Identification of Stat5 Target Genes by siRNA-mediated knockdown
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE10389 | GEO | 2008/06/23
SECONDARY ACCESSION(S): PRJNA108135
REPOSITORIES: GEO
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Project description:STAT5A and STAT5B proteins belong to the family of signal transducers and activators of transcription. They are encoded by 2 separate genes with 91% identity in their amino acid sequences. Despite their high degree of conservation, STAT5A and STAT5B exert non-redundant functions, resulting at least in part from differences in target gene activation. To better characterize the differential contribution of STAT5A and STAT5B in gene regulation, we performed single or double knock-down of STAT5A and STAT5B using small interfering RNA. Subsequent gene expression profiling and RT-qPCR analyses of IL-3-stimulated Ba/F3-beta cells led to the identification of putative novel STAT5 target genes. Chromatin immunoprecipitation assays analyzing the corresponding gene loci identified unusual STAT5 binding sites compared to conventional STAT5 responsive elements. Some of the STAT5 targets identified are upregulated in several human cancers, suggesting that they might represent potential oncogenes in STAT5-associated malignancies. Experiment Overall Design: Ba/F3-beta cells transfected with either control (scramble I) or Stat5A and Stat5B siRNAs were stimulated with IL-3 for either 30 minutes or 2 hours. Unstimulated Ba/F3-beta cells transfected with scramble I siRNA were used as a control. Experiment Overall Design: Total RNA was isolated using the RNeasy Maxi kit, including an on-column DNase I treatment to eliminate genomic DNA contamination, according to the manufacturerâs protocol (Qiagen, Valencia, CA). DNase-treated total RNA (5 µg) was synthesized into biotinylated cRNA probe using one-cycle target labeling and IVT labeling (Affymetrix, Inc., Santa Clara, CA) according to manufacturerâs instructions. Fifteen µg of biotinylated cRNA probe from each sample was hybridized onto murine U74A, U74B, U74C Affymetrix Microarray chips. The hybridized chips were then washed using the Affymetrix GeneChip Fluidics 400 station and scanned in the Affymetrix GeneChip Scanner 3000 (Affymetrix, Inc., Santa Clara, CA) according to manufacturerâs instructions. The quality of cRNA probe synthesis and efficiency of hybridization was analyzed in the GeneChip Operating System for each Affymetrix chip once scanning was complete. Microarray data were normalized using MAS5 and then analyzed using the Genespring (Agilent, Palo Alto, CA). Experiment Overall Design: Out of the 36,767 genes, 14,553 passed our noise and P/M/A call filters. Genes were further selected for being stimulated at least 2-fold by IL-3 after 30 minutes (164 genes) and 2 hours (553 genes) stimulation compared to the unstimulated cells (ScI-transfected cells). Then, IL-3 induced genes that were down-regulated at least 2-fold in the STAT5A/B siRNA-transfected cells at 30 minutes and 2 hours (12 and 29 respectively) were identified as putative STAT5 target genes and further investigated.
2008-10-20 | E-GEOD-10389 | biostudies-arrayexpress
Project description:Two highly conserved transcription factors STAT5A and STAT5B play an identical role in the intracellular signaling pathway upon cytokine stimulation, while gene deletion experiments have revealed separable and overlapping functions of STAT5. This questions whether the phenotypic differences in the organ development observed in the individual knockout mice result from isoform-specific functions or quantitative differences in the expression levels of each STAT5 isoform among tissues. To elucidate the redundancy and isoform-specificity of STAT5 for development at molecular levels, mice carrying only a single allele of either Stat5a or Stat5b were generated. Both of these mice overcame the lethal anemia observed in Stat5ab-null mice, indicating that development of erythroid cell lineage was totally dependent on the dosage of STAT5. The blocked progression of B cell lineage at the pre-pro B cell stage in Stat5ab-/- mice was rescued in the presence of a single allele of either Stat5a or Stat5b, while the number of total B220+ cells in bone marrow was smaller in Stat5abnull/Stat5b- mice than Stat5abnull/Stat5a- mice. The paucity of alveolar progenitor cells in the Stat5ab-null mammary epithelium was rescued by a single allele of either Stat5a but not Stat5b, suggesting cell-type dependent isoform-specific function. Genome-wide gene expression analyses revealed that different steps of cell lineage progression require different gene sets which expression requires the different isoform of STAT5 in a dose-dependent manner in the mammary epithelium. Taken together, this study demonstrates that dose-dependent isoform specificity of STAT5A and STAT5B controls progression and differentiation of each cell lineage. Six days after observation of a plug, mammary tissues from three of each Stat5a-/- mice, Stat5ab+/null mice, Stat5abnull/Stat5b- and Stat5abnull/Stat5a- mice were collected, frozen in liquid nitrogen, and stored at -70 °C
2012-04-30 | E-GEOD-29045 | biostudies-arrayexpress
Project description:STAT5 proteins are vital for lymphocyte development and function. Tyrosine phosphorylation of a C-terminal tyrosine is the key event in cytokine activation of STAT5A and STAT5B. However, the role of STAT5 tyrosine phosphorylation has not been assessed in vivo. Here we generated Stat5a and Stat5b tyrosine mutant knock-in (KI) mice and found that these animals had greatly reduced CD8+ T cell numbers. These cells exhibited profoundly diminished proliferation in response to IL-2, correlating with greatly reduced IL-2-induced pRB and a block in the G1-->S phase transition. The mutant cells also exhibited decreased IL-2-mediated activation of pERK and pAKT, which can in part be attributed to diminished IL-2-induced expression of IL-2R-beta and IL-2R-gamma. Our findings highlight that the tyrosine phosphorylation of both STAT5A and STAT5B is essential for maximal IL-2 signaling and elucidate the molecular basis for achieving an optimal mitogenic effect of IL-2 on CD8+ T cells.
