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Trans Generational Epigenetic Inheritance of Longevity in Caenorhabditis Elegans (2011)

Trans Generational Epigenetic Inheritance of Longevity in Caenorhabditis Elegans (2011)

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Published by James Clement
Chromatin modifiers regulate lifespan in several organisms, raising the question of whether changes in chromatin states in the parental generation could be incompletely reprogrammed in the next generation and thereby affect the lifespan of descendants. The histone H3 lysine 4 trimethylation (H3K4me3) complex, composed of ASH-2, WDR-5 and the histone methyltransferase SET-2, regulates Caenorhabditis elegans lifespan. Here we show that deficiencies in the H3K4me3 chromatin modifiers ASH-2, WDR-5 or SET-2 in the parental generation extend the lifespan of descendants up until the third generation. The transgenerational inheritance of lifespan extension by members of the ASH-2 complex is dependent on the H3K4me3 demethylase RBR-2, and requires the presence of a functioning germline in the descendants. Transgenerational inheritance of lifespan is specific for the H3K4me3 methylation complex and is associated with epigenetic changes in gene expression. Thus, manipulation of specific chromatin modifiers only in parents can induce an epigenetic memory of longevity in descendants.
Chromatin modifiers regulate lifespan in several organisms, raising the question of whether changes in chromatin states in the parental generation could be incompletely reprogrammed in the next generation and thereby affect the lifespan of descendants. The histone H3 lysine 4 trimethylation (H3K4me3) complex, composed of ASH-2, WDR-5 and the histone methyltransferase SET-2, regulates Caenorhabditis elegans lifespan. Here we show that deficiencies in the H3K4me3 chromatin modifiers ASH-2, WDR-5 or SET-2 in the parental generation extend the lifespan of descendants up until the third generation. The transgenerational inheritance of lifespan extension by members of the ASH-2 complex is dependent on the H3K4me3 demethylase RBR-2, and requires the presence of a functioning germline in the descendants. Transgenerational inheritance of lifespan is specific for the H3K4me3 methylation complex and is associated with epigenetic changes in gene expression. Thus, manipulation of specific chromatin modifiers only in parents can induce an epigenetic memory of longevity in descendants.

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Published by: James Clement on Oct 22, 2011
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ARTICLE
Transgenerational epigenetic inheritanceof longevity in
Caenorhabditis elegans 
Eric L. Greer
1,2
, Travis J. Maures
1
, Duygu Ucar
1
, Anna G. Hauswirth
1
, Elena Mancini
1
, Jana P. Lim
1
, Be´re´nice A. Benayoun
1
,Yang Shi
2
& Anne Brunet
1
Chromatinmodifiersregulatelifespaninseveralorganisms,raisingthequestionofwhetherchangesinchromatinstatesintheparentalgenerationcouldbeincompletelyreprogrammedinthenextgenerationandtherebyaffectthelifespanof descendants.ThehistoneH3lysine4trimethylation(H3K4me3)complex,composedofASH-2,WDR-5andthehistonemethyltransferase SET-2, regulates
Caenorhabditis elegans 
lifespan. Here we show that deficiencies in the H3K4me3chromatinmodifiersASH-2,WDR-5orSET-2intheparentalgenerationextendthelifespanofdescendantsupuntilthethird generation. The transgenerational inheritance of lifespan extension by members of the ASH-2 complex isdependent on the H3K4me3 demethylase RBR-2, and requires the presence of a functioning germline in thedescendants. Transgenerational inheritance of lifespan is specific for the H3K4me3 methylation complex and isassociated with epigenetic changes in gene expression. Thus, manipulation of specific chromatin modifiers only inparents can induce an epigenetic memory of longevity in descendants.
Transgenerationalepigeneticinheritancehasbeendescribedforsometraits, including flower symmetry and colour in plants
1–3
, progeny production in worms
4
, heat stress response and eye colour in
Drosophila
5–7
, and coat colour in mammals
8–10
. However, the trans-generational epigenetic inheritance of longevity, and more generally of complex traits, is largely undefined. Chromatin modifiers havebeen shown to regulate longevity in several species
11–18
, raising thepossibility that chromatin changes in parents might not be entirely reset between generations and thereby also regulate longevity indescendants. Deficiencies in the H3K4me3 regulatory complex com-posed of ASH-2, WDR-5 and SET-2 extend lifespan in
C. elegans
12
.We asked if perturbation of members of the H3K4me3 regulatory complex (ASH-2, WDR-5 and SET-2) only in the parental generationcould regulate the lifespan of descendants in subsequent generationsin
C. elegans
.
