Copyright ? 2008 by the Genetics Society of America
Saccharomyces cerevisiae Yta7 Regulates Histone Gene Expression
Angeline Gradolatto,* Richard S. Rogers,†Heather Lavender,* Sean D. Taverna,‡
C. David Allis,‡John D. Aitchison†and Alan J. Tackett*,1
*Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205,
†Institute for Systems Biology, Seattle, Washington 98103 and‡The Rockefeller University, New York, New York 10065
Manuscript received December 28, 2007
Accepted for publication February 22, 2008
The Saccharomyces cerevisiae Yta7 protein is a component of a nucleosome bound protein complex that
maintains distinct transcriptional zones of chromatin. We previously found that one protein copurifying
with Yta7 is the yFACT member Spt16. Epistasis analyses revealed a link between Yta7, Spt16, and other
previously identified members of the histone regulatory pathway. In concurrence, Yta7 was found to reg-
ulate histone gene transcription in a cell-cycle-dependent manner. Association at the histone gene loci
appeared to occur through binding of the bromodomain-like region of Yta7 with the N-terminal tail of
histone H3. Our work suggests a mechanism in which Yta7 is localized to chromatin to establish regions
of transcriptional silencing, and that one facet of this cellular mechanism is to modulate transcription of
of these pathways are centered around the regulation
of chromatin and the histone epigenetic status (Berger
2007; Li et al. 2007). ATP-dependent and -independent
chromatin remodeling provides for the physical rear-
rangement of nucleosomes and accessibility to un-
derlying DNA (Cairns 2005). Furthermore, histones
can be dynamically cycled out of chromatin to define
chromosomal features such as active promoters and
chromatin boundary elements (Dion et al. 2007; Jamai
et al. 2007; Rufiange et al. 2007). They can even be
replaced by histone variants to regulate transcription
(Li et al. 2007). A more combinatorial process of tran-
scriptional regulation is the post-translational modifica-
2007). Certain histone PTMs serve as binding sites
for recruitment of ‘‘effector’’ protein complexes that
modulate gene transcription. For example, the Yng1
component from the NuA3 histone acetyltransferase
binds to lysine 4 trimethylated histone H3 to provide
for histone H3 lysine 14 acetylation and transcriptional
activation (Taverna et al. 2006). Transcriptional regu-
lation through chromatin is undoubtedly a complex
system of events that remain to be fully explored.
role for the yeast tat-binding analog 7 (Yta7) (Tackett
et al. 2005). We found that Yta7 maintains the transition
region (or barrier) between heterochromatin and eu-
IFFERENT regulatory mechanisms are involved in
the cell cycle control of gene transcription. Many
chromatin upstream of the silent HMR locus (Tackett
et al. 2005). Deletion of the YTA7 gene results in spread-
ing of the silent state from HMR and silencing of sur-
rounding genes, demonstrating that Yta7 can regulate
that Yta7 functions in a similar manner downstream of
the silent region at HMR (Jambunathan et al. 2005).
Yta7 contains both an AAA ATPase domain and a
bromodomain-like region (Yta7bd). An affinity-tagged
version of Yta7 shows avid copurification of all core
histones, suggesting that activity may be propagated
through nucleosome association (Tackettet al. 2005).
Yta7bd can mediate the association with histones; how-
ever, the precise region of histone interaction and post-
(Jambunathan et al. 2005). Yta7 also appears to be
Caenorhabditis elegans, where its deletion leads to embry-
onic lethality (Tsenget al. 2007). Additionally, Yta7 has
been linked to telomere maintenance and has shown
an interaction with Rad53 upon DNA damage (Askree
et al. 2004; Smolka et al. 2005). These studies suggest
that Yta7 may be associating with chromatin to modu-
late transcription and to function in numerous cellular
In previous work, we observed that Yta7 copurifies
with transcriptional and chromatin modifying proteins
(Tackettet al. 2005). Intriguingly, we noted that Spt16
is one of many proteins that copurified with Yta7
(Tackettet al. 2005). The Spt16 protein is involved in
transcription and nucleosome regulation via its role
with the yFACT complex (Formosa et al. 2002). Spt16
appears to be essential for displacement of histones to
W. Markham St., Slot 516, Little Rock, AR 72205.
Genetics 179: 291–304 (May 2008)
promote initiation of transcription (providing for pas-
sage of RNA polymerase) and for reassembly of nucle-
osomes after elongation (Formosa et al. 2002; Biswas
et al. 2005). Since Yta7 regulates zones of transcription
to explore the functional significance of the observed
MATERIALS AND METHODS
Yeast strains: Yeast strains are listed in Table 1. Gene
replacements of YTA7 with LEU2 were done by PCR amplifi-
cation of LEU2 from the pUG73 plasmid with 45 bp of
sequence upstream and downstream of the YTA7 gene,
genomic incorporation by homologous recombination and
conformation by PCR. Gene replacements of YTA7 with KAN
were done by PCR amplification of KAN from yta7TKAN
BY4742 (Open Biosystems) genomic DNA with 100 bp of
sequence upstream and downstream of the YTA7 gene,
incorporation into the genome by homologous recombina-
tion and confirmation by PCR. To make strain ATY146, we
used marker swap plasmid M4755 to change the KAN marker
from a bar1TKAN BY4741 strain (Open Biosystems) to a LEU2
marker (Voth et al. 2003). For strains ATY223 and ATY232,
YTA7 was internally tagged by insertion of PrA or Myc9as
described (Gauss et al. 2005).
