MOLECULAR AND CELLULAR BIOLOGY, Feb. 2008, p. 1393–1403
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 4
Spn1 Regulates the Recruitment of Spt6 and the Swi/Snf Complex
during Transcriptional Activation by RNA Polymerase II?†
Lei Zhang,1‡ Aaron G. L. Fletcher,1Vanessa Cheung,2Fred Winston,2and Laurie A. Stargell1*
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523,1and Department of
Genetics, Harvard Medical School, Boston, Massachusetts 320032
Received 20 September 2007/Returned for modification 12 October 2007/Accepted 27 November 2007
We investigated the timing of the recruitment of Spn1 and its partner, Spt6, to the CYC1 gene. Like TATA
binding protein and RNA polymerase II (RNAPII), Spn1 is constitutively recruited to the CYC1 promoter,
although levels of transcription from this gene, which is regulated postrecruitment of RNAPII, are low. In
contrast, Spt6 appears only after growth in conditions in which the gene is highly transcribed. Spn1 recruit-
ment is via interaction with RNAPII, since an spn1 mutant defective for interaction with RNAPII is not targeted
to the promoter, and Spn1 is necessary for Spt6 recruitment. Through a targeted genetic screen, strong and
specific antagonizing interactions between SPN1 and genes encoding Swi/Snf subunits were identified. Like
Spt6, Swi/Snf appears at CYC1 only after activation of the gene. However, Spt6 significantly precedes Swi/Snf
occupancy at the promoter. In the absence of Spn1 recruitment, Swi/Snf is constitutively found at the promoter.
These observations support a model whereby Spn1 negatively regulates RNAPII transcriptional activity by
inhibiting recruitment of Swi/Snf to the CYC1 promoter, and this inhibition is abrogated by the Spn1-Spt6
interaction. These findings link Spn1 functions to the transition from an inactive to an actively transcribing
RNAPII complex at a postrecruitment-regulated promoter.
For a large number of well-characterized genes, the rate-
limiting step in the transcription process is the formation of the
preinitiation complex at the promoter. At these genes, the
recruitment of TATA binding protein (TBP) and RNA poly-
merase II (RNAPII) to the promoter correlates strongly with
transcriptional output (35, 41, 67). Indeed, delivery of TBP and
RNAPII appears to be sufficient for gene activation in many
contexts (for reviews, see references 63 and 64). However,
there are a growing number of genes that have been found to
be regulated at a step after the recruitment of RNAPII. Such
genes include the yeast CYC1 gene, the Drosophila heat shock
genes, and mammalian human immunodeficiency virus type 1
and the c-Myc proto-oncogene (3, 39, 41, 51, 77). Indeed,
whole-genome studies suggest that a significant portion of the
human genome may be regulated postrecruitment of RNAPII
(26, 35). As such, these mechanisms have the potential to
impact the expression of thousands of human genes. Our un-
derstanding of these mechanisms and the rate-limiting steps
involved is incomplete, but these observations suggest that
functions critical for a high level of transcription are either
inhibited or absent under noninducing conditions. To deter-
mine the nature of these functions, further characterization of
genes regulated after the assembly of the general transcription
machinery is imperative.
The yeast CYC1 gene encodes iso-1-cytochrome c, a protein
involved in the electron transport chain in the mitochondria
(75). In the presence of a fermentable carbon source (such as
dextrose), CYC1 gene expression is inhibited and transcrip-
tional levels are extremely low (24, 25). When cells are grown
on a nonfermentable carbon source (such as lactate or etha-
nol), CYC1 is activated and transcriptional output is induced
approximately 10-fold. In contrast to these dramatic changes in
transcriptional output, the occupancy of TBP and RNAPII at
the CYC1 promoter changes very little during the carbon
source change (41, 51). Thus, CYC1 gene expression is regu-
lated at a step after the recruitment of these two essential
members of the general transcription machinery. Postrecruit-
ment mechanisms of gene regulation have been observed in all
organisms, ranging from bacteria to yeasts to flies to humans
(3, 35, 39, 41, 51, 67). Thus, knowledge gained regarding post-
recruitment regulation of gene expression in the highly ame-
nable yeast system has the potential to reveal universal mech-
SPN1, a gene that is essential in yeast and highly conserved
throughout evolution, appears to play a critical role in regu-
lating transcription after assembly of the general transcription
machinery. Mutation of SPN1 results in elevated levels of tran-
scription from the CYC1 gene under noninducing (dextrose)
conditions (19). Moreover, Spn1 genetically interacts with TBP
and Spt4 and physically interacts with Spt6, factors with known
roles in transcription initiation, elongation, processing, and
chromatin remodeling (19, 21, 38, 46). Therefore, we set out to
determine the functional requirement for Spn1 at the postas-
sembly-regulated CYC1 gene.
We show that Spn1, like TBP and RNAPII, is constitutively
recruited to the CYC1 promoter. Moreover, TFIIH, capping
enzyme, and serine-5 phosphorylation of the C-terminal do-
main of Rpb1 are also present at the CYC1 promoter prior to
* Corresponding author. Mailing address: Department of Biochem-
istry and Molecular Biology, Colorado State University, Fort Collins,
CO 80523. Phone: (970) 491-5068. Fax: (970) 491-0494. E-mail: Laurie
† Supplemental material for this article may be found at http://mcb
‡ Present address: Department of Dermatology, School of Medi-
cine, University of Colorado Health Sciences Center, Aurora, CO
?Published ahead of print on 17 December 2007.
induction of transcription. Spn1 is targeted to the promoter via
interaction with RNAPII, since an spn1 mutant defective for
interaction with RNAPII fails to occupy CYC1. Spt6 appears at
CYC1 only after the gene is activated. In the absence of Spn1
promoter occupancy (in the spn1 mutant strain), Spt6 is no
longer recruited to the CYC1 promoter, indicating that Spn1 is
necessary for Spt6 recruitment. In addition, the results from a
genetic screen reveal that the Swi/Snf chromatin-remodeling
complex and Spn1 have strong antagonizing functions and that
recruitment of Swi/Snf is also impacted by Spn1. Taken to-
gether, these studies indicate that Spn1 is essential for coordi-
nating the recruitment of chromatin-remodeling factors for the
proper expression of the postassembly-regulated CYC1 gene.
