The SET Domain Protein, Set3p, Promotes the Reliable Execution of Cytokinesis in Schizosaccharomyces pombe

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DOI: 10.1371/journal.pone.0031224 · Source: PubMed
Abstract
In response to perturbation of the cell division machinery fission yeast cells activate regulatory networks that ensure the faithful completion of cytokinesis. For instance, when cells are treated with drugs that impede constriction of the actomyosin ring (low doses of Latrunculin A, for example) these networks ensure that cytokinesis is complete before progression into the subsequent mitosis. Here, we identify three previously uncharacterized genes, hif2, set3, and snt1, whose deletion results in hyper-sensitivity to LatA treatment and in increased rates of cytokinesis failure. Interestingly, these genes are orthologous to TBL1X, MLL5, and NCOR2, human genes that encode components of a histone deacetylase complex with a known role in cytokinesis. Through co-immunoprecipitation experiments, localization studies, and phenotypic analysis of gene deletion mutants, we provide evidence for an orthologous complex in fission yeast. Furthermore, in light of the putative role of the complex in chromatin modification, together with our results demonstrating an increase in Set3p levels upon Latrunculin A treatment, global gene expression profiles were generated. While this analysis demonstrated that the expression of cytokinesis genes was not significantly affected in set3Δ backgrounds, it did reveal defects in the ability of the mutant to regulate genes with roles in the cellular response to stress. Taken together, these findings support the existence of a conserved, multi-protein complex with a role in promoting the successful completion of cytokinesis.
The SET Domain Protein, Set3p, Promotes the Reliable
Execution of Cytokinesis in
Schizosaccharomyces pombe
Stefan Rentas
1
, Reza Saberianfar
1
, Charnpal Grewal
1
, Rachelle Kanippayoor
1
, Mithilesh Mishra
2
, Dannel
McCollum
3
, Jim Karagiannis
1
*
1 Department of Biology, University of Western Ontario, London, Ontario, Canada, 2 Temasek Life Sciences Laboratory, The National University of Singapore, Singapore,
Singapore, 3 Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America
Abstract
In response to perturbation of the cell division machinery fission yeast cells activate regulatory networks that ensure the
faithful completion of cytokinesis. For instance, when cells are treated with drugs that impede constriction of the
actomyosin ring (low doses of Latrunculin A, for example) these networks ensure that cytokinesis is complete before
progression into the subsequent mitosis. Here, we identify three previously uncharacterized genes, hif2, set3, and snt1,
whose deletion results in hyper-sensitivity to LatA treatment and in increased rates of cytokinesis failure. Interestingly, these
genes are orthologous to TBL1X, MLL5, and NCOR2, human genes that encode components of a histone deacetylase
complex with a known role in cytokinesis. Through co-immunoprecipitation experiments, localization studies, and
phenotypic analysis of gene deletion mutants, we provide evidence for an orthologous complex in fission yeast.
Furthermore, in light of the putative role of the complex in chromatin modification, together with our results demonstrating
an increase in Set3p levels upon Latrunculin A treatment, global gene expression profiles were generated. While this
analysis demonstrated that the expression of cytokinesis genes was not significantly affected in set3D backgrounds, it did
reveal defects in the ability of the mutant to regulate genes with roles in the cellular response to stress. Taken together,
these findings support the existence of a conserved, multi-protein complex with a role in promoting the successful
completion of cytokinesis.
Citation: Rentas S, Saberianfar R, Grewal C, Kanippayoor R, Mishra M, et al. (2012) The SET Domain Protein, Set3p, Promotes the Reliable Execution of Cytokinesis
in Schizosaccharomyces pombe. PLoS ONE 7(2): e31224. doi:10.1371/journal.pone.0031224
Editor: Juan Mata, University of Cambridge, United Kingdom
Received December 1, 2011; Accepted January 4, 2012; Published February 8, 2012
Copyright: ß 2012 Rentas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Sciences and Engineering Research Council of Canada, the University of Western Ontario Academic
Development Fund, and the Canada Foundation for Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: jkaragia@uwo.ca
Introduction
The successful completion of cytokinesis requires the intricate
interplay of gene products ranging from signaling molecules to
elements of the cytoskeleton. Taken together these regulatory
mechanisms ensure that cytokinesis occurs in a faithful and
dependable manner once every cell cycle and over wide-ranging
growth conditions. In Schizosaccharomyces pombe, just as in more
developmentally complex eukaryotes, cytokinesis is critically
dependent on the assembly and constriction of a contractile
actomyosin ring [1,2]. The assembly of the ring occurs at the onset
of mitosis through the formation of cortical nodes comprised of
proteins with roles in nucleating and assembling actin filaments
[3]. Once assembled into a contractile ring, the constriction of the
filaments is controlled through the action of a regulatory module
referred to as the septation initiation network (SIN). The SIN is
comprised of a GTPase-regulated signaling cascade that is
involved in proper ring assembly, temporal co-ordination of ring
constriction, as well as the deposition of the division septum [4,5].
Given the importance of cytokinesis in cellular growth and
development, it is not surprising that evidence supporting the
existence of a cytokinesis monitoring system has emerged in S.
pombe. This monitoring system has the capacity to 1) halt cell cycle
progression at G2/M transition, and 2) stabilize the contractile
actomyosin ring upon perturbation of the cell division machinery
[6,7,8,9,10,11]. Together, these mechanisms ensure that cytoki-
nesis is complete before a new cell cycle begins. Important
regulators of the cytokinesis monitoring system include the SIN
itself, the Cdc14 family phosphatase, Clp1p, the 14-3-3 protein,
Rad24p, and the kinase, Lsk1p [8,9,10,12,13,14].
The critical role of these components can be observed
experimentally through the treatment of fission yeast cells with
low doses of the actin depolymerising drug, Latrunculin A (LatA)
[8,10,12,13,14,15]. At the concentrations used (20–50 times less
than that needed to completely depolymerize the actin cytoskel-
eton) such treatment leads to a Clp1p and Rad24p dependent
delay in mitotic entry and the extended activation of the SIN. This
leads to a prolonged cytokinesis-competent state that is charac-
terized by continuous repair and re-establishment of the
actomyosin ring [8,10,11,13]. Abrogation of this system in clp1D
or rad24D mutant backgrounds results in cytokinesis failure and
the subsequent generation of inviable, multi-nucleate cells [8,10].
In order to identify other genes with roles in promoting the
reliable execution of cytokinesis, a library of S. pombe gene deletion
mutants was screened for hyper-sensitivity to LatA (M. Mishra and
D. McCollum, unpublished). One of the identified genes defined
by the annotated S. pombe ORF, SPCC1235.09 encoded a WD
repeat protein orthologous to human TBL1X, which is known to
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form part of a histone-deacetylase complex with both the MLL5
and NCOR2 proteins [16]. Since knockdown of TBL1X, MLL5,or
NCOR2 results in cytokinesis failure in HeLA cells [16] we were
interested to determine if an orthologous complex might also exist
in fission yeast.