Epistasis analysis: Temperature sensitivity: cells were grown
to saturation at 25?, 10-fold serially diluted, spotted on YEPD
plates and incubated at various temperatures (25?, 30?, 32?,
34?, and 37?). Damage assays: cells were grown to saturation at
25?, 10-fold serially diluted and spotted on YEPD plates
containing methyl methanesulfonate (MMS, 0.01, 0.03, 0.1,
S. cerevisiae strains used in this study
MATa his3D1 leu2D0 lys2D ura3D0
MATa his3D1 leu2D0 met15D0 ura3D0
MATa rtt109TKAN his3D1 leu2D0 met15D0 ura3D0
MATa yta7TKAN his3D1 leu2D0 lys2D ura3D0
MATa sas3TKAN his3D1 leu2D0 lys2D ura3D0
MATa sas3TKAN yta7TLEU2 his3D1 leu2D0 lys2D ura3D0
MATa hir1TKAN his3D1 leu2D0 lys2D ura3D0
MATa hir1TKAN yta7TLEU2 his3D1 leu2D0 lys2D ura3D0
MATa hir2TKAN his3D1 leu2D0 lys2D ura3D0
MATa hir2TKAN yta7TLEU2 his3D1 leu2D0 lys2D ura3D0
MATa asf1TKAN his3D1 leu2D0 met15D0 ura3D0
MATa asf1TKAN yta7TLEU2 his3D1 leu2D0 met15D0 ura3D0
MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100
ATY223MATa YTA7T1200-PrA ade2 LYS URA3-HMR ppr1THIS3 ROY508 (Isogenic
ATY232MATa YTA7T1200-Myc9ade2-1 ura3-1 his3-11,15 trp1-1
MATa rtt109TKAN ade2-1 ura3-1 his3-11,15 trp1-1
MATa yta7TKAN ade2-1 ura3-1 his3-11,15 trp1-1
MATa spt16-11 trp1 leu2 ura3 his7
HLY34MATa spt16-11 yta7TKAN ade2-1 ura3-1 his3-11,15 trp1-1
MATa D(hht1-hhf1) D(hht2-hhf2) leu2-3,112 ura3-62 trp1 his3
(plasmid TRP1, CEN, hht2(K56R)-HHF2)
MSY421 from M. M.
MATa bar1TLEU2 his3D1 leu2D0 met15D0 ura3D0
MATa bar1TLEU2 yta7DTKAN his3D1 leu2D0
MATa hta1TKAN his3D1 leu2D0 met15D0 ura3D0
MATa hta2TKAN his3D1 leu2D0 met15D0 ura3D0
MATa htb2TKAN his3D1 leu2D0 met15D0 ura3D0
MATa hht1TKAN his3D1 leu2D0 met15D0 ura3D0
MATa hht2TKAN his3D1 leu2D0 met15D0 ura3D0
aDonze et al. (1999).
bRecht et al. (2006).
292A. Gradolatto et al.
and 0.3%), hydroxyurea (0.1 and 0.2 m) or 6-azauracil (6-AU,
75, 100, and 200 mg/ml). All serial dilutions were performed
Transcriptional assays: Cells were grown to mid-log phase
and blocked with either 50 nm a-factor (bar1D and yta7D
bar1D) or 15 mg/ml nocodazole ½wild-type (BY4742), yta7D,
efficiency, while nocodazole showed .80%. To release the
cell cycle block, cells were washed twice in ice cold water and
resuspended in 30? prewarmed YEPD media. Time point
samples were taken for budding index counting, RNA extrac-
tion and flow cytometer analysis.
Percent new buds was calculated as the percentage of cells
forming new buds relative to the total population. Cell shape
was classified as unbudded single cells, newly formed small
buds or large buds. Cells arrested in nocodazole were con-
sidered large budded, while those arrested in a-factor were
classified as unbudded single cells. We plotted the formation
of newly formed small buds as this is a rough visual represen-
tation of the onset of histone gene transcription and S phase
(Driscoll et al. 2007).
Hot acidic phenol extraction of RNA was performed as
previously described (Tackettet al. 2005). Reverse transcrip-
tion was conducted with the Invitrogen SuperScript first
strand kit according to the providers’ protocol. Transcription
levels of the histone genes were determined by real-time PCR.
ACT1 was chosen as a housekeeping gene. CLN1 mRNA was
used as a marker of the cell cycle. Primer sequences are listed
in Table 2. Primers to differentiate HHF1 and HHF2 tran-
scripts could not be designed due to sequence similarity, thus
both transcripts were simultaneously monitored with a single
primer set. Specificity of primers for HTA1, HTA2, HTB2,
HHT1, and HHT2 transcripts was determined by real-time
PCR. To measure primer specificity, cDNA was prepared from
genomic deletion strains (hta1D, hta2D, htb2D, hht1D, and
hht2D) and analyzed by real-time PCR with the primers cor-
responding to the deleted gene. Nonspecific amplification
with each primer set was measured relative to ACT1 mRNA.
Relative mRNA levels in Figures 3 and 4 were corrected for
primer specificity. We did not determine the specificity of
HTB1 transcript primers because systematic deletion of the
HTB1 gene produces inviable cells (Giaever et al. 2002).
Flow cytometer samples were fixed in 70% ethanol and
stored at 4?. After sonication in 200 mm sodium citrate, cells
were treated with RNase A at 37?. SYTOX green (Molecular
Probes/Invitrogen) was added to a final concentration of
2 mm. Samples were analyzed with a Beckman Coulter EPICS
XL-MCL flow cytometer.
precipitation (ChIP) was performed as previously reported
using an anti-H3K56ac antibody (Upstate/Millipore 07-677)
(Taverna et al. 2006). ChIP to general histone H3 (Abcam
Ab1791) was performed to control for nucleosome occupancy
(Tavernaet al. 2006). Real-time PCR was used to compare the
enriched levels of the intergenic region between the HTA1
and HTB1 gene pair relative to ACT1.
High resolution ChIP-chip analysis of Yta7: ChIP-chip
analysis of Yta7-Myc was performed in triplicate with dye-
swapping as reported (Ren et al. 2000; Taverna et al. 2006).
