Molecular Biology of the Cell
Vol. 19, 608–622, February 2008
Conversion of a Replication Origin to a Silencer through a
Pathway Shared by a Forkhead Transcription Factor and an
S Phase Cyclin
Laurieann Casey,* Erin E. Patterson,†Ulrika Mu ¨ller,* and Catherine A. Fox*†
Department of *Biomolecular Chemistry and†Laboratory of Genetics, University of Wisconsin School of
Medicine and Public Health, Madison, WI 53706
Submitted April 10, 2007; Revised September 19, 2007; Accepted November 20, 2007
Monitoring Editor: Orna Cohen-Fix
Silencing of the mating-type locus HMR in Saccharomyces cerevisiae requires DNA elements called silencers. To establish
HMR silencing, the origin recognition complex binds the HMR-E silencer and recruits the silent information regulator
(Sir)1 protein. Sir1 in turn helps establish silencing by stabilizing binding of the other Sir proteins, Sir2–4. However,
silencing is semistable even in sir1? cells, indicating that SIR1-independent establishment mechanisms exist. Further-
more, the requirement for SIR1 in silencing a sensitized version of HMR can be bypassed by high-copy expression of
FKH1 (FKH1hc), a conserved forkhead transcription factor, or by deletion of the S phase cyclin CLB5 (clb5?). FKH1hc
caused only a modest increase in Fkh1 levels but effectively reestablished Sir2–4 chromatin at HMR as determined by
Sir3-directed chromatin immunoprecipitation. In addition, FKH1hcprolonged the cell cycle in a manner distinct from
deletion of its close paralogue FKH2, and it created a cell cycle phenotype more reminiscent to that caused by a clb5?.
Unexpectedly, and in contrast to SIR1, both FKH1hcand clb5? established silencing at HMR using the replication origins,
ARS1 or ARSH4, as complete substitutes for HMR-E (HMR?E::ARS). HMR?E::ARS1 was a robust origin in CLB5 cells.
However, initiation by HMR?E::ARS1 was reduced by clb5? or FKH1hc, whereas ARS1 at its native locus was unaffected.
The CLB5-sensitivity of HMR?E::ARS1 did not result from formation of Sir2–4 chromatin because sir2? did not rescue
origin firing in clb5? cells. These and other data supported a model in which FKH1 and CLB5 modulated Sir2–4 chromatin
and late-origin firing through opposing regulation of a common pathway.
Chromatin structures vary with genome position, creating
structural heterogeneity along chromosomes that modulates
every aspect of DNA metabolism (Fischle et al., 2003). Spe-
cific DNA sequence elements form the foundation for this
heterogeneity. For example, certain DNA sequences act to
establish centromeric chromatin required for chromosome
segregation (Cleveland et al., 2003; Henikoff and Dalal, 2005).
In addition, some chromatin structures, such as heterochro-
matin, can dominate the functional capacity of DNA se-
quence elements across chromosomal domains or even over
an entire chromosome. Heterochromatin, for example, can
delay or repress initiation by DNA replication origins and
initiation of transcription from promoters (Gomez and
Brockdorff, 2004; Weinreich et al., 2004; Chang et al., 2006).
Thus, a competing balance exists between DNA sequence
elements that perform different functions or establish differ-
ent types of chromatin structures (Kamakaka, 1997; Donze
and Kamakaka, 2002; Valenzuela and Kamakaka, 2006).
Genetic analyses of transcriptional silencing of the HMRa
locus in Saccharomyces cerevisiae reveal that perturbations in
the cell cycle tip the balance between the types of chromatin
that can form at a particular chromosomal domain (Laman et
al., 1995; Fox and Rine, 1996; Ehrenhofer-Murray et al., 1999).
HMRa silencing defines a form of transcription repression
that requires the assembly of a specialized chromatin struc-
ture called silent chromatin (Fox and McConnell, 2005). For-
mation of silent chromatin requires small DNA elements
called silencers that bind a collection of sequence-specific
DNA binding proteins. HMRa contains two silencers,
HMR-E and HMR-I that flank opposite ends of this ?3000-
base pair locus (Loo and Rine, 1995). HMR-E is necessary
and sufficient for HMRa silencing; the importance of HMR-I
can be observed only under conditions in which silencing
has been compromised (Fox et al., 1995; Rivier et al., 1999).
HMR-E, the better characterized of the two silencers, con-
tains a binding site each for the origin recognition complex
(ORC), and the Rap1 and Abf1 proteins. HMRa silencing is
not essential for yeast cell viability, but ORC is because it
functions as the eukaryotic initiator, the protein complex
that marks chromosomal sites as DNA replication origins
(reviewed in Bell, 2002). Rap1 and Abf1 are also essential
multifunctional nuclear proteins (reviewed in Shore, 1994).
The role of the HMR-E silencer-binding proteins is to
recruit, via multiple independent protein–protein interac-
tions, four silent information regulator (Sir) proteins, Sir1, -2,
-3, and -4, nonhistone chromatin-binding proteins dispens-
able for cell viability but necessary for silencing (reviewed in
Gasser and Cockell, 2001; Rusche et al., 2003; Fox and
McConnell, 2005). One important interaction occurs directly
between Sir1 and ORC (Hou et al., 2005; Hsu et al., 2005). The
Sir1-ORC complex helps recruit and/or stabilize the binding
of the three other Sir proteins, Sir2, -3, and -4 to the silencer
(Rusche et al., 2002). Another key set of interactions occurs
between Rap1 and Sir3 and Sir4 proteins (Moretti and Shore,
This article was published online ahead of print in MBC in Press
on November 28, 2007.
Address correspondence to: Catherine A. Fox (firstname.lastname@example.org).
608© 2008 by The American Society for Cell Biology
2001). The multiple protein–protein interactions that recruit
the four Sir proteins to HMR-E define the establishment/
nucleation phase of silent chromatin assembly. In the second
phase, Sir2, an enzyme, deacetylates nucleosomes neighbor-
ing HMR-E; deacetylated nucleosomes in turn promote
binding of additional Sir2–4 complexes until a higher-order
Sir2–4 silent chromatin structure forms at HMRa (re-
viewed in Rusche et al., 2003; Moazed et al., 2004; Fox and
One key feature of this model is that Sir1 differs substan-
tially from Sir2–4 because its role is confined to silencers
where it enhances the assembly and/or reduces the disas-
sembly of Sir2–4 chromatin, but is not intrinsically required
either for its formation or function (Pillus and Rine, 1989,
2004; Xu et al., 2006). Thus, in contrast to Sir2–4, Sir1 is not
a critical structural component of HMRa silent chromatin.
Another notable feature that distinguishes SIR1 from the
other SIRs is that its requirement in HMRa silencing can be
partially bypassed by perturbations in the cell cycle (Laman
et al., 1995). For example, a deletion of the major S phase
cyclin CLB5 that is required for timely S phase progression
(Donaldson et al., 1998; Gibson et al., 2004) can partially
bypass the requirement for SIR1 (Laman et al., 1995). In
addition, mutations in the HMR-E silencer are also rescued
by cell cycle perturbations caused by drugs or certain mu-
tations (Axelrod and Rine, 1991; Laman et al., 1995; Ehren-
hofer-Murray et al., 1999). These data provide evidence that
other molecular interactions can compensate for the role of
Sir1 in transcriptional silencing and reveal that specific per-
turbations in the cell cycle can tip the balance between silent
and permissive chromatin formation at HMRa. Thus, HMRa
silencing serves as an experimentally tractable model for
dissecting how the cell cycle can modulate the structure and
function of a defined chromatin domain.
In a previous study, we identified forkhead homologue
FKH1 as a high-copy suppressor of defects in HMRa silenc-
ing caused by a sir1? mutation (Hollenhorst et al., 2000).
Fkh1 and its paralogue Fkh2 are evolutionarily conserved
transcription factors that bind directly to promoters within a
group of genes named the CLB2-cluster (reviewed in
Breeden, 2000; Futcher, 2000). Transcription of these genes is
repressed in late M, G1, and early S phases but is activated
beginning in late S phase and through G2 and early M
phases (Spellman et al., 1998). Genes within this cluster
encode proteins including the major G2/M phase cyclin
Clb2 that drive progress through M phase. Fkh2 is the
primary regulator of CLB2-cluster genes, repressing tran-
scription of these genes in G1 phase and stimulating their
transcription in late S and G2/early-M phase. (Koranda et
al., 2000; Kumar et al., 2000; Pic et al., 2000; Zhu et al., 2000;
Reynolds et al., 2003). It is less clear how Fkh1 normally
functions in CLB2-cluster transcription. FKH1 can partially
compensate for loss of FKH2 because fkh1?fkh2? cells exhibit
a substantially greater reduction in CLB2-cluster transcrip-
tion than FKH1fkh2? cells, indicating that Fkh1 can activate
CLB2 transcription. However, paradoxically, the normal role
for Fkh1 in wild-type cells seems to be as a negative regu-
lator of CLB2-cluster transcription during G2/M, thus atten-
uating the level of transcription that can be achieved by
Fkh2-mediated activation (Hollenhorst et al., 2000, 2001;
Sherriff et al., 2007). Although it is unclear how the role(s) of
FKH1 at the CLB2-cluster is related to its ability to affect
silencing, it is known that the DNA binding domain of Fkh1
is critical for it ability to modulate SIR1-bypass HMR silenc-
ing (Hollenhorst et al., 2000).
In this report, we addressed the mechanism(s) by which
FKH1hcbypasses the requirement for SIR1 in silencing. Un-
expectedly (Hollenhorst et al., 2000), the genetic and cell
cycle data reported here provide evidence against the role of
FKH1 in CLB2 transcription serving as the primary factor in
FKH1hc-dependent silencing. Specifically, although FKH1hc
reduced CLB2 transcription, reductions in CLB2 were insuf-
ficient to mimic FKH1hc. Instead, the data generated through
a combination of genetic analyses, Sir3-directed chromatin
immunoprecipitations (ChIPs) and two-dimensional (2-D)
origin mapping experiments were most consistent with a
model in which FKH1 and the S phase cyclin CLB5 act as
opposing regulators that converge on a common target(s)
that inhibit late replication origin firing and promote Sir2–4
chromatin assembly. The data were also consistent with the
idea that this pathway favors distinct SIR1-independent mo-
lecular interactions that contribute to Sir2–4 protein associ-
ation with HMR.
