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In Saccharomyces cerevisiae, the Nrd1-Nab3-Sen1 pathway mediates the termination of snoRNAs and cryptic unstable transcripts (CUTs). Both Nrd1 and the Set1 histone H3K4 methyltransferase complex interact with RNA polymerase II (Pol II) during early elongation, leading us to test whether these two processes are functionally linked. The deletion of SET1 exacerbates the growth rate and termination defects of nrd1 mutants. Set1 is important for the appropriate recruitment of Nrd1. Additionally, Set1 modulates histone acetylation levels in the promoter-proximal region via the Rpd3L deacetylase and NuA3 acetyltransferase complexes, both of which contain PHD finger proteins that bind methylated H3K4. Increased levels of histone acetylation reduce the efficiency of Nrd1-dependent termination. We speculate that Set1 promotes proper early termination by the Nrd1-Nab3-Sen1 complex by affecting the kinetics of Pol II transcription in early elongation.
From initiation through termination, transcript synthesis by RNA polymerase II (Pol II) is coupled to RNA processing and quality control. One mechanism for the temporal coordination of these processes is via the modification of Pol II itself. The C-terminal domain (CTD) of the Pol II subunit Rpb1 consists of multiple tandem repeats of the sequence YSPTSPS. Close to the promoter during early elongation, the serines at positions 5 (S5) and 7 (S7) of the repeat are phosphorylated by the kinase subunit of the basal factor TFIIH (reviewed in reference 8). Later in elongation, serine 2 (S2) is phosphorylated by the yeast Bur1 and Ctk1 kinases or their metazoan homologs Cdk9 (13, 28, 31, 44) and Cdk12 (4). The dynamic CTD phosphorylation patterns direct the recruitment of the appropriate RNA-processing and chromatin modification factors at different stages of transcription.
snoRNAs, some short mRNAs, and the recently discovered class of cryptic unstable transcripts (CUTs) are terminated in yeast via the Nrd1-Nab3-Sen1 pathway (3, 25, 51, 52, 56). Unlike mRNAs that undergo 3′ polyadenylation, transcripts terminated by the Nrd1-Sen1 pathway are directed to the nuclear exosome complex for 3′-end trimming or degradation (59). It is thought that the Sen1 helicase mediates termination after being brought to the transcript by the sequence-specific RNA binding proteins Nrd1 and Nab3 (10, 50). The Nrd1 and Nab3 recognition sequences are short, and their presence and frequency vary between Nrd1 target genes. However, these sites also appear in genes that are not terminated by the Nrd1-Sen1 pathway. Further biasing the complex to short genes is the interaction of the Nrd1 CTD interaction domain (CID) with the S5-phosphorylated CTD in promoter-proximal regions (17, 60). Therefore, it is the combination of the CTD interaction and RNA recognition that ensures the proper loading of the complex and subsequent transcription termination. Defects in the recruitment of the Nrd1-Sen1 complex result in read-through transcription and 3′-processing defects (60).
Transcription initiation and elongation can be influenced by histone modifications and chromatin dynamics. Examples include the deacetylation of histones by Sir2 to create repressive heterochromatin (37, 38) and the methylation of histone H3 at lysine 36 (H3K36) by Set2, which recruits the Rpd3S histone deacetylase (HDAC) complex to suppress cryptic initiation and slow elongation within mRNA genes (11, 24, 30). There are suggestions that chromatin may also play a role in the transcription termination of polyadenylated mRNA genes (2, 39). Since Nrd1-dependent termination occurs in the promoter-proximal region, we asked whether histone modifications in this region might affect snoRNA or CUT termination.
