Download Evidence that the Localization of the Elongation Factor

Copyright 2007 by the Genetics Society of America
DOI: 10.1534/genetics.106.067140
Evidence that the Localization of the Elongation Factor Spt16 Across
Transcribed Genes Is Dependent Upon Histone H3 Integrity in
Saccharomyces cerevisiae
Andrea A. Duina,*,†,1 Anne Rufiange,‡ John Bracey,† Jeffrey Hall,†
Amine Nourani‡ and Fred Winston*,2
*Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, †Biology Department, Hendrix College,
Conway, Arkansas 72032 and ‡Centre de Recherche en Cancérologie de l’Université Laval, L’Hôtel-Dieu de
Québec (CHUQ ), Québec, Québec, Canada G1R 2J6
Manuscript received October 22, 2006
Accepted for publication June 25, 2007
ABSTRACT
A previous study of histone H3 in Saccharomyces cerevisiae identified a mutant with a single amino acid
change, leucine 61 to tryptophan, that confers several transcriptional defects. We now present several lines
of evidence that this H3 mutant, H3-L61W, is impaired at the level of transcription elongation, likely by
altered interactions with the conserved factor Spt16, a subunit of the transcription elongation complex
yFACT. First, a selection for suppressors of the H3-L61W cold-sensitive phenotype has identified novel
mutations in the gene encoding Spt16. These genetic interactions are allele specific, suggesting a direct
interaction between H3 and Spt16. Second, similar to several other elongation and chromatin mutants,
including spt16 mutants, an H3-L61W mutant allows transcription from a cryptic promoter within the
FLO8 coding region. Finally, chromatin-immunoprecipitation experiments show that in an H3-L61W
mutant there is a dramatically altered profile of Spt16 association over transcribed regions, with reduced
levels over 59-coding regions and elevated levels over the 39 regions. Taken together, these and other
results provide strong evidence that the integrity of histone H3 is crucial for ensuring proper distribution
of Spt16 across transcribed genes and suggest a model for the mechanism by which Spt16 normally
dissociates from DNA following transcription.
T
HE basic unit of chromatin is the nucleosome, a
structure consisting of 146 nucleotides of DNA
wrapped around a protein octamer composed of pairs of
each of the four core histone proteins (Luger et al.
1997). In addition to directing the condensation of DNA,
histone proteins also play crucial and active roles in the
regulation of cellular processes that use chromatin as
their substrate (Luger 2006). Studies from many laboratories employing a variety of experimental approaches
have converged into a general model for chromatin
function in which dynamic alterations in chromatin
structure are of central importance to processes such as
gene transcription and DNA replication.
The presence of nucleosomes over transcription units
can pose a structural barrier that is inhibitory to transcription initiation and elongation (Sims et al. 2004;
Workman 2006). Several mechanisms have been described that can overcome the repressive nature of
nucleosomes in initiation, including covalent chemical
1
Present address: Biology Department, Hendrix College, 1600 Washington Ave., Conway, AR 72032.
2
Corresponding author: Department of Genetics, Harvard Medical
School, 77 Ave. Louis Pasteur, Boston, MA 02115.
E-mail: [email protected]
Genetics 177: 101–112 (September 2007)
modifications of specific histone residues, ATP-dependent
remodeling of nucleosomes, and the removal of histones (Berger 2002; Narlikar et al. 2002; Boeger et al.
2003; Reinke and Horz 2003). Furthermore, recent studies have demonstrated that regulatory regions of genes
are inherently low in nucleosome density and that histone loss at promoter regions, a process known to be
mediated at some genes by the Asf1 histone chaperone,
is associated with active transcription (Boeger et al. 2003;
Reinke and Horz 2003; Adkins et al. 2004; Bernstein
et al. 2004; Ercan and Simpson 2004; Lee et al. 2004;
Sekinger et al. 2005; Yuan et al. 2005; Korber et al. 2006;
Segal et al. 2006). These and other studies have revealed
the dynamic nature of chromatin over gene promoters as
it relates to the transcription state and have provided insights into how the factors that mediate these chromatin
alterations relate to each other.
The role of chromatin structure in transcription
elongation has been the focus of intense research in
the last several years. This work has shown that as transcription proceeds across a gene and eventually terminates, the composition of the RNA polymerase II (Pol II)
elongation complex changes, with different factors
arriving at and departing from the complex in a highly
coordinated fashion. These changes are dependent, at
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A. A. Duina et al.
least in part, on the phosphorylation status of the Cterminal domain (CTD) of the largest subunit of Pol II
(Hartzog 2003; Bentley 2005; Buratowski 2005;
Eissenberg and Shilatifard 2006; Rosonina et al.
2006). As the elongation complex traverses a transcription unit, the underlying chromatin undergoes a set of
histone modifications, including changes in methylation, acetylation, and ubiquitylation (Workman 2006).
In addition to covalent modifications, histones are also
believed to be physically displaced in front of an
elongating Pol II complex and reassembled in the wake
of Pol II passage by a number of factors, including the
FACT complex, the elongation factors Spt4, Spt5, and
Spt6, and the histone chaperone Asf1 (Hartzog et al.
1998; Formosa et al. 2002; Belotserkovskaya et al. 2003;
Kaplan et al. 2003; Kristjuhan and Svejstrup 2004; Lee
et al. 2004; Schwabish and Struhl 2004, 2006).
The FACT complex is among the better-characterized
transcription elongation complexes studied thus far.
