FRET and scanning force microscopy help unravel DNA compaction mystery
Information could eventually benefit those with illnesses tied to DNA compaction
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
“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
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
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
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
MORE FROM PHOTONICS MEDIA