DNA Replication Origin Function Is Promoted by H3K4 Di-methylation in Saccharomyces cerevisiae.
ABSTRACT DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.
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ABSTRACT: In the genome of eukaryotic cells, DNA synthesis is initiated at multiple sites called origins of DNA replication. Origins must fire only once per cell cycle and how this is achieved is now well understood. However, little is known about the mechanisms that determine when and where replication initiates in a given cell. A large body of evidence indicates that origins are not equal in terms of efficiency and timing of activation. Origin usage also changes concomitantly with the different cell differentiation programs. As DNA replication occurs in the context of chromatin, initiation could be influenced by multiple parameters, such as nucleosome positioning, histone modifications, and three-dimensional (3D) organization of the nucleus. This view is supported by recent genome-wide studies showing that DNA replication profiles are shaped by genetic and epigenetic processes that act both at the local and global levels to regulate origin function in eukaryotic cells.Current opinion in genetics & development 03/2013; · 8.99 Impact Factor
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ABSTRACT: In Saccharomyces cerevisiae, all H3K4 methylation is performed by a single Set1 Complex (Set1C) that is composed of the catalytic (Set1) and seven other subunits (Swd1, Swd2, Swd3, Bre2, Sdc1, Spp1 and Shg1). It has been known for quite some time that trimethylated H3K4 (H3K4me3) is enriched in the vicinity of meiotic double-strand breaks (DSBs), but the link between H3K4me3 and the meiotic nuclease Spo11 was uncovered only recently. The PHD-containing subunit Spp1, by interacting with H3K4me3 and Mer2, was shown to promote the recruitment of potential meiotic DSB sites to the chromosomal axis allowing their subsequent cleavage by Spo11. Therefore, Spp1 emerged as a key regulator of the H3K4 trimethylation catalyzed by Set1C and of the formation of meiotic DSBs. These findings illustrate the remarkable multifunctionality of Spp1, which not only regulates the catalytic activity of the enzyme (Set1), but also interacts with the deposited mark, and mediates its biological effect (meiotic DSB formation) independently of the complex. As it was previously described for Swd2, and now for Spp1, we anticipate that other Set1C subunits, in addition to regulating H3K4 methylation, may participate in diverse biological functions inside or outside of the complex.Epigenetics: official journal of the DNA Methylation Society 03/2013; 8(4). · 4.58 Impact Factor
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ABSTRACT: The target of rapamycin complex 1 (TORC1) is an evolutionarily conserved signal transduction pathway activated by environmental nutrients that regulates gene transcription to control cell growth and proliferation. How TORC1 modulates chromatin structure to control gene expression, however, is largely unknown. Because TORC1 is a major transducer of environmental information, defining this process has critical implications for both understanding environmental effects on epigenetic processes and the role of aberrant TORC1 signaling in many diseases, including cancer, diabetes, and cardiovascular disease. To elucidate the role of TORC1 signaling in chromatin regulation, we screened a budding yeast histone H3 and H4 mutant library using the selective TORC1 inhibitor rapamycin to identify histone residues functionally connected to TORC1. Intriguingly, we identified histone H3 lysine 37 (H3K37) as a residue that is essential during periods of limited TORC1 activity. An H3K37A mutation resulted in cell death by necrosis when TORC1 signaling was simultaneously impaired. The induction of necrosis was linked to alterations in high mobility group (HMG) protein binding to chromatin. Furthermore, the necrotic phenotype could be recapitulated in wild-type cells by deregulating the model HMG proteins, Hmo1 or Ixr1, thus implicating a direct role for HMG protein deregulation as a stimulus for inducing necrosis. This study identifies histone H3 and H4 residues functionally required for TORC1-dependent cell growth and proliferation that are also candidate epigenetic pathways regulated by TORC1 signaling. It also demonstrates a novel role for H3K37 and TORC1 in regulating the binding of select HMG proteins to chromatin and that HMG protein deregulation can initiate a necrotic cell death response. Overall, the results from this study suggest a possible model by which chromatin anchors HMG proteins during periods of limited TORC1 signaling, such as that which occurs during conditions of nutrient stress, to suppress necrotic cell death.Epigenetics & Chromatin 08/2013; 6(1):29. · 4.19 Impact Factor
DNA Replication Origin Function Is Promoted by
H3K4 Di-methylation in Saccharomyces cerevisiae
Lindsay F. Rizzardi,* Elizabeth S. Dorn,†Brian D. Strahl,*,†and Jeanette Gowen Cook*,†,1
*Curriculum in Genetics and Molecular Biology and†Department of Biochemistry and Biophysics, University of North Carolina,
Chapel Hill, North Carolina 27599
ABSTRACT DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin
structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important
for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and
those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required
for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth
of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found
that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains
bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and
tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially
affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore,
histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and
sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-
translational modifications to support robust genome duplication.
replicated from many individual origins to ensure complete
and precise genome duplication during each cell division
cycle. Individual origins vary both in the likelihood that they
will initiate replication, or “fire,” in any given S phase and in
the firing time within the S phase (Weinreich et al. 2004).
Highly efficient origins fire in most cell cycles, whereas in-
efficient origins fire in only some cycles and are usually
passively replicated by forks emanating from neighboring
efficient origins. Although highly efficient origins that sup-
port initiation in most cell cycles have been identified in
many genomes, the chromosomal determinants of origin
location and function are still incompletely understood.
Strikingly, while DNA sequence elements can be necessary,
it is clear that sequence alone is insufficient to fully specify
eukaryotic origin location and activity (Méchali 2010).
NA replication initiates at discrete genomic loci termed
“origins of replication.” Each eukaryotic chromosome is
Like all DNA-templated processes, replication occurs on
chromatin. Recent progress in the field has demonstrated
that the chromatin structure surrounding origins plays an
essential role in controlling origin activity. For example, the
positioning of nucleosomes near origins can either stimulate
or inhibit origin function (Simpson 1990; Crampton et al.
2008; Berbenetz et al. 2010; Eaton et al. 2010). The major
protein components of chromatin, the histone proteins, can
also be post-translationally modified by acetylation, methyl-
ation, phosphorylation, ubiquitination, and sumoylation
(Kouzarides 2007). These modifications can alter DNA ac-
cessibility and serve as recognition sites for other proteins.
Importantly, several individual histone modifications af-
fect aspects of origin function. For example, acetylation of
histones H3 and H4 accelerates the timing of origin firing
within S phase and can increase origin efficiency (Aggarwal
and Calvi 2004; Espinosa et al. 2010; Unnikrishnan et al.