2023-11-06 | MSV000093320 | MassIVE
Project description:The signal transducer and activator of transcription 5 (STAT5) is an attractive therapeutic target but successful targeting of STAT5 has proved to be difficult. We report herein the development of AK-2292 as a first, potent and selective small-molecule degrader of both STAT5A and STAT5B isoforms. AK-2292 induces degradation of STAT5A/B proteins with an outstanding selectivity over all other STAT proteins and >6,000 non-STAT proteins, leading to selective inhibition of STAT5 activity in cells. AK-2292 effectively induces STAT5 depletion in normal mouse tissues and human chronic myeloid leukemia (CML) xenograft tissues and achieves tumor regression in two CML xenograft mouse models at well-tolerated dose-schedules. AK-2292 is not only a powerful research tool with which to investigate the biology of STAT5 and therapeutic potential of selective STAT5 protein depletion and inhibition in vitro and in vivo, but also a promising lead compound toward ultimate development of a STAT5-targeted therapy.
2023-01-25 | PXD037895 | Pride
Project description:Boehm2014 - isoform-specific dimerization of pSTAT5A and pSTAT5B
To study STAT5 activation, the authors build a dynamic model of pSTAT5 isoform dimerization. Combinatorial binding of pSTAT5A and pSTAT5B is analysed using model hypotheses and concurrent experiments. Model parameters are derived from the experiments on Ba/F3 cells. Results show that pSTAT5 heterodimerization hypothesis for STAT5 activation favours experimental results.
This model is described in the article:
Identification of isoform-specific dynamics in phosphorylation-dependent STAT5 dimerization by quantitative mass spectrometry and mathematical modeling
Boehm ME, Adlung L, Schilling M, Roth S, Klingmüller U, Lehmann WD
J Proteome Res. 2014 Dec 5;13(12):5685-94
Abstract:
STAT5A and STAT5B are important transcription factors that dimerize and transduce activation signals of cytokine receptors directly to the nucleus. A typical cytokine that mediates STAT5 activation is erythropoietin (Epo). Differential functions of STAT5A and STAT5B have been reported. However, the extent to which phosphorylated STAT5A and STAT5B (pSTAT5A, pSTAT5B) form homo- or heterodimers is not understood, nor is how this might influence the signal transmission to the nucleus. To study this, we designed a concept to investigate the isoform-specific dimerization behavior of pSTAT5A and pSTAT5B that comprises isoform-specific immunoprecipitation (IP), measurement of the degree of phosphorylation, and isoform ratio determination between STAT5A and STAT5B. For the main analytical method, we employed quantitative label-free and -based mass spectrometry. For the cellular model system, we used Epo receptor (EpoR)-expressing BaF3 cells (BaF3-EpoR) stimulated with Epo. Three hypotheses of dimer formation between pSTAT5A and pSTAT5B were used to explain the analytical results by a static mathematical model: formation of (i) homodimers only, (ii) heterodimers only, and (iii) random formation of homo- and heterodimers. The best agreement between experimental data and model simulations was found for the last case. Dynamics of cytoplasmic STAT5 dimerization could be explained by distinct nuclear import rates and individual nuclear retention for homo- and heterodimers of phosphorylated STAT5.
