Small Region of Rtf1 Protein Can Substitute for Complete Paf1 Complex in Facilitating Global Histone H2B Ubiquitylation in Yeast

Small region of Rtf1 protein can substitute for complete Paf1 complex in facilitating global histone H2B ubiquitylation in yeast

Anthony S. Piro1, Manasi K. Mayekar1, Marcie H. Warner, Christopher P. Davis, and Karen M. Arndt2

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260

Edited* by Robert G. Roeder, The Rockefeller University, New York, NY, and approved May 14, 2012 (received for review October 14, 2011) Histone modifications regulate transcription by RNA polymerase II acetyltransferase and deacetylase complexes, thereby governing and maintain a balance between active and repressed chromatin histone acetylation patterns on genes (17–20). states. The conserved Paf1 complex (Paf1C) promotes specific From its position at the top of a histone modification cascade histone modifications during transcription elongation, but the that determines the methylation and acetylation state of active mechanisms by which it facilitates these marks are undefined. We chromatin, H2B monoubiquitylation and the factors that estab- previously identified a 90-amino acid region within the Rtf1 lish this mark are key regulators of gene expression. In addition to subunit of Paf1C that is necessary for Paf1C-dependent histone Rad6 and Bre1, the conserved Paf1 complex (Paf1C) is required modifications in Saccharomyces cerevisiae. Here we show that this for H2B K123 ubiquitylation. Paf1C—which in budding yeast histone modification domain (HMD), when expressed as the only consists of the subunits Paf1, Ctr9, Cdc73, Rtf1, and Leo1— source of Rtf1, can promote H3 K4 and K79 methylation and H2B impacts RNA synthesis at multiple stages (21). Paf1C associates K123 ubiquitylation in yeast. The HMD can restore histone mod- with RNA pol II from the 5′ end of a gene to the poly(A) site (22, ifications in rtf1Δ cells whether or not it is directed to DNA by 23); interacts functionally and physically with the transcription a fusion to a DNA binding domain. The HMD can facilitate histone elongation factors Spt4–Spt5/DSIF, Spt16–Pob3/FACT, and modifications independently of other Paf1C subunits and does not TFIIS (8, 24–27); regulates the phosphorylation state of RNA bypass the requirement for Rad6–Bre1. The isolated HMD localizes pol II (28, 29); and is required for proper 3′ end formation of to chromatin, and this interaction requires residues important for certain transcripts (30–32). Important to this study, deletion of histone modification. When expressed outside the context of full- RTF1 from yeast cells causes dramatic reductions in H2B K123 length Rtf1, the HMD associates with and causes Paf1C-dependent ubiquitylation and H3 K4 and K79 methylation (33–36), and this histone modifications to appear at transcriptionally inactive loci, role in histone modification, like other Paf1C functions, is con- suggesting that its function has become deregulated. Finally, the served in higher eukaryotes (37–40). Rtf1 HMDs from other species can function in yeast. Our findings The mechanisms by which Paf1C promotes histone mod- suggest a direct and conserved role for Paf1C in coupling histone ifications have yet to be elucidated. Deletion of RTF1, CTR9,or modifications to transcription elongation. PAF1 reduces the occupancies of Rad6 and Set1/COMPASS on coding regions (33, 35, 41), and purified Paf1C interacts with transcription-coupled histone modifications | nucleosome Bre1 in vitro (42, 43), suggesting that Paf1C serves as a platform for recruiting histone-modifying enzymes to RNA pol II during n eukaryotes, transcription occurs within the context of a re- elongation. Whether Paf1C has a more direct role in promoting Istrictive, yet