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INVESTIGATION
Insight Into the Mechanism of Nucleosome
Reorganization From Histone Mutants That
Suppress Defects in the FACT Histone Chaperone
Laura McCullough,* Robert Rawlins,* Aileen Olsen,
†
Hua Xin,* David J. Stillman,
†
and Tim Formosa*
,1
*Department of Biochemistry and
y
Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84112
ABSTRACT FACT (FAcilitates Chromatin Transcription/Transactions) plays a central role in transcription and replication in eukaryotes by
both establishing and overcoming the repressive properties of chromatin. FACT promotes these opposing goals by interconverting
nucleosomes between the canonical form and a more open reorganized form. In the forward direction, reorganization destabilizes
nucleosomes, while the reverse reaction promotes nucleosome assembly. Nucleosome destabilization involves disrupting contacts
among histone H2A-H2B dimers, (H3-H4)
2
tetramers, and DNA. Here we show that mutations that weaken the dimer:tetramer
interface in nucleosomes suppress defects caused by FACT deficiency in vivo in the yeast Saccharomyces cerevisiae. Mutating the
gene that encodes the Spt16 subunit of FACT causes phenotypes associated with defects in transcription and replication, and we
identify histone mutants that selectively suppress those associated with replication. Analysis of purified components suggests that the
defective version of FACT is unable to maintain the reorganized nucleosome state efficiently, whereas nucleosomes with mutant
histones are reorganized more easily than normal. The genetic suppression observed when the FACT defect is combined with the
histone defect therefore reveals the importance of the dynamic reorganization of contacts within nucleosomes to the function of FACT
in vivo, especially to FACT’s apparent role in promoting progression of DNA replication complexes. We also show that an H2B mutation
causes different phenotypes, depending on which of the two similar genes that encode this protein are altered, revealing unexpected
functional differences between these duplicated genes and calling into question the practice of examining the effects of histone
mutants by expressing them from a single plasmid-borne allele.
FACT (FAcilitates Chromatin Transcription/Transactions)
is a highly conserved histone chaperone with roles in
both transcription and DNA replication (Reinberg and Sims
2006; Formosa 2008). In Saccharomyces cerevisiae, FACT
is an Spt16-Pob3 heterodimer whose activity is supported
by the High Mobility Group B (HMGB)-like DNA-binding
protein Nhp6 (Brewster et al. 2001; Formosa et al. 2001).
Susceptibility of nucleosomal DNA to digestion by some
restriction endonucleases in vitro increases dramatically in
the presence of FACT (Xin et al. 2009), indicating that
FACT either induces a structural change or stabilizes an ex-
isting alternative nucleosomal structure (Winkler and Luger
2011). We have called this activity “nucleosome reorganiza-
tion”(Formosa 2008). FACT enhances binding of TATA se-
quences within nucleosomes by TATA-binding protein (TBP/
Spt15) (Biswas et al. 2005), suggesting that increasing ac-
cess to DNA through reorganization is a physiologically im-
portant role of FACT.
FACT can induce displacement of H2A-H2B dimers from
nucleosomes under some conditions in vitro (Belotserkovskaya
et al. 2003; Xin et al. 2009). However, increased nuclease
sensitivity does not require H2A-H2B loss (Xin et al. 2009).
Reorganized nucleosomes instead appear to have the same
composition as canonical nucleosomes, but the components
are associated with one another in a substantially different
manner. This change in structure increases the probability of
H2A-H2B dimer loss, but dimer loss appears to be just one
possible result of reorganization, not its mechanism. The
effect of FACT on nucleosomes is not common to other his-
tone chaperones, and FACT-mediated reorganization does
not require ATP hydrolysis so it is unlike ATP-dependent
Copyright © 2011 by the Genetics Society of America
doi: 10.1534/genetics.111.128769
Manuscript received March 16, 2011; accepted for publication May 18, 2011
Supporting information is available online at http://www.genetics.org/content/suppl/
2011/05/30/genetics.111.128769.DC1.
1
Corresponding author: Department of Biochemistry, University of Utah School of
Medicine, 15 N. Medical Dr. East, Room 4100, Salt Lake City, UT 84112. E-mail: tim@
biochem.utah.edu
Genetics, Vol. 188, 835–846 August 2011 835
chromatin remodeling (Orphanides et al. 1998; Clapier and
Cairns 2009). FACT is essential for viability (Formosa 2008;
Lolas et al. 2010), but the detailed nature of reorganized
nucleosomes, the role of FACT in forming and resolving
alternative nucleosome forms, and the importance of H2A-
H2B dimer displacement to FACT activity in vivo remain
poorly understood.
Partial loss-of-function mutations in the genes encod-
ing FACT subunits cause a range of defects in transcrip-
tion, replication, and other processes (Lycan et al. 1994;
O’Donnell et al. 2004, 2009; Formosa 2008). Some of
these defects can be enhanced by decreasing histone acety-
lation (Formosa et al. 2002), by blocking histone H3-K4
methylation (Biswas et al. 2006), by mutating histone genes
themselves (Vandemark et al. 2008), or by inactivating the
Hir/Hpc complex that is involved in regulating histone gene
expression and depositing nucleosomes outside of S phase
(Formosa et al. 2002). Some features of chromatin therefore
support FACT activity in vivo, and their loss makes FACT
defects more difficult to tolerate. In contrast, other chroma-
tin factors oppose FACT activity, as the phenotypes caused
by some FACT gene mutations can be suppressed by pre-
venting methylation of H3-K36 (Biswas et al. 2006) or by
inactivating the chromodomain-helicase Chd1 (Biswas et al.
2008). FACT gene mutations can also either enhance or
suppress defects caused by mutating other chromatin factors
such as the histone H3 or the Swi/Snf remodeling complex
(Malone et al. 1991; Duina et al. 2007). Furthermore, phe-
notypes caused by FACT gene mutations can be either en-
hanced or suppressed by altering the ratio of expression of
H2A-H2B relative to H3-H4 (Formosa et al. 2002). The prop-
erties of chromatin therefore affect the efficiency of FACT
function in vivo, but it remains unclear how these properties
influence the central activity of FACT.