In this report we present the results of the molecular and genetic
analysis of the fission yeast orthologues of TBL1X, MLL5, and
NCOR2, and show that just like their human counterparts they
are indeed important for the reliable execution of cytokinesis.
Furthermore, in light of the predicted role of the complex in
chromatin modification, we performed global gene expression
profiling. While this analysis demonstrated that cytokinesis genes
were not significantly affected, it did reveal that the set3D mutant
was impaired in its ability to modulate the expression of stress
response genes. The relevance of these findings within the context
of understanding the relationship between cytokinesis failure,
aneuploidy, and cancer progression is discussed.
Results
The fission yeast Hif2p, Set3p, and Snt1p proteins share
sequence similarity with the human TBL1X, MLL5, and
NCOR2 proteins, respectively
To identify novel regulators of cytokinesis, a library of fission
yeast gene deletion mutants was screened for hypersensitivity to
LatA (M. Mishra and D. McCollum, unpublished). LatA
treatment results in the depolymerization of actin filaments
through the sequestration of actin monomers [15]. At low
concentration (0.2–0.5
mM, or approximately 20–50 times less
than that needed to completely depolymerize the actin cytoskel-
eton) LatA can be used to impede actomyosin ring constriction
and activate the cytokinesis monitoring system. For these reasons
LatA treatment can be used as a tool in genetic screens to identify
mutants defective in cytokinesis [8,10,12,13].
This reverse genetic approach identified a strain bearing a
deletion in the annotated open reading frame, SPCC1235.09,
which encodes a WD repeat domain protein (Figure 1). Reciprocal
BLAST searches revealed that the encoded gene product was the
likely orthologue of human TBL1X (19% identity and 58%
similarity; Figure S1). Interestingly, TBL1X exists in a histone
deacetylase complex together with the MLL5 and NCOR2
proteins [16]. Even more intriguing was the fact that knockdown
of either the TBL1X, NCOR2,orMLL5 genes in HeLa cells results
in increased rates of cytokinesis failure [16]. Furthermore, the
budding yeast orthologue of the WD repeat protein also exists in a
well characterized histone deacetylase complex [17]. This
suggested the existence of an evolutionarily conserved multi-
protein complex with a role in the faithful and reliable execution of
cytokinesis.
To identify potential S. pombe orthologues of MLL5 and
NCOR2, reciprocal BLAST searches were performed. This
analysis identified two open reading frames, SPAC22E12.11c
and SPAC22E12.19, which encode proteins with significant
similarity to human MLL5 and NCOR2, respectively (Figure 1;
Figure S1). We have named the SPCC1235.09, SPAC22E12.11c
and SPAC22E12.19 open reading frames, hif2, set3 (
set domain
containing), and snt1 (
sant domain containing), respectively.
A representation of the fission yeast Hif2p, Set3p, and Snt1p
proteins and their conserved domains are shown in Figure 1.
These domains typically function in chromatin remodeling. For
instance, the SET domain infers possible histone methyltransferase
(HMT) activity while the plant homeodomain zinc finger (PHD) is
thought to assist in protein interaction with nucleosomes [18,19].
The SANT domain is a motif found in numerous co-repressor
Figure 1. Domain structure of Set3p, Snt1p, and Hif2p. Structures are based upon Uniprot database predictions. Arabic numerals to the right
of each schematic indicate the length of the protein in amino acids. Arabic numerals below each schematic indicate the amino acid position of that
domain within the protein. Schematics are not drawn to scale.
doi:10.1371/journal.pone.0031224.g001
SET Domain Protein Regulates S. pombe Cytokinesis
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proteins and has been proposed to recognize unmodified histone
tails [20]. Lastly, the LisH domain has been shown to be important
in binding to hypoacetylated histone H4 tails [21].
hif2D, set3D and snt1D mutants display cytokinesis
defects upon perturbation of the cell division machinery
If Hif2p, Set3p, and Snt1p act as part of a protein complex with
a common function, then one would expect the respective loss of
function mutants to exhibit similar phenotypes. To determine if
this were the case, hif2D, set3D, and snt1D strains, as well as the
respective double and triple mutant strains, were grown to mid-log
phase in liquid YES at 30uC and 10-fold serial dilutions were
plated onto YES plates containing LatA or DMSO (solvent
control). Interestingly, all mutants displayed a reduced capacity for
growth in the presence of LatA in comparison to wild type
(Figure 2A).
Next, the cells were examined at the microscopic level to
determine if the observed sensitivity to LatA was related to defects
in cytokinesis. The deletion mutants were treated with LatA
(0.5
mM) or DMSO (solvent control) for 5 hours in liquid YES
growth medium, and subsequently fixed and stained with DAPI
and aniline blue to visualize the nucleus and cell wall/septum,
respectively [22]. The cells were classified into four different
phenotypic categories: i) uni-nucleate cells, ii) bi-nucleate cells with
a morphologically normal septum (i.e. the septum completely
bisects the cell), iii) bi-nucleate cells with a fragmented septum (i.e.
the septum is non-functional and does not completely bisect the
cell), and iv) tetra-nucleate cells.
As shown in Figure 2B hif2D, snt1D, set3D and wild-type strains
did not display any obvious growth defects when cultured in the
presence of DMSO. In contrast, large proportions of hif2D, snt1D,
and set3D mutant cells (49–75%) displayed a tetra-nucleate
phenotype when treated with 0.5
mM LatA, whereas only 11%
of wild type were tetra-nucleate (Figure 2B,C). Importantly, in
support of the hypothesis that the three gene-products work within
the same pathway, no significant synthetic effects were observed
when the respective double and triple gene deletion mutants were
assayed using only 0.1
mM LatA (Figure 2C). Moreover, genetic
analysis with strains bearing mutations in genes encoding various
cytokinesis regulators (myo2-E1, cdc12-112, cdc15-140) revealed
specific synthetic interactions between the set3, snt1, and hif2 gene
deletions and the temperature sensitive cdc15-140 mutation. The
presence of either the hif2D, snt1D,orset3D gene deletions reduced
the restrictive temperature of cdc15-140 strains to a similar extent
(but did not affect the restrictive temperatures of myo2-E1 or cdc12-
112 strains) (Figure S2). Taken together, the similarities in
phenotype observed in hif2D, snt1D, and set3D gene deletion
mutants are consistent with a model in which the three proteins
modulate a common biological process that influences the
successful completion of cytokinesis in S. pombe.