dynamic, chromatin environment. The posttrans- fi fi histone modi cations is not known. We previously reported that lational modi cation of histones represents a major mechanism deletions and amino acid substitutions within a small region of by which cells control the structure of chromatin. Some mod- the S. cerevisiae Rtf1 protein dramatically reduce global levels of fi i cations of histones include acetylation, methylation, and ubiq- H3 K4 and K79 dimethylation and trimethylation and H2B K123 fi uitylation. These modi cations can alter the structural properties ubiquitylation, leading us to define residues 62–152 as the histone of nucleosomes and serve as specific effectors for the recruitment modification domain (HMD) of Rtf1 (32, 44). Here we show that of proteins that further modify the chromatin template and reg- the Rtf1 HMD interacts with chromatin and is sufficient to pro- ulate transcription (1). mote H2B K123 ubiquitylation and H3 K4 and K79 methylation. Monoubiquitylation of histone H2B on lysine (K) 123 in Our findings suggest that Paf1C plays an active role in promoting Saccharomyces cerevisiae is a conserved modification that is conserved, transcription-coupled histone modifications. enriched on active genes but plays roles in both transcriptional repression and activation (2–4). Consistent with a repressive Results role, H2B monoubiquitylation stabilizes nucleosomes at yeast Rtf1 HMD Is Sufficient to Promote Paf1C-Dependent Histone Modi- promoters (5), inhibits the association of the RNA polymerase fications. To investigate the role of Paf1C in histone modification, fi (pol) II kinase Ctk1 with genes in yeast (6), and interferes with we asked whether the S. cerevisiae Rtf1 HMD is suf cient to fi the recruitment of the elongation factor TFIIS to genes in hu- promote histone modi cations in the absence of all other parts man cells (7). In other studies, H2B monoubiquitylation has of the Rtf1 protein. Because the HMD is genetically separable been shown to stimulate transcription of chromatin templates (8), promote nucleosome reassembly during transcription elon- BIOCHEMISTRY Author contributions: A.S.P., M.K.M., M.H.W., C.P.D., and K.M.A. designed research; A.S.P., gation (9), and inhibit chromatin compaction (10). H2B mono- M.K.M., M.H.W., and C.P.D. performed research; A.S.P., M.K.M., M.H.W., C.P.D., and K.M.A. ubiquitylation is also a prerequisite for other histone modifications analyzed data; and A.S.P., M.K.M., M.H.W., and K.M.A. wrote the paper. that mark active genes. Ubiquitylation of H2B K123 by the Rad6– The authors declare no conflict of interest. Bre1 ubiquitin conjugase–ligase proteins in yeast (11–13) is re- *This Direct Submission article had a prearranged editor. quired for dimethylation and trimethylation of H3 K4 and K79 1A.S.P. and M.K.M. contributed equally to this work. by the Set1/COMPASS and Dot1 methyltransferases, respectively 2To whom correspondence should be addressed. E-mail: [email protected]. – (14 16). Histone H3 K4 dimethylation and trimethylation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. subsequently stimulate the recruitment and activity of histone 1073/pnas.1116994109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1116994109 PNAS | July 3, 2012 | vol. 109 | no. 27 | 10837–10842 Downloaded by guest on September 25, 2021 from regions of Rtf1 required for association with actively A Myc signal at GAL7 UAS B H3 K4 Me3 at GAL7 UAS transcribing RNA pol II and other members of Paf1C (44), we 35 3.5 decided to direct the HMD to chromatin to test its activity. 30 3 Therefore, we constructed a 2-micron–based plasmid that 25 2.5 expresses a fusion of an 89-amino acid HMD (Rtf1 residues 63– 20 2 152) to the Gal4 DNA binding domain (GBD) and a c-Myc 15 1.5 10 1

