Histone H2B Monoubiquitination Functions Cooperatively with FACT to Regulate Elongation by RNA Polymerase II

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Rushad Pavri,1 Bing Zhu,1 Guohong Li,1 Patrick Trojer,1 Subhrangsu Mandal,1 Ali Shilatifard,2 and Danny Reinberg1,* 1 Howard Hughes Medical Institute, Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 683 Hoes Lane, Piscataway, NJ 08854, USA 2 Department of Biochemistry, Saint Louis University School of Medicine, 1402 South Grand Boulevard, Saint Louis, MI 63104, USA *Contact: [email protected] DOI 10.1016/j.cell.2006.04.029

SUMMARY dense chromatin thus permitting the subsequent binding of factors essential for transcription. In addition, gene acti- Over the past years, a large number of histone vation involves factors functioning independently of chro- posttranslational modifications have been de- matin such as the Mediator complex, which interacts scribed, some of which function to attain a with the activator and several components of the basal repressed chromatin structure, while others fa- transcriptional machinery, such as TFIID and RNA poly- cilitate activation by allowing access of regula- merase II (Lewis and Reinberg, 2003; Malik and Roeder, tors to DNA. Histone H2B monoubiquitination 2000; Rachez and Freedman, 2001; Taatjes et al., 2004). Moreover, several gene-specific cofactors are believed is a mark associated with transcriptional activ- to confer cell- or tissue-specificity to a given promoter or ity. Using a highly reconstituted chromatin-tran- a subset of promoters (Spiegelman and Heinrich, 2004; scription system incorporating the inducible Taatjes et al., 2004). RARb2 promoter, we find that the establishment Although much is known regarding events leading up to of H2B monoubiquitination by RNF20/40 and transcription initiation in a chromatin context, much less is UbcH6 is dependent on the transcription elon- known about the subsequent postinitiation events associ- gation regulator complex PAF, the histone ated with transcription elongation. Efficient elongation on chaperone FACT, and transcription. H2B mono- chromatin entails the removal of the physical barrier im- ubiquitination facilitates FACT function, thereby posed by the nucleosome on the transcribing RNA poly- stimulating transcript elongation and the gener- merase II. The best-characterized factor associated with ation of longer transcripts. These in vitro analy- elongation on chromatin templates is FACT, a dimeric protein that contains an HMG1 domain (Orphanides et al., ses and corroborating in vivo experiments dem- 1998). FACT allows elongation on chromatin templates onstrate that elongation by RNA polymerase II by binding and displacing the H2A/H2B dimer from the through the nucleosomal barrier is minimally core nucleosomes (Belotserkovskaya et al., 2003). The for- dependent upon (1) FACT and (2) the recruit- mation of a hexamer (a nucleosome depleted of one H2A/ ment of PAF and the H2B monoubiquitination H2B dimer) is believed to be essential for the passage of machinery. RNA polymerase II through the nucleosomal barrier (Kir- eeva et al., 2002; Belotserkovskaya et al., 2003). FACT was also shown to be essential for the reestablishment of INTRODUCTION the nucleosome (Belotserkovskaya et al., 2003) and for viability in yeast (Malone et al., 1991; Rowley et al., 1991; Transcription regulation at the level of chromatin is a com- Orphanides et al., 1999), and mutants of FACT subunits plex and tightly regulated process. To date, several fami- in yeast show significant elongation defects (Formosa lies of chromatin-modifying factors have been associated et al., 2002). with gene activation, notably the ATP-dependent remodel- Genetic studies in yeast have implicated the PAF com- ing enzymes (SWI/SNF and NURF) (Glass and Rosenfeld, plex and monoubiquitination of lysine 123 of histone H2B 2000), histone acetyl-transferases (HATs, e.g., p300) (H2BK123ub1) in transcription elongation (Shi et al., (Glass and Rosenfeld, 2000), and histone methyl-transfer- 1996; Dover et al., 2002; Mueller and Jaehning, 2002; ases (e.g., CARM1 and the MLL-related family) (Milne et al., Squazzo et al., 2002; Rondon et al., 2004; Xiao et al., 2002; An et al., 2004). These factors are believed to decon- 2005). Importantly, H2BK123ub1 is dependent on PAF

Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. 703 (Krogan et al., 2003a; Ng et al., 2003; Wood et al., 2003b). mally dependent on FACT, PAF, and H2B monoubiquiti- In yeast, PAF has been shown to interact with RNA poly- nation at lysine 120 (H2BK120ub1). We hypothesize that merase II (Shi et al., 1996; Shi et al., 1997; Mueller and H2B monoubiquitination stimulates FACT-mediated dis- Jaehning, 2002) as well as with the Set1 and Set2 histone placement of an H2A/H2B dimer from the core nucleo- lysine methyltransferases (HKMTases) (Li et al., 2002; Kro- some and that this modification enhances the frequency gan et al., 2003b). Set1 and Set2 methylate lysines 4 and 36 of RNA polymerase II passage through the nucleosome. of histone H3, respectively, and both of these marks are associated with active genes (Briggs et al., 2001; Miller RESULTS et al., 2001; Li et al., 2002; Nagy et al., 2002; Noma and Grewal, 2002; Krogan et al., 2003b; Schaft et al., 2003). A Fully Reconstituted Chromatin Transcription Moreover, methylation of lysine 4 of histone H3 is contin- System: FACT Is Required for RAR-Dependent gent upon H2Bub1 at lysine 123 in yeast (Dover et al., Transcription 2002; Sun and Allis, 2002). However, the function of these As in our previous study, we used a plasmid template com- modifications in elongation is unknown. Importantly, FACT posed of the natural promoter of the endogenous RARb2 and PAF have been shown to genetically and physically in- gene fused to b–globin (Figure 1A) (Dilworth et al., 1999). teract in yeast (Formosa et al., 2002; Krogan et al., 2002). Five RAR binding elements (RAREs) are located within However, in contrast to FACT, neither the PAF complex the promoter. Chromatin was assembled using the RSF nor H2BK123ub1 is essential for viability in yeast, although system and purified core histones derived from HeLa cells mutants in PAF or the monoubiquitinating factors, Rad6 (Figure 1B) or bacteria (Figure 2C) as described in Experi- and Bre1, do show elongation defects (Shi et al., 1996; mental Procedures. Shi et al., 1997; Wood et al., 2003a). Using a completely reconstituted transcription assay We previously used the naturally inducible RARb2 pro- composed of highly purified human general transcription moter as a paradigm to study the mechanisms of activa- factors (GTFs), human RNA polymerase II, human Media- tor-dependent transcription in vitro (Pavri et al., 2005). tor, and PARP-1, ligand-dependent transcription requires The retinoic acid receptor (RAR) is a member of the nu- SWI/SNF and p300 chromatin modifying factors when the clear receptor superfamily of transcription factors and ex- chromatin template is assembled in Drosophila embryo ists as a dimer with the retinoid X receptor (RXR) (Mangels- extracts (Pavri et al., 2005). However, we failed to detect dorf and Evans, 1995). The RAR/RXR heterodimer is such transcription from RSF-assembled templates even repressive in its unliganded state, but it is converted to in the presence of SWI/SNF and p300 (Figure 1C, lanes an activator via a conformational change upon binding 7 and 8). The crude extracts previously used to assemble of its cognate ligand, retinoic acid (RA). RAR-mediated chromatin likely contained one or more activities essential ligand-dependent transcription from this promoter re- for transcription. We tested if FACT was one of these miss- quires a novel cofactor, poly (ADP-ribose) polymerase-1 ing activities. (PARP-1), in conjunction with Mediator in vitro and in vivo. Upon addition of purified recombinant FACT, we ob- PARP-1 is responsible for switching Mediator to its active served modest levels of ligand-dependent transcription conformation upon induction through the release (or con- (Figure 1C, lanes 9–16). Importantly, the FACT-dependent formational change) of the negative module of Mediator transcription required SWI/SNF and p300 (Figure 1C, that includes Cdk8 (Pavri et al., 2005). Although this sys- lanes 3–6) as well as Mediator, RAR, and PARP-1 (Figure tem was largely reconstituted with highly purified factors, 1C, lanes 17–24), as previously established in vivo (Pavri chromatin was assembled with crude Drosophila embryo et al., 2005). Yet the transcription signals were weak rela- extracts, precluding the complete identification of the tive to those obtained previously utilizing crude S190 ex- minimal set of factors required for transcription through tracts, suggesting other factor(s) were still missing. chromatin. We now developed a fully reconstituted chromatin sys- PAF Complex and H2BK120ub1 Cooperatively tem using highly purified human factors that integrates Stimulate FACT-Dependent Transcription a recombinant chromatin assembly system comprised of We next tested whether the PAF complex and recombinant human RSF (LeRoy et al., 1998). RSF con- H2BK120ub1 might stimulate FACT-dependent transcrip- tains an ATPase subunit (SNF2H) and a larger subunit tion based on their reported interaction (Sims et al., (Rsf1) of unknown function (LeRoy et al., 1998; Loyola 2004).We previously reported the purification of human et al., 2003). This recombinant RSF-chromatin reconsti- PAF complex (Zhu et al., 2005a) as well as the complex tuted system has two major advantages: (1) in association that mediates H2BK120ub1 composed of the ubiquitin with our reconstituted human transcription system, it al- E3 ligases RNF20/40 (Figure 2A) and the E2 ubiquitin con- lows us to determine the minimal set of factors required jugating enzyme UbcH6 (Zhu et al., 2005b). to facilitate transcription through chromatin, and conse- Upon addition of either the purified PAF complex quently (2) it allows us to determine the specific role(s) of (Figure 2A) or the H2B monoubiquitination factors to the re- these factors and their underlying mechanisms. action containing FACT, there was no detectable stimula- Using this highly defined human system, we demon- tion of transcription (Figure 2B; compare lanes 3 and 4 with strate that efficient elongation through chromatin is mini- lanes 15 and 16 or 17 and 18, respectively). However, the

704 Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. Figure 1. A Fully Reconstituted Chromatin System: Requirements for p300, SWI/SNF, and FACT (A) Diagram of the p(DR5)5b2G template plasmid with the RARb2 promoter used for in vitro transcription assays. The position of the primer used for primer extension analysis is shown. (B) Microccocal nuclease digestion profile of the chromatinized template assembled with the RSF/NAP-1 system. (C) In vitro reconstituted transcription assay on chromatin templates shows a requirement for FACT. RA denotes retinoic acid. addition of both PAF and the H2B monoubiquitination fac- tamers reconstituted with bacterially purified histones tors resulted in robust levels of transcription (Figure 2B, was assembled and analyzed by micrococcal nuclease lanes 5–12). This effect was dependent on the presence digestion (Figure 2C) and in transcription. As in the case of ubiquitin (Figure 2B, lanes 19 and 20), strongly suggest- with native core histones (Figure 1C), the DNA template ing that H2BK120ub1 was essential for the stimulation. contained 5S nucleosome positional sites which allow This cooperative stimulation by PAF and H2BK120ub1 for chromatin assembly independent of histone tails was also strictly dependent on the presence of FACT and thus independent of their associated modifications (Figure 2B, lanes 13 and 14) and, as previously demon- (An et al., 2002). As with native histones, ‘‘recombinant strated (Zhu et al., 2005b), required lysine-120 of human chromatin’’ showed weak transcription in the presence H2B (see below). of SWI/SNF, p300, and FACT (Figure 2D, lanes 3 and Neither FACT, PAF, nor the factors required for 4), and only upon addition of both PAF and the factors H2BK120ub1, alone or in combination, were effectual in mediating H2BK120ub1 was transcription robust transcription from our previously described naked DNA (Figure 2D, lanes 11 and 12). FACT, SWI/SNF, p300, transcription system composed of GTFs, RNA polymer- and ligand were required (Figure 2D). Importantly, ‘‘re- ase II, Mediator, and PARP-1 (Pavri et al., 2005), indicating combinant chromatin’’ containing an H2BK120A substi- that their involvement in transcription is at the level of tution did not show transcription stimulation upon addi- chromatin (data not shown). tion of PAF and the H2BK120ub1 enzymes (Figure 2E). To test whether the presence of preexisting covalent Therefore, the H2BK120 residue, the site of ubiquitination modifications on the histones might be contributing to by RNF20/40 and UbcH6 (Zhu et al., 2005b), was transcription, ‘‘recombinant chromatin’’ composed of oc- required.

Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. 705 Figure 2. RNF20/40 and PAF Cooperatively Stimulate Ligand-Dependent Transcription in a FACT-Dependent Manner (A) Silver stain analyses of puriﬁed RNF20/40 and PAF complexes from epitope-tagged HeLa cell lines. (B) Reconstituted transcription assay performed as in Figure 1. Puriﬁed PAF and RNF20/40 were added to the transcription reaction in various com- binations as shown. (C) Microccocal nuclease digestion analysis of the template assembled with recombinant histone octamers by the RSF/NAP-1 method.

706 Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. Figure 3. Ubiquitination of the RARb2 Promoter In Vitro Is Dependent upon FACT, PAF, and Transcription Initiation (A) Diagram of the template used for ChIP showing the positions of probes P1–P4. (B) Transcription-coupled in vitro ChIP assay. The top panel shows the reaction scheme. Transcription reactions were performed as in Figure 2Band subjected to IP and dot-blot as shown in the scheme. Individual factors were omitted from the transcription reaction as indicated. (C) In vitro ChIP assays performed as in (B). Blots were probed with the primers P1–P4 shown in (A).

H2BK120ub1 Is Dependent on the PAF Complex, random H2BK120ub1, were ineffectual in transcription ac- FACT, and Transcription tivation (compare Figures 2B and 3B). To test for the levels of H2BK120ub1 at different regions of Robust ligand-dependent H2BK120ub1 at the promoter the transcription unit, the transcription reactions were was abolished upon omission of either FACT or PAF subjected to chromatin immunoprecipitation (ChIP) analy- (Figure 3B, lanes 7–10, and data not shown) and, surpris- ses using antibodies to ubiquitin (Figure 3; see Experimen- ingly, upon omission of NTPs (Figure 3B, lanes 11 and tal Procedures) and probes P1–P4 (Figure 3A). 12), demonstrating that H2BK120ub1 occurs coupled to Robust ligand-dependent H2BK120ub1 was detected transcription. Using these same preparations of PAF at the promoter region using probe P2, which hybridizes and monoubiquitination enzymes, we previously demon- around the start site (Figure 3B, upper panel, lanes 1 strated H2Bub1 at lysine 120 on a chromatinized template and 2). Omission of either RNF20/40 or ubiquitin resulted in vitro and that PAF stimulated this reaction (Zhu et al., in a loss of signal (Figure 3B, lanes 3–6), indicating that 2005b). In the present study, FACT-dependent and co- H2B was not premonoubiquitinated. Importantly, a weak transcriptional H2BK120ub1 was robust using 6- to 8- signal was observed at the promoter in the absence of fold lower amounts of the monoubiquitination enzymes transcription (-RA) (lane 1) and within vector sequences (data not shown), indicating a potent stimulation upon (Figure 3B, bottom panel, lanes 1, 2, and 7–12), each of transcription. As well, the H2BK120ub1 mark propagated which were lost upon the omission of ubiquitin or the E3 to downstream nucleosomes in the coding region in a tran- enzymes. These weak signals, likely due to nonspeciﬁc, scription-dependent manner and as a function of the

(D) Transcription assay on chromatin assembled with recombinant octamers. Assays were performed as in (B) except that chromatin was assembled on a template with the transcription unit ﬂanked by nucleosomal position sites. (E) Transcription assays performed as in (D) using chromatin assembled with recombinant octamers containing H2BK120A in lieu of wild-type H2B.

Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. 707 presence of ligand, as evidenced using primers down- cruitment accompanies that of TFIIH and not that of RNA stream of the promoter (Figure 3C, probes P3 and P4). polymerase II at this promoter. This is likely due to the active recruitment of the monoubi- Although RNF20/40 interacts with p53 (Kim et al., 2005), quitination enzymes to the promoter and downstream re- we did not detect interaction between RNF20/40 and RAR, gions via their interactions with FACT and/or PAF (see be- regardless of the presence of ligand in vitro (Figure 4C), nor low). In contrast, the sequences upstream of the promoter was RNF20/40 detectable with RAR that is constitutively and on the vector that are not sites of such recruitment bound to the RARb2 promoter in vivo in PARP-1/ cells show only nonspecific monoubiquitination (Figure 3B, (Figure 4B). Thus, RAR is insufficient to recruit RNF20/40. probe P1 in Figure 3C, and see below). On the other hand, RNF20/40 interacts with UbcH6, and UbcH6 interacts with the purified PAF complex (Zhu FACT, PAF, and RNF20/40 Are Recruited to the et al., 2005b). Although PAF and RNA polymerase II have Endogenous RARb2 Gene upon Transcription been found in association (Shi et al., 1996; Shi et al., Activation 1997; Mueller and Jaehning, 2002), we did not detect We expanded our previous ChIP analyses of the endoge- Paf1 with the stalled PolII transcription complex (Fig- nous RARb2 promoter in P19 cells as a function of RA ure 4A). However, the purified PAF complex interacts treatment (Pavri et al., 2005) using antibodies to the with the Spt16 subunit of FACT (Figure 4D). We speculate FACT subunit Spt16, the PAF subunit Paf1, and to that it is FACT that recruits PAF, which in turn recruits the RNF20/40. FACT appeared to be present at low levels in H2B monoubiquitination machinery. This is in agreement the resting state, but its recruitment to the promoter was with our in vitro transcription results wherein FACT is strongly pronounced upon RA addition (Figure 4A). Subse- a prerequisite for transcription stimulation by PAF and quently, PAF and RNF20/40 were also recruited (Figure H2BK120ub1 (Figure 2B), with H2BK120ub1 being depen- 4A). Importantly, RNA polymerase II, FACT, PAF, and dent on FACT and PAF (Figure 3B) and with FACT recruit- RNF20/40 were detectable in the coding region (Figure ment preceding that of PAF and RNF20/40 (Figure 4A). 4A). As shown previously, the repressive complex NCoR and the Mediator repressive module Cdk8 were present RNF20/40 or Paf1 Depletion Delays RARb2 Induction at promoter sequences prior to RA induction but could In Vivo not be ChIPed at the promoter after induction (Figure We next depleted RNF20/40 or PAF using siRNAs to deter- 4A). On the other hand, the HAT, p300, was recruited to mine their roles in RARb2 expression in vivo. Cells treated the promoter after induction (Figure 4A). Specificity of the simultaneously with siRNAs to both RNF20 and RNF40 ChIP was demonstrated by the inability to detect p300 showed a significant decrease in RNF20/40 expression and the Med6 Mediator subunit within the transcribed compared to mock-transfected cells (Figure 5A). The region, which instead contained readily detectable levels treated cells also exhibited a significant delay in RARb2 ex- of RNA polymerase II, FACT, PAF, and the RNF20/40 pression after induction by RA (Figure 5A, upper panel, and complex (Figure 4A, see Exon 3). quantified in Figure 5E). Since PARP-1 is necessary to attain a transcription When cells were depleted of RNF20 or RNF40 individu- competent complex at the RARb2 promoter in vitro and ally, the untargeted subunit was stable and there was a sig- in vivo (Pavri et al., 2005), we next tested for recruitment nificant delay in RARb2 expression in each case (Figures of FACT, PAF, and RNF20/40 in PARP-1/ cells. In the 5C and 5D) similar to that observed in RNF20/40-depleted WT background (PARP-1+/+), the recruitment profiles of cells (quantified in Figure 5E). Thus, RNF20 and RNF40 are all factors tested were similar to those seen in P19 cells not redundant. Similar results were obtained using siRNA (Figure 4B). However, there was no detectable recruitment to Paf1, a subunit of the PAF complex (Figure 5B and quan- of PAF or RNF20/40 in PARP-1/ cells, although FACT tified in Figure 5E). Thus the loss of RNF20 or RNF40 or was detectable at the promoter at low levels (Figure 4B). PAF was deleterious to optimal RARb2 expression. Consistent with our previous observations, the negative Upon prolonged RA induction (36 hr), RARb2 levels in Cdk8 module of Mediator remains associated after the RNF20/40, RNF20, RNF40, or Paf1 depleted cells ap- RA induction in the absence of PARP-1 (Figure 4B) and peared to recover and approach the levels seen in un- transcription is impaired (data not shown; see Pavri treated cells (Figure 5E). This observation has important et al., 2005), leading to severe defects in the recruitment implications in the context of the conclusions we reached of elongation-associated factors like FACT, PAF, and and agrees with results in yeast demonstrating that these RNF20/40 in vivo. Transcription is impaired due to the in- activities are not essential. ability to recruit TFIIH (Pavri et al., 2005). Previous biochemical studies demonstrated that TFIIE FACT, PAF, and H2BK120ub1 Function after interacts with RNA polymerase II (Flores et al., 1989) and Transcription Initiation TFIIH (Flores et al., 1992; Lu et al., 1992) and regulates To assess the temporal order of factor requirement for TFIIH enzymatic activities (Drapkin et al., 1994). TFIIE transcription from a chromatin template in vitro, we per- was not present at the stalled transcription complex but formed order-of-addition assays. The normal reaction was recruited after RA stimulation along with TFIIH in scheme was slightly altered such that selected factors a PARP-1-dependent manner (Figure 4B). Thus, TFIIE re- were added along with the NTPs, i.e., after preinitiation

