Copyright ? 2011 by the Genetics Society of America
Cohesin Plays a Dual Role in Gene Regulation and Sister-Chromatid
Cohesion During Meiosis in Saccharomyces cerevisiae
Weiqiang Lin, Mian Wang, Hui Jin and Hong-Guo Yu1
Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4370
Manuscript received August 18, 2010
Accepted for publication January 22, 2011
Sister-chromatid cohesion mediated by cohesin ensures proper chromosome segregation during cell
division. Cohesin is also required for postreplicative DNA double-strand break repair and gene
expression. The molecular mechanisms of these diverse cohesin functions remain to be elucidated. Here
we report that the cohesin subunits Scc3 and Smc1 are both required for the production of the meiosis-
specific subunit Rec8 in the budding yeast Saccharomyces cerevisiae. Using a genetic approach, we depleted
Scc3 and Smc1 independently in cells that were undergoing meiosis. Both Scc3- and Smc1-depleted cells
were inducible for meiosis, but the REC8 promoter was only marginally activated, leading to reduced
levels of REC8 transcription and protein production. In contrast, the expression of MCD1, the mitotic
counterpart of REC8, was not subject to Scc3 regulation in vegetative cells. We provide genetic evidence to
show that sister-chromatid cohesion is not necessary for activation of REC8 gene expression. Cohesin
appears to positively regulate the expression of a variety of genes during yeast meiosis. Our results suggest
that the cohesin complex plays a dual role in gene regulation and sister-chromatid cohesion during
meiotic differentiation in yeast.
eukaryotes. In the single-celled organism Saccharomyces
program in response to starvation. A signal transduc-
tion cascade, which leads to changes in gene expres-
sion, initiates meiosis (Mitchell 1994; Kupiec et al.
1997). Consequently, the expression of meiosis-activating
genes is increased and that of meiosis-repressing genes
is decreased. The positive regulators of meiosis, of which
many are transcriptional factors, then activate the ex-
pression of early, middle, and late genes that are re-
quired for recombination, chromosome segregation,
and spore formation. Regulation of meiotic differen-
tiation is facilitated by chromosome structural re-
organization, which can be achieved by the actions of
histone modifiers and ATP-dependent chromatin-
remodeling complexes (Kassir et al. 2003). Additional
chromosomal factors might be required for activating
meiotic gene expression.
The evolutionarily conserved protein complex cohe-
sin, which is composed of Smc1, Smc3, Mcd1/Scc1, and
Irr1/Scc3 in the budding yeast, mediates sister-chromatid
cohesion (Onn et al. 2008; Nasmyth and Haering
2009). Rec8 largely replaces Mcd1 and is the only
EIOSIS is a developmentally regulated cell di-
vision required for sexual reproduction in
meiosis-specific cohesin subunit in yeast of which the
encoding gene is expressed early in meiosis (Chu et al.
1998). Cohesin binds to the yeast chromosome at
discrete loci (Blat and Kleckner 1999; Laloraya
et al. 2000; Glynn et al. 2004; Lengronne et al. 2004),
and the purified cohesin complex forms a ring-shaped
structure (Gruber et al. 2003). The tripartite cohesin
ring made of Smc1, Smc3, and Mcd1 (probably Rec8) is
sufficient for topologically entrapping a pair of sister
chromatids to generate cohesion in yeast (Haering
et al. 2008). Meanwhile, Scc3, which is called SA/STAG
in animals, has been implicated in cohesin oligomeri-
zation (Zhang et al. 2008) and is critical for cohesin
release from the chromosome (Hauf et al. 2005).
Cohesin is important for establishing both the mitotic
and meiotic chromosome architecture (Hirano 2006;
Onn et al. 2008; Nasmyth and Haering 2009).
In addition to mediating sister-chromatid cohesion,
cohesin appears to have a broad influence on chromo-
some metabolism that includes postreplicative DNA
double-strand break repair and gene expression (Strom
et al. 2004; Unal et al. 2004; Dorsett et al. 2005;
Horsfield et al. 2007). Functional analysis of cohesin
and its loading factor, the Scc2 and Scc4 complex,
generate a chromatin boundary that insulates the
transcriptional activity of surrounding genes in yeast
and fly (Donze et al. 1999; Rollins et al. 1999; Dorsett
et al. 2005). Cohesin also plays a role in cell differenti-
ation by modulating gene expression as demonstrated
1Corresponding author: 89 Chieftan Way, Florida State University,
Tallahassee, FL 32306-4371. E-mail: firstname.lastname@example.org.
