Molecular Cell
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Resetting for the Next Generation
Michael Cowley1 and Rebecca J. Oakey1,*1Department of Medical and Molecular Genetics, King’s College London, Eighth Floor Tower Wing, Guy’s Hospital, London SE1 9RT, UK*Correspondence: [email protected]://dx.doi.org/10.1016/j.molcel.2012.12.007
In this issue of Molecular Cell, Seisenberger et al. (2012) refine DNA methylation mapping to interrogate theepigenetic reprogramming of primordial germ cells, defining the timings of methylation loss, linking to plurip-otency, and identifying potential routes to transgenerational epigenetic inheritance.
The totipotent zygote gives rise to all of
the differentiated cell types of the adult
by embarking on a program of cell division
and specification that progressively limits
cellular potential. Epigenetic marks, in-
cluding DNA methylation, are critical for
mediating this program. At fertilization,
the genome undergoes global methy-
lation erasure, creating a blank canvas
onto which epigenetic profiles can be
painted as somatic cell lineages are
specified. However, one fundamental
question is this: what happens to the
cells that will form the germline of the
developing embryo? Primordial germ
cells (PGCs) must achieve an epigenetic
state distinct from somatic cells, retaining
the plasticity necessary for producing
gametes and ultimately a totipotent
zygote in the next generation (Hackett
et al., 2012). Since PGCs are derived
from cells already primed for a somatic
fate, they must be reprogrammed by
another wave of demethylation. This is
crucial for ensuring that epigenetic infor-
mation is reset in each generation. In this
issue of Molecular Cell, Seisenberger
et al. (2012) chart the dynamics of DNA
methylation during PGC development,
revealing insights into reprogramming
mechanisms and demonstrating that
some sequences can be resistant to
demethylation, providing the strongest
candidates yet for carriers of trans-
generational epigenetic inheritance in
mammals.
Murine PGCs are induced in the poste-
rior epiblast by external signals around
e6.5. From e9.5, the cells migrate to the
genital ridge, and sex-specific develop-
ment in the gonads occurs from e12.5.
Seisenberger et al. (2012) optimized
bisulfite conversion coupled to next-
generation sequencing (BS-Seq) for small
input quantities, enabling assessment at
base pair resolution of DNA methylation
in e6.5 epiblasts (selected to represent
the ground state prior to PGC specifi-
cation) plus PGCs isolated from later
developmental stages. The first striking
finding is the dramatically reduced global
levels of DNA methylation in e9.5 PGCs
comparedwith thee6.5 epiblast, revealing
that most erasure occurs within this
developmental window (Figure 1). Some
specific sequences reproducibly escape
this initial loss of methylation but become
demethylated after e10.5. These ‘‘late
demethylaters’’ include the CpG islands
associated with differentially methylated
regions (DMRs) of imprinted loci. It is
noteworthy that DMRs escape methy-
lation erasure altogether during the post-
fertilization demethylation wave (Figure 1),
and the authors suggest that their
delayed demethylation in PGCs might
arise from the persistence of this protec-
tive mechanism, involving binding of the
zinc finger protein Zfp57 (Li et al., 2008).
However, the dynamics of Zfp57 activity,
and how these might be coupled to re-
programming in PGCs, remain unclear.
A second key observation is that while
reprogramming is linked to transient
activation of the pluripotency gene net-
work, global demethylation is not coupled
to promiscuous transcriptional activity.
This implies that other mechanisms,
likely histone modifications, play the lead
role in PGC transcriptional control. The
nature of these mechanisms, and their
relevance in other contexts, warrants
further exploration.
This study complements recent work
by Smith et al. (2012) which explores
methylation dynamics from fertiliza-
tion to the e7.5 epiblast (Figure 1).
Using Reduced Representation BS-Seq
(RRBS), which informs on DNA methyla-
tion at base pair resolution but does not
Molecular Cell 48, D
provide complete genome coverage,
Smith et al. (2012) confirmed two key
transitions in early mouse development:
first, the rapid methylation erasure of
the paternal genome and the passive
erasure of the maternal genome after
fertilization; and second, the genome-
wide gain of methylation between the
inner cell mass of the blastocyst and the
early epiblast. These are the marks that
must then be reset in PGCs. Smallwood
et al. (2011) also exploited RRBS to
interrogate methylation dynamics during
oocyte development. Together, these
studies enable us to build a compre-
hensive picture of the timings of methy-
lation dynamics during development
that underpin totipotency and differentia-
tion (Figure 1).