The trimethylation of histone 3 at lysine 4 (H3K4) by the histone methyltransferase Set1 (6) is highly enriched in promoter-proximal regions of actively transcribed genes, while H3K4me2 peaks slightly further downstream (26, 42). Set1 is recruited to early transcription elongation complexes coincident with the S5-phosphorylated CTD (41). Given that the peak of H3K4 trimethylation (H3K4me3) coincides with the region of Nrd1 recruitment, we tested whether H3K4 methylation affects Nrd1-dependent termination and found that the deletion of SET1 exacerbates the termination defects of nrd1 mutants. Set1-mediated H3K4 trimethylation contributes to Nrd1-dependent termination by promoting Nrd1 recruitment. The recognition of H3K4 trimethylation by several PHD finger proteins helps maintain balanced histone acetylation levels, which influence the kinetics of Pol II transcription and thereby affect the efficiency of termination by the Nrd1-Nab3-Sen1 pathway.
Saccharomyces cerevisiae strains used in this study are listed in Table 1. Plasmid pJC643 was obtained from J. Corden (16), and plasmids pRS416-SET1(1-1080), pRS416-SET1(ΔRRM), and pRS416-SET1(780-1080) were obtained from S. Briggs (6, 18). A list of the oligonucleotides used is given in Table 2. The yeast plasmids expressing Set1 with an H422A or H1017K point mutation were made by inverse PCR of pRS416-SET1(1-1080) with primers Set1 H422A up and Set1 H422A down or with primers Set1 H1017K up and Set1 H1017K down.
Northern blot experiments were performed as previously described (25, 59). Cultures grown overnight at room temperature were diluted and shifted to 30°C in the morning. The cultures were then grown until an optical density (OD) of 0.5 was reached and were harvested for RNA extraction. This temperature shift is especially important for experiments using nrd1 partial deletion strains since they grow only very slowly at 30°C or higher for extended periods of time. A total of 5 to 20 μg of total RNA was resolved on 1.2% formaldehyde-agarose gels. PCR primers described in Table 2 were used to generate probes. The PCR-amplified DNA fragment was gel purified and labeled by using the Megaprime DNA labeling system (Amersham Bioscience).
Chromatin immunoprecipitation (ChIP) experiments for tetra-acetylated H4 (catalog number 06-866; Upstate) and H3 (ab1791; Abcam) were performed as described previously (23, 26). Five micrograms of anti-Nrd1 antibody, kindly provided by J. Corden, was used per ChIP experiment. Monoclonal antibody against Rpb3 (1Y26; Neoclone) was used as previously described (1). All yeast strains were grown to an OD at 600 nm (OD600) of ~0.5 in synthetic complete (SC) medium supplemented with 2% glucose. Primers for ChIP for SNR13 were previously described (25). Twenty-six PCR cycles were used for anti-Rpb3, anti-Nrd1, anti-H3K4me3, anti-H3K4me2, and anti-H3 ChIPs. Anti-acetyl H4 ChIPs were analyzed by using 23 cycles to keep amplification in the linear range.
One microgram of total RNA was used in a Superscript II reverse transcriptase (RT) (Invitrogen) reaction with gene-specific primers (Table 2). One-fiftieth of the cDNA was amplified by using the ChIP PCR protocol with various cycle numbers to achieve linear-range amplification (30 cycles for snoRNA read-through transcripts and CUTs, 20 cycles for full-length snoRNA, and 26 cycles for snR13 read-through transcripts). Note that the data were expressed by dividing the read-through signal by the total signal without correction for different cycle numbers. Therefore, these ratios are relative and not absolute measures of termination read-through. Quantitative PCR analyses to verify a key subset of RT-PCR results were performed by using 50-fold-diluted cDNA in a Roche Light Cycler 480 sequence detector with Eva-Green detection agent. In all cases, similar results were obtained with the two approaches.
Native elongating transcript sequencing (NET-Seq) was performed with wild-type (WT) and set1Δ cells as previously described (14).
