Download May 24, 2002 - The Rockefeller University

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TODAY’S EVENTS
Eric Lander on genome
Eric Lander, founder and director
of the Whitehead Center for
Genome Research, will give a
talk on “The Human Genome
and Beyond” at the Friday lecture today (May 24) at 3:45 p.m.
in Caspary Auditorium.
A member of the Whitehead
Institute and professor of biology
at The Massachusetts Institute of
Technology, Lander has been
one of the principal leaders of
the Human Genome Project.
Lander is a geneticist, molecular
biologist and mathematician,
with research interests in human
genetics, mouse genetics, population genetics and computational
and mathematical methods
in biology.
Lander and his research group
have developed many of the
tools of modern genome
research — including genomic
maps of the human, mouse and
rat genomes in connection with
the Human Genome Project and
techniques for genetic analyses
of complex, multigenic traits. He
has applied these techniques to
understanding cancer, diabetes,
hypertension, renal failure and
dwarfism.
He first came to the Whitehead
Institute as a Whitehead Fellow
in 1986, while still an assistant
professor of managerial economics at the Harvard Business
School. Lander was a Rhodes
Scholar and received his D.Phil.
in mathematics from Oxford
University in 1981.
Lander’s many honors and
awards include the MacArthur
Foundation Prize Fellowship
and the Woodrow Wilson Prize
from Princeton University. A
fellow of the American Association
for the Advancement of Science,
Lander is a member of the
National Academy of Sciences
and its Institute of Medicine.
news notes
THE NEWSLETTER FROM THE ROCKEFELLER UNIVERSITY’S OFFICE OF COMMUNICATIONS AND PUBLIC AFFAIRS
3-D images shed light on first steps
of RNA synthesis
The first three-dimensional
images of the initiating form of
the molecular machinery in bacteria that “transcribes” genetic
information from DNA into
RNA — the crucial first step for
making proteins — is reported
in a pair of papers in the May 17
issue of the journal Science.
used a technique called X-ray
crystallography to reveal the
structure of the holoenzyme, the
core enzyme bound to the sigma
subunit.They also used this
research tool to visualize the
structure of the holoenzyme
bound to a short piece of DNA
containing a promoter sequence.
These research findings of The
Rockefeller University scientists
illuminate how the process of
transcription begins in bacteria.
Understanding this process may
provide clues for developing
new drugs to treat bacterial
infections.
In X-ray crystallography, X-rays
are fired at crystallized proteins,
or complexes, of proteins bound
to other molecules. Analysis of
the data from the diffracted Xrays yields information about the
three-dimensional structure of
the molecule. Some molecules
can be visualized at the resolution of a few angstroms (a typical
atom is roughly two angstroms in
diameter). But Darst notes that
the crystals his group used did
not diffract well enough to produce a high resolution structure.
So, he and his colleagues used a
novel hybrid approach.
The DNA-transcribing machinery
is called the RNA polymerase
(RNAP).The RNAP comprises a
core enzyme, which can synthesize RNA from a DNA template,
but cannot initiate transcription
by itself. Initiation requires another
protein, the sigma subunit, which
binds the core enzyme to form
the holoenzyme.
The sigma subunit “tells” RNAP
where in the genomic DNA to
start transcription. It does this by
recognizing a section of the gene’s
DNA called the promoter region
and “melting” the DNA to
expose one of the double-helical
strands the RNAP will use as the
template to synthesize RNA.
The Rockefeller University
researchers, led by Seth Darst,
studied RNAP from a bacterium
called Thermus aquaticus, a member of a family of organisms that
thrive at high temperatures.They
“From previous and parallel work
in our lab, we had high-resolution structures of the RNA polymerase and parts of the sigma
subunit,” says Darst, who is the
Jack Fishman Professor and head
of laboratory at Rockefeller.“We
fitted the high-resolution structures into the lower-resolution
structure to obtain the structure
of the holoenzyme. And since the
structure of DNA is known, we
used that to solve the structure of
the holoenzyme bound to the
promoter fragment.”
continued on page 2
These are the first 3-D images of the molecular machinery in bacteria that initiates the
process of transcribing DNA into RNA. A short piece of DNA containing a promoter
sequence (green and white double helix) binds to a bacterial “holoenzyme” (multicolored structures).
