Pol II CTD kinases Bur1 and Kin28 promote Spt5 CTR-independent recruitment of Paf1 complex
2012; Springer Nature; Volume: 31; Issue: 16 Linguagem: Inglês
10.1038/emboj.2012.188
ISSN1460-2075
AutoresHongfang Qiu, Cuihua Hu, Naseem A. Gaur, Alan G. Hinnebusch,
Tópico(s)Signaling Pathways in Disease
ResumoArticle13 July 2012free access Pol II CTD kinases Bur1 and Kin28 promote Spt5 CTR-independent recruitment of Paf1 complex Hongfang Qiu Hongfang Qiu Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Cuihua Hu Cuihua Hu Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Naseem A Gaur Naseem A Gaur Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Alan G Hinnebusch Corresponding Author Alan G Hinnebusch Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Hongfang Qiu Hongfang Qiu Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Cuihua Hu Cuihua Hu Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Naseem A Gaur Naseem A Gaur Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Alan G Hinnebusch Corresponding Author Alan G Hinnebusch Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA Search for more papers by this author Author Information Hongfang Qiu1, Cuihua Hu1, Naseem A Gaur1 and Alan G Hinnebusch 1 1Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA *Corresponding author. Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Building 6, Room 230, Bethesda, MD 20892, USA. Tel.:+1 301 496 4480; Fax:+1 301 496 6828; E-mail: [email protected] The EMBO Journal (2012)31:3494-3505https://doi.org/10.1038/emboj.2012.188 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment. Introduction The Paf1 complex (Paf1C) is an important cofactor for elongating RNA Polymerase II (Pol II), comprised of five subunits: Paf1, Rtf1, Cdc73, Ctr9, and Leo1, which is recruited to the coding sequences (CDS) of actively transcribed genes (Kim et al, 2004; Sims et al, 2004; Mayer et al, 2010). Paf1C orchestrates the co-transcriptional ubiquitylation of histone H2B on Lys-123 and methylation of histone H3 on Lys-4, which requires H2B ubiquitylation, and also the methylation of H3 on Lys-36 and Lys-79 (Sims et al, 2004; Smith and Shilatifard, 2010). Paf1C also promotes correct 3′ end formation by its role in recruiting termination factors (Penheiter et al, 2005; Sheldon et al, 2005; Nordick et al, 2008; Rozenblatt-Rosen et al, 2009; Kim and Levin, 2011), and enhances Pol II recruitment to promoters that depend on activator SBF (Swi4/Swi6) (Kim and Levin, 2011). Thus, efficient Paf1C recruitment is crucial for events occurring throughout the transcription process and spanning the entire CDS; however, the mechanism of Paf1C recruitment by elongating Pol II is not well understood. Multiple factors have been implicated in Paf1C recruitment, including the FACT, Ccr4-Not, and Spt4/Spt5 (DSIF) complexes, and the cyclin-dependent kinases (Cdks) Kin28 (cyclin Ccl1) and Bur1 (cyclin Bur2) (Laribee et al, 2005; Wood et al, 2005; Pavri et al, 2006; Qiu et al, 2006; Mulder et al, 2007; Liu et al, 2009; Zhou et al, 2009). Kin28 phosphorylates the heptad repeats (Y1S2P3T4S5P6S7) in the C-terminal domain (CTD) of Pol II subunit Rpb1 on Ser5 and Ser7 (Akhtar et al, 2009; Glover-Cutter et al, 2009; Kim et al, 2009), and this activity stimulates recruitment of mRNA processing factors, histone modifying enzymes and elongation factors near the promoter (Phatnani and Greenleaf, 2006; Buratowski, 2009; Govind et al, 2010). As inhibiting Kin28 does not impair Spt4 recruitment, it appears that Spt4/Spt5 acts directly in Paf1C recruitment (Qiu et al, 2006). Indeed, strong evidence was presented that Bur1 stimulates Paf1C recruitment by phosphorylating the C-terminal repeats (CTRs) of Spt5 (Liu et al, 2009; Zhou et al, 2009). Similar to the role of the phosphorylated Pol II CTD in cofactor recruitment, the hexad repeats in Spt5, with consensus sequence S1A2W3G4G5Q6, could provide a platform