MATERIALS AND METHODS
Yeast strains. The deletion collection strains (83) and the parental BY4741
strain (MATa his3?1 ura3?0 leu2?0 met15?0) were purchased from Research
Genetics. A subset of these deletion strains was chosen to study the genetic
interactions between SPN1 and the genes deleted in these strains (see Table S1
in the supplemental material). For phenotypic studies, genomic SPN1 in these
strains was deleted and covered by either wild-type SPN1 or spn1K192Non
The snf2? strain (MAT? ada2? ada3? leu2?1 ura3?0 snf2::Kanr) was kindly
provided by Caroline Kane (15). The spt16-11 and pob3-7 mutants and their
parental strain (MATa trp1 leu2 ura3 his3) were provided by Timothy Formosa
Yeast medium. Yeast media used to analyze phenotypic changes were made as
described in the literature (27). 5-Fluoroorotic acid plates were made as de-
scribed previously (7). YPGal and YPEG plates were made by replacing the
dextrose in YPD with 2% galactose, 2% ethanol, and 2% glycerol. Plates con-
taining sorbitol, NaCl, and H2O2were made by supplementing YPD medium
with 1 M sorbitol, 1 M NaCl, and 4 mM H2O2. Medium lacking inositol (Ino?)
was made as described (30). Mycophenolic acid (MPA) plates were made by
supplementing SC-U plates with 20 ?g/ml MPA.
Plasmid construction. A 2.2-kb fragment of the LEU2 gene containing its
promoter, coding region, and terminator was amplified from yeast genomic DNA
and subcloned into the pJF201 (TRP1 CEN) plasmid (19) to replace the SPN1
open reading frame (ORF). The resulting plasmid, pLT-1, has the LEU2 gene
flanked by the SPN1 promoter and terminator and was used to produce the
SPN1::LEU2 fragment for genomic SPN1 deletion. An SPN1-covering plasmid
(pUS-1) was created by ligation of the TOA1 promoter, SPN1 ORF, and TOA1
terminator and subcloning into pRS316 (URA3 CEN). Two 1.7-kb fragments
containing the SPN1 promoter and terminator and either the wild-type SPN1
ORF or spn1-K192N were isolated from pJF201 or pJF202, respectively, and
subcloned into pRS313 (HIS3 CEN) to generate pHS-1 (wild type) and pHS-2
Genetic screen. To combine the spn1-K192N allele with the deletion mutants
of various RNAPII transcriptional factors, strains were transformed first with a
URA3-marked engineered SPN1-encoding cover plasmid (pUS-1, which contains
the promoter and terminator from TOA1). Subsequently, the genomic SPN1
ORF was deleted by LEU2 replacement using the insert from pLT-1 and ho-
mologous recombination with sequences within the promoter and terminator of
SPN1. The use of the TOA1 promoter and terminator on the plasmid-borne copy
of SPN1 targeted the one-step disruption solely to the genomic copy of SPN1.
The deletion of genomic SPN1 in the mutant strains was confirmed by PCR (data
not shown). The SPN1 gene (wild type; pHS-1) or the spn1-K192N derivative
(pHS-2) on a HIS3-marked plasmid was then introduced by plasmid shuffling.
The expression levels of the plasmid-borne wild-type SPN1 and spn1-K192N
molecule are comparable to that of genomic SPN1 (data not shown). To assay
the genetic interactions of SPN1 with different transcription factors, the strains
were grown under 10 different conditions. Phenotypic changes were scored by
comparing the growth of strains covered by SPN1 versus spn1-K192N under the
following conditions: 30°C, 38°C, and 14°C; 1 M NaCl, 1 M sorbitol, 4 mM H2O2,
and Ino?media; glucose versus galactose or ethanol/glycerol as a carbon source;
50 mM aminotriazole (AT); or growth on 20 ?g/ml MPA.
Transcriptional assays. S1 nuclease assays were conducted as described pre-
viously (19). For CYC1 induction, cultures grown overnight in rich medium
containing 2% glucose were washed three times in medium lacking glucose,
diluted into medium containing 3% ethanol, and cultured at 30°C for 6 h. For
uninduced samples, cells were grown in YPD for 6 h at 30°C until the optical
density reached 0.8 to 1.0. Yeast cells were then harvested, and total RNA was
isolated by hot-phenol extraction. Hybridizations with excess probe were nor-
mally done with 25 to 30 ?g of RNA samples hybridized with excess32P-labeled
probe overnight at 55°C. S1 nuclease digestion was performed on the hybridized
samples for 30 to 45 min at 37°C. Band intensity was normalized to the intensity
of the tRNAwband.
Coimmunoprecipitation experiments. Coimmunoprecipitation experiments
were performed with the indicated strains (Table 1) as described previously (55)
with a few modifications. Cultures were grown to an optical density (600 nm) of
about 1.0 in rich medium containing 2% dextrose. Cell extracts (300 ?g) were
used immediately following preparation and were precleared by incubation with
50 ?l plain protein A-Sepharose beads (Pharmacia) for 1 h at 4°C. A small
sample was taken after the preclear step to provide a load control. Antihemag-
glutinin (anti-HA) (Santa Cruz), polyclonal anti-Spn1, and anti-Rpb1 (8WG16;
Covance, Inc.) antibodies were coupled to protein A-Sepharose beads, and the
remaining extract was incubated with 50 ?l of these coupled beads for 2 h at
room temperature with occasional stirring. After six washes, the beads were
boiled in loading buffer and 15 ?l was loaded for sodium dodecyl sulfate-
polyacrylamide gel electrophoresis, followed by immunoblot analysis.
