authors.lastname: Rothenberg
mesh_terms: D000818:Animals; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D002460:Cell Line; D050990:GATA3 Transcription Factor / Q000235:genetics / Q000276:immunology*; D051379:Mice; D022423:Myeloid Cells / Q000166:cytology / Q000276:immunology; D054504:Precursor Cells, T-Lymphoid / Q000166:cytology / Q000276:immunology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000276:immunology; D051880:Receptors, Notch / Q000235:genetics / Q000276:immunology; D012097:Repressor Proteins / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D013629:Tamoxifen / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000276:immunology; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics / Q000276:immunology*
chemical_list: D018931:Antineoplastic Agents, Hormonal; C511143:Bcl11b protein, mouse; D050990:GATA3 Transcription Factor; C494748:Gata3 protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D051880:Receptors, Notch; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; D013629:Tamoxifen; C052617:Bcl2 protein, mouse