Daniel E. Sabath, M.D., Ph.D.
Associate Professor of Laboratory Medicine
Adjunct Associate Professor of Medicine
University of Washington
Department of Laboratory Medicine
Seattle Cancer Care Alliance Mail Stop G7-800
825 Eastlake Ave. E.
Seattle, WA 98109
Phone: (206) 288-7125
FAX: (206) 288-7127
Cell: (206) 719-7956
E-mail: dsabath@u.washington.edu
Laboratory Medicine
Research Program:
Regulation of z-globin gene expression
Dr. Sabath's main research interest is regulation of gene expression in hematopoietic cells. The long-term goal of this research is to
determine the molecular mechanisms responsible for hematopoietic gene expression, and then to determine how these mechanisms become
deregulated in the setting of hematopoietic neoplasia. The model gene being studied is the z -globin gene, which is expressed only
during embryonic yolk sac erythropoiesis. The major research activities are:
- Study of z-globin gene expression in stably transfected cells. Previous work in the laboratory identified two major regulatory regions of the z-globin promoter: the proximal 128 bp of the promoter, and a regulatory region located approximately 240 bp 5' to the RNA transcription start site. The proximal promoter appears to be involved in developmental-specific expression of z-globin, whereas the upstream element is required for high-level promoter activity in transfected cell lines. All these experiments were performed in a transient transfection system, so we do not know which of these elements are important in the context of chromatin. Preliminary studies showed that z-globin promoter activity is highly dependent on the site of insertion of the transfected gene. Therefore, we have been developing a system where single copies of transfected genes are inserted into a defined chromosomal location. This is accomplished by Cre recombinase-mediated in vivo recombination, where a transfected gene is targeted to a genomic location containing loxP sites. This system will allow us to alter z-globin promoter structure, and then to determine the effects of these changes on promoter activity in a chromosomal context.
- Characterization of DNA-binding proteins binding to z-globin regulatory sequences. The regulatory sequences described above have been found to bind specific DNA-binding factors. Our experiments indicate that unidentified DNA-binding proteins interact with a site in the upstream regulatory element, and in the proximal promoter at the -93 CCACC site and the -65 CCAAT site. We have identified candidate DNA-binding proteins for the CCACC and CCAAT sites by screening a lambda expression library with binding site probes. We are also using the yeast one-hybrid system to screen for factors that interact with z-globin regulatory sequences. Finally, we are preparing to purify factors that cannot be cloned using the above two expression cloning strategies.
Structure-function studies of the extracellular domain of Mpl
Mpl is the cell surface receptor for the growth factor thrombopoietin, which is implicated in regulation of megakaryocyte growth and differentiation
and platelet production. It has been shown previously that a form of Mpl lacking its distal extracellular domain can confer growth-factor independent
proliferation of cell lines that ordinarily require growth factors for survival. Dr. Sabath's laboratory is currently testing the hypothesis that the
distal extracellular domain of Mpl has a growth inhibitory function by making mutant forms of Mpl and testing the function of the mutants in growth
factor-dependent cell lines and in primary mouse bone marrow cells.
Minimal residual disease detection in breast cancer
Dr. Sabath is recipient of a 3-year grant from the Department of Defense to develop methodology to detect small numbers of brest cancer cells in the
peripheral circulation. A quantitative real-time RT-PCR assay is being developed to detect carcinomal cells using cytokeratin 19 as a molecular target.
Once the assay has been optimized, it will be used to quantitate K19 mRNA in the peripheral blood of breast cancer patients to determine if the quantity
of this mRNA correlates with disease stage at presentation or probability of disease progression.
Gene expression profiling of low grade lymphomas
In work that has been funded by the Washington Technology Center in collaboration with local biotech companies RationalDiagnostics, Inc. and VizX Labs,
LLC, Dr. Sabath's laboratory has been working toward developing a diagnostic tool for lymphoma diagnosis. Using high-density microarrays, a set of
approximately 200 genes has been identified that can distinguish among small lymphocytic, mantle cell, and follicular lymphomas as well as benign reactive
lymph nodes. Current efforts are directed toward developing olignonucleotide probes for these genes that can be used in a custom gene expression array.
Representative Publications:
Sabath, D. E. and Shim, M.-H. (2000) Use of green fluorescent protein/Flp recombinase fusion protein and flow cytometric sorting to enrich for cells undergoing Flp-mediated recombination. Biotechniques. 2000 May;28(5):966-72, 974.
Sprouse, J. T., Werling, R., Hanke, D. C., Lakey, C., McDonnel, L., Wood, B. L. and Sabath, D. E. (2000) T cell clonality determination using polymerase chain reaction (PCR) amplification of the T cell receptor g-chain gene and capillary electrophoresis of fluorescently labeled PCR products. Am. J. Clin. Pathol., 113, 838-850.
Sabath, D. E., Koehler, K. M., Yang, W.-Q., Phan, V., and Wilson, J. (1998) DNA-protein interactions in the proximal z-globin promoter: Identification of novel CCACCC- and CCAAT-binding proteins. Blood Cells Mol. Dis. 24, 183-198.
Sabath, D. E., Koehler, K. M., and Yang, W.-Q. (1996) Structure and function of the z -globin upstream regulatory element. Nucleic Acids Res. 24, 4978-4986.
Sabath, D. E., Koehler, K. M, Yang, W.-Q., Patton, K., and Stamatoyannopoulos, G. (1995) Identification of a major positive regulatory element located 5' to the human z-globin gene. Blood 85, 2587-2597.
Sabath, D. E., Spangler, E. A., Rubin, E. M., and Stamatoyannopoulos, G. (1993) Analysis of the human z-globin gene promoter in transgenic mice. Blood 82, 2899-2905.
Sabath, D. E., Podolin, P. L., Comber, P. G. and Prystowsky, M. B. (1990) cDNA cloning and characterization of interleukin 2-induced genes in a cloned T helper lymphocyte. J. Biol. Chem. 265, 12671-12678.