To test for a genetic interaction between SET1 and NRD1, partial-deletion alleles of NRD1 were combined with a SET1 deletion, and the growth of the resulting double mutant cells was assayed by spotting onto plates and in liquid culture. The Nrd1 CID maps roughly to amino acids 1 to 150, while a region necessary and sufficient for the Nab3 interaction (Nab3 interaction domain [NID]) maps between residues 150 and 214 (16, 50, 60, 65) (Fig. 1A). The deletion of the CID [nrd1(1-164Δ) and nrd1(6-150Δ)] results in slightly slower growth than that of NRD1 cells. However, the nrd1(1-164Δ) set1Δ and nrd1(6-150Δ) set1Δ double mutants showed a much slower growth phenotype than either the nrd1(1-164Δ) or set1Δ mutant alone (Fig. 1B and C). Since nrd1(151-214Δ) cells grew very poorly on plates at 30°C, we monitored growth in liquid culture. The doubling time of nrd1(151-214Δ) set1Δ cells was considerably longer than that observed for nrd1(151-214Δ) or set1Δ cells (Fig. 1C). This synthetic interaction suggests that Set1 might affect an Nrd1-dependent process.
To test whether the observed genetic interaction between SET1 and NRD1 is due to a defect in termination, the expression of the SNR13 gene was assayed by Northern blotting. In wild-type and set1Δ cells, transcription terminates within the downstream TRS31 open reading frame (25, 52) (Fig. 2 A, top). The terminated transcript is then trimmed to yield the mature snR13 transcript. For nrd1(1-164Δ) and nrd1(151-214Δ) cells, a larger dicistronic read-through transcript was also observed (Fig. 2A, bottom). The higher level of read-through transcripts in nrd1(1-164Δ) cells (Fig. 2A, bottom, compare lanes 3 and 5) is likely because the deletion of nrd1(1-164Δ) spans both the complete CID and part of the NID, while nrd1(151-214Δ) affects only Nab3 interactions. When set1Δ was deleted in either of the nrd1 mutant strains, read-through transcript levels increased (Fig. 2A, bottom, and B). This effect is particularly striking with nrd1(151-214Δ). This result suggests that Set1 contributes to efficient Nrd1-dependent transcription termination.
To see if the termination effect of Set1 extends beyond Nrd1 mutant cells, we deleted SET1 in a strain mutated for the Nrd1 partner Nab3. The nab3-11 allele harbors two mutations in its RNA recognition motif (RRM) domain (16) and is defective in Nrd1-dependent transcription termination (51). RT-PCR analysis was performed for nab3-11 and nab3-11 set1Δ cells grown at 30°C using primer sets against the body of snR13 and a downstream read-through region. A range of PCR cycle numbers and cDNA dilutions was tested to ensure linear-range amplification (data not shown and see Fig. S1A in the supplemental material). The deletion of SET1 in a nab3-11 mutant strain increased the accumulation of snR13 read-through transcripts (Fig. 2C), and this result was confirmed by quantitative real-time PCR (qPCR) (Fig. S1B).
The Nrd1-exosome pathway also mediates transcription termination and the degradation of cryptic unstable transcripts (CUTs), including several that emerge from heterochromatic regions of the genome (3, 52, 56, 61). Accordingly, Northern blot analysis of a CUT produced within the ribosomal DNA repeats (NTS1 CUT) was performed (Fig. 2D). No CUT accumulation was observed in wild-type or set1Δ cells. As expected, the cryptic transcript accumulated in nrd1(1-164Δ) and nrd1(151-214Δ) mutants (Fig. 2D, bottom). Consistent with the snR13 results, the levels of the CUT increased markedly when the nrd1 mutants were combined with set1Δ.
We consistently observed that the nrd1(1-164Δ) mutant results in a stronger termination defect than the nrd1(151-214Δ) mutant at SNR13 and the NTS1 CUT (Fig. 2A and D, compare lanes 3 and 5). This is likely because nrd1(1-164Δ) removes both the CTD binding domain and part of the Nab3 interaction domain, whereas the nrd1(151-214Δ) deletion abrogates only the interaction with Nab3 (Fig. 1A). Surprisingly, nrd1(1-164Δ) cells grow better than nrd1(151-214Δ) cells, suggesting that the severity of the growth phenotype could be due to defective termination affecting some essential gene where the Nab3 interaction is more important than CTD binding. Although nrd1 mutants display different levels of read-through transcription, the deletion of SET1 in either nrd1 mutant background always results in increased levels of snR13 read-through or NTS1 CUT transcripts (Fig. 2A and D, compare lanes 4 and 6).
