Promoter regulation by distinct mechanisms of
functional interplay between lysine acetylase
Rtt109 and histone chaperone Asf1
Ling-ju Lin and Michael C. Schultz1
Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7
Edited by Kevin Struhl, Harvard Medical School, Boston, MA, and approved October 5, 2011 (received for review July 14, 2011)
The promoter activity of yeast genes can depend on lysine 56 (K56)
acetylation of histone H3. This modification of H3 is performed by
lysine acetylase Rtt109 acting in concert with histone chaperone
Asf1. We have examined the contributions of Rtt109, Asf1, and H3
K56 acetylation to nutrient regulation of a well-studied metabolic
gene, ARG1. As expected, Rtt109, Asf1, and H3 K56 acetylation
are required for maximal transcription of ARG1 under inducing
conditions. However, Rtt109 and Asf1 also inhibit ARG1 under re-
pressing conditions. This inhibition requires Asf1 binding to H3-H4
and Rtt109 KAT activity, but not tail acetylation of H3-H4 or K56
acetylation of H3. These observations suggest the existence of a
unique mechanism of transcriptional regulation by Rtt109. Indeed,
chromatin immunoprecipitation and genetic interaction studies
support a model in which promoter-targeted Rtt109 represses
ARG1 by silencing a pathway of transcriptional activation that
depends on ASF1. Collectively, our results show that ARG1 tran-
scription intensity at its induced and repressed set points is con-
trolled by different mechanisms of functional interplay between
Rtt109 and Asf1.
amino acid biosynthesis ∣ arginase ∣ RNA polymerase II ∣
phases of transcription. Much of the regulation of transcription
impinges on the proteins responsible for histone acetylation—the
histone-directed lysine acetylases (KATs). One recently discov-
ered KAT being intensively studied from the viewpoint of its
regulation is Rtt109. This yeast protein catalyzes K9, K23, K27,
and K56 acetylation of histone H3. All of these reactions depend,
to a greater or lesser extent, on the conserved H3-H4 chaperone
Asf1. Specifically, Asf1 stimulates H3 K9, K23, and K56 acetyla-
tion by Rtt109 on its own, and K27 acetylation by Rtt109 in com-
plex with histone chaperone Vps75 (1–4). In current models,
transcriptional regulation by Rtt109 is ascribed to its ability to
acetylate H3, and functional interplay between Rtt109 and Asf1
in the regulation of transcription is limited to Asf1 stimulation
of Rtt109 KAT activity.
Here we examine the role of Rtt109 and Asf1 in the regulation
of ARG1, a well-studied metabolic gene of budding yeast. ARG1
is repressed in arginine-replete cells by the ArgR/Mcm DNA
binding complex consisting of Arg80, Arg81, Arg82, and Mcm1
(5–7). Upon arginine limitation, ARG1 is activated by the tran-
scription factor Gcn4 (8, 9). Chromatin reconfiguration, in par-
ticular, acetylation of residues in the amino-terminal tails of H3
and H4, makes an important contribution to the physiological
regulation of ARG1 promoter activity. The enzymes implicated
in this regulation include the KATs Gcn5 and Esa1 (10, 11).
We extended these findings by exploring the contributions of
Rtt109 and Asf1 to ARG1 regulation. In part our results support
the evidence that Asf1-dependent acetylation of H3 K56 by
Rtt109 is important for high transcription (12–15). We also find
that Asf1 and Rtt109 control ARG1 promoter activity under
he acetylation state of nucleosomal histones has a profound
influence on the initiation, elongation, and termination
repressive conditions by an unprecedented mechanism likely
involving Rtt109 inhibition of transcription stimulation by Asf1.
