Comparative genomics is the study of genomes from different species in order
to better understand how species have evolved and what the functions are of
both genes and noncoding sections of the genome. As part of the NIEHS Toxicogenomics
Research Consortium (TRC), researchers at the Duke Center for Environmental
Genomics are focusing on comparative genomics as a way to explore how environmental
stresses affect human health.
The NIEHS established the TRC in November 2001 to serve as the extramural
mechanism by which the NCT applies microarray technology. Funded at $37 million
over five years, the TRC is a coordinated, multidisciplinary effort between
the NIEHS Microarray Center and five academic research institutions (Duke,
the University of North Carolina-Chapel Hill, the Massachusetts Institute
of Technology, Oregon Health & Science University, and the Fred Hutchinson
Cancer Research Center/University of Washington), along with private companies
contracted to assist with microarray development and bioinformatics. The TRC
is intended to significantly advance the pace of discovery in toxicogenomics,
while simultaneously accelerating the development and validation of microarray
technology.
At roughly the halfway point of the initial grant period, the TRC is already
bearing important scientific fruit and appears poised to make substantial contributions
to the field as research supported by rapidly progressing technology produces
new insights into the complex relationship between environmental exposures
and gene expression.
All the Organisms, Two by Two . . .
The Duke investigators believe that comparative genomics--the isolation and
identification of common, conserved genomic responses across different model
species--will have a major impact on advancing useful knowledge within the
field of toxicogenomics. The discipline makes it possible to probe into the
phylogenetic origins of gene families and how they have been altered as species
rose higher; eventually these gene structures and relative functions can be
compared to those of humans, says NCT deputy director James K. Selkirk. Part
of the TRC's mission has been the development of sophisticated, reliable microarrays
of the genomes of model systems such as Caenorhabditis elegans, zebrafish,
yeast, and mouse, which allow rapid, high-throughput screening across species
in a variety of areas of interest.
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image credit: Background: David Schwartz; insets, l–r: David Schwartz;
Dennis Kunkel Microscopy, Inc. |
Ongoing independent exploration by Elwood Linney and Marcy Speer into the
genetics of neural tube development and defects is a case in point. Microbiologist
Linney is working with zebrafish microarrays to identify sets of genes that
might be involved in embryonic neural tube development, or disruption of normal
development by environmental insults. Those candidate genes can then be used
by human geneticist Speer to screen within subject families for polymorphisms
that might correlate with families with neural tube defects such as spina bifida.
Although they have already made substantial strides in the biology, Linney
says, "a major part of the progress to date has been just the technology. When
we started, there weren't any microarrays for zebrafish." With the recent delivery
of a 22,000-gene zebrafish microarray developed in cooperation with TRC contractors, "we're
ready to start generating a variety of different types of data using perturbations
of neural tube development," he says.
Such large-scale genomic screening lends itself to ferreting out the signaling
pathways that might be involved in toxic response and repair mechanisms. Microarray
technology allows researchers to cast a big net over a problem, rather than
focusing on specific genes, says Linney. Once patterns of expression are identified
via microarray, RNA interference (RNAi) or morpholinos (another gene silencer)
can be applied to confirm the function of the genes. "If we think a certain
toxicant is affecting a certain gene product, we can test that by designing
something to knock down that gene product and see if we still get the same
phenotype," he says.
TRC principal investigator David Schwartz is using a similar comparative
genomics approach in his TRC independent project. Schwartz is examining the
genes and genomic responses involved in the immune response to bacterial toxins
such as endotoxin, which are released into the bloodstream during bacterial
infection and can, in themselves, cause a variety of symptoms. "We are using
genomics as a way of highlighting a number of genes that we know are biologically
related to the immune response to bacterial toxins," he explains. This will
help identify genes that may have variants, some of which would predispose
individuals to experience adverse responses when they have various types of
infection.
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image credit: Background: David Schwartz; Insets, l–r: Arnold Greenwell/EHP;
National Human Genome Research Institute |
Schwartz's group has identified a few very promising candidates as a result
of studies with mouse microarrays. "We've found specific areas of the genome
that are clearly associated with the biologic response," he says, "and we've
found a couple that we think may be critical in terms of regulating the response--genes
that had not previously been described as being important or relevant to processing
or responding to bacterial toxins." To further elucidate the potential role
of these genes, Schwartz's team is currently testing them in C. elegans,
as well as looking at loss of function by using RNAi to downregulate their
expression in mammalian cells.
Jonathan Freedman, who heads up the center's Toxicology Core, is the group's C.
elegans specialist. He is currently developing a high-throughput worm
genome-wide microarray to screen compounds and develop signature profiles
of response to a variety of toxicants, including bacterial toxins, chemicals,
metals, and alkylating agents. He believes that such genomewide screening
is the best way to extract useful information. For example, he says, his
group has identified about 300 genes that are upregulated in response to
cadmium. "But we're not going to go through and look at each gene," he says.
Instead, he plans to look at the whole genome to study why cadmium affects
all 300 genes, then link that information to what cadmium can do to cause
cancer and other diseases.
Evolution of a Field
Freedman is enthusiastic about the Duke center's comparative genomics focus. "We
definitely think that's the way toxicogenomics needs to evolve," he says. "There's
just so much power in a lot of these alternative species, especially yeast
and C. elegans and zebrafish. You can do rapid genetics, very rapid
RNAi types of studies, and you can do a lot of linking to the genome."
Besides his own work with C. elegans, Freedman is also working collaboratively
with other member institutes on microarray development and standardization,
a crucial issue within the field. Freedman and colleagues distributed material
to each of the labs to be used in their microarrays, to see whether all the
experiments would come out with the same results. Freedman reports that the
first phase of this cooperative work has been completed and is being prepared
for publication. The study addressed the issue of data reproducibility by standardizing
gene expression experiments across different labs and microarray platforms.
The TRC will continue its collaborative efforts at standardization with a new
project, possibly addressing comparative genomics.
There should be very exciting results emerging from the Duke center's work
in the near future, and from the entire TRC's efforts as well. Schwartz cites
three reasons that the TRC initiative will ultimately prove to be of great
importance to toxicogenomics: "One is that these approaches will undoubtedly
allow us to identify early responses to toxic agents in the environment, and
potentially identify individuals before they develop disease. Secondly, using
genomics as a way of targeting genes may allow us to short-circuit and hasten
the process of identifying which genetic variants are related to susceptibility
to environmental agents. And thirdly, this effort will allow us to more clearly
phenotype diseases into biological categories of disease, as opposed to clinical
or physiological categories of disease that oftentimes lack precision."
Ernie
Hood