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ChIP Analysis of Hepatic Gene Silencing
Mallory J. Stenslik, Lydia R. Bjornbak, Laura J. Hagstrom and
Gary A. Bulla
Department of Biological Sciences, Eastern Illinois University
Abstract
A better understanding of the complex mechanisms
responsible for activating and silencing cellular genes is required to develop
strategies to prevent and treat disease. We have been developing the chromatin
immunoprecipitation (ChIP) assay to examine the binding of liver-enriched
transcription factors to liver-specific gene promoters in cell culture model
systems. To test the applicability of this thus assay, we have used formaldehyde
to crosslink the DNA to the bound protein in the hepatoma cell cultures followed
by isolation and sonication of the DNA-protein complexes. The complexes are then
immunoprecipitated using antibody to the transcription factor HNF1a and the
attached DNA amplified using PCR. Using this technique we show optimization of
key steps in the procedure and verify HNF1a binding to the human alpha-1
antitrypsin promoter in hepatoma cells. We will extend our studies identify
which transcription factors are binding to liver gene promoters in cells which
fail to express liver function despite forced expression of liver-enriched
transcription factors. Thus, the ChIP assay can be utilized to study protein-DNA
interactions on the regulatory regions in our cell culture system to ask
questions concerning the mechanisms of gene silencing and activation.
Introduction
large number of liver-specific and b-cell specific genes (1, see Fig. 1).
Control of gene expression is the foundation of life as organisms develop
and respond to their environment (2). The generally accepted reason for
the silencing of gene expression is simply that they have never been
activated by the body. However newer evidence points out that this is far
too simplistic to explain gene control in many situations. The most likely
explanation for gene silencing is that missing factors that are required
for activation are not present (1). Previous introduction of these factors
has not resulted in gene activation (3-4). However, the ChIP assay allows
an opportunity to study to mechanism of gene silencing that was not
previously available (5-6, and see fig. 2). It allows one to determine
what proteins are interacting with a gene at any given point in time. It
will allow us to determine which specific factors able to bind to a
silenced gene. In order to analyze the a specific gene, genomic DNA has to
be sheered to 200-1000 base pair sizes. .
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Figure 1. Multiple coactivators interact
with HNF1a to activate transcription of the a1-antitrpsin gene
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Figure 2. Models of hepatic gene silencing
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Figure 3. ChIP Assay Progression |
Methods
To carry out the ChIP assay, cultured cells were grown to
80-90% confluency in 100cm culture dishes. DNA was covalently crosslinked to
proteins using formaldehyde. The cells were then lysed using SDS lysis buffer
and scraped into a microfuge tube. Cell lysates (500ul) were then sonicated
using a variety of conditions. Aliquots were taken after each sonication
protocol. In order determine the correct number of sonication pulses, aliquots
were taken after zero, one, two, four and eight rounds of 10-second sonications.
Samples were deproteinated with protease, RNA removed with RNase A, then the
aliquots were run out on an agarose gel with 1000bp and 100bp ladders. The gel
was then analyzed to determine optimal sonication parameters. .
Results
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Fig. 4. DNA Size Fragments After Variable Rounds of
Sonication. Crosslinked DNA was either untreated or sonicated for 1,
4 or 8 rounds of 10 second pulses. DNA was deproteinated, treated
with RNase A then analyzed by agarose gel electrophoresis |
Conclusions
DNA sonication is an reliable method for sheering DNA to the size desired range
for use in the ChIP assay. Results suggest that the optimum DNA length was
produced by sonicating the DNA for eight ten second pulses at one third the
maximum power level. Further experimentation is being conducted to ensure that
the sonication procedure is adequate for immunoprecipitation and final steps of
the ChIP assay. .
References
1. Odem, D.T.et al . (2004) Control of pancreas and liver and pancreas gene
expression by HNF transcription factors. Science 303: 1378-1381.
2. Felsenfeld, G, and Groudine, M. (2003) Controlling the double helix.
Nature 421:448-453.
3. Bulla, G. A. (1999) Extinction of a1-antitrypsin expression in cell hybrids is
independent of HNF1a and HNF4 and involves both promoter and internal
DNA sequences. Nucleic Acids Research 27: 1190-1197.
4. Bulla GA, and Kraus, DA. (2005) Dissociation of the Hepatic Phenotype from
HNF4 and HNF1a Expression, Bioscience Reports. 24:595-608
5. Bulger, M., Sawado, T., Schubeler, D., and Groudine, M. (2002) ChIPs of the
beta-globin locus: unraveling gene regulation within an active domain. Curr
Opin Genet Dev 12: 70-77.
6. Soutoglou, E., and Talianidis, I. (2002) Coordination of PIC assembly
and chromatin remodeling during differentiation-induced gene activation,
Science 295: 1901-1905
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