for Paf1C recruitment when phosphorylated by Bur1. However, it was reported that Paf1C does not bind directly to phosphorylated Spt4/Spt5 complex (Liu et al, 2009), making it unclear how Bur1 and Spt5 promote Paf1C recruitment. Moreover, the reduction in Paf1C recruitment evoked by inhibiting Bur1 exceeded that of Ala substitutions of the Ser1 residue in all 15 hexad repeats of Spt5 (Liu et al, 2009), raising the possibility that Bur1 promotes Paf1C recruitment by an Spt5-independent pathway. In fact, we and others have shown that Bur1 also phosphorylates Ser2 of the Pol II CTD (S2P) in vivo, augmenting the activity of the major, but non-essential, S2P kinase Ctk1 (Liu et al, 2009; Qiu et al, 2009). We found that Bur1 is recruited to the 5′ end of the CDS at ARG1 in a manner stimulated by Kin28 and the pCTD-interaction domain (PCID) in the Bur1 C-terminus. Moreover, Bur1 contributed significantly to S2P formation near the ARG1 promoter and in the fraction of bulk elongating Pol II highly enriched in Ser5P (S5P), while Ctk1 was responsible for the majority of S2P at promoter-distal ARG1 sequences and in bulk Pol II (Qiu et al, 2009). An auxiliary role for Bur1 in S2P formation in the CDS was also detected by genome-wide measurements of S2P occupancy (Tietjen et al, 2010; Bataille et al, 2012). Interestingly, Tietjen et al (2010) further implicated Bur1 in S7P formation at sites distal from the promoter; although Bataille et al (2012) found that Bur1's contribution to S7P was most pronounced under conditions of Kin28 inhibition. Thus, it appears that Bur1 contributes to the formation of both Ser2P and Ser7P in elongating Pol II. Considering our previous finding that Kin28 promotes Bur1 recruitment (Qiu et al, 2009), it was possible that the stimulatory role of Kin28 in Paf1C recruitment (Qiu et al, 2006) merely reflects the positive effect of Kin28 on Bur1 recruitment and attendant phosphorylation of Spt5 CTRs. We set out to test the alternative hypothesis that Kin28 acts more directly by phosphorylating the Pol II CTD on Ser5 and Ser7 to provide an alternative platform for Paf1C recruitment. In addition to phosphorylating the CTD directly, Kin28 could also cooperate with Bur1 to produce S2,S5- or S5,S7-diphosphorylated CTD repeats, and this collaboration with Bur1 in CTD phosphorylation, as well as Spt5 CTR phosphorylation by Bur1, would be enhanced by Kin28's stimulatory role in Bur1 recruitment. Our results indicate that Paf1C binds specifically to both types of diphosphorylated CTD repeats and to phosphorylated Spt5 CTRs (pCTRs) in vitro, and they provide strong evidence that Kin28 and Bur1 can promote Paf1C recruitment independently of Spt5 CTR phosphorylation. Accordingly, we envision a dual mechanism of Paf1C recruitment via diphosphorylated Pol II CTD repeats and Spt5 pCTRs, with both pathways stimulated by Kin28 and Bur1. Results Paf1C can bind directly to phosphorylated Rpb1 CTD repeats Considering that Paf1C recruitment is stimulated by both Kin28 and Bur1 (Laribee et al, 2005; Qiu et al, 2006; Liu et al, 2009), and the evidence cited above that these Cdks phosphorylate the Pol II CTD on Ser5 and Ser7 (Kin28) or Ser2 and Ser7 (Bur1), we investigated whether Paf1C can bind directly to Pol II CTD repeats in a manner stimulated by S2P, S5P, or S7P. To this end, Paf1C was purified from a CDC73-TAP strain through the first step of tandem affinity purification, that is, prior to cleavage of the calmodulin binding domain (CBD) (Puig et al, 2001; see Supplementary Figure S1A), and tested for binding to biotinylated peptides comprised of three CTD repeats, either unphosphorylated, monophosphorylated at Ser2, Ser5, or Ser7, or diphosphorylated at either Ser2,Ser5 or Ser5,Ser7, in all three repeats. (Note that S2P,S7P-diphosphorylated peptides were not successfully synthesized.) Cdc73-CBD and co-purifying Rtf1 show no binding to unphosphorylated