Sharper 3D-View on Human DNA

A new technique enables 3-D visualization of chromatin – DNA plus associated proteins – structure and organization within a cell nucleus by painting the chromatin with a metal cast and imaging it with electron microscopy. (Source: Salk Inst.)

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? 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.

The Salk researchers identified a novel DNA dye that, when paired with advanced micro­scopy in a combined tech­nology, ChromEMT, allows highly detailed visuali­zation of chromatin structure in cells in the resting and mitotic 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. “It is of eminent impor­tance, for this is the biolo­gically relevant structure of DNA that determines both gene function and acti­vity.”

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 in diameter. These nucleo­some 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 Uni­versity of Cali­fornia, San Diego, professor and micro­scopy expert Mark Ellisman, 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 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. Horng Ou, a Salk research asso­ciate says: “Chromatin that has been extracted from the nucleus and subjected to processing in vitro may not look like chromatin in an intact cell, so it is tremen­dously important to be able to see it in vivo.”

With their 3D microscopy recon­structions, the team was able to move through a small volume of chromatin’s twists and turns, and envision how a large molecule like RNA poly­merase, which transcribes 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. (Source: Salk Inst.)

Reference: H. D. Ou et al.: ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells, Science 357, eaag0025 (2017); DOI: 10.1126/science.aag0025

Link: National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, USA • Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, USA

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