ChIP analysis. Chromatin immunoprecipitations (ChIPs) were performed
with the indicated strains (Table 1) as described previously (79), with a few
modifications. Cells (150 ml) were grown to an optical density at 600 nm of 0.8
to 1.0. Cells were treated with a final concentration of 1% formaldehyde for 15
min, with occasional swirling of the flasks at intervals of 5 min. Glycine was added
to a final concentration of 125 mM at room temperature and left for 5 min to
stop cross-linking. Cells were collected and washed twice in ice cold Tris-buffered
saline. Cells were resuspended in FA-lysis buffer (500 ?l of FA-lysis buffer for
a total of 50 ml of cell culture). Chromatin was sheared by sonication using a
Branson W-350 Sonifier (10 times at 10 seconds each on continuous pulse at a
microtip power setting of 6). A portion (10%) of the chromatin material used for
the immunoprecipitation was processed as the input after reversing the cross-
links and purifying the DNA. About 500 ?l of the chromatin material was
incubated with approximately 5 ?l of either anti-Spn1, anti-HA (Santa Cruz),
anti-Myc (Upstate), anti-RNAPII (8WG16; Covance, Inc.), or anti-serine
5-phosphorylated C-terminal domain (CTD) (H14; Covance, Inc.) antibodies by
rotation overnight at 4°C. A 50-?l portion of protein-A Sepharose beads (Phar-
macia; prepared as a slurry as per the manufacturer’s directions) was further
incubated with the chromatin material for 2 h at 4°C. The beads were spun down,
and the antigen-antibody complexes bound to the beads were recovered and
further treated with Tris-EDTA-sodium dodecyl sulfate buffer for 15 min at 65°C
to elute the complexes. Protein-DNA cross-links were reversed by incubation
overnight at 65°C, and the DNA was purified by phenol-chloroform extraction
and used for the PCR analysis.
PCRs were carried out in a total volume of 25 ?l. Each reaction mixture
contained 1 ?l of a 1/100 dilution of 10-mCi/ml32P-labeled dATP. Different
dilutions of each input and immunoprecipitated samples were used to determine
the linear range of the PCR. The PCR products were run on 5% native poly-
acrylamide gels in 0.5? Tris-borate-EDTA buffer. The gels were dried and
exposed to a phosphorimage screen. Images were scanned by STORM and
quantified using ImageQuant software analysis to detect the strengths of various
signals. The primers used to amplify the promoter region of CYC1 (?234 to ?79)
TABLE 1. Yeast strains used for coimmunoprecipitation and
SK1 .....................MAT? ura3-52 trp1-?63 his3?200 leu2?2::PET56
JF101?.................SK1 SPT6-3HA::Kanrspn1::LEU2 YCp22-myc-
JF102 ..................SK1 SPT6-3HA::Kanrspn1::LEU2 YCp22-myc-
LZ101.................BY4741 SNF2-3HA::HIS3 spn1::LEU2/YCp50-SPN1
FY2134...............MATa his4-912? lys2-128? leu2?1RPB3-HA::LEU2
FY2135...............MATa his4-912? lys2-128? leu2?1RPB3-HA::LEU2
1394ZHANG ET AL.MOL. CELL. BIOL.
were 5?AGGCGTGTATATATAGCGTGGAT3? and 5?CCACGGTGTGGCA
TTGTAGACAT3?. The signal strength ratio between the immunoprecipitated
sample and the input after subtracting the signal of a no-antibody control was
used as an indication of the occupancy of the protein. Each experiment was
repeated (from independent cultures of cells) a minimum of three times.
Spn1 is found in association with Spt6 (21, 38), a factor with
important functions for transcription through chromatin (2, 8,
31). To examine the mechanistic relationship between Spn1
and Spt6 at a promoter that is regulated postassembly of TBP
and RNAPII, we examined their occupancy levels on the CYC1
gene via ChIP analysis. Occupancy levels of Spn1 on the CYC1
promoter are comparable under uninduced (partial repres-
sion) and induced (activation) conditions (Fig. 1A and C).
Thus, Spn1 is constitutively recruited to CYC1, like TBP and
RNAPII (41, 51), In contrast to the case for Spn1, TBP, and
RNAPII, occupancy levels of Spt6 are low under uninduced
conditions and increase more than fivefold under inducing
conditions. Thus, Spn1 and Spt6 have separable functions prior
to activation of CYC1, and the appearance of Spt6 correlates
with the transition to an actively transcribing RNAPII com-
Diminished Spn1 occupancy results in failure to recruit
Spt6 to CYC1. In the spn1-K192N mutant (where lysine at
position 192 is replaced with asparagine), CYC1 transcription
levels are elevated under noninducing conditions and are also
abnormally high under inducing conditions (19). These alter-
ations in CYC1 expression suggest that RNAPII is in a tran-
scriptionally more active state at CYC1 in the mutant back-
ground and that a rate-liming step due to Spn1 functions has
been removed. Occupancy levels of Spn1 and Spt6 were thus
determined in the spn1-K192N background. Strikingly, the oc-
cupancy level of Spn1 is diminished under noninducing condi-
tions in the spn1-K192N mutant, and this low level of occu-
pancy is unchanged under fully inducing conditions (Fig. 1B
and C). Occupancy levels of Spt6 are also diminished in the
spn1 mutant background under both noninducing and inducing
conditions. This indicates that the recruitment of Spt6 is de-
pendent on the presence of Spn1 and that neither Spn1 nor
Spt6 is required to activate transcription of CYC1. Indeed,
transcription levels of CYC1 are elevated under noninducing
conditions in the spn1mutant background (19). This suggests
that Spn1 is required to maintain the diminished activity of
RNAPII at the postassembly-regulated CYC1 gene.
Distinct functions of Spn1 and Spt6 during RNAPII tran-
scription. To investigate the requirement for Spt6 at CYC1, we
utilized a well-characterized mutant allele of SPT6, spt6-1004
(31), and examined CYC1 transcription. We found that activa-
tion of CYC1 in a spt6-1004 mutant was decreased by 30% (Fig.
2A). A decrease in CYC1 transcription in the spt6-1004 strain
suggests a positive role of Spt6 in regulating RNAPII transcrip-
tion at CYC1. This is in striking contrast to the fivefold increase
in CYC1 transcription observed in a strain containing the spn1-
K192N mutation (19).
Although Spt6 can stimulate elongation in vitro on naked
DNA (16), it also binds histones and has critical functions for
maintaining and modifying chromatin during transcription
elongation in vivo (2, 8, 31, 56). One molecular aspect of these
chromatin-related functions of Spt6 is in reestablishing chro-
matin structure after transcription (31). For example, loss of
the proper chromatin structure in the spt6-1004 mutant results
in transcription initiation by RNAPII from cryptic TATA sites
within the coding regions of certain genes, such as FLO8 (31).