Set1 has two predicted RRM domains in its N-terminal region, the second of which exhibits RNA binding activity in vitro (58). The in vivo relevance of this activity remains to be elucidated. The C-terminal half of Set1 contains a histone methyltransferase SET domain (6, 18, 46, 58). To determine which domains and functions of Set1 are required to support Nrd1-dependent termination, various set1 mutants (Fig. 3 A) were combined with the nrd1(1-164Δ) mutation and tested for synthetic growth phenotypes. The deletion of RRM1 [set1(230-335Δ)] or both RRMs [set1(1-779Δ)] disrupts H3K4 trimethylation (H3K4me3) but not dimethylation or monomethylation (18, 26, 46). These Set1 deletion mutants resulted in the same enhancement of the nrd1 mutant growth and termination defect as that of set1Δ (Fig. 3B to D), suggesting that H3K4me3 is the histone modification that is relevant for efficient snoRNA termination.
To test whether the genetic interaction between NRD1 and SET1 is dependent on the SET domain or RNA binding activities of Set1, two point mutants of Set1 were assayed for termination defects. The set1(H422A) mutation abolishes RNA binding in vitro without affecting in vivo histone methylation levels (see Fig. S2 in the supplemental material) (58). In contrast, the set1(H1017K) catalytic-site mutation eliminates H3K4 methylation activity in vivo (46). The set1(H422A) mutant showed no additional growth defect when combined with an nrd1 mutant (Fig. 3B). In contrast, the active-site mutant showed the same growth defect enhancement as that of set1Δ. In agreement with the growth phenotypes, snR13 read-through transcript and NTS1 CUT levels increased when an nrd1 mutant was combined with set1(H1017K) or set1(230-335Δ) (Fig. 3C and D). In contrast, set1(H422A) caused little or no additional accumulation of these transcripts.
The monoubiquitylation of histone H2B K123 by the Rad6-Bre1 complex is important for higher levels of methylation of H3K4 by Set1 (53). In rad6Δ cells, di- and trimethylation of H3K4 are lost, while monomethylation is intact (29). The combination of rad6Δ with a nrd1 mutation increased the read-through transcription of snR13 (Fig. 3E). Confirming the connection between Nrd1 and H2B K123 ubiquitylation, it was recently reported that a low level of increased snoRNA read-through was seen in rad6Δ, bre1Δ, or H2BK123R mutant cells (57). Similarly, an increased accumulation of snR13 read-through transcripts was observed for a wild-type NRD1 strain carrying an H3K4R mutation (Fig. 3F), supporting the significance of H3K4 methylation. Taken together, these results indicate that Set1 and H3K4 trimethylation are important for efficient Nrd1-dependent termination, while Set1 RNA binding is not.
Nrd1 can be recruited to target genes via its RNA binding domain, via its CTD interaction, or via its interaction with Nab3. Because Nrd1 termination defects correlate closely with reduced recruitment, we compared the chromatin immunoprecipitation (ChIP) signals of Nrd1 in nrd1(151-214Δ) and nrd1(151-214Δ) set1Δ cells (Fig. 4 B). Nrd1 cross-links to the SNR13 transcribed region (PCR products 2 and 3) (Fig. 4A and B). The Nrd1 ChIP signal was reduced when set1Δ was combined with the nrd1 mutant. This reduction was not due to changes in overall Nrd1 protein levels (Fig. 4C). ChIP of Rpb3 showed that reduced Pol II recruitment was also not responsible for the reduced Nrd1 cross-linking (Fig. 4D). Indeed, a slight increase in Rpb3 recruitment further downstream of SNR13 (PCR products 3 to 5) was observed for nrd1(151-214Δ) set1Δ cells, as expected due to increased termination read-through (Fig. 2A).