MATERIALS AND METHODS
Strains and Plasmids
Yeast strains used in this study (Table 1) were constructed using standard
yeast molecular genetics and recombinant DNA techniques (Sambrook et al.,
1989; Guthrie and Fink, 1991). Five different plasmids were used in this study
as indicated in the figure legends: pRS426 (Sikorski and Hieter, 1989), pCF99
(SIR1 in pRS316; Gardner et al., 1999; Hollenhorst et al., 2000), pCF345 (SIR1
in Yep24; Hollenhorst et al., 2000), pCF480 (FKH1 in pRS426; Hollenhorst et al.,
2000), and pCF942 (FKH1 in pRS316). Cells were grown at 30°C in standard
rich medium (YPD), in minimal medium supplemented with casamino acids
(CAS) (to select for URA3-containing plasmids), or in synthetic media (SC)
lacking defined supplements to select for diploids and/or plasmids as appro-
priate and described in the text (Guthrie and Fink, 1991). All strains were
isogenic to W303-1A except the MATa cells used for mating lawns.
Semiquantitative Mating Assay
The MAT? cells examined for silencing by mating efficiency were grown in
log phase for 2 d, and then they were mixed with an excess of MATa cells
(JRY19). The most concentrated samples of MAT? cells analyzed were 5 ? 106
cells/ml in a total volume of 50 ?l. Tenfold serial dilutions of this concentra-
tion were generated in 50-?l final volumes. Six microliters of each dilution
was analyzed per drop on either YPD or CAS solid agar medium, as appro-
priate, to determine cell counts. To the remainder of the cells, an excess of
MATa cells (15 ?l of log-phase cells concentrated to 5 OD) were added and
mixed. Eight microliters of this mixture was plated to synthetic solid agar
medium appropriately supplemented to select for diploids (and the retention
of a URA3 plasmid, if appropriate). Plating efficiencies indicated that equiv-
alent numbers of cells were being compared for every mating comparison
RNA was isolated from yeast as described previously (Fox et al., 1995), and 15
?g of total RNA was analyzed per lane. RNA blot hybridization was per-
formed with multiprime-labeled DNA probes complementary to a1 mRNA,
CLB2 mRNA, or SCR1 RNA. The probes for a1 mRNA and SCR1 RNA were
described previously (Fox et al., 1995, 1997). Template for the CLB2 probe was
generated by PCR by using the following primer pair: forward, GTCCAAC-
CCAATAGAAAACAC and reverse, CATGCACCGTCTGTCTCTGATG. This
probe was also used in a previous study (Hollenhorst et al., 2000).
Anti-Sir3 monoclonal antibodies were raised against full-length Sir3 purified
from baculovirus-infected Sf9 cells as described previously (Georgel et al.,
2001), and they are available from Neoclone at http://www.neoclone.com/
(the antibodies used to make the ChIP cocktail were 184A, 185A, and 184C).
Anti-Fkh1 polyclonal antibodies were raised in rabbits (Harlan Labs, Madi-
son, WI) to a 6xHIS–Fkh1 fusion protein encoding the N-terminal 302 amino
acids of Fkh1 (pCF1564) expressed in Escherichia coli (BL21 cells) and purified
using ion exchange and nickel-affinity chromatography. These antibodies
were affinity purified and used at a 1:200 dilution for protein immunoblotting
(Harlow and Lane, 1999). Anti-hemagglutinin (HA) monoclonal antibodies
were from Covance Research Products (Princeton, NJ).
ChIP was performed as described previously (Strahl-Bolsinger et al., 1997)
except: Sir3 antibodies were cross-linked to protein A-Sepharose beads (GE
Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) by using
standard methods before ChIP (Harlow and Lane, 1999). 1/50 of the immu-
FKH1 and CLB5 in Silencing and Replication
Vol. 19, February 2008609
noprecipitated DNA and 1/500 of the total DNA were subjected to 26 cycles
of polymerase chain reaction (PCR) by using primers specific for HMRa or
ADH4 or the appropriate ARS (Gardner and Fox, 2001). Primers to X/Ya,
Ya/Z, and boundary elements were as described previously (Rusche et al.,
2002). PCR products were separated on a 1% agarose gel, and band intensities
were quantified using video densitometry analysis and Labworks analysis
software (UVP, Upland, CA).
To compare levels of Fkh1 expressed from a chromosomal copy of FKH1 and
from a 2? plasmid (FKH1hc) quantitatively (Figure 2), crude yeast extracts
were prepared as described previously (Gardner et al., 1999) from wild-type
cells transformed with either empty vector (pRS426; pCF225) or with a 2?
plasmid expressing FKH1 (FKH1hc; pRS426 with FKH1; pCF480) and from a
fkh1? cells transformed with vector. To avoid distortions in the gel that could
affect quantification, lysates from fkh1? cells were used to dilute the lysates
from FKH1 or FKH1hccells, ensuring that the same amount of total protein
was present in each lane. Four microliters of crude lysates was examined per
lane by SDS-polyacrylamide gel electrophoresis (PAGE) by using a 10%
acrylamide gel and standard protein blotting methods were used. To deter-
mine whether protein transfer to the blot was the same for all lanes, the blot
was prestained with Poncea S before incubation with antibody.
Cell Cycle Arrest and Release Experiments
Cells were grown and harvested in log phase (23 or 30°C) from CAS liquid
medium and arrested in G1 with ?-factor for 150 min (5 ?M for bar1? cells; 25
?M for BAR1 cells). The cells were then released from ?-factor arrest by
washing with and releasing into fresh medium. Every 15 min, aliquots were
harvested for analysis of bud morphology and scored as indicated in Figure
2. Alternatively cells were harvested for analyses of DNA content by flow
cytometry as described previously (Weinreich et al., 1999) except that Sytox
Green (Invitrogen, Carlsbad, CA) at 1 ?M was used to label DNA.
Two-Dimensional Origin Mapping
2-D origin mapping was performed as described previously (Fox et al., 1995).
Cells were grown at 23°C in 2 liters of complete rich medium, or, if cells
contained a URA3 plasmid, at 30°C in 2 liters of CAS medium. DNA from
each sample was analyzed for replication intermediates (RIs) by digestion
with HindIII for HMR-E::ARS1 and NcoI for ARS1. DNA was enriched for RIs
with BND cellulose after restriction digest. DNA was separated in two di-
mensions and examined by DNA blot hybridization by using primers specific
for each region (Fox et al., 1995). Probes were created using Megaprime DNA
labeling system (GE Healthcare).
FKH1 is a high-copy suppressor of an HMRa silencing defect
caused by a sir1? mutation (Hollenhorst et al., 2000). The
role of FKH1, together with FKH2, in controlling CLB2-
cluster transcription raised the possibility that FKH1-medi-
ated silencing was related to perturbations of the cell cycle.
To address this possibility and better define how Fkh1 mod-
Table 1. Strains used in this study
MATa his4 leu2 trp1 ura3
MATa ade2-1, his3-11, 15 leu 2-3,112 trp1-1 ura3-1 can1-100
MAT? ade2-1, his3-11, 15 leu 2-3,112 trp1-1 ura3-1 can1-100
MATa his4 leu2 trp1 ura3
JRY3009 HMR-SSa sir1?::LEU2
JRY3009 HMR-SSa sir1?::TRP1
JRY3009 HMR-SSa sir3?::LEU2
JRY3009 HMR-SSa sir1?::LEU2 sir2?::TRP1, LEU2::sir2N345A
JRY2334 HMR-SSa ?I bar1?::HIS3
JRY2334 HMR-SSa ?I bar1?::HIS3 fkh2?::HISG
JRY3009 HMR-SSa sir1?::LEU2 fkh1?::TRP1
JRY3009 HMR-SSa sir1?::LEU2 fkh2?::HIS3
JRY3009 HMR-SSa sir1?::LEU2 clb1?::HIS3
JRY3009 HMR-SSa sir1?::LEU2 clb2?::TRP1
JRY3009 HMR-SSa sir1?::LEU2 clb5?::kanMx
HMR-SSa sir1?::LEU2 clb6?::kanMx
JRY3009 HMR-SSa sir1?::LEU2 clb5?::HIS3 clb6?::kanMx
JRY3009 HMR-SSa sir1?::LEU2 clb5?::kanMx fkh1?::TRP1
JRY3009 HMR-SSa sir1?::LEU2 CLB5–3xHA
JRY3009 HMR-SSa ?I sir1?::TRP1
JRY3009 HMR-SSa rap1 sir1?::TRP1
JRY3009 HMR?E::ARSH4 sir1?::LEU2
JRY3009 HMR?E::ARS1 sir1?::TRP1
JRY3009 HMR-SSa ?I
JRY3009 HMR-SSa ?I sir1?::LEU2 clb5?::kanMx
JRY3009 HMR-SSa rap1
JRY3009 HMR-SSa rap1 sir1?::TRP1 clb5?::kanMx
JRY3009 HMR?E::ARSH4 sir1?::LEU2 clb5?::kanMx
JRY3009 HMR?E::ARS1 sir1?::LEU2 clb5?::kanMx
JRY2334 HMR?E::ARS1 clb5?::kanMx sir1?::LEU2 sir2?::TRP1
JRY2234 hml?::URA3 sir2?::LEU2
JRY2334 hml?::URA3 sir2?::LEU2 clb5?::kanMx
JRY2334 hml?::URA3 clb5?::kanMx HMR?E::ARS1
Gift from Jasper Rine yeast collection
Gift from Jasper Rine yeast collection
Gift from Jasper Rine yeast collection
Gift from Jasper Rine yeast collection
Rusche et al. (2002)
Gibson et al. (2004)
Gibson et al. (2004)
Strains were from the laboratory collection or constructed during the course of this study. The strains noted were gifts from or derived from
strains from the sources in the Reference column.
L. Casey et al.
Molecular Biology of the Cell610
ulated silencing, we compared high-copy FKH1-dependent
(FKH1hc-dependent) and SIR1-dependent silencing at the
molecular and genetic levels.
