In vivo study of the DNA-binding requirements of the haematopoietic transcription factor SCL (stem cell leukaemia) in erythropoiesis

• Main Author: Kassouf, Mira
• Published: University of Oxford 2006
• Subjects: 612.1
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492059

The bHLH (basic helix-loop-helix) transcription factor SCL (Stem Cell Leukaemia) is required for HSC (haematopoietic stem cell) specification and for differentiation of the megakaryocytic and erythroid lineages. bHLH proteins bind DNA as dimers and this has been thought to be a prerequisite for their function. However, this concept has been recently challenged as SCL DNAbinding activity was shown to be dispensable for some of its functions, in in vitro haematopoietic differentiation assays. In my thesis, I studied the in vivo requirements for SCL DNA-binding activity in mice with a germ line DNA-binding (SCL-RER) mutation. In contrast to SCL knock-out embryos that die at E9.5 from absence of haematopoietic development, specification of haematopoietic progenitors was observed in SCLRERIRER yolk sacs. Lethality was first observed at E14.5. However 5% of homozygous mice were born from heterozygous crosses. A specific defect in erythropoiesis was documented. Anaemia was observed throughout development in homozygous embryos and adult mice. A combination of complementary phenotypic and cellular analyses of primitive and definitive erythropoiesis showed that specification of erythroid progenitors occurs but revealed defective erythroid maturation. To understand the molecular defects underlying the phenotype, expression levels of candidate target genes were assessed in fetal liver cells enriched for early erythroid progenitors or late normoblasts. We observed decreased or increased mRNA expression of some genes tested in SCLRERIRER erythroid populations when compared to controls, suggesting that SCL DNA-binding activities are required for both activation and repression of target genes. We then pursued the analysis of SCL-mediated transcriptional regulation by studying the recruitment of SCL to known cl.s-regu Iatory regI.Ons 0 f genes W.ith perturbed expressI.On pattern I.n SCLRER/RER erythro'dl populations by ChIP assay. Our data showed that SCL recruitment to specific hypersensitive sites was compromised on chosen targets such as a-globin, the transcription factor GATAl, and the red cell membrane protein band 4.2. In conclusion, this in vivo model confirms the dispensability of SCL DNA-binding activity for specification of primitive erythroid progenitors and definitive HSCs. As for erythroid development, it is the first in vivo proof in mammals that SCL DNA-binding activity is not required for specification of progenitors but is crucial for their differentiation into mature red blood cells. Finally, through our molecular studies, SCL appears as a multifunctional transcriptional regulator of erythropoiesis that can act as either an activator or a repressor of gene expression depending on the target and the maturation stage.