The components of FACT were first discovered through
a number of genetic and biochemical experiments in
Saccharomyces cerevisiae (Kolodrubetz and Burgum 1990;
Malone et al. 1991; Rowley et al. 1991; Wittmeyer
and Formosa 1997) and have since been found in
several larger eukaryotes. The human FACT complex,
originally identified as a biochemical activity from HeLa
cells that facilitates the passage of Pol II across nucleosomal templates in vitro, is composed of two factors,
hSpt16 and SSRP1, which possess distinct but overlapping biochemical activities (Orphanides et al. 1998,
1999). Specifically, hSpt16, but not SSRP1, directly
interacts with H2A–H2B dimers and mononucleosomes, whereas SSRP1, but not hSpt16, binds to H3–
H4 tetramers (Belotserkovskaya et al. 2003). However,
both subunits are needed for the in vitro deposition of
the four core histones onto DNA templates (Belotserkovskaya et al. 2003). Yeast FACT (yFACT) is composed
of Spt16, Pob3 (the yeast homolog of SSRP1), and Nhp6,
which supplies yFACT with an HMG1 DNA-binding
motif found in SSRP1 but absent from Pob3 (Brewster
et al. 1998, 2001; Formosa et al. 2001). Biochemical
studies have shown that yFACT also associates directly
with nucleosomes and reorganizes these structures in
such a way as to alter the patterns of DNAse I-sensitive
sites of DNA molecules within nucleosomes (Formosa
et al. 2001; Rhoades et al. 2004). Recent work has
provided evidence for a functional relationship between
histone H2B monoubiquitination and FACT activity
during transcription elongation, thereby establishing a
mechanistic link between histone modifications and
FACT function (Pavri et al. 2006).
A role for FACT in transcription elongation in vivo is
supported by several studies in S. cerevisiae, Drosophila,
and mammalian systems, including: (i) immunolocalization studies showing association of the Drosophila
melanogaster FACT complex across actively transcribed
regions on polytene chromosomes (Saunders et al.
2003), (ii) chromatin immunoprecipitation (ChIP)
experiments in yeast showing association of FACT subunits over transcribed regions of active genes (Mason
and Struhl 2003; Kim et al. 2004), (iii) experiments
showing that spt16 mutations result in aberrant transcription from cryptic promoter sites within coding
regions of certain genes (Kaplan et al. 2003; Mason
and Struhl 2003), and (iv) genetic studies implicating
yFACT in the reconstitution of proper chromatin structure in the wake of Pol II passage (Formosa et al. 2002).
These and other findings have converged on a model
that proposes that FACT first alters the structure of
chromatin to facilitate the movement of Pol II across
transcribed genes and then reestablishes proper nucleosomal structure following the passage of Pol II
(Reinberg and Sims 2006). The level of reliance on
FACT function for efficient transcription appears to be
determined by the level of chromatin organization of
specific genes, as shown by experiments indicating that
genes with positioned nucleosomes located at the 59
region of the transcribed unit are more dependent on
FACT activity than genes with less stable nucleosomes
( Jimeno-González et al. 2006). In addition to transcription elongation, the FACT complex is involved in other
cellular functions including regulation of transcription
initiation, DNA replication, response to DNA damage,
and the prevention of heterochromatin spreading in
flies (Reinberg and Sims 2006; Nakayama et al. 2007).
In spite of the extensive characterization of FACT and
its roles in transcription elongation, there is little understanding of what might regulate its association with
transcribed chromatin and whether it interacts directly
with nucleosomes in vivo. In this report, we describe
studies of histone H3 that provide new information
regarding its role in transcription elongation in vivo, in
particular as it relates to its functional relationship with
Spt16. We present evidence that a single amino acid
substitution within the globular domain of histone H3
significantly alters the distribution of Spt16 across transcribed genomic regions, resulting in a dramatic accumulation of Spt16 in the 39-untranslated regions
(39-UTRs) of transcribed genes. These and other results
indicate that the integrity of histone H3 is crucial for
proper Spt16 localization across genes and provide initial
insights into the molecular mechanisms that regulate
the dynamic association of Spt16 with chromatin in vivo.
MATERIALS AND METHODS
Yeast strains, genetic methods, and media: All S. cerevisiae
strains used in this study (Table 1) are GAL21 derivatives of the
S288C strain background (Winston et al. 1995) . The experimental procedures for the integration of the hht2-11 allele into
the genome and the replacement of the HHT1-HHF1 and
HHT2-HHF2 loci with the different markers have been previously described (Duina and Winston 2004). Construction
of the snf 2DTLEU2 allele has been described elsewhere
(Cairns et al. 1996). The spt16-197, spt6-1004, and spt4D2THIS3
Histone H3–Spt16 Interactions
103
TABLE 1
Saccharomyces cerevisiae strains
Strain
yAAD108
yAAD476
yAAD482
yAAD563
yAAD587
yAAD994
yAAD1046
yAAD1048
yAAD1049
yAAD1052
yAAD1053
yAAD1060
yAAD1061
yAAD1063
yAAD1118
yAAD1119
yAAD1121
yAAD1127
yAAD1128
yAAD1134
yAAD1135
yAAD1159
yAAD1200d
yAAD2000d
yAAD2005
yAAD2006
yAAD2013
yAAD2015
Genotype
MATa his3D200 leu2D1 ura3-52 trp1D63 lys2-128d (hht1-hhf1)DTLEU2 (hht2-hhf2)DTHIS3 Ty912D35-lacZThis4
ÆpAAD11æ
MATa his3D200 leu2D1 ura3-52 trp1D63 lys2-128d (hht1-hhf1)DTLEU2 (hht2-hhf2)DTHIS3 Ty912D35-lacZThis4
ÆpAAD11æ
MATa his3D200 leu2D1 ura3-52 trp1D63 lys2-128d (hht1-hhf1)DTLEU2 (hht2-hhf2)DTHIS3 Ty912D35-lacZThis4
SPT16-857 ÆpAAD11æ
MATa leu2D1 ura3-52 trp1D63 lys2-128d (hht1-hhf1)DTLEU2 (hht2-hhf2)DTHIS3 Ty912D35-lacZThis4 ÆpAAD11æ
MATa his3D200 leu2D1 ura3-52 trp1D63 lys2-128d (hht1-hhf1)DTLEU2 (hht2-hhf2)DTHIS3 Ty912D35-lacZThis4
SPT16-790 ÆpAAD11æ
MATa his3D200 leu2D1 ura3-52 trp1D63 lys2-128d (hht1-hhf1)DTHIS3 (hht2-hhf2)DTHIS3 snf2DTLEU2 ÆpDM9æ
ÆpAAD11æ
MATa his3Da leu2Db ura3 c trp1D63 lys2-128d met15D0 (hht1-hhf1)DTLEU2
tkl2DTURA3 can1DTMFA1pr-HIS3 SPT16-857
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11 SPT16-857
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11 SPT16-857
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11 SPT16-790
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht2-hhf2)DTHIS3 SPT16-857
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht2-hhf2)DTHIS3
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 SPT16-857
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11 SPT16-790
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11 SPT16-790
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 SPT16-790
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht2-hhf2)DTHIS3 SPT16-790
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht2-hhf2)DTKANMX4
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 spt16-197 ÆpCC58æ
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11 spt16-197 ÆpCC58æ
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11 spt6-1004 ÆpDM9æ
MATa his3D200 leu2D1 ura3-52 lys2-128d (hht1-hhf1)DTLEU2 hht2-11 ÆpDM9æ
MATa his3D200 leu2D1 ura3-52 trp1D63 lys2-128d (hht1-hhf1)DTLEU2 hht2-11 spt4D2THIS3 ÆpDM9æ
MATa his3D200 leu2Db ura3c lys2-128d (hht1-hhf1)DTLEU2 hht2-11 dst1DTNATMX ÆpDM9æ
a
The allele at this locus is either his3D200 or his3D1.