FIG. 1. Spn1 and Spt6 are not coordinately recruited to CYC1, al-
though Spt6 occupancy is Spn1 dependent. ChIP analysis using no anti-
body (noAb), anti-Myc antibody (Myc), or anti-HA antibody (HA) was
performed on strains grown for 6 h under uninduced (2% dextrose) or
induced (3% ethanol) conditions. (A) Spn1 continually occupies CYC1,
while Spt6 occupies CYC1 only during activation. A control strain con-
taining no tagged factors (untagged) was compared to a strain containing
Myc-tagged Spn1 (Myc-Spn1) and HA-tagged Spt6 (HA-Spt6) via a rep-
resentative ChIP assay. (B) Spn1 and Spt6 do not occupy CYC1 in the
spn1-K192N background. A control strain containing no tagged factors
K192N (Myc-spn1-K192N) and HA-tagged Spt6 (HA-Spt6) via a repre-
sentative ChIP assay. (C) Quantification of the relative Spn1 and Spt6
occupancy levels observed under uninduced and induced conditions. IP
denotes the factor that was immunoprecipitated, whereas Strain indicates
either the wild-type (WT) or spn1-K192N (MT) background. The protein
occupancy level is represented as the ratio of signal from IP samples to
that of the input minus background of a no-antibody control (n ? 4; P ?
0.005). Error bars indicate standard deviations.
VOL. 28, 2008Spn1 AND TRANSCRIPTIONAL REGULATION1395
To determine if Spn1 is involved in this process, we tested the
spn1-K192N strain for short transcripts at FLO8. Unlike the
spt6 mutant strain, the spn1 mutant strain showed no changes
in FLO8 transcription (Fig. 2B). Thus, although the Spn1/Spt6
complex is involved in regulation of CYC1 gene expression,
these two proteins are also likely to have additional and sep-
arable functions in overall regulation of gene expression.
Spn1 is recruited to the CYC1 gene via interaction with
RNAPII. How is Spn1 recruited to the CYC1 promoter? Likely
candidates for recruitment of Spn1 are TBP and RNAPII, since
both are constitutively recruited to the CYC1 gene even when it is
partially or fully repressed (41, 51). Although SPN1 genetically
interacts with TBP, Spn1 was not found to coimmunoprecipitate
is recruited to CYC1 by interacting with RNAPII. Indeed, we
found that Spn1 coimmunoprecipitates with RNAPII (Fig. 3).
These results are consistent with other studies that demonstrate
an interaction between RNAPII and Spn1 by coimmunoprecipi-
tation and by tandem affinity chromatography (38, 46). Strikingly,
we found that the mutant form of Spn1, Spn1-K192N, fails to
coimmunoprecipitate with RNAPII (Fig. 3). This loss of interac-
tion with RNAPII, coupled with the observation that Spn1 is no
longer present at the CYC1 promoter in the spn1-K192N strain,
indicates that the Spn1-RNAPII interaction is essential for tar-
geting Spn1 to the CYC1 promoter.
Additional members of the preinitiation complex are
present on the CYC1 promoter under noninducing conditions.
To investigate the potential rate-limiting step impacted by
Spn1, we further characterized the promoter-bound complex
found at the CYC1 promoter. As previously noted (51), RNA-
PII occupies the CYC1 promoter prior to induction, with very
little change upon transcriptional activation (Fig. 4). Typically,
the CTD of Rpb1 of RNAPII is hypophosporylated prior to
initiation and becomes hyperphosphorylated on serine 5 dur-
ing the transition from initiation to elongation (13, 37). Thus,
this phosphorylation event may be a good candidate for a
rate-limiting step impacted by Spn1 functions. Rpb1 specifi-
cally phosphorylated on serine 5 can be immunoprecipitated
with the monoclonal antibody H14 (9, 59). Strikingly, ChIP
assays using H14 antibodies show that the CTD of Rpb1 is
phosphorylated under noninducing conditions at the CYC1
promoter (Fig. 4). Interestingly, this occupancy level drops
when transcriptional output is high (under inducing condi-
tions), which may reflect the recruitment of hypophosphory-
FIG. 2. Different effects of SPT6 and SPN1 mutations on RNAPII
transcription (A) Effect of spt6-1004 on the regulation of CYC1 acti-
vation. Total RNA from wild-type (WT) and spt6 mutant strains grown
under partially repressed (in medium containing 2% dextrose) and
activated (in medium containing 3% ethanol) conditions were ana-
lyzed by S1 nuclease assay using32P-labeled CYC1 and tryptophan
tRNA probes. tRNAwsignal was used as a loading control to normal-
ize the signal of CYC1 transcripts. A representative gel is shown. For
quantification, the transcription level of CYC1 in the wild-type strain
was set to 100%, and the values for the spt6 mutant strain from three
independent experiments (?4%) are indicated. (B) Effect of an spn1
mutation on FLO8 transcription. Total RNA from wild-type and spn1
or spt6 mutant strains, grown at 30°C or after an 80-minute shift to
37°C, was subjected to Northern blot analysis for FLO8 RNA. The
position of the transcript generated from the cryptic TATA element is
FIG. 3. Spn1 associates with the RNAPII complex, whereas Spn1-
K192N is diminished for this interaction. Protein extracts from strains
were immunoprecipitated (IP) with protein A-Sepharose beads cou-
pled with antibodies as indicated. Proteins of interest were detected by
using corresponding antibodies or epitope tags and immunoblot anal-
yses. Immunoprecipitation with anti-Spn1 antibodies (?-Spn1) pulled
down RNAPII, and conversely, immunoprecipitation with antibodies
to Rpb1, the largest subunit of RNAPII (?-RNAPII), pulled down
Spn1. However, in the spn1-K192N mutant background, RNAPII as-
sociation is significantly reduced with Spn1-K192N. Loads represent
10% of the input material for the IP (100% was loaded).
