Researchers at the
McKusick-Nathans Institute of Genetic Medicine at Johns
Hopkins have invented a cost-effective and highly efficient
way of analyzing what many have termed "junk" DNA and
identified regions critical for controlling gene function.
And they have found that these control regions from
different species don't have to look alike to work alike,
as traditionally thought.
The study was published online at Science Express on
The researchers developed a new system that uses zebra
fish to test mammalian DNA and identify DNA sequences,
known as enhancers, involved in turning on a gene. In
studying RET, the major gene implicated in Hirschsprung
disease and multiple endocrine neoplasia, the team
identified DNA sequences that can control RET but had not
been identified using standard methods. Hirschsprung
disease, also known as congenital megacolon, is a
relatively common birth defect marked by bowel obstruction.
Multiple endocrine neoplasia, or MEN2, is an inherited
predisposition to neuroendocrine cancers.
The notion that mutations in enhancers play a role in
human disease progression has been difficult to confirm
because usually enhancers are located in the 98 percent of
the human genome that does not code for protein, termed
noncoding DNA. Unlike DNA sequences that code for protein,
noncoding DNA, sometimes referred to as "junk" DNA, follows
few rules for organization and sequence patterns and
therefore is more difficult to study.
"The difficulty with human genetic approaches to
common disease is that we lack the power to precisely
localize DNA sequences that are associated with disease,
often leaving us immense stretches of DNA to look at," said
one of the study's corresponding authors, Andy McCallion,
an assistant professor in the McKusick-Nathans Institute.
Most often one is limited to looking in the most obvious
places, which may not yield the best results. "Until now,"
he said, "we've only been able to look under the lamplights
for the car keys."
Traditionally, DNA sequences are thought to have to
look similar to function similarly; this is how scientists
identify genes in other species, a strategy best used for
studying similar species. From an evolutionary standpoint,
the last common ancestor of human and zebra fish lived more
than 300 million years ago. Because DNA sequences in each
species have changed over time, traditional methods of
comparing DNA sequences between humans and zebra fish have
failed to identify any potential enhancers around the RET
gene because the DNA sequences differ too much.
That drove the Johns Hopkins researchers to seek and
develop this new system, by which virtually any DNA
sequence can be tested for its ability to turn on a marker
gene in zebra fish embryos. The system is a significant
advance over current methods in this model species,
allowing researchers to study more sequences in a shorter
period of time. Using this, they identified several human
enhancers able to control expression consistent with the
zebra fish RET gene.
Zebra fish have become the ideal system for doing
these types of large-scale studies. They are small —
only about a half inch in length — they grow quickly
and are relatively inexpensive to maintain compared to mice
or rats. "Zebra fish are the only vertebrate embryo you can
even think about doing this type of work in," said Shannon
Fisher, the study's first author and an assistant professor
in cell biology in Johns Hopkins' Institute for Basic
The researchers' next steps are further study of the
RET enhancers they found to identify other mutations that
might contribute to Hirschsprung disease and MEN2, and to
entice other investigators to collectively build a database
of human enhancers.
"If there's one thing we've learned here, it's that we
are not very good at recognizing enhancers. We just don't
know what they look like," Fisher said. "We are anxious for
others to use this technology on their favorite genes."
The researchers were funded by the March of Dimes and
the National Institutes of Health.
Authors on the paper are Fisher, Elizabeth Grice, Ryan
Vinton, Seneca Bessling and McCallion.