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GEO help: Mouse over screen elements for information. |
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Status |
Public on Oct 16, 2023 |
Title |
Defining STAT5 target genes in thymic precursor cells at pre- and post-commitment stages |
Organism |
Mus musculus |
Experiment type |
Expression profiling by high throughput sequencing
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Summary |
STAT5 is critical for inducing lineage specifying transcription factors, establishing effector gene programs, upregulating IL-2Rα (also known as CD25), and supporting cell survival in response to environmental cues in mature T cells. Although it has been shown that IL-7-induced STAT5 signaling is also essential for survival of thymic precursor cells, the exact roles of STAT5A and STAT5B and their target genes that contribute to the developmental program in early thymic development have been unknown. To define STAT5 target genes in these early T-cell progenitors, we have performed CRISPR-Cas9 mediated acute deletion of Stat5a and Stat5b in combination. We first carried out preliminary experiments to determine the stages in which IL-7 could induce maximal STAT5 phosphorylation in thymic double negative (DN) cells (B. Shin, unpublished data). We then focused on genes responding to loss of STAT5 in two different stages: CD25+ Bcl11b- pre-commitment stage, and CD25+ Bcl11b+ post-commitment stage. Of note, to generate these early pro-T cells in OP9-DLL1 coculture, we exploited bone-marrow precursor cells expressing a Bcl2 transgene to minimize survival defects due to loss of STAT5A and STAT5B. Our data show that STAT5 is necessary, in both pre- and post-commitment stages, for optimal expression of the IL-2Rα gene (Il2ra), which is constitutively expressed in normal DN2 and DN3 pro-T cells. This dataset further defines a distinct set of additional genes responding more strongly to loss of STAT5 in pro-T cells. The linked manuscript by R. Spolski et al. and other datasets referenced therein include data documenting the genomic occupancy of STAT5B in corresponding stages. In summary, our data define STAT5-responsive target genes during early T development.
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Overall design |
Bone-marrow progenitor cells from B6.CRISPR-Cas9;Bcl2;Bcl11b-mCherry/mCherry transgene expressing mice were incubated with IL-7, Flt3-ligand (Flt3l), and SCF for overnight. Three sgRNAs against Stat5a and three sgRNAs against Stat5b were retrovirally introduced, and infected cells were cultured on OP9-DLL1 stromal cells with IL-7 and Flt3l. After 6 days post infection, Lineage marker (Lin)- infection marker+ CD25+ mCherry- (Bcl11b reporter negative, pre-commitment stage) and CD25+mCherry+ (Bcl11b reporter negative, post-commitment stage) cells were FACS sorted and subjected to bulk RNA-seq.
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Contributor(s) |
Shin B, Li P, Spolski R, Oh J, Leonard WJ, Rothenberg EV |
Citation(s) |
37922339 |
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Submission date |
Sep 27, 2021 |
Last update date |
Feb 13, 2024 |
Contact name |
Boyoung Shin |
E-mail(s) |
boyoung@caltech.edu
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Organization name |
Californial Institute of Technology
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Lab |
Ellen V. Rothenberg
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Street address |
1200 E. California Blvd
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City |
Pasadena |
State/province |
California |
ZIP/Postal code |
91125 |
Country |
USA |
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Platforms (1) |
GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
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Samples (8)
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Relations |
BioProject |
PRJNA766644 |
SRA |
SRP338901 |
Supplementary file |
Size |
Download |
File type/resource |
GSE184845_DN2_Stat5ab_FPKM.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
GSE184845_DN2_Stat5ab_count.txt.gz |
1.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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