epitope tag under the control of the ADH1 promoter. We GAL7/TEL-VIR

transformed this plasmid into an rtf1Δ gal4Δ strain and analyzed 5 H3 K4 Me3/ 0.5 recruitment of the fusion protein and restoration of Rtf1-de- 0 0 ﬁ

pendent histone modi cations at the GAL7 promoter under GBD GBD repressing conditions by chromatin immunoprecipitation (ChIP). GBD-Rtf1 GBD-Myc GBD-Rtf1 Myc-HMD GBD- GBD-Myc Using an antibody to the Myc tag, we detected enrichment of the GBD- Myc-HMD GBD–Myc–HMD protein at the GAL7 upstream activation sequence (UAS) (Fig. 1A). The increased level of occupancy by the C GAL7 UAS D PYK1 GBD–Myc–HMD protein relative to the similarly expressed H3 K79 Me2 at H3 K4 Me3 at 3 GBD–Myc control protein (Fig. S1A) indicates a potential role 8 2.5 7 for the HMD in facilitating chromatin association, a point dis- 6 2 cussed below. Interestingly, ChIP analysis revealed a modest but 5 reproducible recovery of H3 K4 trimethylation and H3 K79 1.5 4 dimethylation at the GAL7 UAS in strains expressing the GBD– 1 3 H3 K79 Me2/ H3 K4 Me3/ 2 – 0.5 Myc HMD (Fig. 1 B and C). Total H3 levels at the GAL7 UAS 1 were similar in all strains (Fig. S1B). As judged by a comparison 0 0 with a telomere-proximal region, RNA pol II levels across the GBD GAL7 UAS were very low in all strains (Fig. S1C). These results GBD GBD-Rtf1 fi GBD-Rtf1 GBD-Myc GBD-Myc Myc-HMD indicate that the Rtf1 HMD is suf cienttodirecthistonemod- GBD- Myc-HMD GBD- ifications when recruited to DNA and that it may do so independently of active transcription. E F To test whether the activity of the GBD–Myc–HMD required directed recruitment to DNA, we analyzed histone modification H3 K79 Me2 at PYK1 4 levels at the 5′ end of an active gene, PYK1, which lacks known GBD-Myc-HMDGBD-MycGBD-Myc-Rtf1 – – 3.5 Gal4 binding sites (45). Surprisingly, the GBD Myc HMD re- 3 H3 K4 Me3 stored H3 K4 trimethylation and H3 K79 dimethylation at the 2.5 PYK1 5′ region in an rtf1Δ strain (Fig. 1 D and E). Expression of 2 H3 K4 Me2 this plasmid-encoded HMD did not influence H3 or RNA pol II 1.5 1 H3 K79 Me2

levels at PYK1 compared with cells transformed with control H3 K79 Me2/ 0.5 vectors (Fig. S1 D and E). Importantly, the GBD–Myc–HMD, 0 Total H3 which was expressed comparably to full-length GBD–Myc–Rtf1 (Fig. S1F), also partially restored global H3 K4 and K79 meth- GBD G6PDH Δ GBD-Rtf1

ylation in rtf1 strains (Fig. 1F). GBD-Myc GBD- Myc-HMD To rule out the possibility that the GBD–Myc–HMD protein 1 23 was binding nonspecifically throughout the genome via the GBD, Fig. 1. A GBD–HMD fusion protein is sufficient to promote H3 K4 and K79 we constructed a 2-micron HMD expression plasmid in which we methylation. (A–E) ChIP analysis of GBD–Myc–HMD or control protein oc- replaced the GBD with a nuclear localization signal (NLS). We cupancy at the GAL7 UAS (A), H3 K4 trimethylation at the GAL7 UAS (B)or5′ confirmed that the NLS–Myc–HMD protein was expressed to end of PYK1 (D), and H3 K79 dimethylation at the GAL7 UAS (C)or5′ end of a similar level as a plasmid-encoded, Myc-tagged, full-length PYK1 (E). An rtf1Δ gal4Δ strain was transformed with plasmids that express Rtf1 protein (Fig. S2 A and B). Remarkably, the NLS–Myc– the indicated proteins. Mean values and SD of three biological replicates are shown. IP/input signals for Myc at GAL7 are presented relative to a TEL–VIR HMD strongly complemented the H3 K4 and K79 methylation fi Δ – subtelomeric region. IP/input signals for histone modi cations are presented defects of an rtf1 strain, both at PYK1 and globally (Fig. 2 A C). relative to total H3. (F) Immunoblot analysis of histone modifications in Mutational studies indicated a primary role for the Rtf1 HMD strains expressing the indicated proteins. in controlling H2B K123 ubiquitylation (32). We therefore asked whether the NLS–Myc–HMD could restore H2B K123 ubiquitylation in an rtf1Δ strain. For these experiments, we used expression from low-copy plasmids (Fig. S3A) or the chromo- strains in which the only source of H2B carried a FLAG tag and somal locus (see Fig. S5B). To test whether restoration of his- the gene encoding the Ubp8 ubiquitin protease was deleted to tone modifications by the NLS–Myc–HMD required the high enrich for the H2B–K123 ubiquitin signal (46, 47). In ubp8Δ levels of expression supported by the 2-micron plasmid, we – – – strains expressing either full-length Myc Rtf1 or the NLS Myc constructed two low-copy expression plasmids that differed in HMD, recovery of H2B K123 ubiquitylation was observed, as the promoter. Under the control of the RTF1 promoter on indicated by the slower mobility band in an anti-FLAG immu- a CEN/ARS plasmid, the NLS–Myc–HMD was expressed at very noblot (Fig. 2D). This band was not detected in samples pre- low levels, and we were unable to detect recovery of histone pared from cells that carried the NLS–Myc vector, lacked the modifications in an rtf1Δ strain (Fig. S3 A and B). However, when FLAG tag on H2B, or expressed FLAG–H2B–K123R (Fig. 2D). The NLS–Myc–HMD also led to recovery of chromatin-associ- expressed from the ADH1 promoter on a CEN/ARS plasmid, – – ated, ubiquitylated H2B in an rtf1Δ ubp8Δ strain, as measured by NLS Myc HMD levels were reduced compared with 2-micron fi a sequential ChIP assay (Fig. S2C). Together with our genetic expression levels but were suf cient to promote histone mod- data (32), these results show that the HMD is necessary and ifications (Fig. S3 A and B). Importantly, overexpression of the sufficient for promoting H2B K123 ubiquitylation in vivo. NLS–Myc–HMD from the 2-micron plasmid, which we used for The 2-micron plasmids used above caused overexpression of the remainder of the study, could not promote H3 K4 and K79 the NLS–Myc–HMD and Myc–Rtf1 proteins compared with methylation in the absence of Rad6, Bre1, or H2B K123 (Fig. S4).