Results and Discussion
H3 K56acFavorsHigh Transcriptionof ARG1.We studied the mechan-
ism of ARG1 transcriptional regulation under two steady-state
conditions: repression in arginine-replete medium (yeast extract,
bactopeptone, dextrose, YPD), and induction (or activation) in
arginine-free minimal medium (composition in Table S1, M1D)
(Fig. 1A). Compared to repression, the induced configuration
of ARG1 promoter chromatin is characterized by lower H3 con-
tent and enrichment of H3 K56ac (Fig. 1 B and C). H3 K56ac
occupancy is sensitive to deletion of RTT109 and ASF1 in cells
cultured in either arginine-replete or arginine-free medium
(Fig. 1D), whereas H3 occupancy has little dependence on ASF1
(Fig. 1B). Therefore, (i) ARG1 promoter nucleosomes are
marked by H3 K56ac whether the gene is active or repressed,
(ii) high H3 K56 acetylation is a hallmark of the induced state,
and (iii) Asf1 is not uniquely required to maintain H3 promoter
occupancy under repressing or inducing conditions. Consistent
with published evidence that H3 K56 acetylation is favorable
for transcription, ARG1 expression is dampened under inducing
conditions by the H3 K56R mutation which mimics deacetylation
(Fig. 1E). Conversely, repression is dampened (ARG1 is “in-
duced”) by the K56Q and K56A mutations, which mimic the
charge state conferred by lysine acetylation (Fig. 1F).
The effects of RTT109 and ASF1 deletion on activated tran-
scription are consistent with regulation of ARG1 by a mechanism
that involves H3 K56 acetylation, as described for other genes
(12, 13). Specifically, in arginine-free minimal medium ARG1
transcription is lower in rtt109Δ and asf1Δ cells than wild type
(Fig. 1G), and the Asf1 V94R mutation which compromises bind-
ing to H3-H4 and H3 K56 acetylation (16, 17) phenocopies the
ASF1 null (Fig. 1H). These results suggest that the shared func-
tion of Rtt109 and Asf1 in the regulation of H3 K56 acetylation is
important for activated transcription of ARG1. Consistent with
this interpretation, simultaneous deletion of RTT109 and ASF1
has no greater effect on activated transcription than either dele-
tion alone (Fig. 1G), and the effect of ASF1 deletion is similar
in magnitude to, and nonadditive with, the H3 K56R muta-
tion (Fig. 1I).
ARG1RegulationbyRtt109and Asf1UnderRepression.The H3 K56R
mutation which mimics deacetylation has no effect on ARG1
Author contributions: M.C.S. and L.-j.L. designed research; L.-j.L. performed research; L.-j.L.
contributed new reagents/analytic tools; L.-j.L. and M.C.S. analyzed data; and M.C.S.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/
www.pnas.org/cgi/doi/10.1073/pnas.1111501108PNAS ∣ December 6, 2011 ∣ vol. 108 ∣ no. 49 ∣ 19599–19604
transcription under repressing conditions (Fig. 1F). Therefore,
deletion of RTT109 and the resulting global deacetylation of
H3 K56 were not expected to affect ARG1 transcription in YPD-
grown cells. Surprisingly, however, deletion of RTT109 causes
sixfold induction of ARG1 mRNA (Fig. 2A, Left; compare to
dampening of induction under arginine limitation, Right). ARG1
induction is likely due to elevated transcription initiation because
RNA polymerase (RNAP) II cross-linking to its promoter is
elevated in rtt109Δ in arginine-replete medium (Fig. 2B). Consti-
tutive DNA damage signaling in rtt109Δ cells (12) cannot account
for ARG1 induction because ARG1 is not controlled by the DNA
cells in inducing minimal medium, relative to transcription in repressive
YPD medium (latter set to one). (B) ChIP analysis of H3 cross-linking to
the promoter of ARG1 in wild-type and asf1Δ cells, under repressing and in-
ducing conditions. Occupancy in wild-type cells subject to repression is set to
one. Average of two experiments; the error bar shows the range. (C) ChIP
analysis of H3 K56ac at the promoter of ARG1 under repressing and inducing
conditions. All data points are normalized to H3 occupancy, and occupancy
under repression is set to one. (D) ChIP analysis of H3 K56ac dependency on
RTT109 and ASF1. ARG1 promoter chromatin was probed under repressing
and inducing conditions. Analysis as in C. (E) ARG1 transcription in H3 K56
mutants relative to wild type (H3 K56K), under inducing conditions. Average
of two experiments; the error bar shows the range. (F) As in E, under repres-
sing conditions. (G) Effect of RTT109 and ASF1 deletion (alone and in com-
bination) on ARG1 transcription under inducing conditions. (H) ARG1
transcription in asf1Δ and asf1V94Rcells under inducing conditions. Wild-type
transcription is set to one. (I) Effect of ASF1 deletion and H3 K56 mutation on
ARG1 transcription under inducing conditions.