Set3p works in parallel to other regulators of the
cytokinesis monitoring system
The cytokinesis failure observed in hif2D, snt1D, and set3D
mutants was reminiscent of that seen in the previously character-
ized cytokinesis regulators, clp1D and lsk1D [8,13]. It was thus of
interest to determine if these proteins functioned within the same
pathway or in parallel to Clp1p (a Cdc14 family phosphatase) or
Lsk1p (a Ser-2 CTD kinase), respectively. If Set3p functioned in a
linear pathway with Clp1p or Lsk1p, one would expect set3D clp1D
and set3D lsk1D double mutants to exhibit similar hyper-sensitivity
to LatA relative to the respective single mutants. Conversely, if
Set3p functioned in parallel, one would expect set3D clp1D and
set3D lsk1D double mutants to exhibit a more severe sensitivity to
LatA than either of the respective single mutants. To differentiate
between these two possibilities, a wild-type strain, as well as the
respective single and double deletion mutants, were treated with
LatA (0.1
mM) or DMSO for 5 hours in liquid YES growth
medium. This was followed by fixation and staining with DAPI
and aniline blue to visualize the nucleus and cell/wall septum,
respectively. Interestingly, both set3D clp1D and set3D lsk1D double
mutants showed a significant increase in sensitivity to LatA
(Figure 3). At a concentration of 0.1
mM both double mutants
displayed a large proportion of tetra-nucleate cells with fragment-
ed septa. In contrast, the phenotypes of single mutants at this dose
were far less severe. These results are consistent with a model in
which Set3p functions in parallel to both Clp1p and Lsk1p. Thus,
set3 defines a novel regulatory pathway governing the successful
completion of cytokinesis in fission yeast.
Set3p, Hif2p and Snt1p form a nuclear-localized complex
Bioinformatics suggested that the hif2, set3, and snt2 gene
products exist as part of a conserved histone deacetylase complex.
In order to function in chromatin remodeling, one would expect
Hif2p, Set3p, and Snt2p to localize to the nucleus. To test this
prediction, strains expressing GFP-tagged fusion proteins
integrated at their normal genomic loci and under the control of
their native promoters were constructed. Consistent with a role
in chromatin modification, all three proteins localized to the
nucleus (Figure 4A). The localization of the fusion proteins was not
altered as a function of cell cycle position or as a function of LatA
concentration in the growth medium (data not shown).
Next, to determine if Hif2p, Set3p, and Snt1p physically
interacted in vivo, co-immunoprecipitation experiments using
Myc- or HA-epitope tagged alleles were performed. Interestingly,
Set3-HA and Hif2-HA fusion proteins could be detected in anti-
Myc immuno-precipitates of Snt1-Myc Set3-HA and Snt1-Myc
Hif2- HA extracts (Figure 4B, top and middle panels). Likewise,
Hif2- HA proteins could be detected in anti-Myc immun o-
precipitates of Set3-Myc Hif2-HA extracts (Figure 4B, bottom
panel). Taken together, these data demonstrate that Hif2p, Set3p,
and Snt1p exist as part of a nuclear-localized protein complex in
S. pombe.
Protein levels of Set3p, Hif2p and Snt1p increase upon
LatA treatment
Since hif2D, set3D, and snt2D mutants display cytokinesis defects
upon LatA treatment, we next asked if the expression levels of
these proteins might be responsive to the presence of LatA in the
growth medium. To this end, protein extracts were made from
LatA (0.5
mM) or DMSO treated strains expressing Set3-HA,
Snt1-HA and Hif2-HA fusion proteins. The levels of Set3-HA,
Snt1-HA and Hif2-HA were then quantified by western blotting.
Remarkably, while no significant changes were noted in DMSO
treated cells, the protein levels of Set3p, Snt1p, and Hif2p
increased 2–3 fold upon LatA treatment (Figure 5A–C). Increasing
the dose of LatA to 1
mM did not increase the level of induction
(compare Figure 5A, left and right panels). Thus, the magnitude of
the increase is not dosage dependent in this range of LatA
concentrations.
To ensure that changes in protein level were not due to
genotype specific delays at a particular cell cycle stage (since wild-
type cells engage the cytokinesis monitoring system, whereas the
respective gene deletion mutants have this system abrogated) we
examined the levels of the respective epitope tagged proteins upon
release from a G2/M block produced through the use of the
temperature sensitive cdc25-22 mutation (Figure S3A). The
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efficiency of the block and release was monitored by examining the
level of bi-nucleate cells every 30 minutes after shifting from 36uC
to 25uC (Figure S3B,C). The levels of Set3-HA, Snt1-HA, Hif2-
HA were not significantly altered as a function of cell cycle
position (Figure S3A). Taken together, these results are consistent
with a model in which the increased activity of the Set3p-Snt1-
Hif2p complex is required for a proper cellular response to LatA
induced stress.
Microarray expression profiling
Histone deacetylase complexes have well defined roles in
regulating transcription through chromatin modification [23,24].
This suggested a model in which the Set3p complex might affect
the transcription of genes involved in regulating cytokinesis or
the cytoskeleton. To further explore this hypothesis, expression
profiles were generated for both wild-type and set3D mutants.
Total RNA was extrac ted from wild-type or set3D cultures
Figure 2.
hif2
,
set3
, and
snt1
deletion mutants are hyper-sensitive to LatA treatment. (A) Ten-fold serial dilutions of logarithmically growing
cells of the indicated genotype were plated onto YES plates containing 0.5
mM LatA or DMSO (solvent control) at 30uC for 3 d. (B) Cells of the
indicated genotype were grown to mid-log phase at 30uC and then treated with 0.5
mM LatA for 5 h before being fixed and stained with DAPI (nuclei)
and aniline blue (cell wall/septa). Bar, 10 mm. (C) Quantitation of phenotypes of cells treated as in B. Between 200 and 500 cells were counted for each
genotypic class.
doi:10.1371/journal.pone.0031224.g002
Figure 3.
set3
functions in a pathway parallel to those defined by
clp1
and
lsk1
. Cells of the indicated genotype were grown to mid-log
phase at 30uC and then treated with 0.1
mM LatA for 5 h before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Two
hundred cells were counted for each genotypic class.
doi:10.1371/journal.pone.0031224.g003
SET Domain Protein Regulates S. pombe Cytokinesis
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treated with LatA (0.5 mM) or DMSO for 3 hours. The RNA
samples were then use d in microarray hybridizati ons using Yeast
Genome 2.0 Gene Chips purchased from Affymetrix. Three
replicates of each strain u nder each growth condition (plus or
minus LatA) were obtained for a t otal of 12 samples. After data
processing (see Materials and Methods) the 12 samples were
grouped according to two parameters: genotype (wild-type or
set3D) and drug (LatA treated or DMSO treated). The complete
data set, showing log
2
normalized intensity values for all genes is
included in File S1.
Next, to assess whether the expression data was providing
meaningful biological results, the expression levels of genes known
to play a role in the S. pombe core environmental stress response
(CESR) were analyzed in wild-type cells [25]. The CESR defines a
group of 240 genes whose expression is affected by a variety of
different stresses. These stresses include extremes of temperature,
osmolarity, salt concentration, as well as treatment with oxidizing
or DNA damaging agents [25]. The expression of these genes is
thought to define a transcriptional profile characterizing exposure
to environmental stresses in S. pombe. Since LatA treatment would
Figure 4. Hif2p, Set3p, and Snt1p form a nuclear-localized complex. (A) Cells expressing the indicated fusion proteins were grown to mid-
log phase at 30uC in YES media, fixed, and then stained with DAPI and observed using the DAPI and GFP filter sets. Bar, 5
mm. (B) Cells expressing the
indicated fusion proteins were grown to mid-log phase in YES, lysed under native conditions, and subjected to anti-Myc immunoprecipitations. Both
total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the HA epitope.
