| INVESTIGATION

Histone H2B Ubiquitylation Regulates Histone Expression by Suppressing Antisense Transcription in Fission Yeast

Viviane Pagé,* Jennifer J. Chen,* Mickael Durand-Dubief,† David Grabowski,* Eriko Oya,† Miriam Sansô,‡ Ryan D. Martin,* Terence E. Hébert,* Robert P. Fisher,‡ Karl Ekwall,† and Jason C. Tanny*,1 *Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada, †Department of Biosciences and Nutrition, Karolinska Institute, Stockholm 17177, Sweden, and ‡Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, Mount Sinai School of Medicine, New York, New York 10029 ORCID IDs: 0000-0002-8556-4459 (M.D.-D.); 0000-0003-3495-6150 (J.C.T.)

ABSTRACT Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone in the fission yeast Schizosaccharomyces pombe. H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2+—a gene encoding a GATA-type that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specificE3 ubiquitin ligase Brl2 had elevated levels of ams2+ antisense transcripts and reduced Ams2 levels. These defects were reversed upon inhibition of Cdk9—an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)—indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein- coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle- dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.

KEYWORDS Antisense; Cdk9; H2Bub1; histone genes

ISTONE H2B monoubiquitylation (H2Bub1) is a con- these enzymes requires Cdk9—the kinase component of pos- Hserved and multifunctional histone mark with direct itive transcription elongation factor b (P-TEFb) (Tanny roles in RNA polymerase II transcription elongation, DNA 2014). Ubiquitylation by these factors occurs on a conserved replication, and DNA repair (Moyal et al. 2011; Nakamura lysine on the nucleosome surface (K120 in ; K119 in et al. 2011; Trujillo and Osley 2012; Fuchs et al. 2014). fission yeast), which promotes the methylation of specific H2Bub1 is catalyzed by the E2 ubiquitin-conjugating enzyme sites on histone H3 by Dot1 and COMPASS-related com- Rad6 and E3 ubiquitin ligases related to budding yeast Bre1 plexes (Kim et al. 2013; Anderson et al. 2019; Worden (Fuchs and Oren 2014). During transcription, activity of et al. 2019). H2Bub1 also acts independently of downstream histone methylation by influencing activity of a number of

Copyright © 2019 by the Genetics Society of America additional factors, including histone chaperones and chroma- doi: https://doi.org/10.1534/genetics.119.302499 tin-remodeling factors (Sansô et al. 2012; Fuchs and Oren Manuscript received May 10, 2019; accepted for publication July 23, 2019; published 2014). How these functions are coordinated to regulate tran- Early Online July 24, 2019. Supplemental material available at FigShare: https://doi.org/10.25386/genetics. scription, replication, or DNA repair is not well understood. 8977568. H2Bub1 is required for normal cell cycle progression in 1Corresponding author: Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir William Osler, Room 132, Montreal, Quebec yeast and in mammalian cells. H2Bub1 has direct roles in a H3G 0B1, Canada. E-mail: [email protected] variety of cell cycle events, such as DNA replication fork

Genetics, Vol. 213, 161–172 September 2019 161 Table 1 Strains used in this study Strain number Genotype Source