peptides, little or no binding to S2P and S7P peptides, and relatively weak binding to S5P peptides (Figure 1A). Remarkably, the purified Paf1C binds much more strongly to both S2P,S5P and S5P,S7P peptides, with consistently slightly greater binding for the S2P,S5P peptides (Figure 1A, lanes 1–25 and data not shown). As expected if Cdc73-CBD and Rtf1 bind as subunits of the same complex, the extent of depletion of Cdc73-CBD in the supernatant (S) fraction using S2P,S5P peptides relative to the ‘mock’ no-peptide reaction was comparable to that observed for Rtf1 (Figure 1A, cf. lane 14 versus 10 and 25 versus 17). (The depletion of signal in the supernatant does not equal the increase in signal in the bound fraction (B) because a larger proportion of the latter was examined.) These findings suggest that native Paf1C interacts with CTD repeats in a manner stimulated moderately by S5P and strongly by S2,S5 or S5,S7 diphosphorylation. Figure 1.Paf1C binds to phosphorylated CTD peptides. (A, B) Biotinylated CTD peptides (1.5 μg) phosphorylated on Ser2 (S2P), Ser5 (S5P), Ser7 (S7P), Ser2 and Ser5 (S2P S5P), Ser5 and Ser7 (S5P S7P), unphosphorylated (CTD) were adsorbed to streptavidin-coated magnetic beads. Native Paf1C purified from a CDC73-TAP strain was incubated with beads alone (Mock) or beads bearing peptides at 4°C. 1/40 input (Inp), 1/40 supernatants (S) and bound (B) proteins were subjected to Western analysis with anti-TAP and anti-Rtf1 antibodies. For lanes 24–29, immobilized (S2P S5P) peptides were treated with CIP in the presence or absence of PhosSTOP prior to incubation with purified Paf1C. (B) As in (A) except CTD peptides with Ser2 and Ser5 substituted by Asp were also used. Lanes 1–3 and 4–7 derive from different portions of the same blot as described in Supplementary Figure S10A. (C, D) As in (A, B) except using the indicated purified recombinant GST fusion proteins (500 ng) in place of Paf1C and anti-GST antibodies in Western analysis. Also, 1/10 input (Inp) and supernatants (S) (1/20 input and supernatant in (D) for GST-Ctr9(594–855) were examined. Figure source data can be found with the Supplementary data. Download figure Download PowerPoint To confirm that enhanced binding of Paf1C to the phosphorylated peptides is dependent on the phosphate groups, the S2P,S5P peptides were pre-treated with calf intestine phosphatase (CIP), in the absence or presence of phosphatase inhibitor PhosSTOP, before incubation with purified Paf1C. Indeed, specific binding of Cdc73-CBD and Rtf1 to S2P,S5P peptides was abolished by CIP treatment and fully recovered by inclusion of PhosSTOP (Figure 1A, lanes 24, 26, and 28). Similar results were observed for the S5P peptides (Supplementary Figure S2A). Second, we tested purified Paf1C for binding to phosphomimetic peptides containing Asp2 and Asp5 in all three repeats, and observed no detectable binding (Figure 1B). Accordingly, Paf1C binding to phosphorylated CTD repeats likely involves specific recognition of the phosphate dianion in a manner inefficiently mimicked by monoanionic carboxylate groups. The results in Figure 1A and B support the possibility that Paf1C is recruited by Pol II CTD repeats diphosphorylated on Ser5/Ser2 or Ser5/Ser7. Cdc73, Ctr9, and Rtf1 each contains a putative PCID In an effort to determine which Paf1C subunits can mediate binding to phosphorylated CTD peptides, GST fusions to each subunit were purified from E. coli and used in binding assays. As the full-length GST-Ctr9 fusion was not well expressed, we analysed fusions containing different segments of Ctr9. Interestingly, GST-Rtf1 binds only to the S2P,S5P peptides. By contrast, the GST fusion containing residues 594–855 of Ctr9 binds weakly to S2P and S5P, but tightly to both kinds of diphosphorylated peptides, with a slight preference for S2P,S5P over S5P,S7P (Figure 1C; Supplementary Figure S2B). The GST-Cdc73 fusion