Genetics 187: 1041–1051 (April 2011)
in neuron morphogenesis in flies (Pauli et al. 2008;
Schuldiner et al. 2008). These studies provide insights
into the understanding of the noncanonical role of
cohesin in the regulation of gene expression. Cohesin
function in gene expression is further supported by
recent findings in vertebrates that cohesin subunits
physically interact with the transcriptional factor CTCF
and that they colocalize with CTCF on chromosomes
(Parelho et al. 2008; Rubio et al. 2008; Wendt et al.
yet to be answered. For example, how does cohesin
regulate gene expression during cell differentiation? Is
this regulatory mechanism conserved in eukaryotes? Is
the cohesin holocomplex or individual subunit re-
quired for gene regulation? Is the primary role of
cohesin in sister-chromatid cohesion separable from
that of gene regulation?
genetic analysis of cohesin function in many model
tional mutant alleles. Using a previously proven genetic
approach (Lee and Amon 2003), we have created
conditional alleles of SCC3 and SMC1 that specifically
deplete Scc3 and Smc1 in yeast meiotic cells. In both
Scc3- and Smc1-depleted cells, the level of the meiosis-
specific subunit Rec8 is significantly lowered by a re-
duction of REC8 gene transcription. Our work suggests
that the cohesin complex plays an important role in
positively regulating the REC8 promoter when vegeta-
tive yeast cells differentiate into meiosis.
MATERIALS AND METHODS
Yeast strains and culture conditions: Yeast strains used in
this study are listed in supporting information, Table S1. We
used the CLB2 promoter to replace the endogenous pro-
moters of SCC3 and SMC1 by a PCR-based method as pre-
viously described (Jin et al. 2009). The PMET1-DEGRON-SCC3
We used plasmids pHG40 (Jin et al. 2009) and pHG105 to
create PCUP1REC8 and PREC8GFP alleles by standard yeast
of the REC8 start codon, which included the 59 UTR, by PCR
and placed it in front of the GFP open reading frame to create
pHG105. We used the DMC1 promoter to replace the REC8
endogenous promoter to generate PDMC1REC8 using a similar
been reported previously (Xu et al. 1995; Keeney et al. 1997;
Klein et al. 1999). The tetO array was inserted into the URA3
locus on chromosome V, and tetR-GFP at the LEU2 locus on
chromosome III, as previously described (Michaelis et al.
create C-terminal tags of the following alleles: SCC3-3HA,
were confirmed by colony PCR. PCR primer information
appears in Table S2.
Synchronous meiosis was performed as previously de-
scribed (Yu and Koshland 2005). Briefly, yeast cells were
grown in yeast extract, peptone, acetic acid (YPA) overnight at
30? to an optical density (l ¼ 600 nm) of ?1.6, washed once
with H2O, and resuspended in 2% KoAC for induction of
meiosis. Toinduce PCUP1REC8, 60 mm CuSO4 was addedto the
sporulation medium. The PMET1-DEGRON-SCC3 strain was
grown in methionine-dropout synthetic medium at 25?. Cells
were treated with a-factor (10 ng/ml) for 2 hr at 25? and
washed twice with H2O. This culture was split into two equal
halves; one was incubated at 25? in methionine-dropout
medium and the other at 37? in complete medium.
Meiotic nuclear spreads and fluorescence microscopy:
Yeast surface nuclear spreads were performed as previously
described (Jin et al. 2009). Rec8-3HA, Scc3-3HA, and Smc3-
3HA were detected by an anti-HA antibody (12CA5, Roche).
FITC-conjugated goat anti-mouse was used as secondary anti-
body (Jackson ImmunoResearchLaboratories). Chromosomal
DNA was stained with DAPI. Fluorescence images were
acquired with a 3100 objective lens (NA ¼ 1.40) mounted on
a motorized microscope (AxioImager, Zeiss). Acquired mono-
tone images were merged by AxioVision software (Zeiss). For
assay of sister-chromatid cohesion, yeast aliquots were with-
drawn at 2-hr intervals and fixed with 1% formaldehyde. Green
fluorescent protein (GFP) foci were visualized by fluorescence
microscopy. At least 100 cells were counted at each time point.
Western blot: Yeast aliquots were collected at 2-hr intervals
and processed by the trichloroacetic acid (TCA) method for
total protein extraction as previously described (Jin et al.
2009). Standard SDS-PAGE and Western blot procedures were
followed (Sambrook and Russell 2001). Rec8-3HA and Scc3-
3HA were detected by an anti-HA antibody (12CA5, Roche).
Smc3-V5 was detected by an anti-V5 antibody (Invitrogen).
Dmc1 and Mcd1 were detected by protein-specific antibodies
(gifts of D. Bishop, University of Chicago, and V. Guacci,
Carnegie Institution). GFP was detected by a GFP-specific
antibody (Ab290, Abcam). The level of Tub2 (b-tubulin)
served as a loading control.
Northern blot and RT-PCR: Yeast aliquots were collected at
intervals after induction of meiosis or after G1-phase release.
We extracted total RNA and performed standard Northern
blots (Sambrook and Russell 2001). Gene-specific probes
were used to detect the mRNA of genes of interest. Labeled
blots were scanned with the Storm PhosphorImager (GE).
Signal intensity was quantified with the IPLab software
(Scanalytics). We used the RNeasy kit (Qiagen) to extract
and purify mRNA. Purified mRNA was reverse-transcribed to
cDNA (Invitrogen), and a semiquantitative PCR method was
specific primers (primer information appears in Table S2).
Chromatin immunoprecipitation: Yeast cells were induced
to undergo synchronous meiosis, fixed with 1% formaldehyde
for 2 hr at room temperature, and then subjected to a
chromatin immunoprecipitation (ChIP) procedure as de-
scribed previously (Yu and Koshland 2005). We used an anti-
V5 antibody (Invitrogen) for ChIP of Rpb3-V5-tagged yeast
strains. A semiquantitative PCR-based method was used to
detect the enrichment of Rpb3 at the REC8 and DMC1 genes.