FKH1hc-dependent Silencing of HMRa Required
Recruitment of Sir3 and the Catalytic Activity of Sir2
MAT? HMR-SSa sir1? yeast cells are unable to mate because
the sensitized synthetic silencer (HMR-SSa) requires SIR1 to
silence the a-mating type genes at HMRa (McNally and Rine,
1991). Thus, these cells provide a genetically useful tool for
examining SIR1 function and for identifying other mecha-
nisms capable of transcriptional silencing. Expression of
FKH1 on a 2? vector (FKH1hc) partially restores silencing to
these sir1? cells as seen by a reduction in a1 mRNA ex-
pressed from HMR-SSa (Hollenhorst et al., 2000; Figure 1A),
thus creating SIR1-bypass silencing. Notably, even expres-
sion of FKH1 from a CEN plasmid contributed to a repro-
ducible, albeit lower level of SIR1-bypass silencing (Figure
1A, lane 4) that was also evident in mating assays (Casey,
To address the mechanism by which FKH1hcbypasses the
requirement of SIR1 in silencing HMR-SSa, we first deter-
mined whether FKH1hcreestablished SIR-dependent chro-
matin (requiring SIR2–4) in sir1? cells (Figure 1B). These
experiments were important because in some genetic back-
grounds HMRa silencing can be established in the absence of
Sir2–4 proteins (Rusche and Rine, 2001). To test whether
Sir2–4 chromatin was reestablished by FKH1hcin sir1? cells,
we performed ChIPs with a cocktail of monoclonal antibod-
ies raised against Sir3 (?-Sir3). Sir3 binding requires the
other Sir proteins, Sir2 and Sir4, and it is thus a good
measure of association of the SIR-complex (Sir2–4) with a
region of chromatin (Hoppe et al., 2002; Luo et al., 2002;
Rusche et al., 2002).
These experiments provided evidence that FKH1hc-depen-
dent silencing restored assembly of a Sir2–4 chromatin do-
main at HMRa in a substantial fraction of sir1? cells. Sir3-
directed ChIPs specifically enriched HMR-SSa from SIR1 but
not sir1? cells, indicating that Sir3-association with HMR-
SSa required Sir1 as expected based on previous character-
izations of HMR-SSa (Gardner and Fox, 2001; Bose et al.,
2004) (Figure 1B). Significantly, FKH1hc-dependent silencing
in sir1? cells, although less efficient than SIR1-dependent
silencing (Figure 1A), reestablished measurable Sir3 binding
to HMR-SSa (Figure 1B). The enrichment of HMR-SSa in
these experiments required SIR3 as none was observed in
sir3? cells, indicating that the Sir3 antibodies had the appro-
priate specificity (Figure 1B).
In SIR1-dependent silencing, Sir2–4 complexes bind re-
gions of HMRa beyond the defined silencers (Rusche et al.,
2002). This binding to distal regions, termed spreading, re-
quires the deacetylase activity of Sir2, an NAD-dependent
histone deacetylase (Denu, 2003). To further examine the
molecular nature of FKH1hc-dependent silencing, we com-
pared binding of Sir3 to multiple regions within and outside
HMR-SSa by ChIPs (Figure 1C). Both SIR1 and FKH1hcpro-
ment of Sir3 and the catalytic activity of Sir2.
(A) Steady-state levels of a1 and SCR1 RNAs
were measured in MAT? HMR-SSa sir1? cells
(CFY762) harboring a 2? plasmid (lane 1, vec-
tor; pCF225), a CEN plasmid with SIR1 (lane
2, SIR1-CEN; pCF99), a 2? plasmid with FKH1
(lane 3, FKH1-2?; pCF480), or a CEN plasmid
with FKH1 (lane 4, FKH1-CEN; pCF943). The
average ratio of a1/SCR1 and SD for three
independent experiments is indicated below
each lane. FKH1-CEN also reproducibly en-
hanced silencing slightly as measured by mat-
ing assays (Casey, unpublished data). (B) Sir3
association with HMR-SSa and the control lo-
cus ADH4 was measured by ChIP with a
monoclonal antibody cocktail against Sir3 (?-
Sir3). Three isogenic MAT? HMR-SSa strains
were analyzed that differed only in terms of
their SIR1 or SIR3 genotype: SIR1 sir3?
(CFY1819), SIR1 SIR3 (CFY345), and sir1?
SIR3 (CFY762). ChIPs were performed on
each strain containing either a 2? plasmid
(vector, gray; pCF225) or a 2? plasmid with
FKH1 (FKH1hc, black; pCF480). The percent-
age of specific PCR fragment obtained in the
immunoprecipitate was determined by video-
densitometry analysis using Labworks analy-
sis software (UVP). Data and standard devi-
ations are reported for three independent
ChIP experiments. (C) ChIPs were performed
as described in B except that multiple regions
within the HMR-SSa locus were examined us-
ing primer pairs at the positions indicated in
the cartoon of HMRa above the figure as de-
scribed previously (Rusche et al., 2002). Two
isogenic MAT? HMR-SSa sir1? strains were
analyzed that differed only in terms of their
SIR2 genotype; SIR2 (CFY762) or sir2N345A
(CFY1827). Each strain harbored a plasmid containing SIR1 (pCF99) or a 2? plasmid containing FKH1 (FKH1hc, pCF480).
FKH1hc-silencing required recruit-
FKH1 and CLB5 in Silencing and Replication
Vol. 19, February 2008 611
duced similar patterns of Sir3 binding over the HMRa locus.
Moreover, efficient Sir3 binding required the Sir2 deacety-
lase activity. Specifically, Sir3 binding was examined in cells
harboring a mutant version of SIR2, sir2N345A, that pro-
duces a catalytically defective version of Sir2 (Imai et al.,
2000). In these sir2N345A mutant cells, regardless of whether
the silencing was mediated through SIR1 or FKH1hc, Sir3
failed to associate with regions distal to the silencer, al-
though it retained some ability to bind to the silencer (Figure
1C). Together, these data provided evidence that FKH1hc-
and SIR1-dependent silencing resulted in similar Sir2–4 si-
lent chromatin domains at HMRa that initiated at the
Fkh1hcProlonged the Cell Cycle and Attenuated the CLB2
mRNA Expression Peak
Several observations provide evidence against the possibil-
ity that FKH1hcaffects HMR silencing directly. First, Fkh1-
3xHA does not bind HMRa as measured by ChIPs (Simon et
al., 2001; Hollenhorst, unpublished data), and tethering a
Fkh1–Gal4 fusion protein directly to HMRa via an engi-
neered Gal4 binding site fails to restore silencing (Fox, un-
published data), although Gal4-fusions with other proteins
known to function directly at the silencer (e.g., Sirs) or to
recruit HMRa to the nuclear periphery do (Chien et al., 1993;
Lustig et al., 1996; Andrulis et al., 1998, 2004). Second, certain
cell cycle perturbations enhance silencing in strains contain-
ing mutations in SIR1 or within the HMR-E silencer (Axel-
rod and Rine, 1991; Laman et al., 1995; Ehrenhofer-Murray et
al., 1999), and FKH1 and its closest paralogue FKH2 are
direct regulators of transcription of the CLB2-cluster of
genes (reviewed in Breeden, 2000) that includes CLB2, the
major G2/M phase cyclin. Third a deletion of FKH1 (fkh1?)
causes a small but measurable effect on cell cycle progres-
sion and CLB2 mRNA levels (Hollenhorst et al., 2000). There-
fore, we hypothesized that FKH1hcwould cause changes in
cell cycle progression and CLB2 expression. To test this idea,
cell bud index and CLB2 mRNA levels were monitored
during cell cycle arrest-and-release experiments (Figure 2, A
MATa bar1? cells harboring either an empty 2? plasmid
(vector) or a 2? plasmid with FKH1 (FKH1hc) were arrested
in G1 with ?-factor, released from arrest into fresh medium,
and at 15-min intervals the cell population was monitored
by counting the number of cells in G1(no buds), S (small
buds), and G2/M (large buds) (Figure 2A). CLB2 mRNA
levels were also measured at each interval by RNA blot
hybridization (Figure 2B). Compared with the vector con-
trol, FKH1hcentered S phase with similar kinetics but it
exhibited a lengthened time in S phase, consistent with the
slower growth rate of FKH1hccells (Figure 2A). For example,
although both the control cells (vector) and FKH1hccells
entered S phase 60 min after ?-factor release, at 120 min after
?-factor release ?60% of the FKH1hccells seemed to remain
in S phase or early G2/M phase, whereas only ?30% of the
control cells did (Figure 2A). Differences between the vector
and attenuated the CLB2 mRNA expression
peak. (A) MATa bar1?::HIS3 (CFY1265) cells
pRS426), a 2? plasmid containing FKH1
(FKH1hc; pCF480), or a deletion of FKH2
(fkh2?; CFY2016) and a 2? plasmid (vector)
were harvested in log phase from medium
lacking uracil and arrested in G1 with ?-fac-
tor. The cells were then released from ?-factor
arrest into fresh medium, and every 15 min
aliquots were harvested for analysis of bud
morphology or CLB2 mRNA levels (see B).
Unbudded cells were scored in G1 phase,
small-budded cells were scored in S phase,
and large-budded cells were scored in M
phase. A representative graph for each exper-
iment is shown. (B) The ratio of CLB2 mRNA
to SCR1 RNA was determined for each of the
strains and time points indicated in A. Total
RNA (15 ?g) as determined by absorbance at
260 ? was analyzed per lane, CLB2 mRNA
and SCR1 RNA were probed independently,
and the signals quantified by Phosphor-
Imager analysis. Data from a representative
experiment are shown. (C) Mating analysis of
MAT? HMR-SSa sir1? cells containing either
wild-type FKH2 (CFY762) or a deletion of
FKH2 (fkh2?; CFY603) and harboring 2? plas-
mids with SIR1 (SIR1hc; pCF345), no insert
FKH1hcprolonged the cell cycle
(vector; pCF225) or FKH1 (FKH1hc; pCF480). The cells were mixed with an excess of MATa cells (CFY616) and 10-fold serial dilutions were
plated on medium to select for the growth of a/? diploids as described in Materials and Methods. The most concentrated samples contained
5 ? 103cells/?l, and 8 ?l was analyzed in each spot. Corresponding growth controls indicated that an equal number of cells were being
compared between strains (LC; data not shown). (D) Levels of Fkh1 in MAT? HMR-SSa sir1? (CFY1649) cells were determined by protein
immunoblot with a polyclonal antibody raised against Fkh1 (?-Fkh1). Cells, harboring either an empty 2? plasmid (pRS426; chromosomal;
lanes 1–3) or a 2? plasmid containing FKH1 (FKH1hc; pCF480; lanes 4–6), were harvested in log-phase growth in liquid CAS medium.
