Search Menu
Photonics Media Photonics Buyers' Guide Photonics EDU Photonics Spectra BioPhotonics EuroPhotonics Industrial Photonics Photonics Showcase Photonics ProdSpec Photonics Handbook
More News
Email Facebook Twitter Google+ LinkedIn Comments

FRET and scanning force microscopy help unravel DNA compaction mystery

Oct 2006
Information could eventually benefit those with illnesses tied to DNA compaction

Kevin Robinson

DNA is long — unbelievably long — considering that it is stuffed inside cells. In fact, DNA is longer than a meter and, in many cases, approaches 2 m. How cells coil and compact DNA is still something of a mystery, however. Recently, researchers from the German Cancer Research Center in Heidelberg used Förster resonance energy transfer (FRET) to determine how salt concentrations in solutions affect the ability of proteins called histones to compact DNA. This information may have long-term implications in cancer treatment and research.

Inside a living eukaryotic cell, DNA is tightly coiled by the linking of four types of histones. The histones work in pairs, with four pairs making an octamer. A histone octamer wraps 160 to 220 DNA base pairs. The central 145 to 150 base pairs are coiled around the octamer, and the remaining two ends of the DNA dangle free like loose ends of a ball of twine. Together, the octamer DNA unit is called a nucleosome.

The loose ends, or the linker, of the DNA join with the loose ends from other nucleosomes to create the chromatin fiber, which is about 30 nm in length and is itself tightly packed. The central DNA wraps around the histone octamer less than two times, which is not enough to explain how 2 m of DNA are folded into a 30-nm chromatin fiber. The nucleosome chain also must fold upon itself.

“The path of the linker DNA between the core particles determines the structure of the chromatin fiber,” explained Katalin Tóth, a member of Jörg Langowski’s laboratory. She added that several factors determine how the linker DNA twists and turns, including the interaction with the molecular tails of the histone proteins, the level to which the histone proteins are acetylated, and the surrounding ions in the cell. “Our aim is to know more about the linker DNA and [through it to learn] more about the organization of the chromatin fiber,” she explained.

As detailed in the Sept. 12 issue of Biochemistry, the researchers assessed how ionic strength and acetylation affect the compacting of linker DNA by varying each and using FRET to determine compacting. They examined pieces of chromatin called mono- and trinucleosomes, which consist of a single nucleosome or of three bound together, respectively. Because they are not in a complete chromatin fiber, the mono- and the trinucleosomes have free linker DNA ends. However, the trinucleosome structure is likely to be the smallest structure capable of showing how the linker DNA folds, causing the chains of nucleosomes to become a compact chromatin fiber.

“The free ends may represent a situation slightly different from that in the chromatin, but they can be labeled easily with fluorescent dyes and observed,” Tóth said.

The scientists determined that regardless of the acetylation state of the different histones, increasing the ion concentration induced compaction. However, Tóth said that their results show that histone acetylation does not always inhibit compaction. “At least for mono- and trinucleosomes, the acetylation of [histone] H4 has a compacting effect,” she explained. Because the expression of certain genes can be related to DNA compactness, this may explain why acetylating certain histones can lead to hyper- or hypoactivation of some genes.

The group also coupled the FRET study with scanning force microscopy, which provides complementary information on the conformation of the trinucleosome. The mononucleosomes are too small for scanning force microscopy. “The steady-state FRET in bulk solutions measures an average distance on a lot of particles,” Tóth said. The fluorescent dyes limit the measurable distances to between 2 and 12 nm. Likewise, the samples must be carefully prepared to avoid or quantify free DNA — or superfluous histones — that could cause the nucleosomes to clump and throw off the FRET measurements.

Scanning force microscopy images show 608-base-pair DNA (A), the same DNA reconstituted with unmodified octamers in the absence of linker histones (B) and in the presence of linker histone H1 (C), and with acetylated octamers without linker histone (D). Below are magnified areas of DNA in B, C and D. Reprinted with permission of Biochemistry.

For scanning force microscopy to work, the nucleosomes must be adhered to a surface, even when imaging the particles in solution. “We don’t know whether all conformations [of the nucleosomes] adhere with the same probability,” she said. “The adhesion may also cause some different deformation.”

However, despite this and the inherent challenges of the technique, including broken tips and volumes of data that the technique creates, Tóth said that it allows them to see the forms of the particles and their diversity, contrary to the averaged distance value obtained from a FRET measurement.

The researchers assembled the trinucleosomes in solution from free DNA and histone octamers. This allowed them to control DNA length and to label the free ends with rhodamine X or Alexa 488. For FRET studies, they used a fluorescence spectrometer to measure the fluorescence emission spectra, exciting the donor fluorophore at 495 nm and the acceptor at 585 nm. To record the scanning force images, they employed an atomic force microscope from Digital Instruments Inc. equipped with type NP-S20 silicon nitride probes from Veeco Instruments Inc.

From here, Tóth said the group is continuing with similar studies to probe the structural questions of DNA compaction. And it is introducing single-molecule techniques to investigate events at the molecular level.

Although this work does not have immediate practical application, Tóth hopes it will help build a fundamental understanding of the forces that compact DNA. “Several illnesses are related to the compactness of genetic material,” she said. Eventually, this work may lead to the ability to control specific aspects of genetic compacting. Under the theory that acetylation is partly responsible for chromatin compaction, researchers elsewhere are studying the potential of deacetylase inhibitors for cancer treatment. The results of the Heidelberg researchers’ work revealed that acetylation of the H4 histone aids compacting. Tóth said that more study is needed, however, to better relate cancer to specific patterns of histone acetylation.

Understanding how acetylation is related to compactness could help determine the appropriate histones to be targeted, she said. And, eventually, more knowledge about the compaction factors and mechanisms, together with precise and highly gene-selective targeting techniques, could open interesting avenues in epigenetic therapy.

Terms & Conditions Privacy Policy About Us Contact Us
back to top

Facebook Twitter Instagram LinkedIn YouTube RSS
©2016 Photonics Media
x Subscribe to BioPhotonics magazine - FREE!