From the Howard Hughes Medical Institute, Department
of Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, Pennsylvania 16802-4500, and the
§ Department of Cell Biology, The Scripps Research
Institute, La Jolla, California 92037
Received for publication, November 29, 2002, and in revised form, January 2, 2003
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ABSTRACT |
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The histone methyltransferase Set2, which
specifically methylates lysine 36 of histone H3, has been shown to
repress transcription upon tethering to a heterologous promoter.
However, the mechanism of targeting and the consequence of
Set2-dependent methylation have yet to be demonstrated. We
sought to identify the protein components associated with Set2 to gain
some insights into the in vivo function of this protein.
Mass spectrometry analysis of the Set2 complex, purified using a tandem
affinity method, revealed that RNA polymerase II (pol II) is
associated with Set2. Immunoblotting and immunoprecipitation using
antibodies against subunits of pol II confirmed that the phosphorylated
form of pol II is indeed an integral part of the Set2 complex. Gst-Set2
preferentially binds to CTD synthetic peptides phosphorylated at serine
2, and to a lesser extent, serine 5 phosphorylated peptides, but has no
affinity for unphosphorylated CTD, suggesting that Set2 associates with
the elongating form of the pol II. Furthermore, we show that set2 Nucleosomal structure, once thought to be quite static, turns out
to be extremely dynamic and tightly regulated by various nonhistone
proteins (1). These protein modulators can be generally categorized
into two groups: those that utilize the energy released from ATP
hydrolysis to alter histone-DNA contacts (2), and those that are
capable of modifying histones covalently. To date, the known histone
modifications include acetylation, methylation, phosphorylation,
ubquitinylation, and ADP-ribosylation (3, 4). The consequence of
complex histone modification patterns has yet to be fully understood.
However, based on a growing body of evidence, a "histone code"
hypothesis has been proposed suggesting that these modifications either
directly alter chromatin structure (5) or create a series of molecular
"bar codes" at the nucleosome surface for other proteins to
recognize (3, 6).
Most, if not all, chromatin modulators have a somewhat weak intrinsic
affinity for their nucleosomal substrates in vitro. Nevertheless, the specific pattern of histone modifications and the
particular loci where chromatin remodeling occurs throughout the genome
strongly suggest that these activities are primarily targeted to
certain regions by either DNA bound activators or repressors (7, 8).
Acidic activators have been shown to be able to target SWI/SNF or
Spt-Ada-Gcn5-acetyltranferase to local promoters, thereby facilitating
transcription from nucleosomal templates (9-12). Likewise, the
repressor Ume6 or co-repressor Ssn6/Tup1 can recruit histone
deacetylase complexes to remove the acetylation marks from histone
tails, facilitating transcriptional repression (13-17). In addition to
activator/repressor-dependent targeting of
chromatin-modifying complexes, histone modifications, as proposed in
the "histone code" hypothesis, can also provide recognition sites
for protein complexes and enzymatic activities. For instance, the
binding of Spt-Ada-Gcn5-acetyltranferase or SWI/SNF to promoter
nucleosomes is facilitated by bromodomain recognition of acetylated
histone tails (18, 19). Moreover, phosphorylation of histone H3 serine
10 stimulates K14 acetylation by Gcn5 on the same tail (20, 21),
suggesting that cross-talk can occur between different modifications.
More strikingly, modifications of different histones can also influence
each other in a specific manner. Ubiquitinylation of histone H2B is
required for both methylation of lysine 4 by Set1 and lysine 79 by
Dot1, but not vice versa (22-24).