JTB204 h- ade6-M216 Tanny et al. (2007) JTB86-3 htb1-K119R::kanMX6 ade6-M216 h- Tanny et al. (2007) JTB331 h- brl2D::hphMX4 ade6-M210 Tanny et al. (2007) JTB321 cdk9-T212A::kanMX6 leu1-32 ura4-D18 his3-D1 ade6-M210 h+ Sansô et al. (2012) JTB335 cdk9-T212A::kanMX6 brl2D::hphMX4 leu1-32 ura4-D18 his3? ade6 Sansô et al. (2012) JTB429 cdk9-T212A::kanMX6 htb1-K119R::kanMX6 leu1-32 ura4-D18 his3? ade6 Sansô et al. (2012) JTB362 leu1-32 ura4-D18 his3-D1 ade6-M210 h+ Sansô et al. (2012) JTB98-1 htb1-K119R::kanMX6 ade6 leu1-32 ura4-D18 h- Tanny et al. (2007) JTB425 cdk9as::natMX6 leu1-32 ura4-D18 his3-D1 ade6-M210 h+ This study JTB508 cdk9as::natMX6 htb1-K119R::kanMX6 ade6 leu1? ura4? h? This study JTB854 ams2-13myc::kanMX6 ade6-M216 leu1-32 ura4-D18 his2 h- Kim et al. (2016) JTB913 ams2-13myc::kanMX6 htb1-K119R::hphMX6 ade6 leu1? ura4-D18 his2? h? This study JTB914 ams2-13myc::kanMX6 cdk9as::natMX6 ade6 leu1-32 ura4-D18 his? h? This study JTB902-5 ams2-13myc::kanMX6 brl2D::hphMX4 ade6 leu1? ura4? his2? h? This study JTB916 ams2-13myc::kanMX6 cdk9as::natMX6 htb1-K119R::hphMX6 ade6 leu1? ura4-D18 his? h? This study progression, operation of the S phase checkpoint, DNA dou- was synthesized by Zamboni Chemical Solutions (Bellini Life ble-strand break repair, sister chromatid cohesion, and assem- Sciences Complex, McGill University) and used at a final concen- bly of centromeric chromatin (Trujillo and Osley 2012; tration of 20 mM. Cycloheximide (Sigma) was added to supple- Sadeghi et al. 2014; Hung et al. 2017; Zhang et al. 2017). mented YE media at a final concentration of 100 mM. Standard Several studies have also implicated H2Bub1 in the regula- genetic crosses and tetrad dissection were used to create double- tion of cell cycle-dependent genes (Shema et al. 2008; mutant strains (Moreno et al. 1991). Zimmermann et al. 2011; Jääskelainen et al. 2012; Fuchs Synchronization in S phase with hydroxyurea and Oren 2014). Thus, H2Bub1 function during the cell cycle likely requires coordination among multiple different mech- Cells were grown to OD600 of 0.2 in 300 ml EMM at 30° and anisms. In the fission yeast Schizosaccharomyces pombe, loss hydroxyurea (HU) was added to 12 mM; incubation contin- of H2Bub1 causes defective cell separation and increased cell ued for 4 hr. Cultures were harvested by centrifugation at size, but the origin of these effects is unclear. These pheno- 25°, washed once with prewarmed EMM, resuspended in types are largely reversed upon reduction in the activity of 300 ml prewarmed EMM, and returned to growth. Samples Cdk9, suggesting that they result from aberrant transcription were collected upon return to growth and at 20-min intervals that is Cdk9-dependent (Sansô et al. 2012). To gain insight thereafter. For fluorescence-activated cell sorting (FACS) into the roles of H2Bub1 in cell cycle progression, we have analysis, 1 ml of culture was pelleted in a microfuge, resus- examined its function in cell-cycle-regulated gene expression pended in ice-cold 70% ethanol, and stored at 4°. For RNA in the model eukaryote S. pombe, and we identify a specific analyses, 10 ml of culture was pelleted, washed with 1 ml role for this modification in regulating expression of replica- sterile dH2O and stored at 280°. tion-dependent histone genes. We provide evidence that this RNA analyses arises from a role of H2Bub1 in suppressing Cdk9-dependent antisense transcripts. These results offer new insight into Total RNA was extracted from frozen cell pellets using a hot gene regulatory mechanisms of H2Bub1 and its role in cell- phenol method (Tanny et al. 2007). For quantitative reverse cycle progression. transcription PCR (RT-qPCR), 1 mg RNA was reverse tran- scribed to cDNA (AMBG Easyscript) with oligo-dT primers, which was then amplified with the primers listed in Table 2 Experimental Procedures and a SYBR Green qPCR master mix (Bio-Rad) in a Bio-Rad CFX96 qPCR instrument. Levels of histone mRNAs were Yeast strains and media expressed relative to act1+. Strand-specific RT-qPCR was car- S. pombe strains used in this study are listed in Table 1. S. ried out similarly using gene-specific primers (Table 2). pombe strains were cultured in YE (0.5% yeast extract, 3% FACS analysis dextrose) or in EMM [Edinburgh minimal media; (Moreno et al. 1991)] supplemented with adenine, leucine, uracil, and Samples fixed in 70% ethanol were washed once with 1 ml histidine (0.25 g/liter each). Strains harboring plasmids 50 mM sodium citrate and then resuspended in 0.5 ml expressing ams2+ (Takayama et al. 2016) were cultured 50 mM sodium citrate containing 0.1 mg/ml RNase A. After in supplemented EMM lacking leucine and thiamine. Trans- a 4-hr incubation at 37°, 0.5 ml 50 mM sodium citrate con- formation of plasmids was carried out using a lithium acetate taining 4 mg/ml propidium iodide was added. Samples method (Bähler et al. 1998). Thiabendazole (TBZ; Sigma) were sonicated briefly and analyzed on a FACSCalibur-I in- was added to plates at a concentration of 15 mg/ml; 3-MB-PP1 strument (BD Biosciences). Data were processed using FloJo