The allele at this locus is either leu2D1 or leu2D0.
c
The allele at this locus is either ura3-52 or ura3D0.
d
For these strains, the allele at the HIS4 locus is either HIS4 or his4-912d.
b
mutations have been described in previous studies (Malone
et al. 1991; Basrai et al. 1996; Kaplan et al. 2003). The
dst1DTNATMX allele was created by a one-step PCRtransformation method that resulted in the replacement of
the DST1 open reading frame with the NATMX4 cassette
(Goldstein and Mccusker 1999). The Ty912D35-lacZThis4
reporter gene is a derivative of the Ty912D44-lacZThis4 allele
described previously (Dudley et al. 1999). The techniques
used for mating, transformation, sporulation, and tetrad analysis have been described previously (Rose et al. 1990). Detailed
descriptions of rich yeast extract-peptone-dextrose (YPD),
synthetic dextrose (SD), synthetic complete (SC), omission
(SC), 5-fluoroorotic acid (5-FOA), galactose, and sporulation
media are presented elsewhere (Rose et al. 1990). Selection for
cells containing the NATMX cassette was done on YPD media
containing 100 mg/ml clonNAT (Werner BioAgents, Jena,
Germany), whereas selection for kanamycin-resistant cells was
performed using media containing 200 mg/ml of active G418
(Sigma, St. Louis). Media containing canavanine (SC 1 Can)
contained 50 mg/ml canavanine.
Plasmids: pDM9 is a centromeric, URA3-marked plasmid
harboring the HHT1-HHF1 region. pDM18 is a centromeric, TRP1-marked plasmid carrying the HHT2-HHF2 region.
pAAD11 is a centromeric, TRP1-marked plasmid carrying the
hht2-11-HHF2 region. The details on the construction of these
three plasmids have been presented previously (Duina and
Winston 2004). The generation of pCC58, a URA3-marked
centromeric plasmid containing a 4.6-kb restriction fragment
that includes the SPT16 locus, has been described elsewhere
(Malone et al. 1991).
Identification and analysis of the SPT16-790 and SPT16-857
mutations: Mutations that suppress the H3-L61W Cs phenotype were isolated by replica plating patches of either strain
yAAD108 or strain yAAD476 grown on YPD plates at 30 to
fresh YPD plates and incubated at 14 for several weeks.
Papillae originating from these patches were isolated and further analyzed. The two intragenic mutations and the SPT16790 mutation were derived from strain yAAD108, whereas the
tco89 and SPT16-857 mutations were derived from strain
yAAD476. Thus, the SPT16-790 and SPT16-857 alleles (strains
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A. A. Duina et al.
TABLE 2
Dominance analysis of SPT16-790 and SPT16-857
Relevant genotypea
SPT16/SPT16
SPT16/SPT16-790
SPT16/SPT16-857
Spt phenotypeb
Cs phenotypec
1
/1
1
1
a
Shown are the SPT16 alleles for each test. For testing the
Spt phenotype, the diploid strains were also homozygous for
(hht2-hhf2)D and for lys2-128d. The SPT16-790 and SPT16-857
mutations confer an Spt phenotype only in the context of
reduced levels of histones H3 and H4. For testing the Cs
phenotype, the diploid strains were also homozygous for
(hht1-hhf1)D and hht2-11. See materials and methods for additional details on the strains used in these tests.
b
The Spt phenotype was determined with respect to suppression of the insertion mutation lys2-128d (Simchen et al. 1984).
An Spt1 phenotype indicates lack of suppression (Lys) and an
Spt phenotype indicates suppression (Lys1).
c
The Cs phenotype was determined by incubation of plates
at 14.
yAAD587 and yAAD482) are of independent origin. Initial
evidence that SPT16-790 and SPT16-857 represent mutations
in a single gene was obtained by the observation that the Cs1
phenotype segregated 2:2 in tetrads from crosses between
strains yAAD587 and yAAD482 with strain yAAD563. The
strains used to determine the dominant Spt phenotypes
conferred by SPT16-790 and SPT16-857 shown in Table 2 were
obtained by mating the following strains: SPT16/SPT16 ¼
yAAD1119XyAAD1159, SPT16/SPT16-790 ¼ yAAD1135
XyAAD1159, and SPT16/SPT16-857 ¼ yAAD1118XyAAD1159.
The strains used to determine the dominant suppression of
the H3-L61W Cs phenotype by the SPT16-790 and SPT16-857
alleles shown in Table 2 were obtained by mating the following
strains: SPT16/SPT16 ¼ yAAD1049XyAAD1048, SPT16/SPT16790 ¼ yAAD1049XyAAD1062 (yAAD1062 has the same relevant genotype as strain yAAD1127 in Table 1), and SPT16/
SPT16-857 ¼ yAAD1061XyAAD1048.
As the hht2-11 suppressors are dominant, we first determined their genetic map position using a modified systematic
genetic analysis (SGA) approach with the ordered array of
yeast deletion mutants described previously (Tonget al. 2001).
