, 1142 (2003);
et al.Ao-Lin Hsu,
DAF-16 and Heat-Shock Factor
Regulation of Aging and Age-Related Disease by
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29 January 2003; accepted 14 April 2003
Regulation of Aging and
Age-Related Disease by DAF-16
and Heat-Shock Factor
Ao-Lin Hsu, Coleen T. Murphy, Cynthia Kenyon*
The Caenorhabditis elegans transcription factor HSF-1, which regulates the heat-
shock response, also influences aging. Reducing hsf-1 activity accelerates tissue
aging and shortens life-span, and we show that hsf-1 overexpression extends life-
span. We find that HSF-1, like the transcription factor DAF-16, is required for
daf-2–insulin/IGF-1 receptor mutations to extend life-span. Our findings suggest
this is because HSF-1 and DAF-16 together activate expression of specific genes,
including genes encoding small heat-shock proteins, which in turn promote lon-
gevity. The small heat-shock proteins also delay the onset of polyglutamine-ex-
process to this type of age-related disease.
Heat-shock factor activates transcription of heat-
shock genes, which encode chaperones and pro-
teases, in response to heat and other forms of
stress. Previous studies have implicated heat-
shock proteins (HSPs) in aging. For example,
mild heat stress can cause a period of decreased
mortality rate in Drosophila, and hsp70 has been
implicated in this effect (1). In addition, expres-
hsp70F increases the life-span of C. elegans (3).
Previously, we showed that reducing the
activity of C. elegans HSF-1 causes a rapid-
aging phenotype and shortens life-span (4).
Conversely, we found that animals carrying
additional hsf-1 gene copies (5), which were
resistant to heat and oxidative stress (fig. S1, A
and B), lived approximately 40% longer than
normal (Fig. 1A). Thus HSF-1 activity pro-
The FOXO transcription factor DAF-16,
which functions in the C. elegans insulin/
Department of Biochemistry and Biophysics, Univer-
sity of California, San Francisco, CA 94143–2200,
*To whom correspondence should be addressed. E-
Fig. 1. HSF-1 promotes longevity. (A) Blue, sur-
vival of wild-type animals grown on control bac-
teria containing vector alone; green, animals
grown on bacteria expressing hsf-1 dsRNA; red,
animals carrying additional copies of hsf-1
(CF1824). Two additional HSF-1 overexpressing
lines were obtained and were found to extend
life-span (table S1). (B) hsf-1 overexpression ex-
tends life-span in a daf-16–dependent manner.
Adult life-spans of wild-type and hsf-1–overex-
pressing (CF1824) animals grown on daf-16 RNAi
bacteria (green and blue lines). In addition, hsf-1
overexpression extends the life-span of animals
treated with daf-2 RNAi (red and orange lines).
(C and D) RNAi of hsf-1 completely prevents the
1(qm150) or eat-2(ad1116) mutations, from ex-
tending life-span. Animals were grown on hsf-1
RNAi bacteria from the time of hatching. Adult
life-spans of wild-type (N2) (blue), daf-2(e1370)
(red), eat-2(ad1116) (orange), and isp-1(qm150)
(green) animals grown on (C) control bacteria or
(D) hsf-1 RNAi bacteria. All experiments have
been repeated more than once with similar ef-
fects. For statistical data, see table S1.
R E P O R T S
16 MAY 2003VOL 300 SCIENCEwww.sciencemag.org
on November 14, 2007
CORRECTED 27 JUNE 2003; SEE LAST PAGE ?
IGF-1 signaling pathway (6, 7), also pro-
motes longevity (8–10). As with hsf-1, inhib-
iting daf-16 activity shortens life-span, and
elevating daf-16 activity increases life-span.