FIG. 4. Determination of the occupancy levels of additional initia-
tion complex components at CYC1. ChIP analysis was performed using
antibodies for Rpb1 (RNAPII), the serine 5 phosphorylation-specific
antibody (Ser5-P), or Myc antibodies and the indicated Myc-tagged
strains for helicase subunits of TFIIH (Rad3 and Ssl2) or a capping
enzyme subunit (Ceg1), under uninduced and induced conditions. The
protein occupancy level is represented as the ratio of signal from
immunoprecipitation samples to that of the input minus background
control (irrelevant antibodies for RNAPII and serine 5 phosphoryla-
tion and an untagged strain for the remainder). In each case n ? 3,
with P ? 0.005. Error bars indicate standard deviations.
1396ZHANG ET AL.MOL. CELL. BIOL.
lated RNAPII during multiple rounds of transcription initia-
tion. The presence of serine 5 phosphorylation on the CTD
suggests that TFIIH also occupies the promoter, since it pos-
sesses CTD kinase as well as helicase activities (18, 48, 69, 73,
74). Indeed, tagged derivatives of two different subunits of
TFIIH (Rad3 and Ssl2, the two helicases) were clearly detected
at the CYC1 promoter prior to activation of gene expression
(Fig. 4). A subsequent step in the transcription process is the
capping of the transcript (33, 37). Thus, we next tested for the
occupancy of Ceg1, a subunit of the yeast mRNA capping
enzyme that has been shown to directly contact the phosphor-
ylated form of the CTD (70, 71). Ceg1 was also found to
occupy the promoter prior to activation, and upon induction,
Ceg1 occupancy increased slightly (less than twofold) (Fig. 4).
Taken together with the previous observations that RNAPII
and TBP occupy the CYC1 promoter prior to transcriptional
activation, these results with serine 5 phosphorylation, TFIIH,
and capping enzyme occupancy indicate a remarkably com-
plete preinitiation complex at the CYC1 promoter. Moreover,
we must expand the screen for potential candidates for the
rate-limiting step impacted by Spn1 function.
Genetic interactions of SPN1 with RNAPII transcription
factors. To investigate the functional relationship between
Spn1 and other proteins involved in the regulation of gene
expression, a targeted genetic screen was designed to identify
transcription factors that genetically interact with SPN1. Eighty
different strains were chosen from the yeast deletion collection
(see Table S1 in the supplemental material). These strains
were selected due to the annotated functional activity of the
deleted gene product in some stage of the transcriptional pro-
cess. The factors include subunits of chromatin-remodeling
complexes (SWI/SNF, ISW, FACT, RSC, and CHD1) and
-modifying complexes (SAGA, NuHAT, and histone deacety-
lases), Mediator complex, and elongation factors, activators,
and additional SPT genes. Since these factors have defined
functions in the transcription process, combining this subset of
mutants with the spn1-K192N allele has the potential to reveal
SPN1 functions during RNAPII transcription. Each of the
strains containing a unique deletion was combined with the
spn1-K192N allele. Since SPN1 is essential for viability and
spn1-K192N is a recessive allele, introduction of spn1-K192N
into each deletion strain was accomplished via a genomic
knockout of SPN1 and plasmid shuffling (for details, see Ma-
terials and Methods). Strains with a deleted gene and spn1-
K192N were compared to the parent deletion strain containing
wild-type SPN1 or a strain containing spn1-K192N. Eleven
different conditions, including various temperatures, different
carbon sources, and a number of stress-inducing agents, were
assayed for phenotypic changes. A phenotypic change indicates
a genetic interaction between SPN1 and the deleted gene. Of
the 81 deletion strains tested, 6 showed genetic interactions
with spn1-K192N (Table 2 and Fig. 5).
spn1-K192N is synthetically lethal with a deletion in SPT4
and exacerbates the phenotypes of deletions in RTF1 or DST1.
As has been previously reported for another allele of SPN1
(46), deleting SPT4 results in cell inviability when combined
with the mutation in SPN1 (Table 2). Since Spt4 and the
essential gene product Spt5 are known to interact genetically
and physically with Spt6 (29), this result is not unexpected.
Human Spt4 and its binding partner Spt5 comprise the positive
transcription elongation factor DRB sensitivity-inducing fac-
tor, which binds directly to RNAPII (80, 84). Two other dele-
tion strains showed synthetic growth defects when combined
with the spn1-K192N allele. Deletion of either RTF1 or DST1
when combined with the spn1-K192N allele resulted in mild
growth defects under a variety of conditions (Fig. 5). For the
RTF1 deletion strain these conditions included growth on eth-
anol/glycerol-containing media and Ino?media. Similar to the
case for SPN1 (19), RTF1 was originally identified in a screen
for suppressors of a TBP mutant (78). Rtf1 is a member of the
Paf1 complex (57). Exacerbation of several observable growth
defects was also observed when spn1-K192N was combined
with a deletion in DST1 (Fig. 5). DST1 encodes the general
transcription factor TFIIS (66). In contrast to these effects for
SPT4, RTF1, and DST1, none of the other SPT genes, elonga-
tion factors, or Paf complex members tested exhibited genetic
interactions with SPN1. Thus, the specific genetic interactions
between SPN1, SPT4, RTF1, and DST1 suggest an important
linkage to later steps in the initiation process and the early
steps in elongation.
spn1-K192N synthetically rescues the effects of deletions in
Swi/Snf subunits. In contrast to the genetic effects observed
TABLE 2. Observed synthetic interactions between SPN1 and specific gene products involved in RNAPII-mediated transcription
Protein category and
Change in phenotype under the following conditiona:
1 M NaCl1 M sorbitol 4 mM H2O2
YPGal YPEG 50 mM ATMPA
Subunits of Swi/Snf complex
aDeletion strains (as indicated) were combined with the K192N derivative of SPN1, and alterations in growth properties under 11 different conditions were assayed.
N, no change in phenotype from that observed in either of the two parental strains (the knockout strain or the K192N strain); Ex, phenotype exacerbated in the double
mutant strains; Sup, suppression of mutant phenotypes in the double mutant background; SL, synthetic lethality in the double mutant background.
bSynthetic interactions between SNF2 and SPN1 were observed by mutating SPN1 in strain CMKy42 (15).