binds to all three peptides harbouring monophosphorylated serines but, similar to the above findings on other recombinant subunits and purified Paf1C, it binds much better to the S2P,S5P- and S5P,S7P-diphosphorylated peptides. Based on the fractions of input bound, and the ratios of signals in the B versus S fractions, recombinant Cdc73 binds more tightly than does Ctr9 or Rtf1 to all peptides to which the latter two subunits can bind (Figure 1C; Supplementary Figure S2B). The fusions to Paf1, Leo1, and three other segments of Ctr9 (1–491, 414–800, and 701–1077) showed no binding to any CTD peptides (Figure 1C and data not shown) using comparable levels of fusion proteins in the binding reactions (Supplementary Figure S1B). As shown above for purified Paf1C, dephosphorylating the S2P,S5P peptides greatly reduced binding by GST-Cdc73, GST-Ctr9(594–855), and GST-Rtf1 in a manner restored with PhosSTOP (Supplementary Figure S2C). Furthermore, GST-Rtf1 and GST-Ctr9(594–855) showed little or no binding to the S2D,S5D phosphomimetic peptides (Figure 1D), and GST-Cdc73 showed reduced binding to the S5D and S2D,S5D peptides compared to the cognate mono- and diphosphorylated peptides (Supplementary Figure S2D). Together, these findings suggest that Cdc73, Rtf1, and Ctr9 each contains a PCID that binds more tightly to one or both kinds of diphosphorylated CTD repeats than to monophosphorylated repeats and that also prefers dianionic phosphate groups over monoanionic acidic residues. These results can account for the higher affinity of native Paf1C for diphosphorylated versus monophosphorylated repeats (Figure 1A), and they support our hypothesis that Bur1 stimulates Paf1C recruitment, in part, by phosphorylating Ser2 or Ser7 in CTD repeats already phosphorylated on Ser5 by Kin28. The presence of PCIDs in Cdc73 and Rtf1 is consistent with the known requirements for these two subunits in Paf1C recruitment in vivo (Mueller et al, 2004; Nordick et al, 2008). Given the strong evidence that Rtf1 acts directly in Paf1C recruitment (Warner et al, 2007), we examined whether native Rtf1 can interact with pCTD peptides independently of the other two Paf1C subunits harbouring PCIDs by purifying Rtf1-CBD from an RTF1-TAP cdc73Δ ctr9Δ strain. As Rtf1 association with Paf1C requires Cdc73 (Nordick et al, 2008), the Rtf1-CBD preparation should also be devoid of Paf1 and Leo1. As shown in Supplementary Figure S2E, Rtf1-CBD bound only to the S2P,S5P peptides, and the proportion of input recovered in the bound fraction was considerably smaller than that seen for intact Paf1C (cf. Supplementary Figure S2E, lanes 15–16 versus Figure 1A, lanes 13–14). Thus, while native Rtf1 can interact with S2P,S5P peptides independently of Cdc73 and Ctr9, it appears that Cdc73 or Ctr9 contributes to the relatively stronger pCTD-binding activity of intact Paf1C and also to its interaction with S5P,S7P peptides. Based on the relative affinities of recombinant Cdc73 and Ctr9 for S2P,S5P peptides (Figure 1C), it is likely that Cdc73 makes the greater contribution to Paf1C interaction with this diphosphorylated species of CTD heptad. Evidence that Paf1C binds directly to phosphorylated Spt5 CTRs There is strong evidence that Bur1 promotes Paf1C recruitment by phosphorylating Ser1 in the 15 CTRs of Spt5 (Liu et al, 2009; Zhou et al, 2009); however, evidence was lacking for direct interaction of Paf1C with phosphorylated Spt5 (Liu et al, 2009). To determine whether Paf1C can interact with Ser1-phosphorylated CTRs, we conducted peptide binding assays with purified Paf1C and biotinylated peptides consisting of four repeats of the Spt5 consensus sequence S1A2W3G4G5Q6, synthesized with Ser1 unphosphorylated (CTR), phosphorylated (pCTR), or substituted with aspartic acid (CTRD). The Paf1C purified from the CDC73-TAP strain described above displayed strong, phosphorylation-dependent binding to the Spt5 