2010). In addition, histone H3 lysine 36 mono-methylation
(H3K36me1) by the Set2 methyltransferase has been impli-
cated in the recruitment of the replication initiation protein,
Cdc45 (Pryde et al. 2009). In metazoan genomes, PR-Set7-
catalyzed histone H4 lysine 20 mono-methylation (H4K20me1)
Copyright © 2012 by the Genetics Society of America
Manuscript received June 8, 2012; accepted for publication July 18, 2012
1Corresponding author: Biochemistry and Biophysics, University of North Carolina, 120
Mason Farm Rd., CB#7260, Chapel Hill, NC 27599. E-mail: email@example.com
Genetics, Vol. 192, 371–384October 2012
stimulates the loading of the core replicative helicase (Tardat
et al. 2007, 2010).
It is clear that no single histone modification is absolutely
required for origin function since loss of individual histone-
modifying enzymes does not impact cell viability. This
observation suggests that a combination of histone modifi-
cations facilitates efficient DNA replication in the form of
a “histone code” similar to the combinations known to reg-
ulate transcription (Strahl and Allis 2000). While some ele-
ments of this code have been identified (e.g., H3 and H4
acetylation, H4K20 mono-methylation, and H3K36 mono-
methylation), the complexity of DNA replication led us to
hypothesize that additional histone modifications that im-
pact origin activity remain to be discovered. We therefore
sought to identify those histone modifications and chroma-
tin modifiers that are integral to this process. We conducted
a genetic screen to identify histone modifications that are
required for the fitness of a hypomorphic replication yeast
mutant, cdc6-1. This screen revealed a previously unidenti-
fied positive DNA replication role for histone H3 lysine 4
(H3K4) methylation by the COMPASS complex, and our
subsequent analysis indicates that H3K4 di-methylation is
the relevant modification for this activity. These findings
contribute to elucidating the pattern of chromatin features
that determines origin activity in eukaryotic genomes.
Materials and Methods
Yeast strains and growth conditions
The Saccharomyces cerevisiae strains used in this study are
listed in Table 1, and any additional genotype information is
available upon request. Construction of de novo gene dele-
tion strains was performed by PCR-mediated disruption, and
some double-mutant construction was performed by mating
as indicated in Table 1.
All plasmids used in this study are listed in Table 2.
Synthetic genetic array screen
Synthetic genetic array (SGA) analysis was carried out as
previously described (Tong and Boone 2006). Briefly, 63
deletion strains (Table 3) were mated to the temperature-
sensitive cdc6-1 strain (JCY332), and haploids carrying both
mutations were isolated by growth on selective media. All
of the deletion strains originated from the Yeast Knock
Out library (Open Biosystems) except strains lacking SET1
or DOT1; the set1D strains were created de novo while
the dot1D strain was previously published (Gardner et al.
2011). Additionally, SET1 and BRE1 deletions were recre-
ated de novo in the cdc6-1 mutant in the BY4741 background
(yLF058). All of the resulting double mutants were spotted
in fivefold serial dilutions with an initial OD600of 0.5 onto
YPD, grown for 3 days at 32?, and growth was compared to
that of the cdc6-1 single mutant. Double mutants displaying
a synthetic growth phenotype were confirmed by analyzing
three independent isolates. The fold change in growth is
denoted by a score from 1 to 3 indicating an approximate
5- to 125-fold change compared to cdc6-1 alone. Negative
values indicate growth defects, while positive values indi-
cate enhanced growth or rescue. No genetic rescue was ob-
served in any double-mutant strain.
Minichromosome maintenance assays
Minichromosome (or plasmid) maintenance assays were per-
formed as described previously (Tye 1999). Briefly, yeast strains
containing YCplac33, YCplac111, or YCplac33+2XARS209
were grown to log phase in the appropriate selective
media and 100–200 cells were plated on both selective
and nonselective media to establish an initial percentage of
plasmid-bearing cells. These cultures were also diluted to
a concentration of 1 · 105cells/ml in 5 ml of nonselective
media and grown for 8–10 generations before once again
plating on both selective and nonselective media. Precise
generation numbers were calculated using the following
formula: n = log(CF/CI)/log(2), where CFrepresents the
final number of cells as measured by OD600and CIrepre-
sents the starting cell number of 105cells/mL. After 2 days
of growth, colonies were counted, and the plasmid loss rate
(L) per generation (n) was calculated using the following
formula: L = 1 2 (%F/%I)(1/n), where %F is the final per-
centage of cells that retained the plasmid and %I is the
initial percentage of cells that contain the plasmid.
Whole-cell extracts were prepared by extraction with tri-
chloroacetic acid (TCA). Cell growth was halted by the
addition of TCA to a final concentration of 5%, and the cell
pellets were frozen at 280?. Pellets were resuspended in
200 ml TCA buffer (10 mM Tris–HCl, pH 8.0, 10% TCA,
25 mM NH4OAc, 1 mM EDTA) and broken by glass-bead
lysis. Proteins were precipitated by centrifugation, resus-
pended in 100 ml resuspension buffer (0.1 M Tris–HCl, pH
11.0, 3% SDS), and boiled for 5 min. Samples were centri-
fuged, and the supernatant was quantified using the Dc
Assay (BioRad). Equal concentrations of lysates were loaded
onto 15% SDS-PAGE gels and transferred onto PVDF. The
following antibodies were used: anti-H3 (1:10,000; Active-
Motif 39163), anti-H3K4me1 (1:2000; Millipore 07-436), anti-
H3K4me2 (1:2000; Abcam 32356), anti-H3K4me3 (1:10,000;
gift from M. Bedford), anti-H2B (1:5000; ActiveMotif 39237),
and anti-LexA (1:5000; Millipore 06-719).
Yeast strains were cross-linked with 1% formaldehyde for
15 min at room temperature and quenched with 250 mM
glycine for 5 min at room temperature. Forty OD600units
of cross-linked cells were harvested by centrifugation and
washed thoroughly, and the pellets were stored at 280?.