162 V. Pagé et al. Table 2 Oligonucleotide primers used in this study available at NCBI BioProject (PRJNA382240). Supplemental Name Sequence material available at FigShare: https://doi.org/10.25386/ genetics.8977568. hht-FW TCGGCCAAGATTTCAAGACTG hht-RV CGCCACGGAGACGACGAG hhf-FW ATTCGCGATGCCGTCACCTA hhf-RV TAACCACCGAAACCATAAAT Results hta-FW CTTCGCCGCCGTTTTGGAATA hta-RV CGTTACGGATGGCGAGTTGAAGAT H2Bub1 regulates histone gene expression htb1-FW AGCCATGCGTATCTTGAACTCTTT htb1-RV GGTAACGGCGTGCTTGGCTAACT To examine the role of H2Bub1 in cell-cycle regulated gene hht1-FW CTGTCACCCTTTGATATGTTG expression in fission yeast, we synchronized wild-type or htb1- hht1-RV TAACACATATCCGTTCCCATC K119R mutant cells in early S phase using HU and monitored hht2-FW GCATTGATTGCCTAATATTTTATTTG cell cycle progression over time after release into fresh me- hht2-RV AAATTAATATGCTAAACCCGAC dium. The htb1-K119R mutant encodes histone H2B lacking cdc18-FW TCCCTCGTTTACGAACAAAG cdc18-RV CAGCATGCTGAGATACAAC the conserved ubiquitylation site (Tanny et al. 2007). FACS cdc22-FW CAGGTAGAGGGTACATATG analysis showed that both wild-type and htb1-K119R cells cdc22-RV TGAGATGTTGAAGCAGTAGGC arrested with a G1 DNA content after HU treatment and mik1-FW GGGATTATTGCAGGTCATGG progressed through S phase with similar kinetics after release mik1-RV ATCAACCATCGAGGAGACCGG (completing replication after 60 min) (Figure 1A). A small ace2-FW GAATTCCTCCGGAGACAATG ace2-RV AAGTCACAGCGATACGGACG shoulder peak in the arrested htb1-K119R population ams2-FW (antisense) GAGCCTTTATCCGAAATTGGG (t = 0) likely represents septated cells with two G1 nuclei, ams2-RV (sense) GCAAACAGGCAGAGTTGGC which are observed with increased frequency in this mutant act1ChIP5 CCACTATGTATCCCGGTATTGC (Tanny et al. 2007). We examined expression of cell-cycle- act1ChIP6 CAATCTTGACCTTCATGGAGCT regulated genes by RT-qPCR; the constitutive act1+ gene was used as a control. In wild-type cells, expression of the G1/S + + + software as described (Knutsen et al. 2011), and were im- markers cdc18 , cdc22 , and mik1 were high in HU (rela- + ported into R for visualization. tive to act1 ), decreased upon release as cells entered S phase, and began to rise again 80 min postrelease. Entry into Immunoblotting mitosis and the subsequent G1 phase began at 80 min, as + Whole-cell extracts were prepared with trichloroacetic acid indicated by strong induction of the mitotic gene ace2 (Fig- (TCA) as described (Sansô et al. 2012). SDS-PAGE and im- ure 1B). The htb1-K119R mutation did not affect expression munoblotting were carried out as described (Sansô et al. of G1/S marker genes after release and through the comple- 2012). Antibodies used were monoclonal anti-Myc (clone tion of S phase. We observed a pronounced reduction in ex- + + 9E10, 05-419; Millipore) and TAT1 monoclonal antibody pression of ace2 (as well as a smaller decrease for cdc22 )at against tubulin (a gift from K. Gull). Band intensities were later time points. This is likely a result of delayed and de- quantified using ImageJ software. fective entry into mitosis, consistent with previously de- scribed phenotypes in this mutant (Figure 1B) (Tanny et al. Microscopy 2007; Sansô et al. 2012). Chromosome segregation defects were scored by 49,6-diami- We previously found synthetic lethal interactions between dino-2-phenylindole (DAPI) staining and tubulin immunoflu- the htb1-K119R mutation and deletion of hip1+ or slm9+— orescence as described (Sadeghi et al. 2014). At least genes that encode chromatin assembly factors that comprise 50 anaphase cells were scored for each strain in each exper- the HIRA complex and that regulate expression of histone iment. Septation defects were scored by staining with DAPI genes (Blackwell et al. 2004; Tanny et al. 2007; Kurat et al. and calcofluor as described (Sansô et al. 2012; Mbogning 2014). We thus examined whether htb1-K119R was also in- et al. 2013); .100 cells were scored for each strain in each volved in histone gene regulation. Histones H3 and H4 are experiment. each expressed from three nearly identical genes in S. pombe (hht1+/hhf1+, hht2+/hhf2+, and hht3+/hhf3+), whereas Chromatin immunoprecipitation histone H2A is expressed from two genes (hta1+ and Chromatin immunoprecipitation (ChIP) for methylated his- hta2+), and a single gene encodes histone H2B (htb1+). tone H3 lysine 9 was carried out as described (Sadeghi et al., These genes show similar cell-cycle-dependent expression 2014). profiles with peaks coincident with DNA replication, with the exception of hht2+ and hhf2+ (which show no cell-cycle Data availability oscillation) (Takayama and Takahashi 2007). We performed Strains and plasmids are available upon request. The authors RT-qPCR with primer pairs that amplify the coding regions of affirm that all data necessary for confirming the conclusions of each of the four core histone genes and thus detect all gene the article are present within the article, figures, and tables. copies for each. In wild-type cells, histone gene expression Supplemental figures available at FigShare. RNA-seq data are peaked as cells progressed through S phase, and begin to rise

H2B Ubiquitylation Regulates Antisense 163 Figure 1 H2Bub1 promotes expression of histone genes in S. pombe cells synchronized with hydroxyurea (HU). (A) Fluorescence-activated cell sorting (FACS) analysis of wild-type (JTB204) and htb1-K119R (JTB86-3) cells blocked with HU and released into fresh media. Samples were removed for analysis at the indicated time points (right). Asynchronously growing cells (AS) mark the position of G2 DNA content. Sorted cells were binned based on PI-A intensity and the percent of total cells in each bin is plotted. (B) Steady-state RNA levels of the indicated genes were quantified by RT-qPCR at the indicated times after release from HU block and normalized to act1+ mRNA levels. For each gene, the wild-type expression level at t = 0 was set to 1. Error bars denote SD; asterisks indicate significant differences from wild type (n = 3, unpaired t-test, * P , 0.05).