For this analysis, we took advantage of the observation that
both mutations also confer a strong Spt phenotype (growth
on SC Lys media in a lys2-128d background) in strains that
contain a deletion of the HHT1-HHF1 locus. We used this
phenotype to test linkage between one of the suppressor
mutations (now referred to as SPT16-857) and each strain in
the deletion set.
For these experiments we made lawns of yeast cells on YPD
plates using strain yAAD1046 and mated these cells to the yeast
deletion set strains (MATa his3D1 leu2D0 ura3D0 met15D0
orfDTKANMX) by replica plating. The diploids resulting from
these crosses were selected on diploid-selective media (SC
Trp Ura). The resulting diploids were stimulated to undergo meiosis by replica plating to sporulation media. Sporulated cultures were replica plated to SC His Arg
1canavanine to select for recombinant MATa spores as the
can1DTMFA1pr-HIS3 reporter gene present in yAAD1046
results in expression of HIS3 exclusively in haploid MATa cells.
Recombinant MATa spores were then replica plated to SC His
Arg Leu Ura 1canavanine 1G418 to select for those
spores containing the (hht1-hhf1)DTLEU2, lys2-128d (which is
linked to the tkl2DTURA3 allele), and orfDTKANMX alleles.
Finally, the selected cells were replica plated to SC His Arg
Leu Ura Lys 1Can 1G418 to determine their Spt
phenotype, which would in turn allow us to monitor for the
presence of the suppressor mutation. This analysis resulted in
the identification of a region on chromosome VII that showed
genetic linkage to the suppressor mutation. The identification
of the exact location and nature of the two suppressor mutations was carried out as described in the results section.
Reconstitution of the SPT16-857 allele: To create a strain
containing a newly generated SPT16-857 allele, a 746-bp PCR
product (from position 12083 to 12829 within the SPT16
open reading frame) containing the base-pair substitution
G2569C amplified from an SPT16-857 strain was cloned into
the integrative yeast plasmid pRS406 (Brachmann et al. 1998).
The resulting plasmid was analyzed by DNA sequencing to
ensure that the SPT16 fragment cloned into it contained only
the desired mutation. The plasmid was then linearized using
the HpaI restriction enzyme and transformed into a homozygote SPT16 wild-type diploid strain. The previously described integration and excision gene-replacement method (Rothstein
1991) was then used to generate a strain heterozygous for the
SPT16-857 allele. In subsequent genetic analyses, the newly
generated SPT16-857 allele showed suppression of H3-L61W
phenotypes.
RNA analysis: Northern blot analyses of FLO8 transcripts
were performed as described previously (Kaplan et al. 2003).
Briefly, total RNA from logarithmically growing cells was
collected using the hot- phenol method (Ausubel et al. 1991)
on a 1% agarose gel and transferred to a nylon membrane.
Hybridizations were performed using a radioactively labeled
FLO8 probe specific to a region toward the 39 end of the
transcript (from position 11595 to 12349).
Chromatin immunoprecipitation experiments: Most chromatin immunoprecipitation (ChIP) experiments were performed as previously described (Martens and Winston
2002). Spt16 ChIP assays were conducted using a rabbit polyclonal antibody specific for Spt16 (a gift from Tim Formosa),
the Rpb3 ChIP data were obtained using a Rpb3-specific mouse
monoclonal antibody (Neoclone, no. W0012), and the histone
H3 ChIP experiments were performed using a rabbit polyclonal antibody specific for histone H3 (AB1791, Abcam). The
dependence of the ChIP signals on the specific antibody used
was confirmed by performing mock ChIP experiments in the
absence of antibody. As a control, an untranscribed region of
the genome was assayed (Komarnitsky et al. 2000). In the
ChIP experiments presented in Figure 5, cultures were grown
in either glucose or galactose media overnight as indicated and
the DNAs were quantified by qPCR using the Light Cycler 480
and the qPCR kit (Light Cycler 480 Cyber Green I Master;
Roche, Indianapolis). For each DNA, the reaction was done in
duplicate. Prior to their use in the qPCR the primers were
analyzed for linearity, range, and efficiency. The primer pairs
used for the experiments presented in Figure 5 are as follows:
GAL1 59ORF and 39-UTR, OAD306–OAD307 and OAD345–
OAD346, respectively; GAL2 59ORF and 39-UTR, AO369–
AO370 and OAD347–OAD348, respectively; and PMA1 59ORF
and 39-UTR, F01810–FO1811 and FO1820–FO1821, respectively. The sequences for these and for all other primers used in
this work are listed in supplemental online material at http://
www.genetics.org/supplemental/.
RESULTS
A functional interaction between histone H3 and
Spt16 revealed through the isolation of extragenic
suppressors: Our studies began with the isolation of
Histone H3–Spt16 Interactions
Figure 1.—Analysis of new SPT16 mutants. (A) Suppression of the hht2-11 phenotypes by the SPT16 mutations.
The strains used were as follows: wild type (yAAD1052),
hht2-11 (yAAD1048), hht2-11 SPT16-790 (yAAD1063), and
hht2-11 SPT16-857 (yAAD1061). Note that each of the strains
also harbors a deletion of the HHT1-HHF1 locus such that the
HHT2 allele provides the sole source of histone H3 (see Table
1). Approximately 105 cells were spotted on YPD-based media
and 104 cells on SD-based media. The plates were incubated
at either 30 or 14 (where indicated) for the following times:
YPD 30, 2 days; YPD 14, 14 days; YPD 1 formamide (form), 4
days; SD, 2 days; and SD with no inositol (SD-ino), 3 days. (B)
Nature and location of the amino acid changes present in the
mutant Spt16 proteins. Shown is an alignment of several
amino acids within a region spanning residues 785–862 of
the S. cerevisiae Spt16 protein with those present within the
corresponding region of Spt16 homologs from several other
organisms. Each of the Spt16 mutants described in this study
harbors a single amino acid substitution (indicated in boldface type). In both cases, the change is from a highly conserved acidic residue to a nonacidic amino acid (see the
boxed residues). Note that the two mutations are relatively
near each other, possibly defining a domain involved in functional interactions with histone H3.
suppressors of hht2-11, which encodes the histone H3L61W mutant. This mutation was previously shown to
cause several phenotypes, including a defect in the
ability of the Swi/Snf chromatin-remodeling complex
to interact normally with gene promoters (Duina and
Winston 2004). The hht2-11 mutant also has several
other phenotypes, including an inability to grow at 14
(Cs phenotype), suppression of transcription defects
resulting from insertion of Ty elements and Ty longterminal repeats into certain genes (Spt phenotype),
sensitivity to formamide (For s phenotype), and the
inability to grow in the absence of inositol (Ino
phenotype) (Figure 1A and Duina and Winston 2004).
