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: email@example.com
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.
phosphatase assay. This work was supported by a grant from the
National Institutes of Health (GM64475) to J.K.T. J.K.T. is a Leukemia
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Communicating editor: S. Henikoff
610B. A. Tamburini et al.