(sir2D) has identified the full complement of genes
that are subject to repression by Sir2 (11). Sep-
arately, RNA sequencing data have been published
from matched young and old cells, including RNA
expression data from these Sir2-regulated loci (12).
We reanalyzed these data to ascertain whether
Sir2-regulated genes show age-associated changes
in transcription that could reflect loss of Sir2 func-
tion. Genome-wide, we found no evidence that
Sir2-dependent gene regulation was related to
age-dependent gene regulation (Fig. 2C). Further-
more, telomeric open reading frames repressed
directly by Sir2 showed no evidence of an age-
dependent increase in transcription (fig. S2). In
short, we found no evidence that the transcrip-
tional program in sir2D cells was similar to that
in old yeast cells.
Having shown that HML and HMR were silenced during aging, we reinvestigated the sterility
phenotype reported for old cells (3, 13). We treated
young and old MA Ta cells with various amounts
Fig. 3. Old cells required a higher pheromone dose
than young cells to form a mating projection.
(A) Schematic of the experimental approach (YPD,
yeast extract, peptone, and dextrose). (B) Young and
old (on average, 14 divisions old) MATa cells (y YB4172)
were purified from 2- and 20-hour cultures, respectively, and their response to pheromone was assayed
on agar pads containing indicated a-factor concentrations. The fraction of cells not responding to a-factor
at 10 ng/ml increased with age; however, all cells responded to higher concentrations of a-factor. (C) Young
and old cells of the y YB6829 (hmlD) strain were tested
for pheromone response to a-factor (10 ng/ml). Both
wild-type and hmlD mutant cells lost pheromone sensitivity to a similar extent with age; however, hmlD cells
were more sensitive to pheromone than the corresponding wild-type cells were [y YB4172; data from
(B) are repeated for comparison]. All the plots show
mean values ± SEM; dots represent independent experiments (n ≥ 30 cells). P values were calculated
using a two-tailed t test.
Fig. 4. Formation of Whi3 aggregates contributes to the loss of pheromone
sensitivity with age. (A) The left panel shows an example sequence of an old
mother cell (green star) and its progeny exposed to a-factor (10 ng/ml). The old
mother cell buds instead of responding to pheromone, but its daughters arrest
in G1 and form mating projections. Scale bar, 5 mm. Shown on the right is the
quantification (mean values ± SEM) of the pheromone response of the first
three daughters of pheromone-insensitive old y YB4172 mothers from Fig. 3B.
(B) Old and young MATa cells were exposed to a-factor (10 ng/ml). Pheromone
insensitivity increased with age in the wild-type strain (3.2-fold between young
and old cells), whereas this effect was reduced in whi3-DpQ cells (1.9-fold
increase). Young cells were about five divisions old, and old cells were between
15 and 20 divisions old. Bars show mean values ± SEM (n > 200 total young
cells, n > 170 total old cells). P values were calculated using an unpaired two-
tailed t test. (C) Whi3 forms aggregates in old cells. Scale bars, 5 mm. (D) Quan-
tification of the fraction of young and old cells containing Whi3-3GFP aggregates.
Each dot represents an independent experiment, bars represent means, and
the P value is from a one-tailed t test. (E) Survival curves of wild-type and whi3-
DpQ strains (n = 58 wild-type y YB14326 cells, n = 78 whi3-DpQ y YB14325 cells).
Deletion of the glutamine-rich domain of Whi3 extends life span. The P value
was calculated using the log-rank (Mantel-Cox) test.