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Previous Article | Next Article 
Molecular and Cellular Biology, January 2007, p. 709-720, Vol. 27, No. 2
0270-7306/07/$08.00+0 doi:10.1128/MCB.01627-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Abhijit Shukla,2,
Jessica Schneider,1
Jung Shin Lee,1
Julie D. Stanton,3
Tiffany Dzuiba,1
Selene K. Swanson,4
Laurence Florens,4
Michael P. Washburn,4
John Wyrick,3
Sukesh R. Bhaumik,2 and
Ali Shilatifard1,5*
Department of Biochemistry, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, Missouri 63104,1 Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, 1245 Lincoln Drive, Carbondale, Illinois 62901,2 School of Molecular Biosciences, Washington State University, Pullman, Washington 99164,3 Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110,4 Saint Louis University Cancer Center, Saint Louis University School of Medicine, St. Louis, Missouri 631045
Received 31 August 2006/ Returned for modification 5 October 2006/ Accepted 24 October 2006
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ABSTRACT |
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INTRODUCTION |
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During active transcription, the histone H3K4 methyltransferase COMPASS (complex of proteins associated with Set1) associates with the elongation factor Paf1 complex (Paf1C) to interact with Pol II and chromatin (26, 31, 32, 50). The Paf1C appears to function as a platform for the recruitment of several methyltransferases such as COMPASS and Set2 (16, 17, 26, 44). Histone methylation by COMPASS is predominantly associated with the early 5' coding regions of active genes, whereas Set2-mediated methylation is mostly found in the mid-to-late bodies of transcribed genes (26, 44). As Pol II initiates transcription, COMPASS can mono-, di-, and trimethylate H3K4 in the early 5' coding regions of active genes (26, 32, 40, 50). Shortly after promoter clearance, the histone methyltransferase Set2 engages with the elongating Pol II to methylate H3K36 on the bodies of actively transcribed genes (27, 28, 29, 38, 44, 52). During the transition from the early elongating form to the processively elongating form, the phosphorylated residue of the Pol II carboxyl-terminal domain (CTD) shifts from serine 5 to serine 2. The CTD kinase I complex (Ctk complex), the kinase responsible for serine 2 phosphorylation in yeast, mediates the association of Set2 with Pol II through its ability to phosphorylate the CTD (9, 27, 34, 52).
While attempting to define the molecular mechanism of histone H3 methylation by COMPASS, we discovered that Ctk complex components regulate histone H3K4 methylation patterns. Deletion of any of the subunits of the Ctk complex not only abolishes Set2-meditaed methylation of H3 K36 but also affects the methylation state of H3K4. Systematic analysis of bulk histones in CTK1, CTK2, or CTK3 deletions demonstrated that the loss of these factors leads to global reduction of histone H3K4 monomethylation and elevated K4 di- and trimethylation levels. The Ctk complex does not appear to substantially regulate Rad6/Bre1-mediated histone H2B monoubiquitination (which is required for COMPASS function) (12, 48, 49). It was also recently demonstrated that monoubiquitination of histone H2B is not required for full histone H3 monomethylation by COMPASS (11, 40, 43). Our studies demonstrate that loss of Ctk1 activity can result in altered patterns of histone H3 eviction from transcribed regions.
Recent studies demonstrate that histone methylation (mono-, di-, or trimethylation) patterns may play a role in the regulation of gene expression or proper response to developmental or environmental signals (25, 37, 40, 41). Therefore, transition from mono- to dimethyl or from di- to trimethyl moieties on chromatin is likely to be a highly regulated event. Evidence presented here indicates a possible role for the Ctk complex in proper regulation of the pattern of H3K4 mono-, di-, and trimethylation.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ChIP.