Scc3 is required for sister-chromatid cohesion and
nuclear division during yeast meiosis: To deplete Scc3
in meiosis, we replaced the endogenous SCC3 promoter
with the CLB2 promoter, which is expressed only in
vegetative yeast cells (Lee and Amon 2003). Semiquan-
titative analysis of Scc3 by Western blot showed ?85%
depletion of Scc3 in PCLB2SCC3 cells during meiosis
(Figure 1A; t ¼ 8 hr). This conditional scc3 mutant allele
was competent for meiotic DNA replication (Figure S1)
1042W. Lin et al.
and permitted us to determine whether Scc3 is required
and C). We marked the centromere of one homolog of
chromosome V with GFP to assay sister-chromatid co-
hesion (Michaelis et al. 1997). In wild-type cells, sister
chromatids were cohesive and formed one GFP spot
chromatids were not associated after DNA replication,
forming two GFP spots (Figure 1B). We incorporated an
ndt80D mutation to arrest the cells at prophase I (Figure
1C; Xu et al. 1995). Less than 4% of ndt80D cells showed
two GFP spots because chromosomes did not segregate
and sister chromatids remained cohesive. In contrast,
86% of PCLB2SCC3 ndt80D cells formed two GFP spots
conclude that Scc3 is required for sister-chromatid co-
hesion during yeast meiosis.
Next, to determine whether Scc3 is required for
chromosome segregation, we monitored meiotic nu-
clear divisions (Figure S2). In wild-type cells, 12 hr after
induction of meiosis, 80% of cells had finished both
meiosis I and meiosis II nuclear divisions (Figure S2A).
In contrast, less than 5% of PCLB2SCC3 cells were able to
complete either division (Figure S2B). To determine
whether PCLB2SCC3 cells are blocked by the recombina-
Y135F; Keeney et al. 1997) to bypass the checkpoint
(Figure S2, C and D). More than 55% of PCLB2SCC3
spo11-Y135F cells were able to complete at least one
nuclear division when double-strand break formation
was eliminated (Figure S2D). Together, these data sug-
gest that Scc3-depleted cells are competent for meiosis
initiation but are arrested primarily by the recombina-
Reduced Rec8 protein level in Scc3-depleted meiotic
cells: In loss of sister-chromatid cohesion and failure to
complete nuclear division, the mutant phenotypes of
PCLB2SCC3 resemble those of rec8D (Klein et al. 1999).
We therefore localized Rec8 in Scc3-depleted cells by
immunofluorescence (Figure 2A). As previously shown,
in wild-type cells at pachytene of prophase I, Rec8 was
localized along the length of chromosomes that re-
vealed well-defined rod-shaped structures (Figure 2A,
left panels for both WT and PCLB2SCC3). In Scc3-
depleted cells, chromosomes became amorphous, and
only traces of chromosome-associated Rec8 were ob-
served above the background noise (Figure 2A, right
observation, we found very low levels of total Rec8
protein in Scc3-depleted cells by Western blot (Figure
2B). In contrast, the meiosis-specific protein Dmc1 was
produced on time and in quantities similar to those in
wild-type and PCLB2SCC3 cells, although its degradation
was delayed in PCLB2SCC3 cells because these cells were
blocked at prophase I (Figure 2B). Thus, by two
different means, immunofluorescence and Western
blot, we showed that the Rec8 protein level is dramat-
ically lowered when Scc3 is absent in meiosis.
Next, we determined whether Rec8 is required for
maintaining the Scc3 protein level. By immunofluores-
cence, we found that Scc3 remains to be chromosome
associated in the absence of Rec8 (Figure 2C). By
Western blot, we found that the total amount of Scc3
was present at a wild-type level in rec8D cells during
meiosis (Figure 2, C and D). Therefore, Scc3 is required
for mediating a normal level of Rec8 protein in meiosis,
but not the reverse. Our data suggest that Scc3 can bind
to the chromosome without formation of the meiotic
cohesin complex during meiosis. Alternatively, a re-
Figure 1.—Requirement for Scc3 in sister-
chromatid cohesion during yeast meiosis. (A)
Protein levels of Scc3 during yeast meiosis.
Yeast cells were induced for synchronous meio-
sis, and aliquots were withdrawn at indicated
times. Total protein extracts were prepared by
the TCA method for Western blots, which were
probed by anti-HA (12CA5) and anti-b-tubulin
antibodies. The level of Tub2 (b-tubulin)
served as a loading control. Note that Scc3
was largely depleted in meiosis in PCLB2SCC3
cells. MT, mitosis. Protein extracts were pre-
pared from cells grown asynchronously in
YPD medium. Wild-type (WT), strain 3072;
PCLB2SCC3, strain 3200. (B and C) Assay of sis-
ter-chromatid cohesion in strains 3078C, 3206,
HY2130, and HY1472. Yeast aliquots were with-
drawn at indicted time points and fixed for
fluorescence microscopy. An array of tetO was
inserted at the URA3 locus, ?35 kb from centro-
mere V. Expression of tetR-GFP generated a
GFP signal that could be visualized as a dot by fluorescence microscopy. Cohesed sister chromatids formed only one GFP
dot. At least 100 cells were counted at each time point.
