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The fine art of packaging DNA

Novel method throws light on mechanisms underlying the organization of DNA

Munich, 05/19/2011

Seen in the electron microscope the DNA in a cell nucleus looks like an array of beads on a string. The beads are the so-called nucleosomes – made up of DNA associated with histone proteins - and are linked by stretches of free DNA. Active expression (transcription) of a gene requires nucleosome-free DNA regions. Thus, the actual positions of nucleosomes modulates the activity of genes. The packaging of DNA therefore contributes to the regulation of all cellular functions and is of great interest to researchers. Dr. Philipp Korber and his doctoral student Christian Wippo of the Adolf-Butenandt-Institut at Ludwig-Maximilians-Universität (LMU) Munich, in cooperation with Professor Franklin Pugh’s group at Pennsylvania State University (USA), have now achieved a decisive breakthrough in the field. Using an artificial cell-free system, they have succeeded in reconstituting the biochemical process that directs nucleosome positioning throughout the yeast genome. The nucleosome distribution they obtained recapitulates that observed in the living cell. Furthermore, it emerged that main aspects of current models about how the organization of nuclear DNA is achieved are unlikely to be correct. “This is a real milestone, because we can now study the mechanism of genomewide nucleosome positioning at the biochemical level under fully controlled conditions, which would not be possible in living cells,” says Korber. (Science, 20.5.2011)

DNA, the hereditary material, is a thread-like molecule, but in the cell nucleus it is found in packaged form. Rather like strands of hair wrapped around curlers, stretches of nuclear DNA are wound around particles formed by histone proteins. The “curlers”, called nucleosomes, are linked by regions of nucleosome-free DNA. As nucleosomes inhibit the transcription of genes, this basic three-dimensional organization of the DNA is crucial in controlling which genes are active and therefore in determining what proteins can be synthesized in a given cell. “Genomewide maps giving the positions of the nucleosomes with respect to the DNA sequence have been generated in the course of the past six years,” says Korber. These have shown that nucleosomes are not distributed at random. On the contrary, most of them are found at clearly defined locations. Since the distribution of nucleosomes controls the accessibility of genomic information, the mechanism underlying nucleosome positioning is of fundamental significance for all DNA-dependent processes. Several theoretical models have been proposed to explain nucleosome positioning but none has been adequately tested or even confirmed by experiment. Three hypotheses are particularly prominent in the current discussion. The first invokes a “genomic positioning code”, which postulates that sequence-dependent structural features in the DNA itself influence the positioning of nucleosomes. The second suggests that nucleosomes are passively (“statistically”) aligned relative to a set of fixed barriers on the DNA, like railroad cars being shunted between buffers in a marshalling yard. A third possibility is that the process of gene transcription has an ordering effect that serves to position nucleosomes.

So far it has not been possible to test these models critically in living cells. Nucleosome organization is such a basic aspect of nuclear structure that experimental perturbations have immediate and deleterious effects on cells, which lead to secondary effects that are difficult to interpret. The teams led by Korber and Pugh have now made a crucial breakthrough. They began with an in-vitro biochemical system which is capable of assembling nucleosome beads on naked yeast DNA. Most importantly, when they subsequently added a yeast extract to this material, the nucleosomes came to occupy the same positions as they do in living yeast cells. The yeast extract, together with ATP (which supplies energy), plays the crucial role in the positioning reaction. In the absence of either, the nucleosome distribution found in cells is not recapitulated. This demonstrates that, in addition to DNA and histones, accessory factors present in the extract are necessary for proper positioning of nucleosomes. Moreover, this cannot be an entirely passive process, since it requires an energy source. The observations are not compatible with a positioning code and also argue against a purely statistical distribution of nucleosomes. A second experiment also failed to support the latter model. “Since the system we use is completely cell-free, for the first time we can control the reaction conditions and vary them at will. We were able, for example, to reduce the concentration of nucleosomes by half, which is not possible with living cells. The statistical nucleosome positioning model, which we had favored at the outset, would predict that the average distance between nucleosomes should increase under such conditions. But, to our surprise, it remained the same as before. Something in the yeast extract actively keeps the nucleosomes together,” says Korber. Finally, the last theory, the transcription-based model, can probably be excluded too, since the cell-free system does not support transcription.  As Korber points out: “Now that we have challenged, or even refuted, the most popular models, the search for the factors that implement our newly proposed active packing mechanism is top priority.”

The new experimental system provides an indispensable tool for this. “Reconstructing such complex processes in cell-free systems is not a trivial undertaking,” says Korber, “and our in-vitro system raises biochemical reconstitution to a new level, from which we will gain a better understanding of a basic principle of genome organization.” (göd/PH))

The study was carried out under the auspices of the Collaborative Research Center "Transregio 5" (Chromatin: Assembly and Inheritance of Functional States) and was also supported by the EU-funded Epigenome Network of Excellence.

Publication:
"A packing mechanism for nucleosome organization reconstituted across a eukaryotic genome"
Z.Zhang#, C.J. Wippo#, M. Wal, E. Ward, P. Korber*, B.F. Pugh*.
Science, 20 May 2011
doi: 10.1126/science.1200508
#,* Equal first and last authors respectively

Contact:
Dr. Philipp Korber
Adolf-Butenandt-Institute
Molecular biology
Phone: +49 (0) 89 / 2180 - 75435
Email: philipp.korber@med.uni-muenchen.de
Website: www.molekularbiologie.abi.med.lmu.de/ueber_uns/korber

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