Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans.

Abstract

The plasticity of ageing suggests that longevity may be controlled epigenetically by specific alterations in chromatin state. The link between chromatin and ageing has mostly focused on histone deacetylation by the Sir2 family, but less is known about the role of other histone modifications in longevity. Histone methylation has a crucial role in development and in maintaining stem cell pluripotency in mammals. Regulators of histone methylation have been associated with ageing in worms and flies, but characterization of their role and mechanism of action has been limited. Here we identify the ASH-2 trithorax complex, which trimethylates histone H3 at lysine 4 (H3K4), as a regulator of lifespan in Caenorhabditis elegans in a directed RNA interference (RNAi) screen in fertile worms. Deficiencies in members of the ASH-2 complex-ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2-extend worm lifespan. Conversely, the H3K4 demethylase RBR-2 is required for normal lifespan, consistent with the idea that an excess of H3K4 trimethylation-a mark associated with active chromatin-is detrimental for longevity. Lifespan extension induced by ASH-2 complex deficiency requires the presence of an intact adult germline and the continuous production of mature eggs. ASH-2 and RBR-2 act in the germline, at least in part, to regulate lifespan and to control a set of genes involved in lifespan determination. These results indicate that the longevity of the soma is regulated by an H3K4 methyltransferase/demethylase complex acting in the C. elegans germline.

Full text

Page 1

LETTERS
MembersoftheH3K4trimethylationcomplexregulate
lifespan in a germline-dependent manner in C. elegans
Eric L. Greer1,2, Travis J. Maures1, Anna G. Hauswirth1, Erin M. Green4, Dena S. Leeman1,2, Ge ´raldine S. Maro3,
Shuo Han1, Max R. Banko1, Or Gozani2,4& Anne Brunet1,2
The plasticity of ageing suggests that longevity may be controlled
epigenetically by specific alterations in chromatin state. The link
between chromatin and ageing has mostly focused on histone
deacetylation by the Sir2 family1,2, but less is known about the role
ofotherhistonemodificationsinlongevity.Histonemethylationhas
a crucial role in development and in maintaining stem cell pluri-
potency in mammals3. Regulators of histone methylation have been
associatedwithageinginworms4–7andflies8,butcharacterizationof
their role and mechanism of action has been limited. Here we
identify the ASH-2 trithorax complex9, which trimethylates histone
H3 at lysine 4 (H3K4), as a regulator of lifespan in Caenorhabditis
elegans in a directed RNA interference (RNAi) screen in fertile
worms. Deficiencies in members of the ASH-2 complex—ASH-2
itself, WDR-5 and the H3K4 methyltransferase SET-2—extend
worm lifespan. Conversely, the H3K4 demethylase RBR-2 is
required for normal lifespan, consistent with the idea that an excess
ofH3K4trimethylation—amarkassociatedwithactivechromatin—
is detrimental for longevity. Lifespan extension induced by ASH-2
complex deficiency requires the presence of an intact adult germ-
line and the continuous production of mature eggs. ASH-2 and
RBR-2 act in the germline, at least in part, to regulate lifespan and
to control a set of genes involved in lifespan determination. These
results indicate that the longevity of the soma is regulated by
an H3K4 methyltransferase/demethylase complex acting in the
C. elegans germline.
Genome-wide RNAi screens for genes that regulate lifespan in
C. elegans have been performed previously in worms in which
progeny production was inhibited4,10,11, which could mask the effect
of some genes on lifespan. We performed a targeted RNAi screen
in fertile worms byselecting genes that encode known worm methyl-
transferases, proteins containing the enzymatic domain of methyl-
transferases (SET domain), or orthologues of regulators of histone
methylation. As reported previously4, set-9 and set-15 knock-
down extended worm lifespan (Fig. 1a). We also found that ash-2,
set-2andset-4knockdownextendedfertilewormlifespan,withash-2
knockdown having the most significant effect (23.1–30.9%,
P,0.0001) (Fig. 1a, Supplementary Fig. 1a). ASH-2 is a member
of an H3K4 trimethylation (H3K4me3) complex in yeast, flies and
mammals9,12,13and is important for the conversion of H3K4
dimethylation(H3K4me2)toH3K4me3inmammals14.ash-2knock-
down decreased global H3K4me3 levels at larval stage L3 (Fig. 1b),
indicating that ASH-2 promotes histone H3 trimethylation at lysine
4 in C. elegans. WDR-5/TAG-125 is a WD40-repeat protein that
interacts with ASH-2 in mammals, and is important for the mono-,
di-andtrimethylationofH3K4inmammalsandworms15,16.wdr-5/tag-
125 knockdown or wdr-5/tag-125 deletion also decreased H3K4me3
levels (Fig. 1b) and significantly extended C. elegans lifespan (,30%,
P,0.0001and16–28%,P,0.0001,respectively)(Fig.1c,Supplemen-
tary Fig. 1b). Thus,ASH-2and WDR-5 promote H3K4 trimethylation
and normally limit lifespan in C. elegans.
In mammals, ASH-2 and WDR-5 form a complex with several
H3K4me3 methyltransferases of the SET1/mixed lineage leukaemia
(MLL) family17. There are four SET1/MLL orthologues in C. elegans:
SET-1,SET-2,SET-12andSET-16(SupplementaryFig. 1c). Ofthese,
only SET-2, which is similar to mammalian SET1A/SET1B, regulated
worm lifespan (set-2 knockdown: ,20%, P,0.0001; set-2 deletion
set-2(ok952):17–26%,P,0.0001)(Fig.1a,d;SupplementaryFig.1d,
e). As reported previously16, set-2(ok952) mutant worms or worms
treatedwithset-2RNAihadreducedH3K4me3levels(Fig.1e).SET-2
was relatively specific in regulating lifespan and H3K4me3 in worms,
as neither SET-9 nor SET-15 affected global H3K4me3 levels (Sup-
plementary Fig. 1f), even though they both regulate lifespan4. The
worm SET-2 methyltransferase domain expressed in bacteria directly
methylated histone H3 at lysine 4 in vitro (Fig. 1f). SET2 generated
H3K4me2 but not H3K4me3 in vitro (Supplementary Fig. 1g), con-
sistent with the fact that ASH-2 is required for the conversion of
H3K4me2 to H3K4me3 (ref. 18). To test if ASH-2, WDR-5 and
SET-2acttogethertoregulatelifespan,weperformedepistasisexperi-
ments. Lifespan extension by ash-2 or wdr-5/tag-125 knockdown was
significantly less pronounced in set-2(ok952) mutants than in wild-
type worms (Fig. 1g, combined two-way ANOVA P,0.0001).
Similarly, ash-2 knockdown did not extend further the lifespan of
wdr-5/tag-125(ok1417) mutant worms (Supplementary Fig. 1h,
P50.1534). Thus, ASH-2, WDR-5 and SET-2 probably act in the
same pathway, perhaps in a complex, to limit lifespan. As ash-2 and
wdr-5/tag-125 knockdown slightly extended set-2(ok952) mutant
worm lifespan (Fig. 1g, Supplementary Table 2), other methyltrans-
ferasesmayalsocomplexwithASH-2andWDR-5toregulatelifespan.
RBR-2 is an H3K4me3 demethylase involved in vulva formation in
worms19andishomologoustohumanRBP2andPLU-1(alsoknownas
KDM5AandKDM5B,respectively),twoH3K4me3demethylasesofthe
JARID family19. Consistent with a published report19, rbr-2(tm1231)
mutant worms showed increased H3K4me3 levels (Fig. 2a). Both rbr-
2(tm1231) mutation and rbr-2 knockdown decreased lifespan signifi-
cantly (15–25%, P,0.0001 and 10–24.2%, P,0.0001, respectively)
(Fig.2b,SupplementaryFig.2a),indicatingthatRBR-2isnecessaryfor
normal longevity. The decrease of H3K4me3 induced by ash-2 knock-
down was less pronounced in rbr-2(tm1231) mutant worms than in
wild-type worms (Fig. 2c). In addition, ash-2 knockdown no longer
extended rbr-2(tm1231) mutant worm lifespan (Fig. 2d, P50.4673).
Similarly, the lifespan of wdr-5/tag-125(ok1415); rbr-2(tm1231) and
set-2(ok952); rbr-2(tm1231) double mutants was similar to that of
1Department of Genetics, Stanford University, 300 Pasteur Drive, Stanford, California 94305, USA.2Cancer Biology Graduate Program, Stanford University, Stanford, California
94305, USA.3Departments of Biology and Pathology, Stanford University, Stanford, California 94305, USA.4Department of Biology, Stanford University, Stanford, California 94305,
USA.
doi:10.1038/nature09195
1
Macmillan Publishers Limited. All rights reserved
©2010