In the top photo, the holoenzyme specifically recognizes the sequence of the promoter
and forms a structure called a closed complex — closed because the DNA is not
“melted.” Once the sequence is recognized, the holoenzyme goes through a series of
steps and actually melts DNA (bottom photo) to expose one of the double-helical
strands the core enzyme will use as the template to synthesize RNA. These structures
“provide fuel for a model to understand the steps in bacterial transcription.”
Leprosy bug provides clues to early nerve degeneration
Possible insight to multiple sclerosis and neurodegenerative diseases
that destroy nerve cell “insulation”
Scientists at The Rockefeller
University and New York
University School of Medicine
have found that the nerve damage that leads to a loss of sensation and disability in people
with leprosy occurs in the early
stages of infection. The finding
was reported in the May 3 issue
of Science.
The nerve damage, a hallmark of
leprosy previously thought to be
a byproduct of the immune system’s response to the leprosy bacteria, now is thought to be a
direct result of the leprosy bug
attaching itself to specialized
nerve cells — Schwann cells —
the glial, or supporting, cells of
the peripheral nervous system
(PNS).
The findings suggest that the
body’s immune response does
not play a significant role in the
early stage of neurological
injury.The damage is characterized by the disruption of the
myelin sheath, the insulation on
nerve cell connections that helps
transmit rapid signals between
the brain and the peripheral
organs, for example, skin and
muscles. Myelin is arranged in
segments around axons and sep-
arated by nodes. At and near the
nodes, sodium and potassium
channels are clustered in high
density in the axon membrane
so that they can transmit an
electric charge called the action
potential. Damage to myelin
dramatically decreases such
action potential and nerve conduction velocity, and eventually
causes loss of sensation, disability
and paralysis.
Using laboratory cell tissue cultures and mice genetically manipulated to lack two key immune
system cells, the research team, led
by Rockefeller University micro-
biologist/cell biologist Anura
Rambukkana, showed that
Mycobacterium leprae (M. leprae), the
bacterium that causes leprosy,
destroys the protective myelin
sheath that surrounds nerve fibers
and then hides out in the supporting cells that enclose nonmyelin nerve fibers, poised to initiate later attacks.
“What we show here is a novel
mechanism of inducing demyelination by a bacterial pathogen,”
says Rambukkana, principal
author and research assistant professor in the Laboratory of
continued on page 2
RNA synthesis continued
goes inside the polymerase toward
the active site and back out,
producing a very unusual
protein-protein interaction.
Seth Darst and his team used X-ray
crystallography to visualize the machinery
in RNA synthesis.
The holoenzyme structure offers
some clues to how transcription
begins in bacteria. Research from
Darst’s lab published recently in
Molecular Cell revealed that the
sigma subunit contains four protein “domains” connected to one
another by short stretches of
amino acids called “linkers.”With
information from the holoenzyme structure published in
Science, the researchers show that
the domains of the sigma subunit
spread out and bind to one face
of the core enzyme, which positions sigma to bind to DNA.
The most interesting finding, says
Darst, involves the position of one
of the linkers.This linker, containing about 33 amino acids, is much
longer than the others.This linker
“These sigma domains sit on the
surface of the RNA polymerase,
but this very long linker is buried
inside the polymerase, near the
active site of the enzyme,” he
says.“In order to establish this
complex, there need to be a lot
of conformational changes to get
the linker inside.”
In bacteria, the first step in transcription is binding of RNA
polymerase to a nucleotide, one
of the four building blocks that
make up DNA.The researchers
hypothesize that the linker participates in binding the initiating
nucleotide, and once the RNA
polymerase begins synthesizing
RNA, the sigma subunit is no
longer needed.
the path of the RNA,” says Darst.
“As the RNA transcript begins
to be elongated, it has to push
this linker out from a channel
inside the polymerase.The linker’s not completely pushed out
until the RNA becomes about
12 nucleotides long.”
Scientists have known from biochemical experiments that a
transition occurs when the
RNA transcript reaches a length
of 12 nucleotides: the complex
becomes much more stable and
the elongation phase begins.
