Histone H3K4 demethylation is negatively regulated by histone H3 acetylation 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 lysine 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 methylation 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 histones, 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 chromatin 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 nucleosome 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.