The cell pellets were resuspended in 300 mM FA-lysis buffer
(50 mM HEPES-KOH, pH 7.5, 300 mM NaCl, 1 mM EDTA,
1% TritonX-100, 0.1% Na-deoxycholate) with protease
L. F. Rizzardi et al.
inhibitors and broken by glass-bead lysis, and fixed chro-
matin was sheared by sonication using a Branson sonifier
250. Average DNA fragment lengths were 100–300 bp
determined by gel analysis. After centrifugation and quanti-
fication via Bradford Assay (BioRad), 0.5 mg of soluble chro-
matin was incubated with 2 ml of antibody [anti-H3
(ActiveMotif), anti-H3K4me2 (Abcam), or anti-H3K4me3
(Millipore)] in 1.5-ml tubes overnight at 4? and immuno-
precipitated with 10 ml Protein A Dynabeads (Invitrogen)
for 1 h at 4?. The beads were washed sequentially with
300 mM FA-lysis buffer, twice with 500 mM FA-lysis buffer
(50 mM HEPES-KOH, pH 7.5, 500 mM NaCl, 1 mM EDTA,
1% TritonX-100, 0.1% Na-deoxycholate), once with LiCl so-
lution (10 mM Tris–HCl, pH 8.0, 250 mM LiCl, 0.5% NP-40,
0.5% Na-deoxycholate, 1 mM EDTA), and once with TE, pH
8.0. After washing, the chromatin was eluted from the beads
in 200 ml elution buffer (0.1 M NaHCO3, 1% SDS) for
30 min at room temperature. The eluted material was trea-
ted with RNAse A and Proteinase K before de-cross-linking
at 65? overnight. The DNA was purified using Genesee
UPrep spin columns and eluted in 100 ml water. Immuno-
precipitation (IP) samples and IP controls (set1D) were used
undiluted while input samples were diluted 1:10. Samples
were analyzed by quantitative PCR on the ABI 7900 HT
(AppliedBiosystems) using SYBR Green master mix with
Rox (Fermentas). Primer sequences are available upon re-
quest. Signals from the immunoprecipitates are reported as
a percentage of the input and normalized to H3. Error bars
Table 1 Strains used in this study
MATa met15D0, cdc6-1::hph
MATa met15D0, cdc6-1::hph, set1D::HIS3MX6
MATa met15D0, set1D::HIS3MX6
MATa met15D0, bre1D::kanMX
MATa met15D0, set1D::kanMX
MATa met15D0, bre2D::kanMX
MATa met15D0, swd1D::kanMX
Mata can1Δ::STE2p_Sp.his5+, lyp1Δ::STE3p_LEU2
Mata can1Δ::STE2p_Sp.his5+, lyp1Δ::STE3p_LEU2, cdc6-1::hph
Mata lyp1Δ::STE3p_LEU2, cdc6-1::hph, swd1Δ::kanMX
Mata lyp1Δ::STE3p_LEU2, cdc6-1::hph, bre2Δ::kanMX
Mata lyp1Δ::STE3p_LEU2, cdc6-1::hph, rad6Δ::kanMX
MATa met15D0, rad6D::kanMX
MATa met15D0, cdc6-1::hph, bre1Δ::kanMX
MATa can1D::STE2pr-Sp_his5, lyp1D::STE3pr-LEU2, cdc7-4::natMX
MATa lyp1D::STE3pr_LEU2, cdc7-4::natMX
MATa can1D::STE2pr_Sp-his5, cdc7-4::natMX, set1D::kanMX
MATa can1D::STE2pr-Sp_his5, lyp1D::STE3pr-LEU2, cdc7-1::natR
MATa can1D::STE2pr-Sp_his5, lyp1D::STE3pr-LEU2, cdc7-1::natMX, set1D::kanMX
MATa can1D::STE2pr-Sp_his5, lyp1D::STE3pr-LEU2, cdc45-27::natMX
MATa his3D1, ura3D0
MATa his3D1, ura3D0, set1D::kanMX
MATa his3D1, ura3D0, set1D::kanMX, cdc45-27::natMX
MATa his3D1, ura3D0, cdc45-27::natMX
MATa hht1-hhf1::LEU2, hht2-hhf2::kanMX3, [YCp-URA3(HHT2-HHF2)]
MATa hht1-hhf1::LEU2, hht2-hhf2::kanMX3, cdc6-1::hph, [YCp-URA3(HHT2-HHF2)]
MATa mfa::MFA1p_HIS3, ORC6-rxl::LEU2, URA3::GALp_CDC6DNT-HA
MATa mfa::MFA1p_HIS3, ORC6-rxl::LEU2, URA3::GALp_CDC6DNT-HA, swd1D::kanMX
MATa mfa::MFA1p_HIS3, ORC6-rxl::LEU2, URA3::GALp_CDC6DNT-HA, bre1D::kanMX
MATa hta1-htb1::LEU2, hta2-htb2::TRP1 [pSAB6 HTA1-HTB1 URA3]
Brachmann et al. (1998)
Schuldiner et al. (2006)
Costanzo et al. (2010)
Costanzo et al. (2010)
Costanzo et al. (2010)
Biswas et al. (2006)
Archambault et al. (2005)
Gardner et al. (2011)
Hirschhorn et al. (1995)
aAdditional genotype: his3D1, leu2D0, ura3D0.
bAdditional genotype: his3D1, leu2D0, ura3D0, met15Δ0, lys2Δ0, LYS2+, cyh2.
cGenerated by mating and only markers listed were confirmed.
dAdditional genotype: can1-100, his3-11,15, leu2-3,112, ura3-1, lys2, trp1-1, ade2-1.
eAdditional genotype: his3Δ200, trp1Δ63, lys2-128d, ura3-52, leu2Δ1.
H3K4me2 Is Required for Efficient Replication
represent the standard deviations of the average signals be-
tween experiments (n $ 3).
Identification of histone modifiers that promote
DNA replication origins in the budding yeast S. cerevisiae
are defined by both sequence elements and local chroma-
tin structure. Although DNA replication is essential for cell
proliferation, the majority of histone modifications and
chromatin-modifying enzymes are not individually required
for yeast cell viability. This observation supports the model
that a combination of histone modifications supports repli-
cation origin function. To identify new histone modifications
that contribute to this combination, we conducted a genetic
screen. We reasoned that individual chromatin elements
that influence replication activity would be revealed as ge-
netic suppressors or enhancers of cell growth in a strain
bearing a hypomorphic mutation in an essential replication
The Cdc6 ATPase plays an essential role at origins in
loading the replicative helicase complex composed of MCM2-7
(Bell and Dutta 2002; Takahashi et al. 2002). The cdc6-1
mutant harbors a G260D mutation in the catalytic domain
resulting in failure to load MCMs at restrictive temperatures
(Feng et al. 2000). Yeast cells harboring the cdc6-1 mutation
produce a Cdc6 protein that functions normally at 29? and
is nonfunctional at temperatures .34?, but retains partial
function at intermediate temperatures between 30? and 33?
(Feng et al. 2000). To identify suppressors or enhancers of
cdc6-1, we deleted genes for most of the known histone
modifiers, chromatin remodelers, and histone chaperones
(63 total; see Table 3) in the cdc6-1 temperature-sensitive
replication mutant strain. Double-mutant strains were tested
for fitness at semipermissive temperatures and compared to
the parent single-mutant strains (Tong and Boone 2006).
The majority of double-mutant strains grew neither better
nor worse than their respective parents under any of the
tested growth conditions, and no null alleles improved the
growth of the cdc6-1 mutant (Table 3). Although a role for
the histone acetyltransferase (HAT) Gcn5 in DNA replication
has been shown (Espinosa et al. 2010), it was not included
in this screen because the null mutant strain has a slow-
growth phenotype that would complicate interpretation of
the double-mutant phenotype.