Two-fold serial dilutions of each protein extract were made using protein extract from fkh1? cells as diluents (lane 7) to ensure that each lane
contained similar total protein amounts that facilitated accurate quantification of Fkh1 levels. The fkh1? cells showed no protein band
corresponding to Fkh1 in the immunoblot (lane 7). Equal amounts of protein were loaded per lane as determined by Ponceau S staining of
the blot before immunoblotting.
L. Casey et al.
Molecular Biology of the Cell612
control and the FKH1hccells were also evident at the level of
CLB2 mRNA expression (Figure 2B). In particular, although
the temporal cycling pattern of CLB2 mRNA was similar in
both types of cells, the FKH1hccells produced lower levels of
CLB2 mRNA relative to SCR1 loading control that were most
evident during the peak of CLB2 mRNA expression. The
overall trend was that FKH1hcdampened the amplitude but
not the timing or duration of the CLB2 mRNA wave.
The aforementioned data were consistent with the idea
that reduced CLB2 levels might explain the effect of FKH1hc
on HMR silencing, but additional analyses of fkh2? cells
provided evidence against this idea. Fkh2 is the primary
transcriptional activator of CLB2-cluster genes (Koranda et
al., 2000; Kumar et al., 2000; Pic et al., 2000; Zhu et al., 2000;
Reynolds et al., 2003), and a deletion of FKH2 (fkh2?) also
prolongs the cell cycle and reduces CLB2 mRNA expression
(Hollenhorst et al., 2000). In addition, in fkh2? cells the level
of Fkh1 bound to the promoters of CLB2-cluster genes in-
creases as measured by ChIPs (Hollenhorst et al., 2001).
Therefore, one reasonable explanation for the effects of
FKH1hcon silencing was that the increased levels of Fkh1
outcompeted Fkh2 for binding to CLB2-cluster promoters,
thus mimicking an fkh2?-like phenotype. This explanation
made two predictions. First, FKH1hcshould perturb the cell
cycle and CLB2 expression similarly to fkh2?. Second, fkh2?
should enhance silencing in MAT? HMR-SSa sir1? cells
similarly to FKH1hc.
Direct tests of these predictions provided evidence against
this explanation. Deletion of FKH2 (fkh2?) caused a cell cycle
defect distinct from that caused by FKH1hc; fkh2? cells
showed delayed entry into S phase, but the duration of the
remainder of the cell cycle was similar in fkh2? and wild-
type cells (Hollenhorst et al., 2000; see Figure 2A, fkh2?). In
terms of CLB2 mRNA expression, fkh2? reduced levels of
CLB2 mRNA to an even greater extent than FKH1hc. In
addition and in contrast to FKH1hc, fkh2? delayed the peak
of CLB2 mRNA expression. Thus, compared with FKH1hc,
fkh2? had qualitatively similar but quantitatively distinct
effects on the timing and amplitude of CLB2 mRNA expres-
sion. Nevertheless, as was true for FKH1hc, the overall effect
of fkh2? was to reduce CLB2 mRNA levels.
Therefore, we tested, in side-by-side comparisons whether
fkh2? had any effect on HMRa silencing in MAT? HMR-SSa
sir1? cells or affected the efficiency of FKH1hc-dependent
silencing (Figure 2C). As determined by semiquantitative
mating assays fkh2? had no substantial effect on silencing in
MAT? HMR-SSa sir1? cells nor did fkh2? enhance FKH1hc-
dependent silencing (Figure 2C). Therefore, FKH1hcwas not
restoring silencing in sir1? cells by creating an fkh2?-like
phenotype. These data also provided evidence that reduc-
tions in CLB2 mRNA expression during the cell cycle were
not sufficient to establish FKH1hc-silencing because fkh2?
cells reduced CLB2 mRNA levels but failed to establish
SIR1-bypass silencing. Thus, reduced levels of Clb2 were
insufficient to explain FKH1hc-dependent silencing.
The modest levels of Fkh1 produced in FKH1hccells were
also consistent with the conclusion that FKH1hccells were
not achieving SIR1-bypass silencing by competing for Fkh2
target sites. Fkh1 levels were compared in wild-type and
FKH1hccells by using semiquantitative immunoblotting
with a polyclonal antibody against Fkh1. These experiments
revealed that FKH1hcled to an approximately fourfold in-
crease in the steady-state levels of Fkh1 protein (Figure 2D),
a relatively modest increase that was unlikely to be sufficient
to compete with Fkh2 for bona fide Fkh2 target sites because
Fkh2 binding, but not Fkh1 binding, is enhanced ?10-fold
through cooperative interactions with Mcm1 (Hollenhorst et
al., 2001). Thus, a modest increase in Fkh1 was sufficient to
cause both the cell cycle and silencing phenotypes. These
data, combined with earlier analyses of fkh1? cells that pro-
duced cell cycle and CLB2 mRNA expression profiles that
were the mirror opposite of those produced by FKH1hc(Hol-
lenhorst et al., 2000) also provided evidence that the pheno-
types detected in FKH1hccells reflected a normal role for
Fkh1 in vivo.
Genetic Analyses Revealed that FKH1 and CLB5 Were
Functioning in a Common Pathway
Although the aforementioned data indicated that reduced
CLB2 expression was insufficient to explain the FKH1hcphe-
notypes, they did not address whether CLB2 was necessary.
In addition, the ability of FKH1hcto perturb the cell cycle
(Figure 2) and the documented connection between cell
cycle perturbations and enhanced silencing meant that the
cell cycle perturbation caused by FKH1hcmight be necessary
for and/or closely associated with FKH1hc-dependent silenc-
ing. Therefore, we asked whether deletions of specific CLB
genes affected FKH1hc-mediated silencing in MAT? HMR-
SSa sir1? cells by using RNA blot hybridization of a1 mRNA
and mating assays (Figure 3). We focused on analyzing the
effects of deletions in the G2/M phase cyclins, CLB1 and
CLB2, because these genes are under direct FKH control. In
addition, we analyzed the effects of deletions in the S phase
cyclins CLB5 and CLB6 because clb5? enhances silencing in
sir1? cells (Laman et al., 1995) and the FKH1hccell cycle
phenotype was consistent with the idea that FKH1hcelon-
gated S phase (Figure 2).
In terms of the G2/M phase cyclins, CLB2 but not CLB1
was required for FKH1hc-dependent silencing (Figure 3, A
and B). Specifically, FKH1hcfailed to enhance silencing in
MAT? HMR-SSa sir1? clb2? cells as determined by a1/SCR1
RNA ratios (Figure 3A, compare vector and FKH1hca1/SCR1
RNA ratios for clb2? and CLB). Mating assays provided
independent evidence for this conclusion (Figure 3B). In
contrast, FKH1hcenhanced silencing in MAT? HMR-SSa
sir1? clb1? cells based upon both RNA blot hybridization
(Figure 3A) and mating assays (Figure 3B). Thus direct
measurements of the a1/SCR1 RNA ratios and mating as-
says provided complementary evidence that CLB2 was nec-
essary for establishing FKH1hc-dependent SIR1-bypass si-
lencing at HMR. However, although CLB2 was necessary it
was not sufficient; clb2? failed to enhance silencing in MAT?
HMR-SSa sir1? as measured by a1/SCR1 ratios (Figure 3A)
or mating assays on cells grown either under selective con-
ditions to retain plasmids (Figure 3B) or under rich condi-
tions (Figure 3C). These data were consistent with the con-
clusion from the cell cycle experiments described in Figure 2
that reductions in CLB2 were insufficient to create the
FKH1hcphenotype. Thus, even if the effect of FKH1hcon
CLB2 expression contributes to FKH1hc-dependent silencing,
or CLB2 functions in conjunction with FKH1hc, other CLB2-
independent targets or mechanism(s) also must be relevant
to FKH1hc-dependent silencing.
An earlier study demonstrated that clb5? could enhance
silencing of sir1? cells (Laman et al.,1995). Although CLB2 is
a direct target of Fkh2, our data provided evidence that
reductions in CLB2 were insufficient to account for FKH1hc-
dependent silencing. Therefore, we postulated that path-
ways or target genes affected by the S phase cyclin CLB5
might be more immediately relevant to FKH1hc-dependent
SIR1-bypass silencing. In particular, if FKH1hcand clb5?
achieved SIR1-bypass silencing through modulation of a
common pathway, then FKH1hcand clb5? should cause
quantitatively similar degrees of SIR1-bypass silencing, and,
FKH1 and CLB5 in Silencing and Replication
Vol. 19, February 2008 613
in addition, the two genes should fail to show additive
interactions in terms of this phenotype. Therefore, we tested
whether clb5? cells affected FKH1hc-dependent silencing and
whether fkh1? cells affected clb5?-dependent silencing.
As expected, clb5? but not clb6? enhanced silencing in
MAT? HMR-SSa sir1? cells (Figure 3A; compare vector
a1/SCR1 RNA ratios for clb5? and clb6? to CLB), consistent
with the findings of a previous study (Laman et al., 1995).
Based on RNA blot hybridization, clb5? and FKH1hcen-
hanced HMR-SSa silencing to similar degrees, and FKH1hc
enhanced levels of silencing in clb5? only slightly but repro-
ducibly (Figure 3A). The mating assays were consistent with
the RNA blot hybridizations; FKH1hcprovided for some
level of silencing that was independent of CLB5 (Figure 3B,
clb5?, compare vector and FKH1hc), but it was unable to
enhance silencing of clb5? sir1? cells as well as it enhanced
silencing of CLB5 sir1? cells. These data were consistent
with the idea that FKH1hcand clb5? were impinging on a
common pathway or target protein, to different extents or
through different mechanisms, that was limiting for HMRa
silencing in sir1? cells.
