Paterson Institute for Cancer Research SCIENTIFIC REPORT 2005

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32 GROUP LEADER Georges Lacaud Stem Cell Biology Group http://www.paterson.man.ac.uk/groups/scbio.jsp The AML1/Runx1 transcription factor is a frequent target of gene rearrangements and mutations in human acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). Consistent with its initial implication in leukemias, Runx1 has been shown to be critical for normal haematopoietic development.The MOZ gene is involved in three independent myeloid chromosomal translocations fusing MOZ to the partner genes CBP, P300 or TIF2. MOZ, CBP and P300 are histone acetyltransferase (HAT) that are required as cofactors by many DNA-binding transcription factors. Using the in vitro differentiation system based on mouse embryonic stem (ES) cells, our goals are to further define the role of Runx1 and Moz in early haematopoietic development and how alterations of their function leads to leukemogenesis. Early haematopoietic development The earliest site of haematopoiesis in the mouse embryo is the yolk sac where blood islands, derived from mesodermal cells, develop at approximately day 7.5 of gestation. The yolk sac blood islands consist of two lineages, a population of primitive erythroid cells surrounded by a layer of angioblasts that eventually form the developing vasculature. The parallel development of these lineages in close association provided the basis for the hypothesis that they arise from a common precursor, a cell called the haemangioblast. Definitive haematopoiesis is defined by the generation of all lineages other than primitive erythroid cells and includes the production of definitive erythroid, myeloid and lymphoid cells. Attempts to identify, isolate and characterize the precursors representing these initial stages of lineage development, including the elusive haemangioblast, have been largely hampered by the inaccessibility of the early mammalian embryo. The differ- POSTDOCTORAL FELLOWS Christophe Lancrin Flor Perez-Campo SCIENTIFIC OFFICER Catherine Gavin GRADUATE STUDENT Kelly Chiang Patrycja Sroczynska entiation of embryonic stem (ES) cells in culture offers a powerful alternative approach to study the development of such lineages that are established very early in embryonic life. When removed from conditions that maintain them in vitro in an undifferentiated state, ES cells will spontaneously differentiate and form colonies or embryoid bodies (EBs) that contain precursors for multiple lineages including those of the haematopoietic and endothelial systems. Several studies have established that the early events of haematopoietic and endothelial commitment in the ES/EB model are comparable, if not identical, to that of the early embryo. Using this model system, a precursor was identified that generates blast colonies containing precursors of endothelial and haematopoietic lineages. The blast colony-forming cells (BL-CFC) that generate these colonies represent a transient population that appears in the EBs prior to the emergence of any other haematopoietic lineage precursors. The characteristics of the BL-CFC suggest that it represents the in vitro equivalent of the haemangioblast and as such the earliest stage of haematopoietic development described to date. More recently the existence of the haemangioblast has been demonstrated in vivo in early embryos. A critical function of Runx1 in haemangioblast development. 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 P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 5
STEM CELL BIOLOGY 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 and suggest that its functional requirement may define subpopulations of BL- CFC precursors. Downstream transcriptional targets of Runx1 Runx1 has been shown to participate in the regulation of expression of a number of different genes including IL-3, myeloperoxidase, neutrophil elastase, M-CSF, GM-CSF and T cell receptors that are involved in relatively late stages of haematopoietic development. Our results have indicated that Runx1 is required for definitive haematopoietic development at the level of the BL-CFC and is therefore likely to regulate a different set of genes at this time of development. To identify these genes, we have compared the patterns of gene expression of haemangioblast-enriched-cell-populations or haemangioblast-derived-cell-populations from either Runx1 deficient or Runx1 competent ES cells. The possibility to isolate large number of cells at the precise stage of the haematopoietic defect represents a unique advantage of the ES/EB system. We have validated the differential expression of several candidates on samples generated from the ES/EB system. We are now establishing the direct regulation of the transcription of some of these genes by Runx1 by promoter assays or chromatin immunoprecipitation and are further determining the function of these genes during early haematopoietic development. Runx1 isoforms Previous studies have shown that Runx1 is expressed as multiple naturally occurring spliced isoforms that generate proteins with distinct activities on target promoters. Recent experiments have indicated that expressions of some of these isoforms are differentially regulated during development. One intriguing hypothesis is that the different isoforms of this transcription factor could fulfill distinct functions at the different stages of the establishment of the haematopoietic system. We are testing this hypothesis by further defining the pattern of expression of the different Runx1 isoforms and evaluating their respective functions. Moz and haematopoietic development. MOZ was found translocated in AML with CREB binding protein (CBP), the nuclear receptor TIF2 or the P300 transcriptional co-activator. All these genes encode enzymes containing a histone acetyl transferase domain (HAT) suggesting that aberrant modification of histones or other factors could provide the first step in the route to oncogenicity. We have created ES cells in which one or both allele of MOZ are mutated for the HAT activity. We have evaluated the consequences of such alteration upon in vitro ES cell differentiation. Our studies have revealed a profound defect in the normal proliferation and differentiation of haematopoietic precursors in absence of the HAT activity of MOZ. These results indicate a critical function of the HAT activity of MOZ during haematopoiesis. We are currently investigating the precise molecular and cellular mechanisms affected and evaluating the consequences of the HAT mutation during in vivo haematopoiesis. Critical functions of Runx1 and Moz in haematopoietic development. Runx1 is essential for the generation of definitive haematopoietic precursors from definitive haemangioblasts. Moz is critical for the proliferation and differentiation of both primitive and definitive haematopoietic precursors. P A T E R S O N I N S T I T U T E S C I E N T I F I C R E P O R T 2 0 0 5 Publications listed on page 61 33
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Page 7 and 8: espect with his peers. He enjoyed l
Page 9 and 10: BIOINFORMATICS age, MIAME VICE is b
Page 11 and 12: CARCINOGENESIS Clinical trials invo
Page 13 and 14: CELL CYCLE the RPC contains a two-s
Page 15 and 16: CELL DIVISION Plo1 regulated its af
Page 17 and 18: CELL REGULATION inactivation of ATF
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Page 25 and 26: GENE THERAPY ued our studies utilil
Page 27 and 28: IMMUNOLOGY tory cells following tum
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