In contrast, 21 of the null alleles (including a positive
control, tom1D) impaired growth in the cdc6-1 strain but
had little effect in otherwise wild-type backgrounds. These
genes represent a wide array of chromatin factors including
HATs, histone deacetylases (HDACs), and histone methyl-
transferases (HMTs) (Table 3). Interestingly, many of these
factors contributed either directly or indirectly to a single
histone modification, H3K4 methylation, which is depos-
ited by Set1, the catalytic subunit of the COMPASS com-
plex (Figure 1A). H3K4 is mono-, di-, and tri-methylated by
Set1 as part of the COMPASS complex. COMPASS consists
of seven additional subunits that promote complex integrity
(Swd1, Swd2, Swd3, Bre2), regulate catalytic activity (Sdc1
and Spp1), or have unknown function (Shg1) (Takahashi
and Shilatifard 2010; Mersman et al. 2011; Takahashi et al.
2011). Unlike other COMPASS members, Swd2 also func-
tions as part of the APT transcription termination complex,
and its role in this complex is essential for cell viability
(Soares and Buratowski 2012). For this reason, Swd2 was
not included in our screen. The absence of Set1, Swd1,
Swd3, Bre2, or Sdc1 impaired the growth of the cdc6-1
strain (Table 3).
COMPASS activity and H3K4 methylation promote
We confirmed the enhancer phenotype of the SET1 deletion
strain by constructing a set1D allele de novo in the cdc6-1
parent strain. Growth of the cdc6-1 strain was only slightly
Table 2 Plasmids used in this study
ARS1, CEN4, URA3
ARS1, CEN4, LEU2
ARS1 and 2 copies of ARS209, CEN4, LEU2
GAL1-LexA, 2m, URA3
GAL1-LexA-SET1, 2m, URA3
GAL1-LexA-set1-DRRM, 2m, URA3
GAL1-LexA-set1-H1017K, 2m, URA3
HTA1-Flag-HTB1, CEN6, HIS3
HTA1-Flag-htb1-K123R, CEN6, HIS3
HTA1-Flag-htb1-R119A, CEN, HIS3
HTA1-Flag-htb1-R119D, CEN6, HIS3
Geitz and Sugino (1988)
Geitz and Sugino (1988)
Nakanishi et al. (2008)
L. F. Rizzardi et al.
impaired at 31? compared to wild-type or the cdc6-1 strain
harboring wild-type CDC6 on a plasmid, but growth was
substantially impaired when SET1 was deleted in this strain
(Figure 1B). Expression of wild-type SET1, but not of the
catalytically dead mutant set1-H1017K, rescued the syn-
thetic growth defect of the set1D cdc6-1 strain (Figure 1B).
Importantly, the synthetic growth defect of the set1D cdc6-1
strain was recapitulated in a cdc6-1 strain in which the only
copy of histone H3 bears the K4R (unmethylatable) muta-
tion (Figure 1C). These findings indicate that the catalytic
activity of Set1 is important for robust growth of the cdc6-1
Set1 functions as the catalytic subunit of the COMPASS
complex. We hypothesized that other members of this com-
plex would display similar phenotypes when deleted in the
cdc6-1 strain. Newly constructed deletions of BRE2, SDC1,
SWD1, and SWD3 each impaired the growth of the cdc6-1
mutant at semipermissive temperatures, but not deletion of
SPP1 or SHG1 (Figure 1D and Table 3). Bre2, Sdc1, Swd1,
Swd2, and Swd3 (along with Set1) are the core structural
components of the COMPASS complex required for full ac-
tivity (Takahashi et al. 2011). These results further support
the conclusion that COMPASS enzymatic activity and H3K4
methylation are important for proliferation when Cdc6 is
The poor growth of these double-mutant strains could
be due to a general exacerbation of the replication defect
caused by Cdc6 perturbation, or it could reflect a specific
interaction between Cdc6 and H3K4 methylation. If Set1
and H3K4 methylation are generally important for efficient
DNA replication, then we would expect similar proliferation
defects from deleting SET1 in other replication mutant
strains. To test this prediction, SET1 was deleted in two
temperature-sensitive cdc7 mutants and one temperature-
sensitive cdc45 mutant. These replication factors are required
for origin firing at the G1/S transition, downstream of Cdc6
activity (Tercero et al. 2000; Labib 2010) . Similar to the effect
of deleting SET1 in the cdc6-1 strain, loss of SET1 in the cdc7-1,
cdc7-4, and cdc45-27 mutants impaired growth at semipermis-
sive temperatures (Figure 2A).
The poor growth of cdc6-1 set1D cells suggests that Set1
promotes replication; we thus predicted that loss of H3K4
methylation in a hypermorphic replication mutant would at
least partially rescue the adverse phenotypes of that mutant.
To test this hypothesis, we introduced the SET1 null allele
into the hypermorphic replication mutant RUY028. This
yeast strain harbors two mutations that deregulate replica-
tion origin licensing, resulting in re-replication, an aberrant
phenomenon in which some origins fire more than once per
cell cycle, leading to DNA damage and poor cell growth. The
ORC6-rxl mutation prevents inhibitory phosphorylation of
the Orc6 subunit of the origin recognition complex (ORC)
by CDK, and the GAL1 pr-CDC6-DNT allele produces a hyper-
stable Cdc6 protein (Archambault et al. 2005). In this
strain, re-replication is induced during growth on galac-
tose, which induces transcription of the CDC6-DNT allele.
Table 3 Results from SGA screen for genetic interaction
E3 ubiquitin ligase
E2 ubiquitin-conjugating enzyme
E3 ubiquitin ligase/positive control
aScore represents change in growth corresponding to approximate 5-fold differ-
ences (1, 5-fold; 2, 25-fold, etc.).
bStrains in a YMS196 cdc6-1 background confirmed in three independent isolates.
cStrains created de novo.
H3K4me2 Is Required for Efficient Replication
As expected, loss of H3K4 methylation upon deletion of
SWD1 or BRE1 partially rescues the poor growth of the re-
replicating strain on galactose (Figure 2B). Together, these
data provide strong evidence that H3K4 methylation promotes
efficient DNA replication, most likely through regulation of
A positive role for H3K4 methylation in replication sug-
gests that this histone modification is enriched near origins
of replication. To test this idea directly, we performed
chromatin immunoprecipitation assays for both di- and tri-
methylated H3K4 in asynchronous wild-type yeast cultures.