“We think that signal to go into
elongation occurs when the
RNA becomes long enough to
fill the channel,” says Darst.“It
pushes the linker peptide out, and
then sigma begins to fall off.”
Gene expression is regulated at
every step, but probably the
major focal point of regulation is
at the initiation of transcription.
According to Darst, the linker
that is buried near the active site
may play a role in telling the
RNA polymerase when to start
synthesizing RNA.
This structure also explains a
curious phenomenon called
abortive initiation, in which
transcription starts and stops.
The polymerase remains at the
promoter site synthesizing short
pieces of RNA, which fall out,
and then the process starts over
again. This can happen hundreds
of times before the process
moves on and a long RNA
transcript is synthesized.
“The linker sticks inside the
polymerase and actually blocks
From the structural information
and other experiments, the
says Salzer.“Such signals could
also be activated in other diseases
that cause demyelination.”
nerve injury in patients
before the immune system comes into play.
The researchers also showed that
M. leprae does not need to be
alive to demyelinate nerve cells.
Similar results were obtained after
cultured Schwann cells were
exposed to bacteria that had been
killed with radiation and to fractions of the bacterium’s cell wall.
Schwann cells also play
a significant secondary
role in providing support for the various
growth factors during
the development of the
spinal motor neurons
and sensory neurons.
Thus, Schwann cells
play a wider role not
only in the normal
development of the
nerve cells in the
PNS, but also in their
regeneration.
researchers now know that
abortive initiation happens
because of a competition
between RNA and the linker
peptide. If the peptide wins, the
RNA falls out and transcription
starts over.
“Every once in a while the RNA
manages to push the peptide all
the way out, and once it’s long
enough to push it all the way
out, it’s done, and there’s no more
abortive initiation,” says Darst.
The structure of the holoenzyme
bound to the promoter fragment,
Darst says, “provides fuel for a
model to understand the steps in
bacterial transcription.”
The holoenzyme specifically recognizes the sequence of the promoter and forms a structure
called a closed complex — closed
because the DNA is not melted.
Once the sequence is recognized,
the holoenzyme goes through a
series of steps and ultimately
forms the open complex, where it
actually melts DNA.
“From this structure and others,
we made a model of the closed
complex and the open complex,
kind of a beginning and an
end,” says Darst. “What we’re
really interested in now is the
in-between steps.
“We think the models are pretty
good, but they don’t provide a
lot of insight into how it gets
from A to B.”
A better understanding of transcription in bacteria could lead
to new drug targets to treat bacterial infections, particularly
those that are resistant to current
antibiotics. Previous research
from Darst’s lab showed that
rifampicin, one of the two drugs
most effective against tuberculosis, kills the microbe that causes
TB by physically blocking that
bacterium’s RNA polymerase.
The Rockefeller scientists who
conducted this research are postdoctoral fellow Katsuhiko
S.Murakami, research scientist
Shoko Masuda, postdoctoral fellow Elizabeth A. Campbell and
research scientist Oriana Muzzin.
This research was supported in
part by the National Institute
of General Medical Sciences,
part of the federal government’s
National Institutes of Health,
the Norman and Rosita Winston
Foundation and the Human
Frontiers Sciences Program.
— Joseph Bonner
Leprosy continued
Bacterial Pathogenesis.“This may
unravel clues for early molecular
events of neurodegeneration processes in other diseases, such as
multiple sclerosis, which we currently know nothing about.”
“By using this bacterium we
will be able to obtain novel
insight not only into the mechanism of the early demyelination
process, but also how the complex molecular architecture of
the myelinated fiber is disrupted,” adds Rambukkana.
Using a “co-culture” system
developed by co-author James
Salzer at the NYU School of
Medicine, in which myelinated
nerves form normally in cell culture, the researchers found that
M. leprae produced significant
damage to the myelin sheaths 24
hours after attaching to the
nerves. Myelin damage is the earliest effect observed, followed by
the degeneration of nerve axons
that carry the nerve impulses. M.
leprae does not harm any other
parts of the cell, and there is no
sign that it causes cell death.
The researchers found that, unexpectedly, M. leprae does not need
to enter the cell to cause degeneration of the myelin sheath.
