H3K4 demethylation is negatively regulated by in Saccharomyces cerevisiae

Vicki E. Maltbya, Benjamin J. E. Martina, Julie Brind’Amourb,1, Adam T. Chruscickia,1, Kristina L. McBurneya, Julia M. Schulzec, Ian J. Johnsona, Mark Hillsd, Thomas Hentrichc, Michael S. Koborc, Matthew C. Lorinczb, and LeAnn J. Howea,2

Departments of aBiochemistry and Molecular Biology and bMedical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada V6T 1Z3; cCenter for Molecular Medicine and Therapeutics, Child and Family Research Institute, Vancouver, BC, Canada V5Z 4H4; and dTerry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada V5Z 1L3

Edited by Kevin Struhl, Harvard Medical School, Boston, MA, and approved September 12, 2012 (received for review February 6, 2012) Histone H3 4 trimethylation (H3K4me3) is a hallmark of of lysine-specific HDMs have been identified: amine oxidases, transcription initiation, but how H3K4me3 is demethylated during such as LSD1, and the JmjC (Jumonji C) domain–containing gene repression is poorly understood. Jhd2, a JmjC domain protein, demethylases. This latter class of demethylases can be split fur- was recently identified as the major H3K4me3 histone demethylase ther into several subfamilies, including the evolutionarily con- (HDM) in Saccharomyces cerevisiae. Although JHD2 is required for served JARID1 family of demethylases, which is characterized JHD2 removal of upon gene repression, deletion of does not only by a JmjC domain but also by JmjN, AT-rich interactive, not result in increased levels of H3K4me3 in bulk , indicating C5HC2 zinc finger, and PHD finger domains (6). In the yeast S. that this HDM is unable to demethylate histones during steady-state cerevisiae, the lone member of the JARID1 family of demethy- conditions. In this study, we showed that this was due to the nega- fi tive regulation of Jhd2 activity by histone H3 lysine 14 acetylation lases that has been identi ed is Jhd2. Previous studies have identified Jhd2 as a demethylase capable (H3K14ac), which colocalizes with H3K4me3 across the yeast ge- – nome. We demonstrated that loss of the histone H3-specificacetyl- of demethylating H3K4 in vivo (7 9) and in vitro (10). Although fi transferases (HATs) resulted in genome-wide depletion of H3K4me3, several studies have focused on the identi cation and substrate and this was not due to a transcription defect. Moreover, H3K4me3 specificity of Jhd2, little is known about how it is targeted to levels were reestablished in HAT mutants following loss of JHD2, specific regions of the genome or how its enzymatic activity is which suggested that H3-specific HATs and Jhd2 serve opposing regulated. Repression of the GAL1, SUC2, and INO1 genes is functions in regulating H3K4me3 levels. We revealed the molecular accompanied by loss of H3K4me3, which is dependent on JHD2 basis for this suppression by demonstrating that H3K14ac negatively (10, 11). Additionally, overexpression of JHD2 results in global regulated Jhd2 demethylase activity on an acetylated peptide in loss of H3K4me3 (7–9), however deletion of JHD2 does not vitro. These results revealed the existence of a general mechanism result in a major increase of H3K4me3 in bulk histones, in- for removal of H3K4me3 following gene repression. dicating that under steady-state conditions, Jhd2 is unable to demethylate the majority of H3K4me3 in the cell (9, 10). Gcn5 | histone acetylation | histone methylation | Sas3 Genome-wide analysis of histone PTMs in yeast showed that in addition to H3K4me3, the 5′ ends of active genes contain elevated ukaryotic genomes are packaged as by both histone levels of histone H3 acetylation (5, 12, 13). In yeast, histone H3 is Eand nonhistone proteins. Chromatin plays an important role primarily acetylated by two histone acetyltransferases (HATs), Gcn5 in the regulation of numerous cellular processes, including tran- and Sas3, as part of multiprotein complexes (14, 15). The colocali- scription, DNA replication, and repair. The basic unit of chro- zation of H3K4me3 and histone H3 acetylation suggests that there matin is the core particle, which comprises two copies may be “cross-talk” between these marks, whereby one PTM influ- each of histones H2A, H2B, H3, and H4, around which is wrapped ences the establishment or maintenance of another (16). Consistent 147 base pairs of DNA (1). The amino terminal tails of histones with this, mutation of histone H3 lysine 14 (H3K14) results in a loss are exposed on the surface of the nucleosome and serve as the of H3K4me3 in bulk histones (17), however the molecular basis for main sites for histone posttranslational modifications (PTMs). PTMs, such as acetylation and methylation, have been proposed this cross-talk is unknown. In this study, we demonstrated that this to act as docking sites for various chromatin-modifying complexes relationship was due to the negative regulation of Jhd2 activity by and thus play a role in regulating chromatin structure (2). histone H3 lysine 14 acetylation (H3K14ac). These results revealed Histone H3 lysine 4 trimethylation (H3K4me3) is one of the the basis for the genome-wide colocalization of H3K14ac and most commonly used hallmarks of transcriptional activity. In H3K4me3, and explained why H3K4me3 is susceptible to deme- Saccharomyces cerevisiae, mono-, di-, and trimethylation of H3K4 thylation only after gene inactivation. is catalyzed by the histone methyltransferase (HMT) COMPASS (complex proteins associated with Set1), with Set1 as the catalytic subunit (3). Several auxiliary proteins within COMPASS are re- Author contributions: V.E.M., B.J.E.M., and L.J.H. designed research; V.E.M., B.J.E.M., J.B., quired for Set1 to catalyze the transition from mono- to di- and A.T.C., and K.L.M. performed research; I.J.J. contributed new reagents/analytic tools; V.E.M.,

