ABSTRACT
Telomerase is a ribonucleoprotein complex that maintains the length and integrity of telomeres, and thereby enables cellular proliferation. Understanding the regulation of telomerase in hematopoietic cells is relevant to the pathogenesis of leukemia, in which telomerase is constitutively activated, as well as bone marrow failure syndromes that feature telomerase insufficiency. Past studies showing high levels of telomerase in human erythroblasts and a prevalence of anemia in disorders of telomerase insufficiency provide the rationale for investigating telomerase regulation in erythroid cells. Here it is shown for the first time that the telomerase RNA-binding protein dyskerin (encoded by DKC1) is dramatically upregulated as human hematopoietic stem and progenitor cells commit to the erythroid lineage, driving an increase in telomerase activity in the presence of limiting amounts of TERT mRNA. It is also shown that upregulation of DKC1 was necessary for expansion of glycophorin A+ erythroblasts and sufficient to extend telomeres in erythroleukemia cells. Chromatin immunoprecipitation and reporter assays implicated GATA1-mediated transcriptional regulation of DKC1 in the modulation of telomerase in erythroid lineage cells. Together these results describe a novel mechanism of telomerase regulation in erythroid cells which contrasts with mechanisms centered on transcriptional regulation of TERT that are known to operate in other cell types. This is the first study to reveal a biological context in which telomerase is upregulated by DKC1 and to implicate GATA1 in telomerase regulation. The results from this study are relevant to hematopoietic disorders involving DKC1 mutations, GATA1 deregulation and/or telomerase insufficiency.
Subject(s)
Cell Cycle Proteins/metabolism , Erythroblasts/metabolism , GATA1 Transcription Factor/metabolism , Nuclear Proteins/metabolism , Telomerase , Cell Cycle Proteins/genetics , GATA1 Transcription Factor/genetics , Humans , Nuclear Proteins/genetics , Telomerase/genetics , Telomerase/metabolism , Up-RegulationABSTRACT
The bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin, in vitro reporter assay, and in vivo transgenic data to identify and validate Smad1+63 and the Smad5 promoter as tissue-specific cis-regulatory elements that are active in the developing endothelium. The activity of these elements in the endothelium was dependent on highly conserved ETS, GATA, and E-box motifs, and chromatin immunoprecipitation showed high levels of enrichment of FLI1, GATA2, and SCL at these sites in endothelial cell lines and E11 dorsal aortas in vivo. Knockdown of FLI1 and GATA2 but not SCL reduced the expression of SMAD1 and SMAD5 in endothelial cells in vitro. In contrast, CD31(+) cKit(-) endothelial cells harvested from embryonic day 9 (E9) aorta-gonad-mesonephros (AGM) regions of GATA2 null embryos showed reduced Smad1 but not Smad5 transcript levels. This is suggestive of a degree of in vivo selection where, in the case of reduced SMAD1 levels, endothelial cells with more robust SMAD5 expression have a selective advantage.
Subject(s)
Endothelium/embryology , GATA2 Transcription Factor/metabolism , Gene Expression Regulation, Developmental , Proto-Oncogene Protein c-fli-1/metabolism , Smad1 Protein/genetics , Smad5 Protein/genetics , Animals , Base Sequence , Cell Line , Endothelium/metabolism , GATA2 Transcription Factor/genetics , Gene Knockdown Techniques , Mice , Molecular Sequence Data , Promoter Regions, Genetic , Proto-Oncogene Protein c-fli-1/geneticsABSTRACT
Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the "heptad" complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs.
Subject(s)
Cell Transdifferentiation/genetics , Endothelial Cells/metabolism , Gene Expression Profiling/methods , Hematopoietic Stem Cells/metabolism , Receptors, G-Protein-Coupled/genetics , Animals , CHO Cells , COS Cells , Cell Line , Cells, Cultured , Chlorocebus aethiops , Cricetinae , Cricetulus , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Endothelial Cells/cytology , Female , Gene Ontology , Hematopoietic Stem Cells/cytology , Humans , In Situ Hybridization , Mice, Inbred C57BL , Microscopy, Confocal , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNA/methods , Up-RegulationABSTRACT
Interlinked gene regulatory networks (GRNs) are vital for the spatial and temporal control of gene expression during development. The hematopoietic transcription factors (TFs) Scl, Gata2 and Fli1 form one such densely connected GRN which acts as a master regulator of embryonic hematopoiesis. This triad has been shown to direct the specification of the hemogenic endothelium and emergence of hematopoietic stem cells (HSCs) in response to Notch1 and Bmp4-Smad signaling. Here we employ previously published data to construct a mathematical model of this GRN network and use this model to systematically investigate the network dynamical properties. Our model uses a statistical-thermodynamic framework to describe the combinatorial regulation of gene expression and reconciles, mechanistically, several previously published but unexplained results from different genetic perturbation experiments. In particular, our results demonstrate how the interactions of Runx1, an essential hematopoietic TF, with components of the Bmp4 signaling pathway allow it to affect triad activation and acts as a key regulator of HSC emergence. We also explain why heterozygous deletion of this essential TF, Runx1, speeds up the network dynamics leading to accelerated HSC emergence. Taken together our results demonstrate that the triad, a master-level controller of definitive hematopoiesis, is an irreversible bistable switch whose dynamical properties are modulated by Runx1 and components of the Bmp4 signaling pathway.
Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression Regulation, Developmental/physiology , Gene Regulatory Networks/genetics , Hematopoiesis/physiology , Hematopoietic Stem Cells/metabolism , Models, Theoretical , Signal Transduction/physiology , Animals , Bone Morphogenetic Protein 4/metabolism , Computer Simulation , Gene Expression Regulation, Developmental/genetics , Mice , Mice, Knockout , Receptor, Notch1/metabolism , Signal Transduction/genetics , Smad1 Protein/metabolism , Smad6 Protein/metabolism , Thermodynamics , Transcription Factors/metabolismABSTRACT
Haematopoiesis is one of the most intensively studied and best described systems in the body. However, knowledge of cell signalling pathways that drive haematopoietic stem cell (HSC) development and how they interact with each other and with transcription factors that specify HSCs is still rudimentary. In this review, we will highlight recent advances in the field relating to these questions and identify gaps that need to be filled. To this end, we will briefly describe the developmental ontogeny of HSCs and examine in detail the cell signalling pathways that have been implicated in HSC specification. In conclusion we will discuss how this knowledge has led to the generation of blood cells in the laboratory and consider the implications of that advancement.