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constitute an environmental stress, the expression of the CESR
genes was analyzed in wild-type cells to determine if the
microarray analysis could correctly detect changes in CESR gene
expression.
The CESR is sub-divided into two groups: 136 genes that are
up-regulated in response to stress (CESR-UP), and 104 that are
down-regulated in response to stress (CESR-DN). When the
expression of these genes was examined it was apparent that the
majority of CESR-DN genes (96 out of 104) were down-regulated,
whereas the majority of CESR-UP genes (120 out of 136) were up-
regulated in LatA treated cultures relative to DMSO treated
controls (Figure 6). Taken together these results indicated that the
microarray analysis was providing accurate and biologically
meaningful data.
For subsequent analysis, the data was grouped into four
categories for comparison: 1) wild type, DMSO treated, 2) wild
type, LatA treated, 3) set3D, DMSO treated and 4) set3D, LatA
treated. In order to identify genes differentially regulated by
genotype, volcano plots (p-value,0.05; fold change .1.5) were
used to compare the gene expression profiles of wild-type and
set3D mutant strains in both DMSO and LatA treated conditions.
We first examined the expression of 333 genes with known roles in
cytokinesis and/or the cytoskeleton (File S1 for a complete list).
None of the genes were identified as being differentially expressed
in response to LatA treatment. Furthermore, scatterplots compar-
ing wild-type and set3D strains treated with either DMSO or LatA
(Figure 7) showed a strong correspondence of expression for the
vast majority of cytokinesis genes. Thus, the gene expression data
did not support a model in which the Set3p complex plays a role in
modulating the transcription of genes with roles in cytokinesis
and/or the cytoskeleton.
Since genes with roles in cytokinesis were not affected, we next
examined the global data set to determine the genes that were
differentially regulated by genotype. First, the expression profiles
of set3D and wild-type strains were compared upon DMSO
treatment. Surprisingly, only three genes were identified as being
differentially regulated (p-value,0.05; fold change .1.5)
(Figure 8A). As expected, this list included set3 itself, as well as
mfm2, and the uncharacterized ORF, SPAC186.05c. Thus, under
normal growth conditions where the set3D mutant shows no
obvious phenotype gene expression patterns were not altered to
a great extent.
We next examined the difference in expression profiles in wild-
type and set3D strains treated with LatA. Under these conditions
73 genes were identified as being differentially regulated (p-
value,0.05; fold change .1.5) (Figure 8B). To determine if any of
these genes shared any common functions, GO annotations were
inspected. The gene ontology (GO) project is a bioinformatics
initiative aimed at characterizing the attributes of genes and gene
products across databases. This initiative describes all gene
products with respect to their biological functions, molecular
functions, and their cellular component (www.geneontology.org/).
Interestingly, of the 73 differentially regulated genes, 40% (29 out
of 73) were annotated by GO as having a role in the cellular
response to stress (see File S1 for a complete list).
To further explore the role of Set3p in affecting stress response
genes, we analyzed the expression of the CESR genes in LatA or
DMSO treated wild-type cells. As expected 144 out of 240 genes
(,60%) were differentially expressed upon LatA treatment
(Figure 9A). However, when the expression of CESR genes in
LatA treated set3D cells was examined, only 1 out of 240 (,1%)
were differentially regulated (Figure 9B). Next, we expanded the
analysis to include any gene annotated by GO as having a cellular
response to stress. In this sub-set, 113 out of 561 genes (,20%)
were differentially regulated upon LatA treatment in wild-type
cells, while only 7 out of 561 genes (,1%) were differentially
regulated in set3D backgrounds (Figure 10A).
Lastly, we performed scatter plot analysis on a combined set of
genes that included both the CESR and genes annotated by GO as
having a cellular response to stress. Interestingly, scatter plot
analysis of the stress genes in set3D mutants revealed that the
distribution of data points was closely clustered around median
levels (Figure 10B). In wild-type, on the other hand, a similar
analysis of the distribution revealed the existence of genes that
were either up- (Figure 10B, blue oval) or down-regulated
(Figure 10B, pink oval) in response to LatA. Taken together these
data indicate that set3D cells are severely impaired in their ability
to mount a proper transcriptional response to LatA treatment.
Discussion
Schizosaccharomyces pombe has proven to be an excellent model for
understanding the regulatory modules that ensure the faithful and
reliable execution of cytokinesis [1,2,3,4,6,7,8,9,10,12,13,22]. In
this report we further expand our knowledge in this area by
Figure 5. Set3p, Snt1p and Hif2p levels increase two- to three-fold upon treatment with low doses of LatA. (A) Strains expressing the
indicated fusion proteins were grown to early log phase in YES at 30uC and treated with 0.5
mM LatA (left panel) or 1 mM LatA (right panel). Extracts
were subjected to SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-HA antibody. Tubulin was used as a loading control. (B
and C) Strains expressing the indicated fusion proteins were grown to early log phase in YES at 30uC and treated with 0.5
mM LatA. Extracts were
subjected to SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-HA antibody. Tubulin or the RNA pol II carboxy-terminal
domain (CTD) were used as loading controls. Signal intensities relative to the loading controls of three independent trials were quantified using
ImageJ 1.41 Gel Analyzer software and are plotted below representative blots. Error bars indicate sd.
doi:10.1371/journal.pone.0031224.g005
Figure 6. The core environmental stress response genes (CESR)
respond to LatA treatment in wild-type cells. Volcano plot
analysis of the expression of the CESR genes in wild-type strains treated
with 0.5
mM LatA. Blue diamonds represent CESR genes normally up-
regulated in response to multiple stresses. Red squares represent CESR
genes normally down-regulated in response to multiple stresses.
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identifying three components of a histone de-acetylase complex
(Hif2p, Set3p, and Snt1p) whose function is required to ensure the
successful completion of cytokinesis upon perturbation of the cell
division machinery (Figures 1 and 2). Moreover, through the
creation of set3D lsk1D and set3D clp1D double mutants, we
demonstrate that the complex functions through a novel branch
of control, independently of both Lsk1p and Clp1p (Figure 3). This
is consistent with genetic data indicating that, unlike the lsk1 gene
deletion [13], set3D mutations are incapable of suppressing the lethal
cytokinesis phenotype associated with SIN hyperactivation (data not
shown). Lastly, through phenotypic analysis, co-immunoprecipita-
tion data, and the analysis of intracellular localization, we provide
support for a model in which Hif2p, Set3p, and Snt1p act together
in a physical complex (Figures 2 and 4).