The effects of Set1 RRM deletions on snR13 and NTS1 CUT read-through (Fig. 3) suggest that H3K4 trimethylation contributes specifically to Nrd1-dependent termination. Therefore, Nrd1 recruitment was assayed in nrd1(151-214Δ) set1(230-335Δ) double mutant cells. As with set1Δ, the combination of set1(230-335Δ) with the nrd1 mutant decreased the recruitment of Nrd1 to SNR13 (Fig. 4B; for Rpb3 levels see Fig. 4D). Since set1(230-335Δ) specifically abolishes H3K4 trimethylation while leaving di- and monomethylation intact, these results suggest that it is H3K4 trimethylation that enhances Nrd1 recruitment to chromatin.
We hypothesized that protein complexes that recognize H3K4 methylation might mediate the effects of Set1 on Nrd1-dependent termination. A previous large-scale genetic interaction study (15) showed that deletions of PHO23 or other members of the Rpd3L (Rpd3 large) HDAC complex cause synthetic slow growth in combination with a hypomorphic nrd1 mutant allele. We confirmed a strong genetic interaction between pho23Δ and nrd1(1-164Δ) (Fig. 1B). Rpd3L is typically thought of as a corepressor that is recruited to a small set of promoters by sequence-specific binding partners. However, Rpd3L contains two PHD finger proteins, Pho23 and Cti6, that preferentially bind H3K4-methylated histone tail peptides in vitro (49). Therefore, we considered Rpd3L as a candidate for affecting Nrd1-dependent termination.
RNA analysis showed that pho23Δ increased snR13 read-through in nrd1(151-214Δ) cells (Fig. 5 A, compare lanes 4 and 5). We also quantitated the termination defect of set1Δ and pho23Δ in the nrd1(151-214Δ) background using RT-PCR (Fig. 5F) and quantitative real-time PCR (see Fig. S1C in the supplemental material). These results confirm the effects that we observed by Northern blot analyses. It should be noted that the Northern blots show the stable, polyadenylated snR13-TRS31 read-through transcript. However, in RT-PCR experiments, we were able to include the quantitation of transcripts that correspond to all Pol II termination events prior to the downstream TRS31 termination site, including rapidly degraded, nonpolyadenylated species. Therefore, small termination defects in set1Δ and pho23Δ cells that were below the detection limit of the Northern blots were observable (Fig. 6).
To test whether the Rpd3L HDAC affects SNR13 chromatin, levels of histone H4 acetylation in WT and pho23Δ cells were analyzed (Fig. 5B). Despite the fact that SNR13 is transcriptionally very active and not known to be a target for Rpd3L, a small increase in H4 acetylation levels was reproducibly observed at the SNR13 promoter in pho23Δ cells. In agreement with a direct effect, a ChIP with microarray technology (ChIP-chip) trace for the histone deacetylase Rpd3 showed enrichment over the snR13 region (Fig. 5C) (data from reference 7).
If increased histone acetylation underlies the enhancement of the nrd1 mutant termination defect by pho23Δ, we reasoned that the deletion of an opposing histone acetyltransferase (HAT) might suppress the pho23Δ read-through phenotype. The NuA3 HAT complex is recruited to promoter regions, at least in part by its subunit Yng1, a PHD finger protein that specifically binds H3K4me3 (19, 32, 35, 55). The deletion of YNG1 reduced the termination defect at SNR13 observed for nrd1(151-214Δ) pho23Δ cells (Fig. 5D). This suppression suggests a model in which H3K4 trimethylation targets both NuA3 and Rpd3L complexes to SNR13 via Yng1 and Pho23, respectively. The proper balance of acetylation and deacetylation might affect early elongation and thereby influence the Nrd1 early termination pathway. Accordingly, H4 acetylation levels were decreased at SNR13 in yng1Δ cells (Fig. 5E). This model can also explain why read-through in the nrd1(151-214Δ) pho23Δ strain is greater than that seen for nrd1(151-214Δ) set1Δ cells (Fig. 5A, compare lanes 4 to 6). Whereas pho23Δ would reduce the recruitment of the HDAC Rpd3L, the deletion of SET1 would lead to the loss of both the HDAC and the antagonistic HAT complex. Therefore, the increase in histone acetylation levels is less broad and pronounced in set1Δ cells than in pho23Δ cells (Fig. 5E).
