No 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.

Erythroid and megakaryocyte progenitors express a number of transcription factor genes in common, including the Stem Cell Leukemia (SCL) gene. The SCL gene encodes a 45 kDa protein that contains two distinct functional domains, a basic DNA-binding domain (b) and a helix-loop-helix domain (HLH) required for heterodimerisation with E proteins. We have used a conditional SCL-knockout mouse strain (SCLloxP) to demonstrate that SCL is essential for the growth of erythroid (BFU-E) and megakaryocyte (Mk-CFU) progenitors. To further address the role of SCL in these two related lineages, we have used bone marrow cells from SCL-conditional knockout mice as a source of SCL-null progenitors to perform structure-function analyses. Consistent with the absence of BFU-E, SCL-null bone marrow cells were unable to generate erythroblasts when grown in erythroid cultures (stem cell factor, erythropoietin, insulin-like growth factor 1 and dexamethasone). We subsequently infected SCL-null bone marrow cells with retroviruses expressing wild type or mutant forms of SCL. Expression of wild-type SCL completely rescued the erythroid growth defect of SCL-null bone marrow cells. A truncation mutant containing only the bHLH region was also sufficient for complete rescue of erythropoiesis. However, an HLH only mutant, which lacked the DNA binding domain was unable to rescue erythroid growth, suggesting that SCL directly regulates target genes required for erythropoiesis. To further define the function of SCL in erythropoiesis, we generated immortal cell lines with erythroid potential from SCLloxP mice. In vitro deletion of SCL at the time of initiating erythroid cultures led to rapid cell death that was not rescued by expression of BclXL, suggesting that SCL was not functioning to promote erythroid cell survival. In contrast to the effects on early erythroid cell growth, deletion of SCL after the establishment of erythroblasts had no significant effect on cell growth or survival. This indicates that SCL is required for the initiation or growth of early erythroid progenitors but is dispensable for more mature erythroid progenitor cells. Parallel experiments examining megakaryocytic progenitor growth yielded contrasting results to the erythroid progenitors. Complete rescue of in vitro megakaryopoiesis was observed with both the bHLH and HLH only mutants, with the DNA-binding mutant exhibiting more potent activity than wild-type SCL in this context. The differential DNA-binding requirements of erythroid and megakaryocyte progenitors were even more evident when the HLH only mutant was expressed in wild type cells. In this setting, the DNA-binding mutant increased growth of Mk-CFU but actively inhibited erythroid cell growth. Together, these results indicate that SCL functions to promote erythroid and megakaryocyte cell growth by differing mechanisms. In early erythroid cell growth, SCL functions as a classical transactivator of key target genes, while in megakaryocyte growth, it functions by either sequestration of repressor E proteins, or as a critical, but non DNA-binding, component of a transactivation complex.