B.J.E.M., J.B., A.T.C., J.M.S., M.H., T.H., M.S.K., and M.C.L. analyzed data; and V.E.M. and L.J.H. GENETICS from di- to trimethylation, suggesting that the diverse states of wrote the paper. methylation are differentially regulated (4). Although H3K4me3 is The authors declare no conflict of interest. ′ primarily found at the 5 region of transcriptionally active genes, di- This article is a PNAS Direct Submission. and monomethylation are found in the mid- and 3′ regions of Data deposition: The ChIP-seq data reported in this paper have been deposited in the transcribed genes, respectively (5). These patterns of methylation Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. are thought to be a consequence of the interaction of COMPASS GSE41424). with the elongating form of RNA polymerase II (3). 1J.B. and A.T.C. contributed equally to this work. Although H3K4me3 was once thought of as a stable histone 2To whom correspondence should be addressed. E-mail: [email protected]. PTM, the dynamic nature of lysine methylation became apparent This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with the discovery of histone demethylases (HDMs). Two classes 1073/pnas.1202070109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1202070109 PNAS | November 6, 2012 | vol. 109 | no. 45 | 18505–18510 Downloaded by guest on September 23, 2021 Results increase and 306 genes (5.4%) having a greater than twofold de- H3K4me3 Was Dependent on Acetylation of the Histone H3 Tail. crease in transcript levels in the mutant. Importantly, none of the Studies examining the genome-wide localization of histone PTMs genes required for regulation of H3K4me3 (i.e., those encoding in S. cerevisiae, Drosophila, and human cells showed that levels of Jhd2, components of COMPASS, the PAF complex, or compo- H3K4me3 positively correlate with that of histone H3 acetyla- nents of the H2B ubiquitylation pathway) were found to be mis- tion, suggesting that there may be cross-talk between these mod- regulated, and the protein levels of Set1 and Jhd2 were not affected ifications (5, 12, 18, 19). To test whether H3K4me3 was dependent (Fig. S3B). Thus, although histone H3 acetylation is a generally on H3 acetylation, we used immunoblot analysis of S. cerevisiae ubiquitous mark of transcribed genes, it was only important for whole cell extracts to examine levels of H3K4me3 in a mutant expression of a subset of genes under the growth conditions used. strain lacking the HATs responsible for histone H3 acetylation. These results were consistent with a recent study (21) that dem- In previously published work, we demonstrated that concomitant onstrated a similar phenomenon for many histone PTMs. deletion of ADA2 and SAS3 abolishes the majority of histone H3 To confirm that genome-wide levels of H3K4me3 were de- acetylation, without disrupting acetylation of the nonhistone pendent on histone H3 acetylation, protein–DNA complexes con- substrate Rsc4 (20). ADA2 encodes a noncatalytic component of taining H3K4me3 were immunoprecipitated from wild-type and all Gcn5-dependent HATs, whereas SAS3 codes for the catalytic ada2Δ sas3Δ strains, and the coprecipitating DNA subjected to subunit of the NuA3 HAT complex. It should be noted that there Illumina-based sequencing. Read counts were plotted around the was still residual H3 acetylation in the ada2Δ sas3Δ double transcription start sites (TSSs) of 4,637 genes (22). To correct for the mutant (Fig. S1A), but this was not due to Elp3, a putative H3- global change in H3K4me3, immunoblot data were used to nor- specific HAT, or ADA2-independent Gcn5 HAT activity, as malize the total read count as described (13). A cumulative count demonstrated in Fig. S1 B and C. The histone H3 hypoacetylation plot revealed that, on average, genes showed a loss of H3K4me3 in an ada2Δ sas3Δ strain allowed us to test the requirement of following disruption of the H3-specificHATs(Fig.1E), which was H3 acetylation for H3K4me3. When we measured the levels of confirmed by chromatin immunoprecipitation (ChIP)–quantitative H3K4me3 in an ada2Δ sas3Δ double mutant, we found that this polymerase chain reaction (qPCR) at specificloci(Fig. S4A). strain exhibited levels of methylation that were 41% of wild type (Fig. 1 A and B), suggesting that histone H3 acetylation was re- Loss of Histone H3 Acetylation Promotes H3K4me3 Demethylation by quired for the establishment or maintenance of H3K4me3 levels. Jhd2. The induction of heterochromatin at the HMR locus results Deletion of ADA2 or SAS3 alone did not have as severe of an in a rapid loss of histone acetylation, followed by loss of effect, indicating that these genes played redundant roles in the H3K4me3 with slightly slower kinetics (23). This, when taken maintenance of wild-type levels of H3K4me3 (Fig. 1 A and B). together with our data demonstrating that H3K4me3 levels were In contrast to the loss of H3K4me3, H3K4me2 levels were in- dependent on the H3-specific HATs, supported an intriguing creased in an ada2Δ sas3Δ mutant (Fig. 1 C and D), which was hypothesis that in order for Jhd2 to demethylate H3K4me3, consistent with the fact that the histones that lost H3K4me3 in an histone H3 must be deacetylated. If this was true, then deletion ada2Δ sas3Δ strain were instead dimethylated. Because H3K4me2 of JHD2, the major H3K4me3 demethylase, from an ada2Δ is also associated with transcriptionally active genes (5), these latter sas3Δ strain should result in rescue of H3K4me3 levels. We results indicated that the loss of H3K4me3 seen in the ada2Δ sas3Δ tested this using quantitative immunoblot analysis and found that mutant was not due to a transcription defect. Moreover, genome- when JHD2 was deleted from an ada2Δ sas3Δ mutant, H3K4me3 wide expression profiling showed that despite having significant returned to near wild-type levels (Fig. 2 A and B). To confirm that growth defects (Fig. S2), the ada2Δ sas3Δ mutant exhibited these results were not due to increased H3K4me3 levels at ab- changes in transcript levels at a relatively limited number of genes errant loci, protein–DNA complexes containing H3K4me3 were (Fig. S3A), with 320 genes (5.6%) showing a greater than twofold immunoprecipitated from an ada2Δ sas3Δ jhd2Δ strain, and