In contrast to CLB5 and CLB2, the CLB6 genotype had no
substantial impact on FKH1hc-dependent silencing; clb6? did
not enhance silencing in MAT? HMR-SSa sir1? cells nor did
it perturb the ability of FKH1hcto enhance silencing in these
cells (Figure 3A, clb6?), consistent with an earlier analysis of
the effect of clb6? on silencing (Laman et al., 1995). Interest-
ingly, within the detection ranges of a1 mRNA, clb6? con-
sistently reduced silencing by a small amount (Figure 3A,
compare vector in clb6? to CLB cells), perhaps by enhancing
the relative activity of CLB5 in vivo. Analyses of cells con-
taining deletions of both CLB5 and CLB6 provided evidence
that CLB5 was the S phase cyclin with the most significant
impact on FKH1hc-silencing; clb5? clb6? produced silencing
phenotypes similar to those produced by clb5? alone (Figure
3, B and C).
In these studies mating efficiencies were ?10-fold lower
and somewhat more variable when measured for cells con-
taining plasmids and grown under selective growth condi-
tions. Therefore, as an independent and additional assess-
ment of the effect of CLB genotype on silencing, mating
assays were performed on the same set of yeast cells de-
scribed above that were instead grown on rich nonselective
growth medium and in the absence of plasmids (Figure 3C).
The data from these experiments were consistent with those
obtained with cells harboring plasmids and grown on selec-
tive medium (compare to Figure 3B, vector).
These data provided evidence that FKH1hcaffected silenc-
ing similarly to a clb5? and might function in a common
and CLB5 were functioning in a common path-
way. (A) a1/SCR1 RNA ratios were determined
by quantitative RNA blot hybridization for an
isogenic series of MAT? HMR-SSa sir1? strains
that differed in terms of their CLB genotype (CLB
(CFY762); clb1? (CFY1614); clb2? (CFY1124);
clb5? (CFY2104); clb6? (CFY2279) and in terms
of the plasmid they harbored (vector [pRS426],
SIR1hc[pCF34], and FKH1hc[pCF48]). A nor-
malized ratio of a1 mRNA/SCR1 RNA is
shown on the y-axis with a value of 1.0 as-
signed to the a1/SCR1 ratio calculated for
CLB cells transformed with a plasmid harbor-
ing SIR1 in one experiment. (B) Mating assays
in an isogenic series of MAT? HMR-SSa sir1?
strains that differed in terms of their CLB ge-
notype and in terms of the plasmid they har-
bored as described in A. In these experiments
clb5?clb6? (CFY2268) cells were also ana-
lyzed. (C) Because mating assays were more
robust on rich media, the same cells analyzed
in B were also analyzed in the absence of
Genetic analyses revealed that FKH1
exogenous plasmids after growth in YPD. (D) An isogenic series of MAT? HMR-SSa sir1? cells differing in their CLB5 and FKH1 genotypes
(CLB5 FKH1 [CFY762]; clb5? FKH1 [CFY2104]; and clb5? fkh1? [CFY2120]) were assessed for silencing by mating assays as described above.
Mating was assessed after growth of MAT? cells on either synthetic CAS medium to retain a URA3 plasmid (vector) or rich medium in the
absence of a plasmid (no plasmid) before selection for diploids. (E) The same cells were assessed for silencing by RNA blot hybridization of
a1 mRNA. The a1 mRNA/SCR1 RNA ratio was normalized as in (A). The CLB5 FKH1hccells were CFY762 containing a 2? plasmid with FKH1
(pCF480). The other cells tested in this experiment contained an empty 2? plasmid (pCF225).
affect Fkh1 levels. (A) Levels of Clb5-3xHA protein were determined
by protein immunoblot with anti-HA antibodies in CLB5 (CFY762),
clb5? (CFY2104), and CLB5–3xHA (CFY2446) cells transformed with
either an empty 2? plasmid (vector) or a 2? plasmid containing
FKH1 (FKH1hc; pCF480). We analyzed 0.25 OD (optical density at
600 ?) cell equivalents per 4? lane, and 2? and 1? lanes contained
twofold and fourfold reductions in that amount of extract, respec-
tively. (B) Levels of Fkh1 protein were determined by protein im-
munoblot with anti-Fkh1 antibodies in CLB5 (CFY762) and clb5?
(CFY2104) cells, as indicated (lanes 1–4) and in clb5? cells trans-
formed with an empty 2? plasmid (vector [pCF225]; lanes 5–7) or
CLB5 cells transformed with a 2? plasmid containing FKH1 (FKH1hc
[pCF480]; lanes 8–10). fkh1? cells (CFY527) containing vector
(pCF225) were analyzed as a negative control (lane 11). We ana-
lyzed 0.25 OD cell equivalents per 4? lane and 2? and 1? were
twofold and fourfold reductions, respectively, in that amount of
FKH1hcdid not affect Clb5 levels nor did CLB5 genotype
L. Casey et al.
Molecular Biology of the Cell 614
E-silencer variations used in these experiments. HMR?E:ARS contains either ARS1 or ARSH4 in the orientation shown as a complete
substitute for HMR-SS. (B) Mating assays were performed with an isogenic series of MAT? sir1? strains that differed in terms of their HMR-E
(E) and/or HMR-I (I) silencers at HMRa, as indicated to the left of the figure and described in the text. The cells contained the following
silencers: HMR-SSa (CFY1649) in place of an 800-base pair deletion that includes HMR-E (McNally and Rine, 1991; Palacios DeBeer and Fox,
1999); HMR-SSa as described above, and a 335-base pair deletion of that includes HMR-I (CFY110) (Fox et al., 1995); HMR-SSa with a mutation
in the Rap1 binding site (CFY2221) (McNally and Rine, 1991); a deletion of an 800-base pairs region, including HMR-E (CFY2133); deletion
of an 800-base pairs region including HMR-E replaced with ARSH4 (CFY2071) or ARS1 (CFY2237) as shown in A. Each strain was
transformed with a 2? plasmid containing SIR1 (SIR1hc[pCF345]), an empty 2? plasmid (vector [pCF225]), or a 2? plasmid containing FKH1
(FKH1hc[pCF480]). Mating was assessed by drop tests in which 10-fold dilutions of MAT? cells being tested were mixed with an excess of
MATa cells (CFY616) and grown on medium that selected for diploids retaining the plasmids. (C) Sir3-directed ChIPs were performed on
MAT? sir1? HMR?E::ARS1 ? HMR-I cells (CFY2071) or on MAT? sir1? HMR-SSa ? HMR?I (CFY35) cells transformed with either SIR1 or
FKH1hcas indicated. (D) Sir3-directed ChIPs were used to determine Sir3 association with a number of ARSs compared with HMR-SSa in
sir1? cells transformed with vector, SIR1, or FKH1hc. (E) Mating assays were performed with an isogenic series of MAT? cells that differed
in terms of their silencers at HMRa (as indicated on left of figure and described in A) and their SIR1 or CLB5 genotypes (as indicated at the
FKH1hcor clb5? could use nonsilencer replication origins in place of the HMR-E silencer. (A) Structures and sequences of the
FKH1 and CLB5 in Silencing and Replication
Vol. 19, February 2008 615
pathway because FKH1hconly slightly enhanced the levels
of silencing achieved in clb5? cells (Figure 3, A and B). A
simple model was that FKH1hcsomehow reduced the levels
or activity CLB5. In this model, CLB5 function in silencing
formally occurred downstream of FKH1 and if correct, a
deletion of FKH1 (fkh1?) would have little or no effect on
silencing achieved by a deletion of CLB5 (clb5?). To test this
idea, we compared silencing of HMR-SSa in sir1? cells that
were either clb5? FKH1 or clb5? fkh1? (Figure 3D). clb5?-
dependent silencing was consistently ?10-fold better in
FKH1 cells compared with fkh1? cells as measured by mat-
ing assays (Figure 3D). As described above, mating assays
were ?10-fold more efficient when cells were grown on rich
medium in the absence of plasmids before selective mating.
Nevertheless, regardless of how cells were grown before the
mating assay (harboring an empty plasmid and grown an
selective media to retain the plasmid [vector] or grown on
rich media in the absence of a plasmid [no plasmid]), the
mating assays revealed that clb5?-dependent silencing was
reduced ?10-fold in fkh1? cells (Figure 3D). However, clb5?
still clearly enhanced silencing in fkh1? cells. This effect was
also evident in RNA blot hybridization experiments (Figure
3E); chromosomal FKH1 was required for the full level of
clb5?-silencing. Thus these data provided evidence that
chromosomal levels of FKH1 could affect HMRa silencing.
Furthermore, the epistasis analyses provided evidence that
CLB5 and FKH1 impinged on HMR silencing at least in part
through a common pathway or target protein.
FKH1hcDid Not Affect Clb5 Levels nor Did CLB5
Genotype Affect Fkh1 Levels
One possible explanation for some of the genetic data de-
scribed above was that the FKH1hcreduced levels of Clb5
protein and thereby contributed to a clb5?-like phenotype.
Conversely, perhaps clb5?-enhanced silencing was medi-
ated by increases in the steady-state levels of Fkh1 protein.
To test whether FKH1hcaffected Clb5 protein levels, Clb5-
3xHA was monitored by protein immunoblotting in MAT?
sir1? HMR-SSa cells harboring either an empty 2? plasmid
(vector) or a 2? plasmid containing FKH1 (FKH1hc) (Figure
4A). These data provided evidence that FKH1hchad no effect
on Clb5-3xHA levels. To test the converse possibility that
clb5?-dependent silencing worked through increasing Fkh1
levels, Fkh1 was monitored in CLB5 or clb5? cells by protein
immunoblotting (Figure 4B). Fkh1 levels were unaffected by
a clb5? (Figure 4B, compare levels of Fkh1 in CLB5, lanes 1
and 2) to clb5? cells (lanes 3 and 4) and compare levels of
Fkh1 in clb5? cells transformed with an empty plasmid
(vector, lanes 5–7) to CLB5 cells transformed with FKH1hc
(lanes 5–11). These data provided evidence that clb5?-de-
pendent silencing did not result from increased levels of
Fkh1 and that FKH1hc-dependent silencing did not result
from reduced levels of Clb5.