The carboxyl-terminal domain
(CTD)1 of Rpb1, the largest
subunit of the RNA polymerase II, plays an important role in
transcription regulation (25-27). It contains multiple repeats of a
heptapeptide sequence with the consensus of YSPTSPS (27) and is
responsible for the integrity of the RNA polymerase II holoenzyme (28), preinitiation complex formation (29, 30) and binding of various mRNA processing factors (31-33). Phosphorylation of the CTD is one
of the major regulatory events during the transcription cycle. It is
acutely controlled by four reported CTD kinases: Kin28 (34, 35),
Srb10/Srb11 (36, 37), CTD kinase I (with Ctk1 being the catalytic
subunits) (38), Bur1/Bur2 (39), and only one known CTD phosphatase,
Fcp1 (40). Unphosphorylated pol II interacts with the mediator
complex and is assembled into the preinitiation complex during
transcription initiation (30). The CTD is then phosphorylated by CTD
kinases to start promoter clearance and elongation, causing
dissociation of some factors responsible for preinitiation complex
formation (41) and allowing other factors necessary for elongation and
termination to bind (25). Phosphorylation sites predominantly reside at
serine 5 and serine 2 of the CTD repeats. Chromatin
immunoprecipitation experiments have shown that serine 5 phosphorylation is localized to promoter regions at initiation/early
elongation stages, whereas serine 2 phosphorylation is associated with
the coding region during the elongation phase (42). Interestingly,
recent studies have shown that the pol II holoenzyme possesses certain
intrinsic chromatin-modifying activities that may help pol II elongate
through chromatin. Elp3, a subunit of pol II elongating holoenzyme, has
histone acetyltransferase activity (43). Human RNA polymerase II
complex directly interacts with the histone acetyltransferases p300 and
PCAF. p300 specifically recognizes the unphosphorylated form and
p300/CBP associated factor (PCAF) associates with the
phosphorylated form (44).
Histone methylation occurs at many residues within all four histones,
implying that it could have a critical impact on chromatin structural
regulation (5). Recent identification of novel histone methyltransferases have implicated histone methylation in many important biological processes, such as heterochromatic silencing, transcriptional activation, and transcriptional repression (7, 45). The
catalytic domain of SUV39, the first identified histone methyltransferase, locates within an evolutionarily conserved "SET"
domain and is flanked by two other cysteine-rich domains (46). These
structural motifs have been used as a standard to search for other
histone methyltransferase candidates. In the yeast, Saccharomyces
cerevisiae, there are 6 SET domain proteins. Set1 and Set2 can
methylate histone H3 lysine 4 and lysine 36 (47-51). In addition, a
high-copy disruptor of silencing, Dot1, which has very weak homology
with other methyltransferases, has also been found to methylate histone
H3 at lysine 79 in a nucleosomal context (52-55). The
methyltransferase activities of these enzymes are essential for their
related biological function, but how these chromatin modifying enzymes
are tethered to specific loci remains largely unknown. To investigate
the potential targeting mechanism of histone methyltransferases, we
chose to first identify the proteins that are associated with Set2
in vivo. Mass spectrometry analysis of the Set2 complex
purified using a tandem affinity method revealed that Set2 physically
associates with RNA polymerase II. Immunoblotting and
immunoprecipitation with specific antibodies against different
phosphorylated states of the pol II CTD showed that Set2
methyltransferase activity primarily associates with the elongating
form of the RNA polymerase II that is hyperphosphorylated. This implies
that methylation mediated by Set2 may be involved in regulating
transcription and elongation. Genetic interaction between Set2 and
transcription elongation factor-TFIIS further strengthened this
argument. Moreover, the CTD plays an important role in H3 K36
methylation in vivo. These data are consistent with the
results of three other groups who independently discovered the
interaction of Set2 with the
phosphorylated form of RNA polymerase II
(56).2,3
Yeast Strains and Genetic Manipulations--
YBL102
(Set2- TAP::TRP1) and YBL139
( Protein Purification--
The TAP purification was performed
essentially as described elsewhere (59, 60) with minor modification.