To extend our results beyond SNR13, several other snoRNAs and CUTs were analyzed. Interestingly, while the deletion of SET1 strongly increased the levels of the ribosomal DNA (rDNA) NTS1 CUT in nrd1(151-214Δ) cells, no such enhancement was seen with the deletion of PHO23 (Fig. 5G). In fact, pho23Δ in nrd1(151-214Δ) set1Δ cells actually reduced the accumulation of the NTS1 CUT to nearly WT levels. These results may be due to the fact that repression within the rDNA heterochromatin is maintained by the Sir2 HDAC rather than an Rpd3 complex. Indeed, consistent with our findings, previous studies showed that the deletion of Rpd3L complex members enhanced heterochromatic silencing (33, 54).
We assayed the termination of four additional snoRNAs and three CUTs by reverse transcription and PCR. The deletion of either SET1 or PHO23 in the nrd1(151-214Δ) background resulted in the increased read-through transcription of snR128 (Fig. 6A). The direct involvement of the Rpd3L complex is supported by the strong cross-linking of Rpd3 to this region (7) (see Fig. S3A in the supplemental material). Using this more sensitive RT-PCR assay, we also noted a slight increase in levels of read-through transcripts in set1Δ or pho23Δ cells, even in the wild-type NRD1 background. Termination defects caused by set1Δ or pho23Δ were also seen for snR3, snR71, snR9, and the RPR2 CUT (Fig. 6C and D). Interestingly, the NEL025C and FMP40 CUT levels were increased by the deletion of SET1 but not PHO23 (Fig. 6B and D). In agreement with this, H4 acetylation levels were unaffected by pho23Δ in these regions (Fig. S3B). Similarly, no Rpd3 recruitment to the NEL025C CUT region was observed (Fig. S3C). It is possible that other HAT or HDAC complexes determine acetylation levels at these promoters. These results suggest that Set1 generally contributes to termination at snoRNAs and CUTs. Pho23 also appears to be important for snoRNA termination but perhaps less so at CUTs.
Since the read-through transcription of SNR13 takes Pol II through the promoter region of TRS31, it could be argued that the termination defect in set1Δ or pho23Δ cells is due to changes at the TRS31 promoter. However, SNR3, SNR71, SNR9, and SNR128 are transcribed convergently with their downstream genes yet are still sensitive to the deletion of PHO23 or SET1 (Fig. 6). This finding rules out the possibility that the termination effects are due to Set1-mediated chromatin changes at a downstream promoter.
Our results are most consistent with a role for Set1 in snoRNA termination rather than 3′-end processing. ChIP experiments with nrd1 set1 double mutant cells showed increased Rpb3 levels downstream of the normal termination site relative to the promoter, which is indicative of a termination defect (Fig. 4D). Additionally, the polyadenylated snR13-TRS31 read-through transcript is not subject to Nrd1/exosome-dependent processing but shows increased accumulation in set1Δ cells (Fig. 2A). To definitively demonstrate a role for Set1 in promoting Nrd1-dependent termination, nascent elongating transcript sequencing (NET-Seq) was carried out on SET1 versus set1Δ cells. This technique identifies the precise 3′ ends of transcripts associated with elongating Pol II complexes, before 3′-end processing or degradation complicates analyses (14). The deletion of SET1 had little effect on the abundance of sequence reads within the body of genes (mRNA or snoRNA) or downstream of mRNA termination sites (data not shown). In contrast, a notable increase in transcription downstream of snoRNA termination sites was observed for set1Δ cells (Fig. 7 A). Similarly, there was also increased transcription downstream of the NEL025C CUT and the FMP40 CUT (Fig. 7B). These NET-Seq results confirm that the ChIP (Fig. 4D) and RT-PCR (Fig. 6) results reflect a role for Set1 in promoting Nrd1-dependent termination.