Chromatin immunoprecipitation (ChIP) was performed with wild-type (WT) and 
ctk1 strains. ChIP experiments were then performed as previously described (4). Briefly, after formaldehyde fixation, cells were collected by centrifugation and lysed by agitation with glass beads. The cells were then sonicated to yield chromatin fragments between 300 and 500 bp in length. After clarification of the chromatin solution via high-speed centrifugation, cross-linked proteins were immunoprecipitated (IP) with the indicated antibodies. Following elution of the bound proteins, the protein was digested away from the DNA with proteinase K. Decrosslinking was then carried out at 65°C overnight, and the next day the DNA was isolated by phenol-chloroform extraction, followed by ethanol precipitation. The DNA fragments were then used for PCR with primers directed against the indicated gene regions. The primers used for PCR of the PHO84 and ADH1 loci as shown in Fig. 3 and 5 have been previously described (22, 23). The primers used for PCR were GAL1 (Core) (5'-ATAGGATGATAATGCGATTAGTTTTTTAGCCTT-3' and 5'-GAAAATGTTGAAAG TATTAGTTAAAGTGGTTATGCA-3'), GAL1 (ORF1) (5'-CAGTGGATTGTCTTCTTCGGCCGC-3' and 5'-GGCAGCCTGATCCATACCGCCATT-3', GAL1 (ORF2) (5'-CAGAGGGCTAAGCATGTGTATTCT-3' and 5'-GTCAATCTCTGGACAAGAACATTC-3'), GAL1 [poly(A)] (5'GCATCACAAAATACGCAATAATAACGAG and 5'TTTTGTCCCTGTGTTTTAAAGTTTGTGG), ADH1 (ORF1) (5'-CTGGTTACACCCACGACGGTTCTT-3' and 5'-GCAGACTTCAAAGCCTTGTAGACG-3'), ADH1 (ORF2) (5'-CGGTAACAGAGCTGACACCAGAGA-3' and 5'-ACGTATCTACCAACGATTTGACCC-3'), PHO84 (ORF1) (5'-TAGCTGATATTGTTGGTCGTAAGAG-3' and 5'-TACCAATACCCATGACAAAACGGTA-3'), and PHO84 (ORF2) (5'-TCTGCAGACATTTTGGTCAATGGAA-3' and 5'-AAACGTTTTTGGAACCGGCATAAC-3').
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For ChIP done during 6-azauracil (6AU) treatment, the WT strain was grown in YPR to an OD600 of 0.9 and then transferred to YPG containing 6AU (112 µg/ml) for 90 min of induction prior to formaldehyde cross-linking. Immunoprecipitation was performed with 8WG16 antibody against Rpb1 and H3K4 dimethyl and trimethyl antibodies as indicated previously. IP DNAs were analyzed by PCR with specific primer pairs targeted to different regions of the GAL1 locus as indicated.
For analysis of histone H3 occupancy, both the WT and CTK1 deletion mutant strains were grown in YPR to an OD600 of 0.9 and then transferred to YPG for various induction times prior to formaldehyde cross-linking. Immunoprecipitation was performed with an anti-H3 antibody (Abcam 1791). IP DNAs were analyzed by PCR at the GAL1 core promoter, ORF1, and ORF2. Percent immunoprecipitation (%IP) of H3 at all induction time points from WT and CTK1 deletion mutant strains was calculated. The maximum %IP was set to 100%, and other %IP values were normalized with respect to the maximum %IP.
ChIP-on-chip methods.
ChIP-on-chip analysis was performed as described previously (36), with slight modifications. Briefly, triplicate cultures of WT and ctk1 strains were grown to mid-log phase (OD600, 0.4 to 0.5) and cross-linked with 1% formaldehyde. Cells were lysed with glass beads and sonicated to generate DNA fragment sizes of 150 to 400 bp. Cross-linked DNA fragments were enriched by immunoprecipitation with anti-histone H3 trimethyl K4 antibody (Abcam) bound to magnetic beads (Dynal Biotech). The beads were washed and then eluted, and the cross-links were reversed. IP DNA was amplified and labeled with Cy5 dye (Amersham Biosciences) by ligation-mediated PCR. As a control, a sample of whole-cell extract (WCE) DNA that was not IP was amplified by ligation-mediated PCR and labeled with Cy3 dye (Amersham Biosciences). Labeled PCR products were hybridized to yeast 6.4K ORF arrays (University Health Networks, Toronto, Ontario, Canada) and scanned with a ScanArray 4000XL scanner (Packard Bioscience).
