Defective Stem Cell Factor Expression in c-myb Null Fetal Liver Stroma Submitted 01/31/01 (Communicated by M. Lichtman, M.D., 02/08/01) Catherine Sicurella, 1 Ruth Freeman, 1 Sue Micallef, 1 Michael L. Mucenski, 1,2 Ivan Bertoncello, 1 and Robert G. Ramsay 1,3 ABSTRACT: High levels of c-Myb are observed in immature precursor myeloid and lymphoid cells, while downregulation of c-myb accompanies terminal differentiation to a mature phenotype. This has established c-Myb as a crucial transcription factor for hematopoiesis. Further evidence for this is the embryonic death of the c-myb homozygous mutant mouse at ED15 due to defective fetal liver erythropoiesis. Cells from fetal liver of wild-type and c-myb-/- embryos were examined in detail for their hematopoietic potential and the capacity of the stroma to support wild-type hematopoiesis. The c-myb-/- fetal liver was shown to harbor sevenfold fewer spleen focus-forming cells and a similarly lower number of cells with long-term repopulating capacity (high proliferative potential cells). However, shorter term repopulating cells were not substantially reduced. c-myb-/- stromal cells were unable to support the proliferation of wild-type bone marrow lineage-negative cells. This was found to be partly due to a decrease in stem cell factor (SCF) expression while partial rescue of the stromal cell cultures was achieved through the addition of exogenous SCF. DNA binding studies for two sites within the SCF promoter demonstrated an in vitro interaction between the SCF promoter and c-Myb and transient transfection studies demonstrated that c-Myb could substantially transactivate the SCF promoter in HEK293 cells. These data explain why the c-myb-/- embryos are so impaired in their ability to establish hematopoiesis. © 2001 Academic Press Key Words: c-Myb; stem cell factor; stromal cells; knock-out mice. INTRODUCTION c-myb is one of the many genes required for the development and maintenance of the defin- itive hematopoietic system. Immature avian and human hematopoietic progenitors express high levels of c-myb (1). Thymic lymphocytes and erythroid cells also express relatively high lev- els of c-myb, which fall as these cells mature (2). Treating human bone marrow hematopoi- etic cells with c-myb antisense oligonucleotides provided the first demonstration of a functional role for c-Myb in hematopoietic cells. This resulted in an inhibition of cellular proliferation (3). Other experiments with various hematopoi- etic lineages and c-myb antisense oligonucleo- tides gave similar results (4). In contrast, over- expression of c-Myb inhibits erythroid and myeloid differentiation (5, 6). The c-myb-/- mouse provides further evidence of the impor- tance of c-Myb in the hematopoietic system. It dies at about ED15, due to a failure of all hematopoietic lineages, except megakaryo- cytes, to proliferate and populate the fetal liver (7). Recent work has shown T cell development is also compromised in this mouse (8). The high level of c-myb expression normally seen in hematopoietic progenitor cells suggests the phenotype of the c-myb-/- mouse is due to This paper summarizes a presentation made at the Second International Workshop on Myb Genes, held in Melbourne on November 22–24, 2000, sponsored in part by the Leukemia & Lymphoma Society (U.S.A.). 1 Peter MacCallum Cancer Institute, Melbourne 8006, Australia. 2 Division of Pulmonary Biology, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039. 3 Correspondence and reprint requests to: Robert G. Ramsay, Differentiation and Transcription Laboratory, Peter MacCallum Cancer Institute, Locked Bag #1, A’ Beckett Street, Melbourne, 8006, Australia. Fax: 61-3-9656-1411. E-mail: r.ramsay@pmci.unimelb.edu.au. Blood Cells, Molecules, and Diseases (2001) 27(2) Mar/Apr: 470 – 478 Sicurella et al. doi:10.1006/bcmd.2001.0407, available online at http://www.idealibrary.com on 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved 470