Cohesin Regulates Gene Expression1043
sidual level of the mitotic cohesin complex remained in
Smc1 and Smc3 are the other subunits of the meiotic
cohesin, and as an example, we show that Smc3 was
present at similar levels in wild-type and Scc3-depleted
meiotic cells (Figure 3A). Therefore, Scc3 plays a
specific role in maintaining a normal level of the
meiosis-specific cohesin subunit Rec8. In Scc3-depleted
cells, however, Smc3 failed to bind to meiotic chromo-
somes (Figure 3B), suggesting that Scc3 is required for
Smc3 chromosome association.
To determine whether the prophase block of Scc3-
depleted cells led to lowered levels of Rec8, we assayed
the total protein level of Rec8 in spo11-Y135F and
PCLB2SCC3 spo11-Y135F double-mutant cells by Western
blot (Figure 3C). Wild-type and spo11-Y135 cells did not
differ in the production and degradation of Rec8
(Figure 2B and Figure 3C, left panels). In contrast,
Rec8 protein level remained low in PCLB2SCC3 spo11-
Y135F cells (Figure 3C, right panels). Therefore, re-
duced Rec8 level in Scc3-depleted cells is not caused by
prophase I block.
Scc3 regulates REC8 gene expression by increasing
REC8 promoter activity: We hypothesized that Scc3
regulates REC8 gene expression in yeast meiosis. To
determine the level of REC8 mRNA, we harvested yeast
cells undergoing synchronous meiosis and performed
Northern blots (Figure 4A). In wild-type cells, REC8
transcripts appeared after 2 hr, peaked at ?4–6 hr, and
diminished 12 hr after induction of meiosis (Figure 4A).
of the wild type (Figure 4A). Quantitative analysis by RT-
PCRrevealed that REC8 mRNA inScc3-depleted cellswas
65% lower than that of the wild type 6 hr after induction
of meiosis (Figure 4B). In contrast, the expression of the
meiosis-initiating gene IME1 was reduced by only ?20%
in REC8 gene expression in yeast meiosis.
Our Northern blots also showed that the expression
of SCC3 was largely abolished in PCLB2SCC3 cells during
meiosis (Figure 4A). On the other hand, the level of
SCC3 transcripts in rec8D remained comparable to that
of wild type (Figure 4A), which is consistent with the
rec8D cells (Figure 2D). Therefore, Rec8 is not required
for meiotic expression of SCC3.
To determine whether Scc3 is responsible for REC8
gene transcription during yeast meiosis, we assayed the
density of RNA Pol II binding to the REC8 gene by ChIP
(Figure 4C). Using thePol II subunit Rpb3 as a readout,
we found that the association of Rpb3 with the REC8
gene was reduced by ?70% in PCLB2SCC3 cells after
normalization of Rpb3’s binding to the DMC1 gene
Figure 2.—Reduced Rec8 protein level in
Scc3-depleted cells. Yeast cells were induced to
undergo synchronous meiosis as in Figure 1.
(A) Chromosome association of Rec8 in wild-
type (2824) and PCLB2SCC3 (HY2294) cells. Yeast
aliquots were collected 6 hr after induction of
meiosis, and surface nuclear spreads were pre-
pared for immunofluorescence with an anti-HA
antibody. Red, DNA; green, Rec8. (B) Rec8 pro-
tein level in wild-type and PCLB2SCC3 cells in mei-
osis. Yeast aliquots were collected at indicated
times and prepared for Western blot as in A.
An anti-Dmc1-specific antibody was used to de-
tect the level of Dmc1. (C) Chromosome associ-
ation of Scc3 in wild-type (3072) and rec8D
(HY1495) cells. Surface yeast nuclear spreads
were prepared as in A. Note that Scc3 remains
chromosome-bound in rec8D cells. Red, DNA;
green, Scc3. Bar, 2 mm. (D) Scc3 protein level
in wild-type and rec8D cells. Western blots were
prepared as in B. Note that the level of Scc3 re-
mains normal in rec8D cells.
1044 W. Lin et al.
(Figure 4C). Our data therefore suggest that the de-
crease in REC8 mRNA level in Scc3-depleted meiotic
cells is a result of transcriptional inactivation of the
REC8 promoter during yeast meiosis.
To determine further how Scc3 regulates REC8 gene
transcription, we developed a heterologous reporter
assay by using the REC8 promoter to drive the expres-
sion of GFP (Figure 4D and our unpublished data). The
expression of PREC8GFP, which was inserted at the REC8
locus, essentially mirrored that of the REC8 gene in wild-
type cells (Figure 4, A and D). In contrast, the level of
GFP transcripts from PREC8GFP was low in Scc3-depleted
cells during meiosis, ?60% lower than in the wild type
REC8 promoter still occurred in Scc3-depleted cells
(Figure 4), our data suggest that Scc3 is required for
increasing the REC8 promoter activity but not for its
results from residual Scc3 activity in PCLB2SCC3 cells.
