Copyright ? 2006 by the Genetics Society of America
Dominant Mutants of the Saccharomyces cerevisiae ASF1 Histone Chaperone
Bypass the Need for CAF-1 in Transcriptional Silencing by Altering
Histone and Sir Protein Recruitment
Beth A. Tamburini,* Joshua J. Carson,†Jeffrey G. Linger†and Jessica K. Tyler†,1
*Molecular Biology Graduate Program,†Department of Biochemistry and Molecular Genetics,
University of Colorado Health Sciences Center, Aurora, Colorado 80045
Manuscript received December 15, 2005
Accepted for publication March 22, 2006
Transcriptional silencing involves the formation of specialized repressive chromatin structures. Previous
studies have shown that the histone H3–H4 chaperone known as chromatin assembly factor 1 (CAF-1)
contributes to transcriptional silencing in yeast, although the molecular basis for this was unknown. In this
work we have identified mutations in the nonconserved C terminus of antisilencing function 1 (Asf1) that
result in enhanced silencing of HMR and telomere-proximal reporters, overcoming the requirement for
CAF-1 in transcriptional silencing. We show that CAF-1 mutants have a drastic reduction in DNA-bound
histone H3 levels, resulting in reduced recruitment of Sir2 and Sir4 to the silent loci. C-terminal mutants
of another histone H3–H4 chaperone Asf1 restore the H3 levels and Sir protein recruitment to the silent
loci in CAF-1 mutants, probably as a consequence of the weakened interaction between these Asf1
mutants and histone H3. As such, these studies have identified the nature of the molecular defect in the
silent chromatin structure that results from inactivation of the histone chaperone CAF-1.
repeating units termed nucleosomes. The nucleosome
is composed of ?147 bp of DNA wound almost twice
around an octamer of histone proteins with two mol-
ecules each of histones H2A, H2B, H3, and H4 (Luger
et al. 1997). The packaging of the DNA into chromatin
has a profound influence on transcriptional regulation
(Peterson and Laniel 2004; Cairns 2005). A clear ex-
ample of this is provided by heterochromatin or si-
lenced chromatin, where additional proteins bind to
the nucleosomal array to generate a specialized chro-
matin structure that is transcriptionally ‘‘silent.’’
The formation of silent chromatin structures has
been extensively studied in budding yeast (Rusche
that are transcriptionally silenced: the mating-type loci
HML and HMR, the rDNA, and the telomere-proximal
regions. Here we focus on the mating-type loci and
loci and telomere-proximal regions is established by the
recruitment of the Silent information regulator (Sir)
proteins to the silencers, which are DNA sequences that
dictate the region to be silenced. Sir4 preexists in a
soluble complex with Sir2, while Sir3 is thought to be
HE eukaryotic genome is packaged into chroma-
tin, the foundation of which is a regular array of
et al. 2001; Hoppe et al. 2002). Once bound to the
silencer, the Sir proteins spread throughout the silent
locus, with Sir3 and Sir4 binding to the unacetylated
N-terminal tails of histones H3 and H4 (Hecht et al.
1995; Carmen et al. 2002). The enzymatic activity of
Sir2 as a NAD-dependent histone deacetylase promotes
spreading of the silent chromatin structure at the
telomere-proximal regions and mating-type loci by
deacetylating the histones to create high-affinity bind-
ing sites for Sir3 and Sir4 (Smith et al. 1998; Imai et al.
2000; Landry et al. 2000). The extent of spreading of
the Sir proteins is limited by boundaries between si-
lenced and expressed chromatin. One mechanism by
which the boundaries of silent chromatin structure
appear to be set is the localized recruitment of histone
acetyltransferases, resulting in acetylated histones that
are refractory to binding of Sir proteins (Donze and
Kamakaka2001).Consistent with thisidea, Sir3 spreads
further from a telomere when the gene encoding the
histone acetyltransferase Sas2 is deleted (Kimura et al.
2002; Suka et al. 2002). Similarly, the binding of bro-
modomain factor 1 (Bdf1) to acetylated chromatin pre-
vents deacetylation by Sir2 and therefore prevents the
spreading of the Sir proteins (Ladurner et al. 2003).
The silent chromatin structure is maintained and
inherited through cell division. Every time the DNA
replicates, the histones are reassembled onto the newly
replicated DNA (Cairns 2005). This process is mediated
in part by histone chaperones. The histone chaperone
chromatin assembly factor 1 (CAF-1) was discovered by
1Corresponding author: Department of Biochemistry and Molecular
Genetics, UCHSC at Fitzsimons, Mail Stop 8101, PO Box 6511, Aurora,
CO 80045. E-mail: firstname.lastname@example.org
Genetics 173: 599–610 ( June 2006)
its biochemical ability to deposit histones H3 and H4
CAF-1 is also likely to assemble chromatin following
DNA replication in vivo, as it localizes to sites of DNA
replication (Marheineke and Krude 1998) and is
found in a complex with histones that are specifically
assembled following DNA replication (Tagami et al.
2004). Furthermore, bulk chromatin from yeast lacking
CAF-1 is more accessible to digestion by micrococcal
nuclease and DNAseI, and the endogenous 2m plasmid
is less supercoiled, consistent with a role for CAF-1 in
global chromatin assembly in vivo (Hoek and Stillman
2003; Adkins and Tyler 2004; Nabatiyan and Krude
2004). In vitro, CAF-1-mediated chromatin assembly
following DNA replication is dependent on another
histone H3–H4 chaperone termed antisilencing func-
tion 1 (Asf1) (Tyler et al. 1999). Like CAF-1, Asf1 also
localizes to DNA replication forks in vivo (Schulz and
Tyler 2006). The binding partners and phenotypes of
yeast lacking ASF1 have implicated Asf1 in many pro-
cesses. These binding partners include the DNA damage
checkpoint protein Rad53, the bromodomain factor
complex in addition to CAF-1 and histones H3 and H4
(Tyler et al. 1999, 2001; Meijsing and Ehrenhofer-
Murray 2001; Osada et al. 2001; Sharp et al. 2001;
Chimura et al. 2002; Mello et al. 2002). Yeast lacking
ASF1 are sensitive to DNA damaging agents and repli-
cational stress and show transcriptional defects (Le et al.
1997; Singer et al. 1998; Tyleret al. 1999; Sutton et al.