164 V. Pagé et al. Figure 2 H2Bub1 preferentially activates the cell cycle-regulated copies of genes encoding histone H3. Steady-state RNA levels of the indicated genes were quantified by RT-qPCR at the indicated times after release from HU block and normalized to act1+ mRNA levels. For each gene, the wild-type ex- pression level at t = 0 was set to 1. Asterisks indicate significant dif- ferences from wild type (n =3, unpaired t-test, * P , 0.05).

again 20 min following the increase in G1/S markers. In the twofold in htb1-K119R and brl2D strains grown asynchro- htb1-K119R cells, histone H2B and H4 mRNA levels were nously (Figure 3, A and B) (Tanny et al. 2007; Zofall and decreased by twofold in S phase cells, whereas histone Grewal 2007). H2A and H3 mRNA levels were not significantly changed As part of a project to profile gene expression in the absence (Figure 1B). Consistent with defective mitotic entry, tran- of H2Bub1 or Cdk9 activity [M.S., V.P., J.C.T., and R.P.F., scripts for hta, hht, and hhf failed to rise in htb1-K119R cells unpublished results (Sansô et al. 2017)], we performed after completion of S phase. Decreases in mRNA levels for strand-specific RNA-seq analysis on asynchronously growing histones H2B, H3, and H4 were also observed in htb1-K119R wild-type and htb1-K119R cells (raw data available from and brl2D (lacking an E3 ligase essential for H2Bub1) cells NCBI BioProject PRJNA382240). The htb1-K119R mutation grown asynchronously, indicating that these effects are not caused $twofold changes in the levels of 179 protein-coding artifacts of HU treatment (Supplemental Material, Figure transcripts (80 down, 99 up) and $twofold increases in S1). Thus, H2Bub1 is a positive regulator of histone gene 538 antisense transcripts, indicating a major effect of expression during S phase in fission yeast. H2Bub1 on antisense suppression (M.S., V.P., J.C.T., and Since the hht2+ and hhf2+ genes are not cell-cycle-regulated, R.P.F., unpublished results). The ams2+ gene was among we tested whether H2Bub1 differentially affects individual those with increased antisense transcript levels in the htb1- histone gene copies. We performed RT-qPCR using primer K119R strain. Increased antisense signal was detected across pairs that amplify the 39-untranslated regions, which the entire ams2+ transcription unit in htb1-K119R cells, in- allowed us to distinguish between hht1+ and hht2+.Expres- cluding a region spanning the transcription start site, sion of hht1+ in wild-type cells peaked in S phase as expected whereas sense signals were similar in wild-type and mutant in the HU time course, whereas hht2+ expression levels did not cells (Figure S2). To determine whether increased antisense vary (Figure 2). The htb1-K119R mutation caused diminished correlated with reduced levels in Ams2 protein, we quanti- expression of hht1+ throughout the cell cycle, but had no effect fied ams2+ transcripts by strand-specific RT-qPCR in strains on hht2+. This indicates that H2Bub1 specifically activates harboring ams2-myc (Figure 3C; see also Figure S3). Consis- cell-cycle-dependent histone gene expression. tent with the RNA-seq data, htb1-K119R, and brl2D muta- tions enhanced ams2+ antisense transcripts in this H2Bub1 regulates the levels of the histone gene background. We did not observe a difference in sense tran- activator Ams2 script levels between wild-type and strains lacking H2Bub1 The most well-characterized activator of histone gene expres- (Figure 3C). Thus, the absence of H2Bub1 leads to a concom- sion in fission yeast is Ams2—a GATA-typetranscription factor itant increase in transcripts antisense to ams2+, and a de- that is also required for incorporation of the centromere- crease in Ams2 protein expression. specific histone variant CENP-A into centromeric nucleosomes The loss of H2Bub1 was found to increase ams2+ antisense (Chen et al. 2003; Takayama and Takahashi 2007; Takayama levels in the presence of the C-terminal myc tag, which is et al. 2010). Since loss of H2Bub1 is associated with a re- inserted along with a kanMX6 marker gene 39 of ams2+ duction in histone gene expression (Figure 1), as well as (Kim et al. 2016). This argues that sequences in the ams2+ impaired centromeric chromatin assembly and increased lag- 39UTR are not necessary for antisense production, and that ging in anaphase (Sadeghi et al. 2014), we the transcripts originate from within the ams2+ coding surmised that H2Bub1 may promote the function of Ams2. region. The specific effect of htb1-K119R on expression of the cell- To investigate whether interplay between sense and cycle-regulated hht1+ transcript is also consistent with the antisense transcription might be a feature of ams2+ reg- known function of Ams2 in histone gene regulation ulation in wild-type cells, we performed strand-specific (Takayama and Takahashi 2007). Indeed, we found that lev- RT-qPCR on samples derived from cells synchronized with els of myc-tagged Ams2 protein were reduced by around HU. Previous gene expression analyses (performed without