To further characterize the Cs phenotype, we selected for spontaneous suppressors that allowed hht2-11
105
cells to grow at 14 (see materials and methods). Of
five isolates chosen for further characterization, two
were found to harbor intragenic suppressor mutations,
both resulting in a change of the H3-W61 residue
encoded by the hht2-11allele to a cysteine (H3-W61C).
The remaining three suppressors were found to be
unlinked to the hht2-11 gene. One of these isolates was
found to harbor a recessive mutation in TCO89, a gene
encoding a subunit of the TOR complex 1 (Reinke et al.
2004) and was not characterized further. The other two
suppressor mutations were analyzed extensively and are
the focus of the studies presented here. These two
mutations were shown to suppress a subset of hht2-11
phenotypes (Figure 1A). Whereas neither suppressor
mutation displays any detectable mutant phenotype in
an otherwise wild-type background, both mutations confer a strong Spt phenotype in the context of reduced
intracellular levels of histones H3 and H4 (data not
shown). Both mutations were shown to segregate 2:2,
indicating that each is caused by a single mutation, and
each one was shown to be dominant (Table 2). Furthermore, these two suppressor mutations were shown to be
tightly linked to each other (data not shown), suggesting that they are in the same gene.
The gene identified by these dominant suppressor
mutations was determined in a series of steps beginning
with linkage analysis. To do this, we crossed one of the
suppressor mutants to a yeast strain set containing
deletions in all of the nonessential genes, testing for
linkage between each individual deletion and the
suppressor mutation (see materials and methods).
These crosses defined a genomic region on chromosome VII. The initial crosses were followed by additional
crosses to refine the position of the mutations, and then
by DNA sequence analysis of several genes in this region.
These experiments led to the discovery that each of the
two suppressor mutations consists of a distinct singlebase-pair change in the SPT16 gene, each predicted
to cause a different single amino acid change in the
carboxyl-terminal region of the protein, E790K and
E857Q (Figure 1B). Evidence that these alleles are
responsible for the suppression of H3-L61W phenotypes came from the demonstration that a reconstituted
SPT16-857 allele (see materials and methods) confers
suppression of hht2-11 phenotypes (data not shown)
and that a perfect correlation between the suppression
phenotypes and the presence of the corresponding
SPT16 allele was observed in all the strains tested. As
shown in Figure 1B, both suppressor mutations are
predicted to affect highly conserved acidic residues. The
fact that the two affected residues are relatively close to
each other suggests that they define an Spt16 domain
important for functional interactions with histone H3.
Interestingly, both of these residues are located in a
region of Spt16 previously shown to be a distinct
structural domain of the protein (O’Donnell et al. 2004;
Vandemark et al. 2006), and a different amino acid
106
A. A. Duina et al.
Figure 2.—Molecular and genetic evidence
that H3-L61W affects transcription elongation.
(A) Northern analysis of the FLO8 RNA transcripts in H3 WT SPT16 (yAAD1052), H3 WT
SPT16-857 (yAAD1121), H3 WT SPT16-790
(yAAD1134), H3-L61W SPT16 (yAAD1049), H3L61W SPT16-857 (yAAD1061), and H3-L61W
SPT16-790 (yAAD1128) strains. In the top section, the top arrow indicates the location of the
full-length transcript and the bottom arrow indicates the location of the short transcript. The intensities of the rRNA bands (bottom section)
reflect the amount of total RNA loaded per sample. The data shown are representative of two independent experiments for all of the strains
tested except for the H3-L61W SPT16 and H3L61W SPT16-857 strains, which have been tested
in three independent experiments, and the H3
WT SPT16-790 strain, which has been tested in
a single experiment. The samples shown were analyzed on the same gel; the order of the lanes has
been rearranged to clarify the presentation. (B)
H3-L61W cells (strain yAAD1049) were spotted in a 10-fold dilution series from 2.8 3 108 cells/ml to 2.8 3 105 cells/ml (4 ml
per spot) onto either synthetic complete (SC) medium (top) or SC medium containing 20 mg/ml MPA (bottom) and incubated
at 14 for 28 days. (C) Approximately 1.8 3 106 cells with the indicated genotypes were spotted on either SC media lacking uracil
(SC Ura) or 5-FOA media (SC 1FOA) and incubated for 3 days at 30 (the hht2-11 allele encodes the H3-L61W mutant). Strains
whose genotypes include pHHT1 contain a URA3-marked plasmid carrying a wild-type copy of the HHT1 gene to cover for the hht211 mutation, whereas the strain whose genotype includes pSPT16 (second row) contain a URA3-marked plasmid expressing wildtype Spt16 to cover for the spt16-197 mutation. Before spotting, the cells were grown on nonselective media (YPD) to allow strains
containing nonlethal genomic mutations to lose the plasmids and propagate. Inability to grow on 5-FOA media indicates synthetic
lethal mutant combinations. Refer to Table 1 for the full genotypes of the strains used in these experiments (yAAD2006,
yAAD2000, yAAD2005, yAAD2013, yAAD2015, and yAAD994).
change at position 857 (E857K) has been previously shown
to affect Spt16 function in vivo (O’Donnell et al. 2004).
The SPT16 mutations isolated in this study are likely
to encode gain-of-function versions of Spt16 as both are
dominant for suppression of hht2-11 and for the Spt
phenotype in the context of reduced levels of intracellular histones H3 and H4 (Table 2). In sharp contrast
to the suppression effects shown by these novel spt16
mutations, a partial loss-of-function mutation in SPT16
is synthetically lethal in combination with the hht2-11
mutation (see below). The fact that the genes encoding
histone H3 and Spt16 display allele-specific interactions
supports the notion that the functions of these proteins
are intimately related to each other.