H3K4me3 A B 1.2 C H3K14ac H3K4me3 1.0 H3K4me3 H3K14ac 0.8 H3K4me2 0.6 H3 H3K4me1 PTM 0.4 ADA2 H3K4me0 + - + - 0.2 relative to WT H3 SAS3 + + - - 0.0 ADA2 + - + - ADA2 + - SAS3 + + - - SAS3 + - D H3K4me3 H3K4me1 E 120 H3K4me2 H3K4me0 100 WT 2.0 1.8 ada2 sas3 80 1.6 1.4 1.2 60 1.0 0.8 40 0.6

0.4 percent total 0.2 20 relative to WT K4 methylation 0.0 corrected read count ADA2 + - 0 SAS3 + - Distance from TSS (bp)

Fig. 1. Histone H3-specific acetyltransferases were required for H3K4me3. (A and C) Whole cell extracts from the indicated strains were subjected to immunoblot analysis for H3K4me3, H3K4me2, H3K4me1, H3K4me0, H3K14ac, or H3. (B and D) Immunoblots shown in A and C were repeated with three independent cultures and the results quantified after normalization for histone H3. Shown is H3K4me3 (red) or H3K14ac (blue) normalized for histone H3. Error bars indicate mean ± the SE. (E) Corrected cumulative count plot (SeqMonk) of H3K4me3 levels relative to the TSS of 4,637 yeast genes in the indicated strains.