Tiled microarrays covering the entire Saccharomyces cerevisiae
Microarray data were independently analyzed with both
SignalMap (NimbleGen Systems) and Mpeak version 2.0
software (http:/ /www.stat.ucla.edu/?zmdl/mpeak/). Binding
regions were considered relevant if .2 SD from the mean.
We utilized a strain containing an internal Myc9tag that was
inserted at amino acid position 1200 of Yta7 (Gauss et al.
approaches analyzing four different positions: N-terminal,
insertion at position 106 (prior to the AAA ATPase and
bromodomains), insertion at position 1200 (near the C ter-
minus), and C-terminal. Each tagged strain was tested for
maintenance of barrier activity at HMR and for immuno-
purification of histones (Tackett et al. 2005). The results
showed that only the insertion of the tag at amino acid 1200
preserved barrier maintenance and provided for histone
immunopurification (A. J. Tackett and B. T. Chait, un-
Yta7bd binding to histones and histone H3 peptide mimics:
region (Yta7bd) with a N-terminal GST fusion were expressed,
purified, and confirmed by mass spectrometry (Tavernaet al.
For histone binding studies, 40 mg of GST-Yta7bd (or GST
control) was incubated with 5 mg of acid-extracted T. thermo-
phila histones (purified as described in Taverna et al. 2007)
150 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 1% Triton X-100,
10 mm sodium butyrate, 1:100 Sigma yeast protease inhibitor
cocktail) in a final volume of 500 ml for 1 hr at room
temperature. GST-Yta7bd or GSTalone was collected for 1 hr
at room temperature with EZview red glutathione affinity gel
(Sigma), washed three times in binding buffer with 300 mm
NaCl, washed once with low salt buffer (4 mm HEPES, pH 7.9,
10 mm NaCl), and copurifying histones were released by
Sequences of the primers used for real-time PCR
HHF1 and HHF2
HTB1 and HTA1
Yta7 Regulates Histone Gene Transcription293
heating to 95? in SDS–PAGE sample buffer. Bound histones
were resolved with 4–20% SDS–PAGE and individually visual-
ized by immunoblotting.
coated Dynabeads M280 (250 mg) were incubated with 1.2 mg
or in-house synthesized). Peptide amounts were standard-
ized by dot-blotting and visualization with streptavidin-HRP.
Binding assays were performed by incubation of Yta7bd with
the peptide coated beads for 1 hr at room temperature in
20 mm HEPES, pH 7.4, 150 mm NaCl, 0.2% Triton X-100.
Beads were washed three times with 20 mm HEPES, pH 7.4,
300 mm NaCl, 0.2% Triton X-100. Proteins were resolved by
4–12% SDS–PAGE and visualized by Coomassie staining.
Binding was quantified with ImageJ software (http:/ /rsb.info.
Full-length Yta7 binding to histone H3 peptides: Yta7-PrA
was immunopurified on IgG-coated Dynabeads as previously
described except that the concentration of NaCl was raised
from 0.3 to 1.5 m such that cellular histones no longer
copurified (Tackett et al. 2005). Yta7-PrA was released from
the resin with a nondenaturing elution peptide (Strambio-
De-Castillia et al. 2005; Tackettet al. 2005). Binding studies
ofYta7-PrAwere performed followingthesameprotocolas for
Yta7bd binding experiments with the following biotinylated
peptides: H3 1-20, H3 48-63, and H3 48-63 K56ac. Bound Yta7-
PrA was visualized by immunoblotting for the PrA tag.
Immunopurification of Yta7-PrA and associated histones:
Purification of Yta7-PrA with in vivo associated histones on
IgG-coated Dynabeads was done as previously reported
(Gauss et al. 2005; Tackett et al. 2005). Sodium butyrate
(50 mm) was included during immunopurifications to inhibit
Mass spectrometric analysis of H3K56ac: The gel band
containing histone H3 (that purified with Yta7-PrA) was
chemically treated with d6-acetic anhydride and digested with
et al. 2005). The d6-acetic anhydride treatment converts all
unmodified lysines to triply deuterated acetyl-lysines (Tackett
et al. 2005); thus allowing one to differentiate unmodified
lysines (145 Da) from in vivo acetylated lysines (142 Da) for a
quantitative measurement of histone acetylation (Tackett
et al. 2005). A mass spectrum of the histone H3 peptides
was collected with a MALDI-prOTOF mass spectrometer
(PerkinElmerSciex). Peptides were confirmed by tandem
mass analysis with a vMALDI-LTQ ion trap mass spectrometer
(Thermo). To quantify the level of acetylation for H3K56,
m-over-z software was used to extract the monoisotopic peak
areas for the heavy and light versions of the histone H3 53-63
peptide. Percent acetylated was the percentage of light area
relative to the total percentage of light and heavy areas.
Peptides identified are listed in Table 3.
Yta7 activities overlaps with the activities of other
known histone regulators: Spt16,asubunitoftheyFACT
complex, was foundtocoenrichwithYta7(Tackettetal.
2005). The yFACTcomplex plays an important role dur-
ing the cell cycle, helping the passage of polymerases
et al. 2002). When subunits of the yFACT are mutated
the reassembly of nucleosomes can still be handled by
the Hir/Hpc complex, but simultaneous mutation of
proteins from each complex leads to strong synthetic
defects (Formosa et al. 2002). For instance, a combina-
tion of hir1D spt16-11(T8281I P859S) caused synthetic
temperature sensitivity of the cells, demonstrating de-
pendence of the yFACT pathway with the Hir pathway
(Sutton et al. 2001; Formosa et al. 2002). In conse-
quence, we decided to investigate the effect of double
deletions of the YTA7 gene with the point mutation
of two genes encoding proteins in the Hir/Hpc com-
plex: HIR1 and HIR2. The spt16-11 mutant was chosen
because cells carrying the genomic deletion of SPT16
Additionally, the genes encoding Asf1 and Sas3
were deleted in WT and yta7D strains and cells were
subjected to restrictive growth conditions. Asf1 is a
histone chaperone protein that is widely involved in
replication-coupled nucleosome assembly, response to
DNA damage, repression of histone gene transcription
and heterochromatic silencing (reviewed in Mousson
et al. 2007). Asf1 mediates its activity through interac-
tions with proteins including the Hirs (Sutton et al.
