Surrounding the small islands of genes within the human
genome is a vast sea of mysterious
DNA. While most of this noncoding DNA is junk, some of it
is used to help genes turn on and off. As
reported online in December in Genome Research,
Johns Hopkins researchers have now found that
this latter portion, which is known as regulatory DNA and
contributes to inherited diseases like
Parkinson's or mental disorders, may be more abundant than
we realize.
By conducting an exhaustive analysis of the DNA
sequence around a gene required for neuronal
development, Andrew McCallion, an assistant professor in
the
McKusick-Nathans Institute of Genetic
Medicine, and his team found that current computer
programs that scan the genome looking for
regulatory DNA can miss more than 60 percent of these
important DNA regions.
The current methods find regulatory sequences by
comparing DNA from distantly related
species, under the theory that functionally important
regions will appear more similar in sequence than
nonfunctional regions. "The problem with this approach, we
have discovered, is that it's often throwing
the baby out with the bath water," McCallion said. "So
while we believe sequence conservation is a
good method to begin finding regulatory elements, to fully
understand our genome we need other
approaches to find the missing regulatory elements."
McCallion had suspected that using sequence
conservation would overlook some regulatory DNA,
but to see how much, he set up a small pilot project
looking at the phox2b gene; he chose this gene
both because of its small size and his interest in nerve
development (phox2b is involved in forming
part of the brain associated with stress response as well
as nerves that control the digestive system).
The researchers created what they call a "tiled path,"
cutting into small pieces the DNA
sequence around the phox2b gene, and then inserted each
piece into zebrafish embryos along with a
gene for a fluorescent protein. If a phox2b fragment were a
regulatory element, then it would cause
the protein to glow. By watching the growing fish
embryos--which have the advantage of being
transparent--the researchers could see which pieces were
regulators.
They uncovered a total of 17 discrete DNA segments
that had the ability to make fish glow in
the right cells. The team then analyzed the entire region
around the phox2b gene using the five most
common programs for computing sequence conservation; these
established methods picked up only 29
percent to 61 percent of the phox2b regulators that
McCallion identified in the zebrafish
experiments.
"Our data supports the recent NIH encyclopedia of DNA
elements project, which suggests that
many DNA sequences that bind to regulatory proteins are, in
fact, not conserved," McCallion said. "I
hope this pilot shows that these types of analyses can be
worthwhile, especially now that they can be
done quickly and easily in zebrafish."
McCallion is now planning a larger study of other
neuronal genes. "I think we are only starting to
realize the importance and abundance of regulatory
elements; by regulating the gene activity in each
cell, they help create the diverse range of cell types in
our body."
The research was funded by the National Institutes of
Health and the March of Dimes.
Authors on the paper are David McGaughey, Ryan Vinton,
Jimmy Huynh, Amr Al-Saif, Michael
Beer and McCallion, all of Johns Hopkins.
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