18506 | www.pnas.org/cgi/doi/10.1073/pnas.1202070109 Maltby et al. Downloaded by guest on September 23, 2021 ABH3K4me3 structure. Methylation of histone H3K4 promotes the chromatin 1.2 H3K14ac interaction of multiple factors, including HATs, HDACs, HMTs, 1.0 and HDMs (24). Consequently, H3K4me3 is thought to regulate H3K4me3 0.8 transcription by triggering alterations to chromatin structure. H3 0.6 Because H3K4 methylation and H3 acetylation colocalize, it has PTM always been difficult to tease apart their relative contributions to merge 0.4 transcription. Our data led us to question whether restoration of relative to WT 0.2 JHD2 + - + - H3K4me3 levels could alleviate the effects of histone H3 acet- 0.0 ADA2 + + - - JHD2 + --+ ylation loss. To answer this we identified a set of genes (349) that SAS3 + + - - ADA2 showed reduced transcript abundance (1.5-fold) and reduced + + - - ada2Δ sas3Δ SAS3 - - H3K4me3 levels in the strain and showed rescue of + + H3K4me3 levels upon deletion of JHD2. Upon comparing the 120 levels of transcripts from this set of genes in the ada2Δ sas3Δ and C WT ada2Δ sas3Δ jhd2Δ strains, we found that there was not a sig- 100 ada2Δ sas3Δ nificant change in gene expression. Therefore, H3K4me3 in the jhd2Δ 80 absence of acetylation does not seem to enhance transcription. ada2Δ sas3Δ jhd2Δ 60 This was consistent with the work of others demonstrating that loss of SET1, which encodes the sole H3K4 HMT in yeast, only 40