Additional evidence that hht2-11 affects transcription
elongation: The genetic interaction between hht2-11
and the SPT16 suppressors suggests that one of the
defects conferred by the histone mutant is at the level of
transcription elongation. To obtain additional evidence
that hht2-11 affects transcription elongation, we took
advantage of a recently developed assay that relies on
the finding that mutations that impair many elongation
factors, including Spt6 and Spt16, result in transcription
initiation from cryptic promoters within the coding
region of genes (Kaplan et al. 2003; Mason and Struhl
2003). These findings are consistent with the generally
accepted notion that one role for elongation factors is to
remove the chromatin barrier in front of Pol II to
facilitate transcription elongation, and a second role is
to reestablish proper chromatin structure in the wake of
Pol II passage. Defects in this process would result in the
inability to re-form proper nucleosomal structures
during transcription elongation, thereby allowing use
of cryptic promoters. To test for this defect in hht2-11
mutants, we examined transcription of FLO8, a gene
characterized as having a cryptic promoter (Kaplan
et al. 2003). Our results show that hht2-11 mutants
indeed produce a FLO8 cryptic transcript (Figure 2A,
lane 4). In contrast, the SPT16-857 and SPT16-790 mutations do not cause cryptic initiation at FLO8 in a histone
H3 wild-type background (Figure 2A, lanes 2 and 3). At
the permissive temperature of 30, the SPT16-857 mutation, but not the SPT16-790 mutation, causes partial
suppression of the FLO8 cryptic initiation phenotype
seen in hht2-11 cells (Figure 2A, lanes 5 and 6). However,
partial suppression of this hht2-11 phenotype by both
SPT16-857 and SPT16-790 was observed after a shift to
the nonpermissive temperature of 14 for 24 hr (data
not shown). These results support the notion that the
change in histone H3, H3-L61W, causes transcriptional
elongation defects and that these defects can be suppressed by the SPT16 alleles we have isolated.
A series of genetic experiments also suggests that hht211 affects transcription elongation. In one set of experiments we tested the effects of the drugs mycophenolic
acid (MPA) and 6-azarucil (6-AU), both of which are
thought to affect transcription elongation by reducing
the intracellular pools of nucleotide precursors (Hampsey
Histone H3–Spt16 Interactions
1997). We found that the Cs phenotype of H3-L61W cells
is partially suppressed by the inclusion of either MPA (Figure 2B) or 6-AU (data not shown) in the growth media.
This phenotype is similar to that previously reported for
spt5-242 mutants, whose Cs phenotype was also suppressed by 6-AU (Hartzog et al. 1998). As previously
proposed for spt5-242 (Hartzog et al. 1998), a plausible
interpretation of our observations is that slowing down
transcription elongation through the addition of MPA
or 6-AU to deplete intracellular pools of nucleotide precursors might provide cells with sufficient time to overcome the elongation defects conferred by the H3-L61W
mutant. Regardless of the exact mechanism, the finding
that MPA and 6-AU suppress the H3-L61W Cs phenotype is consistent with the hypothesis that H3-L61W affects transcription elongation.
In addition, we tested for genetic interactions between hht2-11 and mutations in genes encoding known
elongation factors by construction and analysis of
double mutants. Our results show that the hht2-11 mutation causes inviability when combined with partial lossof-function mutations in either SPT16 or SPT6, or a null
mutation in SPT4 (Figure 2C). Since synthetic lethal
interactions between two factors are usually indicative of
a functional relationship between them (Prelich 1999),
these data support the idea that H3-L61W causes defects
in transcription elongation. Interestingly, deletion of
DST1, the gene encoding the general elongation factor
TFIIS, only mildly impairs growth when combined with
hht2-11 (Figure 2C), showing that hht2-11 does not indiscriminately interact with mutations in all elongationfactor genes. As previously shown, a mutation that removes
the Snf2 component of the transcription-initiation complex Swi/Snf does not result in significant synthetic
growth defects in the context of H3-L61W (Figure 2C
and Duina and Winston 2004). These results provide
genetic evidence in support of the notion that hht2-11
confers transcription elongation defects.
H3-L61W affects localization of Spt16 across transcribed genes: The allele-specific interactions uncovered between hht2-11 and SPT16 mutations suggest that
the functions of Spt16 and those of histone H3 are
closely related. One interpretation of our data is that
H3-L61W-containing nucleosomes are defective for
functional interactions with Spt16 and that certain
mutations in Spt16 can alleviate these defects. To learn
more about possible effects of H3-L61W on Spt16
functions, we used ChIP assays to examine whether
Spt16 association along transcribed genes is affected by
the presence of the H3-L61W mutant.
In the first set of experiments, the distribution of
Spt16 over the highly transcribed gene PMA1 was
compared between an H3 wild-type strain and an hht211 mutant. Our results demonstrate that H3-L61W
dramatically alters the distribution of Spt16 over the
PMA1 gene. Whereas Spt16 is localized predominantly
over the PMA1 coding region in a wild-type strain,
107
(Figure 3A, black bars, and see Mason and Struhl
2003; Kim et al. 2004), Spt16 accumulates toward the 39UTR in the hht2-11 mutant (Figure 3A, red bars). In
contrast, hht2-11 does not cause an altered distribution
of RNA Pol II along PMA1 (Figure 3B), suggesting that
H3-L61W does not generally affect the distribution of
transcription factors across transcribed regions. These
latter observations are consistent with our data that hht211 causes only minor effects on PMA1 mRNA levels
(Duina and Winston 2004 and data not shown). To test
if the allele-specific interactions between hht2-11 and the
SPT16 suppressor mutations were reflected by this assay,
we also did ChIP experiments on the Spt16 mutant
proteins. Interestingly, the defect in Spt16 distribution
seen in the hht2-11 mutant is significantly suppressed
when cells express either one of the two Spt16 mutants
described above (Figure 3A, green and yellow bars). To
determine if the defect conferred by H3-L61W on Spt16
localization is general, we tested three additional highly
transcribed genes. All three showed very similar patterns
to what was observed for PMA1 (Figure 4, A–C). Thus,
the effects of H3-L61W on the localization of Spt16
across genes and the suppressive effects of the Spt16
mutants are not confined to the PMA1 locus but appear
to be widespread across the genome.