10838 | www.pnas.org/cgi/doi/10.1073/pnas.1116994109 Piro et al. Downloaded by guest on September 25, 2021 − Therefore, although overexpression of the HMD is likely required transformed with the NLS–Myc vector exhibited a strong Spt for high levels of histone modifications, it does not bypass the (His+) phenotype (Fig. 2E). The NLS–Myc–HMD plasmid com- − canonical pathway for H2B K123 ubiquitylation and downstream plemented the Spt phenotype of the rtf1Δ strain, albeit to a lesser H3 modifications. extent than the Myc–Rtf1 plasmid. This result is consistent with the Null alleles of RTF1 or mutations within the HMD-coding conclusion that the HMD constitutes a functional domain in vivo. region suppress the transcriptional defects caused by a Ty-δ el- HMD Can Support H2B Ubiquitylation in the Absence of Other Paf1C ement insertion mutation within the HIS4 promoter, his4-912δ, − Members. We investigated the extent to which other Paf1C sub- and thus confer a Suppressor of Ty or Spt phenotype (32, 44). − Δ δ units are required for the activity of the HMD. Previous studies We examined the Spt phenotype of an rtf1 his4-912 strain showed that Paf1 and Ctr9 are important for Paf1C stability and – – – that was transformed with the NLS Myc HMD or Myc Rtf1 integrity (26, 28). To eliminate the residual Paf1C that forms in 2-micron expression plasmids. As expected, the rtf1Δ strain the absence of Rtf1 (29) and test the effect on HMD activity, we transformed the NLS–Myc–HMD expression plasmid into rtf1Δ paf1Δ and rtf1Δ ctr9Δ double mutants. Remarkably, even in the PYK1 PYK1 A H3 K4 Me3 at B H3 K79 Me2/3 at absence of Paf1 or Ctr9, the HMD behaved similarly to full- 4 1 fi 3.5 0.9 length Rtf1 and was suf cient to direct H2B K123 ubiquitylation, 0.8 3 H3 K79 dimethylation/trimethylation, and H3 K4 dimethylation, 0.7 2.5 0.6 although at a reduced level compared with strains containing 2 0.5 both Paf1 and Ctr9 (Fig. 3 A and B). In contrast, H3 K4 trime- 1.5 0.4 paf1Δ ctr9Δ 0.3 thylation was greatly reduced in the and backgrounds H3 K4 Me3/ 1 0.2 (Fig. 3A). Although a slight reduction in Rtf1 and HMD protein 0.5 H3 K79 Me2&3/ 0.1 levels in the paf1Δ and ctr9Δ strains may contribute to this effect 0 0 (Fig. 3B), these results suggest a stimulatory role for other Paf1C subunits in the pathway toward H3 K4 trimethylation. Expres- sion of the NLS–Myc–HMD or full-length Rtf1 in cdc73Δ rtf1Δ Myc-Rtf1 NLS-Myc- HMD NLS-Myc NLS-Myc- Myc-Rtf1 HMD NLS-Myc strains also partially restored H3 K79 dimethylation/trimethylation and H3 K4 dimethylation, but not H3 K4 trimethylation, C D whereas deletion of LEO1 had no obvious effect (Fig. S5). We conclude that the HMD is the principal effector of H2B ubiquitylation-dependent histone modifications within Paf1C and Myc-Rtf1NLS-MycNLS-Myc-HMD - + - + - + FLAG-H2B K123R that other subunits play an important regulatory role. NLS-MycNLS-Myc-HMDMyc-Rtf1FLAG-H2B: FLAG-H2B-Ub H3 K4 Me3 Rtf1 HMD Associates with Chromatin. Two strategies were used to FLAG-H2B investigate whether the HMD promotes H2B ubiquitylation by H3 K4 Me2 Rtf1 interacting with chromatin. First, we performed ChIP analysis of HMD localization at two active genes using rtf1Δ strains trans- H3 K79 Me2/3 formed with the 2-micron NLS–Myc–HMD or Myc–Rtf1 ex- Total H3 pression plasmids. Fortuitously, our Rtf1 antiserum (25) recognizes the HMD and reacts equivalently with the isolated G6PDH HMD and full-length Rtf1 in immunoblots (Fig. S2 A and B).

