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HIGHLIGHTS
HIGHLIGHT ADVISORS
WENDY BICKMORE
MRC HUMAN GENETICS UNIT,
UK
SEAN B. CARROLL
UNIVERSITY OF WISCONSIN,
USA
ADAM EYRE-WALKER
UNIVERSITY OF SUSSEX, UK
JANE GITSCHIER
UNIVERSITY OF CALIFORNIA,
SAN FRANCISCO, USA
RALPH J. GREENSPAN
THE NEUROSCIENCES
INSTITUTE, CALIFORNIA, USA
YOSHIHIDE HAYASHIZAKI
RIKEN GENOMIC SCIENCES
CENTER, JAPAN
PETER KOOPMAN
UNIVERSITY OF QUEENSLAND,
AUSTRALIA
LEONID KRUGLYAK
FRED HUTCHINSON CANCER
RESEARCH CENTER, USA
BARBARA MEYER
UNIVERSITY OF CALIFORNIA,
BERKELEY, USA
LEE NISWANDER
SLOAN-KETTERING INSTITUTE,
NEW YORK, USA
CHRISTOS OUZOUNIS
THE EUROPEAN
BIOINFORMATICS INSTITUTE,
UK
NORIYUKI SATOH
KYOTO UNIVERSITY, JAPAN
MARC VIDAL
DANA-FARBER CANCER
INSTITUTE, BOSTON, USA
VIRGINIA WALBOT
STANFORD UNIVERSITY, USA
DETLEF WEIGEL
MAX PLANCK INSTITUTE FOR
DEVELOPMENTAL BIOLOGY,
GERMANY
LEONARD I. ZON
CHILDREN’S HOSPITAL,
BOSTON, USA
T E C H N O LO G Y
Kitting out the rat
A model organism that lacks reliable
genetic modification techniques is
rather like a tool box without a spanner — somewhat lacking in a key
piece of kit. Although the first transgenic rat was reported 12 years ago,
genetically modifying the rat genome
is still far from routine, predominantly because rat embryonic stem
cells have not yet been successfully
derived or cultured. Now, Kent
Hamra and colleagues have sidestepped this problem by generating
transgenic rats from male germline
stem cells. They show that, when
transplanted into sterile rats, these
cells can form functional spermatozoa. Moreover, they can also be easily
transformed with a lentiviral vector
and subsequently used to produce
transgenic offspring.
The authors obtained spermatogonial stem cells (SSCs) from primary cultures of rat spermatogenic
cells — taken from lacZ-expressing
male rats — using a two-step procedure. In step one, cells cultured for
2.5 days were plated onto a collagen
matrix for 4 hours. Germ cells and
somatic cells were then differentiated
from each other by assaying for the
expression of DAZL, a germ-cellspecific marker, and vimentin, a
somatic cell marker. After harvesting
DAZL+ cells — which remained
unattached to the collagen matrix —
Hamra et al. transplanted them into
the testes of sterile male rats. Around
50% of the offspring of these rats
carried the lacZ transgene, which
they transmitted to the following
generation
at Mendelian
ratios. Step
two involved
another enrichment step on a
laminin matrix, which provided the
authors with a population of cells
that were ~97% DAZL+ and could
colonize rat testes with greater efficiency.
These laminin-enriched cells
were next transduced in culture with
a lentiviral vector that carried an
EGFP reporter gene. The transformed cells were transplanted into
three male rats, where they colonized
the seminiferous tubules, as shown
by the expression of EGFP ~200 days
after transplantation. Although only
one of these males proved to be fertile, he sired 44 pups, five of which
received two copies of the transgene,
eight of which received one. The site
of transgene integration was unique
in each line, and seven lines genotyped so far have transmitted the
transgene to ~50% of their progeny.
NATURE REVIEWS | GENETICS
Transgenic animals were also
generated from SSCs cultured for four days, and SSCs
cultured for seven days
retained stem-cell function,
possibly providing a window
of time for the selection of
drug-resistant targeted cells.
Much like a good tool, to be really
useful, genetic modification techniques need to be simple, efficient
and reliable. This approach appears
to meet these requirements — but
we’ll know for sure once it is in general use.
Jane Alfred
References and links
ORIGINAL RESEARCH PAPER Hamra, F. K.
et al. Production of transgenic rats by lentiviral
transduction of male germ-line stem cells. Proc.
Natl Acad. Sci. USA 21 October 2002
(10.1073/pnas222561399)
WEB SITE
David Garbers’ lab:
http://www.swmed.edu/home_pages/
pharmacology/greencenter/garberslab.html
VOLUME 3 | DECEMBER 2002 | 8 9 9
© 2002 Nature Publishing Group
HIGHLIGHTS
Q U A N T I TAT I V E G E N E T I C S
Sizing up developmental timing
Fruits of wild (top) and
domesticated (bottom) tomatoes.