Figure 7. The expression levels of cytokinesis related genes are not differentially regulated. (A) Cells of the indicated genotypes were
grown to mid-log phase at 30uC in YES, and then treated with DMSO (A) or 0.5
mM LatA (B) for 3 hours. Total RNA was extracted and used in
expression profiling using Affymetrix Yeast 2.0. Genechips. Graphs shows scatter plot analysis comparing the expression of cytokinesis related genes
in wild-type and set3D strains. Green lines represent the threshold for a 1.5 fold change in transcript levels. The color of the squares indicates the level
of expression of that gene in DMSO (A) or LatA (B) treated wild-type cells.
doi:10.1371/journal.pone.0031224.g007
SET Domain Protein Regulates S. pombe Cytokinesis
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The importance of understanding the pathways required for the
dependable execution of cytokinesis in eukaryotes was first
articulated by Theodor Boveri almost 100 years ago [26]. In his
classic work ‘‘Concerning the origin of malignant tumours’’ (1914) Boveri
hypothesized that tetraploid intermediates derived from either
cytokinetic failure or cell fusion might undergo chaotic
multipolar mitoses leading to numerical and/or structural
chromosomal defects. Recent experimental evidence provides
strong support for Boveri’s assertions. First, tetraploid mouse
mammary epithelial cells generated by the inhibition of cytokinesis
display increased rates of aneuploidy and (when transplanted into
nude mice) give rise to malignant tumours at greater rates than
controls [27]. Second, tetraploidy often precedes gross aneuploidy
and is an early event in carcinogenesis [28,29]. Third, aneuploidy
(generated by loss of the kinesin, Kif4) promotes tumorigenesis in
vivo [30]. Lastly, several tumor suppressors (BRCA2, LATS) are
required for the completion of cytokinesis [31,32]. Taken together
these results suggest that mechanisms promoting the dependable
execution of cytokinesis are important in maintaining genomic
integrity and in preventing carcinogenesis [33,34,35]. Thus, in the
broadest sense, an understanding of these pathways may provide a
better understanding of one ‘‘route’’ by which eukaryotic cells
become tumorigenic in multicellular organisms.
Given the importance of cytokinesis in maintaining genomic
integrity it is particularly intriguing to note that orthologues of
Hif2p, Set3p, and Snt1p exist in humans (TBL1X, MLL5, and
NCOR2, respectively). Furthermore as might be expected based
on the selection criteria used in the genetic screen MLL5,
Figure 8. Identification of
S. pombe
genes differentially expressed with respect to genotype. Volcano plot (left panels) and scatter plot
(right panels) analysis of the expression of all S. pombe genes in DMSO (A) or LatA (B) treated cells of the indicated genotype. Horizontal green lines
in volcano plots represent a p-value of 0.05. Vertical green lines in volcano plots represent threshold for a 1.5 fold change in expression. Red squares
in volcano plots indicate differentially expressed genes. Diagonal green lines in scatter plots represent the threshold for a 1.5 fold change in
expression. The color of the squares in scatter plots indicates the level of expression of that gene in DMSO (A) or LatA (B) treated wild-type cells.
doi:10.1371/journal.pone.0031224.g008
SET Domain Protein Regulates S. pombe Cytokinesis
PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e31224
NCOR2, and TBL1X have themselves been shown to play a role
in cytokinesis in human cells. In their study, Kittler et al. (2007)
conducted a genome-wide RNAi screen aimed at identifying genes
with roles in cell division in cultured HeLa cells. They discovered
that the knockdown of MLL5, TBL1X,orNCOR2 resulted in
defects in furrow ingression, cytokinesis failure, and finally the
generation of tetraploid intermediates with twice the normal
number of centrosomes.
While highly speculative it is of interest to note that MLL5 is
found in a region of chromosome seven that is frequently deleted
in myeloid malignancies, and furthermore that decreased MLL5
expression levels correlate with unfavourable outcomes in patients
with acute myeloid leukemia [36,37]. Moreover, the down-
regulation of the NCOR2 gene can induce transformation in
certain immortalized cell lines [38]. While a direct role in tumour
progression via cytokinesis failure has not been shown, it is
Figure 9.
set3D
mutants are impaired in their ability to modulate the expression of CESR genes in response to LatA treatment.
Volcano plot (left panels) and scatter plot (right panels) analysis of the expression of CESR genes in wild-type (A)orset3D (B) strains in response to
LatA treatment. Horizontal green lines in volcano plots represent a p-value of 0.05. Vertical green lines in volcano plots represent threshold for a 1.5
fold change in expression. Red squares in volcano plots indicate differentially expressed genes. Diagonal green lines in scatter plots represent the
threshold for a 1.5 fold change in expression. The color of the squares in scatter plots indicates the level of expression of that gene in DMSO treated
wild-type (A)orset3D (B) cells. Genes differentially expressed upon LatA treatment in wild-type cells are shown as green squares in scatter plots.
doi:10.1371/journal.pone.0031224.g009
SET Domain Protein Regulates S. pombe Cytokinesis
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interesting to speculate as to whether MLL5, NCOR2,orTBL1X
might indeed encode tumour suppressors, and if so, whether the
loss of these genes, and any ensuing cytokinesis defects could be
relevant to carcinogenesis. Regardless, the isolation of known
human regulators of cytokinesis in this screen further supports the
utility of using S. pombe as a model for the study of eukaryotic
genetic regulatory networks.
A second observation of particular significance is the discovery
that the levels of Set3p, Hif2p, and Snt1p increase 2–3 fold when
wild-type cells are grown in the presence of low doses of LatA
(Figure 5). Thus, in addition to the observed LatA hypersensitivity
exhibited by the gene deletion mutants, these data provide further
independent support that activity of the complex is required to
respond properly to the presence of LatA in the growth medium.
Up-regulation probably occurs at the post-transcriptional level
since microarray data did not show strong induction of these genes
in wild-type cells treated with LatA (File S1).
While the above data identifies the Set3p complex as being
required for the proper response to LatA induced stress, it says
little with respect to the mechanism of action. Since histone de-
acetylase complexes have well defined roles in transcriptional
regulation, we considered the possibility that the observed
Figure 10.
set3D
mutants are impaired in their ability mount a proper transcriptional response to LatA treatment. (A) Volcano plot
analysis of the expression of genes annotated by GO as having a cellular response to stress. The response of wild-type cells (left panel) and set3D
mutants (right panel) treated with LatA are shown. Horizontal green lines represent a p-value of 0.05. Vertical green lines represent the threshold for a
1.5 fold change in expression. Red squares indicate differentially expressed genes. (B) Scatter plot analysis comparing the expression of stress
response genes (both CESR genes and genes annotated by GO as having a cellular response to stress) in response to LatA treatment. Blue squares
indicates data points for wild type cells. Red diamonds indicate data points for set3D mutants. Genes up-regulated in response to LatA in wild-type
cells are highlighted with a blue oval. Genes down-regulated in response to LatA in wild-type cells are highlighted with a pink oval.
doi:10.1371/journal.pone.0031224.g010
SET Domain Protein Regulates S. pombe Cytokinesis
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cytokinesis phenotypes were the result of defects in the
transcription of genes involved in cytokinesis and/or the
cytoskeleton (Figure 7). Importantly, while expression profiling
clearly showed that this was not the case, a careful examination of
the microarray data did reveal several interesting findings.