percent total affects expression of a very limited number (55 in total) of

20 genes (21). corrected read count 0 H3K14ac Negatively Regulates H3K4 Demethylation by Jhd2 in Vivo and in Vitro. The above data demonstrated that ADA2 and SAS3 Distance from TSS (bp) protected the histone H3 tail from demethylation by Jhd2. To confirm that this was indeed due to acetylation of the H3 tail and to Fig. 2. Deletion of JHD2 restored H3K4me3 levels in cells lacking histone H3 identify the residue involved, we performed site-directed muta- acetylation. (A) Whole cell extracts from the indicated strains were subjected to genesis of the acetylatable on the H3 tail. We chose to immunoblot analysis for H3K4me3 and H3. A yellow color represented equal mutate the residues to glutamine as opposed to arginine, as glu- B A red and green intensities. ( )Theimmunoblotin was repeated with three tamine substitutions did not have an impact on cell growth. Al- independent cultures and the results quantified. Shown is H3K4me3 (red) or H3K14ac (blue) normalized for histone H3 and shown relative to wild-type (WT) though mutation of lysines 9, 18, or 23 to glutamine did not alter levels. Error bars indicate mean ± SE. (C) Corrected cumulative count plot of H3K4me3 levels, mutation of H3K14 recapitulated the loss of H3K4me3 levels relative to the TSS of 4,637 yeast genes in the indicated strains. methylation observed in the ada2Δ sas3Δ strain (Fig. 3A). The requirement of H3K14 for maintenance of H3K4me3 levels has been documented by others (17), but the mechanism behind this the coprecipitating DNA sequenced. A cumulative count plot dependency was unknown until now. revealed that the average genome-wide localization of H3K4me3 If H3K4me3 is dependent on H3K14ac, then it was expected in an ada2Δ sas3Δ jhd2Δ strain was similar to that of wild type that these marks colocalize on the genome. To test this, protein– (Fig. 2C), indicating that loss of JHD2 restored H3K4me3 levels DNA complexes containing H3K4me3 or H3K14ac were immu- to regions that showed methylation loss in the ada2Δ sas3Δ noprecipitated, and the coprecipitating DNA used to probe strain. These results were confirmed at specificlocibyChIP– Affymetrix high-resolution tiling microarrays. Enriched regions qPCR (Fig. S4B). To rule out the possibility that the restoration were identified by comparing signal intensities of the ChIP to of H3K4me3 levels upon loss of JHD2 was an indirect effect of input DNA as previously described (25). Genes were binned by suppression of transcription-related defects in an ada2Δ sas3Δ length, and the average enrichment scores for the genes in each mutant, we used genetic analysis to demonstrate that deletion of bin were calculated. The profiles of H3K14ac and H3K4me3 were JHD2 did not rescue ada2Δ sas3Δ phenotypes such as slow highly similar (Fig. 3B), and comparison of the MAT scores for growth, temperature sensitivity, or sensitivity to 6-azauracil (Fig. each probe revealed Pearson and Spearman rank correlation S2). Additionally, we carried out genome-wide expression pro- coefficients of 0.77 and 0.75, respectively, between the two data- filing and confirmed that deletion of JHD2 did not change the sets, indicating that H3K4me3 and H3K14ac significantly colo- transcription profile of an ada2Δ sas3Δ strain (Fig. S5). calized on the genome consistent with the requirement for Although the majority of genes showed restored H3K4me3 levels H3K14ac for maintenance of H3K4me3 levels. in the ada2Δ sas3Δ mutant upon loss of JHD2, a subset of genes did There were two possible explanations for why Jhd2 was unable not. Interestingly, one such gene, PMA1, is commonly used to ex- to demethylate H3K4me3 when H3K14 was acetylated. First, amine levels of H3K4me3 by ChIP–qPCR. We examined our data H3K14ac may regulate the interaction of Jhd2 with . for genes that showed similar behavior and identified 1,147 genes We considered this unlikely because previous work has shown that that exhibited incomplete rescue of H3K4me3 levels upon deletion the histone H3 tail is dispensable for the interaction of Jhd2 with of JHD2. On average, these genes had a greater loss of transcript chromatin (11). Additionally, we were unable to detect a stable abundance upon loss of ADA2 and SAS3 compared with all genes (P interaction of Jhd2 with peptides representing the first 23 amino value of 0.0001). Moreover, of the 306 genes that showed a twofold acids of the H3 tail regardless of the modification state (Fig. S6A). GENETICS decrease of transcripts in the ada2Δ sas3Δ strain, over half did not We also performed a chromatin association assay to test if de- show recovery of methylation levels in a ada2Δ sas3Δ jhd2Δ mu- letion of ADA2 and SAS3 affected the interaction of Jhd2 with tant. We interpreted these data to indicate that although the ma- chromatin. Proteins from yeast strains expressing HA-tagged jority of genes lost methylation of H3K4 in the HAT mutant due to Jhd2 were fractionated into a soluble “nonchromatin” fraction increased Jhd2 activity on the hypoacetylated histones, a subset lost (sup) and a pellet, which contained the bulk of the chromatin methylation due to decreased RNA polymerase activity. (chrom) in the cell. Measurement of Jhd2 levels in each fraction Histone acetylation facilitates transcription through two mech- by immunoblot analysis demonstrated that loss of ADA2 and anisms: (i) weakening of histone–DNA contacts and (ii)recruit- SAS3 did not affect the relative levels of Jhd2 bound to chromatin ment of bromodomain-containing factors that remodel chromatin (Fig. S6B).