ARG1 regulation by H3 K56ac. (A) ARG1 transcription in wild-type
(A) Effect of RTT109 deletion on ARG1 transcription under repressive and
inducing conditions. Under each condition, transcription in the mutant is re-
lative to wild type (set to one); this normalization highlights the fold-effect
on steady-state transcription under each condition. (B) ChIP analysis of RNAP
II cross-linking to the promoter of ARG1 under repressing conditions. RNAP II
occupancy in wild-type cells is set to one. Average of two experiments;
the error bar shows the range. (C) Wild-type and rtt109Δ cell cycle profiles
in repressive medium without or with nocodazole. (D) ARG1 transcription in
rtt109Δ cells relative to wild type in mixed populations of cells and G2/M-
arrested cells. (E) ARG1 transcription in asf1Δ and asf1V94Rcells under repres-
sive conditions (wild-type transcription is set to one). (F) ChIP analysis of TBP
cross-linking to the promoter of ARG1 under repressing conditions. Occu-
pancy in wild-type cells is set to one. PGK1 is a control gene not regulated
by arginine (10). (G) ChIP analysis of RNAP II cross-linking to the promoter
of ARG1 under repressing conditions in the presence and absence of Asf1.
RNAP II occupancy in wild-type cells is set to one. Student’s t test was used
to assess significance (?P < 0.05).
ARG1 regulation by Rtt109 and Asf1 under repressing conditions.
www.pnas.org/cgi/doi/10.1073/pnas.1111501108Lin and Schultz
damage sensor kinase Mec1 (18). Possible induction by oxidative
stress (19) is also unlikely. Deletion of RTT109 does not confer
sensitivity to exogenous oxidants (20). It follows that rtt109Δ cells
are not under a higher than normal level of endogenous oxidative
stress, and that oxidative stress signaling pathways are not con-
stitutively activated in rtt109Δ cells. Although ARG1 is normally
induced in G2 (21) and rtt109Δ cells accumulate in G2/M (1), G2/
M arrested rtt109Δcells (Fig. 2C) support higher ARG1 transcrip-
tion than arrested wild-type cells (Fig. 2D). Overall, we conclude
that Rtt109 can repress ARG1 independently of cell cycle cues, by
a mechanism that regulates the transcription process prior to
elongation and does not involve Rtt109 acetylation of H3 K56.
Rtt109 function in transcriptional activation of ARG1 requires
Asf1 (Fig. 1G). Accordingly, we hypothesized that ARG1 repres-
sion by Rtt109 also involves Asf1. Under this hypothesis, deletion
of ASF1 is expected to dampen ARG1 repression. A microarray
study provided evidence in favor of this possibility (5.9-fold relief
of repression in asf1Δ cells) (12), and targeted mRNA analysis
revealed twofold increased ARG1 expression in asf1Δ and asf1V94R
cells grown in YPD (Fig. 2E), associated with increased occu-
pancy of the ARG1 promoter by both the TATA binding protein
and RNAP II (Fig. 2 F and G). We conclude that Rtt109 and
Asf1 have a dual role at ARG1: They both promote transcription
when steady-state physiological conditions trigger high ARG1
expression and dampen promoter activity under physiological
conditions of low steady-state transcription. Repression and ac-
tivation of ARG1 both require robust binding of Asf1 to H3-H4
(Figs. 1H and 2E). Overall, our results reveal an unprecedented
role for Rtt109 in stimulation and inhibition of promoter activity,
and demonstrate that the ability of Asf1 to promote functionally
opposite states of chromatin architecture at an individual locus is
not restricted to elongation-coupled events in coding regions
(22): At an individual promoter, Asf1 can also exert positive and
negative affects on chromatin that impact on transcription.