Transgenerational inheritance of longevity
WefirstfocusedonWDR-5,aconservedregulatorycomponentoftheASH-2 complex 
19
whose depletion decreases H3K4me3 levels
12,20–22
and extends lifespan in worms
12
. To test whether longevity could beinherited in a transgenerational epigenetic manner, we crossedwild-type (
1
/
1
) males with
wdr-5
(
ok1417 
) mutant (
wdr-5
/
wdr-5
)hermaphrodites to generate F1 heterozygous hermaphrodites(Fig. 1a). These F1 heterozygous hermaphrodites were genotypedand then self-crossed to generate F2 hermaphrodites (wild type,heterozygous and homozygous at the
wdr-5
locus), which were geno-typed after they had laid F3 generation progeny. In parallel, wecrossed a wild-type male with a wild-type hermaphrodite to generatepure wild-type descendants and control for any beneficial longevity effects that could come from crossing rather than self-matin(Fig. 1a). Longevity of genetically wild-type descendents from wild-type or
wdr-5
mutant ancestors was compared in the F3, F4 and F5generations. Interestingly, genetically wild-type F3 descendants fromP0
wdr-5
parents (
1
/
1
from P0
wdr-5
parents) still showed a
,
20%extension of lifespan (
,
0.0001) compared to descendants frompure wild-type parents (
1
/
1
from P0 WT parents) (Fig. 1b). This20%lifespanextensionwassimilarinmagnitudetothelifespanexten-sion of pure F3
wdr-5
(
ok1417 
) mutants (
wdr-5/wdr-5
) (Fig. 1b). Thelifespan of genetically wild-type descendants from
wdr-5
(
ok1417 
)mutant parents (
1
/
1
from P0
wdr-5
parents) was still extended inthe F4 generation (Fig. 1c), but was no longer extended in the F5generation (Fig. 1d). Thus,
wdr-5
deficiency only in the parentalgeneration can extend the lifespan of subsequent generations.Because the lifespan of F5 generation wild-type descendants from
wdr-5
mutant parents is no longer extended, the lifespan extensionobserved in the F3 and F4 generations is unlikely to be due to extrane-ous mutations that might have been present in the parental
wdr-5
mutant strain. Instead, the transgenerational inheritance of longevity may be due to epigenetic changes in H3K4me3 itself or in anothermoleculethatcanonlybeinheritedforalimitednumberofgenerations.We next asked if a transgenerational epigenetic heritability of life-span was also observed with SET-2, the H3K4me3 methyltransferaseenzyme that functions together with ASH-2 and WDR-5 to regulateH3K4me3 levels
12,20–22
and longevity in
C. elegans
12
(Fig. 2). Similar towhat we observed for
wdr-5
, genetically wild-type descendants from
set-2
(
ok952
) mutants still had a
,
30% extension of lifespan(
,
0.0001) in the F3 and F4 generations (Fig. 2b, c), but not inthe F5 generation (Fig. 2d). Genetically wild-type F3 descendantsfrom the reverse cross—P0
set-2
(
ok952
) males crossed with wild-typehermaphrodites—were also long-lived (Supplementary Table 1),indicatingthattransgenerationalinheritanceoflongevityisnotlinkedto a particular gender in the parental generation.ASH-2 is important for the conversion of H3K4 dimethylation(H3K4me2) to H3K4me3 (ref. 23). Knockdown of 
ash-2
by RNAinterference (RNAi) in worms decreases global H3K4me3 levels attheL3stage
12,22
andextendslongevity 
12
.Weaskedif 
ash-2
knockdownonly in the parental generation affected the lifespan of severalgenerations of descendants. Wild-type parent worms (P0) wereplaced on plates with bacteria expressing RNAi to
ash-2
from birthto the larval stage L4, then switched every day for 3days onto plates
1
DepartmentofGenetics,StanfordUniversity,300PasteurDrive,Stanford,California94305,USA.
2
CellBiologyDepartment,HarvardMedicalSchoolandDivisionofNewbornMedicine,Children’sHospital,300 Longwood Avenue, Boston, Massachusetts 02115, USA.