2001; Chimura et al. 2002; Adkins et al. 2004; Ramey
et al. 2004; Zabaronick and Tyler 2005). These pheno-
types presumably reflect the role of Asf1 in mediating
chromatin assembly and/or disassembly during replica-
tion, DNA repair, and transcriptional regulation.
Both Asf1 and CAF-1 contribute to transcriptional
silencing, but the molecular basis for this is unknown.
Deletion of any of the three genes encoding the CAF-1
complex, CAC1, CAC2, or MSI1/CAC3, results in a loss
of transcriptional silencing (Kaufman et al. 1997). The
silencing defect in CAF-1 mutants appears to be due to
the transient loss of silencing (Enomoto and Berman
1998) and an increased frequency of switching the
expression state of telomeric reporter genes (Monson
et al. 1997). As such, silencing is established in the
absence of CAF-1, but CAF-1 is important for the
maintenance of silencing through the cell cycle and
the inheritance of silencing through DNA replication.
Overexpression of Asf1 weakens silencing of reporters
at the HMR and telomere-proximal loci (Le et al. 1997;
Singer et al. 1998). Deletion of ASF1 causes a nominal
defect in silencing (Le et al. 1997; Singer et al. 1998),
while deletion of ASF1 in addition to inactivation of
CAF-1 or mutation of the silencing enhancer HMR-E
leads to a further defect in silencing (Tyleret al. 1999;
Meijsing and Ehrenhofer-Murray 2001).
To investigate the molecular basis for the contribu-
tion of CAF-1 and Asf1 to transcriptional silencing, we
screened for insertion mutations in yeast ASF1 that alter
itssilencingabilities.The biochemicaland geneticchar-
acterization of Asf1 mutants that bypass the require-
molecular contribution of the CAF-1 and Asf1 histone
chaperones to the silent chromatin structure. Specifi-
cally, CAF-1 is required to deposit a foundation of nu-
cleosomes for recruitment of the Sir proteins; in the
absence of CAF-1 there is a striking reduction in his-
tone H3 and Sir protein occupancy at the silent loci.
This role for CAF-1 in transcriptional silencing can be
bypassed by dominant Asf1 mutants that result in
increased chromatin assembly and recruitment of Sir
proteins in CAF-1 mutants.
MATERIALS AND METHODS
Transposon insertion mutagenesis: The ASF1 ORF and
promoter inserted into pRS314 were subjected to insertion
mutagenesis using the GPS-LS linker-scanning system (New
England Biolabs, Beverly, MA). One-third of the resulting
15-bp insertions introduced an in-frame stop codon. The
mutagenized plasmids were then transformed into asf1Dcac1D
Silencing assays: Yeast strains (see Table 1) were grown to
spotted on plates in 10-fold serial dilutions on rich media,
4) or 59 fluoroorotic acid (59FOA), or low adenine (low ade).
Yeast were grownfor 2–4days at30?andthenplaced at4?for 7
days to allow for the color to develop. To assay the degree of
sector, and then placed at 4? to develop the colony color.
DNA damage sensitivity: Yeast strains were grown to log
phase and adjusted to an OD600nmof 1.0. Strains were then
spotted on plates in 10-fold serial dilutions on media lacking
TRP (labeled control in Figure 2) and with 0.005 and 0.01%
methyl methanesulfonate (MMS). Yeast were grown for 2–4
days at 30?.
Phosphatase assay: Phosphatase activity was measured
exactly as described previously (Adkins et al. 2004).
Flow cytometry analysis: Approximately 5 3 106cells per
sample were stained with propidium iodide (Stone and
Pillus 1996). Ten thousand cells per sample were scanned
using a Beckman–Coulter XL-MCL machine.
Immunoprecipitation: Immunoprecipitation analyses were
performed exactly as described previously (Tamburini et al.
Chromatin immunoprecipitation: Chromatin immunopre-
cipitation (ChIP) was performed as described previously (Kuo
and Allis 1999) with the following alterations. Yeast cells were
grown overnight in rich media, diluted, and grown 2–3 hr at 30?
until cells reached an OD600nmof 1.0. Each ChIP reaction was
performed in duplicate with 1 3 108cells for H3 and Sir2-HA
immunoprecipitations and with 9 3 108for Sir4 immunopreci-
pitations, with a crosslinking time of 15 min. To immunoprecip-
itate Sir2-HA, Sir4, and H3 we used 4 ml of anti-mouse HA
antibody (Covance), 1 ml of antiserum to Sir4 (Hoppe et al.
2002), and 2 ml of antiserum to the C terminus of H3 (Abcam),
respectively. Samples were analyzed using a 1:625–1:2500
600B. A. Tamburini et al.
dilution of the immunoprecipitated samples and a 1:240,000
dilution of the input samples. The linear range of PCR ampli-
ments, to be 25 cycles. The primer pairs were used as described
previously (Hoppe et al. 2002). Agarose gels (4.5–5%) were used
to analyze small PCR products using a 1:1 mixture of GenePure
LE Agarose (ISC Bioexpress) and the low melting temperature
NuSieve GTG Agarose (Cambrex) certified for the recovery of
nucleic acids ,1 kb. The DNA was resolved on the agarose gels
with 1.5% eithidium bromide and quantitated from an un-
saturated image using Labworks (UVB Bioimaging Systems). To
calculate the percentage of total chromatin bound, immuno-
precipitated chromatin (IP) and total chromatin (input) were
multiplied by the respective dilution factors, and then the ratio
of immunoprecipitated chromatin to total chromatin was calcu-
value. In Figure 5, error bars represent the standard deviation
represent statistical significance, where one asterisk represents a
P-value of ,0.1, two asterisks represent a P-value of ,0.05, and
three asterisks represents a P-value of ,0.001.
Isolation of Asf1 mutants with enhanced silencing
capabilities: To better understand the molecular role of
Asf1 in transcriptional silencing, we used transposon-
insertion mutagenesis of the ASF1 open reading frame
to identify mutations that alter Asf1-mediated silencing.