708 Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. Figure 4. In Vivo ChIP on Endogenous RARb2 Promoter (A) ChIP in P19 cells induced with RA for the indicated periods using a panel of antibodies as shown. Primers amplifying the promoter region and a downstream region corresponding to exon 3 of the RARb2 gene were used. (B) ChIP on the RARb2 promoter in PARP-1+/+ and PARP-1/ cells performed as in (A). (C) IPs performed with purified recombinant Flag-tagged RAR (f-RAR) mixed with purified RNF20/40 in the presence or absence of RA, using anti-RAR antibodies. Input lanes represent 50% of f-RAR and 10% of RNF20/40. (D) IPs performed with purified recombinant FACT and purified PAF complex using antibodies to FACT subunits Spt16 and SSRP1 and to the PAF subunit Paf1. complex (PIC) assembly. As shown in Figure 6A, addition PARP-1, are involved in an early ligand-induced chromatin of FACT, PAF, or RNF20/40 after PIC assembly did not af- remodeling step at the promoter to facilitate recruitment of fect the levels of transcription compared to the normal re- factors involved in the formation of a productive transcrip- action (compare lanes 5–10 with lanes 3 and 4). Thus tion initiation complex. FACT, PAF, and H2BK120ub1 have exclusively postinitiation functions in our system, consistent with H2BK120ub1 Effect of H2BK120ub1 on Single-Round levels being dependent on transcription (Figure 3B). How- Transcription and the Generation of Longer ever, SWI/SNF, p300, Mediator, and PARP-1 were essen- Transcripts tial for PIC formation since their addition with the NTPs We next investigated the role of H2BK120ub1 on a single drastically reduced gene expression (Figure 6A, lanes round of transcription by employing sarkosyl. The addition 11–18). Thus, SWI/SNF and p300, like Mediator and of this detergent after the formation of a transcription

Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. 709 Figure 5. Depletion of PAF and RNF20/40 Causes Significant Delay in RA-Inducible Expression (A) RT-PCR analysis. P19 cells subjected to RNAi against both RNF20 and RNF40 were treated with RA for the indicated times. (B) As in (A), using RNAi against Paf1. (C and D) As in (A), using RNAi against RNF20 (C) or RNF40 (D). (E) Quantification of data shown in (A) to (D). RT-PCR signals for RARb2 expression were quantified by Phosphorimager analysis. For each time point, values plotted are calculated relative to the GAPDH values and averaged for three independent experiments. initiation competent complex prevents further rounds of As shown in Figure 6D, while both the 50- and 150-nu- transcription by impeding subsequent PIC formation cleotide transcripts were detectable in the presence of (Hawley and Roeder, 1985; Kraus and Kadonaga, 1998). FACT, their levels were greatly elevated in the presence In the presence of sarkosyl, transcription signals from of H2BK120ub1. In contrast, we could not detect the the chromatinized DNA template were observed only in 950-nucleotide transcript in the presence of FACT alone. the presence of H2BK120ub1 and FACT (Figure 6B, lanes This was not surprising given its inefficiency in generating 1–8). In the absence of sarkosyl, FACT-dependent tran- detectable transcripts after one round of transcription scription was detectable in the absence of H2BK120ub1 (Figure 6B). The 950-nucleotide product was observed but stimulated in its presence (Figure 6B, lanes 3–8; also only upon the addition of the factors that mediate see Figure 2). H2BK120ub1 likely enhances the ability of H2BK120ub1. We conclude that H2BK120ub1 is required RNA polymerase II to traverse the nucleosome in a for the efficient passage of RNA polymerase II through FACT-dependent manner (see below and Discussion). chromatin along the entire length of the transcription unit. We conclude that the initial passage of RNA polymerase II through chromatin is dependent on FACT and H2BK120ub1 Stimulates Elongation by RNA H2BK120ub1. Polymerase II Thus far, we probed for the products of the transcription We directly analyzed the rate of elongation as a conse- reactions using a primer that generates a 150-nucleotide quence of H2BK120ub1. Reactions were assembled as transcript in primer extension assays (Figure 1A). To study described above with all factors but in the presence and the efficiency of transcript elongation as a function of absence of ubiquitin, and the products of the reaction H2BK120ub1, we used primers to detect elongation prod- were analyzed as a function of time (Figure 6E). An ucts of 50, 150, and 950 nucleotides (Figure 6C). overall comparison of the transcript profiles at identical

710 Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. Figure 6. H2BK120ub1 Enhances the Rate of Elongation by RNA Polymerase II through Chromatin (A) Order-of-addition reactions performed following the scheme shown in Figure 2B except that selected factors were added along with the NTPs as indicated. (B) Reactions were performed in the presence (bottom panel) or absence (top panel) of sarkosyl. (C) Diagram of p(DR5)5b2G showing the location of primers used in primer extension assays in (E). (D) Transcription reactions performed as in Figure 2B. Primers yielded transcripts of 50 nt (top panel), 150 nt (middle panel), and 950 nt (bottom panel) as diagrammed in (C) . (E) Transcription run-off assay using linear chromatinized templates under pulse chase conditions. Size markers are shown on the right side. time-points in the presence and absence of H2BK120ub1 nation (RNF20/40, UbcH6, and PAF) do not affect elonga- shows that the size of the transcripts is signiﬁcantly in- tion per se. creased in its presence (Figure 6E). Thus, H2BK120ub1 stimulates elongation by RNA polymerase II through Histone H3 Lysine 4 Methylation Per Se Is Not chromatin. Required for Elongation through Chromatin We were unable to verify that the sites of RNAPII stalling Trimethylation of lysine 4 of histone H3 (H3K4me3) is as- correlated with nucleosome occupation in the reconsti- sociated with active transcription (reviewed in Margueron tuted system as well as in vivo, suggesting that nucleo- et al., 2005). However, this modiﬁcation has not been somes may not be stably positioned on the RARb2 functionally characterized in transcription systems. We gene. As the only factor omitted from the reactions was enriched for an activity that can trimethylate H3K4 by im- ubiquitin, these results along with those in the previous munoprecipitation of nuclear extracts derived from a sta- section demonstrate that the factors catalyzing ubiquiti- ble cell line expressing Flag-Ash2 (Figures 7A and 7B),

Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. 711 Figure 7. Methylation of Histone H3 at Lysine 4 (H3K4) Has No Effect on Transcription through Chromatin (A) Histone lysine methyltransferase (HKMTase) assay of a Flag-Ash2 (f-Ash2)-containing fraction. The f-Ash2 fraction targeted nucleosomes effi- ciently on histone H3 (upper left panel), and the HKMTase activity was specific for lysine 4 of histone H3 (upper right panel). (B) Upper panel: HKMTase assays performed as in (A) and analyzed by Western blot using antibodies specifically recognizing mono- (m1), di- (m2), and trimethylated (m3) H3K4. Lower panel: HKMTase assays in the presence of competitor H3 tail peptides either mono- (m1), di- (m2), tri- (m3), or unmethylated (m0) at H3K4. (C) In vitro transcription-coupled ChIP assay using antibodies to trimethyl-H3K4 (triH3K4Me). The f-Ash2 fraction was added to all reactions. Specific factors were omitted from the reaction as shown (lanes 3–12). (D) ChIP assays, performed as in (C). Primers are the same as in Figure 3B. (E) Transcription assay performed as in Figure 2B. (F) In vivo ChIP on the RARb2 promoter in P19 cells using anti-H3K4me3 and anti-H3K4me2 antibodies as a function of RNF20/40 depletion by RNAi. a subunit of the SET1 and the MLL families of HKMTases we examined only the 50-coding region of the template, (Roguev et al., 2001; Milne et al., 2002; and our unpub- and consistent with the literature we find H3K4m3 levels lished observations). As evidenced by in vitro ChIP exper- are highest over the transcriptional start site but also pres- iments, this activity successfully mediated H3K4me3 at ent 1000 bp downstream. Importantly, H3K4me3 was de- the promoter under transcription conditions in vitro pendent on H2BK120ub1 (Figure 7C), supporting previous (Figure 7C; primers P1–P4 shown in Figure 3A), and this results in yeast demonstrating that this mark is a prerequi- mark was propagated to nucleosomes located down- site for H3K4me3 (Dover et al., 2002; Sun and Allis, stream of the transcription start site (Figure 7D). Of note, 2002). However, H3K4me3 did not affect transcription in

712 Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. this highly reconstituted system (Figure 7E, compare lanes tion sites (5S) surrounding the promoter as previously de- 11 and 12 with 13 and 14). We did find, however, that the scribed (An et al., 2002), the formation of a transcription endogenous RARb2 promoter contains H3K4me3 upon competent complex at the RARb2 promoter required transcription induction, whereas H3K4me2 appears to p300 and acetyl-CoA. It is important to emphasize that be constitutive (Figure 7F). Importantly, in RNF20/40- the role of p300 appears to be restricted to the promoter, depleted cells a loss of H3K4me3 was observed, and as p300 was not detected within the coding sequence. this was specific, as H3K4me2 was not affected (Figure This finding is in agreement with our previous results dem- 7F), demonstrating a requirement for H2BK120ub1 in onstrating that p300 interacts specifically with the non- H3K4me3 but not in H3K4me2 upon transcription phosphorylated form of RNAPII (Cho et al., 1998), the in vivo. These observations are consistent with others form of RNAPII that associates with the transcription initi- demonstrating that H3K4me3 is involved in recruiting fac- ation complex (Lu et al., 1991). Our data confirm previous tors that coordinate effects downstream of transcription reports that SWI/SNF and p300 are essential in chromatin (Sims et al., 2006; see Discussion). remodeling associated with preinitiation events (Figure 6A). DISCUSSION The situation with histone H3K4 trimethylation is intrigu- ing as it is a mark associated with active genes, yet In this report, we focused on the mandatory factors that H3K4me3 was not per se involved in the elongation pro- thwart the contiguous array of nucleosomal barriers to cess under the conditions used. We did observe that the RNA polymerase II catalysis of an RNA transcript. By em- promoter and coding regions became trimethylated on ploying a reconstituted chromatin assembly system cou- H3K4 upon transcription in vitro and in vivo (Figure 7). pled to a highly reconstituted transcription system, we H3K4me3 can recruit CHD1 through a specific interaction have defined this minimal set of factors and how they involving the CHD1-chromodomains (Flanagan et al., modulate a chromatin template to facilitate RNA polymer- 2005; Sims et al., 2005). Since CHD1 was not present in ase II transit using the RARb2 promoter whose expression our reconstituted system, any possible contribution of is induced by retinoic acid. H3K4me3 to elongation remains an open question. How- Our mechanistic studies for the roles of FACT, PAF, and ever, our studies indicate that H3K4me3 per se does not H2BK120ub1 in elongation showed that (1) H2BK120ub1 influence transcription. is dependent on RNF20, RNF40, UbcH6, PAF, FACT, and transcription; (2) FACT is associated with a stalled The Role of PAF in Elongation through Chromatin transcription initiation complex, and its recruitment to Is Likely Indirect the RARb2 promoter is stimulated upon transcription; (3) Our data provide a molecular basis for the reported genetic PAF and the factors mediating H2BK120ub1 are recruited interaction between PAF and H2BK120ub1 and the re- to the RARb2 promoter upon transcription but peak after ported recruitment of Rad6 and Bre1 by PAF to its target FACT; (4) Spt16, the largest subunit of FACT, interacts in yeast, that is, in promoting transcription elongation (Ng with PAF; (5) PAF interacts with the complex mediating et al., 2003; Wood et al., 2003b). How FACT might fit in H2BK120ub1 (Zhu et al., 2005b); (6) FACT-dependent with PAF and H2BK120ub1 was previously unclear. In- monoubiquitination is required for robust single-round deed, interactions between FACT and H2BK120ub1 en- transcription and for the generation of longer transcripts; zymes have not been documented in yeast. On the other and (7) H2BK120ub1 stimulates the efficiency of elonga- hand, genetic interactions and direct protein–protein inter- tion by RNA polymerase II. FACT alone then is insufficient actions (Figure 4D) exist between FACT and PAF (Formosa in conferring robust transcription, and these associated et al., 2002; Krogan et al., 2002). While H2BK120ub1 is re- factors contribute significantly to the process. These bio- quired to facilitate transcription elongation by RNA poly- chemical findings are supported by ChIP and siRNA-me- merase II through chromatin, the role of PAF in this process diated depletion studies in vivo. Our findings are incorpo- is likely to be indirect through its recruitment of the rated in a model that highlights the role of H2BK120ub1 in H2BK120ub1 enzymes (Zhu et al., 2005b). facilitating the passage of RNA polymerase II through the FACT-disrupted nucleosome to enhance elongation (see FACT, PAF, and Transcription Initiation: Setting below). the Stage for H2BK120ub1 In the case of the RARb2 gene, RNA polymerase II and Histone Modifications Promoting Transcription FACT appear to be constitutively localized at the pro- Elongation through Chromatin moter, while PAF was not detectable. The temporal order Our results indicate that the minimal histone modifications of recruitment of the obligatory factors for transcription required for transcription elongation through chromatin through nucleosomes at the activated endogenous are acetylation (by p300) and H2BK120ub1 (Figure 2). RARb2 promoter indicated that FACT peaked before We could not dissect the role of H2BK120ub1 in the ab- PAF and RNF20/40 (Figures 4A and 4B). While PAF inter- sence of histone acetylation, as it is required for RSF-me- acts with RNA polymerase II (Shi et al., 1996; Shi et al., diated histone deposition (Loyola et al., 2001). Although 1997), it also interacts with FACT, leaving open the possi- we attempted to bypass this by using nucleosomal posi- bility that the recruitment of PAF and RNF20/40, with

Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. 713 Figure 8. Mechanistic Model for the Role of H2B Monoubiquitination in Transcription Elongation through Chromatin See text for details. It is important to emphasize that although we indicated that the H2A-H2B dimer that is monoubiquitinated at H2BK120 is displaced by FACT, we do not have evidence for this. It is possible that only one of the two H2B molecules gets monoubiquitinated and that the unmod- ified dimer is displaced from the nucleosome (see Discussion for details). which it interacts, may be promoter dependent. Indeed, uitination (3) and efficient displacement of one H2A/H2B studies in yeast indicate that the recruitment of Mediator dimer from the nucleosome barrier (4). This dissociated and RNAPII varies depending on the promoter studied nucleosome (hexasome) is now easily traversed by RNA (Bhoite et al., 2001). polymerase II (5). This process repeats itself on succes- Strikingly, H2BK120ub1 was dependent on transcrip- sive nucleosomes resulting in an overall increase in the ef- tion initiation (Figure 3). Given that the transcribing RNA ficiency of elongation. polymerase II stalls at the first nucleosome, this and the This study pinpoints monoubiquitination of histone H2B subsequent nucleosome barriers must be relieved by as a mark that is set once the productive transcription FACT. Since transcription activation of the RARb2 gene in- complex is engaged, aiding in the dynamic passage of volves FACT recruitment, we hypothesize that FACT inter- RNA polymerase II. The propagation of this mark through- action with PAF results in recruitment of the monoubiqui- out the nucleosomes in the transcriptional unit succes- tination machinery. This is in agreement with our ChIP sively weakens their barrier potential. The highly defined experiments performed in vitro and in vivo in P19 and nature of the reconstituted system employed in this study MEF cells. should continue to be fruitful in probing other strategies regulating transcription through chromatin.

Mechanistic Model: H2B Monoubiquitination EXPERIMENTAL PROCEDURES Promotes RNA Polymerase II Elongation through Chromatin Template for Transcription Assays We previously examined the preinitiation events leading The p(DR5)5b2G template has been described (Dilworth et al., 1999). up to transcription initiation of the RARb2 gene (Pavri Transcription assays in Figures 1 and 2 were performed on a template et al., 2005). We have now extended these analyses to derived from p(DR5)5b2G as described in Supplemental Experimental postinitiation events on chromatin as summarized in a Procedures. model proposed in Figure 8. Upon initiation, RNA polymerase II begins transcribing Chromatin Assembly Chromatin was assembled with RSF as previously described (Loyola (1) and halts at the ﬁrst nucleosomal block (2). FACT is et al., 2001). During our studies analyzing the deposition of histone then optimally recruited, resulting in the recruitment of H1 onto nucleosome arrays, we found that RSF requires NAP1 (G.L PAF and the H2BK120 monoubiquitination machinery and D.R., manuscript in preparation), and we observed that although (RNF20/40 and UbcH6), followed by H2BK120 monoubiq- NAP1 is not required for the formation of nucleosome arrays, its