Picture courtesy of Dani Zamir, and
reproduced with permission from
Zamir, D. Nature Rev. Genet. 2,
983–989 © (2001) Macmillan
Magazines Ltd.
Fruit weight and size are agriculturally important traits, but little is
known of their genetic and molecular
bases. Many genetic studies on these
traits have been done in the tomato,
owing to the disparity in size between
the fruits of wild and domesticated
tomatoes (see picture). Quantitative
trait loci (QTL) mapping studies in
this plant have identified nearly 30
tomato QTL that affect fruit weight
and size.
One such QTL is fw2.2, which
accounts for ~30% of the difference
in fruit weight between wild and
domesticated tomatoes. Earlier studies have strongly indicated that
altered gene regulation underlies the
effects of the large- and small-fruit
B E H AV I O U R A L G E N E T I C S
Worms gang up
on bacteria
The nematode worm Caenorhabditis elegans
can be shy or gregarious when feeding time
arrives. New work uncovers some of the
neurons and genes that are involved in
regulating social feeding in the worm, and
points towards multiple systems of
antagonistic signalling that control whether,
and when, the worms aggregate into feeding
groups.
The standard laboratory strain of C. elegans
is a loner, preferring solitary feeding. But
worms with a valine-to-phenylalanine
mutation at residue 215 of NPR-1 — a
putative G-protein-coupled receptor — form
aggregates when they encounter bacteria
(their food source). Worms with a deletion at
the npr-1 locus also aggregate, suggesting that
a valine-containing receptor (NPR-1 215V)
normally suppresses aggregation. In two
studies, de Bono and colleagues take
advantage of the excellent worm genetics and
its small nervous system to delve deeper into
the control of social feeding.
The first study investigated how and where
NPR-1 acts. The authors constructed a
transgene that expressed GFP-tagged NPR-1
900
alleles of fw2.2 on fruit weight. It has
long been believed that such mutations, especially when they affect the
timing of development (‘heterochronic’ mutations), might be a
natural force of evolutionary change
in plants. In a detailed study of fw2.2,
Steven Tanksley’s group now provide
the first experimental evidence to
support this theory.
Because previous studies in
plants and Drosophila have shown
that both cell division and expansion
are essential factors that determine
organ and fruit size, Cong et al.
analysed cell size and mitotic index
(MI) in two nearly isogenic tomato
lines in which either a large- or
small-fruit fw2.2 allele was present.
215V from the npr-1 promoter. When
expressed in worms with an npr-1
deletion, this transgene suppressed
aggregation. By using different promoters to
drive transgene expression in subsets of
neurons, the authors showed that expression
in just three sensory neurons — AQR, PQR
and URX — was sufficient to suppress
aggregation.
These three neurons are exposed to the fluid
in the body cavity. Their ability to mediate
social feeding seems to depend on signalling
through a cyclic GMP-gated ion channel, as
neuron-specific mutations in tax-2 or tax-4,
which encode the subunits of the channel,
also suppressed aggregation. So, it seems
that NPR-1 suppresses aggregation by
antagonizing signalling through TAX-2
and TAX-4 in these sensory neurons.
The second study investigated how external
stimuli might elicit aggregation. A screen for
mutations that suppress aggregation in npr-1deleted animals identified four genes. Two of
these, osm-9 and ocr-2, are thought to encode
subunits of a TRP-related cation channel in
C. elegans chemosensory neurons, and are
required for avoidance of various noxious
stimuli. The other two genes, odr-4 and
odr-8, are required to localize a subset of
chemosensory receptors to sensory cilia.
Analysis of GFP transgene expression in
ocr-2; odr-4 double mutants showed that
their expression is required in specific
nociceptive neurons — those that respond
| DECEMBER 2002 | VOLUME 3
Differences in MI were found
between the fruits of these two lines,
but not in cell size. In the small-fruit
fw2.2 line (TA1144), a rapid but
brief rise in MI occurs immediately
after fertilization. By contrast, a
more gradual and sustained rise in
MI occurs in the large-fruit allele
line (TA1143), indicating that an
extended period of cell division
might underlie larger fruit size in
this line. Next, the authors found
that the fw2.2 alleles differ in the
timing of their peak expression by
around one week. This difference in
expression timing inversely correlated with changes in mitotic activity
during early fruit development, indicating that fw2.2 might negatively
regulate cell division. Moreover, by
~12 days post-fertilization, fw2.2
levels in TA1144 were more than
double those in TA1143. However,
only subtle differences in expression
patterns were evident between the
two lines.
to noxious stimuli — to rescue social feeding.
Laser ablation of these neurons abolished
social feeding, confirming the genetic data.
Another piece of the puzzle came from
studies of osm-3 mutants. OSM-3, a kinesin,
is required for proper formation of sensory
cilia on sensory neurons. Although
removing osm-3 function interferes with
the development of the crucial chemosensory
neurons, it doesn’t suppress social feeding.