To examine the relationship between FACT and nucleo-
some reorganization in vivo, we sought histone gene muta-
tions that could compensate for FACT defects. We reasoned
that deficiency for FACT activity could be counterbalanced
by mutating the histones in a way that makes nucleosomes
easier to reorganize. Such mutations would provide insight
into both the functions of FACT in vivo and the nature of
nucleosome reorganization. Here we report that the temper-
ature sensitivity (Ts) and hydroxyurea sensitivity caused by
the spt16-11 allele of FACT can be suppressed by weakening
the H2A-H2B dimer interface with (H3-H4)
2
tetramers
within nucleosomes, but this does not significantly affect
the Spt
2
phenotype also caused by spt16-11. Purified nucle-
osomes with these mutated histones display elevated rates
of spontaneous reorganization using nuclease sensitivity as
an assay, and this partially compensates for Spt16-11 pro-
tein’s defect in achieving stable reorganization in vitro.
These results provide new insights into the mechanism of
FACT activity in vivo, supporting an important role for the
stability of the histone dimer:tetramer interface in nucleo-
some reorganization and revealing a potential role for re-
organization in replication fork progression.
Materials and Methods
Strains are listed in Table 1 and in the supporting informa-
tion,Table S1. W303 strains were derived from MSY1905
(kindly provided by M. Mitchell Smith, University of Vir-
ginia) by conversion of the KanMX marker replacing
HHT1-HHF1 with HIS3 followed by standard crosses within
the W303 background to introduce other mutations. A364a
strains were constructed by integrating markers down-
stream of the histone genes and then amplifying the marked
genomic locus by PCR using an upstream primer containing
the desired mutation 30 nucleotides from the 59end and
25 nucleotides from the 39end and a downstream primer
200 bp distal to the marker (the strategy is outlined in
Figure 3; the primers used are listed in Table S2; Toulmay
and Schneiter 2006). The PCR product was used to trans-
form a wild-type strain, selecting for transfer of the marker
and then screening for cotransfer of the mutation by se-
quencing the entire histone gene. Standard crosses within
the A364a background were then performed to obtain com-
binations of mutations.
The S. cerevisiae genome contains two copies of each of
the genes that encode the four core histones (Osley 1991).
Plasmids pJH33, M4958, and M4959 carrying the genes
HTA1-HTB1 and HHT2-HHF2 encoding histones H2A, H2B,
H3, and H4 in vectors pRS316 (URA3), pRS315 (LEU2), or
pRS314 (TRP1), respectively (Sikorski and Hieter 1989),
were kindly provided by M. Mitchell Smith (University of
Virginia) (Ahn et al. 2005). Mutagenized histone genes were
obtained by amplifying M4958 using primers that align
400 bp into the vector sequences flanking the histone gene
insert, resulting in a 5279-bp product with 400 bp of ho-
mology at each end to pRS414 or pRS415 (Figure 1). PCR
was performed under standard conditions (100-ml reactions
containing 20 pmol of each primer, 10 ng template, 0.2 mM
each deoxynucleoside triphosphate (dNTP), 10 mMTris–Cl
pH 8.3, 1.5 mMMgCl
2
,50mMKCl; 30 cycles of 1 min at 94,
1 min at 54, 5 min at 72) using Pfu polymerase (a small
number of candidates were generated with a 1:200 mixture
of Pfu:Taq polymerases, but most of these contained multi-
ple mutations). Yeast strains DY10003 and DY10004 with
the spt16-11 allele (Table 1) and carrying pJH33 were trans-
formed with this PCR product mixed with vectors pRS414
(TRP1) or pRS415 (LEU2) (Christianson et al. 1992) that
had been linearized with BamHI and HindIII. This yielded
pLM04 (TRP1) and pTF238 (LEU2) derivatives by recombi-
nation in vivo. About 70,000 transformants were obtained
and replica-plated to medium containing 59-FOA to select
for cells lacking the original wild-type histone plasmid
(Boeke et al. 1987), thus demanding that the mutagenized
plasmids expressed histones adequate for supporting viabil-
ity. Strains surviving with only the mutagenized plasmids
were replica-plated to 37or to medium containing 120 mM
hydroxyurea (HU), conditions nonpermissive for growth of
the parent strains. About 2000 candidates were chosen
for retesting, yielding several hundred suppressed strains.
836 L. McCullough et al.
Plasmids were recovered from 100 of the strongest can-
didates, screened for the expected restriction digestion pat-
tern, and used to transform DY10004 pJH33 to establish
linkage of the suppressing mutation with the plasmid.
Twenty-six of these plasmids were found to contain sup-
pressor mutations and were sequenced using four primers
to fully cover all four histone genes on each plasmid (Table
S2). Eighteen of the plasmids had single mutations affect-
ing 1 of 10 residues in H2A or H2B, distributed as shown in
Table2.Someofthe8plasmidswithmultiplemutations
also affected these same residues, and all plasmids had at
least one mutation in H2A or H2B that mapped to the in-
terface between H2A-H2B and H3-H4, but plasmids with
complex mutations were not analyzed further. The screens
for suppression of temperature sensitivity and HU sensitiv-
ity yielded overlapping results, so the resulting plasmids
were combined for further analysis (Figure 1B).
Nucleosomes were reconstituted in vitro with recombi-
nant histones, labeled with fluorescent dyes, and tested for
binding affinity with FACT, sensitivity to DraI digestion, and
retention of H2A-H2B dimers as described previously
(Ruone et al. 2003; Rhoades et al. 2004; Xin et al. 2009).
The spt16-11 (T828I, P859S) (Formosa et al. 2002), hta1-
V101I, and htb1-A84D mutations were introduced into
expression constructs using the Quikchange strategy (Stra-
tagene), and the proteins were purified as previously de-
scribed (Ruone et al. 2003; Rhoades et al. 2004; Xin et al.
2009).
Results
Defects caused by FACT mutants can be suppressed by
histone mutants
The spt16-11 allele causes sensitivity to elevated tempera-
tures, sensitivity to the replication toxin HU, and the
Spt
2
phenotype (Formosa et al. 2001). The amount of Spt16
protein in an spt16-11 mutant drops about fivefold after a
3-hr incubation at 37(Vandemark et al. 2008), so the Ts
2
phenotype probably reflects simple loss of FACT activity.