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2024-09-02 | BIOMD0000000591 | BioModels
Project description:STAT5 proteins are vital for lymphocyte development and function. Tyrosine phosphorylation of a C-terminal tyrosine is the key event in cytokine activation of STAT5A and STAT5B. However, the role of STAT5 tyrosine phosphorylation has not been assessed in vivo. Here we generated Stat5a and Stat5b tyrosine mutant knock-in (KI) mice and found that these animals had greatly reduced CD8+ T cell numbers. These cells exhibited profoundly diminished proliferation in response to IL-2, correlating with greatly reduced IL-2-induced pRB and a block in the G1-->S phase transition. The mutant cells also exhibited decreased IL-2-mediated activation of pERK and pAKT, which can in part be attributed to diminished IL-2-induced expression of IL-2R-beta and IL-2R-gamma. Our findings highlight that the tyrosine phosphorylation of both STAT5A and STAT5B is essential for maximal IL-2 signaling and elucidate the molecular basis for achieving an optimal mitogenic effect of IL-2 on CD8+ T cells. [doi:10.25345/C5833N851] [dataset license: CC0 1.0 Universal (CC0 1.0)]
2023-12-23 | MSV000093724 | MassIVE
Project description:Two highly conserved transcription factors STAT5A and STAT5B play an identical role in the intracellular signaling pathway upon cytokine stimulation, while gene deletion experiments have revealed separable and overlapping functions of STAT5. This questions whether the phenotypic differences in the organ development observed in the individual knockout mice result from isoform-specific functions or quantitative differences in the expression levels of each STAT5 isoform among tissues. To elucidate the redundancy and isoform-specificity of STAT5 for development at molecular levels, mice carrying only a single allele of either Stat5a or Stat5b were generated. Both of these mice overcame the lethal anemia observed in Stat5ab-null mice, indicating that development of erythroid cell lineage was totally dependent on the dosage of STAT5. The blocked progression of B cell lineage at the pre-pro B cell stage in Stat5ab-/- mice was rescued in the presence of a single allele of either Stat5a or Stat5b, while the number of total B220+ cells in bone marrow was smaller in Stat5abnull/Stat5b- mice than Stat5abnull/Stat5a- mice. The paucity of alveolar progenitor cells in the Stat5ab-null mammary epithelium was rescued by a single allele of either Stat5a but not Stat5b, suggesting cell-type dependent isoform-specific function. Genome-wide gene expression analyses revealed that different steps of cell lineage progression require different gene sets which expression requires the different isoform of STAT5 in a dose-dependent manner in the mammary epithelium. Taken together, this study demonstrates that dose-dependent isoform specificity of STAT5A and STAT5B controls progression and differentiation of each cell lineage.
2012-05-01 | GSE29045 | GEO
Project description:The transcription factor STAT5 has long been recognized as an important mediator of prolactin induced mammary development during pregnancy. We have used mouse genetics and genome-wide analyses to determine how altering concentrations of STAT5A and STAT5B impacts different target promoters. Examination of genome-wide STAT5A, STAT5B, PolII and H3K4me3 in wild type (AABB) and Stat5a-null (BB) mammary tissues.
2012-09-18 | E-GEOD-40930 | biostudies-arrayexpress
Project description:Cytokine-activated STAT proteins dimerize and bind to high-affinity motifs, and N-terminal domain-mediated oligomerization of dimers allows tetramer formation and binding to low-affinity tandem motifs, but the functions of dimers versus tetramers are unknown. We generated Stat5a and Stat5b double knock-in (DKI) N-domain mutant mice that form dimers but not tetramers, identified cytokine-regulated genes whose expression required STAT5 tetramers, and defined consensus motifs for dimers versus tetramers. Whereas Stat5- deficient mice exhibited perinatal lethality, DKI mice were viable, indicating that STAT5 dimers were sufficient for survival. Nevertheless, STAT5 DKI mice had fewer CD4+CD25+ T cells, NK cells, and CD8+ T cells, with impaired cytokine-induced proliferation and homeostatic proliferation of CD8+ T cells. DKI CD8+ T cell proliferation following viral infection was diminished and DKI Treg cells did not efficiently control colitis. Thus, tetramerization of STAT5 is dispensable for survival but is critical for cytokine responses and normal immune function. Genome-wide mapping of STAT5A,STAT5B binding in mouse WT and DKI T cells (cultured with or without IL-2 for 1 hr) was conducted. RNA-Seq is conducted in mouse CD8+ T cells (WT and DKI, non-treated or treated with IL-2/IL-15 for 4 hr, 24 hr, 48 hr and 72 hr)
2012-04-25 | E-GEOD-36882 | biostudies-arrayexpress
Project description: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.
2023-10-16 | GSE184845 | GEO