We generated a library of insertion mutations into a
CEN/ARS plasmid carrying the ASF1 open reading
frame driven by the ASF1 promoter. This ASF1 plasmid
librarywasintroduced intoastrain deleted forASF1and
CAC1; CAF-1, via deletion of CAC1, uncovers the role of
Asf1 in transcriptional silencing (Tyleret al. 1999). To
assay silencing, the strain carried a URA3 gene inserted
proximal to the telomere and an ADE2 gene inserted
into the HMR locus. A defect in silencing of TELVIIL-
TURA3 leads to death on 59FOA, while a defect in
silencing of HMRTADE2 leads to a change in colony
color from pink to white. We visually screened 2500
colonies derived from the ASF1 plasmid library for a
change from the light pink colonies of cac1 mutants on
We identified many isolates from the ASF1 plasmid
library with a defect in transcriptional silencing that
were also sensitive to DNA damaging agents. Sequenc-
ing of the plasmids indicated that many of the inser-
tions introduced a 59 in-frame stop codon generating
Saccharomyces cerevisiae strains used in this study
W303 MATa ade2-1 leu2-3,112, lys5, ura3-52, ade3TGal10THO DHOTAde1, DhmlTADE1 DhmrTade1
W303 MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 Asf1-185T-13mycTKAN
W303 MATa ade2-1 leu2-3,112 his3-11 trp1-1 ura3-1 cac1TLEU2 TELVIILTURA3 HMRaTADE2
W303 MATa ade2-1 can1-100 his3-11 leu2-3,112 trp1-1 ura3-1 Asf1-152T-13mycTKAN
W303 MATa ade2-1 leu2-3,112 his3-11 trp1-1 ura3-1 cac1TLEU2 TEVIILTURA3 459bpT-13mycTKAN
W303 MATa ade2-1 leu2-3,112 his3-11 trp1-1 ura3-1 asf1This51 bdf1TKAN TEVIILTURA HMRaTADE2
W303 MATa ade2-1 leu2-3,112 his3-11 trp1-1 ura3-1 asf1This51 cac1TLEU2 hir1TKAN TEVIILTURA
W303 MATa ade2-1 leu2-3,112 his3-11 trp1-1 ura3-1 cac1TLEU2 TEVIILTURA HMRaTADE2
W303 MATa ade2-1 leu2-3,112 his3-11 trp1-1 ura3-1 cac1TLEU2 TEVIILTURA HMRaTADE2
W303 MATa ade2-1 leu2-3,112 his3-11 trp1-1 ura3-1 cac1TLEU2 TEVIILTURA HMRaTADE2 SIR2-3HATTRP
W303 MATa ade2-1 LYS2 leu2-3,12 his3-11 trp1-1 ura3-1 TELVIILTURA3 HMRaTADE2 can1-100 SIR2-3HATTRP
W303 MATa ade2-1 leu2-3,12 his3-11 trp1-1 ura3-1 asf1This51 bdf1TKAN cac1TLEU2 TELVIILTURA3
W303 MATa ade2-1 leu2-3,12 his3-11 trp1-1 ura3-1 asf1This51 sir2TKAN cac1TLEU2 TELVIILTURA3
W303 MATa ade2-1 leu2-3,12 his3-11 trp1-1 ura3-1 ASF1-152TTKAN cac1TLEU2 TELVIILTURA3
W303 MATa ade2-1 leu2-3,12 his3-11 trp1-1 ura3-1 ASF1-185TTKAN cac1TLEU2 TELVIILTURA3
W303 MATa ade2-1 leu2-3,112 his3-11 trp1-1 ura3-1 cac1TLEU2 TELVIILTURA HMRaTADE2
(Tamburini et al. 2005)
W303 MATa ade2-1 leu2-3,12 his3-11 trp1-1 ura3-1 cac1TLEU2 asf1This51 TELVIILTURA3 HMRaTADE2
can1-100 (Tyler et al. 1999)
W303 MATa ade2-1 LYS2 leu2-3,112 his3-11 trp1-1 ura3-1 cac1TLEU2 TELVIILTURA3 HMRaTADE2
can1-100 (Tyler et al. 1999)
W303 MATa ade2-1 LYS2 leu2-3,112 his3-11 trp1-1 ura3-1 TELVIILTURA3 HMRaTADE2 can1-100
(Tyler et al. 1999)
Asf1 and CAF-1 Regulate Silencing601
extremely short Asf1 truncations and these were not
pursued further. We isolated two Asf1 mutants with an
enhanced ability to silence as compared to the wild-type
Asf1 protein expressed from the same CEN vector. One
of these, isolated five independent times, carried an
insertion mutation, termed ‘‘185T,’’ resulting in an in-
frame stop codon and truncation of Asf1 after amino
acid 185 (Figure 1A). Another Asf1 mutant that had
enhanced silencing, termed ‘‘152I,’’ had a 15-bp in-
sertion encoding amino acids V F L H Y between amino
acids 152 and 153 of Asf1 (Figure 1A). The 185T and
152I mutants had an improved ability to grow on 59FOA
and had a darker pink colony color as compared to cac1
mutants carrying the pAsf1 plasmid (Figure 1B). As
such, the 152I and 185T Asf1 mutants had increased
abilities to mediate transcriptional silencing, as com-
pared to the wild-type Asf1 protein, at the telomere-
proximal and HMR loci. Interestingly, the Asf1 mutants
with enhanced silencing abilities are dominant, as they
mediated enhanced silencing even when the endoge-
nous Asf1 protein was also present (Figure 1C). The
increase in silencing caused by the 152I and 185T Asf1
mutants was not simply due to increased amounts of
Asf1, as an extra plasmid-borne copy of the wild-type
ASF1 gene (pAsf1) had no effect on silencing in our
assays (Figure 1C).
To verify the phenotypes of these mutations, we
ASF1 geneto generateAsf1 truncated at 185 (185T) and
152 (152T) amino acids (Figure 1D). To determine the
degree to which silencing is enhanced by the 185Tand
152I/T Asf1 mutants in cac1D cells, we compared their
ability to silence transcription to that of wild-type cells.
We found that cac1D Asf1-185T, cac1D Asf1-152T, and
cac1D Asf1-152I strains were able to grow as well as wild-
type strains on 59FOA and were nearly as pink as wild-
type strains on low adenine (Figure 1D). This indicates
that the 185Tand 152I/T Asf1 mutants compensate for
the lack of CAF-1 for silencing. 13Myc epitopes were
introduced onto the C terminus of all the Asf1 mutants
to verify equal expression levels to endogenous Asf1.