CTR peptides, binding at high levels to pCTR but not to CTR peptides, and binding in a manner abolished by CIP treatment of pCTR and recovered fully with PhosSTOP (Figure 2A). Moreover, purified Paf1C displayed reduced binding to CTRD versus pCTR peptides (Figure 2A). Similar to our results with Pol II CTD peptides, bacterially expressed GST-Cdc73, GST-Rtf1, and GST-Ctr9(594–855) all bind to pCTR peptides in a manner greatly diminished by CIP treatment and recovered with PhosSTOP (Figure 2B), and show no binding to unphosphorylated CTR peptides (Figure 2C). By contrast, GST-Paf1 and GST-Leo1 do not bind to either pCTR or CTR peptides (Figure 2C). Based on the proportion of input recovered with the pCTR peptides, GST-Cdc73 and GST-Rtf1 bind more tightly than GST-Ctr9(594–855) to pCTR peptides (Figure 2B and C). Consistent with this, Rtf1-CBD purified from cdc73Δ ctr9Δ cells also displays strong binding to pCTR peptides (Supplementary Figure S2F). Figure 2.Paf1C binds to phosphorylated Spt5 C-terminal repeats (CTR) peptides. (A–D) Peptide binding assays were conducted as described in Figure 1 using biotinylated CTR peptides (1.5 μg) with Ser1 unphosphorylated (CTR), Ser1 phosphorylated (pCTR), or Ser1 substituted with Asp (CTRD) or Glu (CTRE), and purified native Paf1C or recombinant GST fusion proteins, except using 1/5 or 1/10 input and supernatants of GST-Rtf1 and GST-Ctr9(594–855), respectively. Lanes 10–18 in (A) derive from the same experiment that produced Figure 1B, and the input and Mock lanes from the latter are reproduced here as lanes 10–12. Figure source data can be found with the Supplementary data. Download figure Download PowerPoint Summarizing the binding data for the recombinant subunits in Figure 1C and Supplementary Figure S2B and C, Cdc73 binds tightly to phosphorylated CTR peptides and to both kinds of diphosphorylated CTD peptides, Rtf1 binds strongly to pCTR but only moderately to S2P,S5P peptides, and Ctr9 binds relatively weakly to both pCTR and both kinds of diphosphorylated CTD peptides. Unlike our findings for purified Paf1C, recombinant GST-Cdc73, GST-Rtf1, and GST-Ctr9(594–855) fragments bound to the phosphomimetic Spt5 CTRD peptides to nearly the same extent as to pCTR peptides (Figure 2D). While this last discrepancy remains to be explained, the results in Figure 2 provide strong evidence that Paf1C can interact specifically with phosphorylated Spt5 CTRs. The Cdc73 PCID promotes Paf1C recruitment in vivo Having found that recombinant Cdc73 displays the strongest affinity for both kinds of diphosphorylated CTD repeats, we sought to identify the PCID in Cdc73 and assess its importance in Paf1C recruitment. To this end, we tested a panel of GST-Cdc73 deletion variants for binding to the S5P-CTD peptides in vitro. As shown in Figure 3A, a C-terminal fragment of residues 201–393 was the smallest Cdc73 segment that bound S5P-CTD peptides indistinguishably from full-length Cdc73 (Figure 3A), and it showed the same relative affinities for different CTD peptides observed for full-length Cdc73, namely S2P,S5P>S5P>S2P>unphosphorylated peptides (cf. Figure 3B, top row (WT) with Figure 1C, GST-Cdc73). These results suggested that the functional PCID spans a relatively large region (of 193 residues) in the Cdc73 C-terminus. Figure 3.Phospho-CTD interaction domain (PCID) of Cdc73 is located in its C-terminal region. (A) GST fusions to full-length (F.L.) Cdc73 or the Cdc73 truncations shown schematically on the right were purified and used in peptide binding assays with the CTD peptide S5P as described in Figure 1. The results are summarized in the schematic. (B–D) Peptide binding assays conducted as in Figure 1 using the indicated CTD peptides (B, C) or Spt5 pCTR peptides (D) and either WT or the indicated mutant versions of GST-Cdc73(201–393). Lanes 1–3 and 4–5 of the W357A panel derive from different sections of the same blot