Analysis of several genomic loci revealed that both H3K4
methylation states are enriched at both an early and a late-
firing origin of replication (ARS315 and ARS822, respec-
tively) relative to a nonorigin, telomere-proximal region
The Rad6/Bre1 ubiquitin ligase complex promotes
Methylation of histone H3K4 by the COMPASS complex
requires prior mono-ubiquitination of histone H2B at lysine
123 (H2BK123) by the Rad6/Bre1 ubiquitin ligase complex
(Lee et al. 2007; Nakanishi et al. 2009). Our original screen
detected a strong synthetic growth defect when the bre1D
library mutant was crossed with the cdc6-1 parent strain. De
novo deletion of either BRE1 or RAD6 in the cdc6-1 mutant
Figure 1 COMPASS and H3K4 are required for robust growth of the temperature-sensitive cdc6-1 replication mutant. (A) COMPASS complex model
adapted from Takahashi et al. (2011). An asterisk denotes a complex member that genetically interacts with cdc6-1. (B) Wild-type (BY4741), cdc6-1
(yLF058), set1D (yLF062), and cdc6-1 set1D (yLF063) were transformed with an empty vector [pGLx2], a vector producing LexA-tagged wild-type Set1
[pGLx2-SET1], or catalytically dead Set1 [pGLx2-set1-H1017K] from a GAL1 promoter construct or a vector producing normal Cdc6 [pRS316-CDC6] from
the CDC6 promoter as indicated. Fivefold serial dilutions were spotted onto SC-URA containing 1% galactose/2% raffinose and grown at the indicated
temperatures for 4 days. (C) The cdc6-1 mutation was introduced into the H3-H4 “shuffle” strain (DY7803) transformed with HHT2 or hht2-K4R
plasmids. Fivefold serial dilutions were spotted onto YPD and grown at the indicated temperatures for 3 days. (D) Fivefold serial dilutions of wild-type
(YMS196), cdc6-1 (JCY332), cdc6-1 swd1D (yLF114), cdc6-1 bre2D (yLF120), bre2D (yLF060), or swd1D (yLF061) were spotted onto YPD and grown at
the indicated temperatures for 3 days.
Figure 2 H3K4 methylation is required
for robust growth of multiple replication
mutants. (A) Fivefold serial dilutions of
the meiotic progeny from the cross of
cdc7-1 (TSQ880), cdc7-4 (TSQ131), or
cdc45-27 (TSQ694) with set1D (yLF062)
were spotted onto YPD and grown at
the indicated temperatures for 3 days.
(B) Fivefold serial dilutions of wild-type
(RUY121), ORC6-rxl CDC6-DNT (RUY028),
swd1D (yLF051), and ORC6-rxl CDC6-DNT
swd1D (yLF049) were spotted onto YP
containing 2% dextrose (no re-replication)
or galactose (re-replication induced) and
grown for 2 days at 30?.
L. F. Rizzardi et al.
resulted in an ?125-fold decrease in growth at semipermis-
sive temperatures, validating this genetic interaction (Fig-
ures 4, A and B). Ectopic expression of wild-type BRE1
under control of its own transcriptional promoter fully res-
cued the exacerbated growth phenotype, but expression of
a catalytically dead Bre1 (bre1-H665A) did not (Figure 4A).
Moreover, BRE1 deletion also partially rescued the growth
impairment of the re-replicating RUY028 strain similar to
deletion of SET1 (Figure 4C). These data suggest that the
Rad6/Bre1 ubiquitin ligase complex is also required for ef-
ficient DNA replication, likely through its involvement in
promoting H3K4 methylation.
H3K4 methylation is required for efficient
To assess DNA replication more specifically than general cell
proliferation, we used a plasmid loss assay that measures
the ability of cells to maintain a minichromosome bearing
a centromere and a single replication origin (Maine et al.
1984). Growth in nonselective medium for several cell
divisions allows cells that failed to initiate replication of
the minichromosome to survive, and these cells are then
counted as colonies on nonselective medium. Strains lacking
either SET1 or BRE1 displayed significantly higher plasmid
loss rates per cell division than wild-type strains did (Figure
5A, P , 0.05). Similar to the phenotype resulting from SET1
deletion, cells expressing the mutant histone H3K4R dis-
played a significant increase in plasmid loss rate (Figure 5B).
Notably, H2B mono-ubiquitination by Rad6/Bre1 is a pre-
requisite not only for H3K4 methylation, but also for H3K79
methylation (Nakanishi et al. 2009). Neither deletion of
DOT1, the histone H3K79 methyltransferase, nor mutation
of H3K79 to arginine had any effect on plasmid maintenance
(Figure 5, A and B). Moreover, loss of Dot1 did not impair
the growth of the cdc6-1 strain (Table 3). Efficient growth
and plasmid maintenance by these strains indicate that the
elevated plasmid loss rate of the BRE1 null strain is likely
due to the subsequent loss of H3K4 methylation rather than
loss of H3K79 methylation. Furthermore, these results dem-
onstrate that inefficient minichromosome maintenance is
not a universal phenotype of strains lacking histone-modifying
enzymes or expressing mutant histones.
If these minichromosome maintenance defects are spe-
cific to perturbations of origin function and not chromosome
segregation or expression of the selectable marker, then
adding multiple origins to the test plasmid should rescue the
elevated loss rates of the set1D and bre1D strains. Additional
origins multiply the chances for a successful origin initiation
event on the plasmid, and success at any one origin allows
replication and transmission to both daughter cells. This
property has been used by others to validate origin-specific
phenotypes (Hogan and Koshland 1992). We modified the
single ARS1-containing plasmid by adding two copies of
ARS209. As before, the plasmid harboring only one origin
was lost more frequently from set1D and bre1D strains than
from wild-type strains, but this effect was reversed with the
addition of multiple origins (Figure 5C). This result supports
the conclusion that H2BK123 mono-ubiquitination by Bre1
and the consequent H3K4 methylation by Set1 promote
DNA replication origin function in yeast.
H3K4 di-methylation is required for efficient
The Set1/COMPASS complex is responsible for mono-, di-,
and tri-methylation of H3K4. These methylation states
are differentially distributed over gene bodies (Pokholok
et al. 2005), and several studies have shown that the differ-
ent methylation states of H3K4 and H3K36 can have differ-
ent functions (Taverna et al. 2006; Pinskaya et al. 2009;
Pryde et al. 2009). The individual H3K4 methylation states
depend on other histone residues and are influenced by
the presence or absence of individual COMPASS subunits
(Chandrasekharan et al. 2010; Mersman et al. 2011). To gain
insight into which H3K4 methylation states were required for
efficient DNA replication, we measured plasmid loss rates in
strains lacking different COMPASS complex members. Like the
set1D strain (Figure 5A), both swd1D and bre2D strains dis-
played significantly elevated plasmid loss compared to their
isogenic wild-type counterparts (Figure 6A). These two pro-
teins are both required for Set1 stability (Takahashi et al.