Functional Interplay Between Rtt109 and Asf1 in ARG1 Repression.
Our analysis of ARG1 suggests previously unknown roles for
Rtt109 and Asf1 in dampening of transcription. This unexpected
outcome prompted us to consider conventional but indirect me-
chanisms that might account for the effects of RTT109 and ASF1
mutations on ARG1 repression. We sought to explain three key
observations, starting with dampening of ARG1 repression in the
absence of either Rtt109 or Asf1. Loss of Rtt109 or Asf1 could
induce ARG1 if these proteins normally support expression of the
ArgR/Mcm repressor (an equivalent mechanism could explain
ARG1 induction in mutants of the SWI/SNF chromatin remodel-
ing complex) (23). If this model is correct, then deletion of
ARG80 which encodes an essential subunit of ArgR/Mcm should
have the same effect on ARG1 transcription as deletion of either
RTT109 or ASF1. Our results do not support this prediction:
arg80Δ has a stronger inducing effect on ARG1 than either
rtt109Δ or asf1Δ, and deletion of either ASF1 or RTT109 has an
additive effect on depression caused by loss of ARG80 (Fig. 3A).
It follows that Rtt109 and Asf1 do not regulate ARG1 under
repressing conditions by modulating previously described me-
chanisms of ARG1 regulation by ArgR/Mcm. Consistent with this
interpretation, ASF1 deletion is not associated with altered
mRNA expression of any ArgR/Mcm component in budding
yeast (microarray analysis of YPD-grown cells) (12) and ARG1
induction in an RTT109 null mutant of Candida albicans is not
associated with misregulation of ArgR/Mcm subunits (24).
We next considered a straightforward explanation for the fact
that rtt109Δ (Fig. 2A) more substantially induces ARG1 transcrip-
tion in arginine-replete medium than asf1Δ (Fig. 2E). Because
histone chaperone Vps75 can regulate Rtt109 activity (25, 26),
we hypothesized that Rtt109 repression of ARG1 in wild-type
cells is imposed by parallel nonredundant pathways which sepa-
rately depend on Asf1 and Vps75. It follows that asf1Δ does not
induce ARG1 to the same extent as rtt109Δ because of residual
Vps75-dependent Rtt109 repression in the ASF1 null. If this
hypothesis is correct, then VPS75 deletion should be associated
with partial induction of ARG1 in arginine-replete medium.
ARG1 however is not induced in vps75Δ cells cultured under
repressive conditions (27). Therefore functional redundancies
between Asf1 and Vps75 do not account for the distinct effects
of rtt109Δ and asf1Δ on ARG1 repression, and the contribution
of histone chaperones to ARG1 regulation under repressing con-
ditions is limited to Asf1.
Finally we sought to explain why we obtained two different
answers to a straightforward question: How does H3 K56ac affect
ARG1 transcription under repressing conditions? That is, we
sought to understand why ARG1 is stimulated by mutations which
mimic H3 K56ac (Fig.1F,H3 K56Q and K56A), and by mutations
which eliminate H3 K56ac (Fig. 2A, rtt109Δ; E–G, asf1Δ and
asf1V94R). This discordance suggests that Rtt109 and Asf1 control
ARG1 under repressing conditions by a mechanism unrelated
to the control of H3 K56ac (installation of H3 K56 mutations
which mimic acetylation presumably override another system
of regulation by Rtt109 and Asf1).
A likely alternative to regulation of H3 K56ac by Rtt109-Asf1
is regulation of H3 K9ac. This alternative is likely in view of the
evidence that tail acetylation of H3 is important for repression
of ARG1 (10), and that Rtt109 working in concert with Asf1
catalyzes H3 K9 (4) in addition to H3 K56 acetylation. If this
ways. (A) Effect of ARG80 deletion on ARG1 transcription in rtt109Δ and
asf1Δ cells cultured under repressive conditions. (B) ChIP analysis of H3 K9ac
at the promoter of ARG1 in wild-type and rtt109Δ cells under repressing con-
ditions. Occupancy in wild-type cells is set to one. (C) ChIP analysis of H3/H4
tail acetylation at the promoter of ARG1 in wild-type and asf1Δ cells, under
repressing conditions. Occupancy in wild-type cells is set to one. H4 acetyla-
tion in asf1Δ cells is significantly different from wild type (Student’s t test,
?P < 0.05). (D) Effect of RTT109 and GCN5 deletion (alone and in combina-
tion) on ARG1 transcription under repressive conditions.