H2B Ubiquitylation Regulates Antisense 165 Figure 3 H2Bub1 promotes expression of the histone gene activator Ams2 and suppresses its antisense transcript. (A) Immunoblotting of whole-cell extracts from the indicated ams2-myc strains (JTB854, JTB913, JTB902-5, respectively) with the indicated antibodies. Two isolates of JTB913 (htb1- K119R) are shown; isolate #1 was used in subsequent experiments. (B) Quantification of the experiment in (A) using ImageJ software. Intensities for the myc immunoblot were normalized to tubulin; wild-type values were set to 1. Error bars indicate SD; asterisks indicate significant differences from wild type (n = 3, unpaired t-test, * P , 0.05). (C) Steady-state ams2-myc RNA levels (sense or antisense) were quantified by strand-specific RT-qPCR in the indicated ams2-myc strains and normalized to act1+ mRNA levels. Error bars indicate SD; asterisks indicate significant differences from wild-type (n =3, unpaired t-test, * P , 0.05). (D) Steady-state ams2+ RNA levels (sense or antisense) were quantified by strand-specific RT-qPCR at the indicated times after HU block and release and normalized to act1+ mRNA levels. The wild-type expression level at t = 0 was set to 1. Error bars denote SD; asterisks indicate significant differences from wild type (n = 3, unpaired t-test, * P , 0.05). strand-specificity) indicated that ams2+ expression is cell- and brl2D (Sansô et al. 2012). It remains unclear how this cycle regulated, peaking in G1/S prior to histone gene activa- opposition operates at the level of expression of individual tion (Takayama and Takahashi 2007). Consistent with these genes. We asked whether the effects of htb1-K119R on ams2+ results, we found that ams2+ sense transcripts peaked in G1/S and histone genes might be opposed by Cdk9 activity. We in both wild-type and htb1-K119R, and diminished as cells used an analog-sensitive allele of cdk9+ (cdk9as), which completed DNA replication. Sense ams2+ transcripts were de- allowed us to specifically inhibit Cdk9 activity using the bulky creased twofold in htb1-K119R relative to wild type in S ATP-analog 3-MB-PP1 (Viladevall et al. 2009; Mbogning et al. phase and in the subsequent mitosis (Figure 3D). In contrast, 2013). Treatment of the cdk9as strain (grown asynchro- antisense ams2+ transcripts were found at constitutively low nously) with 3-MB-PP1 had little effect on either the sense levels in wild-type cells, and were elevated 5- to 10-fold in the or antisense ams2+ transcripts relative to DMSO controls. As htb1-K119R mutant throughout the cell cycle (Figure 3D). expected, ams2+ antisense transcripts were elevated by Thus, antisense transcription does not regulate ams2+ during fivefold in the DMSO-treated cdk9as htb1-K119R strain the cell cycle in wild-type cells, and the effects of H2Bub1 loss compared to cdk9as, but 3-MB-PP1 treatment reversed this on ams2+ antisense levels are not specific to a particular cell increase with little effect on the levels of the sense transcript cycle stage. (Figure 4A). To determine if this effect correlated with the levels of Ams2 protein, we performed a time-course of Cdk9 Interplay between H2Bub1 and Cdk9 regulates ams2+ inhibition in the ams2-myc strain background and monitored antisense and Ams2 protein levels protein levels by immunoblot. Upon 3-MB-PP1 treatment of To determine the functional significance of H2Bub1-mediated the cdk9as htb1-K119R strain, we observed a time-dependent suppression of the ams2+ antisense transcript, we examined increase in Ams2 protein levels relative to the tubulin loading its relationship to the previously described opposition be- control (Figure 4, B and C). Ams2 protein levels were not tween H2Bub1 and Cdk9, in which lowering activity of affected in the DMSO-treated control. In addition, Ams2 lev- Cdk9 suppressed the mitotic defects caused by htb1-K119R els were not significantly affected by 3-MB-PP1 treatment in

166 V. Pagé et al. Figure 4 Increase in ams2+ antisense in htb1-K119R cells is reversed by inhibition of Cdk9. (A) Steady-state ams2+ RNA levels (sense or antisense) were quantified by strand-specific RT-qPCR in the indicated strains (JTB425, JTB508, respectively) after a 2-hr treatment with DMSO (2) or 3-MB-PP1 (+). Values were normalized to act1+ mRNA levels. Error bars denote SD; asterisks indicate significant differences between DMSO and 3-MB-PP1-treated samples (n = 3, unpaired t-test, * P , 0.05). (B) Immunoblots on whole-cell extracts from the indicated ams2-myc strains (JTB914, JTB916, respectively) grown in presence of 3-MB-PP1 (+) or DMSO (2) for the indicated times. Antibodies are indicated on the right. (C) Quantification of the experiment in (B) using Image J software. Intensities for the myc immunoblot were normalized to tubulin. Error bars indicate SD; asterisk indicates significant difference between 1 and 4 hr treatment times (n = 3, unpaired t-test, * P , 0.05). (D) Steady-state mRNA levels for the indicated histone genes were quantified by RT-qPCR in the indicated strains (JTB425, JTB508, respectively) after a 2-hr treatment with DMSO (2) or 3-MB-PP1 (+). Values were normalized to act1+ and those for the cdk9as, DMSO condition were set to 1. Error bars denote SD; asterisks indicate significant differences between DMSO and 3-MB-PP1- treated samples for each strain (n = 3, unpaired t-test, * P , 0.05). the cdk9as strain. This suggests that H2Bub1 regulates Ams2 rates under all of the conditions examined, arguing that nei- levels through suppression of the ams2+ antisense transcript, ther H2Bub1 nor Cdk9 activity affect Ams2 protein stability and that elevated ams2+ antisense upon H2Bub1 loss de- (Figure S4B). pends upon Cdk9 activity. We further asked if removal of ams2+ antisense expression Transcriptional and post-transcriptional mechanisms en- through inhibition of Cdk9 could increase histone gene ex- sure that Ams2 protein levels are restricted to the S phase of pression in the htb1-K119R strain. We assessed expression of the cell cycle. In addition to cell cycle-regulated transcription histone genes by RT-qPCR (without strand specificity) in of ams2+, Ams2 protein is a substrate for ubiquitylation by cdk9as and cdk9as htb1-K119R strains. Treatment of cdk9as both the SCF and APC ubiquitin ligase complexes, leading to with 3-MB-PP1 had no effect or caused a slight decrease in its proteolysis in G1 and G2/M (Takayama et al. 2010; histone gene expression relative to the DMSO control. In Trickey et al. 2013). It is thus possible that cell cycle pertur- contrast, similar treatment of the cdk9as htb1-K119R strain bations caused by htb1-K119R or Cdk9 inhibition could result caused increased expression of histone genes, consistent with in altered Ams2 protein stability that could account for the increased Ams2 levels caused by reduction of the ams2+ an- differences in protein levels that we observe. To test this tisense transcript (Figure 4D). possibility, we treated cdk9as ams2-myc or cdk9as htb1- In addition to activating expression of histone genes, Ams2 K119R ams2-myc cells with either DMSO or 3-MB-PP1 for promotes incorporation of the centromere-specific histone 2 hr, and then blocked protein synthesis with cycloheximide. variant CENP-A into centromeric chromatin to allow accurate Samples were removed at various times after cycloheximide mitotic chromosome segregation (Chen et al. 2003). Mutants addition and analyzed by immunoblotting to assess Ams2 lacking H2Bub1 also show mitotic chromosome segrega- protein levels (Figure S4A). Ams2 levels declined at similar tion defects thought to be caused by a decrement in