HU inhibits ribonucleotide reductase (RNR), causing DNA
replication to stall due to the shortage of dNTPs, but also
Table 1 Strains used
Strain Genotype
Figure 1 W303 background
DY10003 MATaade2 can1 his3 leu2 trp1 ura3 spt16-11 hht1-hhf1-D(::HIS3) hht2-hhf2-Δ(::KanMX)
hta1-htb1-Δ(::NatMX) hta2-htb2-Δ(::HphMX) pJH33 (YCp URA3 HHT2-HHF2,HTA1-HTB1)
DY10004 MATaade2 can1 his3 leu2 trp1 ura3 spt16-11 hht1-hhf1-Δ(::HIS3) hht2-hhf2-Δ(::KanMX)
hta1-htb1-Δ(::NatMX) hta2-htb2-Δ(::HphMX) pJH33 (YCp URA3 HHT2-HHF2,HTA1-HTB1)
Figure 3 A364a background
8127-7-4 MATaura3-Δ0 leu2-Δ0 trp1-Δ2 his3 lys2-128@
8500-10-2 MATaura3-Δ0 leu2-Δ0 trp1-Δ2 his3 lys2-128@HTA1(220,His3MX) HTA2(30,URA3)
8541-3-2 MATaura3 leu2 trp1 his3 lys2-128@hta1-V101I(220,His3MX) hta2-V101I(30,URA3)
8262-11-4 MATaura3 leu2 trp1 his3 lys2-128@spt16-11
8554-5-3 MATaura3 leu2 trp1 his3 lys2-128@spt16-11 HTA1(220,His3MX) HTA2(30,URA3)
8541-4-2 MATaura3 leu2 trp1 his3 lys2-128@hta1-V101I(220,His3MX) hta2-V101I(30,URA3) spt16-11
8127-7-4 MATaura3-Δ0 leu2-Δ0 trp1-Δ2 his3 lys2-128@
8500-10-2 MATaura3-Δ0 leu2-Δ0 trp1-Δ2 his3 lys2-128@HTA1(220,His3MX) HTA2(30,URA3)
8541-3-2 MATaura3 leu2 trp1 his3 lys2-128@hta1-V101I(220,His3MX) hta2-V101I(30,URA3)
8324-2-2 MATaura3-Δ0 leu2-Δ0 trp1-Δ2 his3 lys2-128@pob3-Q308K(LEU2)
8500-2-2 MATaura3-Δ0 leu2-Δ0 trp1-Δ2 his3 lys2-128@HTA1(220,His3MX) HTA2(30,URA3) pob3-Q308K(LEU2)
8555-4-2 MATaura3 leu2 trp1 his3 lys2-128@hta1-V101I(220,His3MX) hta2-V101I(30,URA3) pob3-Q308K(LEU2)
Figure 4 A364a background
8483-9-1 MATaura3-Δ0 leu2-Δ0 trp1-Δ2 his3 lys2-128@HTB1(30,URA3) HTB2(30,His3MX)
8606-3-4 MATaura3 leu2 trp1 his3 lys2-128@htb1-A84D(30,URA3) HTB2(30,His3MX)
8868-5-1 MATaura3-Δ0 leu2-Δ0 trp1-Δ2 his3 lys2-128@HTB1(30,URA3) htb2-A84D(30,His3MX)
8442-4-3 MATaura3-Δ0 leu2-Δ0 trp1-Δ2 his3 lys2-128@htb1-A84D(30,URA3) htb2-A84D(30,His3MX)
8482-5-3 MATaura3 leu2 trp1 his3 lys2-128@HTB1(30,URA3) HTB2(30,His3MX) spt16-11
8606-2-1 MATaura3 leu2 trp1 his3 lys2-128@htb1-A84D(30,URA3) HTB2(30,His3MX) spt16-11
8607-1-1 MATaura3 leu2 trp1 his3 lys2-128@HTB1(30,URA3) htb2-A84D(30,His3MX) spt16-11
8455-8-4 MATaura3 leu2 trp1 his3 lys2-128@htb1-A84D(30,URA3) htb2-A84D(30,His3MX) spt16-11
8625-3-4 MATaura3 leu2 trp1 his3 lys2-128@HTB1(30,URA3) hta2-htb2-Δ(::HIS3) spt16-11
8608-5-4 MATaura3 leu2 trp1 his3 lys2-128@htb1-A84D(30,URA3) hta2-htb2-Δ(::KanMX) spt16-11
Table 2 W303 background
DY9999 MATaade2 can1 his3 leu2 trp1 ura3 hht1-hhf1-Δ(::HIS3) hht2-hhf2-Δ(::KanMX3)
hta1-htb1-Δ(::NatMX) hta2-htb2-Δ(::HphMX) pJH33 (YCp URA3 HHT2-HHF2,HTA1-HTB1)
8264-17-3 MATaade2 can1 his3 leu2 trp1 ura3 pob3-Q308K hht1-hhf1-Δ(::HIS3) hht2-hhf2-Δ(::KanMX3)
hta1-htb1-Δ(::NatMX) hta2-htb2-Δ(::HphMX) pJH33 (YCp URA3 HHT2-HHF2,HTA1-HTB1)
FACT Defects Suppressed by Histone Mutants 837
leading to increased transcription of the genes encoding
RNR. At least some FACT mutants retain normal induction
of RNR gene transcription (Biswas et al. 2008; Formosa
2008), so HU sensitivity often reflects a defect in DNA rep-
lication, but indirect effects due to flawed transcription are
also possible. The Spt
2
phenotype results from inappropri-
ate transcription initiation start-site selection (Clark-Adams
et al. 1988), indicating that spt16-11 causes a defect in tran-
scription even at temperatures permissive for growth where
Spt16 protein levels are normal. spt16-11 was chosen for the
suppressor analysis described here because it causes a broad
range of phenotypes associated with transcription and
replication.
To detect histone gene mutations that can compensate
for the defects caused by spt16-11, we constructed a strain
with this mutation that lacked genomic versions of histone
genes but instead carried a single copy of the genes encod-
ing H2A, H2B, H3, and H4 on a plasmid (Materials and
Methods). PCR amplification of the insert containing all four
histone genes was used to introduce random mutations, and
alleles capable of suppressing the Ts
2
or HU sensitivity phe-
notypes caused by spt16-11 were identified. This approach
yielded 26 suppressing plasmids, 18 of which carried single
mutations that altered 1 of 10 residues in H2A or H2B
(Table 2, Figure 1B). H2B-A84 mutants were isolated five
times (A84D four times and A84V once), but mutations af-
fecting other residues were recovered only once or twice.