2001). The protein Sas3 is a component of the NuA3
histone acetyltransferase complex and interacts with
Spt16, suggesting a role in transcription and/or repli-
cation (John et al. 2000; Tavernaet al. 2006). Sas3 also
copurifies with Yta7 (Tackettet al. 2005).
Cells were plated in serial dilutions and subjected to
increasing temperatures or damaging agents: hydroxy-
urea(stallsDNA replication), methylmethanesulfonate
(DNA damaging agent) or 6-azauracil (inhibits tran-
scriptional elongation) (Figure 1). The 6-AU screen did
not show any synthetic defects (data not shown). Cells
containing a deletion of the RTT109 gene served as a
sensitivecontrol for theDNA damageagents(Driscoll
et al. 2007; Han et al. 2007).
Cells harboring the spt16-11 mutation exhibited sen-
sitivity to both temperature and HU treatments as pre-
viously described(Formosa et al.2002) (Figure 1, Aand
B). Additional deletion of the YTA7 gene increased the
growth defect observed for both treatments. This result
suggests that both Yta7 and Spt16 are acting in two func-
tionally overlapping pathways. No effect was observed
for sas3D and sas3D yta7D mutants under any stress con-
dition (data not shown). However, deletion of the ASF1
gene showed a cell growth defect under temperature
Peptides from histone H3 copurifying with Yta7-PrA that
were identified by MALDI mass spectrometry
Extent of lysine
294A. Gradolatto et al.
and HU conditions. Further deletion of the YTA7 gene
increased the HU effect only, indicating that Yta7 and
Asf1 have overlapping function during times of DNA
synthesis. Furthermore, deletion of the YTA7 gene in-
to temperature increase, HU and MMS—suggesting that
Hir and Yta7 pathways are functionally overlapping. Our
results imply (1) a role of Yta7 in DNA damage response
and (2) that Yta7 operates through a functionally over-
lapping pathway with the Asf1/Hir and Spt16-yFACT
Yta7 is a repressor of histone gene transcription:
Since we observed a genetic interaction with known
histone gene transcriptional regulators (Hirs, Asf1, and
Spt16) and since Yta7 is known to regulate transcrip-
tional zones of chromatin via barrier activity, we in-
vestigated whether Yta7 played a role in transcriptional
regulation of the histone genes. To assay for cell-cycle-
dependent transcriptional defects, we synchronized
wild-type and yta7D cells with a-factor (G1-phase arrest)
or nocodazole (M-phase arrest) and cultures were
sampled at various times throughout a full cell cycle.
We first verified that the cells were growing in syn-
chrony. Measurement of the late G1cyclin CLN1 tran-
script levels revealed that after release from each block,
C). However, cells containing the YTA7 gene deletion
showed a defect in CLN1 transcription at the 90 and 100
min time points post-nocodazole release, which could
indicate a lost of synchrony toward the end of the first
cell cycle (Figure 2C). Following the cell cycle through
the formation of new buds also showed a similar pro-
gression of the cells after release from the blocking
agents (Figure 2, B and D), with a slight delay in the
accumulation of buds at 80 min post-release from no-
codazole for yta7D cells (Figure 2D). Thus, the cells
appeared to be synchronized relative to expression of
a G1cyclin and formation of buds. However, release of
yta7D cells from the nocodazole block did show CLN1
transcription and budding defects at times consistent
with M phase (?90 min post-release).
For the time course transcriptional analyses of yta7D
cells with either a nocodazole or an a-factor block, the
peak of histone transcription appeared as expected,
with previous studies (Figure 2, A and C; Figure 3;
Figure 4A) (Hereford et al. 1981). However, the tran-
scriptional analysis of all the histone genes revealed
strikingly different effects of YTA7 gene deletion with
the different cell cycle blocks (Figures 3 and 4A). After
a-factor block and release, yta7D cells revealed a signif-
icant higher activation of transcription for all the his-
tone genes at the 20 and 30 min time points (Figure 3).
Even though gene transcription seemed to start at sim-
ilartimes(?20 minpost-release), therewasanapparent
20-min window where gene transcription in yta7D cells
was higher than in wild-type, but then gene transcrip-
tion decreased in a similar manner to the control strain
(50 and 60 min time points). This a-factor blocking
experiment was repeated three times with identical
results. We also varied which culture (yta7D or wild-type
cells) was sampled first at each time point, avoiding
sampling discrepancies. One explanation of the appar-
ent early transcription of each histone gene in yta7D
Figure 1.—Yta7 activity overlaps with the activities of other described histone regulators. (A) Temperature sensitivity. Indicated
cells were 10-fold serially diluted and incubated at 25? or 34? for the indicated days (d). (B) Hydroxyurea (HU) sensitivity. Cells
were 10-fold serially diluted and incubated at 25? for the indicated days. (C) Methyl methanesulfonate (MMS) sensitivity. Cells
were 10-fold serially diluted and incubated at 25? for the indicated days.
Yta7 Regulates Histone Gene Transcription 295
cells is that Yta7 serves as a cell-cycle-dependent tran-
scriptional repressor of the histone genes.
The nocodazole block and release experiment re-
vealed a different trend (Figure 4A). For each histone
gene, a prolonged transcription in yta7D cells was ap-
parent after the peak (80–90 min post-release). This
defect in transcription was of similar magnitude to that
observed upon the gene deletion of the known histone
repressor Hir2 at 80 min post-release (Figure 4B).
