MOLECULAR AND CELLULAR BIOLOGY, June 2003, p. 4207–4218
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 23, No. 12
Methylation of Histone H3 by Set2 in Saccharomyces cerevisiae Is
Linked to Transcriptional Elongation by RNA Polymerase II
Nevan J. Krogan,1,2,3Minkyu Kim,4Amy Tong,1,2Ashkan Golshani,1Gerard Cagney,1,2,3
Veronica Canadien,5Dawn P. Richards,5Bryan K. Beattie,5Andrew Emili,1,2,3Charles Boone,1,2
Ali Shilatifard,6Stephen Buratowski,4and Jack Greenblatt1,2,3*
Banting and Best Department of Medical Research1and Toronto Yeast Proteomics Organization,3University of Toronto, Toronto,
Ontario, Canada M5G 1L6; Department of Medical Genetics and Microbiology, University of Toronto, Toronto,
Ontario, Canada M5S 1A82; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 021154; Affinium Pharmaceuticals, Toronto, Ontario, Canada,
M5J 1V65; and Department of Biochemistry and Cancer Center, St. Louis University
School of Medicine, St. Louis, Missouri 631046
Received 13 January 2003/Returned for modification 27 February 2003/Accepted 20 March 2003
Set2 methylates Lys36 of histone H3. We show here that yeast Set2 copurifies with RNA polymerase II
(RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methyl-
ation are associated with the coding regions of several genes that were tested and correlate with active
transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which
contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII
CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered
by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in
slight sensitivity to 6-azauracil and much less ?-galactosidase produced by a reporter plasmid, resulting from
a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained
when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain
elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B
ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of
the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.
In Saccharomyces cerevisiae RNA polymerase II (RNAPII)
initiates transcription in concert with general transcription fac-
tors and a 20-subunit mediator complex (16, 38, 59). During
initiation it becomes phosphorylated by the Kin28 subunit of
the general transcription factor TFIIH on Ser5 of the hep-
tapeptide repeats, YSPTSPS, in the carboxy-terminal domain
(CTD) of its largest subunit, Rpb1, resulting in recruitment of
the mRNA-capping enzyme to the transcription complex (23,
32, 49, 69). Ser5 phosphorylation declines during the early
stages of elongation and is replaced by Ser2 phosphorylation,
mediated mainly by the cyclin-dependent kinase, Ctk1, during
the later stages of elongation by RNAPII (10, 23). General
transcription factors and mediator dissociate from the tran-
scription complex or remain behind at the promoter and are
replaced by various elongation factors during chain elongation
by RNAPII (25, 43). Among these elongation factors are Spt4/
Spt5, Spt6/Iws1, and Spt16/Pob3, whose patterns of genetic
interactions suggest that they participate in transcription on
chromatin templates (66). Another elongation factor is the
Paf1 complex, which consists of Paf1, Rtf1, Cdc73, Ctr9, and
Leo1 and associates with RNAPII (25, 34, 52). Chromatin
immunoprecipitation (ChIP) experiments have shown that all
of these polypeptides are associated with transcribed regions
Histone methylation by SET domain-containing histone ly-
sine methyltransferases has important roles in chromatin struc-
ture and function (44). Lysines 4, 9, 27, and 79 are well-studied
sites of methylation on histone H3, while lysine 20 is the only
known methylated lysine in histone H4 (55, 64). There are at
least six proteins with recognizable SET domains in S. cerevi-
siae. Set1, a component of an eight-protein complex (COM-
PASS), methylates Lys4 of histone H3 (7, 24, 33, 36, 46).
Recruitment of COMPASS to the early transcribed region, as
well as histone H3 Lys 4 trimethylation in this region, requires
both Ser5 phosphorylation of the RNAPII CTD by Kin28 and
the Rtf1, Paf1, and Ctr9 subunits of the Paf1 complex (26, 37,
48). Methylation of H3 Lys4 by Set1, as well as methylation of
H3 Lys79 by Dot1, also requires ubiquitination of histone H2B
by Rad6 and Bre1 (13, 19, 58, 67).
Set2 methylates Lys36 of histone 3 (54). Members of the
mammalian nuclear receptor-binding SET-domain-containing
family (NSD1, NSD2, and NSD3) contain a SET domain that
is highly related to that of yeast Set2. NSD genes have been
implicated in acute myeloid leukemia (21, 47, 53), whereas the
NSD2 gene maps to the region associated with Wolf-Hirsch-
horn syndrome (47), which is characterized by mental retarda-
tion and developmental defects.
In this study, we found that Set2 interacts physically with
RNAPII. Similar observations have been made recently by
other groups (28, 29, 68). We used ChIP assays to show that
Set2 is recruited and methylates histone H3 Lys36 in the cod-
ing regions of actively transcribed genes. Synthetic growth de-
* Corresponding author. Mailing address: Banting and Best Depart-
ment of Medical Research, University of Toronto, 112 College St.,
Toronto ON, Canada M5G 1L6. Phone: (416) 978-4141. Fax: (416)
978-8528. E-mail: firstname.lastname@example.org.
fects were observed when a set2 deletion was combined with
deletions of known or suspected transcriptional elongation fac-
tors, including Chd1, Soh1, and members of the Paf1 elonga-
tion complex, as well as components of the Set1 and Set3
complexes. A deletion of set2 also results in slight sensitivity to
the drug 6-azauracil (6-AU) and a decrease in ?-galactosidase
from a lacZ reporter plasmid, implicating Set2 in transcrip-
tional regulation. Finally, recruitment of Set2 and Lys36 his-
tone H3 methylation relies on components of the Paf1 complex
as well as the RNAPII CTD kinase Ctk1. These results suggest
that binding of Set2 to RNAPII and histone H3 methylation
occur during transcriptional elongation and depend on both
the Paf1 elongation factor and the phosphorylation state of the
CTD. The involvement of SET domain proteins in transcrip-
tional elongation would not be entirely unexpected, because a
translocation that joins part of the human elongation factor
ELL to part of the SET domain protein MLL leads to myelog-
enous leukemia (12).