H2B Ubiquitylation Regulates Antisense 167 Figure 5 Partial suppression of htb1-K119R chromosome segregation phenotypes by reduction in Cdk9 activity. (A) Fivefold serial dilutions of the indicated strains (JTB362, JTB98, JTB321, JTB429, respectively) were spotted on the indicated media and grown for 2–3 days at 30°. (B) Frequency of anaphase chromosome segregation defects was quantified by fluorescence microscopy in the indicated strains (see Materials and Methods). Error bars denote SD (n = 3). At least 50 anaphase cells were counted in each experiment. transcription-coupled CENP-A incorporation and an increase sion of HA-Ams2 in both strains, although we consistently in repressive heterochromatin within the core of the centro- observed lower levels of expression in htb1-K119R cells (Fig- mere (Sadeghi et al. 2014). To test if the decreased levels of ure S6A). In control cells harboring empty vector, RT-qPCR Ams2 may contribute to these defects we assayed htb1- analysis revealed the expected decreases in expression of K119R and cdk9-T212A htb1-K119R double mutants for htb1+, hhf, and hht1+ transcripts associated with the htb1- growth in the presence of thiabendazole (TBZ), a spindle K119R mutation. These defects were fully rescued in Ams2- poison to which mutants with chromosome segregation de- overexpressing cells (Figure S6B). Consistent with the HA fects are particularly sensitive. The cdk9-T212A mutation immunoblots, less histone gene expression was observed in eliminates the site in the T-loop and reduces the Ams2-overexpressing htb1-K119R cells than in the wild Cdk9 activity roughly 10-fold (Pei et al. 2006; Sansô et al. type. We scored septation phenotypes by fluorescence mi- 2012). As was the case with cdk9as, this allele increased ex- croscopy after staining with DAPI and calcofluor (to stain pression of histone genes in htb1-K119R cells (Figure S1). the division septum), and found that Ams2 overexpression Whereas the htb1-K119R mutation alone caused sensitivity did not ameliorate the abnormal septation observed in htb1- to TBZ, consistent with published results, this phenotype was K119R cells, despite the normalized expression of histone rescued in the cdk9-T212A htb1-K119R double mutant, con- genes (Figure S6C). Thus, effects on Ams2 levels are not sistent with improved centromere function (Figure 5A). To sufficient to account for phenotypes caused by loss of assess centromere function more directly, we determined the H2Bub1 or their reversal by Cdk9 inhibition. frequency of lagging chromosomes in anaphase cells using Subset of H2Bub1-regulated antisense transcripts fluorescence microscopy. The htb1-K119R mutation caused are Cdk9-dependent increased frequency of lagging chromosomes in both wild- type and cdk9-T212A backgrounds, although the increase The strand-specific RNA-seq analysis showed that the vast was smaller in cdk9-T212A cells (Figure 5B). This suggests majority of antisense transcripts derepressed in the htb1- partial suppression of the centromere defect caused by htb1- K119R mutant were in fact further derepressed by Cdk9 in- K119R. The lagging chromosome phenotype of htb1-K119R hibition [M.S., V.P., J.C.T., and R.P.F., unpublished results; cells also correlates with an increase in histone H3 lysine (Sansô et al. 2017)]. However, prompted by our findings with 9 methylation (H3K9me, a mark of heterochromatin) within ams2+, we analyzed the RNA-seq data to identify other ex- the central core of the centromere (Sadeghi et al. 2014). To amples of antisense transcripts that were increased by the determine if this effect could be modulated by Cdk9, we htb1-K119R mutation relative to wild type, but for which this performed ChIP analysis. The htb1-K119R and cdk9-T212A effect was partially or completely negated by simultaneous single mutations, as well as both mutations combined, caused inhibition of Cdk9. These were selected based on two criteria: increased H3K9me at the central core, indicating that loss of antisense transcripts increased (by .2-fold over wild type) in H2Bub1 can also alter centromere function independently of htb1-K119R, but not in 3-MB-PP1-treated cdk9as htb1-K119R Cdk9 (Figure S5). cells, or those similarly increased in DMSO-treated cdk9as To determine whether reduced levels of Ams2 is important htb1-K119R cells, but not in 3-MB-PP1-treated cdk9as htb1- for other mitotic defects caused by H2Bub1 loss, we intro- K119R cells. We identified transcripts antisense to 37 pro- duced plasmids expressing HA-tagged Ams2 from the nmt1+ tein-coding genes meeting these criteria. We did not identify promoter (which drives constitutive expression in media any significant enrichment of (GO) terms in lacking thiamine) into either wild-type or htb1-K119R cells this list. However, among this small group are ams2+, mis14+ (Takayama et al. 2016). Immunoblotting confirmed expres- (encoding an essential kinetochore protein), pcf3+ (encoding

