By Rachel Shulman

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.
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