MATERIALS AND METHODS
Yeast strains used in this study. The following yeast strains were used in this
study: NJK581 MATa ura3-1 leu2-3,112 his3-11,15 trp1? ade2-1 can1-100
set2-TAP::TRP1 and NJK742 MATa ura3-1 leu2-3,112 his3-11,15 trp1? ade2-1
can1-100 set2(?476-733)-TAP:TRP1. Strains with the following genes replaced by
a kanamycin resistance cassette were obtained from Research Genetics (http:
CDC73, RTF1, CTK1, and SET2. Tandem affinity purification (TAP)-tagged
versions of Set2 were then made in these backgrounds (4, 17), as follows:
YSB971, MATa ura3?0 leu2?0 his3?1 met15?0 set2-TAP::HIS3 ctk1?::KanMX;
YSB972, MATa ura3?0 leu2?0 his3?1 met15?0 set2-TAP::HIS3 rtf1?::KanMX;
cdc73?::KanMX. Sensitivity to 6-AU was tested by plating strains harboring
pRS316 (51) onto plates lacking uracil and containing 25 ?g of 6-AU per ml.
ChIP assays. ChIP assays were performed essentially as described earlier (23).
Yeast strains were grown at 30°C to an optical density at 595 nm (OD595) of 0.4
to 0.6. For the ChIP with Rpb3 on the lacZ gene, both wild-type and set2? strains
harboring p416GAL1-lacZ were incubated until they reached an OD595of ap-
proximately 1 in medium containing 2% glucose. Cells were harvested, washed
three times with sterile water, and divided into two aliquots. The cells were
reinoculated into media containing either 2% glucose or 2% galactose. After 4 h
at 30°C, an aliquot was removed for the ?-galactosidase assay and the remaining
cells were cross-linked with formaldehyde for ChIP assays. The antibody against
Rpb3 was obtained from Neoclone Biotechnology. Unpublished primers used for
PCR in this study are designated by the name of the gene followed by the
position of its 5? end relative to the translation initiation codon, as follows:
CACCTC); Gal1-lacZ1567 (AATGGCTTTCGCTACCTGGAGAGA); Gal1-
AACAAC); Gal1-lacZ3094(AATGGCGATTACCGTTGATGTTGAA); Gal1-
Purification and analysis of Set2. TAP-tagged Set2 was purified on immuno-
globulin G (IgG) and calmodulin columns from extracts of yeast cells (3 liters)
grown in yeast extract-peptone-dextrose medium to an OD600of 1.0 to 1.5. The
cell pellets (7 to 10 g) were frozen in liquid nitrogen and lysed by grinding with
dry ice in a Krups coffee grinder (model no. 203-70). An equal volume of buffer
(250 mM KCl, 100 mM HEPES-KOH [pH 7.9], 1 mM EDTA, 2.5 mM dithio-
threitol [DTT]) was added, and following centrifugation in a Beckman Ti70 rotor
at 4°C for 2 h at 34,000 rpm, the supernatant was dialyzed against IPP buffer (10
mM Tris-Cl [pH 7.9], 0.1% Triton X-100, 0.5 mM DTT, 0.2 mM EDTA, 20%
glycerol, 100 mM NaCl). After dialysis, the extract was again centrifuged in a
Ti70 rotor at 4°C for 30 min at 34,000 rpm and the supernatant was mixed for 3 h
with 200 ?l of IgG-Sepharose (Pharmacia) equilibrated with IPP buffer. Follow-
ing binding, the IgG-Sepharose was washed with 1 ml of IPP buffer followed by
400 ?l of TEV protease cleavage buffer (50 mM Tris-Cl [pH 7.9], 1 mM DTT,
0.1% Triton X-100, 100 mM NaCl). The beads were then incubated overnight at
his3?1 met15?0 set2-TAP::HIS3
4°C with 100 U of TEV protease (Life Technologies) in 200 ?l of TEV cleavage
buffer. The eluate was combined with a 200-?l wash with TEV cleavage buffer.
To this were added 200 ?l of calmodulin binding buffer (10 mM Tris-Cl [pH 7.9],
10 mM ?-mercaptoethanol, 2 mM CaCl2, 0.1% Triton X-100, 100 mM NaCl) and
200 ?l of calmodulin beads (Pharmacia) equilibrated with the same buffer. After
binding for 1 to 2 h at 4°C, the calmodulin beads were washed with 200 ?l of
calmodulin binding buffer and 200 ?l of calmodulin wash buffer (10 mM Tris-Cl
[pH 7.9], 10 mM ?-mercaptoethanol, 0.1 mM CaCl2, 0.1% Triton X-100, 100 mM
NaCl). The purified protein complexes were eluted from the calmodulin beads
with 5 ? 100 ?l of calmodulin elution buffer (10 mM Tris-Cl [pH 7.9], 10 mM
?-mercaptoethanol, 3 mM EGTA [pH 8.0], 0.1% Triton X-100, 100 mM NaCl).
