The Skaggs Institute
for Chemical Biology
Scientific Report 2005
Training in Molecular and Experimental
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 cycledependent 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 SCFSkp2,
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-ETOs effect on the cell-cycle regulator p21waf1 in a myeloid
cell line, the analysis of the role of AML1-ETO on the p21waf1 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 p21waf1-deficient mice.
An ongoing retroviral bone marrow transplantation approach indicates that the loss of p21waf1
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
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,
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.