The scanned images were quantitated with the QuantArray software (Packard Biosciences). The final spot intensity was calculated by subtracting the local background intensity from the spot intensity and then normalized by a global intensity normalization method. The reported gene intensities are averages of duplicate spots present in the yeast 6.4K ORF array. The average IP/WCE (Cy5/Cy3) ratio for each gene was calculated for the triplicate experimental data sets.
TAP tag purification of protein complexes. Purification of tandem affinity purification (TAP)-tagged proteins was performed essentially as previously described (35). Briefly, 1.5 liters of yeast was grown to an OD600 of 1.2. After collection of the cells via centrifugation, the cells were lysed with glass beads in 25 ml of lysis buffer (10 mM Tris [pH 7.9], 150 mM NaCl, 0.1% NP-40, protease inhibitors [Roche]). After clarification of lysates by centrifugation, TAP-tagged proteins were bound to immunoglobulin G-Sepharose beads overnight at 4°C. Following cleavage of the TAP tag with tobacco etch virus protease, the calcium concentration of the eluent was adjusted to 3 mM CaCl2 and it was incubated with calmodulin-Sepharose 4B (Amersham). Following several washes with calmodulin binding buffer (10 mM Tris [pH 7.9], 150 mM NaCl, 0.1% NP-40, 5 mM ß-mercaptoethanol, protease inhibitors [Roche complete mini, EDTA free], 3 mM CaCl2), bound proteins were eluted by the addition of EGTA elution buffer (10 mM Tris [pH 7.9], 150 mM NaCl, 4 mM EGTA, 5 mM ß-mercaptoethanol). The purity of each fraction was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of the protein complexes.
Kinase assays using Ctk1.
Kinase assays were performed essentially as described before (51). Briefly, IP protein complexes were added to 28 µl of kinase assay buffer (50 mM Tris [pH 7.5], 10 mM MgCl2, 0.5 mM dithiothreitol). After the addition of 5 µCi of 
-32P, the reaction mixtures were incubated for 40 min at 30°C. Each reaction was quenched by the addition of SDS-PAGE loading buffer (preheated to 90°C) and brief heating of the samples at 90°C for 5 min. Reaction products were then immediately resolved by SDS-PAGE on 4 to 20% gradient gels and subjected to autoradiography.
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RESULTS |
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Loss of Ctk complex impacts H3 lysine 4 monomethylation and appears to function in a Rad6/Bre1-independent manner. Although deletion of any of the three components of the Ctk complex leads to reduced histone H3K4 monomethylation levels, K4 dimethylation and trimethylation are not noticeably reduced. This observation indicates that the catalytic properties of COMPASS may shift toward di- or trimethylation in the absence of the functional Ctk complex. To test this, amounts of bulk H3K4 monomethylation and trimethylation in strains with CTK1 and CTK3 deleted were analyzed. Compared to the WT, ctk null strains present an about 60 to 70% reduction in global H3K4 monomethylation (Fig. 2A and B). Additionally, the H3K4 trimethylation found in bulk histone preparations of CTK1 and CTK3 deletion strains is slightly higher than that of WT cells (Fig. 2B). This suggests that COMPASS activity is indeed skewed toward dimethylation and trimethylation of H3K4 in a ctk1 null background.