Page 2

rbr-2(tm1231) single mutants (Supplementary Fig. 2b, c, P50.2121
and P50.6943, respectively). Together, these results indicate that
RBR-2 counteracts the effects of ASH-2/WDR-5/SET-2 on H3K4me3
and lifespan.
Whole-mount immunocytochemistry with a monoclonal ASH-2
antibody (SupplementaryFig. 4a)showedthatASH-2isexpressedin
the nuclei of cells from many tissues, and is highly expressed in the
germline (Fig. 3a). Visualization of an ASH-2–GFP (green fluor-
escent protein) fusion driven by the endogenous ash-2 promoter in
low-copy or high-copy transgenic lines confirmed that ASH-2 is
highly expressed in the germline and in newly formed eggs
(Supplementary Fig. 4b, c). Visualization of an RBR-2–GFP fusion
driven by the endogenous rbr-2 promoter in low-copy or high-copy
transgenic lines showed expression of RBR-2 in the nuclei of cells
from many tissues, although RBR-2 was not particularly enriched in
the germline or eggs (Supplementary Fig. 4d, data not shown). The
H3K4me3markwaspresentinthenucleiofcellsfromalltissues,with
high abundance in the germline in L3 and young adult worms
(Fig. 3b, Supplementary Fig. 4e), consistent with the high levels of
ASH-2 and SET-2 (ref. 20) in the germline. As expected, wdr-5/tag-
125(ok1417) and set-2(ok952) mutant worms had markedly reduced
H3K4me3 levels (Fig. 3b, Supplementary Fig. 4e). Conversely,
rbr-2(tm1231) mutant worms showed increased H3K4me3 levels,
particularly in the germline (Fig. 3b, Supplementary Fig. 4e). These
results confirm that members of the ASH-2 complex and RBR-2
regulate H3K4me3, and that this mark is abundant in the germline.
To determine if the presence of an intact germline is necessary for
lifespan regulation by ASH-2, we examined the effects of ash-2
knockdown in glp-1(e2141ts) mutant worms, which develop only
5–15 meiotic germ cells instead of ,1,500 when shifted to the
restrictive temperature at the L1 stage21,22. ash-2 knockdown did
not extend further the long lifespan of glp-1(e2141ts) mutant worms
(Fig. 3c). We verified that at the permissive temperature, ash-2
knockdown extended glp-1(e2141ts) mutant worm lifespan (Sup-
plementary Fig. 5a, b). Consistent with these observations, ash-2
knockdown did not extend the lifespan of sterile glp-4(bn2ts) or
pgl-1(bn101ts) mutant worms (Supplementary Fig. 5c, d). The pres-
ence of an intact germline was also necessary for lifespan regulation
by WDR-5, SET-2 and RBR-2 (Fig. 3d, Supplementary Fig. 5e–g). In
contrast,lifespanregulationbySET-9andSET-15didnotdependon
an intact germline (Supplementary Fig. 6a, b), indicating that not all
methyltransferases require the germline to control lifespan.
Weperformedagenome-wideanalysistoidentifyASH-2-regulated
genes that are dependent on the presence of an intact germline. ash-2
knockdown led to changes in the expression of 220 genes at the L3
stage and 847 genes at day 8 of life (day 5 of adulthood) in wild-type
worms (Fig. 3e, Supplementary Tables 6–9). The majority of ASH-2-
controlled genes were regulated in a germline-dependent manner, as
their expression was affected in wild-type worms, but not in
glp-1(e2141ts)mutantworms(93.6%atL3and69.8%atday8oflife)
a
–30
–20
–10
0
10
20
30
40
set-1set-2set-3set-4set-5set-6
set-7
set-8set-9
set-10set-11set-12set-14set-15set-16
set-17
set-18set-19set-20 set-22set-24 set-25set-30set-32
blmp-1
lin-59
mes-4
met-1met-2
F54F7.7
ash-2
Y55F3AM.14
Percentage change in
average lifespan
*
*
*
*
*
*
*
bc
Age (days)
0
0.2
0.4
0.6
0.8
1
Fraction alive
0510 1520 2530 35
wdr-5/tag-125(ok1417)
N2 (WT)
H3K4me3
H3
N2 (WT)
wdr-5
/tag-125(ok1417)
EV
ash-2
RNAi:
RNAi:
wdr-5/tag-125
H3K4me2
H3K4me1
e
0
0.2
0.4
0.6
0.8
1
Fraction alive
05 10 15 2025 3035
EV
set-2 RNAi
Age (days)
Age (days)
0
0.2
0.4
0.6
0.8
1
Fraction alive
set-2(ok952) tag-125 RNAi
set-2(ok952) ash-2 RNAi
set-2(ok952) EV
N2 (WT) tag-125 RNAi
N2 (WT) ash-2 RNAi
N2 (WT) EV
05 10 1520 2530
d
gf
3H
Nucleosomes
Nucleosomes: –+
SET-2SET:
++
SET-2
EV
set-2
N2 (WT)
set-2(ok952)
H3
H3K4me3
H3K4me1
H3K4me2
Figure 1 | ASH-2, WDR-5 and SET-2 function together to regulate
H3K4me3 and lifespan in C. elegans. a, Percentage change in average
lifespan in worms treated with RNAi to the indicated genes compared with
empty vector. Mean 6 s.d. of two or three independent experiments is
shown. * P,0.05. b, Western blots on L3 worm extracts (representative of
four independent experiments). EV, empty vector; WT, wild type. c, wdr-5/
tag-125(ok1417) mutant worms have an extended lifespan. d, set-2
knockdownextendslifespan.e,WesternblotsofL3wormextractsafterset-2
knockdown or deletion (representative of three independent experiments).
f, The methyltransferase domain of worm SET-2 (SET-2SET) methylates
histone H3 in vitro. g, ash-2 and wdr-5/tag-125 knockdown slightly extend
the lifespan of set-2(ok952) mutant worms, but significantly less than in the
N2 (WT) background. ash-2 mRNA was efficiently knocked-down by RNAi
in set-2(ok952) mutant worms (Supplementary Fig. 3a). Statistics are
presented in Supplementary Tables 1 and 2.
ab
Age (days)
0
0.2
0.4
0.6
0.8
1
Fraction alive
051015 202530 35
rbr-2(tm1231)
N2 (WT)
cd
Age (days)
0
0.2
0.4
0.6
0.8
1
Fraction alive
05 10 15 20 2530 35 40
rbr-2(tm1231)
EV
rbr-2(tm1231)
ash-2 RNAi
N2 (WT)
EV
N2 (WT)
ash-2 RNAi
N2 (WT)
rbr-2(tm1231)
H3
H3K4me2
H3K4me1
H3K4me3
RNAi:
EV
EV
H3K4me3
H3
ash-2
ash-2
N2 (WT)
rbr-2(tm1231)
Figure 2 | RBR-2 is an H3K4me3 demethylase that counteracts the effect
of the ASH-2 methyltransferase complex. a, Western blots of rbr-
2(tm1231) mutant worms (representative of three independent
experiments). b, rbr-2(tm1231) mutant worms have a decreased lifespan.
c, Western blots of rbr-2(tm1231) mutant worms treated with ash-2 RNAi
(representative of two independent experiments). d, ash-2 knockdown does
not extend the lifespan of rbr-2(tm1231) mutant worms. ash-2 mRNA was
efficiently knocked down by RNAi in rbr-2(tm1231) mutant worms
(Supplementary Fig. 3a). Statistics are presented in Supplementary Table 3.
LETTERS
NATURE
2
Macmillan Publishers Limited. All rights reserved
©2010

