ABSTRACT
The Polycomb group (PcG) protein BMI1 is a key factor in regulating hematopoietic stem cell (HSC) and leukemic stem cell self-renewal and functions in the context of the Polycomb repressive complex 1 (PRC1). In humans, each of the 5 subunits of PRC1 has paralog family members of which many reside in PRC1 complexes, likely in a mutually exclusive manner, pointing toward a previously unanticipated complexity of Polycomb-mediated silencing. We used an RNA interference screening approach to test the functionality of these paralogs in human hematopoiesis. Our data demonstrate a lack of redundancy between various paralog family members, suggestive of functional diversification between PcG proteins. By using an in vivo biotinylation tagging approach followed by liquid chromatography-tandem mass spectrometry to identify PcG interaction partners, we confirmed the existence of multiple specific PRC1 complexes. We find that CBX2 is a nonredundant CBX paralog vital for HSC and progenitor function that directly regulates the expression of the cyclin-dependent kinase inhibitor p21, independently of BMI1 that dominantly controls expression of the INK4A/ARF locus. Taken together, our data show that different PRC1 paralog family members have nonredundant and locus-specific gene regulatory activities that are essential for human hematopoiesis.
Subject(s)
Cell Cycle Proteins/physiology , Gene Silencing , Genetic Loci/genetics , Hematopoietic Stem Cells/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cells, Cultured , Female , Fetal Blood/cytology , Fetal Blood/metabolism , Gene Expression Regulation, Developmental , Gene Silencing/physiology , Hematopoiesis/genetics , Hematopoietic Stem Cells/physiology , Humans , Infant, Newborn , Multigene Family/genetics , Multigene Family/physiology , Pregnancy , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/physiology , Repressor Proteins/genetics , Repressor Proteins/metabolism , Repressor Proteins/physiology , Sequence Homology , Substrate Specificity/geneticsABSTRACT
BACKGROUND: Signal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. However, the manner in which STAT5 responds to early-acting and lineage-restricted cytokines, particularly in leukemic stem/progenitor cells, is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at a single-cell level. In normal cord blood (CB) cells, STAT5 phosphorylation was efficiently induced by TPO, IL-3 and GM-CSF within CD34(+)CD38(-) hematopoietic stem cells (HSCs). EPO- and SCF-induced STAT5 phosphorylation was largely restricted to the megakaryocyte-erythroid progenitor (MEP) compartment, while G-CSF as well IL-3 and GM-CSF were most efficient in inducing STAT5 phosphorylation in the myeloid progenitor compartments. Strikingly, mobilized adult peripheral blood (PB) CD34(+) cells responded much less efficiently to cytokine-induced STAT5 activation, with the exception of TPO. In leukemic stem and progenitor cells, highly distinct cytokine responses were observed, differing significantly from their normal counterparts. These responses could not be predicted by the expression level of cytokine receptors. Also, heterogeneity existed in cytokine requirements for long-term expansion of AML CD34(+) cells on stroma. CONCLUSIONS/SIGNIFICANCE: In conclusion, our optimized multiparametric flow cytometry protocols allow the analysis of signal transduction at the single cell level in normal and leukemic stem and progenitor cells. Our study demonstrates highly distinctive cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells.
Subject(s)
Cytokines/metabolism , Fetal Blood/metabolism , Gene Expression Regulation, Neoplastic , Leukemia/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction , Stem Cells/cytology , ADP-ribosyl Cyclase 1/biosynthesis , Antigens, CD34/biosynthesis , Cell Lineage , Cell Separation , Fetal Blood/cytology , Flow Cytometry/methods , Hematopoietic Stem Cells/cytology , Humans , PhosphorylationABSTRACT
OBJECTIVE: To determine the involvement of Rac signaling in self-renewal and expansion on bone marrow stroma of normal CD34+ cells vs leukemic CD34+ cells from acute myeloid leukemia (AML) patients. MATERIALS AND METHODS: Rac signaling was modulated by retroviral introduction of Racl-N17, Racl-V12, or by using the Rac inhibitor NSC23766. In long-term MS5 cocultures (leukemic) expansion, migration, adhesion, and presence of stem/progenitor cells were monitored in both normal as well as leukemic CD34+ cells. RESULTS: Inhibition of Rac signaling impaired migration and adhesion of cord blood (CB) CD34+ cells on MS5 stroma. Long-term inhibition of Rac during a 5-week coculture period on stroma prevented association of hematopoietic progenitors with the bone marrow stromal cells and resulted in a dramatic decrease in the primitive stem cell frequency (long-term culture-initiating cell) in a dose-dependent manner. Many of these phenotypes were reversed in the presence of activated Racl-V12, including improved migration toward, and association with, MS5 cells. CD34+ AML cells were characterized by elevated levels of Rac activity (five of seven patients) and enhanced migration and adhesion to MS5 bone marrow stroma as compared to CB CD34+ cells. A dramatic decrease was observed in the formation of leukemic cobblestone area-forming cells as well as strongly diminished clonal expansion in the presence of the Rac inhibitor NSC23766. CONCLUSION: Our data indicate that Rac signal transduction is required for the maintenance and expansion of both normal as well as leukemic stem/progenitor cells by mediating their interaction with stromal cells.