Maltby et al. PNAS | November 6, 2012 | vol. 109 | no. 45 | 18507 Downloaded by guest on September 23, 2021 A histone C for H3K4me3 was used to detect loss of this PTM upon incubation mutant with Jhd2. Fig. 3C shows that Jhd2 could not demethylate a K14ac - + - + Jhd2 peptide to the same extent as an unacetylated one, indicating that H3K9Q H3K4me3 the ability of Jhd2 to demethylate H3K4me3 was negatively regu- H3K14Q coomassie lated by histone H3K14ac. H3K18Q peptide ++ ++ H3Kme3 modifications -- + + H3K14ac H3K23Q H3K14ac Regulates H3K4 Demethylation During Gene Repression. JHD2 + + -- From this study we can elucidate a mechanism for the loss of WT H3 + --+ H3K4me3 observed upon transcriptional repression. During ac- mut H3 + -- + tive transcription, H3K14ac prevents Jhd2 from demethylating H3K4me3 (Fig. 4A). Transcriptional repression is initiated by B H3K14ac H3K4me3 5 binding of a repressor protein (TR) to the promoter that medi- 4 Gene Length Gene Length 4 ates recruitment of one or more complexes 3 XS XS S 3 S (HDAC), and H3 acetylation is lost. This allows Jhd2 to deme- 2 M M B ada2Δ sas3Δ L 2 L thylate H3K4me3 (Fig. 4 ). In or H3K14Q mutants, 1 XL XL 1 gene repression is not required before demethylation of H3K4me3 0 fi −1 0 by Jhd2, and thus a signi cant amount of methylation is lost in −2 −1 these strains. To test this model, we examined H3K4me3 loss at the GAL1 GAL1 −3 −2 gene upon repression in dextrose. Induction of Average Enrichment Score Average Enrichment Score Average with galactose triggers significant histone loss, with histones be- −450 1000 2000 3000 −450 1000 2000 3000 Distance from TSS (bp) Distance from TSS (bp) ing quickly restored following repression by dextrose (26). Fol- lowing repression, H3K4 is demethylated over a period of several Fig. 3. H3K14ac negatively regulates demethylation by Jhd2 in vivo and in hours in a JHD2-dependent manner (10). To determine whether vitro. (A) Whole cell extracts were subjected to immunoblot analysis for H3 loss of H3K14 affected methylation loss, we repeated this anal- (red) and H3K4me3 (green) and the images merged. Yellow indicated equal ysis using our histone H3K14Q mutant and found that H3K4me3 red and green intensities. The strains expressed either WT or mutant (mut) levels were greatly reduced at the GAL1 5′ ORF compared with B versions of H3 as shown. ( ) ChIP-on-chip analysis of genome-wide levels of wild type, which supports the model that H3K14ac regulates H3K14ac and H3K4me3 relative to the TSS. All genes with known TSSs were divided into five transcript length classes: very short (XS) ≤ 750 bp, short (S) H3K4 demethylation during gene repression. 750–1,500 bp, medium (M) 1,500–2,250, long (L) 2,250–3,000, and very long (XL) 3,000–3,750. The five groups comprised 542, 1,859, 1,266, 631, and 291 Discussion genes, respectively. All genes in each group were partitioned into 150 bp bins, In this work we uncovered a role for histone H3 acetylation in and the average H3K4me3 enrichment values were calculated and plotted. the maintenance of H3K4me3 levels through the negative regu- (C) Jhd2, purified from yeast, was subjected to histone demethylase assays lation of Jhd2 activity. Our findings explained several incon- using the indicated peptides. Removal of H3K4me3 was analyzed by immu- sistencies observed in previous studies. First, despite being the fi noblot with H3K4me3-speci c antibodies. major H3K4me3 demethylase in yeast, Jhd2 seems unable to demethylate the majority of histones under steady-state conditions (8, 10). We now know that because H3K14ac and H3K4me3 are A second explanation for our observations is that although Jhd2 both targeted to the 5′ ends of transcriptionally active genes, K4me3 fi can bind acetylated histone H3, it is unable to ef ciently deme- will often be found on acetylated histone H3 and as a result be fi thylate it. To test this, Jhd2, puri ed from yeast, was subjected to an protected from the activity of Jhd2. Second, although H3K4me3 is HDM assay using synthetic peptides corresponding to the H3 tail generally confinedtothe5′ regions of transcriptionally active (residues 1–23) that were trimethylated at K4 (H3K4me3), with genes, multiple laboratories have observed Set1 bound to the 3′ and without acetylation at lysine 14 (H3K14ac). Immunoblotting ends of genes (10, 27). Because these regions are normally hypo-

A B C GAL1

Set1 Ada2 Activating Repressive Sas3 Gcn5 HDAC Stimulus Stimulus D 0.06 0.05 me me me ace me me 0.04 4 14 TA 4 14 TR 0.03 0.02

Jhd2 H3K4me3/H3 0.01 Jhd2 0 WT K14Q WT K14Q 0 min 75 min

Fig. 4. H3K14ac regulates H3K4 demethylation during gene repression. (A and B) Models for coordinated control of histone acetylation and methylation on the H3 tail. (A) In the presence of an activating stimulus, transcriptional activators (TAs) bind DNA sequences and recruit HAT complexes, which acetylate histone H3. Subsequent recruitment of Set1 by RNAP II results in generation of all three methylation states of H3K4. H3K14ac prevents Jhd2 demethylase activity on H3K4me3, stabilizing this mark. (B) After loss of an activating stimulus or in the presence of a repressive signal, a transcriptional repressor (TR) binds to its DNA sequence and recruits histone deacetylase (HDAC) complexes. HDACs deacetylate the H3 tail, allowing Jhd2 to demethylate H3K4me3. (C) Schematic representation of the GAL1 gene. The arrow indicates the TSS and the line below shows the PCR product detected in D.(D) Yeast strains, expressing WT or mutant versions of histone H3 as indicated, were grown in galactose before transfer to dextrose-only media for the indicated times. ChIP–qPCR was used to measure H3K4me3 levels. Error bars indicate mean ± SE from three independent cultures.