Unlike the a-factor blocking experiment, a uniform
Figure 3.—Yta7 regulates histone gene transcription. bar1D (solid circles) and yta7D bar1D (open squares) cells were blocked
with a-factor, released, and samples were taken at the indicated times. For each time point, cDNA was prepared and the amount of
indicated transcript (relative to ACT1 transcript) was determined by real-time PCR. Darkly shaded regions emphasize the in-
creased histone gene transcription upon gene deletion of YTA7. Error bars are the standard deviation. Asterisks show significant
differences determined by one-tailed t-testing (P , 0.05).
Figure 2.—Timing of CLN1 gene transcription
and new bud formation in a-factor synchronized
bar1D and yta7D bar1D cells and in nocodazole
synchronized wild-type and yta7D cells. Cells were
blocked with a-factor (A and B) or nocodazole (C
and D), released, and samples were taken at the
indicated times. For each time point, the amount
of late G1-cyclin CLN1 transcript relative to ACT1
transcript was determined by real-time PCR (A
and C). Error bars are the standard deviation.
The formation of new buds was also monitored
(B and D).
296A. Gradolatto et al.
defect in transcriptional regulation was not evident. For
example, the HTA1 and HTB1 and HTA2 and HTB2
gene pairs are known to be regulated in a similar man-
ner (Hereford et al. 1981). In our nocodazole blocking
experiment, the defect observed upon deletion of YTA7
showed a similar defect for HTA1 and HTA2 and HTB1
and HTB2 (Figure 4A). Since we observed prolonged
histone gene transcription and a differential histone
gene transcriptional regulation (relative to the a-factor
blocking experiment), we decided to take a more de-
tailed look into possible cell cycle defects in each of the
In Figure 2, we showed that wild-type and yta7D cells
had similar CLN1 transcript levels and new bud forma-
tion, indicative of synchronous growth. To investigate
cell synchrony in a more detailed manner, we moni-
tored DNA content by flow cytometry (Figure 5).
Following a-factor block, cells were released and grew
observed early transcriptional effect is likely not a
consequence of a cell cycle progression defect (Figure
3). However, the nocodazole block and release ex-
hibited slightly different results. YTA7 gene deletion
impaired the ability of the cells to exit from M phase or
recover from the nocodazole block (Figure 5, C and D).
In Figure 5D, the yta7D cells showed a delay in the
transition from 2N to 1N content relative to wild-type.
This led to two important conclusions. First, the pro-
longed and variable histone gene transcription ob-
served in Figure 4A is likely a product of asynchrony,
and that the true effect of YTA7 deletion on histone
gene transcription is observed in the highly synchro-
Figure 4.—Nocodazole blocking alters the observed yta7D transcriptional defect in histone gene regulation. (A) Wild-type
(solid circles) and yta7D (open squares) cells were blocked with nocodazole and released. Samples were taken at the indicated
times and analyzed as in Figure 3. Darkly shaded regions emphasize the increased histone gene transcription upon gene deletion
of YTA7. Error bars are the standard deviation. (B) HTB1 gene transcription is upregulated upon deletion of HIR2. Wild-type
(solid circles) and hir2D (open squares) cells were blocked with nocodazole and released. Samples were taken at the indicated
times and analyzed as in Figure 3. Error bars are the standard deviation. Asterisks show significant differences determined by one-
tailed t-testing (P , 0.05).
Yta7 Regulates Histone Gene Transcription 297
phase. This could be consistent with Yta7 playing some
chromosome specific role as it is a member of a histone
that Yta7 is not involved in chromosome segregation
during M phase (Dubey and Gartenberg 2007).
3–5 support Yta7 functioning as a histone gene tran-
Yta7 associates with histone loci: In order to better
understand the mechanism by which Yta7 regulates
histone gene transcription, we utilized a chromatin
immunopurification with high resolution DNA micro-
the Yta7 protein associates with these loci (Ren et al.
conventional ChIP because we were interested in ob-
serving the distribution profile of Yta7 across the loci at
high resolution, and because uncovering additional
binding sites could provide further information for
defining the mechanism of Yta7 activity. The ChIP-chip
analysis of Yta7-Myc showed significant binding at 177
discrete regions (supplementary Table S1). We did not
observe any obvious trend toward the type of genes Yta7
was associating with.
We show the array data as a composite profile of the
Yta7-associated DNA occupancy (relative occupancy)
plotted as a function of relative coordinates on an
average gene flanked with intergenic regions as was
previously done in Taverna et al. (2006) (Figure 6A).
Figure 6A revealed that on average the Yta7 protein had
preferential association with intergenic regions of chro-
matin as well as with the extreme 59- and 39-ends of an
ORF. In contrast, a component of the NuA3 histone
acetyltransferase complex, Yng1, was recently showed to
minimal association is observed in the intergenic re-
gions (Taverna et al. 2006). The averaged localization
of Yng1 is consistent with the role of NuA3 in stimu-
lating transcription via H3K14 acetylation, which is a
PTM localized to 59-ends of actively transcribing genes
(Pokholok et al. 2005). Our data in Figure 6A suggest
that the mechanism of Yta7 activity may be mediated
through non ORF associations and that Yta7 binding to
intergenic regions could be a mechanism for transcrip-
Six of the top 10 binding sites from the ChIP-chip
analysis were pairings of histone genes (HTB1 and
HTA1, HHF1 and HHT1, HHT2 and HHF2) (Figure
Figure 5.—FACS analysis of DNA content for bar1D and bar1Dyta7D or wild-type and yta7D cells. (A) bar1D and bar1Dyta7D cells
were blocked with a-factor, released, and samples were taken at the indicated times for FACS analysis. (B) Histograms of FACS data
fromAshowing 1Nand 2NDNAcontentsasapercentageofgated cells. (C) Wild-type and yta7D cellswereblockedwithnocodazole,
released, and samples were taken at the indicated times for FACS analysis. The asterisks highlight the key differences between the
WT and yta7D cells. (D) Histograms of FACS data from C showing 1N and 2N DNA contents as a percentage of gated cells.
