The role of the bHLH transcription factor SCL/Tal-1 in haemopoiesis

Lead Research Organisation: University of Oxford

Abstract

Many inherited and acquired diseases are associated with anaemia in which there are too few red blood cells. Understanding these disorders depends on knowing how blood normally forms in the bone marrow throughout development. Recently there has been considerable accumulation of knowledge about the factors that direct formation of the blood. Our interest has focused on one such factor (called SCL) which plays several critical roles in this process. In the future, we plan to understand how this factor controls the formation of blood.

Technical Summary

Elucidating the molecular mechanisms that control lineage specification and differentiation is a central question in developmental biology. Haemopoiesis provides a good model to enable the analysis of such mechanisms. Many ubiquitous and specifically expressed transcription factors have been shown to play specific roles in orchestrating the differentiation of a haemopoietic pluripotent stem cell into specialised blood lineages. The oncoprotein SCL/Tal-1 has recently emerged as a crucial regulatory protein at very early steps in haemopoietic development as well as later for terminal erythroid and megakaryocytic differentiation. SCL ectopic expression in T cells has been described in up to 60% of T-cell acute lymphoblastic leukaemia (T-ALL). Our aim is to increase our understanding of the molecular mechanisms involved in haemopoietic specification/differentiation by studying different aspects of SCLs function in normal haematopoietic development. This is of particular interest from a developmental point of view and may also be relevant to clinical practice, as these questions lie at the heart of how haemopoietic stem cells are formed.||Our objectives are to characterise the transcriptional networks controlled by this key regulator in erythropoiesis and developmental haemopoiesis through the development of integrated and complementary approaches. Importantly, this will contribute to our understanding of haemopoietic stem cell (HSC) genetic regulatory pathways. We expect characterising general molecular mechanisms, such as activation of a specific developmental programme as well as repression of alternate pathways by a master regulator that will apply to other specification models. Importantly, our initial discoveries (new target genes and protein partners) will open up new avenues of investigation, allowing for the expansion of our research programme. Finally, we anticipate that our lists of target genes and partners will provide useful resources and be of interest to other investigators in the Unit working on the early stages of blood formation. We are also planning to develop the iPS cell system and projects using hES cells, to keep abreast with new technologies.||(1) To complete our characterisation of the molecular pathways and target genes controlled by SCL and partners in erythropoiesis, we have established large-scale genomic studies. Through gene expression/ChIP-sequencing analyses, we will dissect some of the mechanisms engaged by SCL that will give insight into molecular pathways leading to red cell production. We will also characterise pathways/cis-elements with distinct requirements for SCL direct and indirect DNA-binding activity. We anticipate drawing general principles from this study that will apply to other transcription factors.||(2) We previously identified the oncoprotein and co-repressor ETO2 as a partner of SCL in erythroid cells. The function of ETO2 in developmental haemopoiesis is being further investigating in Xenopus, by combining cellular and molecular studies. The data we have accumulated so far uncovers unsuspected signalling pathways leading to HSC specification.||(3) To gain insights into the molecular mechanisms underlying mouse haemopoietic development and HSC specification, we will identify SCLs protein partners and target genes in mesodermal/pre-haemopoietic cells by applying proteomic and large-scale genomic studies and using the mouse ES cells system as our developmental model. We will also develop the iPS cell system and projects using hES cells to keep abreast with new technologies.||(4) Finally, we will solve the crystal structure of the bHLH region of an SCL heterodimer to elucidate the role of functionally crucial residues in SCLs HLH domain, test whether binding to DNA alters the structure of the heterodimer and solve the structure of the SCL core (quaternary) complex.||By focusing on the role of SCL in primitive and definitive haemopoietic precursors and in erythroid cells, this p
 
Description Gordon Pilar studentship
Amount £120,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2010 
End 09/2014
 
Description Project Grant
Amount £179,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2008 
End 03/2011
 
Description Project Grant
Amount £50,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2007 
End 03/2008
 
Description Project Grant
Amount £184,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2009 
End 03/2012
 
Title MKD1 cell line 
Description Generation of a new mouse megakaryocytic cell line 
Type Of Material Cell line 
Year Produced 2012 
Provided To Others? Yes  
Impact Very good alternative to using primary mouse mekagaryocyte progenitors. 
 
Title Mouse model 
Description A mouse model expressing a DNA-binding mutant form of a crucial haemopoietic transcription factor. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2008 
Provided To Others? Yes  
Impact We learnt about unexpected mechanisms of action of this regulator in vivo. This had impact on how other key regulators function. 
 
Title Mouse model 
Description Inducible knock-out model of a key, multifunctional blood regulator 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact By giving access to the different stages of blood development regulated by this key transcription factor, this tool is increasing our molecular understanding of stem cell development and differentiation. 
 
Title X-ray structure of LMO2-LDB1(LID) 
Description Crystal structure of a complex of two transcriptional regulators involved in many critical developmental and differentiation pathways. 
Type Of Material Biological samples 
Provided To Others? No  
Impact A better understanding of the mechanism of action of a key component of a multiprotein complex. This has increased our knowledge on how multiprotein complexes form. 
 
Title X-ray structure of a quaternary protein complex bound to DNA 
Description The atomic structure of a key quaternary protein complex bound to DNA was solved by X-ray crystallography 
Type Of Material Biological samples 
Provided To Others? No  
Impact Better understanding of some of the molecular mechanisms involved in the transcriptional regulation of haematopoiesis and leukaemogenesis. These observations will apply to other tissue-specific transcriptional complexes. 
 
Description Crystal structure 
Organisation University of Oxford
Department Wellcome Trust Centre for Human Genetics
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We have provided them with a very interesting protein complex to study by X-ray crystallography.
Collaborator Contribution They have solved the crystal structure of the transcription factor SCL and its partners. We are now further studying this structure in functional assays.
Impact PMID: 21076045 PMID: 21045296 PMID: 23831025 The structure has provided us with a structural explanation for how this complex operates. This has important implications for our understanding of the transcriptional regulation of haematopoiesis and leukaemogenesis.
Start Year 2007
 
Description GATA1 transcriptional regulation 
Organisation Medical Research Council (MRC)
Department MRC Molecular Haematology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in technologies
Collaborator Contribution Expertise in specific technologies
Impact PMID: 20154211 PMID: 18625887 PMID: 16551635
 
Description LRF grant holder day 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact The meeting was intended for LRF grant holders as well as any other researchers with interests in the subject.

Recognition of our work by other groups working in the field.
Year(s) Of Engagement Activity 2006
 
Description School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 100 pupils attended the talks on biomedical careers. This has inspired pupils to study biology at University.

5th and 6th form pupils came to the lab for work experience
Year(s) Of Engagement Activity 2012