168 V. Pagé et al. Table 3 K119R UP, Cdk9-dependent antisense transcripts identified by strand-specific RNA-seq Systematic ID Name Product description

SPCC290.04 ams2 Cell cycle regulated GATA-type transcription factor Ams2 SPCC338.08 ctp1 CtIP-related endonuclease SPAC7D4.09c dfg10 3-Oxo-5-alpha-steroid 4-dehydrogenase (predicted) SPAC30C2.06c dml1 Mitochondrial inheritance GTPase, tubulin-like (predicted) SPAC19B12.05c fcp1 CTD phosphatase Fcp1 SPAC140.02 gar2 Nucleolar protein required for rRNA processing SPBC32F12.16 gem7 GEMIN7 ortholog SPCC645.02 gep4 Phosphatidylglycerol phosphate phosphatase Gep4 (predicted) SPAC144.07c gpn2 Conserved GTPase Gpn2 (predicted) SPAC1834.03c hhf1 Histone H4 h4.1 SPCC622.09 htb1 Histone H2B Htb1 SPBC3D6.04c mad1 Mitotic spindle checkpoint protein Mad1 SPAC31G5.12c maf1 Repressor of RNA polymerase III Maf1 SPAC688.02c mis14 NMS complex subunit Mis14/Nsl1 SPCP1E11.03 mug170 Arrestin family Schizosaccharomyces specific protein Mug170 SPBC32H8.06 mug93 TPR repeat protein, meiotically spliced SPAC25H1.06 pcf3 CAF assembly factor (CAF-1) complex subunit C, Pcf3 SPAC3C7.10 pex13 Peroxin 13 (predicted) SPCC24B10.22 pog1 Mitochondrial DNA polymerase gamma Pog1 SPAC4D7.03 pop2 F-box/WD repeat protein Pop2 SPAC31G5.15 psd3 Phosphatidylserine decarboxylase Psd3 SPAC1782.08c rex3 Exonuclease Rex3 (predicted) SPAC29A4.11 rga3 RhoGAP, GTPase activating protein Rga3 SPBP8B7.02 rng9 Contractile ring myosin V regulator Rng9 SPAC1D4.09c rtf2 Replication termination factor Rtf2 SPAC18G6.04c shm2 hydroxymethyltransferase Shm2 (predicted) SPBC2D10.09 snr1 3-Hydroxyisobutyryl-CoA hydrolase snr1 SPBC1734.05c spf31 DNAJ protein, splicing factor Spf31 (predicted) SPAC1F7.14c tam6 Mitochondrial DUF4536, human DMAC1 ortholog, possibly has a general role in mitochondrial complex assembly SPAC15A10.12c tca17 TRAPP complex subunit 2-like Tca17 (predicted) SPCC1442.09 trp3 Anthranilate synthase component I Trp3 SPAC13G6.09 trs402 SSU-rRNA maturation protein Tsr4 homolog 2 Tsr402 (predicted) SPCC162.06c vps60 Vacuolar sorting protein Vps60 (predicted) SPAC29A4.06c Splicing protein, human NSRP1 ortholog SPAC977.17 MIP water channel (predicted) SPAC9G1.07 Schizosaccharomyces specific protein SPBC776.03 Homoserine dehydrogenase (predicted) a subunit of the CAF-I chromatin assembly complex), and V.P., J.C.T., and R.P.F., unpublished results). Thus, our genes encoding histones H2B and H4, suggesting that im- analyses point to aberrant transcriptional events, sensitive proper regulation of sense:antisense balance at these genes to Cdk9 inhibition, that mediate a subset of gene expression could contribute to the cell cycle defects in htb1-K119R mu- defects in the absence of H2Bub1. tants (Table 3). Comparison with published NET-seq data (Wery et al. Discussion 2018) indicates that this subset of genes experiences excep- tionally high levels of overlapping antisense transcription in We have shown that H2Bub1 regulates histone genes indi- wild-type cells, with 9/37 having 80–100% overlap and 30/37 rectly through an antisense-based mechanism in fission yeast. having an overlap of at least 40% (Figure 6). The distribu- A role for H2Bub1 in antisense suppression is consistent with tion of these genes among gene groups with high levels of its close connection to transcription elongation and with overlapping antisense transcription is significantly different previous studies demonstrating its role in transcription- from that of protein-coding genes in general (P , 0.0001; Chi coupled chromatin assembly (Batta et al. 2011). H2Bub1 squared test). A second notable property of this group of is thought to influence the chromatin assembly function of genes is the fact that 32/37 are oriented convergently with the FACT complex, although the details of this mechanism respect to a neighboring gene, suggesting that the high levels and how it operates at individual genes have not been of overlapping antisense transcription at these locations is elucidated (Nune et al. 2019). Here, we demonstrate the due to transcriptional readthrough. Only two of the genes impact of faulty antisense suppression at a specificgene exhibit a corresponding decrease in sense transcript levels that partially accounts for the cell cycle-related gene ex- in the htb1-K119R mutant as measured by RNA-seq (M.S., pression defects in H2Bub1 mutants.

