ABSTRACT
Previous studies have indicated that the stem cell leukemia gene (SCL) is essential for both embryonic and adult erythropoiesis. We have examined erythropoiesis in conditional SCL knockout mice for at least 6 months after loss of SCL function and report that SCL was important but not essential for the generation of mature red blood cells. Although SCL-deleted mice were mildly anemic with increased splenic erythropoiesis, they responded appropriately to endogenous erythropoietin and hemolytic stress, a measure of late erythroid progenitors. However, SCL was more important for the proliferation of early erythroid progenitors because the predominant defects in SCL-deleted erythropoiesis were loss of in vitro growth of the burst-forming erythroid unit and an in vivo growth defect revealed by transplant assays. With respect to erythroid maturation, SCL-deleted proerythroblasts could generate more mature erythroblasts and circulating red blood cells. However, SCL was required for normal expression of TER119, one of the few proposed target genes of SCL. The unexpected finding that SCL-independent erythropoiesis can proceed in the adult suggests that alternate factors can replace the essential functions of SCL and raises the possibility that similar mechanisms also explain the relatively minor defects previously observed in SCL-null hematopoietic stem cells.
Subject(s)
Aging/blood , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Erythropoiesis/genetics , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Transcription Factors/deficiency , Transcription Factors/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors , Bone Marrow Transplantation , DNA-Binding Proteins/physiology , Erythroid Precursor Cells/physiology , Erythropoiesis/physiology , Hematopoietic Stem Cells/physiology , Mice , Mice, Knockout , Mice, Transgenic , Proto-Oncogene Proteins/physiology , T-Cell Acute Lymphocytic Leukemia Protein 1 , Transcription Factors/physiologyABSTRACT
The stem cell leukemia (SCL) gene is essential for the development of hematopoietic stem cells in the embryo. Here, we used a conditional gene targeting approach to examine the function of SCL in adult hematopoietic stem cells (HSCs). Flow cytometry of bone marrow from SCL-deleted mice demonstrated a 4-fold increase in number of Lin(neg) c-kit(+) Sca-1(+) cells. Despite this increase in the number of phenotypic HSCs, competitive repopulation assays demonstrated a severe multilineage defect in repopulation capacity by SCL-deleted bone marrow cells. SCL-heterozygous cells also showed a mild repopulation defect, thus suggesting haploinsufficiency of SCL. The transplantation defect of SCL-deleted cells was observed within 4 weeks of transplantation, indicating a defect in a multipotent progenitor or short-term repopulating HSCs. Although the defect persisted in secondary transplants, it remained relatively stable, suggesting that SCL was not required for self-renewal of the HSCs. Generation of SCL-deleted cells within SCL-wild-type mice rescued the early repopulating defect. Together, our results suggest that SCL is required for the normal function of short-term repopulating HSCs.
Subject(s)
DNA-Binding Proteins/physiology , Hematopoiesis , Hematopoietic Stem Cells/physiology , Proto-Oncogene Proteins/physiology , Transcription Factors/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors , Bone Marrow Cells/immunology , Cell Differentiation , Cell Lineage , Flow Cytometry , Graft Survival , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Immunophenotyping , Mice , Mice, Knockout , Mice, Transgenic , Multipotent Stem Cells/cytology , Multipotent Stem Cells/physiology , T-Cell Acute Lymphocytic Leukemia Protein 1ABSTRACT
Stromal interaction molecule 1 (STIM1) is a cell surface transmembrane glycoprotein implicated in tumour growth control and stromal-haematopoietic cell interactions. A single sterile alpha motif (SAM) protein-protein interaction domain is modelled within its extracellular region, a subcellular localisation not previously described for other SAM domain-containing proteins. We have defined the transmembrane topology of STIM1 by determining the sites of N-linked glycosylation. We have confirmed that STIM1 is modified by N-linked glycosylation at two sites within the SAM domain itself, deduced as asparagine residues N131 and N171, demonstrating that STIM1 is translocated across the membrane of the endoplasmic reticulum such that the SAM domain resides within the endoplasmic reticulum (ER) lumen. Both N-linked oligosaccharides remain endoglycosidase H-sensitive, indicating absence of full processing within the ER and Golgi. This immature modification is nevertheless sufficient and critical for cell surface expression of STIM1. We show that STIM1-STIM1 homotypic interactions are mediated via the cytoplasmic rather than the extracellular region of STIM1, excluding an essential role for the SAM domain in these protein interactions. These studies provide the first evidence for an extracellular localisation of a SAM domain within any protein, and the first example of a SAM domain modified by N-linked glycosylation.