Page 3

(Fig. 3f). Interestingly, one of the top gene ontology (GO) enrich-
ment categories for genes regulated by ASH-2 at L3 and day 8
was ‘determination of adult lifespan’ (Fig. 3g, P52.6131028and
P52.4731026, respectively). Furthermore, at both L3 and day 8 of
life, ASH-2-regulated genes were also significantly enriched (twofold,
P5531026and 1.5-fold, P5631026, respectively) for genes
whose expression changed during ageing23.
To determine if members of the ASH-2 complex function in the
germline itself to regulate lifespan, we used rrf-1(pk1417) mutant
worms in which RNAi is efficient in the germline, but not in somatic
cells24.ash-2orset-2knockdownstillextendedlifespaninrrf-1(pk1417)
mutants (23.6%, P,0.0001 and 22.5%, P,0.0001, respectively)
(Fig. 4a, Supplementary Fig. 7a), indicating that ASH-2 and SET-2
function in the germline to regulate lifespan. Conversely, to test if
RBR-2 expression in the germline was sufficient to extend lifespan,
we generated low-copy integrants of RBR-2–GFP driven by the
germline-specific pie-1 promoter (Ppie-1::rbr-2::gfp). Three independ-
ent Ppie-1::rbr-2::gfp lines had an extended lifespan compared to two
independentcontrolPpie-1::gfplines(17.9–30.7%,P,0.005)(Fig.4b),
indicating that expression of RBR-2 in the germline is sufficient to
prolong worm lifespan. High-copy Prbr-2::rbr-2::gfp transgenic worms
also had a modest increase in lifespan (10–14%, P,0.005) (Sup-
plementaryFig.7b).Becausehigh-copytransgenicwormstendtohave
suppression of transgene expression in the germline25, these results
indicate that expressionof RBR-2 insomaticcells may alsocontribute
to lifespan extension, or that very low amounts of RBR-2 expressed in
thegermlineinthesehigh-copytransgenicsmaybesufficienttoextend
lifespan. Thus, the ASH-2 methyltransferase complex and the RBR-2
demethylase act in the germline, at least in part, to regulate lifespan.
TheASH-2complexandRBR-2donotseemtoregulatelifespanby
simply affecting progeny production. ash-2 knockdown worms were
fertile and did not have significant differences in number of eggs laid
(Supplementary Fig. 8a), number of live progeny (Supplementary
Fig. 8b),germline cellnumber (Supplementary Fig. 8c)andgermline
morphology (data not shown). Similarly, set-2(ok952), wdr-5/tag-
125(ok1417) and rbr-2(tm1231) mutant worms did not have signifi-
cantdifferencesineggnumberandweremostlyfertile(Supplementary
Fig. 8d).
We next investigated whether ASH-2 and RBR-2 require an intact
adultordevelopinggermlinetoregulatelifespan.Knocking-downash-2
andrbr-2inyoungadults,afterthegermlinehasdeveloped,stillaffected
lifespan (ash-2: 126%, P,0.0001; rbr-2: 213.5%, P,0.0001) (Sup-
plementary Fig. 9a, b). Furthermore, ash-2 knockdown no longer
extended lifespan in glp-1(e2141ts) mutant worms switched to the
restrictive temperature at the young adult stage (Fig. 4c), whereas
ash-2 knockdown extended lifespan in glp-1(e2141ts) mutant worms
that were switched to the restrictive temperature after the egg laying
period (Supplementary Fig. 9c). Similar observations were made with
glp-4(bn2ts) mutant worms (Supplementary Table 4). Consistently,
ash-2knockdownnolongerextendedlifespaninanotherglp-1mutant,
glp-1(e2142ts),whichhasafullydevelopedgermline,butnoadultgerm-
line stem cells or eggs (Supplementary Fig. 9d). Finally, ash-2 knock-
down did not extend further the long lifespan of worms treated with
5-fluorodeoxyuridine (FUdR), a drug that inhibits the proliferation of
germline stem cells and the production of intact eggs in adults26(Sup-
plementaryFig.9e).Similarly,rbr-2knockdowndidnotdecrease—and
a
ASH-2DAPI
ash-2 RNAi
EV
b
rbr-2
H3K4me3
wdr-5/
tag-125
N2 (WT)
DAPI
cd
0
0.2
0.4
0.6
0.8
1
Fraction alive
05 10
Age (days)
152025
N2 (WT) set-2 RNAi
glp-1(e2141ts) EV
N2 (WT) EV
glp-1(e2141ts) set-2 RNAi
fe
GO enrichment day 2 (L3)
Determination of adult lifespan
ER unfolded protein response
P value
2.61 × 10–8
5.61 × 10–6
GO enrichment day 8
Structural constituent of cuticle
Phosphate transport
Determination of adult lifespan
P value
9.59 × 10–9
1.78 × 10–8
2.47 × 10–6
N2
(WT)
glp-1
(e2141ts)
N2
(WT)
glp-1
(e2141ts)
Day 2 (L3)
EV
–3 –2 –1
0123
–3–2–1
0123
ash-2
EV
ash-2
EV
ash-2
EV
ash-2
Day 8
RNAi:
g
Germline-dependent
Germline-independent
Day 8
69.8%
Day 2 (L3)
93.6%
0
0.2
0.4
0.6
0.8
1
Fraction alive
05 101520 25 30
Age (days)
glp-1(e2141ts) EV
glp-1(e2141ts) ash-2 RNAi
N2 (WT) EV
N2 (WT) ash-2 RNAi
Figure 3 | An intact germline is necessary for longevity and gene
expression control by the H3K4me3 regulatory complex. a, b, Whole-
mount immunofluorescence of young adult worms stained with an ASH-2
antibody (a) or an H3K4me3 antibody (b). Dashed lines mark the germline.
Scale bars, 50mm. c, ash-2 knockdown does not extend further the long
lifespan of glp-1(e2141ts) mutant worms that were shifted to the restrictive
temperature at the L1 stage. d, set-2 knockdown does not extend further the
long lifespan of germline-deficient glp-1(e2141ts) mutant worms that were
shifted to the restrictive temperature at the L1 stage. Statistics are presented
in Supplementary Table 4. e, Microarray clusters of genes that change
significantly on ash-2 knockdown in wild-type worms, but not in glp-
1(e2141ts) mutants. Experimental values are presented in Supplementary
Tables 8 and 9. f, Percentage of genes regulated by ASH-2 only in wild-type
worms,butnotinglp-1(e2141ts)mutants.g,Topgeneontology(GO)terms
for genes regulated by ASH-2 in wild-type worms, but not in glp-1(e2141ts)
mutants.
a
0
0.2
0.4
0.6
0.8
1
Fraction alive
Age (days)
0510 152025 30 35
rrf-1(pk1417) EV
rrf-1(pk1417) ash-2 RNAi
N2 (WT) EV
N2 (WT) ash-2 RNAi
b
0
0.2
0.4
0.6
0.8
1
Fraction alive
0510 15 20 25 30 35 40
Age (days)
LC Ppie-1::rbr-2::gfp (#1)
LC Ppie-1::rbr-2::gfp (#2)
LC Ppie-1::gfp (#1)
LC Ppie-1::gfp (#2)
LC Ppie-1::rbr-2::gfp (#3)
c
0
0.2
0.4
0.6
0.8
1
Fraction alive
05 10152025 30
Age (days)
glp-1(e2141ts) EV
glp-1(e2141ts) ash-2 RNAi
N2 (WT) EV
N2 (WT) ash-2 RNAi
Age (days)
0
0.2
0.4
0.6
0.8
1
Fraction alive
0510152025
N2 (WT) EV
N2 (WT) ash-2 RNAi
fem-3(e2006ts) EV
fem-3(e2006ts) ash-2 RNAi
d
Figure 4 | ASH-2/RBR-2 function primarily in the germline to regulate
lifespanand requirethe continuous productionof matureeggs for lifespan
extension. a, ash-2 RNAi extends the lifespan of rrf-1(pk1417) mutant
worms,whicharedeficientforRNAiinthesoma,toasimilarextentasinN2
(WT) worms. b, Three independent lines of low-copy (LC) integrant
Ppie-1::rbr-2::gfp transgenic worms have increased lifespan compared to two
independent lines of Ppie-1::gfp transgenic worms. c, ash-2 knockdown does
not extend further the long lifespan of glp-1(e2141ts) mutant worms that
were shifted to the restrictive temperature at the young adult stage. d, ash-2
knockdown does not extend the lifespan of fem-3(e2006ts) mutant worms,
which do not produce mature eggs. ash-2 mRNA was efficiently knocked
down by RNAi in fem-3(e2006ts) mutants (Supplementary Fig. 3b).
Statistics are presented in Supplementary Tables 4 and 5.
NATURE
LETTERS
3
Macmillan Publishers Limited. All rights reserved
©2010