We found that the longevity phenotypes pro-
duced by hsf-1(RNAi) were similar to those
produced by daf-16(–) mutations. DAF-16 is
required for daf-2–insulin/IGF-1 receptor
mutations to increase life-span; in the short-
lived daf-16 mutants, daf-2 mutations are
unable to increase life-span (6, 7, 11). HSF-1
appeared to be required as well, because nei-
ther of two daf-2 mutations we examined,
e1370 and mu150, was able to extend the
short life-spans of hsf-1 RNAi-treated ani-
mals (Fig. 1, C and D; table S1). In contrast,
the life-spans of both daf-16(–) mutants (12,
13) and hsf-1 RNAi-treated animals (Fig. 1, C
and D) can be extended by eat-2(ad1116)
mutations [which inhibit pharyngeal pumping
and may cause caloric restriction (12)] and by
isp-1(qm150) mutations [which inhibit mito-
chondrial respiration (13)]. [Control experi-
ments indicated that hsf-1 RNAi sharply de-
creased hsf-1 mRNA levels in all of these
strains (fig. S1C).] Together these similarities
suggested the hypothesis that HSF-1, like
DAF-16, might function in the insulin/IGF-1
system. We also found that daf-16 was re-
quired for hsf-1 overexpression to extend life-
span (Fig. 1B). This, too, suggested that
DAF-16 and HSF-1 might act together to
To investigate this hypothesis, we first asked
whether DAF-16’s activities required HSF-1.
We found that hsf-1(RNAi) did not prevent
DAF-16 from accumulating in the nuclei of
daf-2 mutants (9–11) (fig. S2) or activating ex-
pression of two known downstream genes, the
metallothionein gene mtl-1 (14) and the super-
oxide dismutase gene sod-3 (15) (Fig. 2A). This
indicates that DAF-16 can function indepen-
dently of HSF-1. In addition, it implies that,
without adequate HSF-1 activity, the increased
expression of sod-3 and mtl-1 activity are not
sufficient to increase the life-span of daf-2 mu-
tants [though these and other hsf-1–independent
genes could certainly contribute to longevity
(16)]. Next, we asked whether DAF-16 was
required for HSF-1 to activate gene expression
daf-2 mutants are thermotolerant (17, 18), and
we found that their thermotolerance required
daf-16 (table S2). However, the heat-inducibil-
ity of the genes aip-1, unc-33, and the HSP-70
homolog F44E5.4 (19) was not diminished by
daf-16(–) mutations (Fig. 2B). Thus, HSF-1 can
function independently of DAF-16.
The finding that neither DAF-16 nor HSF-1
was absolutely required for the other’s activity
suggested that the two proteins might function
together to turn on a specific subset of genes.
Using DNA microarrays (16) we asked whether
any known heat-shock genes (19) were regulat-
ed by the DAF-2 pathway. Others have found
(20), and we confirmed (16), that expression of
hsp-70 and hsp-90 did not change in daf-2(–)
adults. In addition, hsf-1 mRNA levels were not
affected by daf-2 mutations (16) (Fig. 2A).
However, the expression of several heat-induc-
ible genes, including four small heat-shock pro-
tein (shsp) genes, hsp-16.1, hsp-16.49, hsp-12.6,
and sip-1, was sharply increased in animals with
Fig. 2. HSF-1 is required for the expres-
sion of a subset of DAF-16 targets in
daf-2 mutants. Likewise, DAF-16 is re-
quired for the expression of a subset of
heat-shock response genes after heat
shock. (A) RT-PCR analysis of hsf-1,
mtl-1, sod-3, and four shsps in wild-
type (N2) (lanes 1 to 3 and 10 to 12),
daf-16(mu86) (lanes 4 to 6) and daf-
2(e1370) (lanes 7 to 9 and 13 to 15)
animals grown on either control or
hsf-1 RNAi bacteria. Shown are RT-PCR
products from serial dilutions of total
RNA isolated from the animals after
RNAi treatment. act-1 (?-actin) served
as an internal control. (B) RT-PCR anal-
ysis of hsf-1, hsp-70, aip-1, unc-33, and
four shsps before (lanes 1 to 3 and 7 to
9) or after (lanes 4 to 6, 10 to 12, and
13 to 15) heat shock. RNA was harvest-
ed on day 1 of adulthood from wild-
type (N2) animals grown on control
bacteria (lanes 1 to 6) or bacteria ex-
pressing hsf-1 dsRNA (lanes 13 to 15)
as well as daf-16(mu86) animals grown
on control bacteria (lanes 7 to 12). The
heat-shock treatment was carried out
less than 5 min before the RNA was
harvested. Only slight variation was ob-
served among replicates.