The screen was therefore not saturated, but all of the mu-
tated residues matched the same physical profile in that they
affected residues that are buried within the histone octamer
core on or near the surface of the H2A-H2B dimer that con-
tacts the (H3-H4)
2
tetramer (indicated in Figure 2 as either
magenta or red residues). These results therefore strongly
suggest that FACT activity in vivo involves disruption of the
dimer:tetramer interface, that the spt16-11 mutation causes
a defect in this function, and that weakening the interface by
mutating residues in the interface reduces the requirement
for efficient FACT. It should also be possible to interfere with
this interface by mutating H3 or H4, so it is puzzling that no
such mutations were identified, but it is possible that mu-
tating the interface from the more highly conserved H3 or
H4 protein side leads to inviability.
Suppressors were isolated for the ability to reverse either
the Ts
2
or the HU sensitivity caused by spt16-11, but each
suppressor was found to suppress both phenotypes to vari-
able but similar extents (Figure 1B). This suggests that both
phenotypes have a common underlying defect. Suppression
of spt16-11 was partially allele-specific, as some of the
mutants weakly suppressed a pob3-Q308K mutation affect-
ing the Pob3 subunit of FACT, but others had no effect and
some even enhanced the defects caused by pob3-Q308K
(Table 2, Figure S1A, Figure S4, and Figure 3). spt16-11
and pob3-Q308K therefore cause distinct defects in FACT
function, and weakening the dimer:tetramer interface is
strongly beneficial only to the cells with the spt16-11 de-
ficiency. The histone gene mutations had little or no effect
in an SPT16 wild-type strain (Table 2, Figure S1B). Altering
the histones therefore caused a significant enough effect on
nucleosome structure that an spt16-11 mutation was
strongly suppressed, but the change was not sufficient to
cause an obvious defect in an otherwise normal cell.
Integration of histone gene mutations reveals
differences among expression contexts
FACT mutants are sensitive to histone gene copy-number
variation (Formosa et al. 2002), and it is difficult to maintain
plasmids at uniform copy number throughout a population
of cells. Furthermore, HTA1-HTB1 and HTA2-HTB2 encode
slightly different amino acid sequences (Figure S3), tran-
scription of each gene is regulated by different factors (Osley
and Lycan 1987; Xu et al. 1992; Dollard et al. 1994; Hess
and Winston 2005), and HTA1-HTB1 is essential for viability
but HTA2-HTB2 is not (Formosa et al. 2002; Libuda and
Winston 2006). Either the sequence differences or their
transcription profiles under different conditions are there-
fore functionally important. To test the effects of the sup-
pressing mutations in a more native context, we integrated
two of the mutations into the genome at the endogenous
Figure 1 Histone gene mutations can suppress defects caused by an
spt16-11 mutation. (A) Scheme for mutagenizing histone genes. A plas-
mid carrying wild-type HHT2-HHF2 (H3-H4) and HTA1-HTB1 (H2A-H2B)
was used as the template for PCR using primers TF04-25 and TF05-28 in
the vector sequence flanking the histone gene insert (Table S2). The
product was mixed with linearized vector DNA and used to transform
DY10003 or DY10004 (Table 1). Recombination in vivo produced muta-
genized histone gene plasmids. (B) Candidate plasmids with the single
mutations indicated were recovered and used to transform strain
DY10004 (Table 1), and then isolates lacking the wild-type histone gene
plasmid were derived. Strains with only the mutated plasmid were grown
to saturation, and aliquots of 10-fold serial dilutions were tested on YPAD
(rich medium; Yeast Extract, Peptone, Adenine, Dextrose) at 25or 34or
on HU (60) (YPAD with 60 mMhydroxyurea) at 25.
838 L. McCullough et al.
loci. We chose H2A-V101I and H2B-A84D for this as they
were two of the strongest suppressors of spt16-11 but they
had opposite effects on the pob3-Q308K allele (Table 2).
We inserted selectable markers downstream of each
gene that encodes H2A or H2B in separate wild-type strains
(Figure S2). We then used genomic DNA from these strains
to amplify each gene with the targeted mutation incor-
porated into one PCR primer (Figure 3A) (Toulmay and
Schneiter 2006). The product carrying the desired mutation
was used to transform a wild-type yeast strain, and trans-
formants were screened by sequencing the entire histone
gene to find strains in which the desired mutation but no
unexpected additional mutation had been integrated into
the genome along with the selectable marker. Finally, stan-
dard crosses were performed to obtain combinations of
mutations.
Figure 3B shows the results obtained with the H2A-V101I
expressed from both HTA1 and HTA2. Neither the integrated
markers themselves (tags) nor the histone mutations af-
fected the phenotypes tested in an SPT16 strain, but the tags
themselves slightly enhanced the HU sensitivity and Spt
2
phenotypes (but not the Ts
2
) caused by spt16-11 (Figure
3B; compare rows 4 and 5: the Spt
2
phenotype is revealed
by growth of these lys2-128@strains on medium lacking
lysine). Marking the wild-type H2A genes therefore causes
a minor defect in their expression; as noted previously and
consistent with these results, a decrease in H2A-H2B expres-
sion is detrimental to some spt16 mutants (Formosa et al.
2002). To control for this effect, subsequent experiments
always include marked versions of the H2A genes, whether
wild type or mutant. This ensures that all comparisons are
between strains that are as genetically matched as possible,
although it remains possible that the markers could have
a differential effect on mutant and wild-type alleles.
The integrated H2A-V101I mutation suppressed both
the Ts
2
and hydroxyurea sensitivity phenotypes caused by
spt16-11, but had only a slight effect on the Spt
2
phenotype
(Figure 3B; compare rows 5 and 6). H2A-V101I therefore
significantly corrects the spt16-11 defect most closely asso-
ciated with DNA replication, but it has less effect on the
transcription defect. Integrated H2A-V101I had no effect in
a wild-type strain (Table 2, Figure S1B), but partially sup-
pressed the Ts
2
and HU sensitivity caused by pob3-Q308K
(Table 2, Figure S1A, Figure 3C), generally recapitulating
the results from the plasmid-based assays.
In contrast, the locus expressing H2B-A84D expression
significantly influenced the effect of this mutant. In a wild-
type strain, expressing H2B-A84D from HTB1 caused a very
mild Spt
2
phenotype (Figure 4A; compare the medium lack-
ing lysine (2lys) growth at 2 and 6 days with the 2-day
incubation of an spt16-11 mutant in Figure 4B). Expressing
H2B-A84D from HTB2 did not cause this phenotype and did
not enhance the effect of htb1-A84D (Figure 4A; compare
rows 2 and 4). In an spt16-11 strain, the htb1-A84D allele
suppressed the Ts
2
and hydroxyurea sensitivity phenotypes
but did not affect the Spt
2
phenotype; this was true in both
an HTA1 htb1-A84D HTA2 HTB2 strain (normal H2B avail-
able from the second copy of the gene) and an HTA1 htb1-
A84D hta2-htb2-Δstrain (only H2B-A84D available; Figure
4B; compare row 1 with 2 or 6). However, the htb2-A84D
allele had no effect when paired with HTB1 (Figure 4B, row
3) and caused variable effects when paired with htb1-A84D
(Figure 4B, rows 3 and 4). (HTB1 is essential for viability
so the effect of htb1-Δhtb2-A84D could not be assessed.)