298A. Gradolatto et al.
loci, the other genomic pair of histone genes (HTA2
and HTB2) was also a significant Yta7 binding site
the average plot (Figure 6A), the HTB1 and HTA1
histone locus showed peak Yta7 association at the inter-
genic region, even though the Yta7 protein was also
binding to the genes themselves (Figure 6B). Yta7
bound the other histone loci HHF1 and HHT1, HHT2
and HHF2, and HTA2 and HTB2 with a similar profile
except that peak association occurred within the ORFs.
Since the transcription of all histone genes is similarly
modified in yta7D cells (Figure 3), the exact nature of
this slightly different binding profile among the loci
remains to be elucidated. Our observed interaction of
the Yta7 protein with the histone loci is suggestive of a
direct transcriptional regulatory mechanism.
Yta7 interacts with chromatin via the N-terminal tail
of histone H3: The above results show that Yta7 asso-
ciated with and transcriptionally regulated histone
genes. Yta7 has also been shown to immunopurify with
Figure 6.—The Yta7 protein localizes to histone gene loci. ChIP-chip analysis of Yta7-Myc from asynchronous cells using 50-bp
resolution tiled arrays (NimbleGen Systems). (A) Yta7 associated DNA occupancy is plotted as a function of relative coordinates on
an average gene flanked with intergenic regions. (B) Shown is Yta7-Myc binding at the four histone loci: HTB1 and HTA1, HHF1
and HHT1, HHT2 and HHF2, and HTA2 and HTB2. The log2intensity ratio of immunoprecipitated to control DNA is plotted as a
function of genomic position. Error bars are the standard deviation of triplicate array analyses.
Yta7 Regulates Histone Gene Transcription299
histone proteins (Tackettet al. 2005). One chromatin-
based regulative pathway that precedes histone gene
transcription is the acetylation of histone H3 Lys56
(H3K56ac) at histone loci (Xu et al. 2005). The Yta7
protein also contains a bromodomain-like region that
shows histone association, but the PTM specificity has
not been determined (Jambunathan et al. 2005).
Therefore, we sought to investigate if the Yta7 protein
association with histones was PTM dependent, and
particularly H3K56ac dependent.
We purified a recombinant version of the Yta7 bromo-
domain-like region (Yta7bd). We first investigated
whether the recombinant version of Yta7bd specifically
bound to a particular histone. An Yta7bd-GST fusion or
GST alone was incubated with acid-extracted histones,
and interacting proteins were isolated on glutathione
resin. Histones associated with Yta7bd were visualized by
immunoblotting (Figure 7A). GST alone showed mini-
mal nonspecific affinity for the histones when compar-
ing the input (I) and bound (B) samples. However, the
Figure 7.—Yta7bd interacts with histone H3. (A) Yta7 protein preferentially interacts with histone H3. Yta7bd-GST fusion, or
GSTalone, was incubated with purified histones and isolated on glutathione resin. Histones associated with Yta7 were visualized by
immunoblotting. Input (I) and bound (B) lanes are shown. (B) Yta7bd interacts with the N-terminal tail of histone H3. Recombi-
nant Yta7bd, or GST alone, was incubated with streptavidin Dynabeads coated with biotinylated peptides representing different
regions and modification states of histone H3 (methylation and acetylation). Bound Yta7bd was resolved by SDS–PAGE and vi-
sualized by Coomassie staining. (C) Quantification of Yta7bd binding from B. Yta7bd binding is shown as a percentage relative to
H3 1-20 binding. (D) Full-length Yta7 protein interacts with the N-terminal tail of histone H3. Purified Yta7-PrA was incubated with
streptavidin Dynabeads coated with biotinylated peptides representing different regions and modification states of histone H3.
Bound Yta7-PrA was resolved by SDS–PAGE and visualized by immunoblotting for the PrA tag. (E) Yta7 associated histone H3 was
acetylated at Lys56. Yta7-PrA was immunopurified and coenriching proteins were resolved by SDS–PAGE/Coomassie staining.
Copurifying histone H3 was treated in-gel with d6-acetic anhydride to isotopically mark unmodified lysines and then digested
with trypsin. Shown is a section of a mass spectrum containing the H3K56 peptide as either acetylated (K56ac) or unacetylated
(K56). In accordance to the monoisotopic peak areas, H3K56 was 33% acetylated.
300A. Gradolatto et al.
GST-Yta7bd fusion protein showed significant binding
of histone H3 as well as minor affinity for H2B.
