NEW YORK (GenomeWeb) – Contrary to what was previously thought, chromatin is a disorganized chain that is packed together at differing densities during interphase and mitosis, according to a new study.
About 2 meters of DNA has to be packaged into a nucleus that is a thousandth of a millimeter across. Its organization is typically presented through a hierarchical model in which 147 bases of DNA loop around 11-nanometer nucleosomes with some 20 bases to 75 bases between each DNA-nucleosome structure. These so-called "beads-on-a-string" then fold into additional fibers to eventually form chromosomes.
However, studies underpinning this model were largely performed in vitro and with highly treated DNA, leaving open the question of how DNA might look in vivo.
Researchers from the Salk Institute and elsewhere developed a new visualization approach that combined a fluorescent DNA dye and electron microscopy tomography that allowed them to glimpse higher-order DNA structure from within cell nuclei. As they reported in Science yesterday, the researchers found that DNA does not organize itself as textbooks say, but rather forms flexible chromatin chains with differing diameters and configurations.
"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," senior author Clodagh O'Shea, an associate professor at Salk, said in a statement. "It is of eminent importance, for this is the biologically relevant structure of DNA that determines both gene function and activity."
O'Shea and her colleagues screened through dyes that would enable them to use electron microscopy tomography to visualize DNA. They found one, DRAQ5, that when excited, oxidizes diaminobenzidine (DAB) and can been seen by electron microscopy (EM).
The researchers dubbed the combination of DRAQ5 DNA labeling, DAB photo-oxidation, and conventional EM osmium tetroxide staining of chromatin ChromEM. They then paired it with multi-tilt EM, which allows for better resolution, calling the combination ChromEMT. ChromEMT, they reported, allows for the direct visualization of chromatin and 3D resolution of its structure.
The researchers applied this approach to examine chromatin of interphase cells as well as of mitotic cells. In particular, they visualized chromatin from within human interphase small-airway epithelial cells (SAECs), metaphase SAECs, and anaphase cells from a human osteosarcoma cell line.
From this, they noted that chromatin is actually a disordered granular chain of DNA nucleosomes with diameters between 5 nanometers and 24 nanometers.
O'Shea and her colleagues did not observe in the living cells the higher-order structures predicted by the hierarchical model.
That chain is then further packed together, but at concentrations that differ during interphase and mitosis, they added. During interphase, the researchers found that chromatin has an extended curvilinear structure — nuclei in this phase have chromatin volume concentrations between 12 percent and 52 percent — while during mitosis, chromatin forms more compact loops — these nuclei have chromatin volume concentrations greater than 40 percent.
These findings led the researchers to propose a new model for chromatin organization. They suggested that the disordered chromatin chains are flexible and bend and fold into different packing densities. This, the researchers said, could explain how chromatin can rapidly condense as well as how epigenetic interactions and structures can be passed on to daughter cells after cell division.
"We show that chromatin does not need to form discrete higher-order structures to fit in the nucleus," O'Shea added. "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."
The researchers added that their findings may also have implications for disease research, particularly cancer in which DNA becomes disorganized.