First, wild-type cells respond to LatA with a general cell stress
response (Figures 6, 8,9,10). This was evident by the strong
induction/repression of the fission yeast CESR genes. The CESR
genes are predicted to modulate cellular metabolic pathways and
to limit growth related processes [25]. It is hypothesized that
activation of the CESR may promote survival against potentially
lethal doses of a given stress and in doing so provide a means for
the cell to adapt to its new environment [25,39]. In stark contrast
to wild-type cells, the set3D mutant exhibited a significantly
reduced capacity to modulate the expression of stress response
genes upon LatA treatment (Figures 8,9,10). Thus, cytokinetic
failure in set3D mutants may be a manifestation of the mutant cells
inability to properly adapt to the presence of LatA leading to direct
and/or indirect effects on the function of the cytokinetic
machinery. It is also of interest to note that, in addition to LatA,
set3D strains show sensitivity to the calcineurin inhibitor, FK506
[40]. Intriguingly, calcineurin mutants in S. pombe have been
shown to affect cytokinesis, cell polarity, and spindle pole body
positioning [41].
A role in the stress response, may be an evolutionarily conserved
feature since the Set3p complex in budding yeast is required to
respond to secretory stress [42]. Furthermore, the budding yeast
class I histone deacetylase Rpd3p, and its associated Rpd3-L
complex, is required for activation and repression of environmen-
tal stress response genes [43]. Taking this into consideration, we
favour a model in which the observed defects in cytokinesis are
related to the impaired ability of the mutants to modulate gene
expression so as to properly counter the effects of LatA induced
stress. This is supported by the observation that the protein levels
of all three complex members increase in response to LatA
(Figure 5), as well as the observation that wild-type cells modulate
the expression of a large sub-set of genes with a role in the stress
response (Figures 6, 9 and 10). In any event, we suspect that future
analysis of this system might translate into a theoretical framework
for understanding how the orthologous MLL5 complex functions
to regulate cytokinesis in human cells, as well as how its
dysfunction might lead to genomic instability.
Materials and Me thods
Yeast Methods
All Schizosaccharomyces pombe strains used in this work (Table S1)
originated from previous studies [12,44,45], were created during
the course of this work, or were purchased from Bioneer
Corporation (Alameda, CA). Schizosaccharomyces pombe cells were
cultured in either YES or Edinburgh Minimal Media (EMM)
(Forsburg and Rhind 2006) with the appropriate supplements
(Adenine, Histidine, Leucine, or Uracil). Liquid cultures were
grown with shaking (200 rpm) at 30uC. Genetic crosses were
performed using standard methods [46]. In experiments involving
Latrunculin treatment, S. pombe cells were grown to mid log phase
(O.D. 0.2) and treated with 0.2–0.5
mM of Latrunculin A (Enzo
Life Sciences International, Plymouth Meeting, Pennsylvania)
dissolved in DMSO. Cells were grown at 30uC with shaking at
200 rpm for 3–6 hrs, before being fixed. All experiments were
repeated a minimum of three times. Plasmid vectors were
transformed into S. pombe using the lithium acetate protocol
according to Forsburg and Rhind [46]. In block and release
experiments, cdc25-22 cells were grown to logarithmic phase in
YES at 25uC, shifted to 36uC for 3 hours to block at the G2/M
transition, and then shifted back to 25uC to effect release of the
block. The cell cycle progress of the strains was subsequently
monitored by determining the level of bi-nucleate cells every
30 minutes after the shift.
Fluorescence Microscopy
S. pombe cells expressing Lsg1-GFP fusions, were fixed using
ethanol fixation [46] and stored in PBS pH 7.4. To observe nuclei
and cell wall/septa material, cells were mixed with 0.02
mg/mL
496,-diamidino-2-phenylindole (DAPI) and 1
mg/mL aniline blue.
Fluorescent images were obtained using Zeiss Axioskop 2
microscope driven by ImageJ 1.41 software (National Institutes
of Health) and Scion CFW Monochrome CCD Firewire Camera
(Scion Corporation, Frederick Maryland) using DAPI and GFP
filter sets.
Cloning Methods
The snt1 gene deletion mutant was created using a PCR based
cloning strategy. A 286 bp region upstream of the snt1 start codon
was PCR amplified using High-Fidelity PCR Enzyme Mix
(Fermentas Life Sciences) with the forward primer 59-ggg ggg
gta cca aat gaa ggg gat tcc ttg g-39 and reverse primer 59-ggg ggc
tcg agt gtc aga gga ggc act aca gc-39 and cloned into the
pSKURA4 vector upstream of the ura4 selectable marker using the
restriction enzymes KpnI and XhoI (Fermentas Life Sciences). Next,
a 259 bp region downstream of the snt1 stop codon was PCR
amplified using the forward primer 59-ggg ggt cta gat gtg tcg ggt
tat gat ggt g-39 and reverse primer 59-ggg ggg agc tca ttt ttg gtg tcg
gtt ttg c-39 and cloned downstream of the ura4 selectable marker in
pSKURA4 using the restriction enzymes XbaI and SacI. Molecular
cloning of the desired fragments was confirmed by restriction
digestion and DNA sequencing. The ura4 selectable marker
flanked by upstream and downstream regions of snt1 was excised
with restriction enzymes KpnI and SacI to isolate a linear dsDNA
deletion cassette. The deletion cassette was transformed into S.
pombe strain MBY1343 (ura4-D18). Ura4
+
integrants were selected
for by growth on EMM lacking uracil and subjected to colony
PCR to identify clones in which the construct had integrated into
the genome via homologous recombination. Strains bearing gene
deletions of set3 and hif2 were purchased from Bioneer
Corporation (Alameda, CA). Genotypes were verified by colony
PCR. The hif2::natMX gene deletion was created using the high
throughput knockout strategy devised by the Kim Nasmyth lab
[47]. Primers, plasmids and a detailed protocol are available at the
S. pombe deletion web server (http://mendel.imp.ac.at/Pombe_
deletion/).