The purified proteins were separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) on gels containing 10% polyacrylamide, and
the proteins were visualized by silver staining. Peptide samples were spotted onto
a target plate with a matrix of ?-cyano-4-hydroxycinnamic acid (Fluka). Matrix-
assisted laser desorption ionization–time of flight (mass spectrometry) (MALDI-
TOF) analysis was conducted by utilizing a Reflex IV (Bruker Daltonics, Bil-
lerica, Mass.) instrument in positive-ion reflectron mode (31, 50). Western
blotting was performed by using standard techniques with the monoclonal anti-
bodies 8WG16, H5, and H14 (6, 40, 60).
SGA analysis. Synthetic genetic array (SGA) analysis was carried out as
previously described (61).
Interaction of Set2 with RNAPII. In order to further char-
acterize S. cerevisiae Set2, we used single-step transformation
to place a TAP tag (45) containing a calmodulin-binding pep-
tide and Staphylococcus aureus protein A, separated by a TEV
protease cleavage site, at the C terminus of Set2. To confirm
the success of the tagging procedure, Western blotting was
performed on extract derived from the tagged strain by making
use of an irrelevant immunoglobulin to recognize the protein
A component of the TAP tag. The tagged protein was then
purified on IgG and calmodulin columns and analyzed by SDS-
PAGE followed by staining with silver. Protein bands absent
from a control preparation and corresponding to the tagged
protein and any associated proteins were then identified by
MALDI-TOF (50). Set2 copurified with substoichiometric
amounts of two larger polypeptides identified as Rpb1 and
Rpb2, the two largest subunits of RNAPII (Fig. 1A). This
physical association was confirmed when Set2 and several elon-
gation factors copurified with RNAPII from a strain harboring
a TAP tag on Rpb1 (data not shown). We were not able to
identify by mass spectrometry any of the other minor polypep-
tides that copurified with the Set2-TAP shown in Fig. 1A, other
than Rrp5 and fragments of Set2. Tagged Rrp5 did not copu-
rify with Set2 (data not shown). Moreover, the minor polypep-
tides other than Rpb1 and Rpb2 that copurified with Set2-TAP
have not been reproducibly detected in other preparations.
RNAPII phosphorylated on Ser2 on the Rpb1 CTD hep-
tapeptide repeats has been shown to localize to transcribed
coding regions and 3? ends of genes, whereas Ser5 phosphor-
ylation on the CTD is found primarily in 5? early elongation
regions (23). Western blotting analyses performed with mono-
clonal antibodies H5 and H14 (6, 40), which recognize the
Rpb1 CTD repeats phosphorylated on Ser2 and Ser5, respec-
tively, showed that the RNAPII copurifying with Set2 was
phosphorylated on both residues (Fig. 1B), suggesting that
Set2 might be associated with coding regions during transcrip-
tion. As a control, Western blotting with H5 and H14 did not
detect Rpb1 when a parallel purification was done by using an
extract from an untagged strain. The Rpb1 that copurified with
Set2 from a Set2-TAP strain was also recognized weakly by the
4208KROGAN ET AL.MOL. CELL. BIOL.
monoclonal antibody 8WG16 (60), which recognizes both un-
phosphorylated and partially phosphorylated Rpb1 (Fig. 1B).
These results suggested that Set2 might be associated with
Localization of Set2 and histone H3 Lys36 methylation to
the coding regions of transcribed genes. ChIP assays in which
proteins were cross-linked to DNA in vivo by using formalde-
hyde were employed to analyze the in vivo distribution of Set2
and histone H3 Lys36 methylation along various transcribed
genes (23). Following isolation and shearing of chromatin,
Set2-TAP or Lys36-methylated histone H3 was immunopre-
cipitated with either rabbit IgG agarose (directed against pro-
tein A on the TAP tag) or antibody directed against methyl-
ated histone H3 Lys36 (Upstate Biotechnology). After reversal
of the cross-links, PCR analyses were performed on the co-
precipitated DNA. Primer pairs directed against promoter
regions, coding regions, and 3? untranslated regions were used
to analyze the ADH1, PYK1, and PMA1 genes, encoding alco-
hol dehydrogenase, pyruvate kinase, and plasma membrane
ATPase, respectively (Fig. 2A). Set2-TAP, of all three genes
that were tested, was found to cross-link most strongly to the
coding regions and not to the promoter or 3? untranslated
regions. Similarly, when ChIP experiments were carried out
with antibody directed against Lys36-methylated histone H3,
the same pattern was observed: an enrichment of cross-linked
DNA corresponding primarily to the coding regions of the
genes that were studied (Fig. 2B).
To directly test whether the presence of Set2 is correlated
with active transcription, ChIP was performed on the galac-
tose-inducible gene, GAL1, which encodes galactokinase. In
the absence of galactose, virtually no Set2 cross-linked to any
part of the GAL1 gene. Upon induction, however, both Set2-
TAP and methylated histone H3 Lys36 were detected primarily
in the coding region of GAL1 (Fig. 2C), providing further
evidence that the presence of Set2 and its histone-methylating
activity require elongation by RNAPII.
Influence of Set2 on transcription by RNAPII. The presence
of Set2 in transcribed regions and its interaction with RNAPII
suggested that Set2 might influence transcription by RNAPII.
To determine whether yeast Set2 influences transcription elon-
FIG. 1. TAP of Set2. (A) Purification of Set2 was carried out with strains containing either no tagged protein or a TAP-tagged version of Set2.
The protein complex was purified in the presence of 100 mM NaCl as described in the text and was then analyzed by SDS-PAGE and silver staining.
