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My major research interest is the study of a
transcriptional factor called Kruppel-like factor 4 (KLF4), a C2H2
zinc-finger-containing nuclear factor implicated in the control of cell
proliferation and differentiation. Expression of KLF4 is upregulated upon DNA
damage. In this process, KLF4 is a critical mediator of the checkpoint functions
of p53 by inducing G1/S and G2/M cell cycle arrest. Recently data also showed
that KLF4 has an antiapoptotic effect upon DNA damage. However, the molecular
mechanism is not fully elucidated. In vivo studies have demonstrated that KLF4
is required for terminal differentiation of goblet cells in the colon, a process
that is regulated by the Notch signaling pathway. Recently data also indicated a
new function of KLF4 in embryonic stem cell self renewal. The following
experiments are designed to understand the molecular mechanism of how KLF4 is
involved in DNA damage response, to study how KLF4 is regulated by the Notch
signaling pathway, and to test the role of KLF4 in stem cell biology and cancer
development.
1. Study the role of KLF4 in DNA damage response pathway
We have identified Tip60, a member of the MYST family of HATs that was initially
identified as a cellular protein that interacts with HIV protein Tat, as a
KLF4-interacting protein by yeast two-hybrid screening. Since Tip60 is one
component of a multiple subunit complex that is involved in DNA damage repair
and apoptosis, the interaction between KLF4 and Tip60 provides a possible
mechanism by which KLF4 and Tip60 cooperatively regulate cell cycle arrest and
apoptosis. The interaction domains in both proteins will be identified and the
significance of these domains will be tested in order to dissect their specific
roles in cell cycle arrest and apoptosis. In addition, the role of p53 will be
further studied in this context since the interactions between KLF4 and p53,
between p53 and Tip60 have been reported. Finally the posttranslational
modifications including phosphorylation, ubiquitination, and acetylation of KLF4
after DNA damage will be examined, and the significance of the modifications
will be evaluated.
2. Study the mechanisms of how KLF4 is regulated by the Notch signaling pathway
The Notch signaling pathway has been shown to play a positive or a negative role
in tumor development depending on the cellular context. The in vivo link between
Notch and KLF4 raises a possibility that KLF4 mediates the function of the Notch
signaling pathway in cancer development. Our preliminary studies indicate that
KLF4 gene expression is inhibited by the Notch signaling pathway both in vivo
using a γ-secretase inhibitor that inhibits the Notch signaling pathway and in
vitro using a constitutively active Notch 1 (ICN1). Further studies identified a
Notch responsive element in the KLF4 promoter. The physiological relevance of
the transcriptional factor will be assessed by in vivo methods that likely
involve the generation of transgenic and/or knockout mouse models. The detailed
mechanism such as the singling pathways that are involved will be delineated by
in vitro assays with cell lines by using γ–secretase inhibitors and ICN1 and
different inhibitors that inhibit different signaling pathways.
3. Study the role of KLF4 expression in stem cells or progenitor cells in the
development of cancer
Recently it has been reported that overexpression of four nuclear factors
including Oct4, c-Myc, Sox2 and KLF4 transformed differentiated fibroblasts into
embryonic stem cell like cells, indicating an important role of KLF4 in stem
cell biology. Since KLF4 also has a function to promote embryonic stem cell self
renewal, we hypothesize that KLF4 is expressed in stem cell or progenitor cell
population, and change of KLF4 gene expression in stem cells or progenitor cells
plays an important role in cancer development that has been linked to cancer
stem cells. We have already generated a KLF4/EGFP transgenic mice model using a
modified BAC (bacterial artificial chromosome) clone as the transgene. Since BAC
construct contains big pieces of fragment in both 5’ and 3’ regions of KLF4 gene
and EGFP has been inserted into the KLF4 start codon, this model will most
likely allow us to monitor endogenous expression of KLF4 by chasing expression
of EGFP. Using the same strategy, we have also generated a KLF4/CRE™ transgenic
mouse model. By crossing these mice with a reporter mouse line such as Rosa 26R
beta Geo or EGFP mice, KLF4 expressing cells will be traced by X-gal staining or
green fluorescence of EGFP after tamoxifen induction. By using both KLF4/EGFP
and KLF4/CRE™ transgenic mouse models, KLF4 gene expression in stem cells in
adult tissues including skin, small intestine, colon, and bone marrow is being
examined. Further studies will be conducted to evaluate a role of KLF4
expression in stem cells in cancer development.
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