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Molecular Analysis of Hepatic Gene Silencing
Thomas Roberts, Vince Luong, Anthony J. Imburgia, Jessica J. Tschampa and Gary
A. Bulla
Department of Biological Sciences, Eastern Illinois University
Abstract
It is well known that genes are turned on (activated) by specific
biochemical processes. Recent results suggest that, for some genes, mechanisms
exist to turn off (silence) gene expression. Our hypothesis is that these
silenced genes are unable to interact with essential proteins. We wish to test
this hypothesis using a powerful newly developed technique called the Chromatin
Immunoprecipitation (or “ChIP”) assay. The ChIP assay provides information that
allows one to construct testable models to explain gene silencing.
We are developing the ChIP assay in order to determine whether a liver protein
termed Hepatocyte Nuclear Factor 1 (HNF1) is allowed to bind to the silenced
liver specific alpha 1 antitrypsin (a1AT) gene. HNF1 is known to be required to
express the a1AT gene in the liver, but HNF1 is incapable of activating
expression of the a1AT gene in certain cultured rat cells that our laboratory
has developed.
A critical step in the ChIP assay requires that the DNA extracted
from our desired cell line be sonicated into fragments that between 200 and 1000
base pairs in length. In order to optimize DNA sonication for the ChIP assay,
samples were sonicated, deproteinated and then analyzed by agarose gel
electrophoresis to determine their size. The most optimal sonication treatment
found was eight rounds of ten second pulses. This optimized procedure is now
being used to optimize additional steps in the ChIP assay. These results will
lead an understanding of mechanisms of gene silencing in mammals.
Introduction
HNF1a
is a liver-enriched transcription factor that activates a large number of
liver-specific and b-cell
specific genes (1, see Figure 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 Figure 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-antitrypsin
gene. |
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Figure 2. Models of hepatic gene silencing. |
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.
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Figure 3. Chip assay progression. |
Results
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Figure 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
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Conclusions
DNA sonication is a 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.
7. 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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