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Enhancer element
Promoter

Gene

Mediator

Chromatin loop

+ NIPBL

+ Cohesin

Transcription factory

Figure 1 | The Mediatorcohesin complex. Kagey


et al.2 find that the cohesin complex interacts with
the Mediator complex to facilitate gene expression.
Recruitment of Mediator to the enhancer or
to other upstream elements results in the
formation of a chromatin loop that brings together
the enhancer and the promoter of the gene to be
transcribed. Subsequent recruitment of NIPBL
and cohesin could potentially stabilize enhancer
promoter interactions by embracing the base of the
chromatin loop and/or by facilitating attachment
of the various units to transcription factories,
which are organized by several simultaneously
transcribed genes10.

each other in chromatin. Moreover, the authors


show that the Mediatorcohesin complexes
promote and/or stabilize the physical proximity between enhancers and promoters of active
genes only (Fig. 1).
That the Mediatorcohesin complexes
occupy about 60% of all active promoters in ES
cells hints that any tampering with this complex will probably affect most of the pivotal
features of ES cells, including pluripotency2.
Although it remains to be determined, active
promoters that do not interact with Mediator
may still depend on cohesin function
perhaps in combination with transcriptional
co-activators other than Mediator.
The conclusion that a significant part of
cohesins function is to facilitate crosstalk
between enhancers and the basal transcriptional machinery bound to the promoter region
throws new light on several human diseases.
Investigations of disorders with mutations in

members of the Mediator complex such as


OpitzKaveggia and Lujan syndromes, and
schizophrenia8 should now also consider
a NIPBL/cohesin component. Conversely,
studies of Cornelia de Lange syndrome, which
features mutations in NIPBL (ref. 9), should
take into account a possible role for the
Mediator complex.
But although these diseases all involve misregulation of gene expression8,9, they do not
seem to have other comparable features. One
explanation for this could be cell-type specificity of Mediatorcohesin function. Indeed, as
Kagey et al.2 demonstrate, there is an extensive
change in the patterns of cohesin and Mediator co-localization on chromatin between ES
cells and cells derived from them mouse
embryonic fibroblasts. In all likelihood, this
reflects epigenetic reprogramming events
that control the availability of lineage-specific
cis-regulatory elements.
Kagey and co-workers report opens a
Pandoras box, raising more questions than
it answers. Pertinent issues include the need
for a better understanding of how the cohesin
complex can be differentially recruited to the
chromatin regions displaying enhancer and
insulator functions, and how the associated
cohesin functions are executed. Among the possible ways in which this complex might facilitate activation of transcription are stabilization
of an open chromatin conformation6 and/or
the anchoring of the transcriptional units to
transcription factories (Fig. 1) nuclear
compartments that can be visualized when several genes, even from different chromosomes,

gather together in the interchromosomal space


to be transcribed simultaneously10.
Given that cohesin is the sole known common
denominator between both activation and inhibition insulation of transcription, it is not
far-fetched to assume a mechanistic relationship
between these two processes. It is of interest
that, on the evolutionary scale, cohesins role in
activation of transcription may have pre-dated
its involvement in CTCF-mediated insulation
of transcription6. Whether this means that a
CTCFcohesin function acting at long range
has emerged in vertebrates to counteract the
enhancercohesin function, thereby preventing
unscheduled communications between enhancers and promoters from neighbouring expression
domains, is another intriguing puzzle.
Rolf Ohlsson is in the Department of
Microbiology, Tumor and Cell Biology,
Karolinska Institute, SE-171 77 Stockholm,
Sweden.
e-mail: rolf.ohlsson@ki.se
1. Taatjes, D. Trends Biochem. Sci. 35, 315322 (2010).
2. Kagey, M. H. et al. Nature 467, 430435 (2010).
3. Max, T., Sogaard, M. & Svejstrup, J. Q. J. Biol. Chem.
282, 1411314120 (2009).
4. Wood, A. J., Severson, A. F. & Meyer, B. J. Nature Rev.
Genet. 11, 391404 (2010).
5. Carretero, M., Remeseiro, S. & Losada, A. Curr. Opin.
Cell Biol. 22, 17 (2010).
6. Dorsett, D. Chromosome Res. 17, 185200 (2009).
7. Schmidt, D. et al. Genome Res. 20, 578588 (2010).
8. Philibert, R. A. & Madan, A. Pharmacogenomics 8,
909916 (2007).
9. Liu, J. et al. PLoS Biol. 7, e1000119 (2009).
10. Schoenfelder, S., Clay, I. & Fraser, P. Curr. Opin.
Genet. Dev. 20, 127133 (2010).