G6PDH Therefore, we used the Rtf1 antiserum to immunoprecipitate the 123 – – – – 1 2 3 4 567 NLS Myc HMD and Myc Rtf1 proteins, and the NLS Myc E vector provided a measure of Rtf1-independent background signals. As expected for a subunit of Paf1C, the full-length – NLS-Myc Myc Rtf1 protein showed high levels of occupancy on both genes (Fig. 4 A and B). Interestingly, levels of NLS–Myc–HMD NLS-Myc-HMD occupancy were also significantly enriched over background levels at PYK1 and PMA1, demonstrating that the isolated HMD Myc-Rtf1 SC-Trp-His can localize to chromatin. In a second approach, a nucleosome-pulldown assay was NLS-Myc used to analyze the chromatin interaction properties of the NLS-Myc-HMD HMD. Strains deleted for RTF1 and containing either untagged or FLAG-tagged H2B were transformed with plasmids Myc-Rtf1 that expressed the NLS–Myc tag (vector) or the NLS–Myc– SC-Trp HMD (Fig. 4C,lanes1–4). To preserve the nucleosomes, cells Fig. 2. An HMD protein lacking a sequence-specific DNA binding domain were exposed to formaldehyde and then mononucleosome- can globally promote histone modifications. (A and B) ChIP analysis of H3 K4 and dinucleosome-enriched samples were prepared by micro- trimethylation (A) and H3 K79 dimethylation and trimethylation (B)atthe coccal nuclease digestion of extracts (Fig. S6A). Following the ′ Δ – – PYK1 5 region in an rtf1 strain transformed with NLS Myc vector or plas- immunoprecipitation of FLAG H2B and reversal of cross- BIOCHEMISTRY mids that express NLS–Myc–HMD or Myc–Rtf1. (C) Immunoblot analysis links by heat treatment, coprecipitated proteins were exam- of histone modifications in rtf1Δ strains containing the indicated plasmids. ined by immunoblotting using Rtf1 antiserum. By using this (D) Anti-FLAG immunoblot analysis of strains expressing FLAG–H2B, as in- approach, the NLS–Myc–HMD specifically coprecipitated dicated, and Myc–Rtf1, NLS–Myc vector, NLS–Myc–HMD, or endogenous Rtf1 – – – fi with FLAG H2B (Fig. 4C, lane 4). The coprecipitation of H3 (lane 7). A FLAG H2B K123R strain provided a speci city control. Rtf1 was with FLAG–H2B indicated that the FLAG–H2B was in- detected with Rtf1 antiserum. A consistently observed Rtf1 breakdown corporated into nucleosomes. Importantly, two mutant forms product may be seen in lanes 1 and 2. The epitope(s) for the Rtf1 antiserum – – – – maps within the HMD (Fig. S2). (E)Anrtf1Δ his4-912δ strain, transformed of the HMD, NLS Myc HMD E104K and NLS Myc HMD − with indicated plasmids, was tested for complementation of the Spt pheno- F123S—which abrogate the histone modification activity of type by growth on SC–Trp–His (4 d) or SC–Trp control (2 d) medium at 30 °C. full-length Rtf1 (32) and are expressed at levels similar to the

Piro et al. PNAS | July 3, 2012 | vol. 109 | no. 27 | 10839 Downloaded by guest on September 25, 2021 A A B