Briefly, 6 liters of yeast cells expressing TAP-tagged Set2 were grown
in yeast extract-peptone-dextrose medium at 30 °C to an optical
density of about 2 at 600 nm. The cell pellet was resuspended in an
extraction buffer (E buffer) (40 mM HEPES pH 7.5; 350 mM NaCl; 10% glycerol; 0.1% Tween 20, 1 mM
sodium orthovanadate; 10 mM NaF; and 1 mM Histone Methyltransferase (HMT) Assay--
In vitro
histone methylation reactions were carried out for 1 h at 30 °C
in a 25-µl system containing 50 mM Tris-HCl (pH 8.0); 50 mM NaCl; 1 mM EDTA; 1 mM
MgCl2; 1 mM dithiothreitol, 1 µl of [3H]methyl S-adenosyl-methionine (80 Ci/mmol,
Amersham Biosciences) with 2 µg of HeLa oligonucleosomes or
recombinant yeast histone octamers as substrates. Half of the reaction
was then spotted on Whatman P-81 filter paper for liquid scintillation
counting and the other half was separated on an 18% SDS-PAGE gel,
stained with Coomassie Blue, and fluorographed with En3hance
(PerkinElmer Life Sciences) as described previously (64).
Peptide Pull-down Assay--
The biotinylated CTD peptides
(about 1.5 µg) were bound to 0.5 mg of streptavidin-coated Dynabeads
M280) in 50 µl of high salt binding buffer (25 mM
Tris-HCl pH 8.0; 1 M NaCl; 1 mM dithiothreitol; 5% glycerol; 0.03% Nonidet P-40) at 4 °C for 2 h. The
protein-bound beads were washed once with high salt binding buffer and
twice with CTD binding buffer (25 mM Tris-HCl pH 8.0; 50 mM NaCl; 1 mM dithiothreitol; 5% glycerol;
0.03% Nonidet P-40) and finally resuspended in 50 µl of CTD binding
buffer. Five hundred nanograms of bacterially overexpressed GST-Set2
protein was mixed with the beads and incubated at 4 °C for 1 h
on a Dyna-Mixer (Dynal). Following washing three times with CTD binding
buffer, beads were then subjected to an in vitro HMT assay.
6-AU Sensitivity Assay--
Wild-type and set2 Histone Methyltransferase Set2 Physically Associates with RNA
Polymerase II--
Histone methylation and HMTs have been
extensively investigated by different research groups, however, the
mechanism regarding how histone methyltransferases are targeted to
specific loci has yet to be carefully addressed. In an effort to
determine a potential targeting mechanism of histone
methyltransferases, we decided to identify proteins associated with a
known histone methyltransferase and gain some insights from these
protein partners. Histone methyltransferase Set2 was chosen to start,
because Set2 possesses very robust HMT activity on nucleosomal
substrates (48, 55) and has been shown to regulate transcription on
tethering to a heterologous promoter (48).
We took advantage of a tandem affinity purification (TAP) technique
(59, 60) to isolate potential partners of Set2 in vivo.
TAP-tag with a TRP1 marker was PCR-amplified from vector pBS1479 and
integrated to the carboxyl termini of the chromosomal copy of
SET2. TAP-tagged Set2 strain was confirmed by PCR and Western blotting with PAP (Sigma). Yeast whole-cell extracts from the
Set2-TAP strain were prepared and subsequently applied to IgG-Sepharose
and calmodulin resin. Purified Set2 complex was then separated on a
SDS-PAGE gel and subjected to protein fingerprinting by mass
spectrometry (Fig. 1A).
Protein identification of the Set2 complex revealed that two of the
largest components in the Set2 complex are Rpb1 and Rpb2, the two
largest subunits of RNA polymerase II. Moreover, the silver staining
pattern of the complex excluding Set2 (as labeled in Fig.
1A) is very similar to that of previously purified RNA
polymerase II, suggesting that the complex we isolated by this approach
is likely a Set2 bound RNA polymerase II complex.