FKH1hcCould Use Nonsilencer Replication Origins in
Place of the HMR-E Silencer
As shown above, SIR1- and FKH1hc-dependent silencing
differed in terms of their relationship to the major G2 and S
phase cyclin genes, indicating that at some level these two
forms of silencing relied on different sets of molecular inter-
actions to ultimately establish the same SIR2–4 chromatin
structures. It is well established that Sir1 performs its silencing
function primarily through its interactions with the HMR-E
silencer, an element with a distinct arrangement of protein
binding sites, most notably a binding site for ORC juxtaposed
next to a binding site for Rap1 (Fox and McConnell, 2005)
(Figure 5A, HMR-SS). Both ORC and Rap1 play important
recruiting Sir3 and Sir4 through direct protein–protein interac-
tions. To gain further insight into how SIR1- and FKH1hc-
dependent silencing differed in terms of their ability to es-
tablish Sir2–4 chromatin, their silencer requirements were
Appropriate MAT? sir1? yeast cells that varied only in
terms of their silencers at HMRa were transformed with an
empty 2? vector (vector) or 2? plasmids harboring SIR1
(SIR1hc) or FKH1 (FKH1hc). The transformed cells were com-
pared for their ability to silence HMRa in mating assays
(Figure 5B). As expected, SIR1hc-dependent silencing did not
require the presence of the HMR-I silencer (Figure 5B, line 2,
SIR1hc). In contrast, FKH1hc-dependent silencing was abol-
ished by deletion of HMR-I (Figure 5B, line 2, FKH1hc). Thus,
the two forms of silencing showed different dependencies on
HMR-I. However, it is important to note that SIR1hc- (and
SIR1 at chromosomal levels) is more effective than FKH1hcat
silencing HMRa (Figure 1A), and that deletion of HMR-I
weakens silencing under a variety of conditions (Fox et al.,
1995; Rivier et al., 1999). Therefore, the different requirement
for HMR-I might simply reflect the ability of SIR1 to silence
more robustly than FKH1hc.To test this possibility, we ex-
amined mutant silencers that we knew were defective in
SIR1-dependent silencing for their ability to support
FKH1hc-dependent silencing (Figure 5B, lines 3–5). Impor-
tantly, these experiments revealed that several silencers ex-
isted that were better at supporting FKH1hc- than SIR1-
First, a mutation in the Rap1 binding site (Figure 5B, rap1)
in HMR-SSa abolishes silencing even when SIR1 is overex-
pressed (McNally and Rine, 1991); Figure 5B), line 3, SIR1hc),
presumably because Rap1 is so critical for stabilizing the
binding of Sir3 and Sir4 proteins (Moretti and Shore, 2001).
It was surprising, given that FKH1hcsilencing clearly re-
quired Sir3 and Sir4, that FKH1hccould restore some silenc-
ing to cells that contained a mutation in the Rap1 binding
site within HMR-SSa, whereas SIR1 could not (Figure 5B,
line 3). It was clear that both FKH1hc- and SIR1hc-dependent
silencing required some feature of an E-silencer because an
entire deletion of E (HMR?E) failed to support either forms
of silencing (Figure 5B, line 4). This experiment established
that FKH1hcdiffered from SIR1 in terms of a requirement for
a Rap1 site in the HMR-E silencer.
As mentioned above, the role for ORC in SIR1-dependent
silencing is well established (Fox and McConnell, 2005). Sir1
shows a high specificity for silencer-bound ORCs in vivo;
Sir1 cannot be detected at several nonsilencer replication
origins such as ARS1, even though such elements bind ORC
(Gardner and Fox, 2001). Furthermore, substitution of the
entire E-silencer with the nonsilencer replication origins
ARS1 or ARSH4 (Figure 5A, sequences of “HMR-E” silencers
used in Figure 5B) fails to support SIR1-dependent silencing
(Fox et al., 1995). Even when SIR1 was overexpressed ARS1
or ARSH4 failed to function as effective “E-silencers” (Figure
5B, lines 5 and 6, SIR1hcand vector). Thus, it was unex-
pected that efficient FKH1hc-dependent silencing could be
achieved with either ARS1 or ARSH4 as substitutes for the
Figure 5 (cont).
experiments were as follows: SIR1 CLB5 with HMR-SSa (CFY345);
HMR-SSa?I (CFY35); HMR containing an 800-base pair deletion that
includes HMR-E replaced with either ARSH4 or ARS1 (CFY325 and
CFY321, respectively). sir1? CLB5 versions with the silencers as
listed for SIR1 CLB5 (CFY1649, CFY110, CFY2071 and CFY2237);
and sir1? clb5? with versions with the same silencers as listed
(CFY2104, CFY2230, CFY2193, CFY2236).
top of the figure). The various strains used in these
L. Casey et al.
Molecular Biology of the Cell616
HMR-E-silencer (Figure 5B, lines 5 and 6, FKH1hc). What
was particularly striking was that the level of FKH1hc-de-
pendent silencing achieved in cells in which either ARS1 or
ARSH4 served as the HMR-E silencer was equivalent to that
achieved in the HMR-SSa silencer background that was used
in the original isolation of FKH1 as a high-copy suppressor
of a sir1? mutation (Hollenhorst et al., 2000) (Figure 5B, line
1). That is, for FKH1hc-dependent silencing and in contrast to
SIR1-dependent silencing, either ARS1 or ARSH4 was as
good as HMR-E in terms of functioning as an E-silencer.
As an independent assessment of the unexpected silencer
requirements described above, a Sir3-directed ChIP experi-
ment was performed (Figure 5C). These data were consistent
with the silencing data; Sir3 could bind to HMR?E::ARS1 in
FKH1hccells but not SIR1 cells. In contrast, Sir3 could bind to
HMR-E within an HMR locus that lacked HMR-I in SIR1
cells but not in FKH1hccells. These ChIP data added sup-
porting evidence to the conclusion that FKH1hccells could
use an ordinary ARS in place of HMR-E for Sir2–4–depen-
The data described above indicated that in FKH1hccells,
the Sir3 protein was exhibiting some affinity for ARS1 or
ARSH4 when present at HMR in place of HMR-E. To test
whether Sir3 was exhibiting some affinity for these and other
ARSs in their native locations, we used ChIP to examine Sir3
binding to a number of ARSs in sir1? (vector), SIR1, and
sir1? FKH1hccells (Figure 5D). These data revealed that the
level of Sir3 binding detected by ChIP to ARSs at their native
location was only slightly above background and far below
the level of Sir3 that could be detected by ChIP at HMR.
Because HMR-I was critical for FKH1hc-dependent silencing
(Figure 5B, lines 7 and 8), these data were not surprising
because HMR-I is an element unique to the HMR locus.
Thus, although FKH1hcmay enhance the affinity for Sir3 at
some nonsilencer ARSs, stable binding of Sir3 to these ele-
ments that is detectable by ChIP requires features unique to
HMR, including but not necessarily only HMR-I.
In summary, FKH1hc- and SIR1hc-dependent silencing dif-
fered measurably in terms of silencer requirements optimal
for producing Sir2–4–dependent chromatin at HMRa. Most
remarkably, FKH1hccould establish Sir2–4 chromatin by
using ARS1 or ARSH4 in place of the HMR-E silencer,
whereas SIR1hccould not.
clb5? Also Could Use ARS1 or ARSH4 as a Substitute for
The data concerning the relationship between clb5? and
FKH1hcin terms of SIR1-bypass silencing (Figure 3) raised
the possibility that clb5? and FKH1hcexerted their effects on
silencing through a common pathway. If this were true, then
clb5?-dependent silencing should share the same unusual
silencer requirements as FKH1hc. To test this idea, we deter-
mined whether clb5?-dependent silencing could be achieved
with the same mutant silencers that effectively established
FKH1hc-dependent silencing (Figure 5E). As was true for
FKH1hc-dependent silencing, clb5?-dependent silencing re-
quired HMR-I (Figure 5E, lines 1 and 2). More strikingly,
however, clb5? cells were able to use ARSH4 or ARS1 as
effective substitutes for the HMR-E silencer (Figure 5E, com-
pare lines 3 and 4 with line 1 for sir1?clb5? column),
whereas SIR1 could not (Figure 5E, compare lines 3 and 4
with line 1 for SIR1CLB5 column). In summary, clb5? and
FKH1hccould each establish SIR1-independent silencing at
HMRa by using replication origins as substitutes for HMR-E.
clb5? or FKH1hcCould Suppress HMR?E::ARS1 Origin
The relationship between origin firing and SIR2–4–depen-
dent chromatin is, in general, antagonistic; wherever SIR2–4
silent chromatin forms, origin firing is suppressed (Steven-
son and Gottschling, 1999; Zappulla et al., 2002). Conversely,
origin firing at the HM loci is inefficient regardless of SIR
genotype, in part because the silencers are intrinsically inef-
fective replication origins (Dubey et al., 1991; Palacios De-
Beer and Fox, 1999; Vujcic et al., 1999; Sharma et al., 2001;
Palacios DeBeer et al., 2003).
CLB5 is the S phase cyclin most critical for activating
replication origins that fire late in S phase (Donaldson et al.,
1998; Gibson et al., 2004). Thus, in clb5? cells early S phase
origins fire efficiently, whereas late S phase origins do not,
and overall S phase progression is slowed because replica-
tion forks emanating from early origins must now replicate
a greater fraction of the genome. In the ARS1 substitution
experiment described above (Figure 5), the entire HMR-E
silencer was deleted and replaced with ARS1. In its native
location, ARS1 fires efficiently during early- to mid-S phase
and is CLB5 independent (Donaldson et al., 1998; Gibson et
al., 2004). Given this collection of observations, we asked
ity. (A) 2-D origin mapping experiments were performed in MAT?