Page 4

even slightly extended—worm lifespan in the presence of FUdR (Sup-
plementary Fig. 9f), consistent with a previous study27. Thus, ASH-2
regulates lifespan in adult worms by a mechanism that depends on the
presenceofadultgermlinestemcellsand/orthecontinuousproduction
of eggs.
To distinguish if the germline stem cells or the continuous pro-
ductionofeggsisrequiredfortheregulationoflifespanbyASH-2,we
used fem-3(e2006ts) mutant worms, which have functional germline
stem cells, but do not produce fertilized eggs28. ash-2 knockdown no
longer extended the lifespan of fem-3(e2006ts) mutant worms
(Fig. 4d), indicating that the production of mature eggs, rather than
germline stem cells, is necessary for ash-2 knockdown to extend life-
span. Furthermore, ash-2 knockdown still significantly extended the
lifespan ofdaf-16(mu86) anddaf-12(m20) mutantworms, twogenes
involved in signalling pathways that are critical for germline stem
cells to regulate lifespan21,22,29(Supplementary Fig. 10a, b). Con-
sistently, set-2(ok952) mutant worms displayed lifespan extension
even in the presence of daf-16 RNAi (Supplementary Table 2).
These results indicate that the ability of members of the ASH-2 com-
plex to regulate lifespan depends on the continuous production of
mature eggs, but not on the germline stem cell signalling pathway.
Here we show that members of an H3K4me3 methyltransferase
complexhaveapivotalroleintheregulationoflongevityinC.elegans.
Our results indicate that the ASH-2 complex and RBR-2 regulate
ageingatleastinpartbycontrollingtrimethylationofH3K4,although
they might also act by controlling the methylation of non-histone
proteins. ASH-2 regulates the expression of a specific subset of genes
enriched for lifespan determination genes, but more global changes
in chromatin state and gene expression—and therefore quantity of
protein produced—may also be responsible for the modulation of
lifespan by ASH-2. The observation that the continuous production
ofmatureeggsthroughoutadulthoodisnecessaryforASH-2toregu-
late lifespan in C. elegans may explain why the ASH-2 trimethylase
complex was not identified in earlier screens for ageing genes in C.
elegans, in which the production of mature eggs was inhibited4,10,11.
ASH-2/RBR-2 may function in the germline—perhaps in the matur-
ing eggs—to control endocrine hormones that would, in turn, regu-
late longevity of the soma. Alternatively, ASH-2/RBR-2 may control
the expression of genes involved in lifespan determination, and addi-
tionalsignalsfromthegermline/matureeggsmayberequiredtoallow
lifespan extension. The regulation of H3K4me3 by specific methyl-
transferase complexes may regulate ageing in a germline-dependent
manner in other species. Male flies heterozygous for the H3K4me3
methyltransferase trx had a normal lifespan8, but trx may not be the
specific H3K4me3 methyltransferase that regulates lifespan, or the
H3K4me3 regulatory complex may be more important in females/
hermaphrodites than in males for controlling longevity. The finding
that ageing can be regulated epigenetically raises the intriguing pos-
sibility that aspects of the ageing process could be reversed.
METHODS SUMMARY
Lifespanassays.Wormlifespanassayswereperformedat20uC,withoutFUdR,as
described previously30unless noted otherwise. For each lifespan assay, 90 worms
perconditionwereusedinthreeplates(30wormsperplate),exceptfortheinitial
RNAi screen, which was performed with 30–60 worms per condition.
Statistical analysis. Statistical analyses of lifespan were performed on Kaplan–
Meier survival curves in StatView 5.0.01 by Logrank (Mantel–Cox) tests. For
statistical comparison of independent replicates, Fisher’s combined probability
tests were performed. To compare the interaction between genotype and RNAi,
two-way ANOVA tests were performed.
Microarray analysis. N2 (wild type) and glp-1(e2141ts) mutant worms were
hatched at 16uC and switched to bacteria with control empty vector or ash-2
RNAi and moved to the restrictive temperature of 25.5uC at the L1 stage. RNA
was isolated at larval stage L3 or day 8 of life. Each condition was done in
independent triplicate with ,1,000 worms per triplicate for L3 worms, and
,200 worms picked every day after adulthood to fresh plates per triplicate for
day 8 worms. Total RNA was isolated using an RNAqueous kit (Ambion).
Microarray hybridization was performed at the Stanford Protein and Nucleic
Acid facility with oligonucleotide arrays (Affymetrix, GeneChip C. elegans
Genome Arrays). Background adjustment and normalization was performed
with RMA (Robust Multiarray Analysis).
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 3 December 2009; accepted 28 May 2010.
Published online 16 June 2010.
1. Blander, G. & Guarente, L. The Sir2 family of protein deacetylases. Annu. Rev.
Biochem. 73, 417–435 (2004).
Dang, W. et al.HistoneH4 lysine 16 acetylation regulates cellular lifespan. Nature
459, 802–807 (2009).
Nottke, A., Colaiacovo, M. P. & Shi, Y. Developmental roles of the histone lysine
demethylases. Development 136, 879–889 (2009).
Hamilton, B. et al. A systematic RNAi screen for longevity genes in C. elegans.
Genes Dev. 19, 1544–1555 (2005).
Li, J. et al. Caenorhabditis elegans HCF-1 functions in longevity maintenance as a
DAF-16 regulator. PLoS Biol. 6, e233 (2008).
McColl, G. et al. Pharmacogenetic analysis of lithium-induced delayed aging in
Caenorhabditis elegans. J. Biol. Chem. 283, 350–357 (2008).
Chen, S. et al. The conserved NAD(H)-dependent corepressor CTBP-1 regulates
Caenorhabditis elegans life span. Proc. Natl Acad. Sci. USA 106, 1496–1501 (2009).
Siebold,A.P.etal.Polycombrepressivecomplex2andtrithoraxmodulateDrosophila
longevity and stress resistance. Proc. Natl Acad. Sci. USA 107, 169–174 (2010).
Wysocka, J., Myers, M. P., Laherty, C. D., Eisenman, R. N. & Herr, W. Human Sin3
deacetylase and trithorax-related Set1/Ash2 histone H3–K4 methyltransferase
aretetheredtogetherselectivelybythecell-proliferationfactorHCF-1.GenesDev.
17, 896–911 (2003).
10. Lee, S. S. et al. A systematic RNAi screen identifies a critical role for mitochondria
in C. elegans longevity. Nature Genet. 33, 40–48 (2003).
11. Hansen, M., Hsu, A. L., Dillin, A. & Kenyon, C. New genes tied to endocrine,
metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans
genomic RNAi screen. PLoS Genet. 1, e17 (2005).
12. Miller, T. et al. COMPASS: a complex of proteins associated with a trithorax-
related SET domain protein. Proc. Natl Acad. Sci. USA 98, 12902–12907 (2001).
13. Papoulas, O. et al. The Drosophila trithorax group proteins BRM, ASH1 and ASH2
are subunits of distinct protein complexes. Development 125, 3955–3966 (1998).
14. Dou, Y. et al. Regulation of MLL1 H3K4 methyltransferase activity by its core
components. Nature Struct. Mol. Biol. 13, 713–719 (2006).
15. Wysocka, J. et al. WDR5 associates with histone H3 methylated at K4 and is
essential for H3 K4 methylation and vertebrate development. Cell 121, 859–872
(2005).
16. Simonet, T., Dulermo, R., Schott, S. & Palladino, F. Antagonistic functions of SET-
2/SET1 and HPL/HP1 proteins in C. elegans development. Dev. Biol. 312, 367–383
(2007).
17. Ruthenburg,A.J.,Allis,C.D.&Wysocka,J.Methylationoflysine4onhistoneH3:
intricacy of writing and reading a single epigenetic mark. Mol. Cell 25, 15–30
(2007).
18. Schneider, J. et al. Molecular regulation of histone H3 trimethylation by
COMPASS and the regulation of gene expression. Mol. Cell 19, 849–856 (2005).
19. Christensen, J.etal.RBP2belongstoafamilyofdemethylases,specificfortri-and
dimethylated lysine 4 on histone 3. Cell 128, 1063–1076 (2007).
20. Xu,L.&Strome,S.DepletionofanovelSET-domainproteinenhancesthesterility
of mes-3 and mes-4 mutants of Caenorhabditis elegans. Genetics 159, 1019–1029
(2001).
21. Arantes-Oliveira, N., Apfeld, J., Dillin, A. & Kenyon, C. Regulation of life-span by
germ-line stem cells in Caenorhabditis elegans. Science 295, 502–505 (2002).
22. Berman, J. R. & Kenyon, C. Germ-cell loss extends C. elegans life span through
regulation of DAF-16 by kri-1 and lipophilic-hormone signaling. Cell 124,
1055–1068 (2006).
23. Budovskaya, Y. V. et al. An elt-3/elt-5/elt-6 GATA transcription circuit guides
aging in C. elegans. Cell 134, 291–303 (2008).
24. Sijen,T.etal.OntheroleofRNAamplificationindsRNA-triggered genesilencing.
Cell 107, 465–476 (2001).
25. Kelly, W. G., Xu, S., Montgomery, M. K. & Fire, A. Distinct requirements for
somatic and germline expression of a generally expressed Caenorhabditis elegans
gene. Genetics 146, 227–238 (1997).
26. Mitchell, D. H., Stiles, J. W., Santelli, J. & Sanadi, D. R. Synchronous growth and
aging of Caenorhabditis elegans in the presence of fluorodeoxyuridine. J. Gerontol.
34, 28–36 (1979).
27. Lee, S. S., Kennedy, S., Tolonen, A. C. & Ruvkun, G. DAF-16 target genes that
control C. elegans life-span and metabolism. Science 300, 644–647 (2003).
28. Haag, E. S., Wang, S. & Kimble, J. Rapid coevolution of the nematode sex-
determining genes fem-3 and tra-2. Curr. Biol. 12, 2035–2041 (2002).
29. Gerisch,B.,Weitzel,C.,Kober-Eisermann,C.,Rottiers,V.&Antebi,A.Ahormonal
signaling pathway influencing C. elegans metabolism, reproductive development,
and life span. Dev. Cell 1, 841–851 (2001).
30. Greer,E.L.etal.AnAMPK-FOXOpathwaymediateslongevityinducedbyanovel
method of dietary restriction in C. elegans. Curr. Biol. 17, 1646–1656 (2007).
2.
3.
4.
5.
6.
7.
8.
9.
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
LETTERS
NATURE
4
Macmillan Publishers Limited. All rights reserved
©2010