The authors propose that, as well as blocking
the ability of these neurons to promote social
GFP expression in the body cavity neurons of C. elegans.
Image courtesy of S. Reichett, MRC Laboratory of Molecular
Biology, Cambridge, UK.
www.nature.com/reviews/genetics
© 2002 Nature Publishing Group
HIGHLIGHTS
These results show that the differences in transcript levels between the
small- and large-fruit alleles of fw2.2
are both quantitative (with the smallfruit allele being more abundantly
expressed) and qualitative (as evident
from the difference in their expression timing). Importantly, these findings provide empirical evidence that
heterochronic regulatory changes in
gene expression can bring about phenotypic, and probably evolutionary,
change in plants. But how fw2.2 actually modulates cell division remains
unknown.
Jane Alfred
References and links
ORIGINAL RESEARCH PAPER Cong, B. et al.
Natural alleles at a tomato fruit size quantitative
trait locus differ by heterochronic regulatory
mutations. Proc. Natl Acad. Sci. USA 99,
13606–13611 (2002)
FURTHER READING Dekkers, J. C. M. &
Hospital, F. The use of molecular genetics in the
improvement of agricultural populations. Nature
Rev. Genet. 3, 22–32 (2002)
WEB SITE
Steven Tanksley’s laboratory: http://www.plbr.
cornell.edu/PBBweb/Tanksley.html
feeding, lack of OSM-3 blocks
antagonistic signals that normally
inhibit this behaviour. Indeed,
removing osm-3 function restores
social feeding in odr-4 or ocr-2
mutants. So, as with the body cavity
neurons, nociceptive neurons might
be involved in a system of
antagonism between signals that
promote and suppress aggregation.
As these neurons are required for
responses to stressful or aversive
stimuli, de Bono et al. propose that
aggregation is a response to an
aversive stimulus that is produced by
bacteria. But what the aversive
stimulus that promotes aggregation
is and how the different control
systems interact to regulate when
social feeding occurs remains
unknown.
Rachel Jones, Senior Editor,
Nature Reviews Neuroscience
References and links
ORIGINAL RESEARCH PAPERS de Bono, M.
et al. Social feeding in Caenorhabditis elegans is
induced by neurons that detect aversive stimuli.
Nature 419, 899–903 (2002) | Coates, J. C. &
de Bono, M. Antagonistic pathways in neurons
exposed to body fluid regulate social feeding in
Caenorhabditis elegans. Nature 419, 925–929
(2002)
FURTHER READING Rankin, C. H. From
gene to identified neuron to behaviour in
Caenorhabditis elegans. Nature Rev. Genet. 3,
622–630 (2002)
FUNCTIONAL GENOMICS
The importance
of networking
Autoregulation
Multicomponent
loop
TF1
TP
TP
TF2
Feedforward
loop
TF1
TP
TF1
TF2
TF1
promoter
TP
For many, defining a single pathway was once
the ultimate goal. No longer satisfied with
understanding individual pathways, researchers
now seek to understand how they interact with
each other to bring about changes in living
organisms. Lee et al. now present their tour de
force approach to mapping transcriptional
regulatory networks in the budding yeast. By
using genome-wide location analysis (GWLA),
they define regulatory motifs, which when
combined with global gene expression data
allow them to construct a complete regulatory
network.
Driven by the desire to know how gene
expression is regulated on a global scale, the
authors reasoned that they would ultimately
need to understand how transcription is
regulated. To this end, they used GWLA — a
method they previously developed and that
allows them to find out which transcription
factors (TFs) bind to which promoters. GWLA
involves crosslinking TFs that are bound to
their target promoters (TPs), recovering the
DNA and identifying the TPs by using genomic
DNA as a reference. The analysis was done
under three growth conditions for 106 out of
141 TFs that could be found in the Yeast
Proteome Database.
Lee et al. found that many yeast promoters
were bound by more than two TFs — a feature
that had been thought to be limited to higher
eukaryotes. The 4,000 or so interactions fell
into six basic regulatory motifs, which the
authors consider to be building blocks of larger
regulatory networks. They classify these
networks as autoregulation, multicomponent
loops, feedforward loops, single-input motifs,
multi-input motifs and regulator chains (see
figure). For example, autoregulation is thought
to be important in quick responses to the
changing environment, and therefore it is
associated with a selective growth advantage.
The authors show that 10% of yeast TFs
autoregulate; by contrast, in prokaryotes, this
figure is thought to be between 52% and 74%.
The structure of the feedforward loop suggests
that it might be important in response to a
sustained rather than a transient signal. It
might also provide a way for temporal control.