To confirm the mass spectrometry identification, we performed
immunoblotting with some of the available antibodies against different
forms of pol II to determine whether the corresponding antibodies can
recognize the components identified by mass spectrometry. Western
blotting using antibody against the Rpb3 subunit of pol II demonstrated
that Rpb3 is an integral part of the Set2 complex. Taf6 antibodies were
used as a negative control (Fig. 1B). The Rpb1 subunit of
pol II has different isoforms in regard to the phosphorylation status
of its CTD. Monoclonal antibodies H5 (against CTD-serine
2-Pi) and H14 (against CTD-serine 5-Pi)
were able to pick up the low abundant signals from whole-cell extracts.
However, they react strongly with the TAP purified fraction.
Conversely, using antibody 8WG16 (which recognizes unphosphorylated
CTD), we found that although the unphosphorylated CTD is relatively more abundant in crude extracts, it was not enriched by our
purification (Fig. 2A). We
believe that the faint signal in the Set2-TAP fraction with 8WG16
antibody is probably due to weak cross-reactivity of this antibody with
phosphorylated forms of CTD (65).
Previously, RNA polymerase II has been purified through a method that
combines conventional chromatograph and immunoaffinity column using
8WG16 antibody (66, 67). This method efficiently enriches
unphosphorylated RNA polymerase II, but it inevitably loses some
protein components that only interact with phosphorylated polymerase
II. This may be the reason why Set2 was not found in previously
isolated pol II. To further characterize the Set2 methylase-bound RNA
polymerase, and rule out the possibility that Set2 might bind separately to a subset of pol II, we thus subjected the purified Set2
complex directly onto a gel filtration column. Eluted fractions were
either subjected to Western blotting to detect the presence of proteins
(Fig. 2A) or assayed for histone methyltransferase activity
(Fig. 2B). The peak fraction containing the bulk of Rpb1 (H5, serine 2-Pi-CTD) and Rpb3 co-fractionate with histone
methyltransferase activity, suggesting that Set2 is an integral
component of a phosphorylated RNA polymerase II complex. Based on the
elution profile of the gel filtration column, we estimate that
molecular mass of this complex is about 700 kDa, which is consistent
with the previously reported molecular mass of pol II (about 550-600
kDa) plus the mass of Set2-CBP (about 95 kDa) (66, 68, 69).
Collectively, these results indicate that Set2 physically associates
with the hyperphosphorylated form of RNA polymerase II.
Set2 Specifically Recognizes the Phosphorylated CTD--
The
histone methyltransferase Set2 primarily associates with the
phosphorylated form of RNA polymerase II but not the unphosphorylated form, implying that the CTD likely contributes to this specific interaction. We next sought to determine whether the CTD is directly involved in this interaction and if the CTD alone is
sufficient to support Set2-pol II interaction. Using a defined
synthetic CTD peptide with different combinations of phosphorylation at serine 2 and serine 5, Ho and Shuman have successfully demonstrated the
interaction between mammalian guanylytransferase and the CTD phosphopeptide (70). We thus adopted a similar system where biotinylated synthetic peptides consisting of 4 tandem repeats of the
CTD heptad sequence (YSPTSPS) are used. The CTD peptide only contains 4 repeats of heptapeptides, whereas CTD-serine 2-Pi and
CTD-serine 5-Pi have a phosphate group on all 4 specific
serines in each peptide. These peptides were bound to
streptavidin-coated magnetic beads in a high salt buffer and washed
extensively to remove unbound proteins. Peptide-bound beads were then
adjusted to a low salt buffer and incubated with purified, bacterially overexpressed GST-Set2 protein. Measurement of histone
methyltransferase activity was subsequently taken for bound fractions
and unbound fraction (supernatant) (Fig.
3). Fluorography showed that both the
serine 2-Pi and serine 5-Pi CTD peptide beads
can efficiently pull down HMT activity from solution, whereas
unmodified CTD fails to do so (Fig. 3A). Using a more
quantitative liquid scintillation counting assay (Fig. 3B),
we demonstrated that the serine 2-Pi CTD peptide displays
relatively stronger affinity to GST-Set2 than the serine
5-Pi CTD peptide, which is consistent with the Western blot
in Fig. 1B. Therefore, we conclude that phosphorylation of
the CTD is sufficient to determine the binding of Set2 to RNA polymerase II in vitro.