Set2 and subunits of RNAPII were identified by trypsin digestion and MALDI-TOF. The asterisk indicates another polypeptide, Rrp5, which was
also identified in this preparation and is most likely a contaminant. (B) Immunoprecipitation with IgG followed by Western blot analysis using the
monoclonal antibodies H5 and H14 (6, 40) demonstrated that Set2 copurified with RNAPII phosphorylated on both Ser2 and Ser5 in the CTD
repeats of Rpb1. The RNAPII that copurified with Set2 also reacted with the antibody 8WG16 (60), which recognizes both partially phosphorylated
and unphosphorylated Rpb1. Western blotting analyses with immunoprecipitates using extracts from an untagged strain were also performed as
controls with all three antibodies. Successive lanes were loaded with 0.5, 1.5, and 2.5 ?g of extract protein.
VOL. 23, 2003 HISTONE H3 METHYLATION BY Set2 AND RNAPII ELONGATION4209
gation by RNAPII in vivo, we initially examined a set2? strain
for sensitivity to the pyrimidine analog 6-AU. Treatment with
6-AU leads to reduction of the UTP and GTP concentrations
in yeast cells and impairs elongation by RNAPII (14). Studies
using 6-AU have helped to show that genes like DST1, which
encodes TFIIS, encode factors having a role in transcriptional
elongation (3). Deletion of SET2 resulted in slight sensitivity to
6-AU, suggesting that Set2 might stimulate elongation by
RNAPII (Fig. 3A). Recently, similar observations have been
made by two independent groups (28, 29).
To substantiate this conclusion, we examined whether a set2
deletion would reduce lacZ expression from the plasmid,
p416GAL1-lacZ, which contains the Escherichia coli lacZ gene
fused to a GAL1 promoter (9). Based on results obtained by
FIG. 2. Set 2 and histone H3 methylation on Lys36 localize to transcribed regions. (A) ChIP was performed to monitor the presence of either
the Set2 protein or Lys36 methylation on histone H3 along the PMA1, ADH1, and PYK1 genes. Chromatin was immunoprecipitated either with
rabbit IgG-agarose from strains containing TAP-tagged versions of Set2 or with antibody against Lys36 methylated histone H3 (Upstate
Biotechnology) from a strain with no tag. The Set2(?476-733)-TAP strain was constructed by recombining in the TAP tag so as to remove the last
258 amino acids of Set2. PCR amplification was carried out by using primer pairs recognizing promoter (lane 1), coding (lanes 2, 3, 4, and 5), and
3? untranslated (lane 6) regions for PMA1; promoter (lane 1), coding (lane 2), and 3? untranslated (lane 3) regions for ADH1; and promoter (lane
1), coding (lanes 2, 3, and 4), and 3? untranslated regions for PYK1. Each PCR contained a second primer pair that amplified a region of
chromosome V devoid of open reading frames (marked by asterisks), thus providing an internal control for background. Input, signal from
chromatin before immunoprecipitation. Primer pairs used are as follows: for PMA1, PMA1?370and PMA1?47(lanes 1), PMA1168and PMA1376
(lanes 2), PMA1584and PMA1807(lane 3), PMA11010and PMA11250(lanes 4), PMA12018and PMA12290(lanes 5), and PMA13287and PMA13500
(lanes 6); for ADH1, ADH1?235and ADH1?13(lanes 1), ADH1844and ADH11013(lanes 2), and ADH11231and ADH11400(lanes 3); for PYK1,
PYK1?288and PYK119(lanes 1), PYK1508and PYK1799(lanes 2), PYK11104and PYK11372(lanes 3), PYK11470and PYK11720(lanes 4), and
PYK11695and PYK11995(lanes 5); for the nontranscribed region, Intergenic V-1 and Intergenic V-2. (B) Quantitation of the ChIP assays. Each
value is calculated by dividing the ratio of the ChIP signal to the input signal for the experimental PCR product by the ratio of the ChIP signal
to the input signal for the control PCR product. (C) Set2 is recruited to active genes. Cells grown overnight in medium containing 2% glucose were
harvested, washed twice with sterile water, and inoculated at an OD595of ?0.02 into 300 ml of medium containing either 2% glucose or 2%
galactose and 1% raffinose. Cells were incubated at 30°C until the OD595reached 0.6 and treated with 1% formaldehyde for ChIP assays. Primer
pairs used for PCR are as follows: GAL1?190and GAL154(lanes 1), GAL1427and GAL1726(lanes 2), GAL11039and GAL11331(lanes 3), GAL11764
and GAL12079(lanes 4), and GAL11921and GAL12153(lanes 5); for the nontranscribed region, Intergenic V-1 and Intergenic V-2.
4210 KROGAN ET AL.MOL. CELL. BIOL.
VOL. 23, 2003HISTONE H3 METHYLATION BY Set2 AND RNAPII ELONGATION 4211
FIG. 3. Set2 influences transcription by RNAPII. (A) Sensitivity of the set2 deletion strain to 6-AU. Strains containing either wild-type SET2
or a set2? allele, as well as the plasmid pRS316 (51), were plated on synthetic dextrose-uracil medium with or without 6-AU (50 ?g/ml) and were
grown at 30°C for 2 to 4 days. WT, wild type. (B) Expression of ?-galactosidase from the lacZ fusion plasmid p416GAL1-lacZ (9) in WT and set2?