0 0 M O N T H 2 0 1 1 | V O L 0 0 0 | N AT U R E | 1
Macmillan Publishers Limited. All rights reserved
 ©2011
 
containing OP50-1 bacteria and streptomycin to selectively preventthe growth of RNAi-expressing bacteria (Fig. 3a). Endogenous
ash-2
messenger RNA and ASH-2 protein levels were significantldecreased in the P0 generation, but returned to normal levels insubsequent generations (Fig. 3b, c), indicating that
ash-2
RNAi isnot itself inherited. The lifespan of worms from the F1, F2 and F3generations in which
ash-2
had been knocked down only in the P0parental generation was still significantly extended (19–27%,
,
0.0001) compared to that of descendants of worms treated withempty vector control in the P0 parental generation (Fig. 3d–g). By contrast, F4 generation descendants no longer had extended lifespan(Fig. 3h). We obtained similar results after bleaching P0 worms toavoid potential carry over of RNAi-expressing bacteria (datanot shown). Thus, alteration of the components of the H3K4me3
bc d
+/+ (from P0
 set-2
parents)
 set-2/set-2
+/+ (from P0WT parents)+/+ (from P0
 set-2
parents)
 set-2/set-2
+/+ (from P0WT parents)+/+ (from P0
 set-2
parents)
 set-2/set-2
+/+ (from P0WT parents)
F300.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
00.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
00.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
0510152025303540 Age (days)0510152025303540 Age (days)30.9%,
P
< 0.0001F5–1.8%,
P
= 0.567405101520253035 Age (days)F427.0%,
P
< 0.0001
a
+/ 
 set-2
+/ 
 set-2
F1+/++/++/ 
 set-2set-2/set-2
+/+F2+/+F3
set-2/set-2
+/++/+F4
set-2/set-2
+/++/+F5
set-2/set-2
+/++/+
 set-2/set-2
+/+P0+/++/+
Figure 2
|
Genetically wild-type descendants from
set-2 
mutant parentshave extended lifespan for several generations.
, Scheme for generating wild-type (
1
/
1
) descendants from
set-2
(
ok952
) mutant worms (
set-2/set-2
).
b
d
, Lifespan of genetically wild-type F3 (
b
), F4 (
c
) and F5 (
d
) descendantsfrom
set-2
(
ok952
) mutant worms (
1
/
1
from P0
set-2
parents) compared todescendants of wild-type worms (
1
/
1
from P0 WT parents). Mean lifespanand statistics are presented in Supplementary Table 1.
a bc d
00.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
00.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
00.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
05101520253035 Age (days) Age (days)
+/+ (from P0
wdr-5
parents)
wdr-5/wdr-5
+/+ (from P0 WT parents)
F319.6%,
P
< 0.0001
+/+ (from P0
wdr-5
parents)
wdr-5/wdr-5
+/+ (from P0 WT parents)
F5
6.8%,
P
= 0.119805101520253035 Age (days)05101520253035
+/+ (from P0
wdr-5
parents)
wdr-5/wdr-5
+/+ (from P0 WT parents)
F4
21.8%,
P
< 0.0001+/ 
wdr-5
+/ 
wdr-5
F1+/++/++/ 
wdr-5wdr-5/wdr-5
+/+F2+/+F3
wdr-5/wdr-5
+/++/+F4
wdr-5/wdr-5
+/++/+F5
wdr-5/wdr-5
+/++/+
wdr-5/wdr-5
+/+P0+/++/+
Figure 1
|
Genetically wild-type descendants from
wdr-5 
mutant parentshave extended lifespan for several generations.
, Scheme for generating wild-type(
1
/
1
)descendantsfrom
wdr-5
(
ok1417 
)mutantworms(
wdr-5/wdr-5
).
b
d
, Lifespan of genetically wild-type F3 (
b
), F4 (
c
) and F5 (
d
) descendantsof 
wdr-5
(
ok1417 
) mutant worms (
1
/
1
from P0
wdr-5
parents) compared todescendants of wild-type worms (
1
/
1
from P0 WT parents). Mean lifespanand statistics are presented in Supplementary Table 1.
RESEARCHARTICLE
2 | N AT U R E | V O L 0 0 0 | 0 0 M O N T H 2 0 1 1
Macmillan Publishers Limited. All rights reserved
 ©2011
 
methyltransferase complex (ASH-2, WDR-5 and SET-2) in parentsaffects the lifespan of descendants, supporting the possibility thattransgenerationalinheritanceoflongevityisduetoepigeneticchangesthat may only be inherited for a limited number of generations.