H2B Ubiquitylation Regulates Antisense 169 Figure 6 High levels of overlapping sense:antisense transcription at genes with H2Bub1-suppressed, Cdk9-dependent antisense transcripts. All sense:anti- sense gene pairs (3455 in total) or the gene group in question (37 genes) sorted according to level of over- lapping transcription as assessed by NET-seq. Distribu- tions were found to differ significantly (P , 0.0001; Chi-squared test).

Previous studies in the budding yeast Saccharomyces cer- the relationship between H2Bub1 and Ams2 expression we evisiae pointed to a direct role for Rad6/Bre1-dependent observe, as the RNA-seq data indicated increased antisense H2Bub1 as a coactivator for the SBF transcription factor that overlapping the ams2+ transcription start site in htb1-K119R. drives the G1/S transition (Zimmermann et al. 2011). A coac- Cdk9 dependence of some of the H2Bub1-induced anti- tivator role is also supported by findings in human cells, in sense transcripts is consistent with the opposing phenotypic which the Bre1 ortholog RNF20 has been shown to enhance relationship between Cdk9 and H2Bub1 that we have pre- transactivation by p53 and the androgen receptor (Kim et al. viously described, and partially accounts for suppression of 2005; Jaaskelainen et al. 2012). Interestingly, there is evi- H2Bub1 chromosome segregation phenotypes by Cdk9 loss of dence that H2Bub1 regulates mammalian histone gene ex- function. Our previous results show that Cdk9 inhibition also pression post-transcriptionally through noncanonical mRNA suppresses cell separation defects in htb1-K119R mutants, 39-end processing (Pirngruber et al. 2009). The data pre- and normalizes an aberrant intragenic distribution of RNA sented here argue that antisense suppression is another im- polymerase II observed in genome-wide chromatin immuno- portant, albeit indirect, mechanism employed by H2Bub1 to precipitation experiments (Sansô et al. 2012). The data we regulate gene expression during the cell cycle. report here, along with prior results, are consistent with a The ams2+ antisense transcripts induced upon loss of model based on the concept of aberrant, Cdk9-dependent H2Bub1 may affect ams2+ transcription or post-transcriptional transcriptional events as drivers of phenotypic effects caused steps in Ams2 expression. In asynchronous cells, H2Bub1 by loss of H2Bub1. Reduction of Cdk9 activity would reduce loss had no effect on levels of ams2+ sense transcripts, the detrimental effects of these aberrant events in the con- whereas htb1-K119R cells released from a HU block showed text of a global decrease in the rate of RNA polymerase II decreased ams2+ sense transcript levels. This discrepancy elongation (Parua et al. 2018). However, these phenotypes may be due to an effect of the htb1-K119R mutation on are unlikely to be fully accounted for by the small group of recovery from the HU block, or may reflect differences in H2Bub1-suppressed, Cdk9-dependent antisense transcripts ams2+ transcript stability between the growth conditions in we identified here, and opposing effects are not apparent for these experiments. In either case, determining the mecha- the vast majority of antisense transcripts increased by either nism for the effects of ams2+ antisense at this locus will re- loss of H2Bub1 or reduction in Cdk9 activity (M.S., V.P., quire direct, strand-specific measurement of transcription. J.C.T., and R.P.F., unpublished results). It is possible that The fact that the antisense transcript is Cdk9-dependent sug- additional opposing effects of H2Bub1 and Cdk9 on gene gests that aberrant RNAPII elongation is involved in mediat- expression would be revealed in global run-on or NET-seq ing the effect on ams2+ expression. Our observation that the experiments carried out at various stages of the cell cycle. H2Bub1-suppressed, Cdk9-dependent antisense transcripts Alternatively, aberrant transcription caused by loss of identified by RNA-seq also experience high levels of overlap- H2Bub1 may interfere with other cell cycle-regulated events ping antisense transcription (as determined by NET-seq) also that occur on chromatin, such as binding of condensin and supports this model. Global analyses of nascent transcription cohesin complexes (Schmidt et al. 2009; Kim et al. 2016). indicate that sense and antisense transcription at a given Given that links between H2Bub1 and cell cycle regulation locus are generally anticorrelated, thus increased elongation are highly conserved in evolution, and are implicated in pro- in the antisense direction would be predicted to have a cor- liferation of a variety of , it will be of interest to responding negative effect on sense elongation (Pelechano determine the extent to which aberrant transcription contrib- and Steinmetz 2013; Mayer et al. 2015; McDaniel et al. 2017; utes to the effects of H2Bub1 loss in mammalian cells (Marsh Wery et al. 2018). This relationship is particularly strong and Dickson 2019). Our results suggest that inhibition of when the antisense transcription overlaps the sense pro- CDK9, already under investigation as a potential therapeutic moter, a condition that correlates with decreased protein ex- strategy in (Olson et al. 2018), could have particular pression (Huber et al. 2016). Such a correlation may explain benefit in cancers that exhibit these defects.

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