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Genome Wide Analysis of Gene Silencing in Mammalian Cell
Hybrids
S. Hickman, K. Ingram and G.A. Bulla
Eastern Illinois University, Department of Biological Sciences
Abstract
For about 40 years it has been known that the fusion of mammalian cells of
distinct origin produce hybrid cells that result in global loss of
tissue-specific gene expression. Despite a great deal of research on this
phenomenon, the mechanism of this process remains elusive. Due to recent
advancements in microarray technology, it is possible to monitor gene expression
of entire genomes in cell fusion experiments. We utilized microarray analysis to
observe whole rat genome expression in rat hepatoma cells, rat fibroblast cells,
and rat hepatoma x fibroblast hybrid cells. We used Rat Genome 230 2.0 array
chips from Affymetrix which were incubated with labeled cDNA molecules derived
from RNA extracted from each cell type using a Qiagen RNeasy kit, and then
samples read on a chip reader. Preliminary results suggest that, in agreement
with previous data, a large number of liver-enriched genes are moderately (5-10
fold) to strongly (>10 fold) repressed (194 and 300 genes, respectively) in the
cell hybrids. A nearly equal number of fibroblast-specific genes were also
repressed in the hybrid cells. Furthermore, 35 genes were activated >5 fold in
the cell hybrids compared to either parental cell line. Thus, gene silencing in
cell hybrids is bi-directional and affects a large portion of parental genomes.
Also, a number of previously silent genes are activated in cell hybrids, some of
which may be involved in the extensive gene silencing phenotype observed in cell
hybrids.
Introduction
Tissue-specific gene expression has been studied by
examining silencing of expression in somatic cell hybrids(1). In hepatoma x
fibroblast cell hybrids, most of hepatic specific genes are silenced (2).
Although in some cases this is reversible, it begs the question as to what
mechanism the cells are using to produce the silencing of tissue-specific gene
expression (2). Despite the fact that this phenomenon is poorly understood, it
does appear that the causation of the phenotype is at the level of transcription
(3). With the advent of microarray technology, we are able to observe
genome-wide expression in parental cells and cell hybrids, and identify gene
products that may contribute to silencing of tissue-specific gene expression.
Methods
Rat fibroblast cells (Rat1), hepatoma cells (FTO2B)
and fibroblast x hepatoma hybrid cells (FR(2)) RNA was extracted from confluent
monolayers the using an RNeasy kit from Qiagen. The RNA was analyzed by gel
electrophoresis for visual integrity and relative concentration. The RNA samples
were then sent to the W.M. Keck Center and Functional Genomics at University of
Illinois in Urbana-Champaign. cDNA was synthesized from the RNA of each cell
type via reverse transcriptase polymerase chain reaction (PCR), labeled and
hybridized to the Affymetrix Rat Genome 230 2.0 Array chip and read on a chip
reader. Two chips were used for each cell type.
Results
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Figure 1. Gel analysis (MOPS) showed integrity
of RNA. 1 x 107 cells (obtained by trypsinization) were lysed with 600ul
RLT, and homogenized using a Qiashredder column. RNA was isolated using an
RNAeasy kit, with the modification of a DNaseI step (15 minutes, RT) . |
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Figure 2. Example of readout of differential
gene expression in hepatoma (FTO2B), fibroblast (Rat1) and cell hybrids
(FR(2)) on the microarrays. Spots circled in lower area are expressed
preferentially in the Rat1 cells. |
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Figure 3. A comparison of genome-wide
expression between hepatoma, fibroblast and cell hybrids. |
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Figure 4. A comparison of total gene
expression between hepatoma and fibroblast cells reveals 294
liver-specific genes (expressed >5 fold higher compared to fibroblasts
levels). |
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Figure 5. A comparison of total gene
expression between hepatoma and fibroblast cells reveals 393
fibroblast-specific genes (expressed >5 fold higher compared to hepatoma
levels). |
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Figure 6. Large scale repression of both
hepatoma-specific and fibroblast-specific genes were observed in the
hybrid cells. |
Discussion
A large number of liver-enriched genes are
moderately (5-10 fold) to strongly (>10 fold) repressed (194 and 300 genes,
respectively) in the cell hybrids
A nearly equal number of fibroblast-specific genes
were also repressed in the cell hybrids
35 genes were activated >5 fold in the cell hybrids compared to either parental
cell line
Gene silencing in cell hybrids is bi-directional and affects a large portion of
parental genomes
A number of previously silent genes are activated in cell hybrids, some of which
may be involved in the extensive gene silencing phenotype observed in cell
hybrids.
Subsequent investigation on candidate genes that may identify genes involved in
tissue-specific gene silencing
References
Massa, S., Junker, S., and Matthias, P. Molecular
mechanisms of extinction: old findings and new ideas, Int J Biochem Cell Biol 32
(2000). : 23-40.
Bulla, Gary A. "Hepatocyte Nuclear Factor-4 Prevents Silencing of Hepatocyte
Nuclear Factor-1 Expression in Hepatoma Fibroblast Cell Hybrids." Nucleic Acids
Research 25 (1997): 2501-2508.
Bulla, Gary A. "Selective Loss of the Hepatic Phenotype Due to the Absence of a
Transcriptional Activation Pathway." Somatic Cell and Molecular Genetics 23
(1997): 185-201.
Acknowledgements
We would like to acknowledge the Proposal Initiative
Fund for providing the funding for us to do this research.
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