Scc3 is not necessary for REC8 translation but is
required for Rec8 chromosome association: To pro-
duce Rec8 in Scc3-depleted cells, we constructed an
inducible allele of REC8 (PCUP1REC8), which served as
theonly source of REC8 in meiosis (Figure 5). Upon the
addition of copper ion at the time of induction of mei-
osis, PCUP1REC8 was expressed in wild-type and Scc3-
depleted cells at comparable levels (Figure 5A). These
REC8 transcripts produced by the CUP1 promoter
appeared to be efficiently translated to produce Rec8
protein (Figure 5B). In wild-type SCC3 cells, ectopically
endogenous Rec8; it peaked at ?6 hr and was degraded
by the end of meiosis (Figure 2B and Figure 5B).
Furthermore, these Rec8 proteins localized to the
meiotic chromosome along its entire length just as the
endogenous Rec8 did (Figure 2A and Figure 5C). In
PCLB2SCC3 cells, PCUP1REC8 was expressed, and these
cells produced amounts of Rec8 similar to those seen in
wild-type cells (Figure 5B). Because PCLB2SCC3 cells
were arrested by the recombination checkpoint at
prophase I, the degradation of Rec8 was delayed in
these cells (Figure 5B). Therefore, in the absence of
Scc3, Rec8 can be produced and remains relatively
stable in meiosis, but ectopically produced Rec8 failed
to bind to the chromosome in PCLB2SCC3 cells (Figure
5C), suggesting that Scc3 is required for Rec8 associa-
tion with the chromosome.
One concern was that the CUP1 promoter perhaps
overexpressed REC8 during meiosis and could obscure
our interpretation. We therefore constructed a PDMC1REC8
allele, which was incorporated at the endogenous REC8
locus and produced Rec8 at a level similar to that of
Dmc1 during meiosis (Figure S3A). The expression lev-
els of PDMC1REC8 in wild-type and Scc3-depleted mei-
otic cells appeared comparable because the two strains
promoter, we constructed a heterologous GFP reporter,
PDCM1GFP, which produced similar amounts of GFP in
wild-type and Scc3-depleted meiotic cells (Figure S3B).
Together, our results suggest that Scc3 is required for
REC8 gene expression because it specifically increases
Figure 3.—Requirement for Scc3 for Rec8 but
not for Smc3 production in yeast meiosis. Yeast
cells were induced for synchronous meiosis, ali-
quots were withdrawn at indicated times, and
protein extracts were prepared for Western blots
probed by anti-V5, anti-HA, and anti-b-tubulin
antibodies. (A) Protein level of Smc3 in wild-type
(HY1510C) and PCLB2SCC3 (HY1566) cells. (B)
Chromosome localization of Smc3 during yeast
meiosis. Yeast cells were collected 6 hr after in-
duction of meiosis and prepared for surface nu-
clear spread as in Figure 2A. Tub1 (a-Tubulin)
was detected by a specific antibody (YOL135).
Red, Smc3; green, Tub1; blue, DNA. (C) Protein
levels of Rec8 in spo11-Y135F (HY1499) and
PCLB2SCC3 spo11-Y135F (HY1483) cells.
Cohesin Regulates Gene Expression1045
REC8 promoter activity during meiosis, but Scc3 is not
necessary for translation of REC8 mRNA.
Scc3 is not necessary for MCD1 gene expression in
proliferating cells: The mitotic counterpart of REC8 is
MCD1, which probably arose from an ancient genome-
duplication event (Kellis et al. 2004). To determine
whether Scc3 plays a similar role in regulating MCD1
transcription in proliferating yeast cells, we generated a
degron allele of scc3 (PMET1-DEGRON-SCC3) to deplete
Scc3 in vegetative cells and observed MCD1 gene
transcription and protein production with Northern
and Western blots (Figure 6). To synchronize yeast
culture, we used a-factor to arrest cells at the G1 phase
deplete Scc3 (Figure 6, A and B). The transcription of
SCC3 was completely shut off in cells that were shifted
to the nonpermissive condition (Figure 6C), and Scc3
became depleted in these cells after G1 phase release
(Figure 6D). In contrast, MCD1 was expressed after G1
release, and its level of expression did not appear to
differ greatly, except that cells expressed MCD1 earlier
at the elevated temperature (Figure 6C). As a result,
Mcd1 protein levels in these two treatments were
comparable (Figure 6D). Scc3 is therefore required
for positively regulating REC8 gene expression in
Mcd1 remains relatively stable when Scc3 is absent in
either meiotic or mitotic cells.
Smc1 has a role similar to that of Scc3 in positively
regulating REC8 gene expression during meiosis: To
determine whether cohesin subunits other than Scc3
using the same CLB2 promoter-replacement approach
REC8 transcript was dramatically reduced in Smc1-
depleted cells (Figure 7B). Consequently, the Rec8 pro-
tein level was very low in mutant cells (Figure 7C). In
at comparable levels in wild-type and PCLB2SMC1 cells
for Rec8 production in meiosis. Using the PREC8GFP
reporter assay, we found that the production of GFP was
dramatically reduced in Smc1-depleted meiotic cells
(Figure 7D). Taken together, our results suggest that
the cohesin complex is required for positively regulating
the REC8 gene transcription during yeast meiosis.