Relative to htb1-A84D HTB2 spt16-11, the htb1-A84D htb2-
A84D spt16-11 strain displayed markedly reduced suppres-
sion of the Ts
2
phenotype, increased suppression of the
Spt
2
phenotype, and enhanced sensitivity to low concentra-
tions of HU (Figure 4B, rows 2 and 4).
The effects of the source of the H2B-A84D mutation
are therefore quite complex and vary with the phenotype
being observed. When challenged with HU or elevated
Table 2 Effects of spt16-11 suppressors in wild-type and pob3-Q308K strains
Histone Mutation No. of isolates Phenotypes with pob3-Q308K Phenotypes in wild type
H2A hta1-A87T 2
H2A hta1-R89G 2Ts
2
HUs (enhanced defect) Slight Ts
2
, Slight HUs, NaCl-s
H2A hta1-N100D 1 Ts+ HUr (weak suppression)
H2A hta1-V101I 1 Ts+ HUr (weak suppression) Slight Fmd-s
H2B htb1-T55S 2 Fmd-s
H2B htb1-N66S 1 Fmd-s
H2B htb1-F68S 2Ts
2
HUs (enhanced defect)
H2B htb1-T78P 1Ts
2
HUs (enhanced defect)
H2B htb1-A80V 2Ts
2
HUs (enhanced defect)
H2B htb1-A84V 4 Ts+ HUr (weak suppression)
H2B htb1-A84D 1Ts
2
HUs (enhanced defect) Mild Spt
2
H2B htb1-Y86C 1 Mixed weak effects Slight Fmd-s, Slight HUs
H2B htb1-I104F 1 Ts+ HUr (weak suppression)
Strains 8264-17-3 (pob3-Q308K) and DY9999 (wild type) were transformed with LEU2 plasmids carrying the mutation indicated, and then transformants lacking the wild-
type histone gene plasmid were screened for growth at elevated temperatures or on media containing 30–150 mMhydroxyurea, 1.2 M NaCl, 3% formamide, 10 mM
caffeine, or 10 mg/ml camptothecin. The pob3-Q308K mutation causes Ts
2
and HUs phenotypes, and these were unaffected, enhanced, or suppressed as indicated. In all
cases, the effects were small compared to other synthetic interactions observed with this allele. Moderate formamide sensitivity was observed where indicated (Fmd-s), with
all other effects in the wild-type strain being weak. The number of times each mutation was isolated in the original screen for spt16-11 suppression is indicated as “No. of
isolates.”HUs, hydroxyurea sensitive; HUr, hydroxyurea resistant; NaCl-s, NaCl sensitive.
FACT Defects Suppressed by Histone Mutants 839
temperature, expressing H2B-A84D only from the HTB1 lo-
cus was strongly advantageous to an spt16-11 strain (Figure
4B) but had no apparent effect in a wild-type strain (Figure
4A). However, supplying H2B-A84D only from the HTB2
locus under these stress conditions had no effect on either
spt16-11 or wild-type strains. htb1-A84D caused the same
effects whether normal HTB2 was available or not, suggest-
ing that HTB2 expression is irrelevant in these tests. How-
ever, cells with both htb1-A84D and htb2-A84D mutations
displayed distinct phenotypes compared to the single
mutants, so HTB2 expression is important in this context.
While both HTB1 and HTB2 are transcribed at similar levels
under standard growth conditions (David et al. 2006), these
results show that the two loci have distinct functions under
some circumstances, as observed previously for the heat-
shock response (Norris and Osley 1987) and the ability to
suppress the effects of different Ty1 delta-element insertions
(Clark-Adams et al. 1988). These differences may result
from the slightly different proteins produced by the two
genes (Figure S3), or they may indicate that the two genes
are differentially activated by stress conditions. In any case,
this shows that testing variants only from a plasmid-borne
copy of HTB1 can produce an incomplete picture.
Histone mutations that suppress spt16-11 form unstable
nucleosomes in vitro
The clustering of spt16-11 suppressors in the nucleosome
structure (Figure 2) suggests that the Spt16-11 protein is
defective in a process that includes disruption of the H2A-
H2B interface with (H3-H4)
2
tetramers. To investigate this
possibility as well as other potential mechanisms, we recon-
stituted nucleosomes containing H2A-V101I or H2B-A84D
and tested their stability in vitro. A 181-bp DNA fragment
including the 146-bp sea urchin 5S rDNA nucleosome posi-
tioning sequence was labeled with Cy5 and assembled into
nucleosomes using recombinant yeast histones expressed in
bacteria as described previously (Xin et al. 2009). The H2A-
Q114C mutation was introduced into the HTA1 gene to
provide a unique cysteine residue for labeling this subunit
with a maleimide derivative of the fluorescent dye Oregon
Green 488 prior to assembly of octamers. The resulting nu-
cleosomes therefore contained two fluorescent dyes that
could be detected independently, allowing us to follow the
DNA and H2A-H2B dimer components of the nucleosome
separately.
To test whether suppressor mutations affect the physical
stability of nucleosomes, we used native polyacrylamide gel
electrophoresis to measure the amount of H2A-H2B dis-
placed from the nucleosomes, the amount of tetrasome
formed, and the amount of free DNA released under various
conditions. Over 90% of a sample of wild-type nucleosomes
remained intact by all three measurements after a 1-hr
incubation at 30in 550 mMNaCl (Figure 5, A and B). In
contrast, nucleosomes constructed with H2A-V101I lost
dimers and formed tetrasomes more readily than wild type,
and these effects were even more pronounced with H2B-
A84D. In the latter case, even nucleosomes not exposed to
high salt migrated aberrantly in native polyacrylamide gels,
consistent with the observed spontaneous loss of dimers
during preparation and storage (Figure 5A, lane 5 and not
shown). Incubation of nucleosomes for 1 hr at 65also
caused low levels of tetrasome formation with wild-type
nucleosomes, but substantially elevated levels for each mu-
tant nucleosome (Figure 5, C and D). Thus, both H2A-V101I
and H2B-A84D cause nucleosome instability in vitro, includ-
ing an increase in dimer displacement.