cells. Cells were grown in medium containing 2% glucose until an OD595of ?1 was reached and washed three times in distilled water; then,
medium containing 2% galactose was added with or without 20 ?g of 6-AU/ml and ?-galactosidase activities were assayed after 4 h of growth at
30°C. (C) Density of RNAPII along the lacZ gene on the GAL1-lacZ plasmid. By employing an antibody that recognizes the Rpb3 subunit of
RNAPII (Neoclone Biotechnology), ChIP was used to monitor the approximate relative concentrations of RNAPII at various positions across the
lacZ gene. Wild-type and set2? strains harboring p416GAL1-lacZ were incubated in medium containing 2% glucose until an OD595of 1 was
reached. Cells were harvested, washed three times with water, and reinoculated into media containing either 2% glucose or 2% galactose. After
4 h of incubation at 30°C, cells were cross-linked with formaldehyde for the ChIP assays. PCR amplification was carried out with primer pairs
recognizing promoter (labeled 1), coding (labeled 2, 3, and 4) and 3? untranslated (labeled 5) regions for GAL1-lacZ. Primer pairs used for PCR
were as follows: 1, GAL1-lacZ-340and GAL-lacZ?70; 2, GAL1-lacZ715and GAL1-lacZ1025; 3, GAL1-lacZ1567and GAL1-lacZ1827; 4, GAL1-
lacZ2247and GAL1-lacZ2465; and 5, GAL1-lacZ3094and GAL1-lacZ3414; for the nontranscribed region, Intergenic V-1 and Intergenic V-2.
deleting genes encoding TFIIS or components of the TREX
complex, it has been argued that a decrease in ?-galactosidase
production with this plasmid correlates with a defect in tran-
scriptional elongation (9, 42, 56; our unpublished data). After
4 h of galactose induction, ?-galactosidase synthesis was re-
duced about threefold in a set2? strain compared to that of a
strain with wild-type SET2 (Fig. 3B). The addition of 20 ?g of
6-AU/ml to a set2 deletion strain harboring the lacZ reporter
plasmid resulted in an approximately 20-fold reduction of
?-galactosidase compared to that of a wild-type strain (Fig.
3B). These results, combined with the slight sensitivity to 6-AU
of a set2? strain, suggested that Set2 is a positively acting
transcription factor, perhaps an elongation factor. To further
study the effect that a set2 deletion had on the amount of
?-galactosidase produced from the reporter plasmid, ChIP was
performed to monitor the approximate concentration of Rpb3,
the third largest subunit of RNAPII, at various positions across
the lacZ gene (Fig. 3C). As has been previously observed, there
was an apparent decline in the density of RNAPII from the 5?
end to the 3? end of the gene even in a wild-type strain (23),
and there was a similar 5?-to-3? decline in a set2? strain. How-
ever, the apparent concentration of RNAPII in the promoter-
proximal region was lower in a set2? strain than in a wild-type
strain, suggesting that transcriptional initiation might be de-
fective when SET2 is deleted. The uniform decline of RNAPII
across the lacZ gene in a set2? strain suggests either that Set2
does not affect elongation or else that Set2 uniformly acceler-
ates elongation without preventing the release of RNAPII at
particular sites on the lacZ gene.
Evidence for cotranscriptional methylation of histone H3
Lys36 by Set2. In order to test whether Set2 functions during
transcriptional elongation, the recruitment of Set2, as well as
the presence of Lys36 H3 methylation, was monitored on the
PMA1 gene when several known elongation factors were de-
leted. The Paf1 complex, which contains five subunits and
associates with RNAPII, is thought to be an RNAPII elonga-
tion factor on the basis of both biochemical and genetic criteria
(25, 34, 52). In particular, it colocalizes with RNAPII in the
coding regions of various genes (25, 43). Interestingly, deletion
of genes encoding either of two components of the Paf1 com-
plex, Rtf1 or Cdc73, resulted in a marked decrease in the
recruitment of Set2 across PMA1 (Fig. 4A) and abolished
Lys36 H3 methylation (Fig. 4B), demonstrating that the Paf1
complex is important for the recruitment of Set2 and essential
for its methylation activity.
The RNAPII CTD kinase Ctk1 (27) phosphorylates Ser2 on
the CTD heptapeptide repeats during transcriptional elonga-
tion (10). Deletion of the gene encoding Ctk1 also nearly
eliminated the recruitment of Set2 and its histone H3 Lys36
methylation activity on the PMA1 gene (Fig. 4A and B). The
RNAPII CTD consists of proline-rich heptapeptide repeats
(2). Set2 contains a WW domain in its C-terminal region (Fig.
2A), and WW domains are known to recognize proline-rich
peptides (30). Consistent with this, we found that deleting the
C-terminal portion of Set2, including its WW domain, signifi-
cantly reduced the recruitment of Set2 to the PMA1, ADH1,
and PYK1 genes, especially toward their 3? ends and virtually
eliminated histone H3 Lys36 methylation even though the cat-
alytic SET domain of Set2 was still intact (Fig. 2A and B).
These data suggest that an interaction between the WW do-
main or C-terminal region of Set2 and the Ser2-phosphory-
lated CTD might stabilize the association of Set2 with elon-
gating RNAPII and trigger Lys36 methylation of histone H3.
Genetic evidence relating Set2 function to elongation by
RNAPII. To further evaluate the function of Set2, we used a
method for systematic construction of double mutants, termed
SGA analysis (61), in which the set2? mutation was crossed to
an array of ?4,700 mutant strains, each carrying a unique gene
deletion. Inviable or slow-growing double-mutant meiotic
progeny identify functional relationships between genes. The
set2 deletion was first introduced into a haploid starting strain
of mating type MAT? and then crossed to the array of gene
deletion mutants of the opposite mating type, MATa. Sporu-
lation of the resulting diploid cells led to the formation of
FIG. 4. Effects of deleting genes encoding elongation factors on
Set2 recruitment and histone H3 Lys36 methylation. (A) Recruitment
of Set2 to an actively transcribed gene in strains containing deletions of
genes encoding elongation factors. ChIP was used in strains containing
TAP tags on Set2 to monitor the presence of Set2 along the PMA1
gene. Recruitment of Set2 is severely compromised when genes en-
coding subunits of the Paf1 complex, Rtf1 and Cdc73, or the RNAPII
CTD kinase, Ctk1, are deleted. (B) The presence of Lys36 methylation
on histone H3 in strains containing deletions of genes encoding elon-
gation factors. Deletions of CDC73, RTF1, and CTK1, as well as SET2,
eliminate Lys36 methylation on the PMA1 gene.