Although the plasmid-borne 152I mutation expressed
similar levels of Asf1 protein as compared to the wild-
type Asf1 protein, we were able to detect only small
amounts of expression of the integrated 152I mutants
(Figure 1E, data not shown). Therefore, all further
studies of the 152I Asf1 mutant used the plasmid-borne
copy of this mutation. The plasmid-borne 152I and
integrated 152T and 185T Asf1 mutants expressed the
same protein levels as wild-type Asf1 (Figure 1E).
Wherever it was experimentally possible, further studies
used the integrated ASF1 mutants 152Tand 185T. Since
the Asf1 152T and 152I mutants were phenotypically
identical we used 152Tand 185T to assess the function
of the Asf1 C-terminal region. Because the integrated
ASF1 mutations also result in enhanced silencing we
conclude that the altered silencing was due to the
mutations in ASF1, not due to secondary mutations
elsewhere in the genome or altered expression levels of
Figure 1.—Mutations in Asf1 alter transcriptional silenc-
ing. (A) Schematic of Asf1 mutants and their observed phe-
notype for transcriptional silencing. The purple bars
represent positions of the insertion mutations. (B) Asf1 mu-
tants with enhanced silencing abilities of TELVIILTURA3 and
HMRTADE2 cac1Dasf1D yeast (ROY1169) carrying the wild-
type pAsf1 plasmid, mutant pAsf1 plasmid (as indicated),
or the empty vector (vector) were plated in 10-fold serial di-
lutions onto rich media (control), media with only 25% of the
normal amount of adenine (low ade), or 59FOA. (C) The 152I
and 185T Asf1 mutants are dominant. cac1D yeast (JKT0049)
carrying the wild-type pAsf1 plasmid, mutant pAsf1 plasmid
(as indicated), or cac1Dasf1D yeast (ROY1169) were plated
in 10-fold serial dilutions onto rich media (control) and
plates with the indicated concentration of 59FOA. (D) The
185T and 152T Asf1 mutants bypass the requirement for
CAF-1 in telomeric and HMR silencing. Yeast strains
ROY1172 (WT), ROY1171 (cac1D), BAT004 (cac1D Asf1-
185T) or BAT055 (cac1D Asf1-185T), BAT006 (cac1D Asf1-
(cac1Dasf1D) were plated in 10-fold serial dilutions onto rich
media (control) or media with 59FOA. (E) Anti-myc Western
blot of yeast strains ACN026 (WT), JKT0049 (no tag), BAT006
(152T), BAT004 (185T), and ROY1169 transformed with the
pAsf1152I vector (152I). A total of 7.5 mg/ml of total protein
was loaded for each sample.
602B. A. Tamburini et al.
To determine whether the effects of the Asf1 mu-
tations were specific to transcriptional silencing, we
examined whether the mutants retained other known
properties of wild-type Asf1. We verified that like wild-
type Asf1 (Sutton et al. 2001), the mutant Asf1 proteins
localized to the nucleus, using immunofluorescence
analysis (data not shown). Furthermore, the Asf1 mu-
tants, by contrast to yeast deleted for ASF1 (Tyleret al.
1999), had no obvious cell cycle defect or sensitivity to
the 185Tand 152T Asf1 mutants do not compensate for
the lack of CAF-1 for resistance to DNA damaging agents
Asf1 mediates the disassembly of chromatin at the
PHO5 promoter to allow production of the acid phos-
phatase encoded by PHO5 (Adkins et al. 2004). We
found that both the Asf1-152T and the Asf1-185T
mutants activate the PHO5 promoter as effectively as
wild-type Asf1, as measured by phosphatase activity
(Figure 2C). These data indicate that the I52I, 152T,
and 185T mutations are not globally disrupting all the
functions of Asf1, but instead are specifically affecting
transcriptional silencing mediated by Asf1.
The 185T and 152I/T Asf1 mutants bypass the re-
quirement for CAF-1 for the maintenance of transcrip-
tional silencing: To understand how the Asf1 mutants
are influencing transcriptional silencing, we examined
the colony sectoring due to expression of HMRTADE2
in more detail. CAF-1 has been shown to be important
for the maintenance/inheritance, but not the establish-
ment, of silencing (Monson et al. 1997; Enomoto and
Berman1998).Thisisapparentfrom the white colonies
with red sectors seen in the cac1 mutant carrying the
HMRTADE2 reporter (Figure 3), as was previously ob-
served for cac1 mutants carrying the TELVRTADE2
reporter (Monson et al. 1997). Colonies of the cac1D
Asf1-185T and cac1D Asf1-152T strains were mostly red
(Figure 3), indicating that they were able to maintain
their silent chromatin structure better than a cac1
mutant. When we observed the Asf1-152I mutation on
a plasmid in this same assay we saw identical results,
further demonstrating that the Asf1-152I and Asf1-152T
resistance, cell cycle progression, or nucleosome disassembly.
(A) Tenfold serial dilution analysis of yeast strains ROY1172
(WT), BAT003 (Asf1-185T), BAT005 (Asf1-152T), ROY1170
BAT006 (cac1D Asf1-152T), and ROY1169 (cac1Dasf1D) were
plated on the indicated amounts of methyl methanesulfonate.
of yeast strains ROY1172 (WT), ROY1171 (cac1D), ROY1169
(asf1D cac1D), BAT004 (Asf1-185Tcac1D), and BAT006 (Asf1-
152T cac1D). (C) Phosphatase activity of the gene product of
PHO5 was measured using a colorimetric assay at increasing
times after phosphate depletion from the media. Yeast strains
measured were ROY1172 (WT), ROY1171 (cac1D), BAT006
(cac1D Asf1-152T), and BAT004 (cac1D Asf1-185T).
Figure 3.—Asf1 mutants bypass the defect in maintenance
of transcriptional silencing due to CAF-1 inactivation. Magni-
fication of colonies is shown for analysis of color and sectoring
due to HMRTADE2. Images of colonies of yeast strains
ROY1172 (WT), ROY1171 (cac1D), ROY1169 (cac1Dasf1D),
BAT055 (cac1D ASF1-152T), and BAT056 (cac1D ASF1-185T)
are shown. Comparison of colony color due to silencing of
HMRTADE2 is shown. Streaks of strains described above
are shown, according to the schematic, with each plate includ-
ing the indicated Asf1 mutant.