“This suggests that binding of M.
leprae to the surface of the myelin
sheath is sufficient to induce
myelin breakdown, presumably by
activating signals inside the cell,”
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Previous research by Rambukkana,
Salzer and their colleagues implicated a major component of the
bacterium’s cell wall called PGL-1
in its predilection for peripheral
nerves, and they now propose
that PGL-1 is a crucial cell wall
molecule directly involved in
nerve damage in leprosy.
The mouse model the researchers
studied is called a “Rag-1 knockout.”This mouse, developed by
Associate Professor Peter
Mombaerts in the early 1990s
as a postdoc in Nobel laureate
Susumu Tonegawa’s laboratory at
MIT, is genetically altered to lack
mature B and T cells (B cells are
responsible for producing the
infection-fighting proteins called
antibodies, while T cells help regulate the body’s immune
response). As in the cell culture
model, direct administration of
both M. leprae and its cell wall to
the peripheral nerves of Rag-1
mice caused significant myelin
damage, providing clues for early
Nerve cells in the PNS Using myelin-forming primary nerve tissue cultures (top left), and mice genetically manipulated to
lack two key immune system cells (bottom left, electron micrograph), researchers led by
are able to regenerate
Rockefeller microbiologist/cell biologist Anura Rambukkana showed that Mycobacterium leprae,
after injury, unlike their
the bacterium that causes leprosy, selectively destroys the protective myelin sheath that insulates
counterparts in the cen- nerve fibers (right-hand panels).
tral nervous system
(CNS). Examination of
M. leprae is known to cause debil- The knowledge gained by such
myelinated and non-myelinated
M. leprae-induced myelin damage
nerve cells infected with M. leprae itating neurological injury in
humans,
but
the
clinical
manifesin the early infectious process
revealed an interesting survival
tation
occurs
years
after
a
slow
provides valuable insights into the
strategy of this bacterium. As the
infectious
process.
According
to
pathologic mechanisms of early
damaged myelinated nerve cells
Rambukkana,
information
about
neurodegenerative diseases in
repair themselves by generating
the
pre-clinical
mechanisms
in
general.
new nonmyelinating Schwann
M.
leprae–induced
demyelination
cells after attack, M. leprae
Rambukkana’s and Salzer’s comay allow researchers to develop
sequesters itself in these nonauthors are George Zanazzi at
therapeutics and common diagmyelinated nerve cells, waiting
NYU and Nikos Tapinos at
for a chance to attack again, once nostic tests for early detection of
Rockefeller.
demylenating diseases of both
they multiply and escape from
— Joseph Bonner
infectious origin and unknown
these cells. Rambukkana hypothetiology,
such
as
multiple
sclerosis
esizes that this phenomenon
accounts for the lapsing/remitting and Guillain-Barré syndrome.
characteristic of leprosy.
news notes
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NEWS BRIEFS
Acting President Sakmar comments on research facilities
Symposium recognizes Bob Roeder,
transcription pioneer
Editor’s note: Acting President
Thomas P. Sakmar responded to
several questions from
News&Notes about the recent
decisions of the Board of Trustees’
Executive Committee about the
proposed 12-story research tower on
the north campus and the large-scale
Roosevelt Island housing project for
faculty and students. On May 17,
Board Chairman Richard Fisher
sent to the Board a brief report
about the actions taken at the committee’s meeting May 8.
number of labs from 72 today to
100.The added people and
resources needed to support such
an expansion would have dramatically changed the university and
significantly increased the university’s overall operating budget.
In the report, he wrote that the
committee had “resolved not to proceed further with this particular plan
for a new research facility, while
recognizing that we may develop
alternative plans for a better supported and more affordable facility
on this prime building site in the
future.We also resolved to end our
consideration of the large-scale
Roosevelt Island Housing Project.”
Since Feb. 11, when I became
Acting President, over $5 million has been contributed by
individuals to signal their support for the university and our
research and educational programs. As we near the end of
the fourth year of our six-year
Centennial Campaign, the university has raised $278 million
toward its $350 million goal.
This is impressive; it is possible
that we may complete the campaign ahead of schedule.