FACT might either induce nucleosomes to reorganize or
selectively bind to and stabilize spontaneously reorganized
nucleosomes. In the first case, a weakened dimer:tetramer
interface would make it easier to overcome resistance to
reorganization, and in the second case, it would provide
a larger subpopulation of complexes for binding. In either
Figure 2 Histone gene mutations that suppress
spt16-11 map to the dimer:tetramer interface.
Histone residues identified in the suppressor
screen are shown within the structure of a yeast
nucleosome (PDB 1ID3) (White et al. 2001) as
rendered in MacPyMOL (DeLano Scientific).
Two orientations are shown (top and bottom
panels) with a full nucleosome (left), H2A-H2B
removed (center), or only H2A shown (right) to
reveal buried sites. Sites of suppressor mutants
are shown in magenta or red, with the latter
indicating residues H2A-V101 and H2B-A84
that were chosen for further analysis.
840 L. McCullough et al.
case, these results show that the stability of the dimer:
tetramer interface is an important element of the core
reaction promoted by FACT in vivo.
FACT(Spt16-11) has a nucleosome-binding defect
in vitro
We used purified nucleosomes mixed with normal and
mutant FACT complexes to examine the nature of the
suppression observed in vivo.Wefirst measured the appar-
ent affinity of FACT for nucleosomes using an electropho-
retic mobility shift assay (EMSA) (Rhoades et al. 2004). The
DNA-binding protein Nhp6 is required for complex forma-
tion in this assay (Formosa et al. 2001; Xin et al. 2009), with
50% saturation occurring at 460 nM Nhp6 (Ruone et al.
2003). This same value was observed for both Spt16-Pob3
and (Spt16-11)-Pob3 with wild-type and mutant nucleo-
somes (not shown), so Spt16-11 and Spt16 proteins each
require about the same amount of Nhp6 to support stable
complex formation. In contrast, Spt16-11 was three- to five-
fold less effective than Spt16 in this assay (Figure 6A). This
could mean that Spt16-11 has a lower affinity for nucleo-
somes, that it is less effective in performing reorganization,
or that it is unable to maintain the reorganized state stably
enough for detection by EMSA. Nucleosomes with H2A-
V101I or H2B-A84D were the same as wild-type H2A in this
assay (Figure S5), so while this reveals a defect in Spt16-11
activity, it does not reveal the mechanism of genetic
suppression.
spt16-11 and histone mutants have opposing effects on
reorganization rates
Reorganized nucleosomes are more prone to losing one or
both H2A-H2B dimers in vitro (Belotserkovskaya et al. 2003;
Xin et al. 2009). Nuclease sensitivity can be detected in
complete octameric nucleosomes, so dimer loss is not a nec-
essary feature of reorganization, but reorganization can lead
Figure 3 H2A-V101I suppresses some phenotypes caused
by two distinct FACT gene mutations. (A) Schematic
showing the method used for integrating the hta2-V101I
mutation into the genome (strategies for other genes
shown in Figure S2 and Table S2). (B and C) Cultures of
strains isogenic with the A364a genetic background and
the additional genotypes shown (see Table 1 for the full
genotypes) were tested as in Figure 1). “C”is complete
synthetic medium; HU (90) and HU (150) are YPAD with
90 or 150 mMhydroxyurea. Growth on medium lacking
lysine (2lys) reveals the Spt
2
phenotype in the strains with
the lys2-128@allele (Simchen et al. 1984). Unless noted,
all strains have marker genes (tags) inserted adjacent to
the normal or mutated histone genes (Figure S2). Strains
labeled “H2A-V101I”express this mutant protein from
both HTA1 and HTA2 loci. Note that the severity of the
Ts
2
and hydroxyurea sensitivity phenotypes caused by
spt16-11 here is lower than in Figure 1 because the initial
screen (Figure 1) was performed in the W303 genetic
background where FACT gene mutations routinely cause
stronger defects, whereas the integrants shown here were
constructed in the A364a genetic background.
FACT Defects Suppressed by Histone Mutants 841
to dimer loss so it serves as an assay for the ability of FACT
to promote this structural change.
We measured the total dimer displacement after a 10-
min incubation. As expected, FACT caused more dimer
displacement than Nhp6 alone, but the levels for Spt16 and
Spt16-11 were comparable (Figure 6B). Histone mutants
displayed increased dimer loss in the presence of Nhp6
alone, consistent with the results described above showing
inherent instability of these nucleosomes. Nucleosomes
with wild-type histones or with the H2A-V101I protein dis-
played slightly lower dimer displacement with Spt16-11
than with Spt16, but nucleosomes with H2B-A84D had
a similarly small but opposite effect. Spt16-11 therefore
appears to have a small effect on dimer loss after a 10-min
incubation.
We next examined the rate of dimer displacement.
Instead of the 181-bp 5S rDNA nucleosomes containing
recombinant yeast histones used above, we used a 255-bp
MMTV (Mouse Mammary Tumor Virus) sequence (Flaus
et al. 2004), recombinant Xenopus laevis histones, and low
Mg
2+
ion concentrations. These conditions increase the total
dimer loss caused by FACT (Xin et al. 2009) and increase the
electrophoretic separation of octameric and tetrameric
nucleosomes. These nucleosomes migrate as one major band
in native gels (Flaus et al. 2004) (Figure 6C, “Nuc”) along
with several minor translational variants, while recon-
structions show that tetrasomes migrate to four main bands
(Figure 6C, “Tet”). FACT does not appear to promote trans-
location of nucleosomes (Rhoades et al. 2004), so we infer
that the multiple forms observed with this larger MMTV
fragment are corresponding pairs of octasomal and tetraso-
mal species occupying the same preferred translational
positions.
Samples taken during a 5-min incubation show that
FACT promotes tetrasome formation more rapidly with
mutant histones than with wild-type histones (Figure 6,
C–E). Conversely, FACT with Spt16-11 caused displacement
more slowly than FACT with wild-type Spt16 protein (Fig-
ure 6E, Figure S6). Combining histone mutants with the
FACT mutant resulted in a slight suppression of the Spt16-11
defect (Figure 6E, Figure S6), but the amount of compensation
was minimal.