The presence of sister chromatids is not necessary
for REC8 gene activation: To determine whether sister-
chromatid cohesion is required for activating REC8
Figure 4.—Scc3 regulates REC8 promoter activity during
yeast meiosis. (A) mRNA levels of IME1, REC8, SCC3, and
ACT1 in wild-type (NH144), PCLB2SCC3 (3200), and rec8D
(HY1495) cells. Yeast cells were induced to undergo synchro-
nous meiosis, and aliquots were withdrawn at the indicated
times and prepared for Northern blots probed by gene-
specific probes. (B) RT-PCR analysis of IME1, REC8, and
ACT1 transcripts. Yeast aliquots were withdrawn 6 hr after in-
duction of meiosis; total mRNA was extracted, reversed to
cDNA, and amplified by gene-specific primers. (Right) Quan-
titative analysis with an average of two independent experi-
ments shown. Error bars show standard deviation. (C) ChIP
of Rbp3 in wild-type (HY3000) and PCLB2SCC3 (HY3003) cells
during yeast meiosis. Yeast cells were induced to undergo syn-
chronous meiosis; aliquots were withdrawn 6 hr after induc-
tion and prepared for ChIP analysis. (Left) A representative
gel image. (Right) Quantitative analysis of Rpb3 binding at
the REC8 and DMC1 genes from two independent experi-
ments. (D) A heterologous reporter assay of REC8 promoter
activity (HY2106 and HY2108). Plasmid pHG105 was digested
with MluI and transformed into the REC8 locus. Yeast cells
were induced to undergo synchronous meiosis, and aliquots
were withdrawn for Northern blots as shown in A. Gene-
specific probes were used to detect the mRNA levels of
IME1, GFP, and ACT1.
1046W. Lin et al.
gene expression, we used a genetic approach to abolish
meiotic DNA replication with the PSCC1CDC6 allele
(Hochwagen et al. 2005). In Cdc6-depleted meiotic
absent (data not shown), but Rec8 was produced effi-
ciently in PSCC1CDC6 cells because its protein level
was comparable to that of the wild type during meiosis
(Figure 7E). In addition, immunofluorescence micros-
copy revealed that Rec8 was localized to the chromo-
somes in Cdc6-depleted cells (Figure 7F). Chromosome
axes in the PSCC1CDC6 cells resembled those from the
wild-type cells even though Cdc6-depleted cells lacked
sister chromatids in meiosis (Figure 7F). These results
suggest that REC8 can be efficiently transcribed in the
absence of sister chromatids, so the presence of sister
chromatids is not necessary for REC8 gene expression.
Additional meiotic genes are subject to cohesin
regulation: To determine whether cohesin globally
regulates gene expression during meiotic differentia-
tion in yeast, we surveyed the gene-expression pattern
using theexpressionmicroarray.Expressionof27 genes
was reduced by .75% in Smc1-depleted meiotic cells
6 hr after induction of meiosis; only 8 genes showed
more than a fourfold increase (data not shown). We
focused on the expression pattern of ?52 meiotic genes
such as REC8 that belonged to the category of ‘‘early
genes’’ in meiosis (Chu et al. 1998). Among them, we
found by microarray analysis that the expression level of
two genes (MRD1 and PAD1) was lowered by ?50% in
the PCLB2SMC1 mutant (Figure S4). The expression
level of REC8 was only slightlyreduced in comparison to
that of DMC1 (Figure S4), demonstrating that our
microarray analysis of meiotic gene expression is qual-
itative at best. Whether the cohesin target genes share
common features is currently unknown, but our pre-
liminary analysis supports the idea that cohesin has a
positive role in meiotic gene expression.
Using a genetic approach, we have shown that
cohesin subunits Scc3 and Smc1 are required for effi-
cient transcription of a target gene, REC8, because they
increase its promoter activity during yeast meiosis. Co-
hesin is a major chromosomal factor required for sister-
chromatid cohesion (Guacci et al. 1997; Michaelis
et al. 1997), but its emerging role in regulation of
gene expression is best known in animal development
2008). Nonlethal mutation in genes that encode cohe-
sin and cohesin-associated factors in humans is directly
linked to developmental disorders collectively called
cohesinopathies, which include Cornelia de Lange
syndrome and Roberts syndrome (Liu and Krantz
2009). The etiology of these human diseases remains
to be elucidated. Our work in yeast meiosis using the
REC8 promoter activity as a readout of cohesin function
in gene regulation lends support to the notion that this
noncanonical cohesin activity is evolutionarily con-
served; it also provides molecular insights into cohesin’s
role in cell differentiation and development.
Four lines of evidence support the idea that the
cohesin complex increases REC8 gene expression by
modulating the REC8 promoter activity during meiosis.
