Salk Scientists Solve Longstanding Biological Mystery of DNA Organization
Researchers image 3D genome in nucleus of living human cell for the first time
LA JOLLA —July 27, 2017 -- Stretched out, the
DNA from all the cells in our body would reach Pluto. So how does each tiny
cell pack a two-meter length of DNA into its nucleus, which is just
one-thousandth of a millimeter across?
Researchers image 3D genome in nucleus of living human cell for the first time
The answer to this daunting
biological riddle is central to understanding how the three-dimensional
organization of DNA in the nucleus influences our biology, from how our genome
orchestrates our cellular activity to how genes are passed from parents to
children.
Now, scientists at the Salk
Institute and the University of California, San Diego, have for the first time
provided an unprecedented view of the 3D structure of human chromatin—the
combination of DNA and proteins—in the nucleus of living human cells.
In the tour de force study,
described in Science on July 27, 2017, the Salk
researchers identified a novel DNA dye that, when paired with advanced
microscopy in a combined technology called ChromEMT, allows highly detailed
visualization of chromatin structure in cells in the resting and mitotic (dividing)
stages. By revealing nuclear chromatin structure in living cells, the work may
help rewrite the textbook model of DNA organization and even change how we
approach treatments for disease.
“One of the most intractable
challenges in biology is to discover the higher-order structure of DNA in the
nucleus and how is this linked to its functions in the genome,” says Salk
Associate Professor Clodagh
O’Shea, a Howard Hughes Medical Institute Faculty Scholar and senior author
of the paper. “It is of eminent importance, for this is the biologically
relevant structure of DNA that determines both gene function and activity.”
Ever since Francis Crick and James
Watson determined the primary structure of DNA to be a double helix, scientists
have wondered how DNA is further organized to allow its entire length to pack
into the nucleus such that the cell’s copying machinery can access it at
different points in the cell’s cycle of activity. X-rays and microscopy showed
that the primary level of chromatin organization involves 147 bases of DNA
spooling around proteins to form particles approximately 11 nanometers (nm) in
diameter called nucleosomes. These nucleosome “beads on a string” are then
thought to fold into discrete fibers of increasing diameter (30, 120, 320 nm
etc.), until they form chromosomes. The problem is, no one has seen chromatin
in these discrete intermediate sizes in cells that have not been broken apart
and had their DNA harshly processed, so the textbook model of chromatin’s
hierarchical higher-order organization in intact cells has remained unverified.
To overcome the problem of
visualizing chromatin in an intact nucleus, O’Shea’s team screened a number of
candidate dyes, eventually finding one that could be precisely manipulated with
light to undergo a complex series of chemical reactions that would
essentially “paint” the surface of DNA with a metal so that its local
structure and 3D polymer organization could be imaged in a living cell. The
team partnered with UC San Diego professor and microscopy expert Mark Ellisman,
one of the paper’s coauthors, to exploit an advanced form of electron
microscopy that tilts samples in an electron beam enabling their 3D structure
to be reconstructed. By combining their chromatin dye with electron-microscope
tomography, they created ChromEMT.
The team used ChromEMT to image and
measure chromatin in resting human cells and during cell division when DNA is
compacted into its most dense form—the 23 pairs of mitotic chromosomes that are
the iconic image of the human genome. Surprisingly, they did not see any of the
higher-order structures of the textbook model anywhere.
“The textbook model is a cartoon
illustration for a reason,” says Horng Ou, a Salk research associate and the
paper’s first author. “Chromatin that has been extracted from the nucleus and
subjected to processing in vitro—in test tubes—may not look like chromatin in
an intact cell, so it is tremendously important to be able to see it in vivo.”
What O’Shea’s team saw, in both
resting and dividing cells, was chromatin whose “beads on a string” did not
form any higher-order structure like the theorized 30 or 120 or 320 nanometers.
Instead, it formed a semi-flexible chain, which they painstakingly measured as
varying continuously along its length between just 5 and 24 nanometers, bending
and flexing to achieve different levels of compaction. This suggests that it is
chromatin’s packing density, and not some higher-order structure, that
determines which areas of the genome are active and which are suppressed.
With their 3D microscopy
reconstructions, the team was able to move through a 250 nm x 1000
nm x 1000 nm volume of chromatin’s twists and turns, and envision how
a large molecule like RNA polymerase, which transcribes (copies) DNA, might be
directed by chromatin’s variable packing density, like a video game aircraft
flying through a series of canyons, to a particular spot in the genome. Besides
potentially upending the textbook model of DNA organization, the team’s results
suggest that controlling access to chromatin could be a useful approach to
preventing, diagnosing and treating diseases such as cancer.
“We show that chromatin does not
need to form discrete higher-order structures to fit in the nucleus,” adds
O’Shea. “It’s the packing density that could change and limit the accessibility
of chromatin, providing a local and global structural basis through which
different combinations of DNA sequences, nucleosome variations and
modifications could be integrated in the nucleus to exquisitely fine-tune the
functional activity and accessibility of our genomes.”
Future work will examine whether
chromatin’s structure is universal among cell types or even among organisms.
Other authors included Sébastien
Phan, Thomas Deerinck and Andrea Thor of the UC San Diego.
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