Both set1Δ and pho23Δ cells are sensitive to 6-azauracil (6-AU), a drug that depletes the cellular GTP and UTP pools (24, 66). This phenotype is often seen with mutations in positive transcription elongation factors. We therefore wondered whether the termination defect caused by Set1 and Pho23 could be related to an effect on the Pol II elongation rate. The rpb2-10 allele has a point mutation in a region important for nucleotide triphosphate binding, resulting in a polymerase that is slow and prone to arrest (36, 43). The combination of rpb2-10 with either the nrd1(151-214Δ) set1Δ or the nrd1(151-214Δ) pho23Δ mutation resulted in increased snR13 read-through (Fig. 8 A and see Fig. S1D in the supplemental material) and a decreased growth rate (Fig. 8B). Therefore, the slower Pol II elongation can decrease the efficiency of Nrd1-dependent termination and suggests a possible model for how Set1 and Pho23 influence this process (see below).
The early stages of RNA Pol II transcription elongation are marked by several events that are linked to CTD Ser5 phosphorylation (8). These events include mRNA capping, higher levels of histone H3K4 methylation, and an early termination pathway in yeast that is mediated by Nrd1, Nab3, and Sen1. The Nrd1 complex mediates both transcription termination and the subsequent recruitment of the nuclear exosome for snoRNA 3′-end trimming (51, 59). This complex can be recruited to genes via multiple mechanisms, including the recognition of specific RNA sequences by Nrd1 and Nab3, or by the interaction of the Nrd1 CID with CTD S5P. It is likely that individual genes will vary in being more or less dependent on each of these recruitment pathways.
Here we demonstrate that H3K4 trimethylation, a mark associated with active transcription, also contributes to efficient termination by the Nrd1/Sen1 pathway. The deletion of SET1 either by itself or in an nrd1 mutant background enhances the termination read-through of several snoRNAs and CUTs tested (Fig. 2A and D, D,5F,5F, F,6,6, and and7).7). Although these effects are much less than those seen for mutants of the actual termination factors themselves, our results indicate that chromatin modifications near promoters can influence the choice of whether to terminate soon after initiation or to continue elongating.
Interestingly, mutations in the Paf1 complex have also been reported to cause snoRNA termination defects by reducing the recruitment of the Nrd1 complex (48). Since the Paf1 complex is required for proper H3K4 methylation by Set1, it is logical to ask whether the Paf1 complex effect might be mediated via Set1. Sheldon et al. (48) concluded that this was unlikely because snR13 read-through transcripts were not seen in a SET1 deletion. While this clearly suggests that the Paf1 complex has additional functions related to snoRNA termination, our observations of read-through transcripts in set1Δ cells make it worth revisiting the idea that some of the Paf1 complex occurs via a disruption of H3K4 methylation. We note that while this paper was under review, a follow-up study by the Arndt laboratory showed that the termination defect observed for Paf1 complex mutants is likely mediated via its effect on H2B ubiquitylation (57), a prerequisite for H3K4 trimethylation.
The contribution to Nrd1-dependent termination by Set1 is clearly related to its histone methyltransferase activity. Histone H3 mutated at lysine 4 (Fig. 3F) or a catalytic-site mutant of Set1 (Fig. 3A to D) produced effects similar to those seen in set1Δ cells. It appears that it is specifically H3K4 trimethylation that promotes Nrd1-dependent termination. The monoubiquitylation of H2BK123 by the Rad6/Bre1 complex, which is required for H3K4 di- and trimethylation by Set1 (29, 53), also promotes efficient Nrd1-dependent transcription termination (Fig. 3E) (57). Similarly, the set1(230-335Δ) allele, which abolishes H3K4 trimethylation but not H3K4 dimethylation (see Fig. S2 in the supplemental material) (18, 26), also exhibits a reduction of Nrd1 recruitment with subsequent termination defects (Fig. 3C and D and and4B).4B). Given that snoRNAs and CUTs are terminated relatively close to the promoter, it makes sense that promoter-proximal H3K4 trimethylation would be linked to the Nrd1-Nab3-Sen1 termination pathway.