HMR?E::ARS1 sir1? cells that contained either CLB5 (CFY2237) or
clb5? (CFY2236). Origin activity at HMRa was assessed with an
HMR-specific probe, whereas origin activity at the native ARS1
locus was assessed with an ARS1-specific probe. Two different
exposures of HMR?E::ARS1 blots are shown (dark and light) to
facilitate comparison of this origin activity in CLB5 versus clb5?
cells. (B) 2-D origin mapping experiments with the cells used in A
were repeated side by side with clb5? sir2? cells (CFY2481). (C) The
DNA content in actively dividing populations of isogenic yeast cells
that varied in terms of their SIR2 or CLB5 genotypes as indicated
was determined by flow cytometry. (D) 2-D origin mapping exper-
iments were performed in MAT? HMR?E::ARS1 sir1? CLB5 cells
containing either an empty 2? plasmid (vector; pCF225) or a 2?
plasmid with FKH1 (FKH1hc; pCF480).
clb5? or FKH1hcsuppressed HMR?E::ARS1 origin activ-
FKH1 and CLB5 in Silencing and Replication
Vol. 19, February 2008617
how the ARS1 origin that replaced HMR-E was affected in
clb5? cells by performing 2-D origin mapping experiments
on ARS1 at its native location and at HMR where it substi-
tuted for the HMR-E silencer (HMR??::ARS1) (Figure 6).
These 2-D origin-mapping experiments revealed that in
wild-type (CLB5) cells ARS1 functioned as a robust, efficient
origin whether it was present at its native location (ARS1) or
at HMR, as a substitute for the deleted HMR-E silencer
(HMR?E::ARS1) (Figure 6A, CLB5). However, only ARS1 at
HMRa (HMR?E::ARS1) was sensitive to CLB5 genotype;
clb5? reduced the ability of ARS1 to function as an origin
when it substituted for HMR-E (clb5?, HMR?E::ARS1)
whereas it had no effect on ARS1 firing at its native locus
(clb5?, ARS1) (Figure 6A, clb5?).
Two different mechanisms could contribute to clb5?-me-
diated suppression of origin firing by HMR?E::ARS1. First,
clb5? reestablished silencing at HMR?E::ARS1 and thus si-
lencing, or Sir2–4 chromatin formation, might be inhibiting
firing by HMR?E::ARS1. If this scenario were true, then a
deletion of SIR2 (sir2?), a gene essential for silencing, should
restore efficient firing by HMR?E::ARS1 in clb5? cells as is
observed for other cases of Sir2–4–mediated origin suppres-
sion (Zappulla et al., 2002). Second, some other SIR-indepen-
dent feature of this chromosomal region might suppress
origin firing by HMR?E::ARS1 in clb5? cells. As discussed
above, the HM loci fail to allow for more robust origin firing
by their native silencers even in sir mutant cells that cannot
silence. To distinguish between these mechanisms, we per-
formed 2-D origin-mapping experiments on HMR?E::ARS1
in clb5? sir2? cells, thereby precluding the ability of clb5? to
reestablish Sir2–4 chromatin at HMRa. The sir2? mutation
failed to restore robust firing to the HMR?E::ARS1 origin in
clb5? cells (Figure 6B), suggesting that SIR2–4 chromatin
was not the cause of CLB5-sensitive firing by HMR?E::ARS1.
However, a complicating factor in interpreting these exper-
iments was that large replication forks were also consistently
underrepresented in sir2? clb5? cells compared with CLB5
SIR2 or clb5? SIR2 cells raising the possibility that sir2? was
having additional effects on genome replication. Neverthe-
less, the replication pattern generated for HMR?E::ARS1 in
sir2? clb5? cells indicated that sir2? did not simply restore
robust replication to HMR?E::ARS1. Thus, the dependence
on CLB5 that ARS1 acquired when it replaced HMR-E was
independent of Sir2–4 chromatin formation.
A recent study raises the possibility that SIR genes, in
particular SIR2, can negatively regulate many replication
origins in the yeast genome, most probably at the level of
pre-RC assembly (Pappas et al., 2004). Therefore, in addition
to examining the effect of a sir2? on CLB5 sensitivity of a
particular origin (Figure 6B), the effect of a sir2? on DNA
replication was examined in clb5? cells by flow cytometry
(Figure 6C). In the simplest case, if the majority of CLB5-
sensitive origins were negatively regulated by SIR2, then a
sir2? mutation would suppress the defects of a clb5?. How-
ever, examination of the DNA content in asynchronous pop-
ulations of cells indicated that a sir2? did not reestablish a
wild-type CLB5 DNA-content profile. In addition, cell-cycle
arrest and release experiments indicated that sir2? clb5?
cells showed a delayed entry into M phase compared with
sir2? CLB5 cells, suggesting that many of the global replica-
tion defects caused by a clb5? were not rescued by a sir2?
(Fox, unpublished data). Together, these data provided ev-
idence that SIR2 likely did not regulate many CLB5-sensitive
origins, because sir2? did not suppress a clb5?, consistent
with our data from directed analyses of HMR?E::ARS1 in
sir2? clb5? cells (Figure 6B).
The genetic and cell cycle data concerning FKH1hcwere
consistent with the notion that clb5? and FKH1hcfunctioned
in part through a shared pathway or target to modulate
HMRa silencing. As another test of this shared relationship,
we determined the effect that FKH1hchad on origin firing by
HMR??::ARS1 (Figure 6D). Significantly, FKH1hcalso re-
duced the efficiency of origin firing by HMR??::ARS1, pro-
ducing a molecular phenotype similar to that caused by a
clb5? (Figure 6A, compare vector with FKH1hc), albeit the
effect of FKH1hcon origin firing by HMR??::ARS1 was less
drastic than the effect of clb5? on this origin. This observa-
tion was consistent with clb5? having a more significant
impact on cell growth compared with FKH1hc(Casey, un-
published data). Nevertheless these data supported the idea
that FKH1hcand clb5? bypassed the requirement for SIR1 in
establishing Sir2–4 chromatin through effects on a similar
target or pathway that can impinge on or is associated with
S phase progression.
In this report, we addressed the hypothesis that Fkh1, an
evolutionarily conserved transcription factor implicated in
transcriptional control of CLB2-cluster genes (reviewed in
Breeden, 2000; Futcher, 2000), modulated Sir2–4 chromatin
formation at HMRa through an effect on cell cycle progres-
sion. Several lines of evidence presented here linked FKH1
and the major S phase cyclin CLB5 to a common pathway(s)
or target(s). First, FKH1hcand clb5? each bypassed the re-
quirement for SIR1 in silencing HMRa to a similar extent.
Second, FKH1hcand clb5? each had the unexpected ability to
use a replication origin, ARS1 or ARSH4, as an effective
substitute for the HMR-E silencer in silencing HMRa. And
third, FKH1hcand clb5? each reduced origin firing by the
normally robust ARS1 origin when it substituted for the
HMR-E silencer. Together, this set of shared phenotypes
supports a model in which FKH1 and CLB5 impinge on a
common pathway relevant to both the control of late-firing
replication origins and the cell-cycle sensitive formation of
Sir2–4–repressive chromatin at HMRa.
It was conceivable that FKH1 and CLB5 were part of a
linear pathway in which FKH1 affected expression of CLB5
or vice versa as such a relationship could explain the similar
phenotypes caused by FKH1hcand clb5?. Fkh1 is, after all, a
transcription factor that could directly or indirectly affect
levels of Clb5. However, direct analyses of Fkh1 and Clb5
protein levels provided evidence against the simplest ver-
sion of this possibility. In particular, FKH1hchad no effect on
the steady-state levels of Clb5, and, conversely, clb5? had no
effect on either chromosomally produced levels of Fkh1 or
the ability to overexpress Fkh1 to levels sufficient to estab-
lish HMRa silencing. Moreover, double mutant analyses also
provided evidence against a simple linear relationship be-
tween FKH1 and CLB5. If FKH1hcenhanced HMRa silencing
entirely through CLB5, we would have predicted that
FKH1hcwould fail to enhance silencing at all in clb5? cells.
However, FKH1hcdid enhance silencing of clb5? cells to a
small but reproducible extent as determined by both mating
assays and RNA blot hybridizations. Similarly, although
clb5? cells required a chromosomal copy of FKH1 for max-
imal HMRa silencing, these cells still silenced HMRa more
effectively than corresponding CLB5 cells. A simple model
consistent with these data describes a forked pathway in
which FKH1 and CLB5 have opposing regulatory roles on
common target(s) (T) in this pathway or at least targets with
shared functions (Figure 7). That is, the final activity of T
depends on contributions from both FKH1 and CLB5. Given
L. Casey et al.
Molecular Biology of the Cell 618
what is known about the proteins encoded by FKH1 and
CLB5, a reasonable idea is that FKH1 controls the levels of T,
whereas CLB5 negatively regulates the activity of T through
phosphorylation (e.g., by the Clb5/Cdc28 kinase). Target T
in turn impinges on the activity of late-firing replication
origins (or at least a subset of these origins) and the assem-
bly of Sir2–4 chromatin.
Role of CLB2
Our genetic and molecular studies provided evidence for
how FKH1 and CLB5 were related, but based on our current
data it is difficult to determine how the M phase cyclin CLB2
fits into the model (Figure 7). Because clb2? can rescue some
HMR silencing defects (Laman et al., 1995) and Fkh1 is a
direct transcriptional regulator of CLB2, it was unexpected
that a clb2? failed to mimic the silencing phenotype caused
by FKH1hc. Although FKH1hcclearly reduced levels of CLB2
mRNA, the inability of fkh2? cells, which also reduced the
levels of CLB2 mRNA, as well as clb2? cells to establish
SIR1-bypass silencing provided strong evidence that reduc-
tions in CLB2 were not sufficient to explain FKH1hc-depen-
dent silencing. The observation that CLB2 was required for
FKH1hc-silencing could be because CLB2 acts positively on
target T through an FKH1-independent mechanism, and that
the effects of FKH1hcon CLB2 mRNA levels were unrelated
to how FKH1 established SIR1-bypass silencing at HMR.