VOL. 23, 2003HISTONE H3 METHYLATION BY Set2 AND RNAPII ELONGATION4213
double-mutant meiotic progeny. This resulted in an ordered
array of double-mutant haploid strains whose growth rate was
monitored by visual inspection and image analysis of colony
size. Putative genetic interactions were then confirmed by ei-
ther tetrad dissection or random sporulation.
There were approximately 60 double-deletion combinations
that resulted in synthetic growth defects. Of these, seven are
currently thought to function in transcriptional elongation,
namely RTF1, CDC73, LEO1, CTR9, PAF1, SOH1, and CHD1
(Fig. 5). Rtf1, Cdc73, Leo1, Paf1, and Ctr9 are components of
the Paf1 complex, which is associated with RNAPII and the
RNAPII elongation factor Spt16/Pob3 (25, 34, 52). SOH1 was
originally identified as a suppressor of HPR1 (15), a compo-
nent of the transcription elongation complex, TREX (56).
Soh1 is also thought to associate with the Mediator initiation
complex in higher eukaryotic cells (18). Chd1 is a member of
the chromodomain-helicase-DNA-binding family. It has been
shown to be involved in ATP-dependent nucleosome remod-
eling (62) and has also recently been implicated in transcrip-
tional elongation (11, 25). These genetic interactions with
known elongation factors implied that Set2 functions during
Synthetic growth defects were also detected between set2?
and all seven components of the Set3 complex, namely CPR1,
HOS2, HST1, SIF2, SNT1, YIL112w, and SET3 (Fig. 5) (41).
The Set3 complex contains two known histone deacetylases,
Hos2 and Hst1, but no specific methylation activity has yet
been attributed to the complex. Similarly, deletions of six of
the eight subunits of the Set1-containing complex, COMPASS
(CPS60, CPS40, CPS35, CPS30, CPS25, and CPS15) (7, 24, 33,
36, 46), were found to be synthetically sick with set2? (Fig. 5).
The remaining two components of COMPASS were not tested,
since the CPS35 gene is essential and no set1? strain was
present in the original deletion array. These results suggested
that the function of Set2 is similar in some way to the func-
tioning of COMPASS and the Set3 complex.
A set2 deletion also generated synthetic growth defects when
combined with a deletion of the gene encoding either of two
components of the histone H2B ubiquitination complex, Bre1
or Lge1 (Fig. 5) (19, 67; our unpublished data), consistent with
observations that histone H2B ubiquitination is essential for
histone H3 methylation by Set1 (13, 58). Interestingly, a set2?
htz1? double mutant also has a synthetic growth defect. Htz1
is a histone H2A variant (20), and this result suggested that
Htz1 might have a role in histone methylation or transcrip-
tional elongation or their consequences.
The specificity of the synthetic genetic interactions uncov-
ered by the SGA screen with SET2 was quite striking. In 64
other full-scale SGA screens, synthetic genetic interactions
were only rarely detected with genes encoding subunits of
COMPASS, the Set3 complex, or the Paf1 complex (e.g., for
the Set3 complex: 1 of 64 were detected for SET3, 2 of 64 for
YIL112W, 2 of 64 for HOS2, 0 of 64 for HST1, 5 of 64 for SIF2,
3 of 64 for SNT1, 2 of 64 for CPR1) and then only when screens
FIG. 5. Genetic interaction network representing the synthetic growth defects identified by SGA analysis. Genes are represented by nodes, and
interactions are represented by lines that connect the nodes. All of the interactions were confirmed by either tetrad analysis (for synthetic lethals)
or random sporulation (for synthetic growth defects). Synthetic lethal interaction are shown as red lines, and synthetic growth defects are shown
as blue lines. There were approximately 40 other genetic interactions with SET2 that were identified in the screen and are not shown in this
4214KROGAN ET AL.MOL. CELL. BIOL.
were done with genes that are likely to be involved in tran-
scriptional elongation (A. Tong and C. Boone, unpublished
data). In total, there were 45 interactions with subunits of
COMPASS, the Set3 complex, or the Paf1 complex in 64
screens, averaging less than one hit per screen. Of the 37 other
synthetic genetic interactions with set2? that were detected in
our screen (data not shown), 17 were open reading frames of
unknown function and none are known to be involved in tran-
scription, although further study may indeed reveal that some
are involved in transcriptional elongation and/or histone meth-
Histone H3 methylation on Lys36 was recently attributed to
the SET domain-containing protein Set2 in S. cerevisiae (54).
In an effort to further characterize this protein, we used single-
step transformation to place a TAP tag on the C terminus of
Set2. Following purification and analyses by SDS-PAGE and
mass spectrometry, we discovered that a substoichiometric
amount of RNA polymerase II (RNAPII) copurified with Set2.
This physical interaction suggested that methylation of his-
tone H3 Lys36 might be intimately linked to transcription.
