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Molecular Mechanisms of Hematopoiesis and Leukemogenesis
During hematopoiesis, the pluripotent stem cells become committed to precursor cells of various lineages and eventually differentiate into functionally and morphologically distinct, nonproliferating, terminally differentiated cells and enter the circulating blood. Therefore, hematopoiesis provides an excellent system for studies of cell proliferation, differentiation, and apoptosis. The process of hematopoiesis involves the gain and loss of enzymatic activities and the appearance or loss of surface markers. All these changes are accompanied by a complex pattern of gene regulation, mediated in part by cytokines. Dysregulated gene expression is directly associated with blocking of cell differentiation and the development of leukemia.
AML1 AND ITS FUSION PROTEIN AML1-ETO IN BLOOD CELL DIFFERENTIATION
Acute myeloid leukemia is a major hematopoietic malignant neoplasm characterized by the proliferation of a malignant clone of myeloid progenitor cells. One of the most common targets of chromosomal translocations implicated in acute myeloid leukemia is the gene AML1. The gene was identified through a study of t(8;21) chromosomal translocation in which the runt homology domain of AML1 was fused to a gene termed ETO (MTG8) to encode a fusion protein called AML1-ETO. Subsequent studies indicated that the protein AML1 is crucial for normal hematopoiesis. We previously discovered that AML1 synergistically activates the expression of a critical myeloid gene, the gene for the M-SCF receptor, with 2 other important transcription factors, C/EBP and PU.1.
To study the effect of AML1-ETO on hematopoiesis, we generated mice in which wild-type AML1 was replaced by AML1-ETO. In these mice, human t(8;21) is recapitulated by substituting the fusion gene for the wild-type allele. We found that a block of definitive hematopoiesis occurred in the heterozygous AML1+/AML1-ETO+ embryos. The embryos died in midgestation because of severe hemorrhaging in the CNS. Currently, we are identifying cofactors involved in the synergy among various transcription factors and are using inducible AML1-ETO-expressing cell lines and transgenic mice to analyze the role of AML1-ETO in the development of leukemia.
A NOVEL UBIQUITIN-SPECIFIC ENZYME, UBP43
Using representational difference analysis to identify genes differentially expressed in AML1-ETO mice, we isolated the gene for a novel ubiquitin-specific enzyme, UBP43 (USP18), which belongs to a family of ubiquitin-specific proteases. UBP43 is upregulated in mice that express AML1-ETO.
Protein ubiquitination is important in a variety of cellular events, including ubiquitin-dependent proteolysis. Proteolysis regulated by the ubiquitin pathway has been implicated in control of the cell cycle, activation of transcription, presentation of antigen, and the fate and growth of cells. The ubiquitin-specific proteases are a family of enzymes that can cleave ubiquitin from a wide range of protein substrates. The high degree of divergence in this large family implies that different members may have unique biochemical properties, substrate specificities, and cellular localizations.
UBP43 is highly expressed in the thymus of normal adult mice and in monocytic cell lines, suggesting that it is involved in hematopoiesis. We are using gene knockout, protein interaction, and gene regulation to study the biological function of this novel protease.