Vector NLS-Myc-HMDMyc-Rtf1Vector NLS-Myc-HMDMyc-Rtf1Vector NLS-Myc-HMDMyc-Rtf1 H3 K4 Me3

H3 K4 Me2

H3 K79 Me2/3

Total H3 C D

G6PDH

1 2 345678 9 - B - -

Myc-Rtf1VectorNLS-Myc-HMDMyc-Rtf1VectorNLS-Myc-HMDMyc-Rtf1VectorNLS-Myc-HMDMyc-Rtf1 FLAG-H2B: - + ++++++++ FLAG-H2B-Ub

FLAG-H2B E F

Rtf1

G6PDH 12345678910

Fig. 3. The HMD functions independently of other Paf1C members. Im- Fig. 4. The HMD interacts with chromatin. (A, B, D, and E) ChIP analysis of munoblot analyses of H3 K4 and K79 methylation (A) or FLAG–H2B ubiq- NLS–Myc, NLS–Myc–HMD, and Myc–Rtf1 localization to the 5′ and 3′ regions uitylation (B)inrtf1Δ, rtf1Δ paf1Δ, and rtf1Δ ctr9Δ strains transformed with of PYK1 (A) and PMA1 (B), a TEL VIR proximal region (D), and chromosome V the indicated plasmids are shown. intergenic region (E) using Rtf1 antiserum. Mean values and SDs from three biological replicates are shown. (C) Immunoblot analysis of a pulldown assay — measuring the association of the indicated HMD proteins with nucleosomes wild-type HMD (Fig. S6B) showed reduced association with – fi – – containing FLAG H2B. The gure is representative of three independent FLAG H2B (Fig. 4C,lanes58). Of the two substitutions experiments. (F) ChIP analysis of GBD–Myc–HMD localization at the PYK1 5′ tested, E104K causes stronger defects, eliminating dimethy- region using Rtf1 antiserum. lation and trimethylation of H3 K4 and K79, whereas F123S only reduces these marks (32). In three experiments, we con- regions of Rtf1 normally direct its association and function to sistently observed decreased nucleosome association of HMD– transcribed genes. E104K relative to HMD–F123S. Therefore, the HMD can associate with chromatin, and this interaction requires resi- Histone Modification Function of the HMD Is Conserved. Sequence dues important for histone modification. conservation of the HMD (44) prompted us to test the activities of We previously identified a central region of Rtf1 that is re- Rtf1 HMD sequences from other species. Using predicted struc- quired for proper recruitment of the Paf1C to ORFs—pre- tural features as a guide, we deleted the HMD coding sequence sumably through an interaction with RNA pol II—and a C- from a plasmid-borne S. cerevisiae RTF1 gene, removing amino – terminal region of Rtf1 that is required for interactions with acids 74 187, and replaced it with those of the Schizosacchar- – – omyces pombe, Drosophila melanogaster, Danio rerio,andHomo other Paf1C subunits (44). The NLS Myc HMD lacks these Δ regions, raising the possibility that the HMD can associate with sapiens RTF1 genes. The S. cerevisiae Rtf1 HMD deletion protein was expressed to the same level as wild-type Rtf1 but was com- chromatin independently of transcription. In support of this pletely defective in supporting histone modifications (Fig. 5, idea, we detected enrichment of the NLS–Myc–HMD and lane 2). Upon introduction of the predicted HMD sequence from HMD-mediated histone modifications at transcriptionally in- S. pombe into the S. cerevisiae Rtf1 protein, recovery of H2B active loci (Fig. 4 D and E and Fig. S6C). In contrast to the ChIP ubiquitylation, H3 K4 dimethylation and trimethylation, and H3 patterns observed at the active PYK1 and PMA1 genes, the K79 dimethylation/trimethylation was observed (Fig. 5, lane 5). – levels of Myc Rtf1 occupancy at TELVI and an intergenic re- Insertion of predicted HMD sequences from other Rtf1 homologs gion of chromosome V were appreciably lower than those ob- resulted in stable proteins that rescued the histone modification served for the NLS–Myc–HMD (Fig. 4 D and E). Consistent defect of the Rtf1ΔHMD protein to varying degrees. All of the with the ability of the GBD–Myc–HMD to direct histone chimeric proteins restored some level of H3 K4 dimethylation and modifications at a region lacking Gal4 binding sites (Fig. 1 D H3 K79 dimethylation/trimethylation (Fig. 5, lanes 6–9), although and E), we also detected enrichment of the GBD–Myc–HMD at the recovery of H2B ubiquitylation was apparently below detection PYK1 (Fig. 4F)andTELVI (Fig. S6D). These results suggest levels. These results indicate that the histone modification func- that the HMD has chromatin-association activity, but other tions of the HMD are evolutionarily conserved.