Asf1 and CAF-1 Regulate Silencing 603
alleles are comparable (data not shown). This result
indicates that the 185T and 152I/T Asf1 mutants can
almost entirely rescue the defect in maintenance/
inheritance of silencing caused by lack of CAF-1.
Enhanced silencing by the 185T and 152I Asf1
mutants requires Sir2: To gain insight into the mech-
anism whereby the 185Tand 152I/T Asf1 mutants lead
to increased transcriptional silencing we sought to
identify other gene products required for the increased
transcriptional silencing. To do this, we deleted several
factors that have been shown to interact with Asf1 and/
or have a known role in transcriptional silencing and
tested their effect on silencing in combination with the
Asf1 mutants. We reasoned that if the mechanism of
increased silencing mediated by the Asf1 mutants
required these other factors, then their deletion should
abrogate the increased silencing. Hir1 binds to the N
of both HIR1 and ASF1 together caused no additional
defect in mating efficiency over each single deletion (in
combination with a CAC1 deletion) (Sharp et al. 2001).
We found that deletion of HIR1 only slightly reduced
the enhanced transcriptional silencing mediated by
the 152I or 185T Asf1 mutants (Figure 4A). This result
indicates that Hir1, or the interaction between Asf1 and
Hir1, may be partially required for the enhanced tran-
scriptional silencing due to the Asf1 mutations.
We performed a similar epistasis analysis with BDF1
encoding one of the missing bromodomains of TAF1,
the yeast counterpart of TAFII250. Asf1 interacts with
Bdf1 via its bromodomain and Bdf1 contributes to tran-
scriptional silencing by defining the boundary between
Ladurner et al. 2003). Contrary to a previous report, we
found that the asf1Dbdf1D strain is viable (Chimura et al.
2002), presumably due to differences within the strain
background. Deletion of BDF1 did not reduce the en-
hanced transcriptional silencing mediated by the 185T
and 152I Asf1 mutants (Figure 4B), indicating that
neither Bdf1 nor the interaction between Bdf1 and Asf1
is required for the enhanced transcriptional silencing
due to the Asf1 mutations.
When we performed the same type of epistasis
that deletion of SIR2 abolishes the enhanced transcrip-
tional silencing that is due to the 185T and 152I Asf1
mutants (Figure 4C). This result indicates that Sir2 is
required for the mechanism whereby the 185Tand 152I
Asf1 mutants increase transcriptional silencing at the
telomere-proximal region and the HMR locus. Upon
finding this result we concluded our search and focused
on the requirement of Sir2 in bypassing a cac1 deletion
for transcriptional silencing.
The 185T and 152I/T Asf1 mutants overcome a
defect in histone deposition and Sir2 recruitment in
yeast lacking CAF-1: To investigate how Sir2 contributes
to the increased silencing by the Asf1 mutants, we
measured Sir2 recruitment to the silent loci. We used
HA epitopes, using PCR to amplify regions at increasing
distances from the telomere and at the silencer HMR-E.
We found that yeast deleted for CAC1 have a signifi-
and HMR loci, as compared to wild-type cells (Figure
5A). Furthermore, when we looked at Sir2 occupancy at
Figure 4.—Sir2, but not Hir1 or Bdf1, is required for the
enhanced silencing due to Asf1 mutants. (A) Hir1 is only par-
tially required for enhanced silencing by the 185T and 152I
Asf1 mutants. All strains except WT have CAC1 deleted. Yeast
strains ROY1172 with the pRS314 empty vector (WT),
BAT034 with pAsf1-185T (hir1D Asf1-185T), BAT034 with
pAsf1-152I (hir1D Asf1-152I), ROY1169 with pAsf1-185T
(Asf1-185T), ROY1169 with pAsf1-152I (Asf1-152I), BAT034
with pAsf1 (hir1D), and ROY1171 with empty vector (vector)
adenine (low ade) or 1.0 mg/ml of 59FOA. (B) Bdf1 is not re-
quired for enhanced silencing by the 185Tand 152I Asf1 mu-
tants. All strains except WT have CAC1 deleted. Yeast strains
ROY1172 with the pRS314 empty vector (WT), BAT052 with
pAsf1-185T (bdf1D Asf1-185T), BAT052withpAsf1-152I (bdf1D
Asf1-152I), ROY1169 with pAsf1-185T (Asf1-185T), ROY1169
with pAsf1-152I (Asf1-152I), BAT052 with pAsf1 (bdf1D), and
ROY1171 with empty vector (vector) were plated in 10-fold se-
rial dilutions onto media with 75% less adenine (low ade) or
1.0 mg/ml of 59FOA. (C) Sir2 is required for enhanced silenc-
ing by the 185Tand 152I Asf1 mutants. All strains except WT
have CAC1 deleted. Yeast strains ROY1172 with the pRS314
empty vector (WT), BAT053 with pAsf1-185T (sir2D Asf1-
185T), BAT053 with pAsf1-152I (sir2D Asf1-152I), ROY1169
with pAsf1-185T (Asf1-185T), ROY1169 with pAsf1-152I
(Asf1-152I), BAT053 with pAsf1 (sir2D), and ROY1171 with
empty vector (vector) were plated in 10-fold serial dilutions
onto media with low adenine or 1.0 mg/ml 59FOA.
604 B. A. Tamburini et al.
increasing distances from the telomere, we did not
observe any significant shift in the location of the
boundary between the regions with Sir2 and without
Sir2 (Figure 5A) in the Asf1 mutants, as previously
et al. 2002; Ladurner et al. 2003). Because Sir2 is
absolutely required for silencing, this reduced Sir2
occupancy in cac1 mutant yeast is likely to be the mo-
lecular reason for the defect in the maintenance/
inheritance of silencing in CAF-1 mutants. Strikingly,
the cac1D Asf1-185T and cac1D Asf1-152T strains had a
Sir2 occupancy that was not significantly different from
that of wild type (Figure 5A). This result indicates that
the 185T and 152T Asf1 mutants can restore Sir2
occupancy to wild-type levels in the cac1 mutant.
