St. Louis, Missouri
December 4, 2008
Opening the "X-files" of
biology
 A
team led by Craig Pikaard, Ph.D.,
Washington University in St.
Louis professor of biology in Arts & Sciences, has made a
breakthrough in understanding the phenomenon of nucleolar
dominance, the silencing of an entire parental set of ribosomal
RNA genes in a hybrid plant or animal.
Since the machinery involved in nucleolar dominance is some of
the same machinery that can go haywire in diseases such as
cancer, Pikaard and his collaborators' research may have
important implications for applied medical research.
Nucleolar dominance occurs when nucleoli, protein-rich, dense
regions of RNA within the nucleus, form on the chromosomes
inherited from one parent, but not on the chromosomes inherited
from the other parent. Expression of ribosomal RNA genes drives
the formation of these nucleoli. The hybrid, a result of a
cross-breeding of two different species, always "chooses" to
express the ribosomal RNA genes of one particular parental
species, regardless of whether that species happens to be the
maternal or paternal parent.
Ribosomal RNAs, or rRNAs, are a major component of the
ribosomes, the protein manufacturers of the cell. Because rRNA
genes are highly redundant, cells use nucleolar dominance to
control the dosage of ribosomes in an organism.
According to Pikaard, if researchers could harness the silencing
machinery involved in nucleolar dominance to limit the
expression of rRNA genes, they could potentially slow the growth
rate of tumor cells and thereby slow the progression of diseases
like cancer.
In cancer cells, nucleoli are conspicuously large because of a
dramatic increase in the transcription of rRNAs, which in turn
leads to an increase in the production of ribosomes. This
escalation in ribosome activity means that the cell can
synthesize proteins at an alarmingly rapid rate, which
contributes to the out-of-control cell proliferation that is the
disease's trademark.
Completely silencing all ribosomal genes would not be a viable
therapeutic approach for cancer patients because ribosomes are
necessary for survival. But Pikaard and his collaborators'
research suggests that small interfering RNAs (siRNAs) can
direct silencing agendas that are much more sophisticated than
an all or nothing approach.
"Dr. Pikaard's study demonstrates the potential of a plant model
system to yield important molecular details on how cells silence
large clusters of genes," said Anthony Carter, Ph.D., who
oversees gene regulation grants at the National Institutes of
Health's National Institute of General Medical Sciences, which
partially supported the research. "His findings on the control
of a major class of RNA found in all cells offer new insights
into gene silencing mechanisms."
Pikaard and his collaborators' work, which was published in
Molecular Cell on Dec. 4, is also one of the first to
demonstrate how siRNAs can play a role in controlling the dosage
of vital genes. The research was supported by the National
Institutes of Health and the National Science Foundation.
The weird and the wacky
Nucleolar dominance is considered an "epigenetic" phenomenon.
Epigenetics refers to heritable changes in gene expression that
arise from changes in the "packaging" of DNA rather than
modification of the underlying DNA sequence itself. Because
these changes do not follow the normal rules of genetics,
Pikaard refers to them as the "X-files of biology," unusual
events that are not easily explained nor predicted.
Although biologists have been studying nucleolar dominance since
the 1920s, this phenomenon remained largely unresolved until
recently, when Pikaard's lab reversed an old dogma. Up until
this point, researchers had presumed that nucleolar dominance
was all about turning on one set of parental ribosomal genes. In
1997, Pikaard and his colleagues made headlines with an
experiment that used chemicals to inhibit the two-pronged method
cells employ to silence genes — DNA methylation, which adds
chemical flags to genes, and histone modification, which alters
the proteins that act as spools for DNA. The chemical inhibitors
of silencing turned on the previously unexpressed set of
parental genes, thereby demonstrating that the underlying
mechanism of nucleolar dominance turns genes off, not on.
Since then, Pikaard and his collaborators have been working to
disentangle the complex machinery behind this epigenetic on-off
switch.
Using RNA to fight RNA
To determine the pathway regulating nucleolar dominance,
Pikaard's team exploited a naturally occurring cellular
mechanism known as RNA interference (RNAi).
Pikaard likens RNAi to a "search and destroy mission." Fragments
of RNA known as small interfering RNAs (siRNAs) prevent specific
genes from being expressed by guiding cleavage of matching RNA
strands. Once these RNA strands are cut into smaller pieces,
they can no longer be translated into proteins. RNAi has high
specificity because the target RNA strand must have a genetic
code that is complementary to the siRNA's nucleotide sequence.
In nature, cells use RNAi to silence "junk DNA," (noncoding
regions of the DNA), and "selfish DNA" such as virus-derived
retrotransposons (jumping genes) that can be detrimental if
activated.
In the lab, Pikaard and his collaborators use RNAi to
"knockdown" expression of target genes.
Using a hybrid of two species of Arabidopsis, the plant version
of a lab rat, Pikaard's team knocked down expression of genes
coding for products that prior research had suggested might be
involved in silencing. By knocking these suspects down one by
one and assessing whether nucleolar dominance had been disrupted
after each knockdown, Pikaard and his collaborators were able to
determine which proteins and RNAs were necessary to keep the
silenced parental genes off.
New clues
The RNAi knockdowns identified several new players necessary for
the silencing machinery in nucleolar dominance to function, and
also highlighted the key role of siRNA.
First in the pathway is RNA-dependent RNA Polymerase 2 (RDR2),
which prepares a stretch of RNA for DICER-LIKE 3 (DCL3), an
enzyme that chops up RNA transcripts into smaller segments.
These smaller fragments of RNA become siRNAs, which then guide
the de novo cytosine methyltransferase , DRM2, to the targeted
genes. DRM2 is required to put a methyl group, a chemical flag
that signals for silencing, on ribosomal genes that had been
active in the parental genome. MBD6 and MBD10, methylcytosine
binding proteins, then adhere to the segments of DNA that have
been methylated by DRM2. At the same time, HDA6, a histone
deacetylase, modifies the proteins that act as spools for the
DNA.
The end result of this convergent, siRNA-mediated pathway is the
large-scale silencing of hundreds of clustered rRNA genes that
span millions of basepairs of DNA.
Nucleolar dominance occurs on a scale second only to
X-chromosome inactivation, a process by which one of the two
copies of the X-chromosome present in female mammals is randomly
inactivated. Although nucleolar dominance is on a
sub-chromosomal scale, it is, at least to date, "the largest
scale gene silencing phenomenon that clearly seems to involve
siRNAs," says Pikaard.
Pikaard explains, "siRNAs are not just regulating the selfish
DNA or the junk DNA, but they're regulating the really essential
genes too."
He believes that siRNAs might be the key to understanding the
choice mechanism underlying which parental genes get switched
off and which get left on, and he and his collaborators plan to
investigate this possibility in future research.
"The truth is out there," says Pikaard. |
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