First, Scc3and Smc1 have similar effects on regulation of
REC8 gene expression; second, Scc3 modulates the
density of Pol II binding to the REC8 gene; third, a
Figure 5.—Ectopic expression of REC8 in mei-
otic cells. (A) The expression level of PCUP1REC8
in wild-type (HY1417C) and PCLB2SCC3 (HY1417)
cells during meiosis. Yeast cells were induced
for synchronous meiosis, and aliquots were with-
drawn at indicated times for Northern blots as
shown in Figure 4A. Note that PCUP1REC8 is ex-
pressed in PCLB2SCC3 cells. (Right) A semiquan-
titative measurement of REC8 transcripts over
those of ACT1. (B) Protein level of Rec8 in
wild-type and PCLB2SCC3 cells. Western blots were
prepared as in Figure 1A to reveal the levels of
Rec8-3HA and b-tubulin. (C) Chromosome asso-
ciation of Rec8 in wild-type and PCLB2SCC3 cells.
Yeast surface nuclear spreads were prepared for
immunofluorescence as in Figure 2A. Note that
Rec8 is produced but does not bind to chromo-
somes in PCLB2SCC3 cells. Red, DNA; green,
Rec8. Bar, 2 mm.
Cohesin Regulates Gene Expression1047
heterologous reporter assay using the REC8 promoter
shows that it is under the influence of cohesin; and
finally, the REC8 open reading frame driven by the
inducible CUP1 or the meiosis-specific DMC1 promoter
can betranscribedand translated atcomparablelevelsin
specific cohesin subunit, feedback control by meiotic
cohesin of REC8 promoter activation is not surprising
(W. Lin, H. Jin and H. Yu, unpublished data). In ad-
formation and its association with the chromosome were
important, the cohesin loader, the Scc2/Scc4 complex,
would have a similar role in meiotic gene activation.
Indeed, our analysis of Scc2 in yeast meiosis shows that it
is required for recruiting cohesin to the chromosome to
activate the cohesin-regulated promoter REC8 (W. Lin,
H. Jin and H. Yu, unpublished data), but our observa-
tion differs from those in the fly, where cohesin and its
loader Scc2 (called Nipped B) apparently have opposite
effects on gene regulation (Rollins et al. 2004). The
reason for this discrepancy is currently unknown.
How, then, does cohesin activate gene transcription
in yeast? In vertebrates, direct binding of cohesin to the
transcriptional factor CTCF, which has been implicated
in insulating gene transcription, may explain cohesin’s
2008; Stedman et al. 2008; Wendt et al. 2008). In yeast,
no equivalent of CTCF is yet known, but currently no
evidence indicates that cohesin binds directly to the
transcriptional machinery. Upon meiotic differentia-
tion, yeast cells reorganize the higher-order chromo-
some structure that necessitates the change of gene
expression pattern (Kassir et al. 2003), of which
cohesin could act as an important chromosomal factor.
complex RSC and also interacts with modified histones
during double-strand break repair (Unal et al. 2004;
Chai et al. 2005). Finally, a recent study in yeast showed
that scc2 and eco1 mutations that mimic human dis-
eases lead to altered chromosome organization (Gard
et al. 2009). Therefore, cohesin-mediated chromosome
organization may facilitate the recruitment of transcrip-
tional factors to the 59 upstream sequences of cohesin-
regulated genes to activate or repress gene expression.
Alternatively, cohesin might directly interact with the
transcriptional factors, for example, with the mediator
(Kagey et al. 2010), to regulate gene expression. These
two possibilities are not mutually exclusive, but this
study does not distinguish between them.
Cohesin is required for REC8 gene expression during
meiotic differentiation but not for that of its duplicated
not seem to regulate meiotic genes universally because
the expression of the meiosis-specific genes IME1,
DMC1, and others is largely unaffected in Scc3- or
Smc1-depleted cells (this report and data not shown).
Figure 6.—Scc3 is not required for MCD1 ex-
pression in vegetative cells. (A) A diagram show-
ing the experimental procedure. (B) Yeast
budding index showing cell progression. Yeast
aliquots were withdrawn at indicated times after
G1 release, fixed, and examined by phase-con-
trast microscopy. (C) mRNA levels of SCC3,
MCD1, and ACT1 after release from a-factor ar-
rest. Yeast aliquots were withdrawn at indicated
times and prepared for Northern blots probed
by gene-specific probes as shown in Figure 4A.
Note that MCD1 is expressed only after release
from a-factor arrest. (D) Protein levels of Scc3
and Mcd1. Yeast aliquots were withdrawn at indi-
cated times and prepared for Western blots
probed by anti-HA, anti-Mcd1, and anti-b-tubulin
antibodies. Note that Mcd1 remains at a normal
level in Scc3-depleted vegetative cells.