We then investigated if the Yta7bd interaction with
histone H3 was directed by H3K56ac (or another his-
tone H3 PTM). Binding of Yta7bd to a series of dif-
ferentially modified histone H3 peptide mimics was
performed (Figure 7B). We observed that Yta7bd did
of histone H3 containing H3K56 or H3K56ac (Figure
7B, lanes 13–16). However, Yta7bd showed a preferen-
tial interaction with the N-terminal 20-amino-acid tail of
histone H3 (Figure 7B, lanes 3 and 4). The interaction
of Yta7bd with the N-terminal tail was favored for the
by the decreased association observed with the addition
capacity .30%, and further acetylation reduced it ?50–
70%, while single acetylation caused a binding reduc-
tion of ?20–40% (Figure 7C). The ability of Yta7bd to
preferentially bind the unmodified histone H3 tail and
to tolerate various single PTMs suggests that the Yta7
protein interaction with chromatin is mediated by a
general interaction with the N-terminal tail of histone
H3. This is not surprising because the bromodomain-
for acetyl-lysine specificity in other bromodomains, but
does retain many of the amino acids needed for bind-
ing to the histone peptide backbone (Jambunathan
et al. 2005). These results suggest that the Yta7 protein
associates with chromatin through an interaction with
the N-terminal tail of histone H3. It is possible that Yta7
binding could be stimulated by combinations of differ-
ent PTMs. A similar analysis with full-length Yta7 also
showed enrichment for the N-terminal tail of histone
with histones was H3K56ac independent. However,
we hypothesized that these two histone gene transcrip-
tional regulation signals could simultaneously coexist
on chromatin to provide for independent, overlapping
transcriptional regulation. To test whether H3K56ac
and Yta7 could be found simultaneously on chromatin,
we isolated an ex vivo complex of full-length Yta7 with
copurifying histones as previously reported (Tackett
et al. 2005). We performed a quantitative PTM analysis
of histone H3 using isotope labeling and MALDI-mass
spectrometry (Tackett et al. 2005). The analysis of
the H3 peptides mass spectrum revealed peptides that
corresponded to 41% sequence coverage of histone
H3 (Table 3). Of particular interest, we observed pep-
tide 53-63, which contained Lys56 (Figure 7E). We
observed two peaks that corresponded to the unacety-
lated and the acetylated peptide. These peptides were
confirmed by tandem MS. The peak areas from the
mass spectrum were used to determine that the popu-
lation of histone copurifying with Yta7 was 33% acety-
lated at H3K56, thus demonstrating that Yta7 could
associate with both H3K56 acetylated or unmodified
show significant acetylation (Table 3). These data sug-
gest that Yta7 and H3K56ac cancoexist on a given piece
The data in Figure 7 show that Yta7 binding to
chromatin is not stimulated by H3K56ac, but the two
entities can coexist. We wanted to test whether acetyla-
tion of H3K56 was dependent on Yta7. To monitor for
this dependence, we utilized ChIP to H3K56ac at the
intergenicregion betweenHTA1 and HTB1 (Figure 8A)
(Xu et al. 2005). H3K56ac ChIP on wild-type and yta7D
cells showed no significant difference (P-value ¼ 0.19).
Cells harboring a H3K56R mutation were used as a
control (Recht et al. 2006). In addition to performing
ChIP to this one region of known H3K56ac, we cor-
related our genomewide Yta7 ChIP-chip data to the
reported sites of H3K56ac, using only enrichments $2
8B). This low correlation is in agreement with results
from Figure 7B that showed no direct affinity of Yta7bd
for H3K56ac. The results in Figures 7 and 8 suggest that
Figure 8.—Yta7 does not direct or
correlate with general H3K56 acetyla-
tion. (A) H3K56ac is not dependent
on Yta7. ChIP to histone H3K56ac was
performed in wild-type, yta7D, and
H3K56R cells. Real-time PCR was used
to measure enrichment at the inter-
genic region between HTA1 and HTB1
relative to ACT1. ChIP to unmodified
H3 was used to control for nucleosome
occupancy. Error bars are the standard
deviation of the mean. The asterisk
shows the significant difference deter-
mined by t-testing (P , 0.01). (B) Ge-
nomewideYta7 bindingdoes not
correlate with H3K56ac. Over the 177 sites identified from ChIP-chip analysis of Yta7, only 6.8% were overlapping with previously
reported H3K56ac-binding sites (Xu et al. 2005).
Yta7 Regulates Histone Gene Transcription 301
expression are independent.
tightly linked to histone gene expression. Maintaining
theproper dosage level ofhistone transcriptsisacrucial
cellular mechanism that is protected through multiple
pathways. These histone gene regulatory pathways ex-
hibit various levels of redundancy and crosstalk of
protein interactions. Our work describes how Yta7, a
protein initially identified as a component of a barrier
chromatin complex, is also involved in histone gene
expression and is connected to otherhistone regulatory
S. cerevisiae contains two copies of the four core his-
tones, which are transcribed during S phase. These
copies exist as pairs on chromosomes 2, 4, and 14.
Preceding transcriptional activation of the histone
genes is a rapid cycle of acetylation on histone H3 lysine
56 (H3K56ac) (Xu et al. 2005). H3K56ac coats the
promoter and extends into the open-reading frame
regions of these genes in a Spt10-dependent manner,
and is needed for proper histone gene transcription
(Xu et al. 2005). In addition to H3K56ac, histone gene
transcription is regulated by the Asf1/Hir complex and
Spt16 containingyFACTcomplexpathways (Sherwood
et al. 1993; Spector et al. 1997; Sutton et al. 2001;
Formosa et al. 2002; Recht et al. 2006; Mousson et al.
2007). Asf1/Hir and yFACT complexes appear to func-
transcription, with both being needed for proper S-
phase transcription of histone genes. The Asf1/Hir
complex functions to maintain cell-cycle-dependent
and hydroxyurea-induced repression of histone gene
transcription (Sutton et al. 2001). The Spt16 yFACT
complex aids in transcriptional initiation and provides
for the proper reassembly of chromatin after polymer-
ase passage (Formosa et al. 2002). Mutations of the
Asf1/Hir and yFACT transcriptional regulators alter,
but do not abolish S-phase histone gene transcription,
suggesting that other mechanisms of gene regulation
exist. Our work links the Yta7 protein to one of these
novel mechanisms of histone gene transcriptional regu-
lation. We favor the hypothesis that Yta7 functions in
concurrence with a multi-pathway cellular program that
regulates histone gene transcription during the cell
cycle. Evidence for our hypothesis is demonstrated at
the level of cellular pathway crosstalk, transcriptional
control and chromatin association.
An epistasis analysis provided evidence that Yta7 ac-
tivity genetically interacts with other known cellular
pathways that regulate histone gene transcription,
namely the Asf1/Hir and Spt16/yFACT pathways (Fig-
ure 1). The synthetic nature of the observed growth
defects indicated that Yta7, Asf1/Hir, and Spt16/yFACT
have overlapping cellular functions. Thus, we identified
a functional significance for our observed copurifica-
tion of Yta7 and Spt16 proteins (Tackett et al. 2005).