Page 5

Acknowledgements We are grateful to A. Fire, K. Helin, S. Kim, K. Shen,
T. Stiernagle and the Caenorhabditis Genetics Center, M. W. Tan and A. Villeneuve
for gifts of strains, reagents and antibodies. We thank S. Kim, G. Seydoux,
C. Slightam and K. Shen for advice on worm transgenesis, and S. Kim, A. Morgan
and Y. Kobayashi for help with microarray analysis. We thank A. Fire, S. Kim,
G. Seydoux, M. W. Tan and J. Wysocka for discussions. We thank members of the
Brunet laboratory, M. Kaeberlein, J. Lieberman, J. Sage and J. Wysocka for critical
reading of themanuscript. This work was supported byNIH grant R01-AG31198to
A.B.;E.L.G.wassupportedbyNIHtraininggrantT32-CA009302,anNSFgraduate
fellowship and by NIH ARRA-AG31198. T.J.M. was supported by NIH grant
T32-HG000044. D.S.L. and E.M.G. were supported by NIH training grant
T32-CA009302. S.H. was supported by a Stanford graduate fellowship. G.S.M.
was supported by a Human Frontier Science Program post-doctoral fellowship.
M.R.B. was supported by NIH fellowship F31-AG032837. O.G. was supported by a
Searle Scholar award.
Author Contributions E.L.G. and A.B. conceived and planned the study. E.L.G.
performed the experiments and wrote the paper with the help of A.B.; T.J.M.
completed Fig. 3a, b, Supplementary Fig. 4c, e and Supplementary Fig. 8c, and
generated alllow-copy integrant transgenicworm lines. A.G.H. helped with Fig.1b,
e and Supplementary Fig. 1f. E.M.G. was advised by O.G. and completed Fig. 1f and
Supplementary Fig. 1g. D.S.L. helped with Fig. 2a. G.S.M. generated all high-copy
transgenic worm lines. S.H. helped with Supplementary Fig. 8c. M.R.B. generated
thePrbr-2::rbr-2::gfp construct.Allauthors discussed the results andcommentedon
the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to A.B. (anne.brunet@stanford.edu).
NATURE
LETTERS
5
Macmillan Publishers Limited. All rights reserved
©2010