To honor Robert Roeder on
his 60th birthday, about 25 colleagues, including former graduate students and postdocs from his
lab, have organized a symposium,
“Three Decades Studying the
Regulation of Eukaryotic
Transcription,” which will occur
9 a.m. to 5:30 p.m., Friday, May
31, in Caspary Auditorium.
As head of the Laboratory of
Biochemistry and Molecular
Biology, Roeder has significantly
improved scientific understanding of how human genes are
switched “on” or “off ” — a crucial life process occurring at all
times in every one of the billions of cells comprising the
human body. Recent research
finding in his lab are described
in an article on page 5.
Lectures at the symposium will
reflect on his initial groundbreaking discovery of the three
proteins responsible for “reading”
human genes as well as his ongoing elucidation of the complex
manner in which this vital process is controlled.
The speakers will focus on the
four major areas in transcription
that are the targets of Roeder’s
lab: activators, general transcription factors, chromatin and
signaling.
A complete schedule can be
found at
www.rockefeller.edu/lectures/
053102.html.
“Designing for the Senses”
No, it’s not an extra who
wandered off the set of Star
Wars. It’s Paul Smith, staff
member in Communications
and Public Affairs, trying on
a “cocoon suit” designed by
Eiko Ishioka for Olympic athletes. The suit, which was
used during the 2002 Winter
Olympics in Salt Lake City,
shuts out sound and other
stimulation and helps athletes concentrate on their
performances.
The suit was exhibited at
“Designing for the Senses,”
a day-long conference that
brought together experts in
science and technology and
the design, culinary and fashion professions.The May 17
event was co-sponsored by
The Rockefeller University
and The Cooper-Hewitt,
National Design Museum of
the Smithsonian Institution.
PEOPLE IN THE NEWS
Is it true that the new
research tower project is
being set aside?
As it is now proposed, yes.
However, a new plan for a
research building and for modernization of current research
laboratories may emerge from
the evaluation of all of the
existing research facilities and
research infrastructure. The
Executive Committee of the
Board has directed me to begin
leading this evaluation process.
Why was the proposed
research building
reconsidered?
It was reconsidered in part
because of the recent updates of
estimated construction budgets
for the proposed building. These
costs are substantially higher
than the estimates when the
Board approved the concept for
the facility.
The proposed research building, if
erected, would have substantially,
and not incrementally, transformed the university. It was based
on a program that would have
increased the university’s lab space
significantly and brought the total
Is there a problem with
fund raising — is that the
reason for the decision?
No. In fact fund raising has
been very successful thus far this
year.
How will the Executive
Committee’s decision
affect the search for the
next president and faculty
recruitment in general?
It’s not likely to have any effect
at all. The university has adequate space for its current faculty and for the active searches
under way. For example, about
24,000 square feet of new space
is coming on line in the
Rockefeller Research Building
and another 7,000 square feet in
the hospital. Looking forward,
we are in very good shape with
respect to funds available for the
new faculty recruitment and
faculty development.
How did Arnold Levine’s resignation affect this decision?
When I took over as Acting
President, I fully intended to
implement the plans that I had
inherited. However, the Board
instructed me to carry out a
careful analysis of the capital
project including the Roosevelt
Island housing development
project. Considering that a
change in the administration
had occurred, it was natural to
take a fresh look at our current
and future needs and see how
much they would cost. This is a
normal process for organizations
under new leadership — a fresh
perspective is always welcome.
When presented with the upto-date picture, the Executive
Committee of the Board
directed me to act accordingly.
How will this decision affect
the university’s research
future?
This decision was made to
insure that the university would
continue to have the resources
and unique environment to provide its excellent faculty and
students with the resources
needed to make the stellar scientific achievements for which
The Rockefeller University is
well known.
What role did the faculty
play in this decision?
The faculty did not play a direct
role in the Executive
Committee’s decision. But, they
will play a very strong role in
the process of determining our
future direction and the research
resources that this campus needs
for the future. It is after all the
scientists who are responsible
for carrying out our scientific
research mission.
The administrative staff and scientific support staff also will be
called on to analyze the effects
of any major changes in our
research program.
What about housing?
Although the proposed major
Roosevelt Island housing development project is being set
aside, the Executive Committee
directed me to act promptly to
insure that we will have excellent housing options for our
students, postdocs and faculty.
We are now actively addressing
this issue.