Relationship of ARG1 repression by Rtt109 and Asf1 to other path-
Lin and SchultzPNAS
December 6, 2011
mechanism underlies ARG1 repression by Rtt109, then RTT109
deletion should be associated with low H3 K9 acetylation of
ARG1 in YPD-grown cells. We observe no such association
(Fig. 3B), and RTT109 deletion does not affect H3 K14, K18,
or K23 acetylation (28). Furthermore, we do not observe loss of
overall H3 or H4 tail acetylation at ARG1 in asf1Δ cells (Fig. 3C).
It follows that neither Rtt109 nor Asf1 controls ARG1 under
repressing conditions by a mechanism that depends on H3 K9ac.
By extension, we reasoned that repression by Rtt109 is not in the
same pathway as repression that depends on Gcn5 acetylation of
H3 K9. Consistent with this proposition, rtt109Δ and deletion of
GCN5 are additive in their stimulatory effect on ARG1 transcrip-
tion under the repressing condition (Fig. 3D). We conclude that
Rtt109 and Asf1 act in parallel to Gcn5-dependent H3 acetyla-
tion to repress ARG1 transcription in arginine-replete medium.
The absence of a compelling conventional explanation for
ARG1 repression by Rtt109 and Asf1 prompted our further
characterization of this regulation. In current models, regulation
of transcription by Rtt109 and Asf1 is mostly ascribed to their
activities off chromatin, specifically their ability to collaborate
in the acetylation of soluble H3. Because Rtt109-Asf1 acetylation
of soluble H3 does not affect ARG1 repression, we turned our
attention to the possibility that Rtt109 and Asf1 control ARG1
promoter activity as components of chromatin. We focused on
Rtt109, because Asf1 occupancy of ARG1 (Fig. S1) is likely to
reflect global, nonspecific association with chromatin (12, 29).
Rtt109 can be cross-linked to the upstream activating region,
promoter, and coding region of ARG1 under arginine-replete
conditions (Fig. 4A). Importantly, Rtt109 promoter occupancy
is (i) higher under repression than under induction (Fig. 4B;
differential enrichment was not observed in the ORF—Fig. S2),
and (ii) dependent under repression on sequence-specific tran-
scription factors that modulate the strength of ARG1 repression
(Fig. 4C Left), namely, the leucine zipper protein Gcn4 and the
zinc finger protein Arg81, which is assembled specifically on the
ARG1 promoter when arginine is not limiting (30). Deletion
of GCN4 and ARG81 has little effect on Rtt109 occupancy of
POL1, a gene not known to be regulated by arginine (Fig. 4C,
Right). These relationships suggest that ARG1 repression de-
pends on promoter-targeted Rtt109 and perhaps Rtt109 regula-
tion of a nonhistone protein directly involved in chromatin
metabolism at the ARG1 promoter.