Together, these results show that FACT containing Spt16-11
protein has significant defects in vitro in forming stable com-
plexes with nucleosomes and in promoting the normal rapid
rate of dimer loss. Mutant histones identified as suppressors
of the spt16-11 allele are lost from nucleosomes at an ab-
normally high rate, but do not strongly reverse the Spt16-11
defects in these assays.
Nuclease sensitivity reveals a potential mechanism
of suppression
We have proposed that FACT promotes equilibration of nu-
cleosomes between canonical and reorganized forms, with
the rate of digestion by nucleases being proportional to the
fraction of time that the nucleosomes spend in the reor-
ganized state (Xin et al. 2009). We therefore used restriction
endonuclease sensitivity to examine whether Spt16-11 and
histone mutants alter the persistence of the reorganized
state.
We measured the rates of DraI digestion of 181-bp 5S
rDNA nucleosomes with yeast histones to probe two distinct
physical contexts [DraI-78, near the dyad of symmetry, and
DraI-140, near an entry/exit point, as described in Xin et al.
(2009)]. Both FACT and FACT(Spt16-11) enhanced the rate
of digestion near the dyad significantly, although FACT
(Spt16-11) consistently produced less of an effect (Figure
6F). The histone mutants had the opposite effect of increas-
ing the rate of DraI digestion. The same overall pattern was
observed near the entry/exit points, although the rates of
digestion were generally higher (Xin et al. 2009) and the
Figure 4 The source of expression of H2B-A84D affects the resulting
phenotypes. Isogenic strains from the A364a genetic background with
the spt16-11 mutation (Table 1) were tested as in Figure 1, with the
concentration of HU indicated. Panel A shows strains with the WT
SPT16 allele and B shows strains with the spt16-11 mutation. Incubation
times are listed in days to allow comparison of the level of growth at
comparable times. In particular, htb1-A84D causes a weak Spt
2
pheno-
type, as very little growth on medium lacking lysine is observed after
2 days but substantial growth is visible after 6 days. This is weak relative
to the effect of spt16-11, which causes substantial growth on medium
lacking lysine after 2 days. Both HTB1 and HTB2 are tagged in all cases,
whether wild-type (+) or mutant (A84D). “Δ”indicates deletion of both
HTA2 and HTB2.
842 L. McCullough et al.
defect of FACT(Spt16-11) was less significant. These results
show that FACT(Spt16-11) can reorganize nucleosomes, but
it either produces a structure with less accessibility to the
DNA or fails to achieve or maintain the reorganized state for
the normal length of time.
Notably, the combination of FACT(Spt16-11) with H2B-
A84D nucleosomes produced a rate of digestion that is as
high or higher than the rate with wild-type FACT combined
with wild-type nucleosomes (Figure 6F). This also appears
to be true for FACT(Spt16-11) with H2A-V101I nucleosomes
at the entry/exit point, but not near the dyad. These two
histones behave differently in vivo and may use overlapping
but distinct mechanisms of suppression. These results sug-
gest that FACT with Spt16-11 protein fails to achieve or
maintain the open reorganized nucleosome form long
enough to allow the normal rate of restriction endonuclease
digestion, but this defect is counterbalanced by histone
mutants that achieve this state more rapidly or maintain it
for a longer amount of time than usual.
Discussion
We have shown that histone gene mutations can compen-
sate for defects in FACT activity, with the spt16-11 mutation
being suppressed by H2A-H2B mutants that destabilize the
interface between these dimers and the (H3-H4)
2
tetramer.
In tests with purified components, these histone mutants
caused increased dimer displacement in the absence of
FACT and more rapid or more persistent nucleosome reor-
ganization in the presence of FACT. Genetic suppression
therefore appears to result from combining a FACT complex
that is inefficient at nucleosome reorganization with nucle-
osomes that are more rapidly or more easily reorganized. By
comparing the properties of the same FACT and histone
gene mutations in vivo and in vitro, these results provide
insight into the mechanism of FACT activity in its physiolog-
ical settings. Initial reports indicated that FACT removes one
H2A-H2B dimer from a nucleosome (Belotserkovskaya et al.
2003), and our experiments showed that dimer displace-
ment can be an outcome of FACT action but is not a neces-
sary feature of the mechanism of reorganization (Xin et al.
2009). Both sets of in vitro investigations indicate that FACT
activity involves disruption of the H2A-H2B:(H3-H4)
2
inter-
face, and the results presented here confirm that this is an
important feature of FACT function in vivo.
FACT activity has been implicated in both DNA replica-
tion and transcription (Formosa 2008). FACT’s ability to
promote equilibration between stable and dynamic forms of
histone:DNA complexes could reduce the barrier to polymer-
ase progression posed by existing nucleosomes, but it could
also promote establishment of chromatin through the reverse
reaction by converting loosely associated components into
Figure 5 Histone gene mutations destabilize nucleosomes
in vitro. (A) Nucleosomes were constructed using recombi-
nant yeast histones (normal or the mutant indicated) and
a 181-bp 5S rDNA fragment with Cy5 at the 59end and
Oregon Green 488 (Molecular Probes) attached to H2A
residue C114 (originally Q114) (Xin et al. 2009). Samples
were prepared in triplicate and incubated for 1 hr at 30in
100 or 550 mMNaCl and then separated by electropho-
resis through a native 4% polyacrylamide gel in 0.25·TBE
as described (Xin et al. 2009). The single gel was then
scanned to detect the fluorescent dyes on the DNA and
the H2A independently using a Typhoon scanner (GE).
One of the three repeats for each condition is shown.
(B) Each lane was scanned and the amount of signal cor-
responding to free DNA, displaced H2A-H2B, or tetrasome
forms was determined as a percentage of the total signal
in the lane. The regions assigned as free DNA and tetra-
somes are shown in the DNA scan in A, as determined
using pure reference samples (tetrasomes migrate as two
main bands using this combination of DNA and histones).
The amount of displaced H2A-H2B was determined by
calculating the signal in the top 40% of the H2A-H2B scan
in A, as described previously (Xin et al. 2009). The change
in the level of each form caused by treatment with high
salt is plotted, with error bars indicating the standard de-
viation among the three samples. (C and D) As in A and B,
except samples were incubated at 30or 65for 1 hr and
only the total amount of tetrasomes detected is displayed.