as described in Supplementary Figure S10B. Figure source data can be found with the Supplementary data. Download figure Download PowerPoint A multiple sequence alignment of Cdc73 from different yeasts and fungi revealed blocks of conserved residues throughout the C-terminal segment (Supplementary Figure S3), including a possible WW motif—a PCID containing two conserved Trp residues (Meinhart et al, 2005)—and other strings of conserved residues with no obvious similarity to previously established PCIDs (Li et al, 2005; Meinhart et al, 2005; Phatnani and Greenleaf, 2006; Vojnic et al, 2006; Lunde et al, 2010; Xiang et al, 2010; Ghosh et al, 2011). We introduced mutations into the GST-Cdc73(201–393) construct to generate Ala substitutions in the conserved Trp residues (W357A and W380A), or in 6–7 consecutive residues in three other conserved blocks, and tested the mutant proteins for peptide binding. As shown in Figure 3B and C, all of the mutations except W357A reduced or abolished binding of the fusion protein to pCTD peptides. Interestingly, all substitutions besides W357A also greatly impaired binding to the Spt5 pCTR peptides (Figure 3D), consistent with the possibility that the same structural elements in the Cdc73 C-terminus responsible for binding to pCTD repeats mediate binding to the Spt5 pCTRs. However, it is possible that these substitutions disrupt folding of the Cdc73 C-terminal region and impair binding to pCTD and/or pCTR peptides by an indirect mechanism. After introducing a subset of these PCID mutations into chromosomal CDC73 (along with a C-terminal myc tag), we discovered by Western analysis of yeast WCEs that they moderately reduce abundance of Cdc73–myc protein. However, for three of the mutants we could mitigate these reductions by introducing a low-copy plasmid harbouring the same mutant allele: W380A; W357AW380A; and 304-309Ala (Supplementary Figure S4A). The resulting strains also display normal levels of Paf1C, as indicted by the fact that nearly WT amounts of Rtf1 (Figure 4A) and HA-tagged Paf1 (Supplementary Figure S4B) co-immunoprecipitated with each cdc73–myc protein. Accordingly, we proceeded to evaluate the effects of the cdc73 mutations on Paf1C recruitment by chromatin immunoprecipitation (ChIP) analysis of the myc-tagged Cdc73 subunits. Figure 4.Substitutions in the Cdc73 PCID eliminating binding to pCTD peptides reduce Cdc73–myc occupancies at ARG1. (A) Untagged WT strain BY4741 and the following CDC73-myc13 strains were cultured in synthetic complete medium lacking leucine (SC-Leu) to log phase (A600 of ∼0.6): HQY1147 (CDC73-myc13); HQY1468 (cdc73-W380A-myc13 harbouring low-copy plasmid pHQ1862 with cdc73-W380A-myc13, henceforth abbreviated as -W380A*); HQY1469 (cdc73-W357A,W380A-myc13 harbouring pHQ1861, abbreviated as -W357A,W380A*); and HQY1471(cdc73-6Ala(304-309)-myc13 harbouring pHQ1952, abbreviated as -304-309Ala*). WCEs were prepared and immunoprecipitated with anti-myc antibodies and subjected to Western analysis with anti-myc and anti-Rtf1 antibodies. The last six lanes derive from a separate blot as described in Supplementary Figure S10C. (B–D) ChIP analysis of Cdc73–myc binding at ARG1. The CDC73-myc13 strains described in (A) and CDC73-myc13 gcn4Δ strain HQY1148 were cultured in SC lacking Ile, Val, and Leu (SC-Ilv-Leu) and treated with sulfometuron (SM) for 30 min to induce Gcn4. ChIP analysis was conducted using anti-myc antibodies for Cdc73–myc (B, C) or anti-Rpb3 antibodies for Rpb3 (D). DNA extracted from immunoprecipitates (IP) and input chromatin (Input) samples was subjected to PCR in the presence of [33P]-dATP with the appropriate primers to amplify radiolabelled ARG1 5′ORF or 3′ORF sequences, or sequences from a chromosome V intergenic region (ChrV) examined as a control. PCR products were resolved by PAGE and visualized by autography, with representative results shown for two biological