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Since COMPASS activity is mediated through its association with Pol II via its interaction with the Paf1C (16, 17, 26), we wanted to ensure that the altered stability of the largest subunit of Pol II (Rpb1) in the absence of Ctk1 phosphorylation was not the cause of the observed H3K4 methylation changes. Therefore, we compared Rpb1 levels from WT and ctk1 null cells. This study indicated no apparent substantial changes in Rpb1 levels (Fig. 2D).
Changes in COMPASS activity do not appear to be the direct result of COMPASS phosphorylation by the Ctk complex. Discrete subunits of COMPASS mediate individual H3K4 methyl marks (40). Phosphorylation of Rad6 and recruitment of the Paf1C to chromatin by the Bur1/Bur2 complex modulate H2B monoubiquitination levels (51, 53). Our observation that loss of Ctk1 activity results in an overall decrease in histone H3K4 monomethylation and an increase in H3K4 trimethylation from bulk histone may indicate that COMPASS processivity increases in the absence of Ctk1. Since COMPASS catalytic activity is altered in the absence of Ctk1, it is possible that Ctk1 activity may be directed toward a subunit of COMPASS. This activity could serve as a repressive mark to inhibit COMPASS activity or prevent the binding of a specific subunit required for proper regulation of histone methylation. To test this idea, COMPASS and the Ctk complex were affinity purified with the TAP tag on Cps60 and Ctk1, respectively, and the presence of subunit components was confirmed by MudPIT analyses (40). Using these reagents, we reconstituted an in vitro kinase assay. Pol II was purified with TAP-tagged Rpb3 as a positive control, since Ctk1 was previously shown to phosphorylate Rpb1 (9). When COMPASS is incubated in the presence of Ctk1, no observable kinase activity toward COMPASS is detected in vitro (Fig. 2E, lanes 3 to 5). However, addition of the Ctk complex to Pol II resulted in phosphorylation of the Rpb1 subunit (Fig. 2E, lanes 7 to 9), indicating that the purified Ctk complex is enzymatically active.
The Paf1C plays an integral role in COMPASS-mediated histone methylation (26, 32, 50). The affinity-purified Paf1C was also used in an in vitro kinase assay to determine if subunits of the Paf1C are targets for Ctk-mediated phosphorylation. While there is no specific Ctk-mediated activity toward any member of the Paf1C, a nonspecific activity was observed when the Paf1C was incubated alone or with the Ctk complex (Fig. 2F, compare lane 11 to lanes 12 to 14). Since the Ctk complex displays no observable specificity for subunits of COMPASS or the Paf1C, it seems likely that the aberrant histone H3 methylation observed in the absence of Ctk complex function may be directly linked to the phosphorylation state of other substrates such as the CTD of Pol II.
Deletion of CTK1 results in accumulation of histone H3K4 trimethylation on chromatin on constitutively active genes. Histone H3K4 methylation and COMPASS are localized predominantly at the promoter and early 5' coding regions of active genes in yeast (26, 33, 37). Since our studies have concentrated on analysis of bulk histones, we wanted to determine whether an increase in histone H3K4 di- and trimethylation occurs on chromatin of actively transcribed genes following the loss of Ctk1. Using antiserum directed against di- and trimethylated histone H3K4, ChIP was performed in a WT strain, as well as strains with CTK1 deleted. Interestingly, ChIP experiments revealed an increase in trimethylation levels in the coding region of the transcriptionally active genes tested (PHO84 and ADH1) (Fig. 3A and B). In the accompanying report, Strahl and colleagues have also demonstrated that an increase in trimethylation levels in a ctk1 null strain is manifested as a "spread" of these methyl marks from the 5' end to the 3' end of individual genes (54). Since Set2 does not associate with the elongating form of Pol II in the absence of Ctk1, it is possible that association of COMPASS with Pol II becomes more stable and COMPASS activity extends throughout active coding regions.