Fig. 3. (A) Model. In DAF-2
pathway mutants and in nor-
mal animals subjected to
HSF-1 bind the regulatory re-
genes, including shsp (and
possibly other) genes and ac-
tivate their expression. It is
possible that DAF-2 pathway
somehow increase the ability
of HSF-1 or DAF-16 (respec-
tively) to activate, in a spe-
cific fashion, the common
target genes (dashed lines),
although this need not be the
case. Only a subset of DAF-
16’s or HSF-1’s downstream
targets require both proteins
for their expression. These
common target genes are re-
quired for the increased lon-
gevity of daf-2 mutants and
mals. Other genes (such as
sod-3 or mtl-1) may also
contribute to longevity; how-
ever, our findings suggest
that without sufficient hsf-1 activity, their increased expression is not sufficient to extend life-span.
(B) Potential DAF-16 (blue) and HSF-1 (red) binding sites, GTAAAc/tA (16, 21) and TTCTa/cGAA
(19), respectively, located upstream of the shsp genes.
R E P O R T S
www.sciencemag.orgSCIENCE VOL 30016 MAY 2003
on November 14, 2007
reduced daf-2 activity and decreased in animals
with reduced daf-16 activity (16).
We investigated the shsp genes in more
detail. First, we confirmed the microarray
data using reverse transcription polymerase
chain reaction (RT-PCR) (Fig. 2A). We then
asked whether HSF-1 was required for in-
creased shsp gene expression in daf-2 mu-
tants. We found that it was (Fig. 2A). Thus,
HSF-1 functions in the insulin/IGF-1 system.
In addition, DAF-16 was required along with
HSF-1 to activate shsp expression after heat
shock (Fig. 2B). Thus, DAF-16 functions in
the heat-shock response.
A simple model to explain these findings is
that in daf-2 mutants and in normal animals
subjected to heat shock, DAF-16 and HSF-1
both bind directly to regulatory sequences in the
shsp (and possibly other) genes (Fig. 3A). We
favor this model because we found sequences
identical to consensus DAF-16 (16, 21) and
shsp genes (Fig. 3B). Heat shock triggers DAF-
16 nuclear localization (9, 11), suggesting that it
may increase the ability of DAF-16 to activate
shsp expression. Thus it is possible that DAF-16
functions as a heat-shock factor to regulate part
of the heat-shock response. Likewise, DAF-2
pathway mutations could potentially increase
the ability of HSF-1 to activate shsp expression
Because HSF-1 and DAF-16 are both re-
quired for the longevity of daf-2 mutants, we
next asked whether their common targets, the
shsp genes, influenced life-span. RNAi of each
gene shortened the life-span of daf-2(e1370)
mutants by approximately 25% (Fig. 4B) and
that of daf-2(mu150) mutants by a similar ex-
tent (16). shsp RNAi also decreased the lon-
gevity of animals overexpressing HSF-1 (Fig.
4C). In addition, like hsf-1 RNAi, shsp RNAi
decreased the life-span of wild type, though to
a lesser extent than for daf-2 mutants (Fig. 4A).
Together these findings suggest that DAF-16
and HSF-1 increase longevity, at least in part,
by increasing shsp expression. Moreover, be-
cause at least some of these sHSPs are likely to
be functionally redundant, together they may
make a substantial contribution to longevity.
The failure of previous workers to observe
increased shsp-gfp expression in DAF-2 path-
way mutants (22) was probably due to low GFP
expression levels. However, this group found
that when such mutants are subjected to tran-
sient heat shock, both shsp expression later in
life and life-span increase substantially (22).
This has suggested the hypothesis that shsp
expression extends life-span (22). Curiously,
treating wild-type animals in the same way does
not increase shsp expression later in life, and
produces only small increases in life-span (22).