FACT Defects Suppressed by Histone Mutants 843
mature nucleosomes. Consistent with a nucleosome assem-
bly function, FACT has been implicated in the deposition of
new nucleosomes after DNA replication (Belotserkovskaya
et al. 2003; Vandemark et al. 2006) as well as in the re-
establishment of repressive chromatin after transcription
(Jamai et al. 2009). Defects in this nucleosome deposition
activity are likely to be the cause of cryptic promoter activa-
tion (Kaplan et al. 2003), failed repression of SER3 expres-
sion (Hainer et al. 2010), and the Spt
2
phenotype (Malone
et al. 1991; Rowley et al. 1991). Most FACT gene mutations
(including spt16-11) cause phenotypes associated with chro-
matin quality defects, indicating that maintaining appropri-
ately repressive chromatin throughout the genome requires
optimal levels of FACT activity. The histone mutants de-
scribed in this report did not suppress the Spt
2
phenotype
caused by spt16-11, so by this assay they do not enhance the
ability of the Spt16-11 protein to assemble normal chromatin.
Essentially all known mutations in FACT genes cause the
Spt
2
phenotype, but only a subset of these mutations causes
the HU sensitivity that is likely to reflect a defect in replica-
tion fork progression or stability. The results described here
suggest that spt16-11 causes HU sensitivity because Spt16-11
protein is unable to promote nucleosome reorganization rap-
idly enough to allow a normal rate of fork progression
Figure 6 Spt16-11 protein and
its suppressors alter the rates of
reactions on the basis of reorga-
nization in vitro. (A) Nucleosomes
constructed as in Figure 5 were
mixed with different concentra-
tions of Spt16-Pob3 hetero-
dimers containing normal Spt16
or Spt16-11 protein, along with
10 mM Nhp6. Samples were in-
cubated for 10 min at 30and
then separated by native PAGE,
and the fraction of the total
DNA signal in FACT–nucleosome
complexes was determined as
described previously (Xin et al.
2009). The nucleosomes shown
contained the H2B-A84D muta-
tion and are typical of results
obtained with wild type and
H2A-V101I. Multiple prepara-
tions of wild-type and mutant
FACT were tested in indepen-
dent experiments; values for half
saturation varied somewhat be-
tween experiments, but the de-
fect for Spt16-11 protein was
reproducible. (B) Nucleosomes
constructed as in Figure 5 were
treated for 10 min at 30with
5mM Nhp6 alone, 5 mM Nhp6
and 200 nM Spt16-Pob3 [“FACT
(Spt16)”], or 5 mM Nhp6 and
(Spt16-11)-Pob3 [“FACT(Spt16-
11)”]. Three independent sam-
ples for each condition were
analyzed as in Figure 5, A and
B, with the average and standard
deviation of the three measure-
ments presented. (C and D)
Nucleosomes were constructed
using recombinant histones from
X. laevis (with normal histones or
the mutation indicated) and a 255-bp MMTV DNA fragment (Flaus et al. 2004) labeled with Cy5. Samples were treated with FACT for the amount of
time indicated, excess unlabeled genomic DNA was added to disrupt the FACT–nucleosome complexes, and then products were separated by native
PAGE (Rhoades et al. 2004). Conversion from initial forms (“Nuc”) to the tetrasomal products (indicated by arrows) was quantitated and plotted in D. (E)
As in D, except FACT(Spt16) or FACT(Spt16-11) were mixed with nucleosomes containing H2A or H2A-V101 as indicated. (F) Nucleosomes were
constructed using wild-type or mutant yeast histones and 181-bp 5S rDNA fragments with DraI recognition sites 78 or 140 bp from the left edge of the
nucleosome (Xin et al. 2009). The initial rate of digestion by DraI in the absence of other factors or with wild-type or mutant FACT was determined by
examining samples taken at 8-min intervals with denaturing PAGE (Xin et al. 2009). Each condition was tested in three independent experiments with
the average and standard deviation shown.
844 L. McCullough et al.
through a normal chromatin template. The additional delay
that results from decreased availability of dNTPs when RNR is
inhibited by HU might be intolerable when FACT is defective.
Histone mutants that promote faster nucleosome reorganiza-
tion by destabilizing the histone dimer:tetramer interface
then diminish the impaired progression and restore viability
under HU stress, but do not resolve the defect in chromatin
quality, so the Spt
2
phenotype persists.
Our tests of an H2B mutation integrated into the genome
reveal a clear functional difference between HTB1 and
HTB2. In normal cells, each gene is transcribed at about
the same level on about the same cell cycle schedule. How-
ever, the A84D mutant is effective only at suppressing the
HU sensitivity (likely to be a replication defect) caused by
spt16-11 if it is expressed from HTB1 alone. Curiously, the
effects of htb1-A84D are similar when it is paired with nor-
mal HTB2 or a deletion of HTB2. In contrast, expressing
H2B-A84D from both loci begins to suppress the Spt
2
phe-
notype (transcription defect) caused by spt16-11, but en-
hances the HU sensitivity (probable replication defect).
These differences could be due to the different primary
sequences of Htb1 and Htb2 proteins, or they could indicate
that the two genes have different expression profiles under
some circumstances such as nucleosome replacement out-
side of S phase. In either case, Htb1 and Htb2 proteins have
different functional roles, and this becomes particularly im-
portant in an spt16-11 strain. It is therefore necessary to
consider the expression context when examining the effects
of histone gene mutations, especially when they affect H2B.
The results presented here provide support for the model
that FACT activity promotes or requires destabilization of
histone dimer:tetramer interaction. The analysis suggests
that promoting the reversible, rapid oscillation of nucleo-
somes between a stable canonical form and a more open
reorganized form is a key function of FACT in vivo, high-
lighting the dynamic nature of nucleosomes under physio-
logical conditions.
Acknowledgments
We thank Mitch Smith for providing the initial strain used in
our screen, Liz Kendall and Peter Winters for technical
assistance, and members of the Formosa and Stillman labs
for valuable input. This project was supported by a grant
from the National Institutes of Health to T.F. and D.J.S.
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Communicating editor: F. Winston
846 L. McCullough et al.
GENETICS
Supporting Information
http://www.genetics.org/content/suppl/2011/05/30/genetics.111.128769.DC1
Insight Into the Mechanism of Nucleosome
Reorganization From Histone Mutants That
Suppress Defects in the FACT Histone Chaperone
Laura McCullough, Robert Rawlins, Aileen Olsen, Hua Xin, David J. Stillman, and Tim Formosa
Copyright © 2011 by the Genetics Society of America
DOI: 10.1534/genetics.111.128769
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