To assess whether the accumulation of Spt16 at 39UTRs in hht2-11 cells is dependent upon transcription,
we performed ChIP experiments to determine the levels
of Spt16 occupancy at the GAL1 and GAL2 genes under
conditions in which these genes are either repressed
(glucose medium) or expressed (galactose medium). As
shown in the top two sections of Figure 5, abnormally
high levels of Spt16 are detected in the 39-UTRs of both
of these genes in hht2-11 cells only when cells were
grown under inducing conditions. In contrast, the
Spt16-39-UTR accumulation phenotype in hht2-11 cells
is observed at the PMA1 gene regardless of the carbon
source present in the growth media (Figure 5, bottom
section). These results strongly suggest that the abnormal accumulation of Spt16 at 39-UTRs is dependent
upon active transcription.
H3-L61W does not greatly affect the distribution of
nucleosomes across transcribed genes: Our studies
have shown that H3-L61W causes Spt16 to accumulate
over the 39-UTRs of transcribed genes. In an effort to
investigate the molecular mechanisms underlying this
effect, we developed two testable models that would be
consistent with these observations. For the first model,
we envision Spt16 as able to normally traverse the length
of transcribed genes in the context of H3-L61W, but
unable to dissociate from the 39-UTRs. In this model,
Spt16 normally receives a signal that causes departure
from the 39 end of genes once the transcription process
is terminated; the H3-L61W mutant might somehow
interfere with this signal, resulting in the accumulation
of Spt16 at the 39 ends of genes. For the second model,
we envision Spt16 as unable to properly remove and
108
A. A. Duina et al.
Figure 3.—Analyses of the association of Spt16
and RNA Pol II across the PMA1 gene in the context of wild-type or mutant versions of histone H3
and Spt16. (A) H3-L61W causes Spt16 to accumulate at the 39-UTR of PMA1, and the two
Spt16 mutants isolated in this study alleviate this
defect. ChIP experiments were performed on
H3 WT (yAAD1052 and yAAD1053), H3-L61W
(yAAD1048 and yAAD1049), H3-L61W Spt16E857Q (yAAD1060 and yAAD1061), and
H3-L61W
Spt16-E790K
(yAAD1127
and
yAAD1128) using an Spt16-specific polyclonal antibody (see materials and methods). Nine
primer sets were used to assess Spt16 binding
across the PMA1 ORF (shaded area) as well as
within the 59- and 39-UTRs in the different genetic backgrounds. The percentage of immunoprecipitation values (%IP) indicated in the bar
graphs for each of the strains at each region
across the PMA1 locus are relative to the corresponding input material. Each %IP value represents the average with standard error from three
independent experiments. As a reference point,
the average levels of Spt16 binding to an untranscribed region (see materials and methods)
were as follows: H3 WT, 0.22% 6 0.006; H3L61W, 0.45% 6 0.009; H3-L61W Spt16-E857Q,
0.47% 6 0.007; and H3-L61W Spt16-E790K,
0.38% 6 0.008. (B) H3-L61W does not appear
to greatly affect the distribution of RNA Pol II
across the PMA1 locus. ChIP experiments were performed on H3 WT (yAAD1052) and H3-L61W
(yAAD1049) strains using a monoclonal antibody
specific for the Rpb3 subunit of RNA Pol II (see
materials and methods). The two regions
assayed are indicated above the PMA1 locus
diagram. The %IP values are relative to the input material and in each case represent the average of two independent experiments. As a reference point, the average levels of Rpb3 binding to the untranscribed region were as follows: H3 WT, 0.09% 6
0.01; and H3-L61W, 0.10% 6 0.01. For both sets of experiments, the analysis and quantitation of the signals were carried out
using the method described by Martens and Winston (2002) (see materials and methods).
redeposit histones in the context of H3-L61W during
the elongation process. This defect might in turn result
in the ‘‘sliding’’ and accumulation of nucleosomes
toward the 39 ends of genes as transcription proceeds.
If this were to take place, Spt16 might also be expected
to accumulate at the 39 ends of genes since its substrate,
the nucleosomes, has been pushed toward the 39-UTRs.
To differentiate between these two models, we performed ChIP experiments to determine the distribution
of nucleosomes across the PMA1 gene in wild-type and
hht2-11 strains. For these studies, we used a histone-H3specific antibody. We note that this antibody was raised
against a peptide encompassing residues 124–135 of
human histone H3 (Rao et al. 2005) and therefore
should still recognize the H3-L61W mutant protein. Importantly, the levels of histone H3 protein in wild type
and the hht2-11 mutant are similar as measured by
Westerns using this antibody (data not shown). The
results from the histone-H3-ChIP experiments show
that H3-L61W has only a minor effect on the nucleosomal distribution across the PMA1 gene, resulting in a
slight increase in the concentration of nucleosomes in
the 39-UTR when compared to wild-type cells (Figure 6).
This minor effect is not expected to be the cause of
Spt16-39-UTR accumulation in H3-L61W cells, since in
these cells we see at least a 15-fold increase in Spt16-39UTR occupancy compared to wild-type cells. These data
are therefore consistent with the first model, raising the
possibility that the H3-L61W mutation interferes with a
signal that normally causes Spt16 to dissociate from the
39-UTR of transcribed genes.
DISCUSSION
In this work we provide evidence indicating that the
integrity of histone H3 is crucial for the normal association of Spt16 along transcribed regions. We have
found that a single amino acid substitution, L61W,
within the globular domain of H3, results in a dramatic
transcription-dependent accumulation of Spt16 at the
39-UTRs of transcribed genes. H3-L61W appears to
affect Spt16 localization in a general fashion, as all the
transcribed genes analyzed thus far are affected in a
similar way by this mutant. Furthermore, two specific
Histone H3–Spt16 Interactions
109
Figure 4.—H3-L61W affects Spt16 binding
across three other transcribed genes and the
Spt16 mutants partially suppress these defects.
