MOLECULAR AND CELLULAR BIOLOGY, Nov. 2005, p. 9447���9459 Vol. 25, No. 21 0270-7306/05/$08.00 0 doi:10.1128/MCB.25.21.9447���9459.2005 Copyright �� 2005, American Society for Microbiology. All Rights Reserved. Dimethylation of Histone H3 at Lysine 36 Demarcates Regulatory and Nonregulatory Chromatin Genome-Wide��� Bhargavi Rao,1 Yoichiro Shibata,2 Brian D. Strahl,1,2 and Jason D. Lieb1,3,4* Curriculum in Genetics and Molecular Biology,1 Department of Biochemistry and Biophysics,2 Department of Biology,3 and Carolina Center for the Genome Sciences,4 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received 6 June 2005/Returned for modification 5 July 2005/Accepted 9 August 2005 Set2p, which mediates histone H3 lysine 36 dimethylation (H3K36me2) in Saccharomyces cerevisiae, has been shown to associate with RNA polymerase II (RNAP II) at individual loci. Here, chromatin immunoprecipita- tion-microarray experiments normalized to general nucleosome occupancy reveal that nucleosomes within open reading frames (ORFs) and downstream noncoding chromatin were highly dimethylated at H3K36 and that Set2p activity begins at a stereotypic distance from the initiation of transcription genome-wide. H3K36me2 is scarce in regions upstream of divergently transcribed genes, telomeres, silenced mating loci, and regions transcribed by RNA polymerase III, providing evidence that the enzymatic activity of Set2p is restricted to its association with RNAP II. The presence of H3K36me2 within ORFs correlated with the ���on��� or ���off��� state of transcription, but the degree of H3K36 dimethylation within ORFs did not correlate with transcription frequency. This provides evidence that H3K36me2 is established during the initial instances of gene tran- scription, with subsequent transcription having at most a maintenance role. Accordingly, newly activated genes acquire H3K36me2 in a manner that does not correlate with gene transcript levels. Finally, nucleosomes dimethylated at H3K36 appear to be refractory to loss from highly transcribed chromatin. Thus, H3K36me2, which is highly conserved throughout eukaryotic evolution, provides a stable molecular mechanism for estab- lishing chromatin context throughout the genome by distinguishing potential regulatory regions from tran- scribed chromatin. In eukaryotic cells, the accessibility of the DNA template is influenced by chromatin structure. For example, in Saccharo- myces cerevisiae, transcription factors have been shown to bind to consensus sequences upstream of genes in preference to identical consensus sequences that occur within the coding sequences of transcribed genes (24, 31). Likewise, transposons preferentially insert into promoter regions (33), and the dou- ble-strand breaks required for meiotic recombination in S. cerevisiae occur preferentially in gene promoters rather than in the coding regions (12, 55). Chromatin context therefore is a major determinant of where on the genomic DNA template many biological phenomena will occur. Regulation of accessibility to the DNA template is likely to be mediated in large part through differential regulation of nucleosome occupancy. Promoter regions of S. cerevisiae ex- hibit reduced nucleosome occupancy genome-wide (2, 26), and these differences in nucleosome occupancy are important for promoter accessibility (47). Furthermore, in S. cerevisiae, pro- moter and nonregulatory chromatin can be biochemically frac- tionated, indicating that those regions have distinct physical properties (36). Nucleosomes can be moved or displaced from specific genomic regions by several general mechanisms, in- cluding nucleosome-remodeling complexes like SWI/SNF and RSC (34), binding of activators to DNA (3, 4, 35), transcrip- tional elongation by RNA polymerase II (RNAP II) (20, 26, 46), and inherent properties of DNA sequence (47). Template accessibility and nucleosome occupancy can also be mediated by posttranslational modification of the N-terminal histone tails, most notably acetylation (27, 48). Although chromatin context may be defined in part by regional differences in his- tone modifications, no chromatin mark has been shown to cor- respond specifically to coding or regulatory regions throughout the genome. Here, we present evidence that dimethylation of histone H3 at lysine 36 (H3K36me2), which is mediated by the methyltransferase Set2p (25, 51), may provide such a mark. Set2p interacts with the C-terminal domain (CTD) of RNAP II (22, 29, 56), and this interaction is regulated by the phos- phorylation state of the CTD. Serine 5 (Ser5) of the CTD repeat is phosphorylated by Kin28p during initiation of tran- scription, while serine 2 (Ser2) and Ser5 are phosphorylated by Ctk1p during elongation (8, 17, 19, 30). Set2p associates pref- erentially with Ser2/Ser5 