The authors wondered whether they could
use these building blocks to construct a network
of interactions. They decided to build a network
for regulators involved in the yeast cell cycle
Single-input motif
Multi-input motif
TF1
TP
TP
TP
TF1
TF2
TF3
TP
TP
TP
Regulator chain
TP
TP
TF1
TF2
TP
TF3
TP
Modified with permission from Lee, T. I. et al. Science 298, 799–804
© (2002) American Association for the Advancement of Science.
because the large amount of information
available for this process would make their
theoretical model easily testable. To construct
their network, the authors used an algorithm
that combines the GWLA data with gene
expression data. As core regulators that share
the same spatial and temporal expression
patterns were defined, more regulators with
the same expression pattern were added, and
so the network grew.
Astonishingly, the algorithm — which
was automated and required no previous
knowledge of biology — assigned all the
regulators to the correct cell-cycle stages.
Moreover, those regulators that had been
poorly characterized were now placed in a
particular position of the network, which
can now be tested experimentally.
All of the interactions are testable, and the
approach is applicable to any organism for
which good genomic and expression data are
available. One important observation that
emerges from this work is that the control of
cellular processes involves transcriptional
regulation of other regulators. This has
important implications for mutation analysis
— if expression profiling is used to characterize
a mutation, it is as likely to reveal direct targets
of a mutated regulator as it is to reveal the
effects of network disruption.
Magdalena Skipper
References and links
ORIGINAL RESEARCH PAPER Lee, T. I. et al. Transcriptional
regulatory networks in Saccharomyces cerevisiae. Science 298,
799–804 (2002)
WEB SITE
Rick Young’s lab: http://web.wi.mit.edu/young
NATURE REVIEWS | GENETICS
VOLUME 3 | DECEMBER 2002 | 9 0 1
© 2002 Nature Publishing Group
HIGHLIGHTS
ETHICS WATCH
GENE MAPPING
Public perceptions and regulatory policy
There are many reasons why the field of biotechnology is
particularly difficult to regulate. It is complex, the relevant science
moves forward quickly, and the risks and benefits that are
associated with it are not always easy to identify or agree on.
However, I believe that the diverse and changing nature of public
perceptions stands as the single greatest regulatory challenge in
this area.
The international debate over “therapeutic cloning” is a good
example of the dilemma. During the past few years, governments
throughout the world have been struggling with how best to
regulate both reproductive and therapeutic cloning. Although
the public clearly endorses a ban on reproductive cloning, the
available opinion data on therapeutic cloning paints a more
complex picture. Most research on public opinion has found
strong support for stem-cell research and, even, a degree of
support for the concept of therapeutic cloning1. However, for
some citizens — about 20% in Canada — no amount of potential
social or scientific benefit will justify this type of research.
As such, policy makers are left without a clear public mandate.
Recently, the US President’s Council on Bioethics explicitly noted
this lack of consensus, and therefore concluded that a ban on all
forms of human cloning was not justified and that a moratorium
should be imposed to give time “to seek moral consensus”2.
(I suspect that this “moral consensus” will remain elusive.)
Public opinion will also change. And, rightly or not, history tells
us that this change is likely to be in the direction of increased
public support (or, at least, increased ambivalence). In vitro
fertilization, sperm donation, the transplantation of human
organs and research involving cadavers were all activities that
were first met with a degree of public resistance.
We should not make laws solely on the basis of opinion polls —
a methodology with inherent limitations. However, we must also
accept that, for many areas of biotechnology, it will be difficult to
justify regulatory policy on broad consensus alone. I believe that
the best way to deal with this inevitable state of affairs is to avoid
the use of rigid statutory prohibitions and, instead, to establish
regulatory bodies with the power to oversee particular areas of
biotechnology 3. The regulatory body should be interdisciplinary,
have the necessary expertise and a public engagement and
education mandate, and be appropriately accountable. Whereas
statutory bans are often difficult to enact or change, a regulatory
approach can accommodate emerging science and new social
concerns. And because a regulatory
body can serve as a forum for
continuing public debate, it can
remain sensitive to the public’s
moral ambiguity concerning much
of biotechnology.
Timothy Caulfield
REFERENCES
1
Timothy Caulfield is at the Health Law
Institute at the University of Alberta,
Canada.
902
Scoffield, H. Canadians favour limited use of clones
for emergencies only, survey finds. Globe and Mail
A2, 16 June (2000) | 2 The President’s Council on
Bioethics. Human Cloning and Human Dignity: An
Ethical Inquiry (Washington, DC, July 2002) [online]
<http://www.bioethics.gov/cloningreport/
fullreport.html> | 3Knowles, L. Science policy and the
law. Reproductive and therapeutic cloning. NYU J.
Legis. Public Policy 4, 13 (2001)
More than just a
pretty face
For nearly 20 years, some sheep
have flaunted what must be every
vain person’s dream: a genetic
variant that confers beautiful
buttocks, commonly known as the
callipyge phenotype. More
prosaically, the single-locus
mutation responsible for the
phenotype causes muscle
hypertrophy, and it does so
through an unusual genetic means,
known as polar overdominance.