10840 | www.pnas.org/cgi/doi/10.1073/pnas.1116994109 Piro et al. Downloaded by guest on September 25, 2021 Discussion To investigate how Paf1C stimulates transcription-coupled histone modifications, we characterized an 89-amino acid HMD of the S. cerevisiae Rtf1 protein. We show that this small fragment of Rtf1 can largely substitute for a complete Paf1C in promoting global H2B ubiquitylation and H3 K4 and K79 methylation. The HMD interacts with chromatin in a manner dependent on amino acids that are required for histone modification, indicating that HMD activity and chromatin association are correlated. The isolated HMD associates with and causes Rtf1-dependent histone modifications to appear at poorly transcribed regions of the genome, suggesting that the functions of the HMD are normally constrained by other parts of Paf1C. Finally, we found that HMD-like sequences from Rtf1 homologs are partially functional in S. cerevisiae. Studies in yeast and metazoans have revealed a critical role for Paf1C in promoting histone modifications during transcription elongation. Defects in Paf1C cause dramatic reductions in H2B ubiquitylation, H3 K4 methylation, and H3 K79 methylation, as well as decreased occupancy of Rad6–Bre1 and Set1/COMPASS on coding regions (33, 35–38, 40, 41). In addition, in vitro assays on chromatin templates demonstrated a requirement for Paf1C in transcription-coupled H2B ubiquitylation (8, 42). Together with direct physical interactions between Paf1C and Bre1 (42, 43), these observations provide support for a model in which Paf1C mediates the interaction between histone modifiers and the RNA pol II elongation machinery. However, the retention of Fig. 5. The HMD is functionally conserved. Immunoblot analysis of histone Rad6 at yeast promoters in rtf1Δ cells without concomitant H2B modifications in strains expressing FLAG–H2B or untagged H2B and empty ubiquitylation previously led to the conclusion that Paf1C is re- vector, an S. cerevisiae Myc-tagged Rtf1 protein deleted for the HMD quired for the activity of Rad6–Bre1 at promoters in addition to its (Rtf1ΔHMD), wild-type Myc–Rtf1 (lanes 3 and 4), or chimeric Myc–Rtf1 pro- – role in recruiting Rad6–Bre1 to transcribed regions (36, 41). Our teins in which amino acids 74 187 of S. cerevisiae Rtf1 were replaced with the predicted HMD of S. pombe, D. melanogaster (two HMD lengths tested), discovery of a small region within Rtf1 that is competent to pro- D. rerio,orH. sapiens. mote H2B ubiquitylation is in agreement with the activation model. Through ChIP and nucleosome pulldown assays, we found – that the HMD can interact, directly or indirectly, with chromatin. residual Paf1C plays a role in enhancing the activities of Rad6 Bre1, Set1/COMPASS, or both. Amino acid substitutions that impair the histone modification Accounting for its broad effects on gene expression, H2B function of Rtf1 (32) also impair the interaction of the HMD monoubiquitylation has been reported to alter the physical with nucleosomes (Fig. 4). This observation suggests a coupling properties of chromatin (5, 10), influence the activities of between the histone modification and chromatin binding func- transcription elongation factors (6–9), and ensure proper tions of the HMD and suggests that Paf1C, through the HMD, transcription termination (32). The balance between gene ac- may transiently associate with chromatin during transcription. tivation and repression mediated by H2B monoubiquitylation is Whether the HMD, either alone or in the context of full-length especially important in human cells, where this modification Rtf1, interacts directly with nucleosomes or whether this asso- promotes transcription of the p53 tumor suppressor gene and ciation is mediated by factors such as Rad6, Bre1, or Set1/ inhibits the expression of protooncogenes (49). Through its COMPASS remains to be elucidated. However, our current data association with RNA pol II and its roles in histone modifica- indicate that deletion of these factors does not eliminate the – tion, RNA pol II phosphorylation, Chd1 recruitment, and HMD chromatin interaction (Fig. S7). Although they do not transcription termination, the multifunctional Paf1C also exclude direct interactions between the HMD and the histone influences the expression of many genes (21). Underscoring the fi modi ers, our results are also consistent with the possibility that significance of understanding the molecular functions of this an interaction between the HMD and nucleosomes, even if complex are its connections to development, cancer, and stem transient, may prepare nucleosomes for catalysis. cell pluripotency (50–53). Our discovery that the function of Our results on the HMD provide insights into the functions of the S. cerevisiae HMD can be partially replaced with those other parts of Paf1C. The isolated HMD associated with and of other eukaryotes highlights the conserved nature of fi promoted histone modi cations at transcriptionally inactive loci this domain. (Fig. 4 and Fig. S6C). In contrast, full-length Rtf1 preferentially occupied transcribed genes. Our HMD constructs lack regions of Methods Rtf1 required for its interactions with other Paf1C subunits and Yeast Strains and Growth. Yeast strains (Table S1) are isogenic to FY2 (54) and transcribed regions of genes (44). We conclude that, because of were generated through transformation or tetrad analysis (55). Rich (YPD) BIOCHEMISTRY the absence of these interactions, the localization of the HMD is and synthetic complete (SC) media were prepared as described (55). To − deregulated and its histone modification function appears to be measure the Spt phenotype, plasmid transformants of strain KY619 were – uncoupled from RNA pol II. Most likely, other regions of Paf1C, grown in SC Trp medium, harvested by centrifugation, and washed with × 8 such as the Rtf1 ORF association/Plus3 domain (44, 48), are sterile H2O. Serially diluted cultures (10-fold; starting with 1 10 cells per milliliter) were spotted onto solid medium. dominant to the HMD in controlling the localization of Paf1C. Δ Δ Δ Based on our analysis of paf1 , ctr9 , and cdc73 mutants, Rtf1 Plasmids. Details of plasmid construction are provided in SI Methods. is the key member of Paf1C with respect to the establishment of H2B K123 ubiquitylation and downstream marks. However, the ChIP Assays. ChIP assays were performed as described (ref. 44; SI Methods). absence of H3 K4 trimethylation in these strains shows that the Sonicatedchromatin was incubatedovernight with primary antibodies, followed