phosphorylated repeats of the RNAP II CTD, and deletion of CTK1 abolishes H3K36me2 (22, 56). Set2p-RNAP II interactions are also dependent on the Paf1 complex (Paf1p, Rtf1p, Cdc73p, Ctr9p, and Leo1p) (22), which also associates with RNAP II (21, 40, 49). This and other biochemical data suggest that Set2p associates with RNAP II specifically during transcription elongation (18, 22, 29, 45, 56). Chromatin immunoprecipitation (ChIP) assays followed by quantitative PCR on a few selected loci have supported this assertion, showing that H3K36me2 is generally restricted to the transcribed regions of RNAP II-regulated genes (1, 18, 22, 45). While there is strong evidence that Set2p is associated with elongating polymerase, the physiological functions of Set2p * Corresponding author. Mailing address: Department of Biology, CB no. 3280, 203 Fordham Hall, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. Phone: (919) 843- 3228. Fax: (919) 962-1625. E-mail: jlieb@bio.unc.edu. ��� Supplemental material for this article may be found at http: //mcb.asm.org/. 9447

and H3K36me2 are still unknown. Evidence suggesting a func- tion for Set2p in transcriptional elongation comes from results showing either sensitivity or resistance of set2 strains to the elongation inhibitor 6-azauracil. These phenotypes are similar to those exhibited by strains defective for genes encoding elon- gation factors like Chd1p, Iswi1p, and Fkh1p (22, 28, 29, 45, 57). A role in transcriptional elongation is also supported by synthetic genetic interactions between set2 and deletions of all members of the Paf1 complex, the chromodomain factor Chd1p, a putative elongation factor Soh1p, and the Bre1p or Lge1p components of histone H2B ubiquitination complex (22). However, whatever role Set2p plays in elongation is ei- ther not essential or redundant, since set2 strains are viable and, in many backgrounds, exhibit very mild phenotypes. To further elucidate the cellular function of H3K36me2, we determined its pattern of distribution throughout the S. cerevisiae genome. We performed additional experiments to determine how the pattern of H3K36me2 changes in response to a change in global transcriptional state and the relationship between the H3K36me2 mark and nucleosome stability. H3K36me2 demar- cates the structurally distinct regulatory and nonregulatory re- gions of yeast genomic chromatin and may serve as an indicator of chromatin context. MATERIALS AND METHODS Nomenclature. Throughout the paper, we use a recently proposed uniform histone modification nomenclature (53). Thus, for example, ���H3K36��� refers to histone H3 lysine at residue 36, and ���H3K36me2��� refers to dimethylation of that residue. Strains and culture conditions. For H3K36me2 and histone H3 ChIPs, strain AS4 (MAT trp1-1 arg4-17 tyr7-1 ade6 ura3) was used (50). For histone H4 ChIPs, a previously described myc-tagged H4 strain constructed in strain UCC1111 [MAT ade2::his3- 200 leu2- 0 lys2- 0 met15- 0 trp1- 63 ura3- 0 adh4::URA3-TEL (VII-L) hhf2-hht2::MET15 hhf1-hht1::LEU2 pRS412 (ADE2 CEN ARS)-HHF2-HHT2] was used (37, 38). Unless otherwise described, yeast was grown to an optical density of 0.8 to 1.0 at 600 nm with shaking at 30��C in 100 ml of yeast extract-peptone-dextrose media (1% yeast extract, 2% peptone, 2% dextrose). Antibodies. Antibodies against histone H3 lysine 36 dimethylation have been described previously (51) and were derived from Upstate (catalog no. 07-369). myc-tagged antibodies were also obtained from Upstate (catalog no. 05-419). The rabbit histone H3 antiserum was obtained from Abcam, Inc. (AB1791), and was raised in rabbits using a peptide corresponding to amino acids 124 to 135 (CGIQLARRIRGERA) of human histone H3. Dot blot. Peptides (KSAPSTGGVKKPHRYKPGTGK-BIOTIN) in which the residue corresponding to H3K36 (underlined) was either mono-, di-, or tri- methylated were resuspended in double-distilled H2O (10 g/ l) and serially diluted in Tris-buffered saline (TBS) (150 mM NaCl, 10 mM Tris, pH 7.6). Aliquots of 100- l peptide-TBS solution were spotted onto polyvinylidene diflu- oride membranes by using a Bio-Rad dot blot apparatus. Membranes were washed in TBS and then blocked in a solution of 2.5% (wt/vol) Carnation nonfat dry milk in TBS-Tween 20 (0.1% Tween 20 in TBS) for 10 min prior to incuba- tion with a 1:10,000 dilution of the specified antibody for 2 h at room tempera- ture. Membranes were washed with TBS-Tween 20 for 10 min three times, incubated with anti-rabbit horseradish peroxidase-conjugated immunoglobulin G for 2 h at room temperature, and then washed again for 10 min three times prior to detection using ECL-Plus from Amersham. ChIP assays. ChIP assays were performed as described previously (23). Briefly, whole-cell extracts were prepared from 1% formaldehyde-fixed