This means that animals only
manifest the phenotype if they are
heterozygous for the callipygous
variant and only if the variant has
been inherited from a particular
parent (in this case, the father).
Freking and colleagues have now
pinpointed the single base change
in the gene, CLPG, that underlies
the callipygous trait. Breeders and
geneticists alike have a stake in this
discovery: leaner meat could be
bred as a result, and we could gain
a better understanding of the
epigenetic mechanisms that
underlie the inheritance of the
phenotype.
Previous efforts to map CLPG
had localized it genetically to a
small (400-kb) telomeric region on
chromosome 18 — small enough
to make a direct-sequencing
approach to finding the variant a
realistic goal. The high level of
background polymorphism in
sheep, however, made it impossible
to detect the causative SNP simply
by comparing affected
heterozygotes to normal
homozygotes. The authors
therefore turned to the pedigree of
the particular flock they were
studying for some help. One
callipygous ram in particular was
key: the critical region of both
copies of chromosome 18 in this
ram were probably identical-bydescent, apart from the presence of
the CLPG mutation on one copy.
Comparing the sequence of the
ram to a panel of informative
genotypes uncovered 616
| DECEMBER 2002 | VOLUME 3
polymorphisms, but only one of
them — an A to G change — could
be uniquely assigned to the
callipygous trait. The G allele was
never found in sheep of diverse
breeds, so validating further the
pedigree-screening approach as the
most robust there is for finding the
causative variant of a phenotype.
It’s taken ten years, but an
important aspect of the callipyge
phenotype has now been found,
heralding the starting point for
understanding what the CLPG
variant does. The CLPG region is
conserved in cattle, human and
mouse genomes, and the variant
might be incorporated into an
RNA transcript, but little else is
known about its function. Initial
attempts to detect whether the
variant has some regulatory effect
— for example, by altering the
imprinting status of the region —
have been unsuccessful. Clearly
more work is needed to get to the
bottom of this trait.
Tanita Casci
References and links
ORIGINAL RESEARCH PAPER Freking, B. A.
et al. Identification of the single base change
causing the callipyge muscle hypertrophy
phenotype, the only known example of polar
overdominance in mammals. Genome Res. 12,
1496–1506 (2002)
www.nature.com/reviews/genetics
© 2002 Nature Publishing Group
HIGHLIGHTS
IN BRIEF
CANCER GENETICS
Women only
Women-only nights can be fun, but
other events that occur exclusively to
women are not so great. Why, for
example, should only women who
inherit a mutation in the BRCA1
tumour suppressor be prone to breast
cancer? Shridar Ganesan et al. have
shown, in the November 1 issue of Cell,
that BRCA1 might be involved in X
inactivation. Perhaps this could explain
this female-specific phenomenon.
BRCA1 was known to localize to
the unpaired X chromosome in
pachytene spermatocytes, and
Ganesan et al. confirmed this by
showing that BRCA1 colocalized
with a component of the XY body.
The XY body shows similarities to
the inactive X (Xi) chromosome in
female somatic cells — both are heterochromatic, silenced and are coated
with the non-coding XIST RNA. So,
does Xi also localize BRCA1?
Immunofluorescence of BRCA1
and fluorescent in situ hybridization
(FISH) of XIST, carried out on
female human cell lines, revealed that
BRCA1 and XIST could colocalize
to a nuclear structure — FISH of an
X-chromosome probe confirmed
that this was one of the X chromosomes. Chromatin immunoprecipitation analysis — using antibodies
to BRCA1 or its binding partner
BARD1 — followed by reversetranscriptase PCR (RT-PCR) of XIST
confirmed this interaction.
The next step was to investigate
what happened in BRCA1-deficient
cells. Frozen sections from sporadic
breast and ovarian cancers were examined, and although the majority had
nuclear BRCA1 and focal XIST (as
opposed to diffuse) staining, those
from BRCA1-deficient women did not.
The HCC1937 human breast cancer
cell line — which contains a germline
mutation in one BRCA1 allele and has
lost the wild-type allele — also lacked
focal XIST staining. This could be
restored by ectopic expression of
wild-type BRCA1, but not of cancerassociated BRCA1 mutants. Similarly,
RNAi of BRCA1 in wild-type cells
decreased focal staining of XIST.
FUNCTIONAL GENOMICS
So how does BRCA1 regulate XIST
— through its localization, synthesis
or stability? The authors carried out
RT-PCR of XIST in HCC1937 cells
that were transfected with either a vector control or with wild-type BRCA1,
to distinguish between these alternatives. The levels of XIST RNA were
equivalent in both transfected lines,
indicating that BRCA1 can influence
XIST localization, but not its synthesis
or stability.
