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Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs
Sakie Ohmura, … , Satoru Takahashi, James Douglas Engel
Sakie Ohmura, … , Satoru Takahashi, James Douglas Engel
Published January 25, 2016
Citation Information: J Clin Invest. 2016;126(3):865-878. https://doi.org/10.1172/JCI83894.
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Research Article Genetics

Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs

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Abstract

The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell–specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte–specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte–specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell–regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell–specific transcriptional activity.

Authors

Sakie Ohmura, Seiya Mizuno, Hisashi Oishi, Chia-Jui Ku, Mary Hermann, Tomonori Hosoya, Satoru Takahashi, James Douglas Engel

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Figure 1

In vivo genome deletion of Tce1.

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In vivo genome deletion of Tce1.
(A) Two CRISPR/Cas9 plasmids expressing...
(A) Two CRISPR/Cas9 plasmids expressing gRNAs that correspond to sequences surrounding the 7.1 kbp that define the boundaries of Tce1 were coinjected into mouse fertilized oocytes. Tce1-deleted mutant allele founder (F0) mice were intercrossed to obtain homozygous deletion mutant mice. (B) Thymocytes, (C) peripheral blood cells, and (D) splenocytes isolated from F2 animals at 5 to 6 weeks of age bearing homozygous (white circle) or heterozygous (black circle) deletions of Tce1 were analyzed for cell surface expression of T cell stage–specific markers. The absolute numbers of Lin–cKithiCD25– (ETP), Lin–cKithiCD25+ (DN2), Lin–cKitlo/–CD25+ (DN3), Lin–cKitlo/–CD25– (DN4), CD4+CD8+ (DP), CD4+CD8–CD3+TCRβ+ (CD4 SP), and CD4–CD8+CD3+TCRβ+ (CD8 SP) thymocytes are shown at the top. Each stage of T cells was isolated by flow cytometry and analyzed for the expression of Gata3 mRNA, as normalized to Hprt by qRT-PCR. Each circle represents an individual mouse, and black bars represent the average for each genotype. Data are representative of the summary of 2 independent experiments. *P < 0.05 by Student’s t test.

Copyright © 2022 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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