RESUMO
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.
Assuntos
Fator de Transcrição GATA3/imunologia , Células Precursoras de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Regulação para Cima/imunologia , Animais , Antineoplásicos Hormonais/farmacologia , Linhagem Celular , Fator de Transcrição GATA3/genética , Camundongos , Células Mieloides/citologia , Células Mieloides/imunologia , Células Precursoras de Linfócitos T/citologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Receptores Notch/genética , Receptores Notch/imunologia , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tamoxifeno/farmacologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
PU.1 and GATA-3 are transcription factors that are required for development of T cell progenitors from the earliest stages. Neither one is a simple positive regulator for T lineage specification, however. When expressed at elevated levels at early stages of T cell development, each of these transcription factors blocks T cell development within a different, characteristic time window, with GATA-3 overexpression initially inhibiting at an earlier stage than PU.1. These perturbations are each associated with a distinct spectrum of changes in the regulation of genes needed for T cell development. Both transcription factors can interfere with expression of the Rag-1 and Rag-2 recombinases, while GATA-3 notably blocks PU.1 and IL-7Ralpha expression, and PU.1 reduces expression of HES-1 and c-Myb. A first-draft assembly of the regulatory targets of these two factors is presented as a provisional gene network. The target genes identified here provide insight into the basis of the effects of GATA-3 or PU.1 overexpression and into the regulatory changes that distinguish the developmental time windows for these effects.