S. pombe strains expressing carboxy-terminal epitope tagged
fusion protein were constructed using a PCR based cloning
strategy. To create the Set3-GFP and Set3-HA expressing strains a
C-terminal fragment of the set3 gene was PCR amplified using
High-Fidelity PCR Enzyme Mix (Fermentas Life Sciences) from S.
pombe genomic DNA with the forward primer 59-ggg ggg aat tct
gaa ata ctt caa gaa gcg aaa aca ag-39 and reverse primer 59-ggg
ggc ccg ggt cgc gta aat gaa ggg tta g-39 and cloned in frame into
the EcoRI and SmaI sites of the pJK210-GFP and pJK210-HA
vectors respectively. Molecular cloning of the desired C-terminal
fragments was confirmed by restriction digestion and DNA
sequencing. Plasmid clones containing the desired C-terminal
fragment were transformed into S. pombe strain MBY1343 (ura4-
D18). Ura4
+
integrants were selected for by growth on EMM
lacking uracil and subjected to colony PCR to identify clones in
which the construct had integrated into the genome via
homologous recombination. To create the Set3-myc expressing
SET Domain Protein Regulates S. pombe Cytokinesis
PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e31224
strain a C-terminal fragment of the set3 gene was PCR amplified
using High-Fidelity PCR Enzyme Mix (Fermentas Life Sciences)
from S. pombe genomic DNA with the forward primer 59-ggg ggg
gta cct gaa ata ctt caa gaa gcg aaa aca ag-39 and reverse primer 59-
ggg ggc ccg ggt cgc gta aat gaa ggg tta g-39 and cloned in frame
into the KpnI and SmaI sites of the pJK210-Myc vector. Plasmid
clones containing the desired C-terminal fragment were trans-
formed into S. pombe strain MBY1343 (ura4-D18). Ura4
+
integrants
were selected for by growth on EMM lacking uracil and subjected
to colony PCR to identify clones in which the construct had
integrated into the genome via homologous recombination.
Snt1-GFP and Snt1-Myc as well as Hif2-GFP and Hif2-HA
expressing strains were created using an analogous strategy as the
epitope tagged Set3p strains. The forward and reverse primers
used to create the Snt1-GFP integrant strain were 59-ggg ggg aat
tct gag gtt ggg atg aaa aag aag aa-39 and 59-ggg ggc ccg ggt aca att
tta tcg ttt ttg gac tg-39 respectively, and the forward and reverse
primers used to create the Snt1-Myc integrant strain are 59-ggg
ggg gta cct gag gtt ggg atg aaa aag aag aa-39 and 59-ggg ggc ccg ggt
aca att tta tcg ttt ttg gac tg-39 respectively. The forward and
reverse primers used to create the Hif2-GFP and Sif3-HA
integrant strains are 5-ggg ggg aat tct gat cta gag gtg atg ctg gtg
c-39 and 59-ggg ggc ccg ggc aga gaa tca tgt aaa aaa tca ca-39
respectively.
Biochemical and Immunological Methods
Cells of the indicated genotype were grown up to the mid-log
phase at 30uC, collected by centrifugation, and resuspended in
STOP buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 50 mM
NaF, 10 mM EDTA, 1 mM NaN
3
). Cell pellets were stored at
280uC up to a maximum of 6 months. Cell pellets were
subsequently thawed, and lysed using vortexing with glass beads
in extraction buffer (1% IGEPAL CA630 (tetr-Octylphenoxy
polyethanol), 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 2 mM
EDTA, 1 mM PMSF (phenylmethanesulphonylfluoride), 2 mM
Benzamidine, 50 mM NaF, 0.1 mM Na
3
VO
4
,50mMB-
glycerophosphate, 15 mM p-nitrophenyl phosphate, J Tablet
Sigma Protease Inhibitors). Total cell extracts were subjected to
SDS-PAGE, transferred to PVDF membranes and immunoblotted
with anti-HA primary antibody (HA.11; Sigma) at a dilution of
1:2000. As a loading control, anti-tubulin (B14; Sigma) was used at
a dilution of 1:2000, or primary antibody specific to the
unphosphorylated C-terminal domain of RNA polymerase II
(8WG16; Covance) was used at a dilution of 1:5000. Peroxidase
conjugated anti-mouse IgG (Sigma) at 1:10000, was used as
secondary antibody for HA, tubulin and RNA polymerase II
blotted membranes. Blots were quantified using ImageJ 1.41 gel
analyzer software (National Institutes of Health).
In co-immunoprecipitation experiments cell extracts were
obtained from the indicated strains as described above. Immuno-
precipitations were performed using Protein G DynabeadsH
(Invitrogen) according to the manufacturer’s protocol. Briefly,
immunoprecipitations were performed by incubating anti-HA
antibodies (HA.11; Sigma) with the Protein G Dynabeads using
extraction buffer as the wash solution. Cell extracts were then
added to the antibody-bound bead slurry. After incubation and
repeated washing with extraction buffer, the bound proteins were
eluted by incubation at 96uC for 5 minutes. The eluted proteins
were then subjected to SDS-PAGE, transferred to PVDF
membranes and probed with anti-Myc antibodies (9E10; Sigma).
Global Gene Expression Analysis
Strains of the indicated genotype were grown to mid-log phase
and treated with 0.5
mM LatA or an equal volume of DMSO for
3 hours at 30uC. Total RNA extraction was performed using the
Ambion RiboPure
TM
Yeast Kit (Ambion Inc). Three replicates
were performed for each strain under each growth condition (plus
or minus LatA). Bioanalysis of all samples showed that the isolated
RNA was of sufficient quality to proceed to hybridizations (data
not shown). GeneChipH Yeast Genome 2.0 Arrays purchased from
Affymetrix were used for hybridization. Hybridizations were
performed by the London Regional Genomic Centre using
standard Affymetrix protocols. The quality of hybridization was
analyzed using duplicate probes for the bioB, bioC, bioD , and cre
genes. During hybridization, transcripts of these genes are
‘‘spiked’’ into the Affymetrix hybridization cocktail at concentra-
tions of 1.5, 5, 25 and 100 pM, respectively. The level of
hybridization as measured by the normalized signal values (data
not shown) was consistent with the level of spiked transcript (i.e.
bioB showed the lowest signal values and cre the highest). This
demonstrated that the hybridizations had been performed
successfully and that the data could be used for further analysis.
Expression profiling data was obtained from London Regional
Genomic Centre as .CEL files, which contained the raw
hybridization signal-intensity values. Analysis of the data was
done using Genespring GX 10.0.1 software provided by Agilent
Technologies Inc. and Strand Life Sciences Pvt. Ltd. The .CEL
files were first normalized with RMA (robust multi-array analysis)
algorithm. RMA uses PM (perfect match) probes from the data
and corrects the background by fitting a model that is the addition
of an exponentially distributed signal and a normally dispersed
background [48]. This generated normalized hybridization signal-
intensity data for all twelve samples. Normalized expression data
was subsequently used in the analysis. Data was grouped into two
categories: genotype (wild type or set3D), and b) drug (LatA or
DMSO treated). Thus, four experimental groups were used for
comparison: 1) wild type, DMSO treated, 2) wild type, LatA
treated, 3) set3D, DMSO treated, and 4) set3D, LatA treated. To
identify differentially regulated genes, data was filtered using
Volcano plot analysis employing the Benjamin-Hochberg multiple
testing correction. Genes showing statistically significant differ-
ences (p-value,0.05) and fold changes greater than 1.5 were
identified as differentially regulated. Data is accessible from the
GEO database under accession number GSE33228).
Supporting Information
Figure S1 ClustalW alignments of the Hif2p, Set3p, and
Snt1p proteins with their human orthologues, TBL1X
(A), MLL5 (B), and NCOR2 (C), respectively. Alignments in
(B) and (C) were performed with conserved segments present in
the N-terminal regions of the respective proteins. Asterisks (*)
indicate identities. Colons (:) indicate conserved substitutions.