VOL. 28, 2008Spn1 AND TRANSCRIPTIONAL REGULATION 1397
with the above-described strains, in which double mutants were
less healthy (or dead), combining the spn1-K192N allele with
deletions in three other genes resulted in cells that grow sig-
nificantly better (Table 2 and Fig. 5). Enhanced growth/syn-
thetic rescue is seen upon combining spn1-K192N with a dele-
tion for genes encoding three different subunits of the Swi/Snf
complex: SNF2, SNF5, and SNF6. Swi/Snf is an ATP-depen-
dent chromatin-remodeling complex (for a review, see refer-
ence 52). Deletion of SNF2 alone causes severe growth defects
under all assay conditions, while snf5? and snf6? mutants are
similar to each other and exhibit growth defects on galactose or
ethanol/glycerol media, Ino?plates, or plates containing hy-
drogen peroxide, AT, or MPA. Importantly, all of these growth
defects are suppressed by spn1-K192N. More strikingly, these
three Swi/Snf mutants also suppress the temperature-sensitive
phenotype of spn1-K192N (Fig. 5). We did not observe genetic
interactions with four other genes that encode products found
in this complex. However, SNF2 encodes the enzymatic
ATPase subunit of the complex (60), and Snf2 requires both
Snf5 and Snf6 for proper function (12, 22, 43). As such, SPN1
genetically interacts with the critical gene products of the Swi/
Snf complex but not with other related factors (see Table S1 in
the supplemental material), such as ATP-dependent chroma-
tin-remodeling factors (such as ISW1, ISW2, CHD1, RAD26,
ITC1, etc.), or chromatin-modifying complexes (such as
SAGA, other HATs, or histone deacetylases).
The strong genetic interaction between Spn1 and compo-
nents of the Swi/Snf complex prompted us to explore the role
of Swi/Snf in CYC1 transcription regulation. Using S1 nuclease
assays, CYC1 transcripts were measured in the parental, spn1-
K192N, snf5?, and snf6? strains, as well as spn1-K192N snf5?
and spn1-K192N snf6? double mutant strains. Consistent with
our previous studies, the K192N mutation in SPN1 results in an
additional fivefold increase in CYC1 transcription under induc-
ing conditions (19). Deletion of SNF5 or SNF6 results in a 50
or 35 percent decrease, respectively, in CYC1 transcription
compared to that in the wild-type parental strain (Fig. 6A).
This indicates that the function of the Swi/Snf complex is
required for normal levels of CYC1 transcription. Mutating
SPN1 in these Swi/Snf deletion strains restored CYC1 tran-
scription to normal levels. These effects were not due to
changes in Spn1 protein levels in the Swi/Snf mutant back-
grounds, as immunoblot analysis indicates no changes in Spn1
levels in the different strain backgrounds (Fig. 6B). Taken
together, the results support our genetic observations and im-
ply a counteracting effect between Spn1 and the Swi/Snf com-
plex at a molecular level.
Snf2, the ATPase subunit of Swi/Snf, is recruited to CYC1
during activated transcription. Since deleting components of
the Swi/Snf complex resulted in transcriptional defects in
CYC1 expression, we next investigated whether this was a di-
rect effect of the functional activity of Swi/Snf at the CYC1
promoter. An epitope-tagged version of Snf2 (the ATPase
subunit) was created and used in a ChIP assay to determine the
occupancy of Snf2 at the CYC1 promoter. We found that Snf2
shows a significant increase (fivefold) in occupancy at CYC1
under inducing conditions (Fig. 7). Thus, like for Spt6, the
appearance of Snf2 at the postassembly-regulated CYC1 gene
correlates with the transition to a highly active RNAPII state.
Diminished Spn1 occupancy leads to autonomous recruit-
ment of Snf2 at CYC1. The genetic suppression between mu-
tants of Spn1 and the Swi/Snf complex and their opposite
FIG. 5. SPN1 genetically interacts with SNF2, SNF5, SNF6, RTF1, and DST1. Yeast cells of the indicated strains were diluted serially and plated
onto the indicated medium. Pictures were taken after 2 to 3 days of growth. The growth defects of the rtf1? and dst1? strains are exacerbated by
the mutation in SPN1. The temperature-sensitive phenotype of the spn1 mutant (spn1-K192N) is suppressed by snf2?, snf5?, and snf6?, and the
growth defects of the Swi/Snf mutants are suppressed by spn1-K192N. WT, wild type; MT, mutant.
1398ZHANG ET AL.MOL. CELL. BIOL.
effects on transcription of the CYC1 gene suggest a simple
model whereby Spn1 antagonizes the function of Swi/Snf. One
way to accomplish this would be for Spn1 to inhibit Swi/Snf
recruitment to the gene until inducing conditions are present.
If correct, then this model predicts that in the absence of Spn1,
the Swi/Snf complex would be constitutively recruited to the
CYC1 promoter. We tested this hypothesis by performing ChIP
assays in a spn1-K192N background, in which Spn1 fails to
occupy the CYC1 promoter. In contrast to the case for wild-
type cells, which showed low levels of Snf2 during partial re-
pression, we found high occupancy levels of Snf2 at the CYC1
gene in the mutant strain (Fig. 7). The autonomous increase in
recruitment of this component of Swi/Snf in the mutant SPN1
strain is in good accord with the elevated levels of transcription
also observed in this strain (19). Taken together, these results
suggest that Spn1 plays an inhibitory role in the recruitment of
the Swi/Snf complex.