— Cathy Yarbrough
Cheers on their 10th and 20th anniversaries
Postdoctoral fellowships
The university’s Faculty Nominating Committee has selected the
recipients of the following postdoctoral fellowships for the coming
academic year.
Charles H. Revson Fellowships in Biomedical Research:
Deepti Jain (Seth Darst lab), Terry Lechler (Elaine Fuchs lab),
Vincent Noireaux (Albert Libchaber lab) and Lei Zheng (Robert
Roeder lab).
FPO
Women & Science Postdoctoral Fellowships:
Sara Buonomo (Titia de Lange lab), Emily Harms (Michael Young lab),
Li Liu (Magda Konarska lab) and Woelsung Yi (Jeffrey Ravetch lab).
The 2002-2003 C. H. Li Memorial Scholarship recipient is
Minghao Zhong (James Darnell lab).The committee also selected
Michael Lampson (Tarun Kapoor lab) as the first recipient of the
Francis Goelet Fellowship, a new three-year award.
3
Massive applause and cheers erupted as Acting President Thomas P. Sakmar announced the names of each faculty and staff
member who had served 10 and 20 years at the university. An article about the May 16 reception for Employee Recognition Day,
and the employees honored, will be in the next issue of News&Notes.
More than just packaging, histones help turn on genes
Rockefeller scientists redefine role of proteins responsible for bundling DNA into cells
Histones, the proteins that help
roll several feet of DNA into the
microscopic span of a single
nucleus, are turning out to be
much more than just packaging
material. Instead, recent studies
indicate that these once underrated proteins actively participate
in switching genes “on” — a
vital life process occurring at all
times in each one of our cells.
Now, new research at The
Rockefeller University shows
that the dangling ends or “tails”
of histones also are less passive
than previously believed. In fact,
the researchers say these tails act
more like “arms” to directly
affect gene activity once the
DNA is unpackaged.
The findings, reported in the
April issue of Molecular Cell, not
only provide fundamental
insight into the complex workings of the human cell, but also
may one day lead to new treatments for diseases in which
genes have been improperly
turned on or off, such as cancer.
Furthermore, this kind of
knowledge may provide new
strategies for combating the
handful of developmental disor-
patterned, with varying combinations of chemical tags that
ultimately dictate which genes
get switched on, and which
remain silent.
“If there is some form of histone code, then this new system
could potentially enable scientists to interpret it,” says Robert
G. Roeder, head of the
Laboratory of Biochemistry and
Molecular Biology at
Rockefeller and principal author
of the report.
Moreover, knowledge of such a
code would allow scientists to
manipulate it for the treatment
of diseases in which gene production has gone awry.
Unraveling DNA
Histones help to package DNA,
the hereditary material of life,
into each cell’s nucleus.The
double-helical strand of DNA
wraps around a ball of histones
consisting of four distinct proteins: H2A, H2B, H3 and H4.
This fundamental unit, called a
nucleosome, is repeated at regular intervals throughout the
length of DNA and, under a
microscope, resembles “beads on
In the laboratory of Robert G. Roeder, Woojin An (left) and Vikas B. Palhan (right)
report that the “tails” of histones, previously thought to play only a passive role in
packaging DNA, in fact, behave more like “arms” to switch on genes once the DNA
becomes unpackaged.
ders associated with malfunctioning histones, such as
Rubinstein-Taybi syndrome and
Coffin-Lowry syndrome.
“We found that if we removed
the tails from histones, genes
could not be activated,” says
Woojin An, a postdoctoral fellow at Rockefeller and first
author of the new research
paper. “Because these tails are
essential to switching genes on,
they are really more like arms.”
In addition to this unexpected
finding, the researchers also have
developed a powerful new biochemical tool for deciphering a
possible “histone code.”
According to this emerging theory, histone tails are coded, or
5
a string.” Compact strings of
nucleosomes coil up further to
form chromatin and even further
to become the familiar X-shaped
chromosomes of human cells.
Hidden within this bundled
mass of histones and DNA are
the genes: the cell’s instructions
for manufacturing its tens of
thousands of proteins. Therefore,
before the cell’s machinery can
“read” or, as scientists say, “transcribe” these genetic instructions
and ultimately produce a protein
of interest, it must somehow
open up or unfold the tightly
wound chromatin.
