The focus of this proposal is the role of homeotic genes in human hematopoietic commitment and differentiation. This gene family determines cell fate and tissue identity during development of the Drosophila embryo. Homeobox gene products contain a highly conserved 60 amimo acid sequence (called the homeobox), which is a DNA binding domain. Homeobox-containing genes are also expressed in temporally and spacially specific patterns in human embryos. We have made the novel observation that at least 5 unique homeobox genes are expressed in lineage-restricted patterns in human hematopoietic cell lines and that expression is modulated during differentiation. This project has five major aims: 1. To demonstrate lineage-restricted patterns of expression of homeoboxcontaining genes in a survey of human leukemic cell lines representing a spectrum of hematologic phenotypes (e.g. erythroid, myeloid, monocytic, and lymphoid). We will perform Northern gel analysis of mRNA isolated from a spectrum of leukemic cell lines using our 5 homeobox probes. 2. To study the modulation of homeobox gene expression during hematopoietic differentiation. We will perform Northern gel analysis of mRNA from a range of inducible leukemic cell lines before and after exposure to a variety of differentiation induc- ers. 3. To study the expression of homeobox genes in normal blood cells and partially purified hematopoietic progenitors from human marrow, and the modulation of that expression by recombinant hematopoietic growth factors. 4. To characterize homeobox-containing cDNAs from human hematopoietic cells. We have cloned and sequenced partial cDNAs for the Hox 2.2 and 2.3 genes from an HEL cell library and a novel homeobox gene (PL1) from U-937. We propose to obtain complete sequence for these three cDNAs. 5. To express homeobox-containing proteins for the study of their intracellular distribution and turnover. Full-length cDNAs will be fused to beta-galactosidase and used to express protein in a bacterial system. Purified homeobox-proteins will be used to produce antibodies to study their role(s) in differentiating hematopoietic cells.