To investigate whether the defect in Sir protein
recruitment in the cac1 mutant was specific to Sir2, we
examined recruitment of Sir4. To do this we performed
ChIP analysis using an antibody specific to Sir4. We
found a significant reduction in Sir4 recruitment to the
HMR-E and telomere-proximal region in the absence of
CAF-1 (Figure 5B). As was the case with Sir2, we found
that Sir4 recruitment was restored to wild-type levels by
the additional mutation of Asf1-152T or Asf1-185T in a
cac1 mutant (Figure 5B). The restored Sir2 and Sir4
occupancy resulting from the 185T and 152T Asf1
silencing (Figures 1 and 3A) and increased mainte-
nance of silencing due to these Asf1 mutants (Figure 3).
Consequently, the enhanced transcriptional silencing
CAF-1 is likely to result from increased Sir2 and Sir4
occupancy at the silent regions.
Next, we wanted to determine the mechanism
whereby the 185T and 152I/T Asf1 mutants restored
Sir protein occupancy at silent regions in CAF-1 mu-
tants. We compared global levels of Sir2 proteins in WT,
cac1D, cac1D Asf1-185T, and cac1D Asf1-152T strains
and found no significant difference (Figure 5D). Fur-
thermore, transcript levels of the SIR genes are not
significantly altered by deletion of CAC2 or ASF1
(Zabaronick and Tyler 2005). Sir2 is recruited to
interaction with histones H3 and H4 on chromatin
B) is due to reduced histone occupancy we measured
Figure 5.—Reduced his-
tone H3 and Sir levels at the
silent loci in CAF-1 mutants
are restored by the 152T and
185T mutations of Asf1. (A)
els. Quantitation of Sir2-HA
ChIP analysis from strains
ROY1172 (No tag), BAT046
(WT), BAT043 (cac1D Asf1-
185T), BAT044 (cac1D Asf1-
152T), and BAT045 (cac1D)
is shown at the indicated re-
gions of the genome; primer
pairs used were as described
2002), where TEL0.35 is 0.35
kb away from the end of TEL-
VIR, etc., and the HMR-E
within the HMRa locus flank-
of total chromatin was calcu-
chromatin precipitated. Error
bars represent the standard
deviation of at least three in-
dependent experiments. (B)
tion (ChIP) analysis of Sir4
levels: quantitation of Sir4 antibody ChIP from the same strains used in A. (C) Chromatin immunoprecipitation (ChIP) analysis of
H3 levels: quantitation of H3 C-terminal Antibody ChIP from the same strains used in A. The ratio of immunoprecipitated DNA
(IP) to input was calculated as a percentage of thetotal chromatin, and the value for the wild-type samples was normalized to 1. Error
bars represent the standard deviation of multiple independent experiments. (D) Western blot analysis of Sir2-HA. Soluble protein
extracts from the yeast strains used in A were Western blotted with either an anti-HA antibody or a GAPDH antibody (as a loading
control) to visualize Sir2 or GAPDH levels, respectively.
Asf1 and CAF-1 Regulate Silencing 605
histone occupancy by ChIP. Our ChIP analyses used an
antibody to the C terminus of histone H3 that has been
used extensively to measure histone occupancy on DNA
(Reinke and Horz 2003; Adkins et al. 2004; Bernstein
and Struhl 2004; Radonjic et al. 2005; Zhang et al.
2005). We found that histone H3 occupancy is greatly
reduced in the cac1 mutant at all regions that we
examined, including the telomere-proximal and HMR
loci and an open reading frame not found within a
silenced region, ALD6 (Figure 5C). Given that histone
H3 is always tightly associated with histone H4, and
given their central location in the nucleosome, the
reduced histone H3 occupancy indicates a reduced
nucleosome density in cac1 mutants. Our results strongly
suggest that the reduced Sir2 and Sir4 occupancy at si-
lent regions and the resulting defect in transcriptional
nucleosome density. When we examined histone H3
occupancy in the cac1D Asf1-185T and cac1D Asf1-152T
strains,wefound no significantdifferencefromwildtype
(Figure5C).Thisresult indicatesthat the185Tand152T
which in turn restores Sir2 and Sir4 occupancy and
The Asf1-histone H3 interaction is disrupted in Asf1-
152T and Asf1185T mutants: To investigate how the
185T and 152I/T Asf1 mutants result in increased
histone occupancy, we first verified that the amount of
total Asf1 protein and total soluble H3 present was
equivalent between samples. This allowed us to rule out
altering expression levels of histones (Figure 6A). Since
the Asf1 and histone levels were comparable between
samples we tested whether the interaction between Asf1
and histone H3 was affected by these mutations. To do
this, we measured the level of histone H3 that coimmu-
noprecipitated with the endogenous wild-type and
mutant Asf1 proteins. We had previously found that
the stringency of our immunoprecipitation conditions
required the addition of an in vivo protein–protein
crosslinker, dithiobis(succinimidyl propionate) (DSP),
to detect the Asf1–histone H3 interaction (Tamburini
et al. 2005) (Figure 6B). However, we found that
even with the crosslinker, we were unable to detect co-
immunoprecipitating histone H3 with the Asf1-152Tor
Asf1-185T proteins (Figure 6B). It is unlikely that the
Asf1 mutants are globally misfolded, because they were
still able to co-immunoprecipitate Rad53 (Emili et al.
2001; Hu et al. 2001) (Figure 6B). Interestingly, the
interaction between Rad53 and Asf1-152Tor Asf1-185T
was weaker than between wild-type Asf1 and Rad53,
suggesting that the unstructured C-terminal tail of Asf1
may act to stabilize protein–protein interactions. We
propose that the interaction between Asf1 and histone
H3 still exists, but that it is less stable, allowing Asf1 to
indicate that the 152T and 185T mutations reduce the
stability of the histone–Asf1 interaction.