The synthetic growth defects with yta7D cells were de-
tectable only under stressful growing conditions (tem-
perature, DNA synthesis inhibition, and DNA damage),
suggesting that Yta7 is linked to mechanisms of DNA
repair. Under stress conditions, such as hydroxyurea
treatment, cells shut down histone gene transcription
(Sutton et al. 2001). Deletions in histone transcrip-
tional regulators, such as Hir1 and Asf1, prevent the
proper deactivation of histone gene transcription in
the presence of hydroxyurea, thus highlighting the sig-
nificance of the observed synthetic growth defects with
yta7D cells under stress (Figure 1) (Sutton et al. 2001).
Our MMS studies are in accordance with previous
studies showing that Yta7 interacted with Rad53 under
DNA damage conditions (Smolka et al. 2005). More-
over, the Yta7 human homolog ATAD2 was down-
regulated in a colon cancer cell line arrested in S phase
after 5-fluorouracil treatment, suggesting a role in DNA
damage and repair (De Angelis et al. 2006). The epis-
tasis analysis revealed that the cellular pathway(s) con-
taining Yta7 were overlapping with known pathwaysthat
can modulate histone transcript levels, thereby linking
Yta7 to a possible role in histone gene transcription.
A direct test of the involvement of Yta7 in the tran-
scriptional regulation of histone gene expression dem-
onstrated that Yta7 functioned as a transcriptional
repressor (Figure 3). Deletion of the YTA7 gene led to
elevated transcript levels for all histone genes prior to
peak transcription. Histone genetranscription was then
downregulated in a similar manner relative to wild-type
cells. In this experiment, we observed a similar mis-
regulation of transcription at each gene, which was
consistent with Yta7 serving a similar repressive role at
each locus (Figure 3). The areas under the peaks of
histone gene transcription showed that deletion of the
the peak level was not higher—rather the peak was
broader. With the decline of both peaks overlapping,
Yta7 appeared to be involved in some early step of
transcriptional regulation. The evidence in Figure 3
showing that Yta7 served as a repressor of histone gene
transcription is in agreement with the epistasis analysis
that linked Yta7 to the histone gene transcriptional
regulatory network (Figure 1). As may be expected,
double deletion of the repressor Yta7 with hydroxyurea-
dependent histone gene repressors (namely Hir1 and
Asf1) caused a synthetic growth defect in cells (Figure
1B). This growth defect could be the consequence of
potentially toxic levels of free histone proteins as pre-
et al. 2005). Our time course transcriptional data did
not indicate whether this repressive effect was direct or
indirect. It is possible that Yta7 acts in an indirect
302A. Gradolatto et al.
manner to modulate histone transcript levels. Rather
than acting as a direct repressor of gene transcription,
Yta7 could play a role in the post-transcriptional turn-
since Yta7 has a putative bromodomain, transcriptional
gene regulatory activity, and associates with chromatin,
we favor a direct mechanism that would involve the
Yta7 protein binding to the histone loci (Tackettet al.
To determine if the Yta7 protein indeed directly
associated with the histone loci, we performed a series
of in vivo and in vitro binding studies. Genomewide
ChIP-chip of Yta7 revealed direct chromatin association
at 177 novel sites (Figure 6). Six of the top 10 binding
sites were the histone loci (Figure 6B). Yta7 binding was
observed across each ORF and intergenic region at the
histone loci. It is possible that such a binding profile
could provide for formation of a chromatin state that
represses transcription. Binding of Yta7 to the histone
loci further strengthens the hypothesis that Yta7 is di-
rectly regulating histone gene transcription. Since Yta7
contains a histone H3 binding bromodomain-like region
(Figure 7A), we investigated whether there was a partic-
ular histone H3 PTM code that promoted Yta7 associa-
tion. Full-length Yta7 and Yta7bd were found to engage
histone H3 at the N terminus with a preferential asso-
ciation in the absence of tested PTMs (Figure 7, B–D).
Purification of in vivo associated histone H3, revealed
significant levels of H3K56ac, but this PTM did not
stimulate Yta7 protein association nor was H3K56ac
dependent upon Yta7 (Figure 7, C and E; Figure 8).
The in vivo purification of H3K56ac with Yta7 would be
regions at histone loci (Figure 6; Xu et al. 2005). Our
chromatin association studies are consistent with the
hypothesis that Yta7 mediated transcriptional repression
occurs through direct association at the histone loci.
The results presented shed light on the cellular pro-
gram needed for transcriptional regulation of the his-
tone genes. There appear to be multiple levels of
regulation that provide for proper histone dosage,
which is not surprising for a key cellular process. We
find that Yta7 serves a key role in a previously unchar-
acterized branch of the histone gene transcriptional
regulatory pathway. Our findings also provide for a
broader understanding of the function of Yta7, which
seems to regulate transcription not only at transition
zones betweensilentand active chromatin, butalsovia a
dynamic mechanism at other chromosomal regions.
We thank Lauren Blair and Cagdas Tazearslan for critical reading,
Piotr Zimniak for access to real-time PCR instrumentation, Andrew
Krutchinsky and Markus Kalkum for assistance with mass spectromet-
ric hardware/software development, Cecile Artaud and Anastas
Pashov for flow cytometry analysis, Tim Formosa for the spt16-11
Institutes of Health (NIH) grants supported this work: P20RR015569
(A.J.T.,co-investigator), GM63959(C.D.A.), RR022220 and
GM076547 (J.D.A.). A.J.T. acknowledges support from the Arkansas
Biosciences Institute and recognizes mass spectrometric support from
Sam Mackintosh and Chris Warthen funded through an NIH IDeA
Network of Biomedical Research Excellence grant (P20RR016460).
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Communicating editor: F. Winston
304A. Gradolatto et al.