One possible explanation is that DAF-16 activ-
ity, which is required for shsp expression, is
elevated in daf-2 mutants. Heat-shocking these
animals provides additional HSF-1 activity,
which then further increases shsp expression
and life-span. Consistent with this, we found
that overexpression of HSF-1 further increased
the life-spans of daf-2(–) animals (Fig. 1B).
How might sHSPs protect cells against
aging? The sHSPs are known to form large
oligomers that bind to unfolded proteins and
prevent them from aggregating (23–25). In
aging animals, this activity may prevent ox-
idized or otherwise damaged proteins from
aggregating before they can be refolded or
degraded (although sHSPs could conceivably
influence longevity in a different way).
A fundamental mystery in biology is how
the normal aging process is coupled to the dis-
eases of aging. At least part of the answer
appears to involve the insulin/IGF-1 pathway.
For example, Huntington’s-like polyglutamine-
Fig. 4. sHSP activity extends life-span and delays polyglutamine protein aggregation. Adult life-spans of
(A) wild type, (B) daf-2(e1370) mutants, and (C) hsf-1 overexpressing animals (CF1824) grown on
control bacteria (green) or bacteria expressing dsRNA of hsp-16.1 (violet), hsp-16.49 (light blue),
hsp-12.6 (yellow), sip-1 (orange), or hsf-1 (red). Dark blue, survival of wild-type (N2) animals grown on
control bacteria containing the vector alone. (D) Fluoresence micrographs of late L4/young adult
transgenic animals expressing a fusion of polyQ (40 repeats) to yellow fluorescence protein (Q40-YFP)
in muscle cells (26). Animals were classified into separate groups according to the numbers of
aggregates they contained. (Top) Animals with less than 20 aggregates. (Bottom) Animals with more
than 60 aggregates. (E) Quantitation of number of aggregates for animals grown on control bacteria or
bacteria expressing daf-16, hsf-1, four shsps, hsp-70, sod-3, ctl-1 (catalase), and mtl-1 dsRNA. Q40-YFP
expressing animals grown on RNAi bacteria from the time of hatching were examined as 1-day-old
daf-16, hsf-1, and four shsp RNAi all significantly accelerated the onset of polyglutamine aggregates,
whereas the other stress-response genes we tested did not. [Unexpectedly, RNAi of two other hsp-70
homologs we tested, hsp-1 and hsp-70F, delayed aggregate formation (table S3). This may be due to
reduced negative feedback regulation of HSF-1 by these HSP-70s (29, 30).] At least 200 animals were
examined in each experiment. Asterisk, P ? 0.0001; pound sign, P ? 0.01 (Chi-square test). Additional
data and statistics are shown in table S3.
R E P O R T S
16 MAY 2003VOL 300SCIENCEwww.sciencemag.org
on November 14, 2007
repeat proteins expressed in C. elegans form
aggregates as the animals age, and this aggrega-
tion is delayed in long-lived insulin/IGF-1 path-
way mutants (26). Because sHSPs are known to
inhibit protein aggregation (27), we asked
whether shsp RNAi might accelerate the onset
of polyglutamine aggregates in C. elegans. We
found that it did (Fig. 4, D and E), whereas
RNAi of the other stress-response genes we
tested did not (Fig. 4). As predicted, daf-16 or
hsf-1 RNAi accelerated aggregation formation
to an even greater extent (Fig. 4E) (28). Thus,
possibly by functioning as molecular chaper-
ones, sHSPs may influence the rates of aging
and polyglutamine aggregation coordinately. In
this model, mutations in the DAF-2 pathway
delay both aging and susceptibility to this age-
related disease, at least in part, by increasing
shsp gene expression.
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31. We thank C.K. lab members for discussions and ad-
vice, J. Morley and R. Morimoto for the Q-40::YFP
lines, and J. Ahringer for the bacterial RNAi clones. D.
