Defining the mechanism of c-Myb involvement in the regulation of haemopoietic stem cells

Lead Research Organisation: University of Birmingham
Department Name: Health and Population Sciences

Abstract

Blood stem cells provide the constant supply of new blood cells throughout life, and they are also important in disease; for example, their ability to replace a patient?s own cells underpins bone marrow transplantation as a therapy for leukaemia and leukaemia-related diseases, whereas on the other hand, mutated versions of blood stem cells are thought to underlie many leukaemias. The unique properties of stem cells depend largely on their profile of active genes, which in turn is controlled by specialised proteins called transcription factors that act as molecular switches. One such protein that appears to be a key in the activation of genes in blood stem cells is c-Myb. This project aims to investigate how and to what extent c-Myb acts as a switch of gene activation in blood stem cells. The work will be performed using stem cells purified from the bone marrow of mice genetically modified to have altered amounts of c-Myb so as to induce changes in the balance of gene activities that it normally controls. The knowledge gained from this study will help to complete the picture of the network of molecular processes that are crucial for normal blood stem cell function, and will have wider implications for the understanding of stem cells in normal and disease situations and perhaps ultimately for their manipulation in a therapeutic context.

Technical Summary

Haemopoietic stem cells (HSCs) reside in small numbers in the bone marrow where they serve as a continuous source of blood cells throughout life. The maintenance of HSCs and their ability to respond to the requirements for differentiated cells requires tight regulation of the balance between cell division (self-renewal) and commitment to differentiate. The regulation of gene expression is central to the behaviour of HSCs, and recently a number of transcriptional regulators have been demonstrated to play a role, although the details of the molecular mechanisms involved and the interplay between different regulators remains unclear. We have identified the c-Myb protein, which is known to be essential for haemopoietic development, as an essential regulator of HSC maintenance. In HSCs, c-Myb appears to influence expression of other transcription factors and signalling molecules that are themselves known to be important in stem cell function. The proposed project aims to investigate the link between c-Myb and the transcription factor Ikaros, and the role that both proteins play in controlling expression of the stem cell receptor tyrosine kinases c-Kit and Flt-3. Purified bone marrow HSCs will be derived from mice genetically modified to express lower levels of c-Myb or undergoing conditional ablation of the c-myb gene. c-Myb-dependent changes in Ikaros expression as well as the overall influence of c-Myb on gene regulation will be probed directly in purified HSCs by in situ immunofluorescence using antibodies against Ikaros or histone modifications characteristic of gene repression or activation. The part played by Ikaros in the overall effect of reduced c-Myb activity will be determined by infection of modified HSCs with Ikaros-expressing lentiviruses. To investigate the mechanism by which c-Myb affects Ikaros gene expression, we will use DNAse hypersensitivity and histone modifications as indicators of chromatin remodelling and transcriptional activity. The initial studies on the Ikaros gene will be performed in a model HSC line, but we will also apply a novel procedure for chromatin immunoprecipitation to investigate epigenetic marks of gene regulation in small numbers of purified HSCs, comparing the Ikaros gene locus in conditions of normal or reduced c-Myb expression.
 
Description Leukaemia Research Specialist Programme Grant
Amount £830,000 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2010 
End 12/2012
 
Description Mechanisms central to the development and maintenance of myeloid disease: The role of Myb family proteins and their potential for therapeutic intervention
Amount £1,396,658 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2013 
End 12/2016
 
Title ES myb kd 
Description myb knockdown ES cells 
Type Of Material Cell line 
Provided To Others? No  
Impact Has enabled generation of chimeras to assess the cell autonomous effects of reduced c-myb expression 
 
Title ES myb kd/ikaros 
Description ES cells homozygous for the myb kd allele constitutively expressing ikaros 2 isoform 
Type Of Material Cell line 
Provided To Others? No  
Impact Chimeras generated from this line have demonstrated that ikaros can rescue many of the stem cell defects caused by decreased c-myb expression 
 
Description Provision of Ikaros reagents/know how 
Organisation Medical Research Council (MRC)
Department MRC Clinical Sciences Centre (CSC)
Country United Kingdom 
Sector Public 
PI Contribution We have investigated to what extent expression of ikaros, a c-Myb target gene, can explain the effects of c-Myb on haemopoietic stem cells
Collaborator Contribution Provision of reagents/expertise in relation to the study of ikaros
Impact It appears that ikaros can rescue many aspects of the c-myb knock down phenotype, confirming its pivotal role as a target of c-Myb
Start Year 2007
 
Description Rescue of c-Myb deficiency through c-myb KO ES cells 
Organisation University of Oslo
Country Norway 
Sector Academic/University 
PI Contribution Provision of ES cells and expertise
Collaborator Contribution Genetic modification of ES cells and assessment of c-Myb function
Impact None yet
Start Year 2012
 
Description The role of protein stability in the control of Myb levels in HSC 
Organisation University of Kumamoto
Country Japan 
Sector Academic/University 
PI Contribution Initial concept and planning for development of new mouse strain, and hosting collaborator to initiate project
Collaborator Contribution Jointly developed and created mouse strain. Now working together on experiments using reporter mouse to monitor Myb levels in primary HSC
Impact Publication in Stem Cells in 2014
Start Year 2008
 
Description Use of c-myb KD ES cells to study smooth muscle differentiation 
Organisation University of Toronto
Department McEwen Centre for Regenerative Medicine
Country Canada 
Sector Academic/University 
PI Contribution Provision of cells and know how regarding their culture etc
Collaborator Contribution Use of the c-myb KD ES cells to study commitment and differentiation processes
Impact None yet
Start Year 2010
 
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 Mixed audience of pupils and parents in a secondary school

None yet apart from lively questioning
Year(s) Of Engagement Activity 2011
 
Description Society of Biology Charter Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Talk lead to extensive questioning and follow up questions by email

Outcomes awaited - only given recently
Year(s) Of Engagement Activity 2014