2011), and in their absence no H3K4 methylation is detectable
(Figure 6B). Unlike Swd1 and Bre2, Spp1 is a COMPASS sub-
unit that is required for H3K4 tri-methylation, but not mono- or
di-methylation (Takahashi et al. 2009) (Figure 6B); loss of
SPP1 had no effect on plasmid maintenance (Figure 6A).
This result is consistent with our earlier observation that
Spp1 loss did not affect proliferation of the cdc6-1 strain
(Table 3). Taken together, these data suggest that H3K4
Figure 3 H3K4 methylation is present at replication origins. Chromatin
immunoprecipitation experiments were performed on asynchronous
wild-type (BY4741) or set1D (yLF062) strains grown at 30?. Immunopre-
cipitates using antibodies to total histone H3, di-methylated H3K4
(H3K4me2) in A and tri-methylated H3K4 (H3K4me3) in B were analyzed
by quantitative PCR for chromosomal DNA fragments from a region near
telomere VI-R and two replication origins, ARS315 and ARS822. Error
bars represent the standard deviations of n $ 3 biological replicates.
Significant enrichment of H3K4 methylation at origins compared to telo-
mere VI-R was determined using the Student’s unpaired t-test (*P ,
H3K4me2 Is Required for Efficient Replication
di-methylation is sufficient for proper origin function and
that H3K4 tri-methylation is dispensable.
To examine the role of H3K4 di-methylation without
perturbing the COMPASS complex, we took advantage of H2B
mutants that were previously shown to differentially affect
H3K4 methylation (Chandrasekharan et al. 2010). Mutation
of H2BK123 to arginine abolished mono-ubiquitination of
this residue and consequently eliminated both H3K4 di-
and tri-methylation (Figure 6D). Importantly, similar to
loss of BRE1, this mutation induced a significantly higher
plasmid loss rate than wild type (Figure 6C). This result,
in combination with the failure of the Bre1 catalytically
dead mutant to complement the growth defect of the bre1D
cdc6-1 mutant, strongly argues that the H2BK123 ubiquiti-
nation function of Bre1 is required for full origin function.
Additionally, it supports theimportance of H3K4 di-methylation
in this process.
A previous study reported that mutational alteration of
H2B arginine 119 to alanine does not prevent H2BK123
mono-ubiquitination, but does affect the degree of H3K4
methylation (Chandrasekharan et al. 2010). As previously
reported, changing H2BR119 to alanine (H2BR119A) re-
duces the chromatin association of Spp1 and therefore
H3K4 tri-methylation, whereas changing H2B119 to aspartic
acid (H2BR119D) results in loss of H3K4 di- and tri-
methylation to the same extent as eliminating H2BK123
mono-ubiquitination (Chandrasekharan et al. 2010). Our
analysis of these histone H2B mutants revealed that strains
expressing htb1-R119D displayed elevated plasmid loss rates
similar to those of the htb1-K123R strain (Figure 6C). Mu-
tation of R119 to alanine abolished H3K4 tri-methylation
as expected, while having only a moderate effect on H3K4
di-methylation (Figure 6D). This H2B mutant showed a
plasmid loss rate similar to the wild-type HTB1 strain. The
Figure 4 H2BK123 mono-ubiquitination promotes robust growth of the temperature-sensitive cdc6-1 replication mutant. (A) Wild-type (BY4741),
bre1D (yLF151), cdc6-1 (yLF058), and cdc6-1 bre1D (yLF150) were transformed with either an empty vector [pRS315], a vector expressing BRE1 [pRS315-
9xMyc-BRE1], or bre1-H665A [pRS315-9xMyc-bre1-H665A] from the native BRE1 promoter. Fivefold serial dilutions were spotted onto SCD-LEU and
grown for 3 days at the indicated temperatures. (B) Fivefold serial dilutions of wild-type (YMS196), cdc6-1 (JCY332), rad6D (yLF154), and cdc6-1 rad6D
(yLF117) were spotted onto YPD and grown for 3 days at the indicated temperatures. As previously reported, the rad6D strain is cold sensitive at
temperatures ,30? (McDonough et al. 1995). (C) Wild-type (RUY121), ORC6-rxl CDC6-DNT (RUY028), bre1D (yLF052), and ORC6-rxl CDC6-DNT bre1D
(yLF050) were spotted onto YP containing 2% dextrose or 1% galactose (re-replication induced) and grown for 3 days at 30?.
Figure 5 H3K4 methylation is required
for efficient origin-dependent mini chro-
mosome maintenance. (A) Plasmid loss
rates of a single-origin-bearing plasmid
[YCpLac33] were measured in a wild-
typestrain (BY4741) and in strains lacking
SET1 (yLF089), BRE1 (yLF151), or DOT1
(YNL037). Loss rates are reported per cell
division. (B) Plasmid loss rates of a single-
origin-bearing plasmid [YCpLac111] were
measured in the histone “shuffle” strain
(DY7803) transformed with plasmids
expressing wild-type HHT2, hht2-K4R,
or hht2-K79R. (C) Plasmid loss rates of
plasmids bearing either a single origin
[YCpLac33] or three origins [YCpLac33
+ 2X ARS209] were measured. For all
experiments, the average loss rates were obtained from at least three independent transformants, and the error bars indicate standard deviations.
Statistics were performed using the Student’s unpaired t-test (*P , 0.05).
L. F. Rizzardi et al.
strict correlation between the ability to produce H3K4 di-
methylation and normal plasmid maintenance underscores
the importance of H3K4 di-methylation, but not necessarily
H3K4 tri-methylation, for origin function (Figure 6C).
Our initial observation that loss of SET1 in a cdc6-1
mutant strain resulted in a proliferation defect, coupled
with the requirement for Set1 for proper minichromosome
maintenance, clearly indicates that H3K4 methylation is
necessary for robust DNA replication. To determine if H3K4
di-methylation is sufficient for DNA replication, we transformed
the cdc6-1 set1D strain with plasmids directing the production
of wild-type Set1, catalytically dead Set1 (Set1H1017K), or
a Set1 protein lacking the RRM domain. The latter mutant
can only di-methylate H3K4 (Schlichter and Cairns 2005).
Each of these Set1 proteins was fused to a LexA tag for
detection on immunoblots (Figure 7B) and expressed from
the GAL1 promoter on galactose-containing medium. Immu-
noblot analysis of the three H3K4 methylation states con-
firmed that the fusions generated the expected methylation
states (Figure 7B). As before, production of active Set1 sup-
pressed the growth phenotype caused by deleting SET1 in
the cdc6-1 strain (Figure 7A). Importantly, production of
Set1DRRM also fully rescued this proliferation defect with-
out the ability to produce H3K4 tri-methylation (Figure 7B).