Importance of the H3K4me3 demethylase and germline
The H3K4me3 demethylase RBR-2 is necessary for the lifespan exten-sion caused by deficiencies in members of the ASH-2 complex 
12
. Weasked if the transgenerational extension of longevity induced by defi-ciencies in members of the ASH-2 complex is dependent on RBR-2.The lifespan of genetically wild-type F3 descendants from P0
wdr-5
parents (
1
/
1
from P0
wdr-5
parents) was no longer extended in thepresenceof 
rbr-2
RNAi(Fig.4a,b).Similarly,F3wild-typedescendantsfrom
set-2;rbr-2
parents (
1
/
1
from P0
set-2;rbr-2
parents) were nolongerlong-lived(SupplementaryFig.1). Together, thesedataindicatethatthetransgenerationalinheritanceoflongevityduetodeficienciesinH3K4trimethylationcomplexmembersisdependentontheH3K4me3demethylase RBR-2. The fact that the longevity of wild-type descen-dantsof 
wdr-5
and
set-2
mutantsisrevertedbydeficienciesin
rbr-2
alsoindicates that this extended lifespan is unlikely to result from extrane-ous mutations in
wdr-5
or
set-2
strains.
rbr-2
mutation or knockdowndid not leadtoa shorteningoflifespanindescendants(SupplementarFig. 2), indicating that by itself, RBR-2 deficiency does not affectlongevity in a transgenerational manner.LongevityduetomodulationoftheASH-2complexisdependentona functioning germline
12
. To test if wild-type descendants of worms
a bgcde fh
  a  s   h  -   2 
  m   R   N   A  r  e   l  a   t   i  v  e   t  o
  p  a  n   a  c   t   i  n
   m   R   N   A
***
NSNS0P0F1F2
   C  o  n   t  r  o   l
  a  s   h  -   2
   C  o  n   t  r  o   l
  a  s   h  -   2
   C  o  n   t  r  o   l
  a  s   h  -   2
   C  o  n   t  r  o   l
RNAi:0.20.40.60.811.21.400.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
00.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
00.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
00.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
00.20.40.60.81.0
   F  r  a  c   t   i  o  n  a   l   i  v  e
05101520253035 Age (days)05101520253035 Age (days)05101520253035 Age (days)05101520253035 Age (days)05101520253035 Age (days)Control RNAi
 ash-2
RNAiP024.6%,
P
< 0.0001Control RNAi
 ash-2
RNAiF219.4%,
P
< 0.0001Control RNAi
 ash-2
RNAiF119.0%,
P
< 0.0001Control RNAi
 ash-2
RNAiF327.2%,
P
< 0.00012
 ash-2
RNAiControl RNAiOP50-1 bacteriaDays:311112ControlControlControlP0F1F2
 ash-2 ash-2 ash-2
P0F1F2
*******
Egg lay for subsequent generation. . .. . .
α
-tubulin ASH-2
F1P0 F2
*
      a       s        h    -       2
   C  o  n   t  r  o   l
      a       s        h    -       2
   C  o  n   t  r  o   l
      a       s        h    -       2
RNAi:
Control RNAi
 ash-2
RNAiF40.4%,
P
= 0.7137
Figure 3
|
Knockdown of 
ash-2 
only in the parental generation extendslifespan for several generations.
, Scheme for generating wild-typedescendants from RNAi-treated parents.
b
,
ash-2
mRNA levels at day 7 indifferent generations of worms treated with
ash-2
RNAi or empty vector(control) only in the P0 generation. Mean
6
s.e.m. of three independentexperiments.
***
5
0.0002 with paired
-test.
c
, ASH-2 protein levels at L3stage in different generations of worms treated with
ash-2
RNAi or empty  vector (control) only in the P0 generation. Representative of two independentexperiments.
*
,non-specificband;arrow,ASH-2;arrowhead,proteinrelatedtoASH-2, possibly a degradation product.
d
h
, Lifespan of P0 (
d
), F1 (
e
), F2(
), F3 (
) and F4 (
h
) generations of worms with RNAi knockdown of 
ash-2
orcontrol RNAi (empty vector) in parents only. Mean lifespan and statistics arepresented in Supplementary Table 2.
ARTICLERESEARCH
0 0 M O N T H 2 0 1 1 | V O L 0 0 0 | N AT U R E | 3
Macmillan Publishers Limited. All rights reserved
 ©2011

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