What is the mechanism for epigenetic
reprogramming in PGCs? This is a
particularly hot topic because of the
recent discovery that Tet3 orchestrates
genome-wide demethylation of the
paternal genome upon fertilization (Gu
et al., 2011). This is an active mechanism
involving the oxidation of 5-methylcyto-
sine to 5-hydroxymethylcytosine. Tet3
protein is not detected in PGCs, sug-
gesting the involvement of alternative
mechanisms (Hackett et al., 2012). Sei-
senberger et al. (2012) provide strong
evidence that PGC demethylation is pre-
dominantly passive, meaning that methy-
lation is not maintained during DNA
replication. To demonstrate this, the
authors make use of hairpin bisulfite
high-throughput sequencing, a method
that keeps double-stranded DNA to-
gether and permits a strand-specific
assessment of methylation. Many CpG
dinucleotides hemimethylated in e9.5
PGCs were completely unmethylated
by e13.5. Moreover, in hemimethylated
sequences, the methylated CpGs were
ecember 28, 2012 ª2012 Elsevier Inc. 819
Figure 1. DNA Methylation Dynamics during Developmental ReprogrammingAfter fertilization, the paternal genome (blue line) is demethylated rapidly by active mechanisms, while the maternal genome (red line) is passively demethylated.Differentially methylated regions (DMRs) associated with imprinted genes are protected from this erasure (dashed green line). De novo methylation occurspostimplantation (black line), but PGCs are not specified until the epiblast stage (shading at top of figure). This methylation must be reset in PGCs. Frome6.5, the figure shows the methylation dynamics in the cells that form the germline only. Most sequences are demethylated in PGCs by e9.5. A subsetof sequences are late demethylaters and are not reprogrammed until after PGC migration. These include, but are not limited to, the imprinted DMRs. IAPsare resistant to demethylation during both the postfertilization and the PGC reprogramming waves. Variably erased CGIs (VECs) can resist erasure duringPGC reprogramming, but their methylation status during postfertilization reprogramming is unclear. Following sex determination, de novo methylation of thegerm cells occurs, but the dynamics are sex specific. Methylation is completed in prospermatogonia before birth, whereas methylation of oocytes is establishedduring the growth phase, after birth. In adulthood, the gametes are appropriately methylated to form a new zygote and restart the cycle of methylation dynamics.Shown below are the developmental windows examined by three key studies, with the specific time points analyzed indicated. blast., blastocyst. d5, day 5oocytes. GV, germinal vesicle oocytes. MII, metaphase II oocytes.
Molecular Cell
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predominantly located on the same
DNA strand, consistent with a failure to
maintain methylation during DNA replica-
tion. While this suggests demethylation
in PGCs is passive, other studies have
shown an involvement of active mecha-
nisms (reviewed in Hackett et al., 2012).
The relative importance of these pro-
cesses requires clarification, but the po-
tential redundancy underlines the funda-
mental importance of reprogramming
in ensuring the integrity of information
inheritance.
Through the analysis of DNA methyla-
tion during PGC development, Seisen-
berger et al. (2012) assess resistance to
genome-wide demethylation, identifying
regions of both consistent and variable
methylation. From this, the authors
propose a potential mechanism for the
much debated transgenerational epige-
netic inheritance. This form of inheritance
refers to non-sequence-based effects on
phenotype passed from one generation
820 Molecular Cell 48, December 28, 2012 ª
to the next via the gametes (Daxinger
and Whitelaw, 2012). The epigenetic
carriers of this information must resist
being cleared during early development
and PGC reprogramming. It is known
that intracisternal A particles (IAPs)
remain methylated in mature gametes,
preimplantation embryos, and PGCs
(Popp et al., 2010), but this new study
catalogs not only sequences that fully
resist reprogramming but also some
that variably escape. Intriguingly, the
latter include some CpG islands (CGIs)
located away from the influence of IAPs,
termed variably erased CGIs (VECs), that
exhibit variable erasure across multiple
stages of development including mature
gametes. These VECs present potential
vehicles for the transmission of epige-
netic information across generations,
and could provide the mechanistic basis
for the observed transgenerational inheri-
tance of metabolic phenotypes, such as
obesity, in animal models of nutritional
2012 Elsevier Inc.
manipulation (Daxinger and Whitelaw,
2012). This is a tantalizing finding, and
these models of transgenerational inheri-
tance should be revisited with this in
mind. Do VECs show different methyla-
tion profiles in the gametes of nutritionally
deprived animals relative to controls?
Later, we can think about how this infor-
mation might be laid down during game-
togenesis in the first place.
