The role of SCL in haematopoiesis and leukaemogenesis

Schuh, Annette Hildegard (2005). The role of SCL in haematopoiesis and leukaemogenesis. PhD thesis The Open University.



The fundamental question of how restriction of differentiation potential and activation of lineage-specific gene expression in progenitor cells are regulated is at the heart of research into the molecular mechanisms of biology. The basic helix-loop-helix (bHLH) transcription factor SCL/TAL-1 serves as a paradigm for a transcription factor with pivotal roles in haematopoietic stem cell (HSC) specification, maturation of haematopoietic lineages as well as T-cell leukaemogenesis. Interestingly, it has been described as both a transcriptional activator and a repressor. However, the molecular mechanisms conferring these opposing functions are currently unknown.

As a first step towards characterising the role of SCL in haematopoiesis and leukaemogenesis, we performed a gain-of-function study using a transgenic mouse model over-expressing SCL. Although no leukaemia was observed, constitutive expression of SCL or a mutant lacking the DNA binding domain in HSCs and early progenitors caused a severe defect in T- and B-lymphopoiesis. This was likely due to an interaction with the important lymphoid regulator and SCL heterodimerisation partner E2A.

Then, we set up a proteomics strategy to identify protein partners of SCL in haematopoiesis. We isolated SCL-containing protein complexes in erythroid cells and megakaryocytes using a novel purification strategy based on in vivo biotinylation of SCL. Mass spectrometry analysis led to the identification of several previously unknown candidate partners including ETO-2, a member of the ETO family of co-repressor proteins and SSDP2, a partner of LDB-1. Using several different approaches, we validated the SCL/ETO-2 interaction in cell lines as well as primary erythrocytes and megakaryocytes. We then identified Gfi-1 b, a critical regulator of erythropoiesis with repressive function and the co-repressor mSin3A as additional partners of SCL and ETO-2 in erythroid cells, but not megakaryocytes. Further characterisation in transactivation experiments and during in vitro differentiation of primary fetal liver cells led us to hypothesise that, in early erythropoiesis, ETO-2 might confer a repressive function to SCL.

In conclusion, this study gives a first insight into how the composition of the multimeric protein complexes including SCL might modulate haematopoietic differentiation and cell fate decision.

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