Page 6

METHODS
C. elegans strains. N2 and daf-16(mu86) strains were provided by M. W. Tan.
glp-1(e2142ts) and rrf-1(pk1417) strains were provided by A. Fire. unc-119(ed3)
strain was provided by S. Kim. wdr-5/tag-125(ok1417), set-2(ok952), rbr-
2(tm1231), fem-3(e2006ts), pgl-1(bn101ts), glp4(bn2ts), glp-1(e2141ts) and daf-
12(m20) strains were provided by T. Stiernagle and the Caenorhabditis Genetics
Center. Double mutants were constructed by crossing rbr-2(tm1231) males
with glp-1(e2141ts) hermaphrodites, set-2(ok952) males with rbr-2(tm1231)
hermaphrodites, wdr-5/tag-125(ok1417) males with rbr-2(tm1231) or pgl-
1(bn101ts) hermaphrodites. Prbr-2::rbr-2::gfp high-copy or low-copy integrant
transgenic lines contain 2.3kilobases of the rbr-2 promoter driving the full-
length rbr-2 cDNA fused to GFP at the carboxy-terminus. Pash-2::ash-2::gfp
high-copy or low-copy integrant transgenic lines contain 1.6kb of the ash-2
promoter driving the full-length ash-2 cDNA fused to GFP at the C terminus.
Ppie-1::rbr-2::gfp low-copy integrant transgenic line contains 2.4kb of the
germline-specific pie-1 promoter31driving the full-length rbr-2 cDNA fused to
GFP at the C terminus. Ppie-1::gfp low-copy integrant transgenic line contains
2.4kb of the germline-specific pie-1 promoter driving GFP.
RNA interference. Escherichia coli HT115 (DE3) transformed with vectors
expressing dsRNA of the genes of interest were all obtained from the Ahringer
library(agiftfromM.W.Tan),exceptRNAitorbr-2,blmp-1,set-4andset-18that
were from the Open Biosystems library (a gift from K. Shen) and were grown at
37uCand seededonstandardnematodegrowthmedium(NGM)platescontain-
ing ampicillin (100mgml21) and isopropylthiogalactoside (IPTG; 0.4mM).
Adult worms were placed on standard NGM plates and removed after 4–6h to
obtain synchronized populations of worms. L1 worms obtained from these syn-
chronized populations were placed on NGM plates containing ampicillin
(100mgml21) and IPTG (0.4mM) seeded with the respective bacteria. Worms
placed on RNAi at different time points were placed on empty vector control
RNAiattheL1stageandshiftedtotherespectivebacteriaattheappropriatetime.
Constructs.pSM,aderivativeofthepPD49.26plasmid32withadditionalcloning
siteswasusedtogeneratetransgenicconstructs.Prbr-2::rbr-2::gfptransgenicplas-
mid included 2.3kb of rbr-2 promoter driving the full-length rbr-2 cDNA fused
to GFP at the C terminus. The rbr-2 promoter was amplified by PCR from wild-
type (N2) genomic DNA using the following primers, F: 59-ATAGCGGCCGCC
CAACATTCTCTGGTGTGTATG-39 and R: 59-TATGGATCCTTTTCAGTTTG
GCTTATAGCTTCATGAG-39 and subcloned into pSM between NotI and
BamHI. The rbr-2 cDNA was amplified by PCR from rbr-2 cDNA19with the
following primers F: 59-ATAGCTAGCATGCGTGCACGTCGTCAAGAGAAC
AG-39 and R: 59-TATACCGGTAAATCGGTGGAAACACTCGAAGTTGGAGA
GTCC-39andsubclonedintopSMcontainingtherbr-2promoter,betweenNheI
and AgeI.
Pash-2::ash-2::gfptransgenicplasmidincluded1.6kbofash-2promoterdriving
the full-length ash-2 cDNA fused to GFP at the C terminus. The ash-2 promoter
was amplified by PCR from wild-type (N2) genomic DNA using the following
primers, F: 59-ATAGCTAGCATCTCGTTCTTCTTCTGCTCACGG-39 and R:
59-TATGTCGACAGTGCTGAAAATCTAGTTTTTTAG-39 and subcloned into
pSM between NheI and AccI. The ash-2 cDNA isoform A (C. elegans ORFeome
library, Open Biosystems) was amplified by PCR with the following primers, F:
59-ATAGTCGACATGAGAAGCTCAAAAGGAGGTCGGGG-39 and R: 59-TA
TGGTACCATAATTTCTTGTTTAATTTCTTTC-39 and cloned into pSM con-
taining the ash-2 promoter, between AccI and KpnI. To generate the Ppie-1::rbr-
2::gfp biolistic bombardment plasmid, rbr-2::gfp was subcloned between SpeI
and XmaI into the pIC26 plasmid33containing 2.4kb of the pie-1 promoter31.
To generate the Pash-2::ash-2::gfp and Prbr-2::rbr-2::gfp biolistic bombardment
plasmids, the unc-119 rescue fragment and a PacI linker: 59-GGGGTTAA
TTAACCCCGC-39 were subcloned between SpeI and PacI in pSM Pash-2::ash-
2::gfp and pSM Prbr-2::rbr-2::gfp. To generate a bacterially expressed form of
SET-2, the SET domain of the set-2 cDNA isoform B (C. elegans ORFeome
library, Open Biosystems) was amplified by PCR with the following primers,
F: 59-ATAGGATCCCAACGTCGATTGCTTACGTCACTTGGTG-39 and R:
59-TATCTCGAGTCAATTAAGATATCCACGACACGTCTTCGC-39 and cloned
into pGEX 4T1 (Promega) between BamHI and XhoI. To generate a bacterially-
expressed fusion protein between maltose-binding protein (MBP) and ASH-2,
full-length ash-2 cDNA was amplified by PCR with the following primers,
F: 59-ATAGAATTCATGAGAAGCTCAAAAGGAGGTCGGGGACG-39 and R:
59-TATTCTAGATTAAATTTCTTGTTTAATTTCTTTCTTG-39 and then sub-
cloned between EcoRI and XbaI into the pMCH vector34. All the fragments
generated by PCR were entirely sequenced to verify that there were no mutations
introduced by the PCR amplification steps.
High-copy transgenic worm generation. Extrachromosomal-array-carrying
transgenic strains were generated using a microinjector, injecting 10ngml21of
the transgene plasmid and 60ngml21of the co-injection plasmid Podr-1::dsred
(ref. 35). Two or three independent transgenic lines for each transgene were
examined for fluorescence expression and lifespan assays. Fluorescence images
