The Skaggs Institute
for Chemical Biology

Scientific Report 2005

Training in Molecular and Experimental Medicine

E. Beutler

As scientists, one of our major duties is to provide the scientific leaders of the next generation, and the Skaggs Institute for Chemical Biology is helping make this possible by supporting the training of 5 young scientists in the Department of Molecular and Experimental Medicine at Scripps Research.

Hepatitis B and C viruses (HBV, HCV) infect more than 500 million persons and cause acute and chronic hepatitis and hepatocellular cancer. Shinichi Asabe is testing the hypothesis that dynamic changes in T-cell function have an important impact on the course and outcome of HBV and HCV infections. Specifically, he is comparing the phenotypic and functional evolution of the CD4⁺ and CD8⁺ T-cell responses to HBV and HCV with the severity and duration of infection in acutely and chronically infected humans and chimpanzees. This information will provide fundamental insight into the immunobiology of these infections, and it may lead to the development of novel immunotherapeutic and antiviral approaches to prevent and treat the associated serious liver disease.

DNA replication in eukaryotic cells is tightly controlled to ensure that DNA is replicated once and only once per cell cycle. Such a mechanism is extremely important for faithful transmission of genetic information from one generation to the next. Either incomplete replication or overreplication of chromosomal DNA would result in cell death or lead to genetic instability that is often associated with human diseases such as cancer. In the past 2 years, Enbo Liu has made great progress in our understanding of how rereplication is prevented in mammalian cells. He showed that cell cycle–dependent regulation of Cdt1, a replication licensing factor, is essential for ensuring a single round of DNA replication per cell cycle. When cells enter S phase, Cdt1 is phosphorylated by cyclin-dependent kinases, and this phosphorylation induces the association of Cdt1 with ubiquitination complex SCF^Skp2, which targets Cdt1 for degradation. Dr. Liu also found that rereplication is induced and cell-cycle checkpoints are activated when Cdt1 is overexpressed, supporting the idea that regulation of Cdt1 activity is an essential means to prevent rereplication.

Jonathan Flanagan has been working on signaling factors that affect iron absorption in mice. Transferrin receptor 1 (TfR1) is a protein expressed on the cell surface that mediates cellular uptake of iron. The amount of circulating soluble TfR1 (sTfR1) is directly related to the amount of iron absorbed. To investigate a potential signaling function of sTfR1, Dr. Flanagan used a hydrodynamic gene transfer technique to express transfected plasmid constructs of human sTfR1 (hsTfR1) and murine sTfR1 (MsTfR1) from the livers of C57Bl6 mice. The efficacy of the hydrodynamic technique was indicated by sustained expression of hsTfR1 in mice, at a level 6-fold higher than the normal level of hsTfR1 in humans. However, despite its attractiveness as a potential modifier of iron absorption, neither hsTfR1 nor MsTfR1 had any effect on iron absorption.

AML1-ETO, the fusion protein derived from the 8;21 translocation associated with M2 phase acute myeloid leukemia, does not promote leukemia in many mouse models. Luke Peterson helped show that deletion of the NcoR/SMRT-interacting domain region of AML1-ETO results in a truncated form that is leukemogenic in mice. In addition, a natural splice form of AML1-ETO, AML1-ETO9a, that produced a shorter version of the protein similar to the AML1-ETO deletion mutant was detected in cell samples from patients with leukemia. After the observation of AML1-ETO’s effect on the cell-cycle regulator p21^waf1 in a myeloid cell line, the analysis of the role of AML1-ETO on the p21^waf1 promoter showed that it promotes the expression of this negative cell-cycle regulator. This finding prompted the study to look at the potential of AML1-ETO in promoting leukemia in p21^waf1-deficient mice. An ongoing retroviral bone marrow transplantation approach indicates that the loss of p21^waf1 cooperates with AML1-ETO in promoting leukemia in these mice, consistent with observations that secondary genetic events are required in AML1-ETO leukemogenesis.

Zhengyi Ye is investigating the transcriptional control of the protein transthyretin, the normal serum carrier of retinol-binding protein charged with retinol and 10% of serum thyroxine. Although transthyretin behaves as a negative acute-phase reactant that is downregulated by IL-6 via the transcription factor HNF 3α, little information is available on its positive control. The gene for transthyretin appears to be similarly regulated in hepatocytes and brain parenchyma, and its transcription is increased by a number of CNS stimuli. Dr. Ye is using a combination of immunohistochemical and molecular biologic techniques to analyze the regulation of the gene under a variety of physiologic conditions. In the past year, he showed that triiodothyronine hyperthyroidism does not increase transthyretin transcription in cerebral cortex, developed a strain of mice transgenic for both a common human transthyretin mutation and genes that predispose to Alzheimer disease, and studied the gene expression profiles in hearts from young and old mice with and without transgenes that cause deposition of transthyretin amyloid.

Publications

Biggs, J.R., Zhang, Y., Peterson, L.F., Garcia, M., Zhang, D.E., Kraft, A.S. Phosphorylation of AML1/RUNX1 regulates its degradation and nuclear matrix association. Mol. Cancer Res. 3:391, 2005.

Peterson, L.F., Boyapati, A., Ranganathan, V., Iwama, A., Tenen, D.G., Tsai, S., Zhang, D.E. The hematopoietic transcription factor AML1 (RUNX1) is negatively regulated by the cell cycle protein cyclin D3. Mol. Cell. Biol. 25:10205, 2005.

Yan, M., Burel, S.A., Peterson, L.F., Kanbe, E., Iwasaki, H., Boyapati, A., Hines, R., Akashi, K., Zhang, D.E. Deletion of an AML1-ETO C-terminal NcoR/SMRT-interacting region strongly induces leukemia development. Proc. Natl. Acad. Sci. U. S. A. 101:17186, 2004.