This work discerns the molecular basis for the
mechanism whereby chromatin assembly factors medi-
ate transcriptional silencing. Our data indicate that
a nucleosome density sufficient to recruit enough Sir
proteins to mediate the formation of a stable silent
chromatin structure. Mutations within the C terminus
of the chromatin assembly factor Asf1 suppress the
requirement for CAF-1 in silencing, presumably via
their weakened interaction with histones that results
in a restored nucleosome density and increased Sir pro-
CAF-1 promotes transcriptional silencing by depos-
iting histones that serve as the binding sites for Sir
proteins: Although the defect in the maintenance/
inheritance of transcriptional silencing in CAF-1 mu-
tants has been well documented, the molecular mech-
anism was unclear (Monson et al. 1997; Enomoto and
Berman 1998). The Sir2–Sir4 complex and Sir3 are
required for the maintenance of silencing and are
Figure 6.—The Asf1–histone H3 interaction is
reduced in Asf1-185T and 152T mutants. The
amount of soluble histones is invariable between
the Asf1 mutant strains used. (A) Western blot
analysis of total Asf1 and histone proteins using
an H3 C-terminal antibody and an antibody that
recognizes H3 acetylated on lysine residues 9 and
14 was performed with soluble protein extracts
from yeast strains ACN026 (WT), ROY1172 (no
tag), BAT004 (Asf1-185T), and BAT006 (Asf1-
152T). The amount of total protein loaded from
each sample was 7.5 mg/ml. (B) Co-immunopre-
cipitation analysis of histones with Asf1. Yeast
strains used in A were immunoprecipitated with an anti-myc antibody and Western blot analysis was used to visualize H3 and
Rad53. ‘‘Crosslinker’’ indicates the use of a protein–protein crosslinker, DSP. Newly synthesized soluble histones are acetylated
on lysine 9 of histone H3; therefore, the anti-H3 acetyl lysine 9/14 antibody was used to detect all soluble histones bound to
Asf1, as used previously (Tamburini et al. 2005).
606B. A. Tamburini et al.
recruited to DNA via interactions with histones H3
and H4 (Hecht et al. 1995). In the absence of CAF-1,
nucleosome occupancy is reduced and the resulting Sir
recruitment is reduced (Figure 5, A–C). It was interest-
ing to note that the level of Sir proteins on the silent
chromatin in CAF-1 mutants was not reduced to the
same extent as the level of histone proteins (Figure 5,
A–C). Sir proteins appear to be limiting for silencing in
wild-type cells (see, for example, Renauld et al. 1993;
Fritze et al. 1997; Smith et al. 1998). As such it is
unlikely that the maximal Sir:histone ratio is reached
at the silent chromatin in wild-type cells. In CAF-1 mu-
tants, histone deposition is reduced compared to that
in wild type (Figure 5C). Total available pools of Sir
protein in the CAF-1 mutant cell are the same as that in
a wild-type cell (Figure 5D). Consequently, CAF-1
mutants have a higher Sir:histone ratio at the silent
chromatin than wild type. Even though the Sir:histone
ratio at the silent chromatin is higher in the CAF-1
mutants, the absolute levels of Sir proteins on the
chromatin are still less than that of wild type (because
that this is the reason for the silencing defect in CAF-1
mutants. The Sir:histoneratio at the silent chromatin in
the CAF-1 mutants, although higher than that in wild
type, still presumably does not reach the maximal Sir:
histone ratio possible, because overexpression of Sir
proteins in CAF-1 mutants restores the silencing defect
(Monson et al. 1997; Enomoto and Berman 1998;
Smith et al. 1999).
Given the reduced histone occupancy and Sir2 and
Sir4 occupancy at the silent regions in CAF-1 mutants
(Figure 5, A–C), we propose that CAF-1-mediated
chromatin assembly following DNA replication gener-
ates a nucleosome density sufficient for recruitment of
enough Sir proteins to mediate the maintenance of the
silent chromatin structure. Because silencing can be
established in CAF-1 mutants, it appears that a silent
chromatin structure with suboptimal stability can be
generated in the absence of CAF-1, but is not stably
maintained. This is likely to be a consequence of the
reduced levels of Sir2 and Sir4 recruitment in CAF-1
mutants because reduced levels of Sir2 or Sir3 lead to a
similar defect in the maintenance of silencing (Holmes
and Broach 1996; Enomoto and Berman 1998). In
accordance with this idea, overexpression of the SIR3 or
SIR2 gene is able to suppress the silencing defect seen
in CAF-1 mutants (Monson et al. 1997; Enomoto and
Berman 1998; Smith et al. 1999) (data not shown).
Overexpression of SIR3 may force Sir3 to the silent
regions, increasing the occupancy of Sir3 protein to a
level sufficient for maintenance of the silent state in
CAF-1 mutants. Furthermore, it has been shown pre-
viously that overexpression of histones H3 and H4 can
partially rescue the silencing defect in CAF-1 mutants
histone occupancy on the DNA, which would facilitate
recruitment of the Sir proteins for silencing. Similarly,
deletion of one copy of the genes expressing histone
H3 and H4 exacerbates the silencing defect of CAF-1
mutants (Kaufman et al. 1998), presumably resulting in
an even lower histone occupancy on the DNA and
further reduced Sir protein recruitment.
Our data provide the first direct indicationof a defect
in histone deposition in CAF-1 mutants, supporting
a role for CAF-1 in chromatin assembly in vivo. The
1 mutants (Figure 5C) is consistent with (i) the bio-
chemical function of CAF-1 as a chromatin assembly
factor that deposits histones H3 and H4 (Smith and
Stillman 1989, 1991), (ii) the increased accessibility of
chromatin upon CAF-1 inactivation to micrococcal
nuclease and DNAse I and reduced supercoiling of
Adkins and Tyler 2004; Nabatiyan and Krude 2004),
and (iii) the increased psoralen accessibility observed
at the rDNA loci in CAF-1 mutants (Smith et al. 1999).
CAF-1 is recruited to sites ofDNA replicationvia its inter-
action with proliferating cell nuclear antigen (PCNA)
(Shibahara and Stillman 1999). Consistent with the
idea that silencing defects in CAF-1 mutants are the
consequence of improper chromatin assembly follow-
ing DNA replication is the fact that PCNA alleles also
have silencing defects that are in the same genetic path-
way as CAF-1 mutants (Zhang et al. 2000).
Mutations that reduce global histone methylation or
acetylation indirectly influence silencing (Lacoste
et al. 2002; Ng et al. 2002; van Leeuwen et al. 2002).
Sir proteins bind preferentially to unmodified histones,
and consequently the loss of histone modifications
appear to be limiting (Buck and Shore 1995; Strahl-
Bolsinger et al. 1997; Smith et al. 1998), the result is a
reduced effective concentration of Sir proteins and
weakened silencing. Although histone methylation has
not been examined in CAF-1 mutants, there is no
detectable defect in histone acetylation in CAF-1 mu-
tants (Adkins and Tyler 2004). This argues against the
possibility that reduced Sir2 occupancy in the silent
regions ofcac1 mutants may bedue to increasedhistone
acetylation at the silent loci preventing Sir binding or
reduced histone acetylation elsewhere in the genome
sequestering the Sir proteins. However, it is possible
that there is increased histone acetylation restricted to
the silent regions that was undetectable by the pre-
vious analysis, causing the decrease in Sir2 recruitment.
