The Set2 Histone Methyltransferase Functions through the Phosphorylated Carboxyl-terminal Domain of RNA Polymerase II*

Bing LiDagger , LeAnn HoweDagger ||, Scott Anderson§, John R. Yates III§, and Jerry L. WorkmanDagger **

From the Dagger  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

    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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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 set2Delta ppr2Delta 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
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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

    MATERIALS AND METHODS
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Yeast Strains and Genetic Manipulations-- YBL102 (Set2- TAP::TRP1) and YBL139 (Delta 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 (ppr2Delta ::URA3) is a gift from Dr. Kane and has been described (61). YBL155 (set2Delta ppr2Delta ) is a haploid strain generated from a cross between YBL150 (set2Delta ::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 his3Delta 200 ura3-52 leu2-3,112 rpb1Delta 187::HIS3 GAL+(pRP112)), then shuffling out plasmid pRP112.

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 beta -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).

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 set2Delta (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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INTRODUCTION
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RESULTS
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REFERENCES

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.


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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).

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).


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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).

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.


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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).

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.


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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.

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 (ppr2Delta ), deletion of SET2 in combination with deletion of TFIIS (ppr2Delta ) 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 set2Delta 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.

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 ppr2Delta clearly grows more slowly than the wild-type on 6-AU plates. Nevertheless, ppr2Delta set2Delta 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

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: CTD, carboxyl-terminal domain; 6-AU, 6-azauracil; TAP, tandem affinity purification; pol II, RNA polymerase II.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

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