Both histone H3K4 dimethylation and trimethylation are increased during active transcription at an inducible gene in the absence of CTK1. ChIP studies performed during galactose induction of the GAL1 gene demonstrated that both H3K4 di- and trimethylation dramatically increase in a CTK1 null strain compared to WT cells (Fig. 3C and D). The increase in histone methylation correlates with heightened times of transcriptional activity at the GAL1 locus, reaffirming the interplay between COMPASS activity and Pol II in H3K4 methylation regulation. Interestingly, although an increase in the levels of trimethylated H3K4 were observed at the constitutive ADH1 and PHO84 loci, the increase in both di- and trimethylation was greatly exacerbated during the switch from an inactive to an induced GAL1 gene (Fig. 3C and D).
Loss of CTK1 results in a global increase in H3K4 trimethylation levels. When global ChIP-on-chip analyses were performed to observe regions where trimethylation increased in the absence of Ctk1 activity, the number of genes with enriched H3K4 trimethylation increased nearly threefold in the ctk1 null strain (378 genes) compared to the WT (132 genes; Fig. 3E and F). A similar increase in H3K4 trimethylation enrichment was observed independent of the cutoff used to identify enriched genes (e.g., 2-fold, 1.75-fold, and 1.5-fold). This result confirms that global levels of histone H3K4 trimethylation on chromatin are elevated in the ctk1 null mutant and that trimethylation is now occurring at loci not normally found to be trimethylated. In contrast, there is a slight decrease in the number of genes with depleted H3K4 trimethylation in the ctk1 null mutant compared to the WT (Fig. 3E and F). In the WT strain, regions depleted for H3K4 trimethylation contained a significant number of genes located in telomere-proximal chromosomal regions (86 genes, P = 2.4 x 10–57), as had been previously observed for H3K4 dimethylation (3). In the ctk1 null mutant, however, the number of telomere-proximal genes depleted for H3K4 trimethylation is decreased by 35% compared to the WT.
Histone occupancy and association of Pol II are altered on an actively transcribed gene in the absence of Ctk1 activity. Since we observed an increase in the levels of H3K4 di- and trimethylation on actively transcribed gene GAL1 in the absence of Ctk1 (Fig. 3), we wanted to determine whether Ctk1 loss could alter the occupancy of histone H3. As shown in Fig. 4A to D, the occupancy of H3 on the core promoter and body of the GAL1 gene is inversely correlated with transcription. Histone H3 is evicted as the GAL1 gene is activated by switching the growth medium from raffinose (noninducing) to galactose (inducing). Such observations on the occupancy of H3 on promoters and transcribed regions of genes have recently been reported (1, 5). However, our ChIP studies demonstrate that histone H3 eviction during active transcription is reduced in a ctk1 null background (Fig. 4A to D). To determine whether this reduction in the rate of histone H3 eviction is through the reduced levels of Pol II recruitment to chromatin, we performed ChIP studies to determine the kinetics of recruitment of Pol II to the GAL1 gene upon activation (Fig. 5A to D). Our studies demonstrate that a reduced amount of Pol II was found to be associated with chromatin (compared to WT strains) when ChIP was used to determine if a buildup of Pol II occurred during the induction of the GAL1 gene in a CTK1 null strain (Fig. 5A to D). Approximately half as much Pol II is associated with the GAL1 locus in the CTK1 deletion cells after 90 min (Fig. 5A). However, in the same time period, H3K4 di- and trimethylation are increased drastically (Fig. 3C and D).
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Ctk1 loss does not appear to specifically alter Pol II elongation properties on the GAL1 gene on the basis of our measurements; however, the overall rate of transcription, including transcription initiation, appears to be slowed down in the absence of Ctk1. Mason and Struhl recently demonstrated that the drug 6AU reduces both the elongation rate and the processivity of Pol II and that this processivity defect is exacerbated by mutations in Spt4, TFIIS, and the Ctk complex (the Ctk kinase complex) (30). Furthermore, induction of transcriptional stress, such as treatment with 6AU, can result in altered nucleosome occupancy on the TEF1 gene in yeast (55). We tested the effect of 6AU treatment on Pol II and nucleosome occupancy and H3K4 methylation on the GAL1 gene (Fig. 4E). Our study indicates that 6AU does not significantly alter the pattern of H3K4 trimethylation on the genes tested. In an accompanying report, Strahl and colleagues also demonstrated that 6AU treatment does not alter H3K4 trimethylation levels from bulk histone (54). Therefore, on the basis of these observations we conclude that Ctk1's regulation of K4 di- and trimethylation appears to be independent of 6AU-induced stress on the GAL1 gene.
