GATA-3 dose-dependent checkpoints in early T cell commitment

J Immunol. 2014 Oct 1;193(7):3470-91. doi: 10.4049/jimmunol.1301663. Epub 2014 Aug 29.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents, Hormonal / pharmacology
  • Cell Line
  • GATA3 Transcription Factor / genetics
  • GATA3 Transcription Factor / immunology*
  • Mice
  • Myeloid Cells / cytology
  • Myeloid Cells / immunology
  • Precursor Cells, T-Lymphoid / cytology
  • Precursor Cells, T-Lymphoid / immunology*
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / immunology
  • Receptors, Notch / genetics
  • Receptors, Notch / immunology
  • Repressor Proteins / genetics
  • Repressor Proteins / immunology
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Signal Transduction / immunology*
  • Tamoxifen / pharmacology
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / immunology
  • Up-Regulation / drug effects
  • Up-Regulation / genetics
  • Up-Regulation / immunology*

Substances

  • Antineoplastic Agents, Hormonal
  • Bcl11b protein, mouse
  • GATA3 Transcription Factor
  • Gata3 protein, mouse
  • Proto-Oncogene Proteins c-bcl-2
  • Receptors, Notch
  • Repressor Proteins
  • Tumor Suppressor Proteins
  • Tamoxifen
  • Bcl2 protein, mouse

Associated data

  • GEO/GSE59215