were obtained using a confocal microscope Zeiss LSM 510 META.
Low-copy integrant transgenic worm generation by biolistic transformation.
Transgenic worm strains were generated using standard biolistic transformation36
by bombarding unc-119(ed3) worms with the constructs containing the indicated
cDNAfusedtoGFPdrivenbyeitherendogenouspromotersorbythepie-1germline
specific promoter, as well as the unc-119 rescue gene. Approximately 2weeks after
bombardment, worms in which the uncoordinated phenotype was rescued were
pickedtoindividualNGMplates.Progenywerekeptat25uCfor3–4daysandwere
examinedforGFPgenomicintegrationbyPCR.GFP-positivewormswereselected
and expanded37. Two to three independent lines were generated per construct.
QuantitativeRT–PCR.TwohundredwormswerepickedtoNGMplateswithE.
coli OP50 overnight two days in a row. Worms were then picked to NGM plates
without bacteria and washed three times with M9 buffer (22mM KH2PO4,
34mM K2HPO4, 86mM NaCl, 1mM MgSO4). Worm pellets were resuspended
in TRIzol (Invitrogen), followed by six freeze-thaw cycles in liquid N2. One
microgram of total RNA was reverse-transcribed with oligo dT primers using
SuperscriptIIreversetranscriptase(Invitrogen)accordingtothemanufacturer’s
protocol. Real-time PCR was performed on a Bio-Rad iCycler using iQ SYBR
green (Bio-Rad) with the following primers: pan-actin F: 59-TCGGTATGG
GACAGAAGGAC-39, pan-actin R: 59-CATCCCAGTTGGTGACGATA-39,
ash-2F:59-CGATCGAAACACGGAACGA-39,ash-2R: 59-TGCCGGAATCTGC
AGTTTTT-39. The experiments were conducted in duplicate and the results
were expressed as 2(2(target gene number of cycles2pan-actin number of cycles)).
Microarray analysis. One-class analysis in significance analysis of microarrays
(SAM)38was performed comparing empty vector to ash-2 RNAi using a 5% false
discovery rate (FDR). Resulting SAM lists of genes regulated by ASH-2 were com-
paredforgenesthatwerespecificallychangedinN2,butnotinglp-1(e2141ts)mutant
worms. A complete linkage hierarchical clustering was performed using Gene
Cluster 3.0 (ref. 39). Clustering results were analysed further with Java Treeview40.
GOstat41wasusedtoidentifyoverrepresentedgeneontologycategoriesinthelistof
ASH-2 regulated genes that were dependent on the presence of the germline.
Antibodies. The monoclonal ASH-2 antibody was generated by injection of a
fusion protein between GST and amino acids 285–572 of C. elegans ASH-2 and
was affinity-purified by Abmart (Shanghai, China). The H3K4me3 antibody
(8580), the H3K4me1 antibody (8895), the H3K9me3 antibody (8898) and
the H3 antibody (1791) were obtained from Abcam. The H3K4me2 antibody
(07-030)wasobtainedfromUpstateBiotechnology.Theaffinity-purifiedmono-
clonal GFP antibody was a gift from A. Villeneuve.
Whole-mount immunofluorescence. Worms were washed several times to
remove bacteria and resuspended in fixing solution (160mM KCl, 100mM
Tris-HClpH7.4, 40mMNaCl,20mMNa2EGTA,1mMEDTA,10mMspermi-
dine HCl, 30mM PIPES pH7.4, 1% Triton X-100, 50% methanol, 2% form-
aldehyde)andsubjectedto tworounds of snap freezingin liquidN2. Theworms
were fixed at 4uC for 30min and washed briefly in T buffer (100mM Tris-HCl
pH7.4, 1mM EDTA, 1% Triton X-100) before a 1h incubation in T buffer
supplemented with 1% b-mercaptoethanol at 37uC. The worms were washed
with borate buffer (25mM H3BO3, 12.5mM NaOHpH9.5) and thenincubated
in borate buffer containing 10mM DTT for 15min. Worms were blocked in
PBST (PBS pH7.4, 0.5% Triton X-100, 1mM EDTA) containing 1% BSA for
30min and incubated overnight with H3K4me3 antibody (1:100), the mono-
clonal ASH-2 antibody (1:100), or with an affinity-purified GFP antibody
(1:1,000) and with Alexa Fluor 594 secondary antibody (1:25–1:100). 49,6-dia-
midino-2-phenylindole (DAPI; 2mgml21) was added to visualize nuclei. The
worms were mounted on a microscope slide and visualized using a Leica SP2
confocal system or a Zeiss Axioskop2 plus fluorescence microscope.
Protein analysis by western blot. Worms were grown synchronously to appro-
priate stages and washed off plates with M9 buffer (22mM KH2PO4,34mM
K2HPO4,86mMNaCl,1mMMgSO4).WormswerewashedseveraltimesinM9
buffer and snap frozen in liquid N2. Sample buffer (2.36% SDS, 9.43% glycerol,
5% b-mercaptoethanol, 0.0945 M Tris-HCl pH6.8, 0.001% bromophenol blue)
was added to worm pellets and they were repeatedly snap frozen in liquid N2.
Worm extracts were sonicated three times for 30s at ,15W (VirSonic 600) and
boiled for 2min before being resolved on SDS–PAGE (14%) and transferred to
nitrocellulose membranes. The membranes were incubated with primary anti-
bodies (H3K4me3, 1:1,000; H3K4me2, 1:2,000; H3K4me1, 1:500; H3K9me3,
1:1,000; H3, 1:2,000) and the primary antibodies were visualized using horse-
radish peroxidase-conjugated anti-rabbit secondary antibody (Calbiochem
401393) and ECL Plus (Amersham Biosciences).
Invitromethylationassays.Methyltransferaseassayswereperformedasdescribed
previously42. Briefly, 10mg of glutathione S-transferase (GST)-purified SET-2SET
wasincubatedwithnucleosomespurifiedfromHeLacellsinthepresenceofeither
0.1mMS-adenosyl-methionine (SAM) or2mCi[3H]SAM at20uC overnightina
doi:10.1038/nature09195
Macmillan Publishers Limited. All rights reserved
©2010