To test if ARG1 repression involves protein acetylation by
Rtt109, transcription was compared in rtt109Δ cells expressing
wild-type or catalytically inactive Rtt109 from a low-copy vector
(28) (Fig. 4D). In repressing medium M2D, ARG1 induction
associated with deletion of RTT109 is suppressed by wild-type
but not catalytically dead rtt109DD287288AA. Therefore protein
acetylation by Rtt109 is important for repression of ARG1. Col-
lectively, our results suggest that ARG1 repression depends on
KAT-dependent regulation of a nonhistone protein by chromatin-
Inhibition of transcription by Rtt109 and Asf1 is not restricted
to ARG1; these proteins also inhibit transcriptional activation
of stress response genes (31). There are however important dif-
ferences between ARG1 and the stress response genes in their
regulation by Rtt109 and Asf1. First, Rtt109 and Asf1 control
the steady-state set point of ARG1 transcription under repressing
conditions, but do not influence this phenotype of the stress
response genes. Second, the rtt109Δ and asf1Δ mutations have
the same effect on activation of the stress response genes, but
significantly different effects on ARG1 repression (Fig. 2 A
and E). Third, Asf1 controls H3 dynamics at stress-induced genes
but not the promoter of ARG1 (Fig. 1B). The notion that ARG1
and the stress response genes differ in their regulation by Rtt109
and Asf1 was confirmed by a genetic interaction experiment. In
this experiment, mRNA expression was measured in rtt109Δ
asf1Δ cells and the corresponding single mutants (Fig. 4E).
The individual mutations have identical and nonadditive effects
on activation of the stress response genes (31). The same muta-
tions have strikingly different effects on ARG1. First, rtt109Δ
ChIP analysis of Rtt109 cross-linking at the ARG1 locus under repressing con-
ditions. The occupancy measurement obtained in no-antibody control ChIPs
is set to one. Average of two experiments; the error bar shows the range. (B)
ChIP analysis of Rtt109 cross-linking to the promoter of ARG1 under repres-
sing and inducing conditions. Analysis as in A. (C) Effect of GCN4 and ARG81
deletion on Rtt109 cross-linking to the promoter of ARG1, and coding region
of POL1, under repressing conditions. Analysis as in A; results are for three
independent experiments. (D) Rtt109 KAT activity is required for ARG1 re-
pression. The averages are for three (RTT109 + and − at left; both strains
harbor the empty vector) or two (“WT” and “KAT-dead”) experiments;
the error bars respectively show the standard error and range. (E) Effect
of RTT109 and ASF1 deletion (alone and in combination) on ARG1 transcrip-
tion under repressive conditions. (F) Model of ARG1 regulation by Rtt109
and Asf1. See text for details.
Interplay between Rtt109 and Asf1 in the repression of ARG1. (A)
www.pnas.org/cgi/doi/10.1073/pnas.1111501108Lin and Schultz
more strongly affects ARG1 repression than asf1Δ (Fig. 4E; also
compare Fig. 2 A and E). Second, deletion of ASF1 suppresses
transcription induction associated with deletion of RTT109, such
that ARG1 expression in the double mutant is identical to expres-
sion in asf1Δ (Fig. 4E). Based on these results, we suggest that
Rtt109 and Asf1 are components of a unique system that
represses ARG1 independently of mechanisms that control H3
acetylation. Below we refer to this system of regulation using
Asf1 not H3Kac yes Rtt109 (ANKYR).
The most important contribution of this work is the description
of previously undiscovered modes of functional interplay between
Rtt109 and Asf1 in a system which contributes to ARG1 regula-
tion under repressing conditions. We propose the following work-
ing model to explain the architecture of this ANKYR system
(Fig. 4F; Fig. S3 explains this model in the context of the tran-
scription phenotypes of the mutants examined). As originally
suggested by Tyler and coworkers, we envisage that a population
of Asf1 molecules interacts nonspecifically with chromatin
throughout the genome (ref. 29; see also ref. 12). This nonspecific
interaction accounts for the presence of Asf1 at ARG1 (Fig. S1).
When associated with chromatin, Asf1 can potentially reconfi-
gure nucleosomes by one of two mechanisms—one that favors
transcription, and one that disfavors transcription (22). We sug-
gest that the pathway of transcription stimulation by ARG1-asso-
ciated Asf1 is intrinsically more potent than the Asf1-dependent
pathway of transcription inhibition. The notion that alternative
pathways of promoter regulation by Asf1 can have different
strengths is well established for PHO5 (13). In our model, dele-
tion of RTT109 has a stronger inducing effect than deletion of
ASF1 because, in wild-type cells, Rtt109 inhibits the positive
effect of Asf1 on ARG1 transcription. The latter regulation is
likely to occur on chromatin, because (i) both proteins occupy
the promoter of ARG1, (ii) Rtt109 occupancy is higher under
repressing conditions than under inducing conditions (Fig. 4B),
and (iii) ARG1 regulation does not involve H3 K56 acetylation
(a reaction that Rtt109 can only perform on soluble H3) (32).