Spt6 is recruited before Swi/Snf during CYC1 activation. If
Spn1 has an inhibitory effect on the recruitment of the Swi/Snf
complex during partial repression, then how is this relationship
altered under inducing conditions, considering that Spn1 still
occupies the promoter? One possibility is that the direct inter-
action of Spn1 with Spt6 abrogates the inhibitory activity. If
this model is correct, than Spt6 would appear at the promoter
prior to Swi/Snf. CYC1 transcription reaches maximum levels
at approximately 6 h of growth in medium containing ethanol
(Fig. 8A). One hour after activation, Spt6 occupancy levels
reach over 70% of the maximum level (Fig. 8B and D). In
contrast, occupancy levels of the Swi/Snf complex increase at
around 2 h after activation and reach the maximum at the 4-h
time point (Fig. 8C and D). As such, Swi/Snf occupancy cor-
relates well with CYC1 transcription levels. The fact that Spt6
is recruited earlier than Swi/Snf upon activation supports a
model whereby the interaction of Spt6 with Spn1 relieves the
inhibitory effect of Spn1 on the recruitment of the Swi/Snf
SPN1 encodes a highly conserved protein with an essential
role in transcription by RNAPII (19, 38, 46). To further define
how Spn1 regulates RNAPII transcription, a targeted genetic
FIG. 6. The Swi/Snf complex is required for full activation of the
CYC1 gene. (A) Quantification of the effects of Swi/Snf mutants on
CYC1 transcription. S1 nuclease assay results show the effects of the
Swi/Snf complex on CYC1 activation. The indicated strains were grown
under uninduced and induced conditions, and total RNA was isolated
and analyzed via S1 nuclease assay. tRNAwsignal was used as a loading
control to normalize signals of CYC1 transcripts. The fold changes in
induction were calculated by dividing the signals of CYC1 transcripts
under inducing conditions by the amount observed under noninducing
conditions. The bar graph shows fold changes (mean ? standard de-
viation; P ? 0.005) of CYC1 levels from each strain of four separate
experiments. (B) Spn1 levels were comparable in all strains tested.
Protein extracts from the indicated strains were analyzed by Western
blotting using polyclonal anti-Spn1 antibody. TBP expression levels
were used as an internal control. WT, wild type; MT, mutant.
FIG. 7. Occupancy of Swi/Snf on the CYC1 promoter during acti-
vation in wild-type and spn1K192Nbackgrounds. ChIP analysis was
performed on strains, as indicated in Fig. 1. (A) Swi/Snf occupies
CYC1 during activation in a wild-type background. (B) The Swi/Snf
complex autonomously occupies CYC1 in the spn1-K192N background.
(C) Quantification of the relative Swi/Snf occupancy levels under un-
induced and induced conditions. The protein occupancy level is rep-
resented as the ratio of signal from immunoprecipitation (IP) samples
to that of the input minus background of a no-antibody control (n ? 4;
P ? 0.005). WT, wild type; MT, mutant. Error bars indicate standard
VOL. 28, 2008 Spn1 AND TRANSCRIPTIONAL REGULATION1399
screen was designed to identify transcription factors that ge-
netically interact with SPN1. We found that SPN1 interacts
with three Swi/Snf genes (SNF2, SNF5, and SNF6), and SPT4,
RTF1, and TFIIS (DST1). A number of lines of evidence sug-
gest that the genetic interactions we identified are biologically
significant. First, the genetic interactions observed were highly
specific: only five transcription factors from over 80 strains
tested show an interaction with SPN1. We did not observe
phenotypic changes upon combining spn1-K192N with other
deletion mutations of the transcription machinery, such as
Mediator components, RNAPII-associated factors, and differ-
ent chromatin-remodeling and -modifying factors. Second, we
observed that SPN1 genetically interacts with only one ATP-
dependent chromatin-remodeling complex, Swi/Snf, and not
with other ATP-dependent chromatin-remodeling or -modify-
ing factors such as Isw1, Isw2, FACT, RSC, or SAGA. Third,
mutations in SPT6 also suppress the transcription defects of
Swi/Snf mutant strains (28, 44, 82) and are synthetically lethal
with a DST1 (TFIIS) null mutant (15, 29). Fourth, synthetically
lethal interactions have been observed between a deletion of
SPT4 and deletions of RTF1 (14, 42) or DST1 (29). Taken
together, these results indicate an extensive set of genetic and
biochemical interconnections between Spn1, Spt6, Swi/Snf,
Spt4, Rtf1, and TFIIS (DST1).
To further characterize the functional aspects of SPN1, we
examined occupancy of a number of components of the tran-
scriptional machinery to arrive at a working model for CYC1
gene activation (Fig. 9). Like TBP and RNAPII (41, 51), we
find that Spn1 occupies the CYC1 promoter under noninducing
conditions. In addition, TFIIH and capping enzyme also ap-
pear to occupy the promoter prior to activation, and consistent
with this, serine 5 of the CTD of Rpb1 is also phosphorylated.
Spn1 appears to be recruited to the promoter via interactions
with RNAPII, since the SPN1 mutant (spn1-K192N), which is
defective for interaction with RNAPII, does not occupy the
CYC1 promoter. Under inducing conditions for CYC1, Spt6
promptly occupies the promoter. Spt6 recruitment is most
likely via interaction with Spn1, since a loss of Spn1 at CYC1
also results in the loss of Spt6 in the spn1-K192N background.
After Spt6 is recruited, the Swi/Snf complex occupies the CYC1
promoter. Swi/Snf recruitment correlates the best with tran-
scriptional output, suggesting that this is an important step in
CYC1 gene expression. Indeed, in the absence of Spn1 (as well
as Spt6), Swi/Snf is constitutively recruited to CYC1, indicating
that Spn1 negatively regulates Swi/Snf recruitment.
The model described above indicates that Spn1 and Spt6
have distinct but dependent functions for CYC1 regulation.
Are Spn1 and Spt6 functions always codependent? Most likely
they are not, since we found that Spn1 does not appear to play
a role in cryptic start site formation, whereas Spt6 does (31). In
addition, others have shown that mutations in SPN1 have no
effect on Spt6-mediated chromatin reassembly at PHO5 (2)
and that patterns of gene perturbations in microarray studies
are fairly distinct for mutations in SPN1 compared to SPT6
(10). All of the above suggests that although Spn1 and Spt6
cooperate for regulation of CYC1 gene expression, they also
have additional and separable functions in overall regulation of
Since Swi/Snf is a chromatin-remodeling complex (for a re-
view, see reference 52), it is likely to be needed at CYC1 to
perturb histone-DNA interactions. Like the majority of yeast
promoters (6, 45, 72), CYC1 is “open” and devoid of histones
(51), and thus it is unlikely that Swi/Snf is required for pro-
moter chromatin remodeling. In contrast to the promoter re-
FIG. 8. Time course of transcription and occupancy levels of Spt6 and
Swi/Snf at CYC1. (A) S1 nuclease assay showing the time course of CYC1
increase of Spt6 occupancy on the CYC1 promoter during 0 to 5 h of activa-
tion. Spt6 occupies the CYC1 promoter within 2 h after activation. (C) ChIP
promoter during 0 to 5 h of activation. Swi/Snf occupancy parallels that of
CYC1 transcription output. (D) Line graph showing the time course of Spt6
and Swi/Snf occupancy levels on the CYC1 gene upon activation. The levels
of Spt6 and Swi/Snf occupancies at 6 h of activation were set as 100%. The
occupancy levels of both factors at each time point were converted to the
percentage of their maximum occupancy levels and graphed (n ? 3; P ?