To determine whether the specific interaction between Set2 and RNA
polymerase II can serve as a signal to regulate the recruitment of
histone methyltransferase activity of Set2 to its target in vivo, we measured the relative methylation levels of histones in
RNA polymerase II CTD truncation mutants. Although removal of all the
CTD repeats are lethal, deletion of a significant number of repeats can
be tolerated by cells (62, 71). Three CTD truncation mutants containing
9, 10, and 14 repeats, respectively and an isogenic wild-type strain
(with 26 repeats) (62) were tested for the histone methylation level by
Western blotting using specific antibodies against the modified
histones (Fig. 4). We found that there
exists a good correlation between the relative level of methylation of
H3 at lysine 36 and the degree of CTD truncation. In particular, the
CTD mutant containing 9 repeats displays a significant reduction of K36
methylation. This correlation is consistent with growth phenotype of
these mutants, where only the mutant containing 9 repeats shows
temperature sensitivity (62). More importantly, this methylation defect
occurs specifically at lysine 36, which is mediated by Set2 (48), but
not at lysine 4 (Fig. 4A) where its corresponding
methyltransferase Set1 does not seem to stably interact with RNA
polymerase II (49-51). This result suggests that CTD truncation does
not cause a general histone methylation defect, and the compromised
methylation level of H3 at lysine 36 is likely caused by reduced
recruitment of the histone methyltransferase Set2 to its chromatin
substrates through the truncated RNA polymerase II CTDs.
Set2 Genetically Interacts with TFIIS and Participates in
Regulating Transcription Elongation--
Set2 specifically binds to
hyperphosphorylated RNA polymerase II, which is believed to be an
elongating form of pol II, suggesting that Set2 mediated histone
methylation may be involved in transcription elongation. A genetic
approach was taken to address this issue in vivo.
Transcription factor TFIIS plays an important role in regulating
elongation. TFIIS can stimulate RNA polymerase II to cleave its nascent
transcripts whenever pol II stalls at the blocks generated by either
DNA or DNA-binding proteins, thereby facilitating RNA polymerase II to
proceed through the DNA templates (72, 73). Moreover, TFIIS has been
shown to genetically interact with many chromatin-modifying factors,
such as the Spt4/Spt5 elongation complex, the elongator complex (with
Elp3 being a histone acetyltransferase), the Spt16/Cdc68 component of
FACT (facilitates chromatin transcription), and SWI/SNF (see
review in Ref. 73). This strongly suggests that chromatin remodeling is
involved in the regulation of transcription elongation. Unlike SWI/SNF,
where deletion of its subunits is synthetically lethal with deletion of
TFIIS (ppr2
The drug, 6-AU has been widely used in a phenotypical test for
transcription elongation defects. When metabolized, 6-AU is converted
to 6-azaUMP, which inhibits the enzyme of de novo nucleotide synthesis and reduces the level of GTP and UTP (74). Nucleotide depletion creates an additional obstacle for the crippled transcription elongation machinery to overcome, thereby making elongation mutants more sensitive to the drug. A yeast strain lacking SET2
displays a very modest 6-AU sensitivity even at relatively high drug
concentration (Fig. 5, 50 µg/ml and 100 µg/ml) whereas
ppr2 We have presented biochemical evidence that the histone
methyltransferase Set2 physically associates with the RNA polymerase II. This interesting observation provides a novel mechanism for the
targeting of histone methyltransferase activity to genes by interaction
with the basal transcription machinery. More importantly, this
association is highly regulated, because Set2 binds to the serine 2 phosphorylated CTD, and the serine 5 phosphorylated CTD to a lesser
extent, but shows little interaction with the unphosphorylated CTD. CTD
phosphorylation is well known to be coordinately controlled by multiple
CTD kinases and phosphatases during a transcription cycle (27). CTD
enters preinitiation complex as an unphosphorylated form at the
transcription initiation stage (28). Serine 5 of the CTD is
subsequently phosphorylated at the promoter clearance and early
elongation stages around promoter regions (42). With pol II reading
along the coding region, serine 2 becomes gradually phosphorylated
whereas the phosphate group at serine 5 is removed (42). Set2
preferentially binds to the elongating form (serine 2-Pi-CTD) of RNA polymerase, suggesting that it might play
a role in pol II elongation through nucleosomal templates. Its
relatively weak interaction with the serine 5-CTD might also make Set2
able to enter the transcription cycle at an early stage. Furthermore, Set2 genetically interacts with transcription elongation factor TFIIS,
providing in vivo evidence for the involvement of histone methyltransferases in elongation. This conclusion is also supported by
the earlier observation that K4 methylated histone H3 is primarily distributed in the coding regions of active genes (76).
