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: rebecca.oakey@kcl.ac.ukhttp://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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REFERENCES

Daxinger, L., and Whitelaw, E. (2012). Nat. Rev.Genet. 13, 153–162.

Gu, T.P., Guo, F., Yang, H., Wu, H.P., Xu, G.F., Liu,W., Xie, Z.G., Shi, L., He, X., Jin, S.G., et al. (2011).Nature 477, 606–610.

Hackett, J.A., Zylicz, J.J., and Surani, M.A. (2012).Trends Genet. 28, 164–174.

Li, X., Ito, M., Zhou, F., Youngson, N., Zuo, X.,Leder, P., and Ferguson-Smith, A.C. (2008). Dev.Cell 15, 547–557.

Popp, C., Dean, W., Feng, S., Cokus, S.J., An-drews, S., Pellegrini, M., Jacobsen, S.E., andReik, W. (2010). Nature 463, 1101–1105.

Seisenberger, S., Andrews, S., Krueger, F., Arand,J., Walter, J., Santos, F., Popp, C., Thienpont, B.,

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Dean, W., and Reik, W. (2012). Mol. Cell 48, thisissue, 849–862.

Smallwood, S.A., Tomizawa, S., Krueger, F., Ruf,N., Carli, N., Segonds-Pichon, A., Sato, S., Hata,K., Andrews, S.R., and Kelsey, G. (2011). Nat.Genet. 43, 811–814.

Smith, Z.D., Chan, M.M., Mikkelsen, T.S., Gu, H.,Gnirke, A., Regev, A., and Meissner, A. (2012).Nature 484, 339–344.

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: sousa@uthscsa.eduhttp://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