wild-type and set2 cells by using lysis buffer (50 mM HEPES-KOH, pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X, and 0.1% sodium deoxycholate) and sonicated to shear the chromatin (0.25- to 1-kb range). Immunoprecipitation was per- formed with anti-H3K36me2, anti-myc, or anti-H3. After cross-link reversal at 65��C, DNA was extracted by using a QIAGEN PCR purification kit according to the manufacturer���s instructions. DNA amplification, labeling, array hybridization, and data processing. ChIP- enriched DNA and reference DNA in all experiments were amplified as de- scribed previously (5). Briefly, two initial rounds of DNA synthesis with T7 DNA polymerase using primer 1 (5 -GTTTCCCAGTCACGATCNNNNNNNNN-3 ) were followed by 25 cycles of PCR with primer 2 (5 -GTTTCCCAGTCACGA TC-3 ). Cy3-dUTP or Cy5-dUTP was then incorporated directly with an addi- tional 25 cycles of PCR using primer 2. Microarray hybridizations were per- formed using standard procedures (16). The arrays were scanned with a GenePix 4000 scanner, and data were extracted with Genepix 5.0 software. Data were normalized such that the median log2 ratio value for all quality elements on each array equaled zero, and the median of pixel ratio values was retrieved for each spot. Only spots of high quality by visual inspection, with at least 50 pixels of quality data (regression R2 of 0.6) and for which intensity of the reference signal was strong ( 350 U), were used for analysis. Arrayed elements that did not meet these criteria on at least half of the arrays were excluded from analysis. All data were log transformed before further analysis. For normalization with the nucleosome occupancy data, the median log2 ratio values of H4-myc ChIP were subtracted from the median H3K36me2-ChIP ratio values. Unless otherwise noted, all data presented are nucleosome occupancy normalized in this way. While many methods of bulk nucleosome normalization are possible, all must contend with the inherent difficulties of combining ChIP data sets produced with two different antibodies (6). The method used here is simplest and provides a more realistic representation of the modification pattern than do unnormalized data. We provide all raw data (see below) so that readers may apply their preferred normalization method. DNA microarray preparation. Open reading frames (ORFs) and intergenic regions from yeast (S288C) were PCR amplified and printed on polylysine- coated glass slides by using a robotic arrayer as described previously (16). ORFs were generally represented by PCR products that extended from start codon to stop codon. Elements representing intergenic regions generally included all DNA be- tween annotated ORFs, with the fragments divided such that PCR products were no longer than 1.5 kb. Locus-specific detection of ChIP enrichment. The sequences of the primers used (see Fig. 2 and 5) are shown in Table 1. Availability of data. All raw microarray data and images are available to the public through the UNC microarray database (https://genome.unc.edu/). Data are also available in Table S2 of the supplemental material and through GEO (accession number GSE 2991). RESULTS Chromatin from ORFs and regions immediately down- stream of ORFs is enriched for histone H3 lysine 36 dimethy- lation. As the initial step in our goal to determine the genome- wide location of H3K36me2 in S. cerevisiae, we characterized a polyclonal antibody directed against H3K36me2. While the general specificity of this antibody for methylation at H3K36 had been previously verified (18, 57), its precise specificities to the different possible H3K36 methylation states (mono-, di-, and trimethylation) were unknown. To determine the specific- ity of this antibody for H3K36 methylation, we performed dot blots against peptides that were either mono-, di-, or tri-meth- ylated at the residue corresponding to H3K36 (see Materials and Methods). As shown in Fig. 1A, the antiserum was specific to dimethylation of H3K36 and did not cross-react with any of the related modifications. Having verified the specificity of this antiserum, ChIP exper- iments were performed using extract from wild-type strains. To assess the relative abundance of genomic fragments enriched by the ChIP, samples were RNase treated and DNA was am- plified and labeled fluorescently. In parallel, total genomic DNA was prepared from input extract, RNase treated, ampli- fied, and labeled with a different fluorescent marker. The two samples were then analyzed by comparative hybridization to DNA microarrays. The microarrays used in this study cover the entire yeast genome, including the coding and noncoding re- gions, at approximately 1-kb resolution (see Materials and Methods). H3K36me2 ChIP-microarray (ChIP-chip) experi- 9448 RAO ET AL. MOL. CELL. BIOL.