Periods (.) indicate semi-conserved substitutions.
(TIF)
Figure S2 Deletion of the set3, snt1,orhif2 genes
reduces the restrictive temperature of cdc15-140 mu-
tants. Cells of the indicated genotype were cultured to
logarithmic growth phase at 25uC. Ten-fold serial dilutions were
subsequently plated onto solid YES media and incubated at 25uC,
31uC, or 36uC for 3 d.
(TIF)
Figure S3 The protein levels of Set3-HA, Snt1-HA, or
Hif2-HA do not change as a function of cell cycle
position. Strains of the indicated genotype were grown to early
log phase in YES at 25uC and shifted to 36uC for 3 hours to arrest
the cells at the G2/M transition. Cells were subsequently released
SET Domain Protein Regulates S. pombe Cytokinesis
PLoS ONE | www.plosone.org 14 February 2012 | Volume 7 | Issue 2 | e31224
from the block by shifting to 25uC and cells collected every
30 minutes. (A) Extracts were subjected to SDS-PAGE, transferred
to PVDF membranes, and immunoblotted with anti-HA antibody.
Tubulin was used as a loading control. (B) To monitor the
efficiency of the block and release, the level of bi-nucleate cells was
quantitated every 30 minutes after shift to 25uC. (C) Represen-
tative micrographs of Set3-HA cdc25-22 cells at various time points
after release. Cells were fixed and stained with DAPI (nuclei) and
aniline blue (cell wall/septa).
(TIF)
Table S1 Yeast strains used in this study.
(DOC)
File S1
(XLSX)
Acknowledgments
We thank David Carter and the London Regional Genomics Centre for
expert technical assistance with microarray hybridizations. We would also
like to thank members of the laboratory and the UWO Biology
Department for helpful discussions and/or critical reading of the
manuscript.
Author Contributions
Conceived and designed the experiments: JK. Performed the experiments:
SR RS CG RK MM JK. Analyzed the data: SR JK. Contributed reagents/
materials/analysis tools: DM. Wrote the paper: SR JK.
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SET Domain Protein Regulates S. pombe Cytokinesis
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    • "Interestingly, this decrease in the restrictive temperature of the cdc15-140 mutation is similar to that caused by the presence of the set3Δ, snt1Δ, and hif2Δ gene deletions in cdc15-140 backgrounds. These data further support a common function for the hos2, set3, snt1, and hif2 genes [19]. Once having established that the Hos2p protein was indeed involved in the regulation of cytokinesis, we explored the possibility that the protein's deacetylase activity was related to its function. "
    [Show abstract] [Hide abstract] ABSTRACT: Background In Schizosaccharomyces pombe the SET domain protein, Set3p - together with its interacting partners, Snt1p, and Hif2p - form a complex that aids in preventing cell division failure upon mild cytokinetic stress. Intriguingly, the human orthologs of these proteins (MLL5, NCOR2, and TBL1X) are also important for the faithful completion of cytokinesis in tissue culture cells. Since MLL5, NCOR2, and TBL1X form a complex with the histone deacetylase, HDAC3, we sought to determine if an orthologous counterpart played a regulatory role in fission yeast cytokinesis. Results In this report we identify the hos2 gene as the fission yeast HDAC3 ortholog. We show that Hos2p physically interacts with Set3p, Snt1p, and Hif2p, and that hos2∆ mutants are indeed compromised in their ability to reliably complete cell division in the presence of mild cytokinetic stresses. Furthermore, we demonstrate that over-expression of hos2 causes severe morphological and cytokinetic defects. Lastly, through recombinase mediated cassette exchange, we show that expression of human HDAC3 complements the cytokinetic defects exhibited by hos2∆ cells. Conclusions These data support a model in which Hos2p functions as an essential component of the Set3p-Snt1p-Hif2p complex with respect to the regulation of cytokinesis. The ability of human HDAC3 to complement the cytokinesis defects associated with the deletion of the hos2 gene suggests that further analysis of this system could provide insight into the role of HDAC3 in both the regulation of cell division, as well as other biological processes influenced by HDAC3 deacetylation.
    Full-text · Article · May 2012
  • [Show abstract] [Hide abstract] ABSTRACT: Eukaryotic cells ensure error-free progress through the cell cycle by monitoring (1) the completion of cell cycle events, (2) damage to critical cellular components, or (3) structural changes such as the attachment of kinetochores to the mitotic spindle. In the presence of damage, or in the face of a reduced capacity to complete essential events, cells are capable of delaying the cell cycle so that damage can be repaired, or previous cell cycle phases can proceed to completion. Although such "checkpoints" have been extensively studied in many organisms-and much is understood with respect to the monitoring of DNA replication and DNA damage-little is known with regards to mechanisms that might monitor the completion of cytokinesis. In this review I summarize recent work from the fission yeast, Schizosaccharomyces pombe, describing the existence of regulatory modules that aid in ensuring the faithful and reliable execution of cytokinesis. Together, these modules promote the maintenance of a "cytokinesis-competent" state characterized by delayed progression into mitosis and the continuous repair and/or re-establishment of the acto-myosin ring. In this way, fission yeast cells are able to increase the likelihood of successful cell division prior to committing to a subsequent cell cycle. The recent demonstration of conservation between S. pombe components of these modules, and human proteins with defined roles in preventing cell division failure, suggest that the lessons learned in S. pombe may be applicable to other eukaryotes.
    Full-text · Article · May 2012
  • [Show abstract] [Hide abstract] ABSTRACT: The SMRT coregulator functions as a dual coactivator and corepressor for estrogen receptor-α (ERα) in a gene-specific manner, and in several studies its elevated expression correlates with poor outcome for breast cancer patients. A specific role of SMRT in breast cancer progression has not been elucidated, but SMRT knock-down limits estradiol-dependent growth of MCF-7 breast cancer cells. In this study, small-interfering RNA (siRNA) and short-hairpin RNA (shRNA) approaches were used to determine the effects of SMRT depletion on growth of ERα-positive MCF-7 and ZR-75-1 breast cancer cells, as well as the ERα-negative MDA-MB-231 breast cancer line. Depletion of SMRT inhibited growth of ERα-positive cells grown in monolayer but had no effect on growth of the ERα-negative cells. Reduced SMRT levels also negatively impacted the anchorage-independent growth of MCF-7 cells as assessed by soft agar colony formation assays. The observed growth inhibitions were due to a loss of estradiol-induced progression through the G1/S transition of the cell cycle and increased apoptosis in SMRT-depleted compared to control cells. Gene expression analyses indicated that SMRT inhibits apoptosis by a coordinated regulation of genes involved in apoptosis. Functioning as a dual coactivator for anti-apoptotic genes and corepressor for pro-apoptotic genes, SMRT can limit apoptosis. Together these data indicate that SMRT promotes breast cancer progression through multiple pathways leading to increased proliferation and decreased apoptosis.
    Article · Jun 2014
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