Silencing defects can also be due to transcriptional
misregulation of gene products required for silencing.
of Sir2 protein upon deletion of CAC1 (Figure 5D), nor
did we observe altered transcript levels of any of the
known silencing proteins by microarray analyses in cac2
or asf1 mutants (Zabaronick and Tyler 2005). The
Asf1 and CAF-1 Regulate Silencing 607
strong correlations between the reduced histone occu-
pancy, Sirproteinoccupancy, and transcriptional silenc-
ing in the CAF-1 mutants and their restoration by the
Asf1-152T and 185T mutants strongly support our pro-
posal that reduced histone deposition is the molecular
basis for the silencing defect in CAF-1 mutants.
The C terminus of Asf1 contributes to the histone
interaction and transcriptional silencing: Our studies
provide the first evidence that the C terminus of Asf1 is
functionally relevant. While other studies have found
that Asf1 is functional without the C terminus of the
protein (Umehara et al. 2002; Daganzo et al. 2003;
Mousson et al. 2005), we have uncovered a role in
transcriptional silencing where mutations in the Asf1
C terminus are able to compensate for the defect in
silencing caused by deletion of CAC1. We observed an
apparent reduced affinity interaction between the Asf1
mutants and histone H3, suggestingthat the Cterminus
of Asf1 may contribute to the stability of the interac-
tion with histone H3. There are at least two possible
scenarios for how a reduced interaction between his-
tone H3 and Asf1 could lead to higher histone levels on
the DNA when CAF-1 is inactive and the resulting
increased Sir2 and Sir4 recruitment and restored
transcriptional silencing that we observed. First, the
mutant Asf1 proteins may be less efficient at removing
the histones from the DNA during chromatin disassem-
bly, resulting in increased histone occupancy on the
DNA. Second, the weakened interaction between histo-
nes and mutant Asf1 may promote histone deposition
onto the DNA. However, only an increased assembly
activity of the mutants could account for the dominant
nature of the mutant Asf1 proteins over the wild-type
Asf1 protein (Figure 1C). Furthermore, a decreased
disassembly activity would yield a silencing phenotype
that is observed with the 185Tand 152I/TAsf1 mutants.
Consequently, we propose that the C-terminal muta-
tions result in Asf1 proteins with increased chromatin
assembly activity. The 185T and 152I/T Asf1 mutants
that bypass the requirement for CAF-1 for silencing are
reminiscent of specific mutations within the N-terminal
tail and core of histones H3 and H4 that also bypass the
requirement for CAF-1 for telomeric silencing (Smith
et al. 2002). It will be interesting to determine whether
those particular histone mutants also lead to increased
histone deposition onto the silent chromatin in CAF-1
mutants, perhaps via reducing their affinity for Asf1.
While the crystal structure and NMR structure of the
conserved N-terminal domain of yeast Asf1 and human
Asf1a, respectively, have been solved, little is known
about the regions of Asf1 that mediate protein inter-
actions important for its function (Daganzo et al. 2003;
Mousson et al. 2005). Mutational analysis directed
against unstructured loop regions within the Asf1
conserved domain has revealed the binding site for
human HirA on Asf1a to be in the cleft of the protein
C-terminal regions of the conserved domain (Daganzo
of Asf1 is important for histone H3–H4 binding, while
the nonconserved acidic C terminus of Asf1 contributes
to binding of histones H3, H4, H2A, and H2B in vitro
(Umehara et al. 2002). Recent data also suggest that the
binding, specifically valine 94, aspartic acid 54, and
arginine 108 (Mousson et al. 2005). Since the 152Tand
185T Asf1 mutants were not able to detectably bind to
histone H3 in our assay, it is possible that the C-terminal
tail ofAsf1 stabilizestheinteraction of histone H3 inthe
‘‘groove’’ of Asf1 (Daganzo et al. 2003). Moreover,
histone binding to Asf1 truncated at amino acid 155 is
easily disrupted in high-salt conditions, suggesting that
the highly acidic C terminus is involved in stabilizing
electrostatic contacts (Daganzo et al. 2003). Interest-
ingly, the interaction between Rad53 and Asf1-152T or
Asf1-185T was weaker than between wild-type Asf1 and
Rad53 (Figure 6B), possibly because the C-terminal tail
of Asf1 is also involved in stabilizing this interaction.
Here we demonstrate that the C-terminal region of
the Asf1 protein has distinct silencing function in vivo.
The Asf1 mutants that had enhanced silencing activity
and weakened histone interaction were insertions
between amino acids 152 and 153, truncation at 152,
and truncation at 185. Amino acid 152 is found at the C
unstructured region (Daganzo et al. 2003). Because
insertion and truncation at amino acid 152 provided
indistinguishable defects in silencing, it would appear
that the sequences around amino acid 152 are impor-
tant for silencing. Furthermore, the silencing defect in
the truncation at amino acid 185 indicates that sequen-
ces C-terminal to amino acid 185 are also important for
silencing, perhaps contributing to the stability of
histone binding. One possibility is that the region of
Asf1 around amino acid 152 and the C terminus of Asf1
are close together in three-dimensional space, where
they both contribute to the same aspect of silencing.
However, such a prediction awaits the structure of the
full-length Asf1 protein.
In summary, CAF-1 contributes to the recruitment of
Sir proteins to the silent loci via the interaction with
histone proteins to mediate transcriptional silencing.
Furthermore, mutations that weaken the interaction
betweenAsf1 and thehistonesresultinrestoredhistone
levels, Sir2 recruitment, and transcriptional silencing
at the silent loci in CAF-1 mutants. These studies pro-
vide molecular insight into the interplay between the
formation of the underlying nucleosome structure and
specialized chromatin structures.
We thank Christine English, Melissa Adkins, and Josh Ramey for
critical reading of this manuscript. We are grateful to Danesh Moazed
for the Sir4 antibody. We thank Judit Kiss for technical assistance with
screening the mutants and Melissa Adkins for assistance with the
608B. A. Tamburini et al.
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