These data demonstrate that H3K4 di-methylation is both
necessary and sufficient for robust growth of the cdc6-1 mu-
tant and suggest that this histone modification plays an im-
portant positive role in origin function.
This study documents a novel role for H3K4 di-methylation
in DNA replication origin function. Yeast strains with hypo-
morphic mutations in multiple replication genes are highly
dependent on H3K4 di-methylation for robust growth. In
these replication mutants (cdc6-1, cdc7-1, cdc7-4, cdc45-27)
under semipermissive conditions, replication activity is re-
duced to the minimum needed for normal growth, and any
further reduction in active origins caused by loss of H3K4
di-methylation results in severely impaired proliferation.
Additionally, normal propagation of a minichromosome con-
taining a single origin requires H3K4 di-methylation even
in an otherwise wild-type strain. Every mutation that pre-
vents H3K4 di-methylation, including loss of the prerequisite
histone H2BK123 mono-ubiquitination, causes a similar
replication phenotype. These data provide clear evidence
that H3K4 di-methylation is important for full origin
Figure 6 H3K4 di-methylation promotes efficient mini-
chromosome maintenance. (A) Plasmid loss rates of the
single-origin-bearing plasmid [YCpLac33] were measured
for wild-type (BY4741), swd1D (yLF061), bre2D (yLF060),
and spp1D (yLF153). (B) Immunoblot analysis of whole-cell
extracts (from strains shown in A) using antibodies to total
H3, H3K4me1, H3K4me2, and H3K4me3. (C) Plasmid loss
rates of the single-origin-bearing plasmid [YCpLac111]
were measured for the H2B “shuffle” strain (FY406) trans-
formed with plasmids expressing HTB1 [pZS145], htb1-
K123R [pZS146], htb1-R119D [pZS473], or htb1-R119A
[pZS145-R119A]. (D) Immunoblot analysis of whole-cell
extracts (from strains shown in C) using antibodies specific
for total H2B, H3, and H3K4 methylation as in B. All plas-
mid loss data represent the mean and standard deviation
of at least three independent transformants. Statistics were
performed using the Student’s unpaired t-test (*P , 0.05).
H3K4me2 Is Required for Efficient Replication
The Set1 histone methyltransferase and the Bre1 E3
ubiquitin ligase have well-established roles in regulating
gene expression (Shukla et al. 2006; Mutiu et al. 2007).
Nonetheless, several lines of evidence suggest that the phe-
notypes that we have detected are due to reduced replica-
tion activity as opposed to altered gene expression. First,
many chromatin-modifying enzymes showed no synthetic
growth phenotype when combined with the cdc6-1 muta-
tion. Some of these non-interacting genes include those with
much more profound effects on patterns of gene expression
than Set1, suggesting that replication phenotypes are not a
general outcome of perturbed gene expression control. In-
terestingly, even the Rpd3 histone deacetylase that regulates
origin firing time within S phase (Knott et al. 2009) did not
genetically interact with cdc6-1 (Table 3), implying that the
synthetic growth phenotypes observed here are relatively
specific for origin function and not origin firing time. Sec-
ond, a genome-wide analysis identified only 55 transcripts
that changed significantly in a set1D strain compared to a
wild-type strain, and none of those genes are predicted to
directly affect origin activity (Lenstra et al. 2011). Third,
the mini-chromosome maintenance phenotypes associated
with loss of H3K4 di-methylation were largely suppressed
by the addition of extra origins to the test plasmid, indicat-
ing that the phenotypes are closely tied to origin function
and not to other biological parameters. Fourth, we directly
detected H3K4 di- and tri-methylation at two yeast origins.
Moreover, our analysis of a published genome-wide H3K4
tri-methylation data set identified peaks of methylation dis-
tinct from nearby transcription-associated peaks (Radman-
Livaja et al. 2010 and our unpublished observations). Finally,
our observation that loss of H3K4 methylation exacerbates
poor growth of a replication hypomorphic strain but suppresses
the poor growth of an origin-firing hypermorphic strain indi-
cates that the replication phenotypes reported here are most
likely direct positive effects of H3K4 di-methylation at origins.
Taken together, these data provide strong evidence that the
role of H3K4 di-methylation in origin function is direct and
separate from any indirect transcriptional effects.
DNA replication is an essential process for proliferation,
yet neither Set1 nor Bre1 are essential gene products. In-
terestingly, very few null mutations in yeast chromatin-
modifying enzymes show significant growth defects despite
their importance for several essential processes, such as tran-
scription, replication, and repair. The function of histone mod-
ifications in transcription control has been described as a
“histone code” in which combinations of post-translational
modifications promote or repress transcription at a given
locus (Strahl and Allis 2000; Oliver and Denu 2010). In this
model, no single histone modification generates an active or
inactive promoter, and thus the effects of mutations that
alter local chromatin structure are cumulative. We propose
that the same concept applies to chromatin structure at
DNA replication origins. Consistent with the idea that, like
promoters, origins can accommodate elimination of a single
positive element of chromatin structure, the kinetics of
S-phase progression are normal in a set1D strain grown un-
der standard conditions (data not shown). Also analogous to
transcriptional control at promoters, we propose that his-
tone H3K4 di-methylation is an important element of origin
chromatin structure, but its absence alone does not severely
inhibit origin function. In fact, origin activity is robust enough
that even substantial reductions in expression of MCM pro-
teins or deletion of many origins from a yeast chromosome
causes no growth defects (Dershowitz et al. 2007; Ge et al.
2007; Blow et al. 2011). Nevertheless, the effect of H3K4 di-
methylation loss over several cell cycles or in combination
with other replication perturbations causes a significant loss
of replication fitness.
How does H3K4 di-methylation promote DNA replication?
We propose two models by which H3K4 di-methylation
could facilitate DNA replication (Figure 8). The first is a re-
cruitment model whereby a replication factor directly inter-
acts with di-methylated H3K4 to associate with replication
origins. This possibility is supported by precedent since
it has been suggested that mono-methylation of H3K36
can recruit the replication initiation factor Cdc45 (Pryde
et al. 2009). In addition, a member of the origin recognition
Figure 7 H3K4 di-methylation is sufficient for robust growth
of the cdc6-1 mutant. (A) Wild-type (BY4741), cdc6-1
(yLF058), set1D (yLF062), and cdc6-1 set1D (yLF063) were
transformed with an empty vector [pGLx2], a vector produc-
ing LexA-tagged Set1 [pGLx2-SET1], or Set1 lacking the RRM
domain [pGLx2-set1-DRRM] from the GAL1 promoter. Five-
fold serial dilutions were spotted onto SC-URA containing
1% galactose and grown at the indicated temperatures for
5 days. (B) Immunoblot analysis of whole-cell extracts from
either wild-type (BY4741) or set1D (yLF062) strains trans-
formed with empty vector [pGLx2] or vectors producing
LexA-tagged normal (“WT”) Set1 [pGLx2-SET1], catalytically
dead (“CD”) Set1 [pGLx2-set1-H1017K], or Set1 lacking the
RRM domain (“RRM”) [pGLx2-set1-DRRM] from the GAL1
promoter. Blots were probed with antibodies specific for
LexA, total H3, H3K4me3, H3K4me2, and H3K4me1. A sin-
gle asterisk represents a likely degradation product unique to
the positive control construct; a double asterisk represents
a nonspecific band detected by the H3K4me1 antibody.
