Role of AML1/Runx1 isoforms in development, self-renewal and differentiation of haematopoietic progenitor/stem cells

The main focus of this research proposal is in the development and regulation of blood stem cells by a gene called AML1/Runx1. Stem cells are one of the main foci of current biology due to their potential to self-replicate and give rise to a variety of mature cells. AML1/Runx1 is critically important for the development of the adult blood system and is frequently involved in the development of human leukaemias. AML1/Runx1 gene produces different forms of the protein which can compete for the influence on other genes which are involved in stem cell development and function as well in the development of leukaemias. We have generated genetically altered embryonic stem (ES) cells and mouse experimental systems which allow AML1/Runx1 gene to be artificially regulated. Regulation of AML1/Runx1 gene is achieved by the use of a modern bi-component tetracycline inducible system. The design of the system enables fine tuning of levels of the AML1/Runx1 protein in cells. The influence of these AML1/Runx1 proteins will be studied in cultured ES cells as well as in developing mouse embryos and in the adult mouse. Two additional genetically modified mice which have fluorescent tagged genes controlled by AML1/Runx1 will also be used in this research to enable accurate identification of cells which express AML1 protein. Better understanding of stem cell regulation and the role of AML1 in this process will have potential implications for the development of protocols for practical regenerative medicine.

The Runx1/AML1 is critically important for development of the definitive haematopoietic system and is one of the most frequent targets in chromosome rearrangements associated with leukaemias. Runx1/AML1 gene encodes for a RUNT homology domain transcription factor which functions as a heteromeric transcription factor complex (AML1:CBFb). Normal haematopoietic development and some forms of leukaemia are AML1 dose dependent. AML1 locus generates a number of splice isoforms which may play different specific roles in cell differentiation. The short isoform of AML1 lacking transcription activation domain is of particular interest since it can be a dominant negative regulator of long AML1 isoforms. Due to this and co-operation with different co-factors AML1 exerts complex regulatory effects on cells which are poorly understood.
In this research proposal the influence of AML1/Runx1 isoforms and their expression levels on the generation and the condition of stem/progenitor cells will be analysed in i) an embryonic stem (ES) cell differentiation system, ii) in embryonic development and in iii) the adult organism. Tetracycline-inducible ES cell and mouse systems generated in the lab will be used for this purpose. The design of these systems allows inducibility and expression levels of AML1 to be monitored. The analysis will be complemented by the use of AML1-YFP non-invasive knock-in reporter mice which will allow identification of AML1-expressing cells in vivo. Furthemore, HoxB4-YFP knock-in reporter mice will be used to monitor the condition of ?stemness? of HSCs upon overexpression of AML1 isoforms.
The proposed research will use methods of in vitro haematopoietic and endothelial differentiation. The influence of AML1 isoforms on haemangioblast and haemogenic endothelium will be investigated in culture and supported by extensive flow cytometry analysis and long-term transplantation studies.