ChIP analyses demonstrated that Set2 localizes primarily to
the coding regions of three genes that were tested, and this
correlated well with the locations of histone H3 Lys36 meth-
ylation. Consistent with the notion that the recruitment of Set2
is linked to elongation by RNAPII, the presence of Set2 was
found to correlate with active transcription when ChIP was
performed on the GAL1 gene following induction with galac-
tose. Strahl et al. tethered Set2 to a heterologous promoter and
demonstrated that, under these conditions, Set2 represses
transcription (54). However, we found little or no Set2 or
histone H3 Lys36 methylation in various promoter regions,
suggesting that Set2 activity is not normally directed towards
this region. Deletion of the SET2 gene caused slight sensitivity
to 6-AU and a large reduction in ?-galactosidase produced by
a reporter gene. Both the sensitivity to 6-AU and the reduced
?-galactosidase synthesis from the reporter gene that we used
in our experiments have been used as indicators that a gene is
involved in enhancing elongation by RNAPII (3, 9, 25, 42, 52).
A similar suggestion has been made based on the enhanced
sensitivity to 6-AU generated when a SET2 deletion is com-
bined with a deletion of DST1, which encodes TFIIS (28).
Follow-up ChIP experiments showed that the concentration of
RNAPII is lower across the lacZ gene in a set2? background
than in a wild-type strain. Therefore, we propose that wild-type
Set2 normally has a positive role in transcription. This exper-
iment did not, however, prove that Set2 specifically stimulates
elongation by RNAPII.
Phosphorylation and dephosphorylation of the CTD of the
largest subunit of RNAPII, Rpb1, are required for gene reg-
ulation (22, 63). Phosphorylation on Ser5 of the CTD hep-
tapeptide repeats, YSPTSPS, by the Kin28 subunit of the gen-
eral transcription factor TFIIH is associated with early
transcriptional elongation, whereas Ser2 phosphorylation by
Ctk1 is found at later elongation steps (10, 23). Our Western
blot analyses demonstrated that both types of RNAPII copu-
rify with Set2, again implying that the recruitment of Set2 to a
transcription unit and histone H3 methylation on Lys36 might
be cotranscriptional. It is striking that the distribution of Set2
along various genes, strong in the coding region and weak in
the promoter-proximal and 3? untranslated regions, is virtually
identical to the distribution of Ser2-phosphorylated CTD (10,
23) and the Ser2 kinase, Ctk1 (M. Kim, S. H. Ahn, and S.
Buratowski, unpublished data). Moreover, deletion of CTK1
results in substantially less recruitment of Set2 to the PMA1
gene and virtually eliminates histone H3 Lys36 methylation,
indicating that Lys36 methylation depends on Ser2 phosphor-
ylation of the CTD during transcriptional elongation.
The WW domains of Ess1/Pin1 and Rsp5 are known to
interact with the proline-rich CTD repeats of RNAPII (8, 35,
57). Since our ChIP experiments showed that deletion of the
C-terminal region of Set2, including its WW domain, reduced
the recruitment of Set2 and essentially eliminated its methyl-
ation activity, we propose that there is a physical interaction
between the WW domain of Set2 and the Ser2-phosphorylated
CTD of RNAPII that stabilizes the association of Set2 with
RNAPII and triggers the histone H3 methylation activity of
Set2. Consistent with this conclusion, Li et al. recently also
found that Set2 copurifies with RNAPII and showed that Set2
can bind to high concentrations of a Ser2-phosphorylated CTD
peptide (29). Although this study found that deletion of the
WW domain of Set2 eliminates the association between
RNAPII and Set2, we have observed that Set2 (?476-733)-
TAP, which lacks the WW domain, still coimmunoprecipitates
with some RNAPII (N. J. Krogan and J. Greenblatt, unpub-
lished data) and is still recruited, albeit less efficiently, to ac-
tively transcribed genes (Fig. 2A and B). Xiao et al. (68) have
found that a C-terminal portion of Set2 that is distal to its WW
domain is sufficient to bind RNAPII. Therefore, a site on
RNAPII other than the Ser2-phosphorylated CTD is also
likely to mediate an interaction with Set2.
To further pursue Set2’s role in transcriptional elongation,
the presence of histone H3 Lys36 methylation and the recruit-
ment of Set2 were examined on the PMA1 gene when genes
encoding subunits of the Paf1 complex were deleted. The Paf1
complex contains five subunits (Paf1, Cdc73, Rtf1, Leo1, and
Ctr9) and has recently been implicated, both biochemically and
genetically, in the process of transcriptional elongation (11, 25,
34, 52). Specifically, deletions of genes encoding components
of the Paf1 complex cause sensitivity to 6-AU and genetically
interact with mutations in genes encoding other known elon-
gation factors. Furthermore, the Paf1 complex interacts phys-
ically with both RNAPII and Spt16/Pob3, the yeast homologue
of the human elongation factor FACT (39). ChIP experiments
have also shown that the Paf1 complex colocalizes with
RNAPII in the coding regions of various genes (25, 43) and,
like Set2, declines in the region beyond the poly(A) signal
(Kim et al., unpublished). We have shown here that when
genes encoding two components of the Paf1 elongation com-
plex, Cdc73 and Rtf1, are deleted, Set2 recruitment is signifi-
cantly reduced and histone H3 methylation is virtually elimi-
Consistent with a functional connection between Set2 and
the Paf1 complex, synthetic growth defects were observed
when a set2 deletion was individually combined with deletions
of genes encoding all five subunits of the Paf1 complex. The
SET2 gene also interacts with the genes encoding two other
putative elongation factors, Soh1 and Chd1. Mutations in
VOL. 23, 2003HISTONE H3 METHYLATION BY Set2 AND RNAPII ELONGATION 4215
SOH1 were originally identified as suppressors of HPR1 (15), a
component of the transcription elongation complex TREX
(56). We have recently found that a soh1 deletion also interacts
genetically with genes encoding a number of other known
elongation factors, including TFIIS, and causes significant sen-
sitivity to 6-AU and a marked decrease in expression of the
lacZ gene on the plasmid p416GAL1-lacZ (Krogan and Green-
blatt, unpublished). The CHD1 gene interacts genetically with
a number of genes encoding elongation factors (11; Krogan
and Greenblatt, unpublished) and was recently implicated in
termination by RNAPII (1). Moreover, we recently found that
Chd1 is associated with casein kinase II and Spt16/Pob3 (25)
and can be effectively cross-linked to the coding regions of a
number of genes (Kim et al., unpublished). The dependence of
Set2 function on the Paf1 complex and the CTD kinase Ctk1,
as well as the many genetic interactions between the SET2
gene and genes encoding known positive elongation factors,
strengthens our conclusion that Set2 function is cotranscrip-
tional and that Set2 may act as an activator of elongation.
