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Adult haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells and must respond to extracellular cues including cytokines.

ABSTRACT: Adult haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells and must respond to extracellular cues including cytokines. The JAK/STAT pathway is a highly conserved pathway, activated by many cytokines, in which tyrosine-phosphorylated STATs (pSTATs) function as transcription factors. STAT5 is a pivotal downstream mediator of several cytokines known to regulate haematopoiesis but its function in the HSC compartment remains poorly understood. Here we show that STAT5-deficient HSCs exhibited an unusual phenotype - reduced multi-lineage repopulation and self-renewal combined with reduced cell cycle entry and increased differentiation. This reflected not only loss of a canonical pSTAT5 transcriptional program but also loss of distinct functions mediated by tyrosine-unphosphorylated STAT5 (uSTAT5). Consistent with this concept expression of an unphosphorylatable STAT5B mutant constrained HSC differentiation, promoted HSC maintenance and upregulated transcriptional programs associated with quiescence and stemness. Moreover, treatment with a JAK1/2 inhibitor (ruxolitinib) increased the uSTAT5:pSTAT5 ratio, constrained HSC differentiation and proliferation and promoted HSC maintenance, thus phenocopying uSTAT5B overexpression. Our results therefore reveal a previously unrecognized interplay between pSTAT5 and uSTAT5 in the control of HSC function. In addition the demonstration that JAK inhibition promotes HSC maintenance has implications for gene therapy using HSCs and may contribute to the failure of JAK inhibitors to eradicate myeloproliferative neoplasms. To evaluate the long-term functional capacity of uSTAT5B overexpressing HSCs, WT CD45.2 ESLAM HSCs were sorted and transduced with lentivirus containing STAT5B-YF, STAT5B-WT and EV in SCF and IL-11 cultures. Five days after infection, cells were sorted for viability (DAPI-, Thermo Fisher) and green fluorescent protein (GFP) expression and processed according to the manufacturer’s protocol for 10x Chromium (10x Genomics, Pleasanton, CA) experiments .

ORGANISM(S): Mus musculus

PROVIDER: GSE260462 | GEO | 2024/05/29

REPOSITORIES: GEO

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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. This model is hosted on BioModels Database and identified by: BIOMD0000000591. To cite BioModels Database, please use: BioModels: Content, Features, Functionality, and Use.. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Adult haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells and must respond to extracellular cues including cytokines.

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