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