bI o Lo GI CA L I M AG I N G

Beyond fluorescence
Nanoparticles that generate light through a mechanism known as second harmonic
generation have been used to image live tissue. The particles overcome many
problems associated with fluorescent probes for bioimaging.
bRuCE E. CoHEN

iologists love optical microscopy. Open


any cell-biology journal and you will
be struck by a collection of colourful
microscopy images worthy of a Seurat retrospective, with fluorescent staining patterns
that help to unravel the complexities of cell
signalling and development, neuroscience
and cancer. But while microscopes have grown
steadily more sophisticated over the years, so
much so that single-molecule bioimaging is no
longer uncommon, many of the fluorescent
probes used for microscopy have remained
unchanged for decades or longer. These traditional organic fluorophores can present problems for biologists increasingly demanding

imaging experiments for example, limits


on probe brightness and stability often curb
the types of data that can be acquired.
Recently, a new and unexpected class of probe
has emerged that overcomes many of the shortcomings of traditional fluorophores: inorganic
nanoparticles. These nanoparticles are based
on elements far beyond carbon in the periodic table, and have optical properties entirely
unlike those of their organic predecessors.
Reporting in Proceedings of the National Academy of Sciences, Pantazis et al.1 describe nanoparticles that convert near-infrared (nIR) light
to visible light, a property that may overcome
several persistent problems in bioimaging. Whats
more, the authors illustrate the utility of their
nanoparticles for live-tissue imaging.