Seisenberger et al. (2012), viewed in
concert with other studies, provide
detailed insights into the timings and
mechanisms of reprogramming events
that are fundamental for resetting infor-
mation for the next generation. The study
provides a solid foundation from which to
further investigate several key concepts,
including the uncoupling of DNA methy-
lation and transcription, the relative
contributions of demethylation mecha-
nisms, and the potential for the transmis-
sion of epigenetic information between
generations.
Molecular Cell
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Li, X., Ito, M., Zhou, F., Youngson, N., Zuo, X.,Leder, P., and Ferguson-Smith, A.C. (2008). Dev.Cell 15, 547–557.
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A Dancer Caught Midstep:The Structure of ATP-Bound Hsp70
Rui Sousa1,*1Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA*Correspondence: [email protected]://dx.doi.org/10.1016/j.molcel.2012.12.008
Hsp70 ATP binding induces substrate release, but the transiency of this state has inhibited its characteriza-tion. In this issue, Kityk et al. determine the Hsp70*ATP structure utilizing engineered disulfide bonds,providing insights into the workings of this essential molecular machine.
The study of transient protein conforma-
tions can be as important as the charac-
terization of long-lived states, as the
functional significance of a particular
conformation is not determined by its
persistence. This is certainly true for
Hsp70s, a family of protein chaperones
whose nucleotide regulated cycles of
protein substrate binding mediate many
of the cellular proteostatic and protein
processing reactions from bacteria to hu-
mans. Hsp70s are comprised of a nucleo-
tide binding domain (NBD) and a protein
substrate binding domain (SBD). When
ADP is bound to the NBD, the SBD holds
tightly to protein substrates, which bind in
a b sandwich pocket (SBDb) capped by
an a-helical lid (SBDa; Figure 1A). Upon
release of ADP and binding of ATP—
a reaction catalyzed by nucleotide ex-
change factors—the SBD releases its
bound substrate. Binding of a new
substrate, presented to the Hsp70 by a J
cochaperone, stimulates ATP hydrolysis,
resulting in a long-lived Hsp70*ADP*sub-
strate complex. Structures of Hsp70s in
apo- or ADP-bound states (Bertelsen
et al., 2009) reveal the SBD in same tight
binding (closed) conformation seen with
an isolated SBD (Zhu et al., 1996), indi-
cating that it is the ATP-bound NBD that
induces conformational changes in the
SBD (Swain et al., 2006). But a crystal
structure of the Hsp70 ATP state had
proven impossible to obtain, until now.
In this issue of Molecular Cell, Mayer
and colleagues captured the HSP*ATP
state for crystallization by engineering di-
sulfide bonds to lock in this normally tran-
sient conformation (Kityk et al., 2012).
This is often a perilous path, as the engi-
neering itself may artificially constrain
the resulting conformation. However,
these investigators also carry out solution
experiments that validate their structure
and provide further insight into the mech-
anism of this molecular machine.
So how does HSP70 work? Befitting
such an ancient protein, the Hsp70 NBD
exhibits the familiar hexokinase/actin
fold, and the ATP-induced conforma-
tional change in the NBD recapitulates
the historically important induced fit
change—predicted by Koshland, seen
by Steitz—driven by glucose binding to
hexokinase. In the case of both HSP70
and hexokinase, binding of the ligand in
the center of the horseshoe-shaped
protein domain induces it to close around
that ligand. With Hsp70, this involves
clockwise rotation (as defined by the
view in Figure 1) of NBD lobe II (subdo-
mains IIA and IIB), resulting in closure of
the space between subdomains IB and
IIB and widening of a crevice between
subdomains IA and IIA. Widening of this
crevice allows the linker that connects
NBD and SBD to bind within it and,
combined with changes in the relative
orientation of subdomains IA and IIA,
creates a surface on the NBD to which
SBDb binds. Binding of SBDb to this
surface displaces interactions between
SBDb and SBDa and sterically pushes
SBDa away from SBDb. The displaced
SBDa settles onto a site on subdomain
IB of the NBD (Figure 1B).
It was the interaction of SBDa and the
subdomain IB of the NBD that Kityk
et al. (2012) took advantage of to create
their locked in ATP conformation. To
guide their engineering, they modeled
their bacterial Hsp70 (DnaK) on yeast
Hsp110 (aka Sse1). Hsp110 is a distant
Hsp70 homolog that acts as nucleotide
exchange factor for Hsp70 and that also
binds misfolded proteins. However, a
ecember 28, 2012 ª2012 Elsevier Inc. 821