Lee and C.K. carried out the life-span analysis of
daf-2(mu150); hsf-1(RNAi). A-L.H. was supported by
the Canadian Institutes of Health Research. C.T.M.
was supported by Bristol-Myers Squibb Fellowship of
the Life Sciences Research Foundation. This work was
supported by grants from the Ellison Foundation and
the NIH to C.K.
Supporting Online Material
Materials and Methods
Figs. S1 to S3
Tables S1 to S3
20 February 2003; accepted 11 April 2003
Regulation of Interferon
Regulatory Factor–3 by the
Hepatitis C Virus Serine Protease
Eileen Foy,1Kui Li,2Chunfu Wang,1Rhea Sumpter Jr.,1
Masanori Ikeda,2Stanley M. Lemon,2Michael Gale Jr.1*
Persistent infections with hepatitis C virus (HCV) are likely to depend on viral
inhibition of host defenses. We show that the HCV NS3/4A serine protease
blocks the phosphorylation and effector action of interferon regulatory fac-
tor–3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3/4A
protease function by mutation or a ketoamide peptidomimetic inhibitor re-
lieved this blockade and restored IRF-3 phosphorylation after cellular challenge
with an unrelated virus. Furthermore, dominant-negative or constitutively ac-
tive IRF-3 mutants, respectively, enhanced or suppressed HCV RNA replication
in hepatoma cells. Thus, the NS3/4A protease represents a dual therapeutic
control of HCV infection.
Persistent HCV infection is a leading cause of
liver disease worldwide and is frequently re-
fractory to current interferon (IFN)-based ther-
apies (1, 2). HCV persistence is facilitated by
the ability of the virus to incorporate adaptive
mutations and to replicate as a population of
genetically distinct quasispecies (3, 4), but is
likely to result from specific disruption of host
immune responses by HCV proteins (5–7). The
HCV genome, a 9.6-kb single-stranded RNA of
Fig. 1. HCV regulation of the IRF-3 pathway. (A) Organization of the genotype 1b HCV RNA
replicating in Huh7 C5B2-3 cells (referred to as Huh7 2-3 cells) (19) and of the genotype 1a
polyprotein expressed conditionally in UHCV11 cells. The NS3/4A coding region is shaded. (B)
Immunostaining for IRF-3 in SenV-infected (SenV?) or mock-infected (SenV–) cells. From top to
bottom, IRF-3 subcellular localization in control Huh7 cells, 2-3 cells, or interferon-cured 2-3c cells
is shown (20), as are UHCV11 cells with (?HCV 1a) or without polyprotein expression (–HCV 1a).
On the right are immunoblots for NS3 and actin in corresponding cell extracts. (C) Huh7 cells, Huh7
2-3 cells, or 2-3c cells were transfected with the indicated promoter–luciferase reporter constructs
and then infected with SenV (black bars) or mock-infected (gray bars). Luciferase activities were
determined in cell extracts (mean ? S.D. from three experiments) (20).
R E P O R T S
www.sciencemag.orgSCIENCEVOL 30016 MAY 2003
on November 14, 2007
www.sciencemag.orgSCIENCEErratum post date 27 JUNE 2003
post date 27 June 2003
C O R R E C T I O N S A N D C L A R I F I C AT I O N S
R RE EP PO OR RT TS S: : “Regulation of aging and age-related disease by DAF-16 and
heat-shock factor” by A.-L. Hsu et al. (16 May 2003, p. 1142). The
authors stated incorrectly that Lithgow’s lab had previously used an
shsp::gfp fusion to assay shsp gene expression. Instead, this group used
an shsp::lacZ fusion and anti-SHSP antisera [G.Walker et al., J. Geron-
tol. 5 56 6A A, B281 (2001)]. In addition, as the Hsu et al. paper was going to
press,Walker and Lithgow reported that overexpression of hsp-16 can
extend C. elegans’ life-span [G.A.Walker, G. Lithgow, Aging Cell 2 2, 131
(2003)], and, using microarrays, McElwee et al. observed an increase in
shsp expression in insulin/IGF-1 pathway mutants [J. McElwee et al.,
Aging Cell 2 2,111 (2003)],as did Hsu et al.
on November 14, 2007