Surprisingly, synthetic growth defects were obtained when a
set2 deletion was combined with deletions of genes encoding
all seven components of the Set3 complex (41). The Set3 com-
plex has a negative effect on the expression of certain meiosis-
specific genes, perhaps because the Set3 complex contains two
histone deacetylases, Hos2 and Hst1, as well as Set3. The
synthetic phenotypes generated when a set2 deletion is com-
bined with deletions of genes encoding components of the Set3
complex suggest, however, that the Set3 complex may also have
a positive role in transcription by RNAPII. This idea is sup-
ported by the recent observation that at least two components
of the Set3 complex, Set3 and, surprisingly, Hos2, are recruited
to actively transcribed genes (65).
A set2 deletion also generated synthetic growth defects when
combined with deletions of genes encoding components of the
Set1 complex, COMPASS. COMPASS methylates Lys4 of hi-
stone H3, a modification that has been shown to be needed for
effective silencing at telomeres and ribosomal DNA loci (7, 24,
33, 36, 46). Interestingly, components of COMPASS as well as
FIG. 6. Model for the coupling of histone methylation to transcriptional elongation and the phosphorylation of RNAPII in S. cerevisiae. See
the text for details.
4216KROGAN ET AL.MOL. CELL. BIOL.
histone H3 Lys4 methylation, like Set2 and components of the
Paf1 complex, localize to the transcribed regions of various
genes and interact genetically with a number of known or
suspected elongation factors (5, 48; our unpublished data).
Moreover, methylation of Lys4 by COMPASS, like methyl-
ation of Lys36 by Set2, also requires components of the Paf1
complex, which associates with COMPASS (26, 37). Genetic
interactions among components of three different Set protein-
containing complexes imply that these complexes are function-
ally redundant and may all associate directly or indirectly with
RNAPII and function during transcriptional elongation.
Our data and other published studies on COMPASS and
Set2 have uncovered a remarkable coordination of RNAPII
phosphorylation with histone H3 methylation during transcrip-
tional elongation, as illustrated in Fig. 6. COMPASS is re-
cruited specifically to the early transcribed region (26, 37), and
this depends on both the Paf1 complex and phosphorylation of
the RNAPII CTD on Ser5 by Kin28. The consequence of
COMPASS recruitment is histone H3 trimethylation on Lys4
in the same region (37). Upon further elongation by RNAPII,
Ser5 phosphorylation declines and is replaced by Ser2 phos-
phorylation mediated by Ctk1 (10, 23). We found that Set2 is
recruited to this region, where it methylates Lys36 of histone
H3, and the recruitment of Set2 depends on both the Paf1
complex and phosphorylation of RNAPII by Ctk1 on Ser2, as
has also been observed by Li et al. (28) and Xiao et al. (68).
Finally, in the region beyond the poly(A) signal, Set2 disap-
pears and histone H3 Lys36 declines in concert with dephos-
phorylation of RNAPII on Ser2 (10, 23) and the disappearance
of the Paf1 complex (Kim et al., unpublished).
Finally, set2? bre1? and set2? lge1? double mutants also
have synthetic growth defects. Bre1, an E3 ubiquitin ligase, is
associated with Rad6 and Lge1, and all three are required for
ubiquitination of histone H2B, a modification which is neces-
sary for methylation of histone H3 on Lys4 by COMPASS (13,
19, 58, 67; our unpublished data). Therefore, disruption of
Lys4 methylation by deleting genes encoding components of
COMPASS or the Bre1/Lge1 complex results in a growth de-
fect when SET2 is also deleted. This further confirms the
functional redundancy between the Lys36 and Lys4 methyla-
tions on histone H3. Interestingly, SET2 also genetically inter-
acts with HTZ1, which encodes a variant histone, H2A (20).
This genetic interaction may predict a role for Htz1 in histone
ubiquitination, histone methylation, and/or transcriptional
We are grateful to A. Aguilera for the gift of the plasmid
p416GAL1-lacZ and to B. Strahl and K. Struhl for sharing unpublished
N.J.K. was supported by a PGS-B Scholarship Award from the
Natural Sciences and Engineering Research Council of Canada
(NSERC) and a Doctoral Fellowship from the Canadian Institute of
Health Research (CIHR). This research was supported by grants to
J.F.G. from the Canadian Institutes of Health Research, the Ontario
Genomics Institute, and the National Cancer Institute of Canada with
funds from the Canadian Cancer Society.
N.J.K. and M.K. contributed equally to this report.
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