ChIP experiments were performed as described
in Figure 3A, except that the genomic regions
tested correspond to the 59 ORF regions and
39-UTRs across the (A) ADH1 locus, (B) FBA1 locus, and (C) PDC1 locus. Each %IP value represents the average with standard error from
three independent experiments.
amino acid changes in Spt16, E790K, and E857Q, partially compensate for the H3-L61W defect. Taken together, these results suggest that a direct Spt16–H3
interaction is required for normal localization of Spt16
on chromatin.
Our results, combined with those from a previous
study (Duina and Winston 2004), suggest a working
model for the role of the Spt16–H3 interaction. Analysis
of the predicted structure of a nucleosome containing
H3-L61W suggests that the L61W substitution may lead
to increased H3–H4 tetramer stability, potentially leading to stabilization of the H3 N-terminal helix (Duina
and Winston 2004). This stabilization, in turn, might
prevent the propagation of a structural change within
nucleosomes, such as histone modifications or nucleosome remodeling, that might normally serve as a signal
Figure 5.—The accumulation of Spt16 at the
39-UTRs of the inducible genes GAL1 and
GAL2 is dependent upon transcription. Spt16
ChIP experiments were carried out on H3 WT
(yAAD 1053) and H3-L61W (yAAD1048) strains
grown in the presence of either glucose (repressive condition) or galactose (activating condition). Two primer sets per gene were used to
assess Spt16 binding to the 59 ORF and to the
39-UTR regions of the GAL1, GAL2, and PMA1
genes. The analysis and quantitation of the signals were performed using real-time PCR technology (see materials and methods). The
%IP values are relative to the corresponding input material and represent the average with standard error from three independent clones. Note
that accumulation of Spt16 in the 39-UTR of the
constitutively expressed gene PMA1 (bottom) is
independent of the carbon source. As a reference
point, in these experiments the average levels of
Spt16 binding to the control untranscribed region were as follows: H3 WT (glucose), 0.49% 6
0.09; H3 WT (galactose), 0.50% 6 0.09; H3L61W (glucose), 1.21% 6 0.13; and H3-L61W
(galactose) 1.15% 6 0.11.
110
A. A. Duina et al.
Figure 6.—H3-L61W does not appear to greatly affect the
distribution of nucleosomes across the PMA1 gene. ChIP
experiments were performed as described in Figure 3A, except that the immunoprecipitations were performed with a
histone-H3-specific antibody (see materials and methods).
Each %IP value represents the average with standard error
from three independent experiments. As a reference point,
the average levels of histone H3 binding to the control untranscribed region were as follows: H3 WT, 2.4% 6 0.32;
H3-L61W, 4.10% 6 0.57. As anticipated, the %IP of histone
H3 at the untranscribed region was relatively high since nucleosomes are present throughout the genome.
for the departure of Spt16 from 39-UTRs after transcription has terminated. Thus, according to this model,
enrichment of Spt16 at 39-UTRs in H3-L61W cells would
be a result of the inability of a ‘‘39-UTR-dissociation
signal’’ to be properly transmitted from the nucleosome
to Spt16. In the context of this model, the amino acid
changes in Spt16 identified here might mediate their
suppressive effects by rendering Spt16 more sensitive to
the weakened 39-UTR dissociation signal in H3-L61W
cells, thereby allowing for more efficient departure of
Spt16 from 39-UTRs in the H3-L61W background.
Our studies provide insights into a general process
that has not been extensively addressed to date: the mechanisms that regulate the dissociation of transcriptionelongation factors during or following gene transcription.
As Pol II travels across the polyadenylation [poly(A)]
sites of transcribed genes, exchange of certain elongation factors (e.g., the PAF and TREX complexes) for
poly(A) factors occurs, while other elongation factors,
including the FACT complex, appear to depart from
genes along with Pol II downstream of the poly(A) sites
(Kim et al. 2004). Whereas it has become clear that processing of nascent RNA transcripts is coupled to transcription termination and the eventual departure of Pol
II from transcribed units, the exact molecular mechanisms responsible for the coupling of these processes
have yet to be completely understood. Similarly, the
processes that control the dynamic exchange of elongation and termination factors on the transcription mach-
inery as it travels across genes are not well- characterized.
The finding that H3-L61W appears to prevent normal
dissociation of Spt16 from transcribed genes, while
apparently not affecting Pol II, raises the possibility that
the signals required for dissociation of Pol II and Spt16
(and perhaps other factors) from 39-UTRs are at least to
some degree separable.
Future studies will take advantage of the experimental
system described in this work to learn more about the
dynamic interactions that occur between chromatin and
Spt16, as well as chromatin and other elongation factors,
in vivo. Experiments to determine the breadth of the
effects of H3-L61W on chromatin association of other
factors will provide important information about the
specificity of the defects associated with this histone
mutant. In addition, isolation of other mutations, both
in histone and in nonhistone proteins, that prevent
proper dissociation of Spt16 from 39-UTRs will provide
insights into the nature of the putative signaling pathway
that regulates Spt16 dissociation from chromatin following transcription.
We are grateful to Mark Hickman and Reine Protacio for helpful
comments on the manuscript and Kacey Swindle for technical assistance. We thank Tim Formosa for providing the anti-Spt16 antibodies.
A.A.D. is grateful to Patricia Wight and members of her laboratory in
the Department of Physiology and Biophysics at the University of
Arkansas for Medical Sciences for providing assistance in the performance of part of this work. This material is based upon work supported
by the National Science Foundation under grant 0543412, the Charles
A. King Trust Fellowship from The Medical Foundation, National Institutes of Health (NIH) grants P20RR16460-03 and P20RR16460-03,
ARIA 0711-03 from the IDeA Networks of Biomedical Research Excellence (INBRE), Program of the National Center for Research
Resources, and start-up funds and a faculty project grant from Hendrix
College to A.A.D. This work was also supported by NIH grant GM32967
to F.W. and a Canadian Institutes of Health Research grant to A.N. A.N.
holds a Tier 2 Canadian Research Chair.
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Communicating editor: M. D. Rose