replicates in adjacent lanes of (B), and quantified with a phosphorimager. The resulting values were expressed as ratios of IP to Input signals and normalized to the corresponding ratio for WT CDC73-myc cells. Error bars correspond to standard errors of the mean derived from four or more independent cultures and two independent immunoprecipitations for each culture. (See Materials and methods in Supplementary data for further details on quantification of ChIP data.) Figure source data can be found with the Supplementary data. Download figure Download PowerPoint We showed previously that the occupancy of myc-tagged Paf1 in ARG1 chromatin is increased on induction of transcriptional activator Gcn4 (by starvation for isoleucine and valine) to levels ∼6-fold above background in GCN4 cells but remains at background levels in gcn4Δ cells (Qiu et al, 2006). As shown in Figure 4B, Cdc73–myc occupancy in the ARG1 CDS is similarly induced by Gcn4, showing much higher levels in GCN4 (WT) versus gcn4Δ cells (lanes 11–12 versus 9–10). Importantly, the W380 and 304-309Ala mutations significantly reduced Cdc73–myc occupancy under inducing conditions (Figure 4B and C). An even stronger reduction was observed for the W357A,W380A double mutant, although the W357A single mutation had little or no effect on its own. The reductions in Cdc73 occupancy occurred without significant decreases in Pol II (Rpb3) occupancy at ARG1 (Figure 4D). These results suggest that the Cdc73 PCID is crucial for efficient Paf1C recruitment at induced ARG1. We also measured Cdc73–myc occupancies at the GAL1 gene under conditions of galactose induction, and at the constitutively expressed genes PMA1 and ADH1. As shown in Supplementary Figure S5A–F, the W380A, W357A, W380A and 304-309Ala mutations all produced significant reductions in Cdc73–myc occupancies, but not Pol II (Rpb3) occupancies, in the coding sequences of these three genes, consistent with a general requirement for the Cdc73 PCID in Paf1C recruitment. Bur1 promotes Paf1C recruitment by Spt5 pCTR dependent and independent pathways The results above indicate that the Cdc73 PCID is required for robust Paf1C recruitment in vivo, but do not reveal whether it mediates interaction with Spt5 pCTRs, Pol II pCTD repeats, or both, to promote Paf1C recruitment. It was shown previously that substituting Ser1 with Ala in all 15 CTRs of Spt5 (spt5-S1-15A) reduced Paf1–myc occupancy at the 5′ ends of ARG1 and PYK1 in vivo (Liu et al, 2009), consistent with the idea that Spt5 phosphorylation by Bur1 promotes Paf1C recruitment. To provide additional evidence supporting this interpretation, we examined whether the phosphomimetic Asp and Glu substitutions in the Spt5 CTRs would have less effect than Ala substitutions on Paf1C recruitment to ARG1. Indeed, Paf1–myc recruitment was reduced by a factor of 3–4 in the non-phosphorylatable spt5-S1-15A mutant, but only by 20–30% in the phosphomimetic mutants spt5-S1-15D and spt5-S1-15E, at the 5′ and 3′ ends of the ARG1 CDS (Figure 5A and B). Thus, even though the Spt5 CTRD and CTRE peptides were inferior to the pCTR peptides in binding Paf1C in vitro (Figure 2), the phosphomimetic Spt5 substitutions can sustain a substantial level of Paf1C recruitment in vivo, supporting the conclusion that phosphorylation of Ser1 in the Spt5 CTRs by Bur1 stimulates Paf1C recruitment (Liu et al, 2009). We speculate that phosphomimetic Spt5 CTRs are highly effective in Paf1C recruitment in vivo because they contain an immutable negative charge, whereas the phosphorylation level of the WT CTRs is likely modulated by protein phosphatases. Figure 5.Inactivating bur1-as reduces Paf1–myc occupancy at ARG1 in spt5 phosphomimetic and non-phosphorylatable CTR mutants. (A, B) Phosphomimetic substitutions in Spt5 CTRs sustain Paf1 recruitment at ARG1. PAF1-myc13 strains with t
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