1048 W. Lin et al.
These observations imply that a complex interplay takes
place between trans-acting factors and cis-acting DNA
sequences in regulating the expression of cohesin-
target genes during meiotic differentiation. Cohesin
associates with the chromosome at specific loci of the
yeast genome, which are predominately located at re-
gions of convergent transcription (Glynn et al. 2004;
Lengronne et al. 2004). These binding sites would
position cohesin toward the 39-end of the transcribed
genes, rather that at promoter-proximal sequences,
which might explain why only a subset of meiotic genes
is subject to cohesin regulation (this study and our
unpublished data). In this regard, our study is consis-
tent with a recent observation of cohesin activity in G1-
arrested vegetative cells, showing that a small number of
genes changed their expression pattern in response to
Our genetic analysis using the cdc6 mutant indicates
that the primary role of cohesin in sister-chromatid
cohesion is not necessary for its regulation of its target
gene. In the cdc6 mutant, cohesin, revealed by Rec8,
is localized to the meiotic chromosome axis in a way
that is similar to that in the wild type. Because sister-
chromatid cohesion is coupled to DNA replication in
yeast (Uhlmann and Nasmyth 1998), our results sug-
gest that chromosomal binding of cohesin is sufficient
for carrying out cohesin’s function in gene regulation.
Therefore, cohesin’s role in sister-chromatid cohesion
Our results also lend support to the notion that reg-
ulation of gene expression by cohesin is independent
of sister-chromatid cohesion in postmitotic and dif-
ferentiating animal cells (Horsfield et al. 2007; Pauli
et al. 2008; Schuldiner et al. 2008; Nativio et al. 2009).
In summary, we have shown that cohesin plays a
positive role in target gene activation during yeast
meiotic differentiation. Lack of cohesin is detrimental
to yeast meiosis in many aspects, including gene tran-
scription, recombination, and chromosome segregation.
Figure 7.—Activation of REC8 promoter requires Smc1 but
not sister-chromatid cohesion. (A) Depletion of Smc1 during
meiosis (2821 and HY1875). Yeast cells were induced to un-
dergo synchronous meiosis, and aliquots were withdrawn at
theindicatedtimes forWesternblotsasinFigure 1A.(B)Tran-
scriptional level of REC8 during meiosis (NH144 and HY1875).
Total RNA was extracted and probed with gene-specific probes
as in Figure 4A. (C) Protein level of Rec8 in wild-type
(HY1503C) and PCLB2SMC1 (HY1868) cells in meiosis. Yeast
protein extracts were prepared for Western blots, which de-
tected the levels of Rec8 and Dmc1 as shown in Figure 2B.
The level of b-tubulin served as a loading control. (D) A het-
erologous reporter assay of REC8 promoter activity in wild-type
(HY2460) and PCLB2SMC1 (HY2460-1) cells in meiosis.
PREC8GFP wasplaced atthe URA3 locusbytransformation. Yeast
cells were induced to undergo synchronous meiosis, and ali-
quots were withdrawn at the indicated times and prepared for
Western blots probed by anti-GFP (Ab290) and anti-b-tubulin
antibodies. (E) Rec8 protein level in wild-type (HY2740)
and PSCC1CDC6 (HY2741) cells during meiosis. Representative
time points are shown. (F) Chromosome localization of Rec8
in wild-type and PSCC1CDC6 cells during meiosis. Yeast cells
were collected 6 hr after induction of meiosis and prepared
for surface nuclear spread as in Figure 2A. Note that chromo-
somes still formed rod-shaped structures in the absence of sis-
ter chromatids. Red, DNA; green, Rec8. Bar, 2 mm.
Cohesin Regulates Gene Expression1049
The identification of cohesin target genes in yeast
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biological significance and mechanism of cohesin func-
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Cohesin Regulates Gene Expression1051
Cohesin Plays a Dual Role in Gene Regulation and Sister-Chromatid
Cohesion During Meiosis in Saccharomyces cerevisiae
Weiqiang Lin, Mian Wang, Hui Jin and Hong-Guo Yu
Copyright ? 2011 by the Genetics Society of America
W. Lin et al.
FIGURE S1.—FACS analysis of meiotic S-phase progression in wild-type (NH144) and PCLB2SCC3 (3200). Yeast cells were
induced to undergo synchronous meiosis, and aliquots were withdraw at indicated times and prepared for FACS determination of
DNA content. Time in hours is shown to the left.
W. Lin et al.
FIGURE S2.—Requirement for Scc3 in nuclear division during yeast meiosis. Yeast cells were induced to undergo synchronous
meiosis; aliquots were withdraw at indicated times, fixed in 4% formaldehyde, stained with DAPI, and visualized by fluorescence
microscopy. (A–D) Meiotic nuclear division in wild-type (WT, NH144), PCLB2SCC3 (3200), spo11-Y135F (HY1499), and PCLB2SCC3
spo11-Y135F (HY1483) cells. At least 100 cells were counted at each time point.
W. Lin et al.
FIGURE S3.—The activity of the DMC1 promoter is not subject to Scc3 regulation in meiosis. (A) Ectopic production of Rec8
in meiosis with PDMC1REC8 (HY2207 and HY2226). Yeast cells were induced for synchronous meiosis, and protein extracts were
prepared for western blots probed by anti-HA and anti-b-tubulin antibodies. Note that Rec8 is produced by PDMC1REC8 in
PCLB2SCC3 cells. (B) A heterologous reporter assay of DMC1 promoter activity in wild-type (HY2464) and PCLB2SCC3 (HY2466)
cells. PDMC1GFP was inserted at the URA3 locus by standard yeast transformation with pHG112, which was digested by AflII.
Yeast cells were induced for synchronous meiosis, and protein extracts were prepared for western blots probed by anti-GFP and