714 Cell 125, 703–717, May 19, 2006 ª2006 Elsevier Inc. presence stimulated this reaction. Therefore, NAP1 was included in An, W., Kim, J., and Roeder, R.G. (2004). Ordered cooperative func- our chromatin reconstitution assays. tions of PRMT1, p300, and CARM1 in transcriptional activation by p53. Cell 117, 735–748. Transcription Assays Belotserkovskaya, R., Oh, S., Bondarenko, V.A., Orphanides, G., Stu- Transcription assays were performed exactly as described (Pavri et al., ditsky, V.M., and Reinberg, D. (2003). FACT facilitates transcription- 2005). The reaction scheme is shown in Figure 1C. Experiments involv- dependent nucleosome alteration. Science 301, 1090–1093. ing sarkosyl were performed essentially as described (Kraus and Ka- Bhoite, L.T., Yu, Y., and Stillman, D.J. (2001). The Swi5 activator re- donaga, 1998), with 0.1% sarkosyl (Sigma) added 10 s after NTP addi- cruits the Mediator complex to the HO promoter without RNA polymer- tion and reactions terminated after 40 min incubation. For the run-off ase II. Genes Dev. 15, 2457–2469. experiments shown in Figure 6E, p(DR5)5b2G was linearized, chromatinized, and subjected to transcription analyses. Reactions were per- Briggs, S.D., Bryk, M., Strahl, B.D., Cheung, W.L., Davie, J.K., Dent, formed by a pulse-chase method as described (Marshall and Price, S.Y., Winston, F., and Allis, C.D. (2001). Histone H3 lysine 4 methyla- 1992), using radiolabeled UTP. At the time points indicated, the reaction is mediated by Set1 and required for cell growth and rDNA silenc- tions were terminated and analyzed as described (Marshall and Price, ing in Saccharomyces cerevisiae. Genes Dev. 15, 3286–3295. 1992). Cho, H., Orphanides, G., Sun, X., Yang, X.J., Ogryzko, V., Lees, E., Na- katani, Y., and Reinberg, D. (1998). A human RNA polymerase II com- In Vitro ChIP plex containing factors that modify chromatin structure. Mol. Cell. Biol. The reaction was performed essentially as described with minor differ- 18, 5355–5363. ences (Dilworth et al., 2000; Dilworth et al., 2004). Briefly, transcription Dilworth, F.J., Fromental-Ramain, C., Remboutsika, E., Benecke, A., assays performed as described above were subjected to microccocal and Chambon, P. (1999). Ligand-dependent activation of transcription nuclease treatment to generate mononucleosomes. Samples were in vitro by retinoic acid receptor alpha/retinoid X receptor alpha hetero- then precleared on protein G beads (containing salmon sperm DNA) dimers that mimics transactivation by retinoids in vivo. Proc. Natl. and incubated with antibody overnight at 4º C. Immune complexes Acad. Sci. USA 96, 1995–2000. were recovered on protein G beads for 1 hr. The beads were washed, Dilworth, F.J., Fromental-Ramain, C., Yamamoto, K., and Chambon, and the bound DNA was eluted and purified. The samples were dena- P. (2000). ATP-driven chromatin remodeling activity and histone ace- tured and dot-blotted onto a nylon membrane followed by hybridiza- tyltransferases act sequentially during transactivation by RAR/RXR tion with radiolabeled probes (P1–P4; see Figure 3A). Blots were In vitro. Mol. Cell 6, 1049–1058. washed, developed, and visualized by autoradiography. The vector probe hybridizes to the E. coli Ampicillin resistance gene. Dilworth, F.J., Seaver, K.J., Fishburn, A.L., Htet, S.L., and Tapscott, S.J. (2004). In vitro transcription system delineates the distinct roles In Vivo ChIP of the coactivators pCAF and p300 during MyoD/E47-dependent ChIP assays were performed as described (Vaquero et al., 2004), and transactivation. Proc. Natl. Acad. Sci. USA 101, 11593–11598. primers for the RARb2 promoter and downstream coding region (exon Dover, J., Schneider, J., Tawiah-Boateng, M.A., Wood, A., Dean, K., 3) have been described (Lefebvre et al., 2002). Johnston, M., and Shilatifard, A. (2002). Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J. Biol. HKMTase Assays Chem. 277, 28368–28371. HKMTase assays were performed as described (Nishioka and Rein- Drapkin, R., Reardon, J.T., Ansari, A., Huang, J.C., Zawel, L., Ahn, K., berg, 2003). Flag-Ash2 was stably expressed in 293 cells, and extracts Sancar, A., and Reinberg, D. (1994). Dual role of TFIIH in DNA excision from these cells were subjected to immunoprecipitation with Flag an- repair and in transcription by RNA polymerase II. Nature 368, 769–772. tibodies, and the eluted material was tested for HKMTase activity as Flanagan, J.F., Mi, L.Z., Chruszcz, M., Cymborowski, M., Clines, K.L., shown (Figures 7A and 7B). Kim, Y., Minor, W., Rastinejad, F., and Khorasanizadeh, S. (2005). Dou- For additional Experimental Procedures,seeSupplemental Data. ble chromodomains cooperate to recognize the methylated histone H3 tail. Nature 438, 1181–1185. Supplemental Data Supplemental Data include Experimental Procedures and References Flores, O., Maldonado, E., and Reinberg, D. (1989). Factors involved in and can be found with this article online at http://www.cell.com/cgi/ specific transcription by mammalian RNA polymerase II. Factors IIE content/full/125/4/703/DC1/. and IIF independently interact with RNA polymerase II. J. Biol. Chem. 264, 8913–8921. ACKNOWLEDGMENTS Flores, O., Lu, H., and Reinberg, D. (1992). Factors involved in specific transcription by mammalian RNA polymerase II. Identification and We thank Drs. J. Lis, L. Vales, and F. Jeffrey Dilworth for helpful com- characterization of factor IIH. J. Biol. Chem. 267, 2786–2793. ments and suggestions. This work was supported by a grant from the Formosa, T., Ruone, S., Adams, M.D., Olsen, A.E., Eriksson, P., Yu, Y., National Institutes of Health (GM37120) and the Howard Hughes Med- Rhoades, A.R., Kaufman, P.D., and Stillman, D.J. (2002). Defects in ical Institute to D.R. SPT16 or POB3 (yFACT) in Saccharomyces cerevisiae cause depen- dence on the Hir/Hpc pathway: polymerase passage may degrade Received: February 9, 2006 chromatin structure. Genetics 162, 1557–1571. Revised: March 23, 2006 Glass, C.K., and Rosenfeld, M.G. (2000). The coregulator exchange in Accepted: April 20, 2006 transcriptional functions of nuclear receptors. Genes Dev. 14, 121– Published: May 18, 2006 141.

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