Page 7

methyltransferase reaction buffer (50mM Tris-HCl pH8.0, 10% glycerol, 20mM
KCl,5mMMgCl2,1mMdithiothreitol(DTT)and1mMphenylmethylsulphonyl
fluoride (PMSF)). Reactions were subjected to SDS–PAGE and either autoradio-
graphy or western blot as described above.
Fertilityassays.Wild-type(N2)wormstreatedwithemptyvectororash-2RNAi
bacteria or set-2(ok952), wdr-5/tag-125(ok1417), or rbr-2(tm1231) mutant
worms were synchronized. From day 3 to day 8 of life, 10 worms were placed
on NGM plates with OP50-1 in triplicate (30 worms total per condition). After
24h,theadultwormswereremovedfromeachplateandthenumberofeggswas
counted.Forash-2RNAi,thenumberofliveprogenywasalsocountedafter48h.
Statistical analyses of fertility were performed using two-way ANOVA tests with
Bonferroni post-tests, or t-tests using the mean and standard error values.
Germlinecellmorphology.N2wormsweregrownuntileitherday4orday8on
plates with bacteria expressing empty vector or ash-2 RNAi. Worms were then
fixed, permeabilized andDAPI-stained as describedabove.Worms wereimaged
on a Leica SP2 confocal microscope. Z-sections of germ cell nuclei were imaged,
compiled to stacks and manually counted in a blinded manner using MetaView
imagingsoftware.Foreachcondition,distalgonadgermcellnumbersfromthree
worms were counted and the results of two independent experiments were
analysed using a two-tailed Student’ t-test.
31. Mello, C. C. et al. The PIE-1 protein and germline specification in C. elegans
embryos. Nature 382, 710–712 (1996).
32. Fire, A., Harrison, S. W. & Dixon, D. A modular set of lacZ fusion vectors for
studying gene expression in Caenorhabditis elegans. Gene 93, 189–190 (1990).
33. Cheeseman, I. M. et al. A conserved protein network controls assembly of the
outer kinetochore and its ability to sustain tension. Genes Dev. 18, 2255–2268
(2004).
34. Venteicher, A. S., Meng, Z., Mason, P. J., Veenstra, T. D. & Artandi, S. E.
Identification of ATPases pontin and reptin as telomerase components essential
for holoenzyme assembly. Cell 132, 945–957 (2008).
35. Klassen, M. P. & Shen, K. Wnt signaling positions neuromuscular connectivity by
inhibiting synapse formation in C. elegans. Cell 130, 704–716 (2007).
36. Praitis, V., Casey, E., Collar, D. & Austin, J. Creation of low-copy integrated
transgenic lines in Caenorhabditis elegans. Genetics 157, 1217–1226 (2001).
37. Merritt, C. & Seydoux, G.In WormBook (ed. The C. elegans Research Community)
doi:10.1895/wormbook.1.148.1 Æhttp://www.wormbook.org/æ (2010).
38. Tusher, V.G.,Tibshirani, R.&Chu,G.Significance analysisofmicroarraysapplied
to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001).
39. Eisen, M. B., Spellman, P. T., Brown, P. O. & Botstein, D. Cluster analysis and
display of genome-wide expression patterns. Proc. Natl Acad. Sci. USA 95,
14863–14868 (1998).
40. Saldanha, A. J. Java Treeview–extensible visualization of microarray data.
Bioinformatics 20, 3246–3248 (2004).
41. Beissbarth, T. & Speed, T. P. GOstat: find statistically overrepresented Gene
Ontologies within a group of genes. Bioinformatics 20, 1464–1465 (2004).
42. Shi, X. et al. Modulation of p53 function by SET8-mediated methylation at lysine
382. Mol. Cell 27, 636–646 (2007).
doi:10.1038/nature09195
Macmillan Publishers Limited. All rights reserved
©2010