, 1312b (2006);
et al. Matt Kaeberlein,
Life-Span Extension by Calorie Restriction"
-IndependentSIR2 Mediates HST2 Comment on "
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Comment on ‘‘HST2 Mediates
Extension by Calorie Restriction’’
Matt Kaeberlein,1* Kristan K. Steffen,2Di Hu,2Nick Dang,2Emily O. Kerr,2
Mitsuhiro Tsuchiya,2Stanley Fields,3Brian K. Kennedy2*
Calorie restriction (CR) increases life span in yeast independently of Sir2. Lamming et al. (Reports,
16 September 2005, p. 1861) recently proposed that Sir2-independent life-span extension by CR
is mediated by the Sir2 paralogs Hst1 and Hst2. Contradictory to this, we find that CR greatly
increases life span in cells lacking Sir2, Hst1, and Hst2, which suggests that CR is not mediated by
Sir2, Hst2, or Hst1.
creases yeast replicative life span and was
proposed to work through a Sir2-dependent
mechanism (1). We recently reported that CR in-
creases life span in the long-lived BY4742 strain
to a greater extent in cells lacking Sir2 than in
wild-type cells, as long as extrachromosomal
ribosomal DNA (rDNA) circles are kept at low
levels by deletion of the gene coding for the rep-
lication fork block protein, Fob1 (2). This dis-
alorie restriction (CR), instituted by re-
ducing the glucose concentration of the
media from 2% to 0.5% or lower, in-
covery has since been confirmed in a report by
Lamming et al. (3), in which they extended our
work and reported that CR fails to increase the
life span of cells lacking Sir2, Hst2, and Hst1.
Based on this finding, Lamming et al. (3) pro-
pose that Sir2-family proteins (sirtuins) are
activated by CR, and a Sir2-redundant function
of Hst2 (and to a lesserextentHst1) as a repressor
of rDNA recombination accounts for Sir2-
independent life-span extension by CR.
To test the model of Lamming et al. (3), we
generated yeast lacking Sir2, Hst1, Hst2, and
Fob1 and determined the effect of CR on life
span. Contradictory to the results of Lamming
et al. (3), we observed a significant life span
extension at 0.5% (23% increase in mean life
span, P 0 0.003), 0.05% (66% increase in mean
life span, P 0 1.3 ? 10–8), and 0.005% (72%
increase in mean life span, P 0 4.9 ? 10–9)
glucose in BY4742 sir2D hst1D hst2D fob1D
mother cells (Fig. 1A). Lamming et al. (3) also
report that a genetic model of CR, deletion of
the gene encoding hexokinase, HXK2, fails to
increase the life span of BY4742 sir2D fob1D
hst1D hst2D mother cells. In contrast, we found
that sir2D fob1D hst1D hst2D hxk2D mother
cells were significantly longer lived (62% in-
crease in mean life span, P 0 6.3 ? 10–7) than
sir2D fob1D hst1D hst2D mother cells (Fig. 1B).
These data are consistent with the model that
Sir2-independent life-span extension by CR is
not mediated by Hst2 or Hst1 (or both).
In addition to BY4742, the W303AR5 strain
was used by Lamming et al. (3) to examine
Sir2-independent life-span extension by CR. In
1Department of Pathology,2Department of Biochemistry,
University of Washington, Seattle, WA 98195, USA.
3Departments of Genome Sciences and Medicine, Howard
Hughes Medical Institute, University of Washington, Seattle,
WA 98195, USA.
*To whom correspondence should be addressed. E-mail:
firstname.lastname@example.org (M.K.); email@example.com
Fig. 1. Life-span extension by CR occurs independently of Sir2, Hst1, and
Hst2. (A) CR significantly increases the life span of BY4742 sir2D fob1D
hst1D hst2D mother cells at 0.5%, 0.05%, and 0.005% glucose. (B)
Deletion of HXK2 significantly increases the life span of BY4742 sir2D
fob1D hst1D hst2D mother cells. (C) CR fails to increase the life span of
W303AR5 mother cells. Mean life spans shown in parentheses.
www.sciencemag.org SCIENCEVOL 312 2 JUNE 2006
on March 6, 2007
previous work from the Sinclair and Guarente Download full-text
labs (4–6), experiments measuring the effect on
life span of CR by growth on low glucose have
been carried out solely in the PSY316 strain
background, with W303AR5 used for rDNA re-
combination analysis and life span experiments
involving SIR2 or FOB1. To the best of our
knowledge, the report by Lamming et al. (3) is
the first to claim life-span extension from growth
on reduced glucose in W303AR5. We therefore
further examined the effect of CR in W303AR
by determining the life span of W303AR5 cells
at different glucose concentrations ranging
from 2% to 0.05%. We found no significant
life-span extension by CR at any reduced
glucose level (Fig. 1C). It is unclear why the
dataofLamminget al. (3) differfromours.Allof
our experiments were carried out using the
(2, 7, 8), withresearchers performing the micro-
dissection blind to the identity of individual
strains within each experiment.
In addition to our findings reported here, the
notion that Sir2 and other sirtuins redundantly
mediate the CR response, as proposed by
Lamminget al. (3), is difficult to reconcile with
several observations. First, CR increases life
span by a greater magnitude in fob1D cells rel-
ative to wild-type cells, irrespective of Sir2,
Hst1, and/or Hst2 activity (2). Second, PSY316,
a strain that shows robust life-span extension in
response to CR, shows no life-span effect in
response to increased expression of Sir2 (9).
Third, the in vivo activity of Sir2, as measured
by telomere silencing, is not enhanced by CR
(7, 8). Finally, CR does not extend life span in a
sir2D FOB1 strain (1), and Lamming et al. (3)
offer no explanation as to why CR does not
activate Hst2 and Hst1 to offset the loss of Sir2
under these conditions. Further, the report by
Lamming et al. (3) that Hst2 inhibits rDNA
recombination in a Sir2-independent fash-
ion contradicts previous findings by Gasser_s
whether sirtuins function to mediate life-span
extension in response to CR. Parallel studies are
ongoing in other model systems such as
Caenorhabditis elegans, Drosophila melano-
gaster, and mice. Our data here demonstrate
that Sir2, Hst1, and Hst2 are not required either
alone or in combination for life-span extension
by CR in yeast, consistent with the model that
CR is not mediated by sirtuins in this organism.
1. S. J. Lin, P. A. Defossez, L. Guarente, Science 289, 2126
2. M. Kaeberlein, K. T. Kirkland, S. Fields, B. K. Kennedy,
PLoS Biol. 2, E296 (2004).
3. D.W. Lamming et al., Science 309, 1861 (2005); published
online 28 July 2005 (10.1126/science.1113611).
4. R. M. Anderson, K. J. Bitterman, J. G. Wood, O. Medvedik,
D. A. Sinclair, Nature 423, 181 (2003).
5. M. Kaeberlein, M. McVey, L. Guarente, Genes Dev. 13,
6. S. J. Lin et al., Nature 418, 344 (2002).
7. M. Kaeberlein et al., Science 310, 1193 (2005).
8. M. Kaeberlein et al., PLoS Genet. 1, e69 (2005).
9. M. Kaeberlein et al., J. Biol. Chem. 280, 17038
10. S. Perrod et al., EMBO J. 20, 197 (2001).
4 January 2006; accepted 4 April 2006
2 JUNE 2006VOL 312 SCIENCEwww.sciencemag.org
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