The question of how Set2-mediated histone methylation influences
transcription elongation still remains unresolved. Based on recent
studies and data presented here, we propose some working models
regarding the function of Set2 in transcription elongation. First, it
was noticed that when yeast RNA polymerase III transcribes through
nucleosomes, it creates an intranucleosomal DNA loop, which causes
polymerase to intermittently stall along the nucleosomal DNA templates
(77, 78). In this sense, methylation of histone tails may alter the
nucleosomal structure in such a way that RNA polymerase II can read
through its template smoothly and efficiently. Second, methylation
marks can also stimulate the binding of other chromatin remodeling
factors and elongation factors so as to facilitate elongation. A
Brm-containing chromatin remodeling complex can recognize the histone
methylation pattern generated by the Drosophila epigenetic
regulator-histone methyltransferase, Ash1, and binds to the methylated
peptide in vitro (79). It would be interesting to determine
whether SWI/SNF prefers to bind to methylated nucleosomes, because its
function has been tightly linked to transcription elongation in an
in vivo study (61). Third, Set2-mediated methylation might also block the addition of other histone modifications or prevent
other chromatin-modifying complexes from accessing nucleosomes. It has
been demonstrated that methylation of K4 of histone H3 can disrupt the
interaction between the histone deacetylase NuRD and the H3 tail (80,
81). In addition, genome-wide hypoacetylation of chromatin overlaps
with H3 K4 hypomethylation (76). Future experiments to address these
possibilities will certainly improve our understanding on the effects
of methylation on transcription elongation.
Histone acetylation is dynamically regulated by histone
acetyltransferases and histone deacetylases (82). However, to date, a
histone demethylase has yet to be discovered, which leaves a controversial debate over whether histone methylation is dynamic or
static (45, 83, 84). It has been proposed that removal of methylation
marks from chromatin is either though proteolytic digestion of histone
tail or total replacement of the methylated histone (3). Accumulating
evidence suggests that methylation might also be dynamically regulated,
based on a series of observations that methylation is involved in many
transcription system in which genes need to be turned on or off in a
rapid fashion or within one cell cycle (47, 57, 58, 85, 86). The data
presented here also point to the fact that at least some methylation
might be reversible, considering the association of Set2 with the basal transcription machinery, which often needs to transcribe the same nucleosomal DNA region for multiple rounds. It would be inefficient to
erase those marks by cleaving the tail or reassembling nucleosomes. To
this end, investigations aimed at finding out the potential mechanism
for the turnover of methylation are an important priority.
ppr2
double mutants
(PPR2 encodes TFIIS, a transcription elongation factor) are
synthetically hypersensitive to 6-azauracil, and that deletions in the
CTD reduce in vivo levels of H3 lysine 36 methylation.
Collectively, these results suggest that Set2 is involved in regulating
transcription elongation through its direct contact with pol II.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
SET2::URA3) were made by PCR-based
tagging strategy with the vector pBS1479 (59, 60). The TAP tag was
integrated at the carboxyl terminus of the endogenous SET2
loci and confirmed by PCR and Western blotting with
peroxidase-anti-peroxidase (Sigma), which recognizes the protein
A module of the tag. Yeast strain CMKy80
(ppr2
::URA3) is a gift from Dr. Kane and has
been described (61). YBL155 (set2
ppr2
) is
a haploid strain generated from a cross between YBL150
(set2
::LEU2) and CMKy80. RNA
polymerase II CTD truncation mutants were provided by J. Corden (62).