18508 | www.pnas.org/cgi/doi/10.1073/pnas.1202070109 Maltby et al. Downloaded by guest on September 23, 2021 acetylated and thus susceptible to demethylation by Jhd2, this may Library construction for the Illumina platform was performed using partially explain why the 3′ regions of genes do not have detectable a custom procedure for paired-end sequencing. Briefly, 2–10 ng of ChIP levels of H3K4me3, despite the presence of COMPASS. Finally, material was end-repaired and A-tailed before being ligated to TruSeq PE induction of heterochromatin by regulated expression of the si- adaptors. In between each reaction, the material was purified using phenol: lencing protein, Sir3, results in loss of both histone H3 acetylation chloroform:isoamyl alcohol extraction followed by ethanol precipitation. fi and H3K4me3 from the HMR locus (23). However, removal of The resulting material was then ampli ed in the Phusion HF master mix H3K4me3 occurs with noticeably slower kinetics than histone (NEB) using TruSeq PE PCR primer 1.0 and custom indexed multiplexing primers [5′ AAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAG- deacetylation, which is consistent with a requirement for deace- ACGTGTGCTCTTCCGATC 3′,where“NNNNNN” corresponds to unique hexamer tylation before demethylation. barcodes]. PCR amplification was performed as follows: denaturation at 98 °C Although these studies were performed in yeast, we expect the for 60 s; eight cycles of (98 °C, 30 s; 65 °C, 30 s; 72 °C, 30 s), and a final extension results to be directly applicable to mammalian systems for several at 72 °C for 5 min. Amplified libraries were purified using 0.8 (vol) Agencourt + reasons. First, in human CD4 T cells, genome-wide H3K14ac and AMPure XP solid phase reversible immobilization paramagnetic beads and H3K4me3 levels were found to positively correlate at the 5′ ends of eluted in 10 mM Tris·HCl pH 8.5. An aliquot of each library was run on an genes, as we have shown here for S. cerevisiae (19). Second, the Agilent High Sensitivity chip to check the size distribution and molarity of the four mammalian H3K4me3-specific demethylases (JARID1A–D) PCR products. show similar domain architecture to yeast Jhd2 (6, 28). Inter- Equimolar amounts of indexed, amplified libraries were pooled, and frag- estingly, JARID1A (RBP2) is a component of a Sin3 HDAC ments in the 200–600 bp size range were selected on an 8% (wt/vol) Novex TBE complex, which coordinately deacetylates and demethylates his- PAGE gel (Invitrogen). An aliquot (1 μL) of the library pool was run on an tones at E2F4 target genes during myogenic differentiation (29). Agilent High Sensitivity chip to confirm proper size selection and measure Although Jhd2 does not seem to be stably complexed with any DNA concentration. The pooled libraries were diluted to 15 nM and their proteins in yeast (7), the idea that it may be recruited during gene concentration was confirmed using the Quant-iT dsDNA HS assay kit and Qubit repression by transient interaction with an HDAC is an intriguing fluorometer (Invitrogen). Libraries were sequenced on the Illumina HiSeq one. Of final note, the H3K4-specific HDM, LSD1, is also found machine at the UBC Biodiversity Research Centre NextGen Sequencing Facility. complexed with an HDAC (HDAC1 or 2), and hyperacetylated Clusters were generated on the cBOT (HiSeq2000) and paired-end 100 nucle- histones were found to be less susceptible to demethylation by otide reads generated using v3 sequencing reagents on the HiSeq2000 (SBS) platform. The hexamer barcode was sequenced using the following primer [5′ recombinant LSD1, as we have seen here with Jhd2 (30, 31). It is GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 3′]. Image analysis, base-call- important to point out, however, that unlike Jhd2, which acts 2+ α ing, and error calibration were performed using Casava 1.8.2 (Illumina). through a Fe -and -oxoglutarate-dependent hydrolysis, LSD1 is S. cerevisiae fl Reads were aligned to the genome using BWA (41) and vi- a avin-monoamine oxidase, and thus parallels between LSD1 and sualized using SeqMonk (v0.21.0: www.bioinformatics.bbsrc.ac.uk/projects/ Jhd2 must be applied with caution. seqmonk). The average gene profiles were generated