Analysis of the role of factors required for histone eviction in the regulation of the pattern of H3K4 methylation. Our studies thus far indicate that loss of Ctk1 results in altered patterns of Pol II occupancy and H3 eviction from chromatin. These changes are accompanied by altered H3K4 methylation patterns. Pol II occupancy and histone eviction are functionally related effects resulting from Ctk1 loss. To determine whether defects in histone H3 eviction alone could cause the observed phenotypes, we tested the effect of an ASF1 deletion in this process. Recently, it was demonstrated that Asf1 specifically mediates histone H3 eviction (42). Therefore, we initially tested the effect of ASF1 deletion on bulk patterns of H3K4 methylation. As shown in Fig. 6A, ASF1 deletion does not significantly alter global H3K4 methylation patterns compared to CTK1, -2, and -3 deletions (compare Fig. 6A to Fig. 1E). To further demonstrate whether ASF1 deletion alters H3K4 methylation patterns on chromatin, we analyzed H3K4 methylation on the GAL1 gene in the presence and absence of Asf1 by ChIP (Fig. 6B). These studies also confirm our observation that Asf1 does not alter H3K4 methylation patterns (compare Fig. 6B to Fig. 3C and D). Furthermore, we did not observe defects in Pol II occupancy in an asf1 null strain (Fig. 6C). These studies together suggest that the defect in histone H3 eviction alone does not explicate the altered H3K4 methylation phenotype seen in the absence of Ctk complex components.
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Although trimethylation of H3K4 by COMPASS does not take place as rapidly as it is reported to do in vivo, this is to be expected as (i) the in vitro reaction is set up with limiting concentrations of enzyme and substrates for the purpose of studying the reaction itself in vitro and (ii) the in vitro methylation reaction with COMPASS and H3 is set up in the absence of monoubiquitinated H2B and chromatin, and therefore COMPASS will not be able to transition to trimethylation as rapidly. However, this study clearly demonstrates that a transition from mono- to dimethylation and then from di- to trimethylation is a sequential process and that COMPASS requires this extended time to process the conversion from unmethylated to monomethylated and then on to di- and trimethylated histone H3. Therefore, as more histone H3 becomes trimethylated, this means that less H3 in the reaction mixture is left in the monomethylated form. Indeed, toward the end of the assay one can observe that trimethylation is steadily increasing as monomethylation is starting to recede (Fig. 6D and E).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since Ctk1 is the predominant CTD serine 2 kinase in yeast, any disruption of Ctk1 activity can have numerous outcomes for transcription efficiency (6, 9, 27, 34). Phosphorylation of the Pol II CTD by Ctk1 serves as a docking site for another histone methyltransferase, the Set2 protein. Deletion of Ctk1 not only abolishes interaction between Set2 and Pol II, it also ablates Set2's interaction with chromatin (27, 28, 29, 47, 52). Set2 histone methyltransferase activity remains associated with elongating polymerase until transcriptional termination (27). Unlike COMPASS-mediated methylation in the early 5' coding regions of active genes, Set2-mediated methylation of histone H3 is linked to transcriptional repression (7, 19, 21, 47). Recently, several groups have shown that the bromodomain of Eaf3 binds methylated lysine 36 of histone H3, and this recruits the Rpd3 histone deacetylase complex (7, 19, 21). Thus, one of the main functions of CTD serine 2 phosphorylation by the Ctk complex is to regulate a subset of transcriptional repressors during the processive stages of transcription. In addition to regulation of Set2 histone methyltransferase activity by the Ctk complex, we suggest that the Ctk complex may also modulate the pattern of histone modifications mediated by Set1/COMPASS within active regions of chromatin. Although the Ctk complex is not capable of phosphorylating either COMPASS or the Paf1C in vitro (Fig. 2E and F), Ctk1 substrates other than the CTD of Pol II may regulate COMPASS-mediated H3K4 methylation patterns. We have recently initiated biochemical and genetic projects in the laboratory searching for a possible substrate.