As XIST is required for X-chromosome inactivation, Ganesan et al.
investigated whether loss of BRCA1
influences the pattern of histone H3
methylation on lysine 9 (H3mK9),
which is associated with transcriptional silencing. In female cells, there is
a large amount of anti-H3mK9 antibody staining on Xi, but this is absent
from HCC1937 cells. Similarly,
H3mK9 immunofluorescence analysis
on frozen sections of sporadic and
BRCA1-deficient breast cancers indicates that BRCA1 is required for focal
staining of H3mK9, and hence gene
silencing.
But is loss of BRCA1 expression
sufficient to reactivate previously
silenced genes? This was tested in a
female mouse cell line in which one X
chromosome carried a non-functional
copy of Xist and the other, inactivated,
X chromosome carried a silenced copy
that was tagged with GFP. RNAi of
Brca1 resulted in the reactivation of
Xist–GFP in a subset of these cells.
The loss of BRCA1 in women
might therefore reactivate genes
that are normally silent on the Xi.
The upregulation of a set of Xchromosomal genes in BRCA1-deficient ovarian cancers lends support
to the importance of this phenomenon in promoting tumorigenesis,
but the establishment of a firm link
remains a future goal.
Emma Greenwood, Senior Editor,
Nature Reviews Cancer
References and links
ORIGINAL RESEARCH PAPER Ganesan, S. et al.
BRCA1 supports XIST RNA concentration on the
inactive X chromosome. Cell 111, 393–405 (2002)
WEB SITE
David Livingston’s lab: http://www.hms.harvard.
edu/dms/bbs/fac/livingston.html
Genome-wide DNA replication profile for Drosophila
melanogaster: a link between transcription and
replication timing.
Schübeler, D. et al. Nature Genet. 32, 438–442 (2002)
It has been speculated that a link exists between the time at
which a genomic region is replicated in S phase and its
transcriptional activity, predominantly because early replication
might allow DNA to be packaged into an ‘open’ chromatin
conformation. A microarray-based approach for associating
replication timing with gene expression found just such a
correlation in Drosophila. This property might distinguish
metazoans from lower eukaryotes, such as budding yeast, in
which no correlation was found.
D E V E LO P M E N TA L B I O LO G Y
Different regulation of T-box genes Tbx4 and Tbx5
during limb development and limb regulation.
Khan, P. et al. Dev. Biol. 250, 383–392 (2002)
The limb identity gene Tbx5 promotes limb initiation by
interacting with Wnt2b and Fgf10.
Ng, J. K. et al. Development 129, 5161–5170 (2002)
T-box genes Tbx4 and Tbx5 have been implicated in hindlimb
and forelimb development, respectively. Khan et al. cloned Tbx4
and Tbx5 cDNAs from newt and showed that, unlike in higher
vertebrates, both genes are expressed in the fore- and the
hindlimbs. Interestingly, the authors found that, during limb
regeneration, their expression is reminiscent of higher
vertebrates’ — Tbx5 is preferentially upregulated in the
forelimbs and Tbx4 in the hindlimb — indicating that
regeneration is not simply a reiteration of development. An
additional role for Tbx5 is revealed by Ng et al., who used gainof-function experiments in chick and zebrafish mutants and
morpholino-based knock-down to show that Tbx5, together
with Wnt2a, is also necessary and sufficient for limb outgrowth.
The authors also show that Tbx5 lies downstream of WNT and
that it acts in a feedback loop with Fgf10.
T E C H N O LO G Y
Production of maternal-zygotic mutant zebrafish by
germ-line replacement
Ciruna, B. et al. Proc. Natl Acad. Sci. USA 27 October 2002
(10.1073/pnas.222459999)
In zebrafish, and other organisms, maternally contributed RNAs
and proteins can mask the effects of zygotic mutations. To
overcome this, Ciruna et al. generated sterile fish by ablating
primordial germ cells (PGCs) using morpholino oligos against
the PGC transcript, dead end. PGCs from homozygous mutant
donors were then transplanted into the sterile fish, where they
repopulated the gonad and gave rise to maternal-zygotic mutants.
This technique could be used to create large clutches of purely
mutant embryos.
NATURE REVIEWS | GENETICS
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HIGHLIGHTS
WEB WATCH
Free associations
• The Genetic Association
Database:
http://www.grc.nia.nih.gov/
branches/rrb/dna/
association
Association studies have
finally found a home of their
own. The steadily increasing
number of these studies —
which report a link between
one or more genetic
polymorphisms and specific
human disorders — is filling
the literature databases. So,
researchers have for some
time needed a single,
comprehensive repository of
all the results that have been
published so far. Such a
resource has now become
available thanks to efforts
headed by Kevin Becker at
the National Institute on
Aging/NIH, who has compiled
a freely accessible web site —
the Genetic Association
Database (GAD) — that
catalogues the results of over
700 association studies. The
site is still under development
and will grow both in content
and utility. Nevertheless, its
features are clear to see. The
studies can be queried
according to disease and
phenotypic information, as
well as according to various
aspects of a study’s design,
such as study sample size or
the statistical significance of
the association. Links are
available, through PubMed, to
the original publication, and
comments that could be
potentially useful to other
investigators can also be
posted for each study.