by aligning reads The fact that H3K4me3 was dependent on histone H3 acetyla- relative to transcription start sites (22). Cumulative read counts were ad- tion is interesting when one considers that both Gcn5 and Sas3 are justed to reflect bulk modification levels as described (13). The list of genes components of complexes that contain H3K4me3 binding motifs that show reduced H3K4me3 in both the ada2Δ sas3Δ and ada2Δ sas3Δ (32–36). Although we did not observe loss of H3K14ac in bulk jhd2Δ strains was generated through SeqMonk by Pearson correlation, with histones in a set1Δ mutant (Fig. S7), this does not rule out the a correlation coefficient of 0.9 to the read density over gene loci. possibility that H3K14ac and H3K4me3 are mutually dependent on each other at some loci. Such a self-reinforcing loop would Expression Microarray Analysis. RNA was isolated by the method of hot + cause a continuous increase in both H3K4me3 and H3 acetylation phenol extraction (37). Poly(A ) RNA was amplified and fragmented using levels during the period when a gene is transcriptionally active. the Message Amp III kit (Ambion) as per the manufacturer’s instructions. Although it is still a matter of debate whether histone PTMs are Hybridizations were performed on a GeneChIP Yeast Genome 2.0 (Affyme- heritable through cell division, an intriguing possibility is that any trix 900553) according to the manufacturer’s instructions. Expression data inheritance that does exist may depend more on the “intensity” of were extracted using Expression Console Software (Affymetrix) with MAS5.0 a specific PTM rather than the actual modification per se. Statistical algorithm. Materials and Methods Jhd2 Purification and HDM Assays. Jhd2 was overexpressed from pBG1805 (Thermo Scientific Open Biosystems Yeast ORF Collection) in a caf1Δ strain by Yeast Strains and Antibodies. All strains used in this study are listed in Table S1. growth in raffinose followed by an overnight induction with galactose. Cell Yeast culture, genetic manipulations, and strain verification were performed extracts were prepared by bead beating in 50 mM Hepes, pH 7.5, 150 mM using standard protocols (37). Genomic deletions were verified by PCR and NaCl, 0.1% (vol/vol) Tween 20, and Jhd2 was immobilized on IgG-Sepharose whole cell extracts were generated as previously described (38). Antibodies fi used for immunoblot analysis are listed in Table S2. (GE Healthcare). The puri ed protein was liberated by treatment with PreS- cission Protease (GE Healthcare) and subjected to demethylase assays (10) us- fi ChIP-qPCR, ChIP-on-Chip, and Chromatin Association Assays. ChIP and ChIP-on- ing synthetic biotinylated peptides corresponding to the rst 23 amino acids of chip were performed as previously described (25, 39). The chromatin asso- the histone H3 tail (Genscript). To detect methylation loss, peptides were run ciation assay was performed as outlined by others (40). on 15% (wt/vol) tricine-SDS-urea gels (42) and probed with anti-H3K4me3 antibodies (Active Motif, 39159). ChIP-seq Library Construction, Sequencing, and Data Analysis. Chromatin im- munoprecipitation was performed essentially as described (5) using bead ACKNOWLEDGMENTS. Support for this work was provided by grants to L.J.H., beating instead of zymolyase to obtain the cell lysate. Briefly, 100 mL of M.C.L., and M.S.K. from the Canadian Institutes of Health Research (MOP- 84431, MOP-77805, and MOP-79442, respectively). V.E.M, J.M.S., and A.T.C. S. cerevisiae was grown to midlog phase and cross-linked with 1% (vol/vol)

were supported by fellowships from the University of British Columbia, the GENETICS formaldehyde for 15 min before collection. Cells were disrupted by bead Child and Family Research Institute, and the Natural Science and Engineering beating and digested to mononucleosomes with 500 units of micrococcal Research Council of Canada, respectively. T.H. was a fellow of the Canadian nuclease. H3K4me3 nucleosomes were immunoprecipitated with 20 μgof Institutes of Health Research/Michael Smith Foundation for Health Research H3K4me3 antibody (Abcam ab1012) and protein G Dynabeads (Invitrogen). Bioinformatics Training Program.

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