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

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.

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.