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RESEaRch NEWS & VIEWS


When fluorescent molecules
such that even a modest continua
b
c
d
absorb light, they enter an excited
ous-wave laser is sufficient to
state and, after a certain lifetime,
make single particles visible7.
can return to the ground state
But it might be possible to reduce
by emitting lower-energy light
the photon flux required for the
that is observed as fluorescence
activity of SHG-active particles
(Fig. 1a). The energy difference
by changing the surface chembetween absorbed and emitted
istry of BaTiO3 nanoparticles,
light is known as the Stokes shift
or by using other nanoparticle
and ensures, for example, that
shapes or compositions.
fluorescein (the fluorophore
Will inorganic nanoparticles
that is the traditional workhave as large an impact on biohorse of bioimaging) and green
imaging as fluorescent proteins
fluorescent protein (GFP) emit
such as GFP have had? The
green light after excitation with
answer mainly depends on how
blue light. There are, however, Figure 1 | One photon or two? Light-emitting probes for cellular imaging can
readily nanoscientists can adapt
two key drawbacks to using be illuminated using several different mechanisms. a, In Stokes-shifted imaging,
them for easy integration into
Stokes-shifted probes for cel- light excites a probe, which then emits lower-energy light as fluorescence. Here,
cells. Quantum dots lumilular imaging. First, cells have green fluorescent protein (GFP) is excited by blue light and emits green light. b, In
nescent semiconducting nanomany Stokes-shifted fluorescent two-photon imaging, two low-energy photons are delivered to a probe almost
particles that have exceptional
molecules of their own, leading simultaneously. The excited probe then emits higher-energy fluorescence. Here,
stability and brightness have
to unwanted background fluor- GFP1is excited by two near-infrared (nIR) photons, and emits green light. c, Pantazis been used in bioimaging for
escence that can be particularly et al. report that barium titanate nanoparticles scatter nIR light to produce
more than a decade. But their
higher-energy blue light, through a mechanism called second harmonic generation.
problematic in sensitive experi- d, When irradiated sequentially with two nIR photons, lanthanide-doped
use has been hampered in part
ments such as single-molecule nanoparticles produce higher-energy (red) phosphorescence, through a process
by difficulties in attaching them
imaging. Second, the intense known as two-photon upconversion68. Lanthanide atoms in the nanoparticles are
to biological molecules and by
light typically used by micro- shown in red and green.
their large size. Pantazis and colscopes to excite fluorescent
leagues BaTiO3 nanoparticles1
molecules can damage cells, especially light no photobleaching or cytotoxic by-products. are even larger, more than twice the diameter
at the ultraviolet (UV) and blue end of the SHG has been used to image repeating asym- of a ribosome.
visible spectrum. Photodamage may also occur metric cellular structures such as cytoskeletal
Recent advances in the synthesis of quantum
indirectly, if the excited state of a fluorescent proteins, as well as other biological systems to dots9 and upconverting nanoparticles10 have
molecule reacts with a nearby molecule instead which certain SHG-active organic dyes have shrunk them to antibody-sized structures
of emitting fluorescence. Adding insult to been added2,3. Previous work has also shown that should be more palatable for biologists.
injury, the fluorophore is often photobleached that SHG works well when nanoparticles made Similar reductions in the size of SHG-active
in this process, rendering it dark and useless.
of noble metals or metal oxides are used as nanoparticles, as well as improvements in surOne way in which microscopists have probes4,5.
face passivation (which makes the particles
circumvented these problems is by building
Pantazis et al.1 now extend SHG to whole- biocompatible) and in methods for attaching
two-photon microscopes, which use pulsed animal imaging. They started by surveying a them to biological targets, could dramatically
nIR lasers powerful enough to deliver two series of nanocrystals for SHG activity in vitro, expand their reach. Cell biologists may not
photons to a probe nearly simultaneously. and found that 30-nanometre barium titanate completely abandon fluorescence in favour of
The additive energy of the photons excites the (BaTiO3) crystals produced the strongest signal, phosphorescence or light scattering by inorfluorophore and leads to anti-Stokes emis- exhibiting none of the undesirable on/off blink- ganic nanoparticles, but the absence of blinksion, in which light is emitted at higher ener- ing behaviour of comparable fluorescent probes. ing, background noise and photobleaching
gies (shorter wavelengths) than the excitation When the authors injected BaTiO3 nanocrystals may be too alluring for some to resist.
source (Fig. 1b). Not only is the nIR light that into live zebrafish embryos, the resulting SHG
is used to stimulate anti-Stokes emission less images showed minimal background signal and Bruce E. Cohen is at the Molecular Foundry,
damaging to cells than UV or visible light, but little apparent loss of brightness over time, con- Lawrence Berkeley National Laboratory,
it also scatters less and penetrates farther into sistent with a lack of photobleaching or chemi- Berkeley, California 94720, USA.
tissue, making it useful for whole-animal imag- cal breakdown of the probe. This suggests that e-mail: becohen@lbl.gov
ing experiments. A drawback of two-photon these particles could be useful in tracking cell
P., Maloney, J., Wu, D. & Fraser, S. E.
excitation is that it is extremely inefficient, so lineages during embryonic development, as the 1. Pantazis,
Proc. Natl Acad. Sci. USA 107, 1453514540
that ultra-high peak powers of pulsed lasers stable particles are passed from cell to cell. It
(2010).
are essential for achieving the large photon flux will be interesting to see for just how long the 2. Nuriya, M. et al. Proc. Natl Acad. Sci. USA 103,
786790 (2006).
needed to produce observable signals.
particles can be tracked in living systems.
J. S. & Cohen, B. J. Phys. Chem. B 112,
In their work, Pantazis et al.1 use a techLike two-photon imaging, SHG requires 3. Salafsky,
1510315107 (2008).
nique called second harmonic generation high-power pulsed nIR lasers, but another 4. Hsieh, C. L., Pu, Y., Grange, R. & Psaltis, D. Opt.
Express 18, 1191711932 (2010).
(SHG; Fig. 1c) to produce an optical signal new class of anti-Stokes-emitting nanocrystal
for bioimaging. In SHG, two photons interact may obviate this need. These crystals, known 5. Nakayama, Y. et al. Nature 447, 10981101
with certain asymmetric materials such as as phosphorescent upconverting nanoparticles 6. (2007).
Park, Y. I. et al. Adv. Mater. 21, 44674471 (2009).
non-centrosymmetric crystals, which lack (Fig. 1d), also display no observable photo- 7. Wu, S. et al. Proc. Natl Acad. Sci. USA 106,
1091710921 (2009).
a central point of symmetry to produce a bleaching or blinking6,7, and have been imaged
single photon with twice the energy (half the in live mice8. A key distinction is that upcon- 8. Salthouse, C., Hilderbrand, S., Weissleder, R. &
Mahmood, U. Opt. Express 16, 2173121737 (2008).
wavelength) of the incident photons. Because verting nanoparticles are visible with about 9. Allen,
P. M. et al. J. Am. Chem. Soc. 132, 470471
SHG is based on scattering, rather than fluor- 1,000-fold weaker illumination intensity than
(2010).
escence, there is no excited state and therefore is needed for SHG of the BaTiO3 nanoparticles, 10.Wang, F. et al. Nature 463, 10611065 (2010).
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