RUNX1 in haematopoietic differentiation.
To investigate the role of Runx1 at the earliest stage of haematopoietic commitment, we have analyzed its expression pattern and function during ES/EB differentiation and in early yolk sac development. Expression analyses indicated that Runx1 is expressed in yolk sac mesodermal cells prior to the establishment of the blood islands and within the BL-CFC in EBs. Embryoid bodies generated from the Runx1-/- ES cells did not contain definitive haematopoietic precursors, reflecting the defect observed in the mutant mice. Analysis of early EBs revealed a profound defect in the potential of the Runx1-/- ES cells to generate blast colonies. Fewer colonies were generated by the mutant ES cells and their potential was restricted to endothelial and primitive haematopoietic development. In contrast, Runx1+/+ blast colonies displayed endothelial as well as both primitive and definitive developmental potential. Altogether these results provide evidence that Runx1 does function at the haemangioblast stage of development.
Targets of RUNX1
Our previous studies revealed a profound defect in the potential of the Runx1-/- ES cells to generate blast colonies and demonstrated that RUNX1 is critical for the generation of definitive haematopoietic from haemogenic endothelium during the formation of blast colonies. RUNX1 is likely to regulate the expression of an important set of genes at this stage of development. To identify these genes, we compared the patterns of gene expression of haemangioblast-enriched-cell-populations or haemangioblast-derived-cell-populations from either Runx1deficient or Runx1 competent ES cells. We further validated the differential expression of candidates on samples generated from the ES/EB system and further documented the regulation by RUNX1 of the transcription of several of these genes by promoter assays or chromatin immunoprecipitation. We are currently evaluating the specific function of some of these genes at the onset of haematopoietic development and testing their potential to rescue haematopoietic development in absence of RUNX1. We have selected previously uncharacterized transcriptional target genes of RUNX1 and have initiated a series of experiments, such as conditional knock-out and knock-in, to determine the pattern of expression and function of these new genes.
In vertebrates, the transcription of the Runx1 gene is under the control of two alternative promoters, distal (P1) and proximal (P2), which generate specificRunx1 transcripts. We investigated the activities of distal and proximal Runx1promoters at the single cell level and tracked the cell populations expressing the respective isoforms. We demonstrated that at the onset of haematopoeisis, bothin vitro and in vivo, the activity of the proximal promoter marks a haemogenic endothelium cell population, whereas the subsequent activation of the distalRunx1 promoter defines fully committed definitive haematopoietic progenitors. Interestingly, haematopoietic commitment in distal Runx1 knock-out embryos appears normal, suggesting that the proximal isoform plays a critical role in the generation of haematopoietic cells from haemogenic endothelium.
RUNX1 and Leukemia
The core binding factors AML1/RUNX1 and CBFβ are the most frequent targets of these genetic alterations in human leukaemia. The t(8;21) translocation resulting in AML1-ETO fusion and the inv(16) generating the SHMMC-CBFβ fusion accounts together for more than 20% of all the AML cases. Animal models have indicated that the full length AML-ETO, expressed either upon viral transfer or as a transgene, is not able to induce alone leukemia in mice. However an alternatively spliced form of AML1-ETO has recently been shown to cause following retroviral transfer a rapid development of leukemia in mice. Based on this new finding, we are developing an animal model in which the expression of this form of AML1-ETO is inducible. This new tool will allow us to study the molecular events leading upon expression of AML-ETO to the development of leukaemia.