Strain RPB1, rpb1 9CTD, 10 CTD, and 14 CTD are constructed
by introducing individual plasmid pY1 (containing wild-type CTD), Y1WT
(9), pY1WT (10), or pY1WT (14) into Z26 (MAT alpha
his3
200 ura3-52 leu2-3,112
rpb1
187::HIS3 GAL+(pRP112)),
then shuffling out plasmid pRP112.
-glycerophosphate, supplemented with complete sets of fresh
proteinase inhibitors) and disrupted in a bead beater (Biospec). Crude
whole-cell extracts were then clarified by ultracentrifugation and
directly applied to IgG-Sepharose and calmodulin resin as described
previously (59, 60), with the exception that all buffers contained 10% glycerol. The purified complex was resolved on a 10% SDS-PAGE gel and
visualized with silver staining. Excised protein bands were subjected
to mass spectrometry as described in previously (63).
(YBL150) strains were transformed with the ARS/CEN plasmid pRS316 prior
to the test. All yeast strains were grown in the synthetic media
lacking uracil to an OD600 of 1, then plated onto
Ura
synthetic drop-out plates supplemented with 25 µg/ml, 50 µg/ml, or
100 µg/ml of 6-azauracil (Sigma). Cells were allowed to grow 2-4
days at 30 °C before photographs were taken.
RESULTS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of the proteins associated
with the histone methyltransferase Set2. A, the Set2 complex
was purified from the yeast strain (YBL102) expressing
carboxyl-termini-TAP-tagged Set2. The purified complex was separated on
a 10% SDS-PAGE and visualized with silver staining. A similar
purification from an untagged strain (w303a) was performed in parallel
and we did not observe any significant bands with silver staining (data
not shown). The excised bands were subjected to mass spectrometry
analysis. Identified peptides from indicated bands are listed in the
boxes. The Rpb3 subunit is solely identified by Western blotting using
an antibody against yeast Rpb3 (see Fig. 2A). The estimate
molecular mass of the bands labeled with asterisks are
matched with the migration pattern of previously purified RNA
polymerase II and are presumably corresponding subunits of RNA
polymerase II. B, yeast whole-cell extract (YBL102) and
TAP-purified Set2 complex were assayed by immunoblotting with the
monoclonal antibodies 8WG16 (against unphosphorylated CTD of Rpb1), H5
(against serine 2 phosphorylated CTD), and H14 (against serine 5 phosphorylated CTD), as well as polyclonal antibodies against the Rpb3
subunit, and Taf6 (as a negative control).
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Fig. 2.
Set2 is an integral component of an RNA
polymerase II complex. TAP-purified Set2 complex was subjected to
gel filtration chromatography on a mini-Superose 6 column using a Smart
system (Amersham Biosciences). All fractions were assayed by
immunoblotting (A) and in vitro HMT assays
(B). Fractions not shown in the figure did not contain
significant signals in either assay. Sizes of the complexes were
estimated by calibrating the column with molecular mass standards.
A, immunoblotting of column fraction with the indicated
antibodies. B, HMT assays with HeLa oligonucleosomes as
substrates. Both recombinant GST-Set2 and the Set2 complex can
efficiently use HeLa oligonucleosomes and recombinant yeast histone
octamers as substrates, but neither can methylate HeLa core histones
(data not shown).
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Fig. 3.
Set2 specifically binds to the
hyperphosphorylated CTD peptide. Biotinylated synthetic CTD
peptides (1.5 µg) were adsorbed to 500 µg of streptavidin-coated
magnetic beads (Dynal M280). About 500 ng of recombinant GST-Set2 was
incubated either with magnetic beads alone (Mock) or beads
with immobilized peptides, as indicated, for 1 h at 4 °C.