Alternatively reduced levels of CLB2 may be an essential
component of FKH1hcsilencing, but the precise pattern and
amplitude of CLB2 mRNA expression caused by FKH1hcis
itself critical, such that a clb2? or the distinct pattern of CLB2
reduction caused by fkh2? does not suffice to create SIR1-
bypass silencing. A better understanding of precisely how
Fkh1 controls CLB2 expression and/or a FKH-independent
mode for regulating CLB2 mRNA levels would help address
Sir2–4 Chromatin Formation and the Cell Cycle
It is well established that perturbations in the cell cycle can
enhance or reduce the efficiency of silencing assessed in
certain mutant backgrounds, but the mechanisms behind
these effects are unclear (Axelrod and Rine, 1991; Laman et
al., 1995; Ehrenhofer-Murray et al., 1999). In addition, studies
in which expression of individual SIR genes is controlled
with inducible promoters or temperature-sensitive muta-
tions indicate that multiple phases of the cell cycle may
modulate different distinct steps required for the ultimate
assembly of functionally silenced chromatin (Lau et al., 2002;
Kirchmaier and Rine, 2006; Matecic et al., 2006). For example,
studies using a temperature-sensitive sir3 allele or an induc-
ible version of SIR1 provide evidence that Sir2–4 can rebind
HMRa in G1 phase yet fail to establish silencing (Lau et al.,
2002; Kirchmaier and Rine, 2006). Rather silencing, as de-
fined by repression of transcription of the a1 gene, is not
established until passage through the following S and M
phases. Thus some of the cell cycle-regulated steps that
ultimately lead to a silenced transcriptional state at HMRa
must occur downstream of the binding of Sir proteins to
HMRa. It was conceivable, therefore, that the effects of
FKH1hcand by inference those of several cell cycle mutants
on HMRa silencing were mediated through similar steps
and that they did not affect the efficiency of Sir binding to
chromatin. Our use of the synthetic silencer (HMR-SSa) (Mc-
Nally and Rine, 1991) allowed us to address this question.
Unlike natural HMRa where substantial levels of Sir3 bind-
ing can be detected by ChIP even in sir1? cells (Rusche et al.,
2002), Sir3 binding to HMR-SSa was completely dependent
on Sir1. This feature allowed us to demonstrate unequivo-
cally that FKH1hcreestablished Sir2–4 nucleation at HMR-E
even in the absence of Sir1. Thus FKH1hc, and by inference
clb5?, bypass the requirement for SIR1 in HMRa silencing at
least in part by promoting efficient Sir2–4 nucleation at a
Linking Late Origin Firing and HMRa Silencing
The close association between silencers and DNA elements
with the capacity to function as origins at the HM loci has
spurred studies over the past two decades aimed at trying to
explain the mechanistic nature of this link. Recent data
focused on HMR-E has raised the possibility that there exists
a competing relationship between the roles of ORC at ori-
gins and silencers and that the close association between
these elements is a by-product of the dual functions of ORC
(Palacios DeBeer and Fox, 1999; Palacios DeBeer et al., 2003;
McConnell et al., 2006). In this model for the silencer–origin
relationship, HML and HMR silencers are weak or inactive
origins at least in part because robust and/or early origin
firing inhibits silencing. Therefore, it was unexpected that
ARS1 and ARSH4, two independent and robust replication
origins with no documented silencer activity, could act as
effective substitutes for the HMR-E silencer. Indeed in a
silencer-trap screen in which the 2? ARS was shown to
possess weak silencer activity, direct tests of ARS1 demon-
strated it could not substitute for HMR-E in silencing HMRa
(Grunweller and Ehrenhofer-Murray, 2002). This observa-
tion was confirmed in this report because neither SIR1 cells
nor sir1? cells could use ARS1 as effectively as they used
HMR-E for silencing HMRa. However, what was new and
surprising was that ARS1 could function as effectively as
HMR-E in sir1? cells that also harbored FKH1hcor clb5?.
Thus, ARS1 became an effective SIR1-independent silencer
under conditions that also, notably, reduced the ability of
ARS1 to act as a replication origin. Therefore, this observa-
tion was consistent with the idea that robust origin and
silencer activity were incompatible and that target T en-
hanced Sir2–4 chromatin formation because it inhibited or-
igin firing by HMR?E::ARS1 (Figure 7A). Sir2–4 chromatin
did not create a late-firing CLB5-sensitive origin at HMRa
because even in the absence of silent chromatin formation
(sir2?) the HMR?E::ARS1 origin remained suppressed in
clb5? cells. However, another explanation consistent with
the observations reported here was that late origin firing and
silencer function were not linked causally but instead could
be regulated in opposing directions by the same target T
activity and silencing at HMRa (A) FKH1 positively regulates (ar-
row) and CLB5 negatively regulates (flat head) a common pathway
or target referred to as T. T in turn negatively regulates origin firing
by normally late-firing replication origins (or origins placed within
chromosomal domains that cause late firing). Robust origin firing is
incompatible with the assembly of Sir2–4 chromatin. (B) FKH1 and
CLB5 regulate target T as described in A, but origin firing is not
causally linked to Sir2–4 chromatin assembly. Rather firing by late
replication origins is negatively regulated, whereas Sir2–4 is posi-
tively regulated by target T.
Two models for how FKH1 and CLB5 modulated origin
FKH1 and CLB5 in Silencing and Replication
Vol. 19, February 2008 619
(Figure 7B). This second version of the model is actually
more consistent with the observation that clb5? clb6? cells
could silence as effectively as clb5? CLB6 cells, because clb5?
clb6? cells restore a normal S phase (Schwob and Nasmyth,
1993), suggesting that suppression of late origin firing is not
essential for Sir2–4 chromatin formation in clb5? cells.
Because silencing of HMRa weakened by silencer muta-
tions can be enhanced by artificially tethering the mutant
HMRa locus to the nuclear membrane (Andrulis et al., 1998),
we propose that a reasonable candidate for target T is a cell
cycle-regulated protein(s) or protein complex that modu-
lates the association of HMRa with the nuclear periphery,
where Sir2,-3, and -4 proteins are concentrated (Palladino et
al., 1993; Maillet et al., 1996; Taddei et al., 2005). In addition,
late-firing replication origins show an enhanced association
with the inner nuclear membrane in both yeast and mam-
mals (Dimitrova and Gilbert, 1999; Heun et al., 2001), which
also supports the hypothesis that target T represents a pro-
tein or complex that affects the association of HMRa with the
nuclear membrane and that this association in turn can affect
both origin firing and silencing.
In this interpretation, ARS1 and ARSH4 were equivalent
to an HMR-E silencer weakened by mutation. That is, these
origins acted as “proto-silencers” (Boscheron et al., 1996), too
weak to function on their own but efficient enough when
combined with other Sir-stabilizing influences, such as an-
other weak proto-silencer, HMR-I. This must mean that
ARS1 and ARSH4 possess an intrinsic affinity, albeit sub-
stantially reduced compared with the native silencer, for the
Sir2–4 protein complex. Thus even in the absence of Sir1,
Sir2–4 proteins must have some inherent affinity for origins,
or at least a component of origins, such as ORC. Consistent
with this possibility, a recent study provides evidence that
SIR2, and less effectively SIR3 and SIR4, can affect the activ-
ity of many genomic origins, suggesting that these proteins
may interact with many nonsilencer origins in the genome
(Pappas et al., 2004). This interaction may be fleeting and
thus not detectable by ChIP. However, when this weak
interaction is combined with additional weak Sir interaction
sites provided by the HMR-I silencer and a higher concen-
tration of Sir proteins that might be provided by association
with the nuclear membrane, the result is sufficient stabiliza-
tion energy that allows Sir2–4 proteins to establish silencing.
Thus HMRa silencing in clb5? (or FKH1hc) cells measured
here might be effectively trapping transient interactions that
occur throughout the genome between Sir2–4 complexes
In summary, the cell cycle perturbation caused by a clb5?
(or FKH1hc) associated with enhanced silencing could in-
volve an increase in the levels and/or activity of a target T.
Although it is possible that target T acts to suppress late-
firing replication origins that in turn directly interfere with
silencers (Figure 7A), it is also possible that both silencers
and late-firing replication origins are connected to the com-
mon target T that in turn regulates the function of each of
these DNA elements in opposing directions and to different
degrees (Figure 7B).
The FKH1hcEffect Involved Gene Targets and/or Target
Sites Distinct from Fkh2 Targets
In terms of gene expression FKH1hchad a modest but repro-
ducible dampening effect on the wave of CLB2-mRNA ex-
pression during the cell cycle. These data were consistent
with the idea, supported by earlier analysis of fkh1? cells
(Hollenhorst et al., 2000) that Fkh1 acts as a negative regu-
lator of CLB2 transcription at least during the stages of the
cell cycle when CLB2 mRNA expression peaks. Because
Fkh2 is a positive regulator of CLB2 transcription, it was
somewhat surprising that an fkh2? affected neither silencing
on its own nor the ability of FKH1hcto enhance silencing.
Therefore, FKH1 must act through gene targets and/or bind-
ing sites distinct from those controlled by FKH2 to cause the
phenotypes documented here. Whether FKH1hcenhances
Fkh1 association with its normal targets or also causes sig-
nificant binding to new targets, and whether one or more of
these targets is also critical for the clb5?-like phenotype
remains to be determined.
Forkhead transcription factors (forkhead box, FOX in
metazoans) have evolutionary conserved roles in cell cycle
regulation, proliferation and differentiation, and modest
changes in forkhead transcription factor levels have clear
and pivotal effects on complex multicellular phenomena
such as organogenesis and disease states (Carlsson and
Mahlapuu, 2002; Gaudet and Mango, 2002). Studies of the
effects of Fkh1 dosage in the single-celled yeast should con-
tribute to unraveling how forkhead transcription factors
contribute to remodeling a eukaryotic transcriptional pro-
gram. Transcriptional silencing serves as sensitive barome-
ter for examining this issue and others associated with cell
cycle regulation of chromosome structure and function.
We thank Zhonggang Hou for help in producing Fkh1 antibodies, Kristopher
H. McConnell and Bonita Brewer (University of Washington, Seattle) for
advice on 2-D origin mapping, Philipp Mu ¨ller for help with figures, and Oscar
Aparicio (University of Southern California) for generously sharing yeast
strains. We also thank Erika Shor for comments on the manuscript. LC was
funded in part by a fellowship from the American Heart Association (AHA)
(0135265Z). EEP was funded in part by the NIH Predoctoral Training Pro-
gram in Genetics to the Laboratory of Genetics (5 T32 GM07133) and an AHA
fellowship (0615552Z). This work was supported primarily by grants from the
American Cancer Society (ACS-RSG-02-164-02-GMC) and the National Insti-
tutes of Health (RO1 GM56890) to CAF.
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