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Regulation of Set9-Mediated H4K20 Methylation by a PWWP Domain Protein

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Regulation of Set9-Mediated H4K20 Methylation by a PWWP Domain Protein Molecular Cell Article Regulation of Set9-Mediated H4K20 Methylation by a PWWP Domain Protein Yu Wang,1 Bharat Reddy,1 James Thompson,2 Hengbin Wang,3 Ken-ichi Noma,4 John R. Yates III,2 and Songtao Jia1,* 1Department of Biological Sciences, Columbia University, New York, NY 10027, USA 2Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA 3Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294, USA 4Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, Bethesda, MD 20892, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.02.002 SUMMARY within specific regions of the genome to organize chromosomes into distinct structural and functional domains. For instance, Methylation of histone H4 lysine 20 (H4K20me) is H3K4me (methylation of histone H3 lysine 4) is usually associ- essential for recruiting checkpoint proteins 53BP1/ ated with actively transcribed genes, whereas H3K9me is Crb2 to DNA lesions and subsequent activation of enriched at transcriptionally repressed heterochromatin struc- a DNA-damage checkpoint. In fission yeast, Set9 tures (Litt et al., 2001; Noma et al., 2001). In addition to control- (spKMT5) catalyzes mono-, di-, and trimethylation ling gene expression, H3K79me and H4K20me are required for of H4K20. However, the mechanisms that regulate efficient recruitment of checkpoint proteins 53BP1/Crb2 to sites of DNA damage (Botuyan et al., 2006; Du et al., 2006; Huyen Set9 function are poorly understood. Here, we iden- et al., 2004; Sanders et al., 2004). The subsequent activation of tified a PWWP domain protein Pdp1 as a Set9-asso- DNA-damage checkpoints transiently arrests the cell cycle to ciated factor. Pdp1 binds to histones and is required allow DNA repair, thus preventing the propagation of DNA for Set9 chromatin localization. Yeast cells without lesions (Rouse and Jackson, 2002; Zhou and Elledge, 2000). Pdp1 were deficient in all three states of H4K20me, The distinct distribution profiles of various histone lysine meth- sensitive to genotoxic treatments, and impaired in ylations across genomes suggest that the enzymes that catalyze Crb2 recruitment. The PWWP domain of Pdp1 binds histone lysine methylation, the histone methyltransferases to H4K20me, and mutations within the PWWP (HMTases), are tightly regulated. Indeed, many HMTases form domain that abrogated this interaction in vitro highly conserved complexes with other chromatin proteins to reduced both the association of Set9 with chromatin regulate their localization or activities. For example, the E3 ubiq- and the extent of H4K20me in vivo. These results uitin ligase complex Cul4-Rik1 associates with the H3K9 HMTase Clr4 and regulates its localization to repetitive DNA elements to demonstrate that the PWWP domain is a new establish heterochromatin (Hong et al., 2005; Horn et al., 2005; methyl-lysine recognition motif that plays important Jia et al., 2005). The H3K27 HMTase E(Z)/EZH2 forms the poly- roles in epigenetic regulation. comb repressive complex 2 (PRC2) that includes ESC/EED and SU(Z)12, which are essential for its HMTase activity (Cao et al., INTRODUCTION 2002; Czermin et al., 2002; Kuzmichev et al., 2002; Muller et al., 2002). In some cases, the associated proteins also regulate the In eukaryotic cells, genomic DNA is folded with histone and processivity of HMTases. For instance, the association of mAM nonhistone proteins into chromatin. The building block of chro- with ESET/SETDB1 is required for trimethylation of H3K9, and matin is the nucleosome, which contains 146 base pairs of WDR5 of the MLL complexes is essential for trimethylation of DNA wrapped around an octamer of histones (two each of H3K4 (Wang et al., 2003; Wysocka et al., 2005). H2A, H2B, H3, and H4) (Luger et al., 1997). Covalent modification The methylation of H4K20 is involved in a diverse array of of histones and DNA along with chromatin remodeling are essen- cellular processes, such as organizing higher-order chromatin, tial features of an intricate epigenetic system for the regulation of maintaining genome stability, and regulating cell-cycle progres- chromatin structure and function. These epigenetic modifica- sion (Fang et al., 2002; Julien and Herr, 2004; Karachentsev tions not only affect compaction of DNA in the