Unbound fraction (Sup) and bound fraction (B)
were subjected by HMT assay with 2 µg of HeLa oligonucleosomes.
Reactions were incubated at 30 °C for 1 h with occasional
gently flicking the tubes. Half of the reactions were loaded on an 18%
SDS-PAGE gel for fluorography (A) and the remainder were
spotted on Whatman P-81 filters for liquid counting
(B).
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Fig. 4.
Methylation of H3 at lysine 36 is compromised
in RNA polymerase II CTD truncation mutants. A, whole-cell
extracts were prepared from yeast strains indicated. RPB1 contains a
wild-type copy of the RNA polymerase II CTD, whereas rpb1 9 CTD, 10 CTD, and 14 CTD harbor only the
plasmid carrying 9, 10, and 14 CTD repeats, respectively. Western blots
were probed with antibodies against dimethylated K36 of histone H3,
dimethylated K4 of histone H3, and Taf6 (as a loading control).
B, the relative levels of K36 methylation in the RNA
polymerase II CTD truncation mutants are quantified based on three
experiments.
), deletion of SET2 in combination
with deletion of TFIIS (ppr2
) are viable and grow
normally on synthetic media (Fig. 5) and
rich media (data not shown).
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Fig. 5.
Set2 is involved in transcription elongation
in vivo. Yeast strains were grown in Ura
synthetic drop-out media to an OD600 of 1. Cultures were
collected and diluted to identical optical densities. Four microliters
of serial dilutions from each culture were spotted on SD
URA plates
or SD
URA supplemented with different concentration of 6-AU.
Photographs of the plates were taken after 2 days for control plates
and up to 4 days for strains on 6-AU. Wild-type and set2
were transformed with pRS316, an ARS/CEN plasmid
to make them URA+, all other strains contain the URA3
marker. SD, yeast synthetic drop-out medium.
clearly grows more slowly than the wild-type on 6-AU
plates. Nevertheless, ppr2
set2
double
mutants are synthetically hypersensitive to 6-AU, even at lower
concentrations (Fig. 5, 25 µg/ml), indicating that SET2 genetically interacts with PPR2. Very similar observations
were made in the case of the elongator complex in that deletion of elongator subunits alone only cause marginal phenotypes, but in combination with deletion of TFIIS result in a more severe sensitivity to 6-AU (75). The genetic interaction between TFIIS and Set2 further
solidified the argument that Set2 plays an important role in
transcription elongation, presumably through its association with RNA
polymerase II.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Steve Buratowski for kindly providing the synthetic CTD peptides. We thank Drs. C. Kane and Joseph Reese for yeast strains and reagents. We also thank the members of Simpson, Reese, and Tan laboratories for comments and advice on this study.
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FOOTNOTES |
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* This work was supported in part by Grant GM47867 from the National Institute of General Medical Sciences (to J. L. W.) and by National Center for Research Resources Yeast Center Grant RR11823.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Postdoctoral associate of the Howard Hughes Medical Institute.
Supported by a postdoctoral fellowship from the Canadian
Institutes of Health Research.
** Associate investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, 306 Althouse Laboratory, Dept. of Biochemistry and Molecular Biology, The Pennsylvania State Univ., University Park, PA 16802. Tel.: 814-863-8256; Fax: 814-863-0099; E-mail: jlw10@psu.edu.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M212134200
2 N. J. Krogan, M. Kim, A. Tong, A. Gorshani, G. Cagney, V. Canadien, D. Richards, B. Beattie, A. Emili, C. Boone, A. Shilatifard, S. Buratowski, and J. Greenblatt, submitted for publication.
3 T. Xiao, H. Hall, K. Kizer, Y. Shibata, M. C. Hall, C. H. Borchers, and B. D. Strahl, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: CTD, carboxyl-terminal domain; 6-AU, 6-azauracil; TAP, tandem affinity purification; pol II, RNA polymerase II.
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