0.005). Error bars indicate standard deviations.
1400ZHANG ET AL.MOL. CELL. BIOL.
gion, the ORF of CYC1 has detectable levels of histone H3,
and deletion of SNF5 results in a two- to threefold increase in
histone occupancy in this region (data not shown). Thus, these
results are consistent with a role of the Swi/Snf complex in
mobilizing histones in the transcribing region. This could en-
hance the transcriptional elongation process. Indeed, Swi/Snf
has established roles in directing remodeling of large chroma-
tin domains encompassing coding regions (36).
It is interesting to note that Swi/Snf has also been implicated
in promoter clearance by RNAPII (23). These results, coupled
with ours demonstrating that Swi/Snf recruitment correlates
with active RNAPII, makes one wonder whether Swi/Snf may
facilitate “remodeling” of some other component in the system
in addition to nucleosomes. Perhaps RNAPII itself may need
to alter its conformation to achieve an active transcribing
and/or elongating state at CYC1. The high-resolution struc-
tures of the preinitiated and elongating polymerase suggest
that conformational changes must occur to accommodate spe-
cific promoter recognition, DNA melting, RNA chain exten-
sion, etc. (11, 32, 81, 85). Moreover, a large number of poised
RNAPIIs (65), as well as partial preinitiation complexes (87),
have been detected at various locations in the yeast genome
without corresponding transcriptional activity. It is unknown
how these inactive complexes are converted into active ones,
but it is interesting to speculate that chromatin-remodeling
complexes may have other fundamental targets besides nucleo-
somes and that these remodeling events may play a role in the
transition to competent elongation complexes.
We also found that spn1-K192N is synthetically lethal with a
deletion in SPT4 (this was also observed by Lindstrom et al.
) and exacerbates the phenotypes of deletions in DST1
(TFIIS) or RTF1. Interestingly, each of these gene products
plays a potential role in the transition to a competent elonga-
tion complex. Spt4 (in combination with Spt5) is implicated in
regulating the elongation process (46). The human homo-
logues of Spt4 and Spt5 comprise the positive transcription
elongation factor DRB sensitivity-inducing factor, which binds
directly to RNAPII and plays a role in release from pausing of
RNAPII (80, 84). TFIIS (DST1) plays a role in the initiation of
transcription (62) and promoter escape (49) and also rescues
arrested RNAPII at pause sites by stimulating the RNAPII to
cleave and realign the nascent transcript (1, 4, 40). All of these
observations place Spt4 and TFIIS firmly in a multifunctional
role that is consistent with the initiation-to-elongation transi-
tion. Likewise, RTF1 has been implicated in a number of stages
in the RNAPII-mediated transcription process, including tran-
script start site selection, elongation, processing, and histone
modifications (14, 29, 50, 58, 76, 78). Like for these other
multifunctional factors with which Spn1 genetically interacts,
our data strongly suggest that Spn1 negatively regulates the
transition to a productive elongation complex. Release of this
inhibition, either by lack of recruitment (in the spn1-K192N
mutant) or via interaction with Spt6, allows for productive
transcription. In addition, Spn1 colocalizes with RNAPII along
the entire ORFs of a number of constitutively active genes (34,
38). Also, Spn1 associates with RNAPII phosphorylated on
serine 5 and serine 2 residues of the CTD of the largest subunit
of RNAPII (46). Phosphorylation of the CTD is thought to
correlate with stages of the transcription process in that hypo-
phosphorylated RNAPII binds to promoters, serine 5 phos-
phorylation occurs during initiation, and serine 2 phosphory-
lation occurs during elongation (for reviews, see references 54
and 61). Consistent with a novel and negative role in the
elongation process, the spn1-K192N mutant does not display
6-azauracil or MPA sensitivity (19), two common phenotypes
shared by many mutants with mutations in factors with positive
roles in elongation (29, 62, 68). These compounds alter the
elongation rate and processivity of RNAPII in vivo (53), due to
depletion of nucleotide pools (17).
The extensive primary amino acid sequence identity between
yeast and human Spn1 (19), as well as the existence of homo-
logues for the other transcription players involved, suggests the
potential for conservation of Spn1 function in higher eu-
karyotes. As in yeast, mammalian Spn1 (also known as Iws1) is
essential for cell viability (47). Moreover, expression of pos-
trecruitment-regulated human immunodeficiency virus type 1
requires functional Spn1 and Spt6, as knockdown and muta-
tional analyses demonstrate defects in transcript production
and processing (86). In addition, human Spn1 is involved in the
expression of the c-Myc proto-oncogene (86), another gene
that is regulated after recruitment of the preinitiation complex
(5, 39). Thus, the exciting and emerging picture is one in which
Spn1 plays a central role in postrecruitment mechanisms in
humans as well as in Saccharomyces cerevisiae.
We thank Timothy Formosa and Caroline Kane for providing yeast
strains and Xu Chen for data analysis and figure preparation.
This work was supported by grants from the National Institutes of
Health to L.A.S. (GM056884) and F.W. (GM32967).
FIG. 9. A model for CYC1 gene regulation. (A) Under uninduced
conditions, Spn1, TBP, RNAPII, TFIIH, and the capping enzyme
subunit, Ceg1, are constitutively recruited to the CYC1 gene. In addi-
tion, serine 5 of the CTD of Rpb1 is phosphorylated. Spn1 occupancy
prevents Swi/Snf interaction with the CYC1 promoter. (B) Under in-
ducing conditions, Spt6 is recruited to the CYC1 promoter via inter-
action with Spn1. (C) Spt6 recruitment is followed by the recruitment
of the Swi/Snf complex, which correlates with induced levels of gene
VOL. 28, 2008Spn1 AND TRANSCRIPTIONAL REGULATION1401
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