Importantly, the database also
includes studies in which no
genetic association was
found.
With 75–100 association
studies published each week,
GAD should attract a lot of
traffic. Anyone can submit the
results of an association
study to the site, provided
that permission is obtained
from the curator. The future of
the database — the only one
of its kind — could be made
even rosier if Becker realizes
his ambition to enrich the site
by integrating it with geneexpression and NCBI
databases.
D E V E LO P M E N TA L B I O LO G Y
Cutting out a pattern
Protein degradation is a key
regulator of the cell cycle,
inflammation and
transcr iption — but
little is
known
about its role in development. Zhu
and Kirschner now report in
Developmental Cell the identification
of Xom — a developmental gene that
is regulated by proteolysis.
In a screen, in Xenopus, to identify
gene products that are differentially
degraded before and after the onset
of midblastula transition, the authors
found two proteins that matched
these criteria — an unknown protein
and Xom, a homeobox transcription
fac tor.
Xom is
stable
during
early
gastrulation but is subsequently
degraded through the
ubiquitin–proteasome
pathway.
Xom has two socalled PEST domains
(proline, aspartate and glutamate,
HUMAN DISEASE
A clear suspect
Approximately 0.05% of the
Western population suffers from
systemic lupus erythematosus
(SLE) — a complex autoimmune
disease. Although several
susceptibility loci for SLE have
been identified, the nature of the
genes and mutations that underlie
this disease have remained
unknown. Now, Prokunina et al.
report in Nature Genetics the
association of the programmed
cell death gene 1 (PDCD1) with
SLE. Importantly, they also
propose how a particular
sequence variant of PDCD1 might
contribute to the disease’s
aetiology.
In a previous study of a Nordic
population, the authors identified
a susceptibility locus for SLE on
chromosome 2. One gene in this
region stood out as a potential
candidate, PDCD1. This is because
PDCD1 encodes an
immunoreceptor that belongs to
the immunoglobulin family and
that is known to regulate
peripheral self-tolerance in T and
B cells. Moreover, Pdcd1−/− mice
suffer from SLE-like symptoms.
By sequencing PDCD1 in five
healthy unrelated individuals and
in five SLE sufferers from the
Nordic population, the authors
discovered seven SNPs in this
gene, three of which constituted a
disease-associated haplotype that
could account for all of the LOD
score seen in the original
serine, or threonine-rich regions),
one of which turned out to be
required for Xom degradation.
Interestingly, this so-called ‘Xom
destruction motif ’ (XDM) resembles the glycogen synthase kinase 3
(GSK3) consensus phosphorylation site, which is conserved in
the known substrates of GSK3dependent proteolysis, such as
β-catenin.
In the XDM, the authors found
two phosphorylation sites at Ser140
and Ser144. In an in vitro assay, an
exogenous peptide of the phosphorylated XDM blocked Xom degradation, paradoxically so did the
unphosphorylated peptide. However,
phosphorylation of XDM seems to be
important for Xom degradation
because the same peptide, when it
contains serine-to-alanine mutations
at positions 140 and 144, cannot
block Xom degradation in vitro,
implying that the embryonic extract
used in these assays contained a
kinase activity, although this turned
out not to be GSK3.
Using in vitro binding assays,
Zhu and Kirschner next found
that the E3 ubiquitin ligase Skp1–
Cullin–F-box complex (SCF),
population sample. These SNPs
were then genotyped in five sets of
families with different ethnic
origins. The results were clear —
only one SNP, which lies in an
enhancer-like region in intron 4 of
PDCD1, consistently associated
with SLE. This region of intron 4
contains binding sites for
transcription factors that are
known to be involved in
haematopoietic differentiation
and in inflammation. In
particular, the SNP disrupts
a putative binding site for
RUNX1, which is inactivated in
translocations that lead to acute
myeloid leukaemia. Using an
electrophoretic mobility shift
assay, the authors confirmed that
RUNX1 indeed binds to this
sequence and that this binding
is abolished by the sequence
change that is associated with
the SNP.
The authors propose that
RUNX1 binding to the wild-type
Tanita Casci
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| DECEMBER 2002 | VOLUME 3
www.nature.com/reviews/genetics
© 2002 Nature Publishing Group
HIGHLIGHTS
which contains the F-box protein
β-TRCP, is responsible for Xom
degradation. And they were able to
show that a dominant-negative
form of β-TRCP could block Xom
degradation in vivo. Intriguingly,
the same E3 ubiquitin ligase complex also mediates the phosphorylation-dependent degradation of
β-catenin.
So, what is the role of Xom
degradation in early Xenopus development? BMP4 is a ventral morphogen that acts together with Xom
in an auto-activating feedback loop
in which Xom is activated by BMP
and vice versa. In addition, Xom
inhibits transcriptional activity of
dorsal-specific genes that, in turn,
inhibit BMP activity. Zhu and
Kirschner hypothesize that the proteolysis of Xom might be needed to
cease Xom-mediated repression of
dorsal-specific genes, such as
goosecoid, during early gastrulation.