Piro et al. PNAS | July 3, 2012 | vol. 109 | no. 27 | 10841 Downloaded by guest on September 25, 2021 by incubation with Protein A- or G-coupled Sepharose (Amersham Biosciences). Nucleosome Pulldown Assay. Yeast cultures were grown to ∼2.5 × 107 cells Following reversal of cross-links and purification of DNA, immunoprecipitated per milliliter in selective medium. Cells were treated with formaldehyde, (IP) and total (input) DNA were analyzed by quantitative real-time PCR using harvested, and transferred to MNase digestion buffer (SI Methods). Fol- SYBR green (Fermentas) detection or by PCR performed in the presence of [α32P] lowing glass bead lysis, cells were treated with MNase (Roche) for 30 min at 37 °C. After stopping the digestion with EDTA and centrifugation, the dATP. For the latter, PCR products were resolved on native polyacrylamide gels supernatant was used in an immunoprecipitation reaction with anti-FLAG and quantified by using a phosphorimager. Mean values of three biological M2 affinity resin (Invitrogen), and precipitated proteins were analyzed replicates with SD are shown unless otherwise noted. by immunoblotting.

∼ × 7 Immunoblotting Analysis. Yeast cultures were grown to a density of 3 10 cells ACKNOWLEDGMENTS. We thank Andrew VanDemark and Sean Kellner for per milliliter in selective medium, and extracts were prepared by glass bead lysis in technical support; Joe Reese, Fred Winston, Brad Cairns, Bob Roeder, Zu- radioimmunoprecipitation assay buffer or trichloroacetic acid as described (44, Wen Sun, Beth Stronach, and Beth Roman for protocols and reagents; and 56). Proteins were resolved by SDS/PAGE, transferred to nitrocellulose, and Rich Gardner and Joe Martens for comments on the manuscript. This work was supported by National Institutes of Health Grant R01GM52593 (to K.M.A.) immunoblotted by standard methods (SI Methods). Extract preparation and im- and by fellowships from the Howard Hughes Medical Institute and the munoblot analysis of FLAG–H2B ubiquitylation were performed as described (32). Beckman Scholars Program (to C.P.D.).

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