Set1 may promote SNR13 termination by affecting histone acetylation levels. Because the deletion of PHO23 also leads to read-through, we suspect that this effect occurs via the Rpd3L HDAC. Although sequence-specific DNA binding proteins such as Ume6 can recruit Rpd3L to repress specific promoters (12, 22, 27, 47), we speculate that the PHD fingers of the Rpd3L subunits Pho23 and Cti6 can bind H3K4me3 to moderate acetylation levels at active promoters. This model is supported by several findings. First, cells lacking Pho23 show a small but significant increase in acetylation at the SNR13 (Fig. 5B) and YEF3 (18, 26) promoters. Additionally, Rpd3 recruitment was observed in SNR13 and SNR128 regions (Fig. 5C and see Fig. S3A in the supplemental material) (data from reference 7) where pho23Δ results in termination defects (Fig. 5A and and6A).6A). Another genome-wide ChIP analysis of Rpd3 showed that, in addition to being recruited to a small group of repressed promoters, the deacetylase was unexpectedly found to cross-link to active promoters proportional to their transcription rate (27). At the time, those authors speculated that Rpd3 might be recruited to active promoters to dampen high levels of acetylation. A similar story emerged from genome-wide mapping of HATs and HDACs in mammalian cells. HDACs are recruited to active genes, and levels correlated positively with H3K4 methylation (63). Therefore, both HATs and HDACs may functionally associate with active promoters. Supporting this view, we show that the blocking of NuA3 HAT recruitment by the deletion of YNG1 reverses the enhancement of the termination defect by pho23Δ (Fig. 5D). Although it may appear paradoxical for H3K4me3 to recruit the antagonistic activities of both NuA3 (via Yng1) and Rpd3L (via Pho23 and Cti6) to the promoter, this mechanism may maintain balanced levels of histone acetylation or perhaps allow cycles of acetylation and deacetylation that are important for proper transcription.
In yeast, the choice between early Nrd1-dependent termination or continued elongation to downstream polyadenylation sites must be made for both snoRNAs and mRNA genes (8). Set1 may influence this decision point by affecting the kinetics of early transcription (Fig. 8). This idea is supported by the sensitivity of set1Δ or pho23Δ cells to 6-AU as well as the genetic interaction with a RPB2 allele that slows elongation. The loss of H3K4 trimethylation may make it slightly harder for Pol II to travel through the promoter-proximal chromatin environment, perhaps due to downstream effects on nucleosome acetylation or remodeling. A slower transit time through this window might affect the modifications of Pol II or the recruitment of necessary elongation factors, resulting in termination defects for snoRNAs and CUTs. The recent observation that another slow Pol II allele, rpb1(N488D), shows reduced capping efficiency (21) supports this concept. Although recent reports failed to detect widespread mRNA changes in the absence of Set1 (18, 46, 62), it is possible that a subset of genes could be affected by this mechanism.
A choice between early or late termination pathways might also underlie the regulation of genes in higher eukaryotes, where higher levels of Pol II accumulate near the 5′ ends of genes relative to further downstream (34). While some of these promoter-proximal polymerase molecules may be paused, others may be destined for early termination (8). It was recently found that bidirectional transcription is an inherent feature of many actively transcribed genes (reviewed in reference 9). In yeast, the reverse-direction promoter-associated transcripts are terminated by the Nrd1-Nab3-Sen1 complex (40). It will be interesting to see whether H3K4 methylation also plays a role in mediating early pausing and termination in metazoans.
We thank G. Fink and S. Bumgarner for sharing their Rpd3-Myc ChIP-chip data, J. Corden (JHMI) for anti-Nrd1 antibody and Nrd1 plasmids, S. Briggs (Purdue) for Set1 partial deletion constructs, and R. Buratowski for help with the tables in the supplemental material. We also thank T. S. Kim, D. Hazelbaker, and M. C. Keogh (AECOM) for critical reading of the manuscript.
L.S.C. was supported by the Damon Runyon Cancer Research Foundation. This work was supported by NIH grants GM46498 and GM56663 to S.B.
†Supplemental material for this article may be found at http://mcb.asm.org/.
Published ahead of print on 27 June 2011.