If true, the auto-activating circuit
would be eliminated, leading to
dorsoventral asymmetry in the
mesoderm and to the loss of BMP
expression on the dorsal side and
high expression on the ventral side
of the embryo.
PDCD1 modulates its
transcription and ensures its
correct expression. Because
PDCD1 contains an
immunoreceptor tyrosinebased inhibitory motif, it
might be involved in
preserving self-tolerance by
inhibiting auto-reactive
cells. It remains to be
confirmed whether, without
RUNX1 binding, PDCD1
dysregulation leads to loss of
self-tolerance and to the
chronic lymphocyte
hyperactivity that is
characteristic of SLE.
Magdalena Skipper
References and links
ORIGINAL RESEARCH PAPER
Prokunina, L. et al. A regulatory
polymorphism in PDCD1 is
associated with susceptibility
to systemic lupus
erythematosus in
humans. Nature
Genet. 28 October
2002
(10.1038/ng1020)
Indeed, luciferase reporter assays
showed that non-degradable Xom
is ~20 times better at inhibiting transcriptional activation of goosecoid
than wild-type Xom. Consistent
with this result, embryos with nondegradable Xom have truncated
heads — which is typical of an
enhanced ventralized phenotype.
This effect is restricted to the dorsal
side, not surprisingly, as Xom’s
effects are restricted to that part of
the embryo.
On the basis of these findings, Zhu
and Kirschner propose that the correct dorsoventral BMP expression
pattern in the mesoderm during gastrulation in the frog depends on the
specifically timed proteolysis of Xom.
But how Xom is stabilized during
early gastrulation and what turns on
its subsequent degradation remain
unknown.
Arianne Heinrichs, Senior Editor,
Nature Reviews Molecular Cell Biology
References and links
ORIGINAL RESEARCH PAPER Zhu, Z. &
Kirschner, M. Regulated proteolysis of Xom
mediates dorsoventral pattern formation during
early Xenopus development. Dev. Cell 3, 557–568
(2002)
WEB SITE
Marc Kirschner’s laboratory:
http://cellbio.med.harvard.edu/faculty/kirschner
IN BRIEF
EVOLUT ION
Variation in gene expression within and among natural
populations.
Oleksiak, M. F. et al. Nature Genet. 32, 261–266 (2002)
By assaying genome-wide gene expression levels in three fish
populations, the authors confirm what neutral theory proposes:
that much of the significant variation in gene expression levels
between populations is probably due to random genetic drift and
reflects within-population variation. But, the authors also found
differences in gene expression levels that were environmentally,
rather than genetically, determined, supporting the theory that
important evolutionary adaptations proceed through variation in
gene expression rather than coding sequence changes.
IMPRINTING
Regional loss of imprinting and growth deficiency in
mice with a targeted deletion of KvDMR1.
Fitzpatrick, G. V. et al. Nature Genet. 32, 426–431 (2002)
Beckwith–Wiedemann syndrome (BWS) predisposes to excessive
growth, and is characterized by loss of maternal-specific imprinting
in a putative imprinting control region, KvDMR1. Fitzpatrick et al.
report that paternal inheritance of a Kvdmr1 deletion reduces
mouse growth and causes the de-repression of six genes in cis of
Kvdmr1, including the cyclin-dependent kinase inhibitor Cdkn1c.
These findings indicate that methylation loss in BWS patients
activates KvDMR1-mediated repression on the maternal
chromosome to cause abnormal CDKN1C silencing.
G E N E R E G U L AT I O N
Thiamine derivatives bind messenger RNAs directly to
regulate bacterial gene expression.
Winkler, W. et al. Nature 419, 952–956 (2002)
Many different post-transcriptional mechanisms for gene
regulation exist. Here, Winkler et al. show that mRNA can also block
its own translation. The authors show that mRNAs that encode
Escherichia coli’s enzymes of vitamin B1 biosynthesis can bind
vitamin B1 or its derivatives in the absence of a protein cofactor.
This complex binds to other mRNAs of the same species and, by
sequestering a ribosome-binding site, prevents their translation.
S E X D E T E R M I N AT I O N
Exploring the envelope: systematic alteration in the
sex-determination system of the nematode
Caenorhabditis elegans.
Hodgkin, J. Genetics 162, 767–780 (2002)
Sex is almost ubiquitous in nature and is determined in various
ways — for example, by chromosomal or maternal cues —
indicating that its regulation might undergo rapid evolutionary
change. Hodgkin has confirmed this hypothesis: by using the
detailed knowledge of the sex-determination system in C. elegans,
he has created a collection of stable worm strains that artificially
mimic many of the sex-determination systems found in nature.
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