Numerous studies in the last few years have investigated the regulation of COMPASS histone methyltransferase activity, and here we indicate a unique role for the Ctk complex in this process. Many of the previously described mechanisms involving Rad6/Bre1, the Paf1C, and the Bur1/Bur2 complex demonstrate a reduction in COMPASS activity. However, deletion of Ctk complex components results in a change in COMPASS-mediated histone methylation and a global shift toward dimethylation and trimethylation of H3K4 as monomethylation levels diminish in vivo. This effect is quite different from that observed when histone H2B monoubiquitination is altered. An increase in histone H2B monoubiquitination results in increased COMPASS activity, and monomethylation becomes enriched (10). It is possible that increased histone monoubiquitination allows transcription to proceed more rapidly; therefore, the time of association between COMPASS and the histone H3 tail is reduced and monomethylation becomes the predominant form of methylated H3K4. This is similar to in vitro studies performed with COMPASS and H3 (Fig. 6).
Early in our studies, it was uncertain whether Ctk1 loss results in an increase in the levels of histone H3K4 trimethylation in a manner by which COMPASS histone methyltransferase activity becomes hyperactivated. Loss of Ctk1 affects several events in the transcriptional cycle, i.e., (i) the histone methyl mark left by Set2, (ii) serine 2 phosphorylation of the CTD, (iii) the timely recruitment of polymerase to activated genes, and (iv) the loss of proper transcriptional efficiency. Additionally, we demonstrated with our ChIP studies that histone H3 eviction during transcription is reduced in a CTK1 deletion strain. However, this delay does not fully explain the observed increase in H3K4 trimethylation levels, as the loss Asf1 (which is also required for histone H3 eviction [42]) does not result in H3K4 monomethylation loss or an increase in trimethylation similar to that of ctk1 null strains. Our in vitro studies demonstrated the necessity of increased times of association between COMPASS and the histone H3 tail in order to properly catalyze the pattern of mono, di-, and trimethylation of H3K4.
On the basis of the above observations, one can envision a scenario in which the transcription initiation, promoter clearance, and transcription elongation stages of the transcription cycle are sluggish in the absence of a functional Ctk complex. Furthermore, Ctk1 loss results in a decrease in the rate of histone H3 eviction and creates an increase in the concentration of the substrate for COMPASS. As a result of the "sluggish polymerase," the local histone H3 concentration can increase, as does the time of association between COMPASS and histone H3 on chromatin.
In addition to the importance of methylation on particular lysine residues within histones, patterns of methylation (such as mono-, di-, or trimethylation) are required for a proper response to extracellular and intracellular signals (25, 37, 40, 41). Therefore, cells must somehow regulate the patterns of methylation on chromatin. Our studies suggest a possible mechanism for the regulation of the pattern of H3K4 methylation via COMPASS by the Ctk complex. If the pattern of H3K4 methylation on chromatin could be regulated by the rate of the elongating Pol II, therefore, elongation factors such as ELL or elongin A, which alter the Vmax of transcription elongation (13, 15, 45), could play a role in the regulation of the pattern of histone methylation. Future genetic and biological studies aiming to define roles for histone mono-, di-, and trimethylation patterns on chromatin and factors required for the implementation of such marks should be very informative.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 6 November 2006.
A.W. and A.S. contributed equally to this report.
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