Because Rtt109 is present in the promoters of numerous genes
(12, 13, 33), and Asf1 is a promiscuous chromatin-binding
protein, it is possible that the ANKYR system operates at multi-
ple loci throughout the genome.
In our model of the ANKYR system, under repressing condi-
tions Rtt109 KATactivity dampens transcriptional stimulation by
Asf1. How protein acetylation by Rtt109 (Fig. 4D) might control
this putative Asf1-dependent stimulation of ARG1 remains
a matter of speculation. There are numerous precedents for
cellular regulation of nonhistone targets by KATs that also modify
histones (34, 35). Although acetylation of cellular Asf1 has not
been reported, Rtt109 has weak KATactivity toward Asf1 in vitro
(1) and a complex containing Rtt109 and Asf1 can be recovered
from yeast cells after chemical cross-linking (36). Perhaps then
Rtt109 acetylation directly modulates Asf1 activity. Alternatively,
autoacetylation of Rtt109 (37, 38) could affect the function
of Asf1. For example, Asf1 stimulation of transcription could
be inhibited when Asf1 comes into contact with autoacetylated
Rtt109. Finally, Rtt109 might regulate another nonhistone pro-
tein involved in Asf1-dependent stimulation of ARG1.
Adkins et al. (29) have proposed that the Asf1 associated
with repressed promoters is not able to drive chromatin toward
a configuration that is permissive for transcription. How is Asf1
prevented from doing so when it is fully capable of promoting
chromatin opening in the context of transcription elongation?
This problem remains unsolved. For example, whether the posi-
tive pathway of chromatin reconfiguration that depends on Asf1
has a default state of low activity, or a default state of high activity
that is restrained under some conditions, has not been studied.
Our results suggest that Asf1 stimulation of transcription (at
ARG1) is restrained by a mechanism that involves Rtt109. This
finding raises the possibility that functional switching of Asf1
between states that close and open chromatin is under physiolo-
gical control by a pathway that depends on Rtt109.
Materials and Methods
Yeast Strains and Culture. Yeast strains (Table S2) were obtained from
published sources or constructed by methods outlined in Minard et al. (12).
The standard repressing medium was yeast extract, bactopeptone, dextrose
(YPD). The standard inducing medium (M1D) was based on yeast nitrogen
base without amino acids. Strains harboring centromere-based plasmids
were cultured in a similar repressing medium with arginine (M2D). Formula-
tions are given in Table S1. Cells were arrested in G2/M with 10 μg∕mL
nocodazole (Sigma) for 2–4 h.
Cell Cycle Profiling. Cellular DNA content was measured by flow cytome-
Chromatin Immunoprecipitation. ChIP was performed as published (12, 40)
using antibodies against the indicated proteins or epitopes. Preferential
recognition of H3 K9ac by the antibody used for the experiment in Fig. 3B
has been reported elsewhere (41). Primers for PCR and antibody suppliers
are listed in Tables S3 and S4, respectively. Quantification of PCR data has
been described elsewhere (12, 39).
RNA Analysis. Total RNA isolation and quantification by real-time RT-PCR
were performed as described previously (12). Primers including loading
controls (SCR1 and ACT1) are listed in Table S5.
Except where indicated, all graphs show the average result of at
least three independent experiments and the error bar represents the
ACKNOWLEDGMENTS. We are grateful to Alain Verreault and Zhighuo Zhang
for some of the genetic reagents used in this study, and Magnus Friis for
critical reading of the manuscript. Operating support for this work was
provided by grants to M.C.S. from the Canadian Institutes of Health Research,
the Alberta Heritage Foundation for Medical Research (AHFMR), and the
Faculty of Medicine and Dentistry, University of Alberta. Personnel support
was provided the Government of Alberta (AHFMR Research Scientist Award
to M.C.S; QEII Scholarship to L.-j.L).
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