L. F. Rizzardi et al.
complex, Orc1, contains a bromo-adjacent homology do-
main that mediates nucleosome binding in budding yeast
and specifically binds to di-methylated H4K20 at origins in
human cells (Müller et al. 2010; Kuo et al. 2012). Such
histone-binding domains in core replication factors or as-yet-
unidentified bridging proteins could link H3K4 di-methylation
to recruitment of the replication machinery at origins.
The second model suggests that H3K4 di-methylation
near origins recruits another chromatin modifier such as
a HAT. Acetylation of nearby nucleosomes resulting in in-
creased origin accessibility would allow more efficient asso-
ciation of replication factors (Figure 8). Acetylation is already
known to affect the efficiency and timing of origin firing
(Vogelauer et al. 2002; Goren et al. 2008). Additionally,
several HAT complexes contain subunits that specifically rec-
ognize H3K4 methylation. The HAT complex SAGA contains
the catalytic subunit Gcn5 as well as two proteins, Chd1 and
Spt8, that genetically interact with cdc6-1 (Table 3). Chd1 is
a chromatin remodeler whose mammalian counterpart is
capable of binding H3K4 methylation (Sims et al. 2005)
while Spt8 impacts transcription by directly recruiting
TATA-binding protein (Sermwittayawong and Tan 2006).
SAGA also contains the Sgf29 subunit that has recently been
reported to bind di- and tri-methylated H3K4 to facilitate
SAGA recruitment to some promoters (Bian et al. 2011).
SAGA is responsible for acetylation of multiple H3 residues
as well as H4K8 and H2BK11 and K16 (Grant et al. 1999).
Two other HAT complexes were also identified in our screen,
NuA3 and NuA4; both contain subunits, Yng1 and Yng2,
respectively, that bind H3K4 methylation (Martin et al.
2006a,b; Taverna et al. 2006; Shi et al. 2007). Further ex-
amination of these and other H3K4 methylation readers will
shed light on the role of H3K4 methylation in the context of
Prior research has identified other histone modifications
that impact origin function. Mono-methylation of H3K36
by Set2 facilitates recruitment of the replication initiation
factor Cdc45 in yeast (Pryde et al. 2009). In addition to
methylation, H3K4 can also be acetylated by both Gcn5
and Rtt109 (Guillemette et al. 2011). At promoters, this
acetylation is typically found just upstream from the peak
of H3K4 tri-methylation. Whether H3K4 acetylation is also
found at origins is currently unknown. Multi-acetylated his-
tones H3 and H4 have been shown by several groups to
impact the ability of origins to fire (Unnikrishnan et al.
2010) and the timing of origin firing in yeast, Drosophila,
and mammalian cells (Vogelauer et al. 2002; Hiratani et al.
2009; Schwaiger et al. 2009). Additionally, the HATs Hat1
and Gcn5 physically interact with the replication machinery
(Suter et al. 2007; Espinosa et al. 2010). In mammalian
cells, Gcn5 acetylates Cdc6 at several lysine residues, and
this acetylation is required for subsequent phosphorylation
by cyclin/CDKs (Paolinelli et al. 2009). In vertebrates, the
H4-specific HAT, Hbo1, binds (Iizuka and Stillman 1999;
Burke et al. 2001) and acetylates several origin-binding pro-
teins and histone H4 (Iizuka et al. 2006; Miotto and Struhl
2010), and this acetyltranferase activity is required for effi-
cient origin licensing. In metazoans, methylation of H4K20
by PR-Set7 (a.k.a. Set8) is cell cycle regulated (Tardat et al.
2010; Beck et al. 2012) and is required for proper S-phase
initiation and prevention of re-replication (Wu and Rice
2011). In S. cerevisiae, histone H3 is phosphorylated at thre-
onine 45 in a cell cycle-dependent manner by the Cdc7-Dbf4
kinase. Mutating this residue to alanine causes sensitivity to
hydroxyurea and camptothecin, indicating its importance in
proper DNA replication (Baker et al. 2010).
Because all organisms except S. cerevisiae lack common
nucleotide sequence motifs at origins, discovering a chroma-
tin signature that promotes origin function is essential to
understanding the location and activity of replication
zones in higher eukaryotes. Given the complexity of meta-
zoan genomes, it may be that several different chromatin
signatures specify origins in different chromosomal domains.
In fact, many histone modifications have been found at
human replication origins, including H3K4me3, H3K56me,
H4K20me1, H4K20me2, and multiple acetylations on H3
and H4 (Rampakakis et al. 2009; Miotto and Struhl 2010;
Tardat et al. 2012; Yu et al. 2012). Our discovery that H3K4
di-methylation is able to promote DNA replication adds to
the growing number of histone modifications that could
function together to confer origin function at discrete geno-
mic loci. Further work will determine which suite of his-
tone modifications is common to all origins or is required
only at specific genomic loci to successfully promote DNA
We thank Zu-Wen Sun for strains and H2B plasmids; Charlie
Boone for the cdc45-27, cdc7-1, and cdc7-4 mutant strains;
Alisha Schlichter and Brad Cairns for Set1 mutant plasmids;
Doug Koshland for plasmids containing the tandem ARS209
Figure 8 Two models by which H3K4 di-methylation
could promote efficient DNA replication. The recruitment
model suggests either direct or indirect recruitment of rep-
lication factors by H3K4 di-methylation, while the accessi-
bility model suggests recruitment of a HAT that acetylates
nucleosomes near the origin, resulting in an open chroma-
tin state that allows replication factors to access and bind
H3K4me2 Is Required for Efficient Replication
insert; Ali Shilatifard for wild-type and catalytically dead
Bre1 plasmids; Fred Cross for RUY121 and RUY028 re-
replication strains; Mark Bedford for the H3K4 tri-methyl
antibody; and members of the Cook and Strahl labs for
many helpful discussions during the course of this work.
This research was supported by National Institutes of Health
(NIH) grants T32 GM07092-34 (L.F.R.), T32 CA071341-15
(E.S.D.), and NIH R01 GM68088 (B.D.S.) and by funding
from the American Heart Association (10PRE3740013 to
L.F.R. and BGIA 0865097E to J.G.C.).
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