But just how the cell’s machinery knows which stretches of
chromatin to break open and
Rockefeller researchers have developed one of the first successful test tube systems for studying the effects of DNA packaging
proteins — histones — on gene activity. Above: reconstituted “chromatin” containing genetically altered histones and DNA. The
new chromatin system led the researchers to the finding that “tails” of histones are essential to switching on genes. In addition,
the system will allow scientists to explore the possibility of a “histone code” — an emerging theory that states that varying patterns of chemical tags along histone tails control which genes become activated, and which remain silent.
read, and which to keep silent,
remains poorly understood.
Some scientists think that the
key to regulating the activity of
genes may lie with the very
proteins that package them.
They propose that the tails of
each histone become coded
with different patterns of chemical tags and thus, like a combination lock guarding a safe full
of genes, only permit access to
those gene-reading machines
that somehow “know” the right
“histone code.” This code is not
inherent to the histones themselves but is generated by other
proteins in the nucleus.
Now, as a twist to this developing
story, Roeder and collegues have
discovered an entirely novel function for tails.They show that
these dangling ends, in addition
to repressing genes through chromatin folding, also play an active
role in switching genes on once
the folding is reversed.
“As counterintuitive as it may
seem, these tails seem to act
both to block and to activate
gene transcription,” says Roeder.
Previously scientists thought
that the tails — which, unlike
the rest of the histone, extend
from the compact body of the
core nucleosome — blocked
transcription of a gene by causing chromatin folding. Gene
activation was thought to occur
after the inhibitory effects of the
tails were neutralized by the
addition of a chemical tag — a
process called acetylation.
“Histone tails are covered with
positive charges and are therefore attracted to the negatively
charged DNA,” says An.
“Scientists believed that acetylation, by masking the positive
charges on tails, caused the
chromatin and underlying DNA
to become less constricted by
the histones and thus more
receptive to unfolding.”
“Something else must be repressing transcription of genes besides
the tails,” says An. “And this must
be the residual nucleosome core
structure.”
A novel biochemical tool
“In addition,” he says, “these
data tell us that the tails are
required for transcription, a surprising finding really when you
consider that for several decades,
these protein tails were thought
to be mainly involved in
repressing genes.”
To test this theory, the researchers
recreated coiled-up chromatin in
a test tube using engineered or
recombinant histones and DNA
— a feat in itself considering that
this type of biochemical tool had
never before been successfully
implemented.They also showed
that by adding a specific gene
activator protein and a “coactivator” protein called p300 they
could coax the chromatin to
open up and permit transcription
of a target gene.
Both a gene activator and a coactivator are required to switch on
a target gene. First the activator
binds to a specific sequence of
DNA in front of the target gene;
next the coactivator binds to the
activator and allows it to communicate with the gene-reading or
transcription machinery, and
finally transcription begins.
Importantly, p300, in addition to
its role as a mediator, enhances
transcription via acetylation of
histone tails.
Next, the researchers repeated the
chromatin assembly experiment
with recombinant histones lacking tails. Because the prevailing
theory at the time stated that the
physical presence of histone tails
acts mainly to prevent transcription of a target gene, they predicted that the tailless histones
would render chromatin unable
to fold and automatically lead to
gene activation. But, to their surprise, the exact opposite was true:
the chromatin, though unfolded,
remained silent and unfit for
gene transcription.
The researchers then asked how
acetylation of histone tails fits
into their new model. If this
process of chemical tagging does
not simply modify the tails to
enable chromatin unfolding, they
wondered, then what is its role?
To address this question, they
again assembled chromatin in a
test tube; only this time they
used histones with tails genetically altered to make acetylation
by p300 impossible. Once more,
the researchers found that the
target gene could not be activated, thereby demonstrating not
only that histone tails are
required for turning genes on,
but, more specifically, that acetylated histone tails are required.
Finally, taking this experiment
one step further, the researchers
showed that p300 prefers to
acetylate histone protein H3,
with its next choice being H4.
In addition, they found that
only a few of the possible acetylation sites on histone tails H3
and H4 seem to be essential for
gene activation.
— Whitney Clavin