Scientists at the Salk Institute and the University of California, San Diego, have developed a technique that enables detailed visualization of the 3D structure of chromatin in the nucleus of living human cells.
Published in Science today, the Salk researchers identified a novel DNA dye that, when paired with advanced microscopy in a combined technology called ChromEMT, facilitates an intricate look at chromatin structure in cells in the resting and mitotic stages.
"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."
X-rays and microscopy showed that the primary level of chromatin organization involves 147 bases of DNA spooling around proteins to form nucleosomes, which 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.
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 University of California, 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. O'Shea's team called the technique, which combines their chromatin dye with electron-microscope tomography, ChromEMT. The team used ChromEMT to image and measure chromatin in resting human cells and during cell mitosis when DNA is compacted into its most dense form.
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.
Not only do the team’s findings potentially upend the classic textbook model of DNA organization, their 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."