nucleus but also et al., 2005; Nishioka et al., 2002; Rice et al., 2002; Sakaguchi play important regulatory roles in almost every aspect of DNA and Steward, 2007; Schotta et al., 2004, 2008). Multiple enzymes metabolism including transcription, replication, recombination, in higher eukaryotes catalyze H4K20me. PR-Set7/Set8 is respon- and DNA repair (Berger, 2007; Downs et al., 2007; Groth et al., sible for H4K20me1 (Couture et al., 2005; Fang et al., 2002; Nish- 2007; Kouzarides, 2007; Li et al., 2007). ioka et al., 2002; Xiao et al., 2005), whereas Suv4-20hs are Lysine methylation is one of the most intriguing histone modi- responsible for H4K20me2 and H4K20me3 (Schotta et al., fications due to its remarkable specificity (Martin and Zhang, 2004, 2008; Yang et al., 2008). In contrast, in fission yeast Schiz- 2005). It is detected on many histone lysines, each of which osaccharomyces pombe, Set9 is the sole enzyme that catalyzes can be mono-, di-, or trimethylated (me1, me2, and me3, respec- all three states of H4K20me (Sanders et al., 2004), and Set9-medi- tively). These highly conserved modifications normally cluster ated H4K20me is required for efficient recruitment of checkpoint 428 Molecular Cell 33, 428–437, February 27, 2009 ª2009 Elsevier Inc. Molecular Cell A PWWP Domain Recognizes Methylated Histones Figure 1. Pdp1 Associates with Set9 A control UV 120 J/m2 UV 150 J/m2 D (A) Serial dilution plating assays were performed to WT Number of peptides & percent coverage set9 measure the survival of yeast strains after treat- ment with indicated doses of UV. Set9-Flag Gel bands Complex (B) Extracts prepared from wild-type and Set9- Set9 5, 12.9% 8, 24.7% Flag strains were used to perform affinity purifica- B C Pdp1 3, 14.8% 9, 21.2% tion with anti-Flag resin, resolved by SDS-PAGE, and stained with Coomassie blue. The identity of protein bands, as determined by mass spectrom- untagged Set9-Flag untaggedSet9-Flag etry, is indicated on the right. (C) A portion of the samples, as in (B), was sub- 250 250 E jected to western blot analysis with Flag antibody. 150 150 (D) Mass spectrometry analysis of proteins associ- 100 100 1 441 ated with Flag-Set9. The number of peptides, as 75 75 Set9 Set9 Set9 SET well as the percentages of each protein these 50 Pdp1 50 peptides cover, is indicated. 37 37 1 359 Pdp1 (E) Schematic diagrams of Set9 and Pdp1. The 25 25 PWWP SET and PWWP domains are indicated. 20 20 15 15 10 10 Coomassie stain -Flag western protein Crb2 to sites of DNA damage (Du et al., 2006; Sanders Set9-Flag cells did not show defects in survival after UV treat- et al., 2004). This process is highly conserved with the recruitment ment and retain H4K20 methylation (Figure 1A and Figure S1 of 53BP1 (a Crb2 homolog) in mammals (Botuyan et al., 2006). available online), indicating that Set9-Flag is functional. Affinity Structural analysis of 53BP1 and Crb2 indicate that they both purification with Flag resin revealed two protein bands that recognize H4K20me2/H4K20me1, but not H4K20me3, because were specifically present in the Set9-Flag purified fraction but their binding cavities contain four aromatic residues that prevent were absent in the untagged control fraction (Figure 1B). Mass the binding of the bulkier trimethylated form (Botuyan et al., 2006). spectrometry analysis of gel-excised bands identified the protein Indeed, biochemical studies demonstrated that 53BP1/Crb2 with a relative molecular mass of 55 KDa as Set9, which was associates only with H4K20me1 and H4K20me2 (Botuyan et al., confirmed by western blot analysis (Figure 1C). The protein 2006; Greeson et al., 2008; Kim et al., 2006), and only with a molecular weight of 40 KDa was identified as H4K20me2 is involved in the recruitment of Crb2 (Greeson SPBC29A3.13, an uncharacterized PWWP domain protein, et al., 2008). However, the mechanism that regulates Set9 to which we named Pdp1. In addition, mass spectrometry analysis perform different degrees of H4K20me is poorly understood. of the Set9-Flag complex by MudPIT analysis (Multidimensional To gain insights into the mechanism underlying the regulation Protein Identification Technology) (Washburn et al., 2001) also of Set9, we affinity-purified Set9 complex from fission yeast. We identified Pdp1 and Set9 as major components of the complex found an uncharacterized PWWP (proline-tryptophan-trypto- (Figure 1D). phan-proline) domain-containing protein, Pdp1 (PWWP domain To confirm the interaction between Pdp1 and Set9 in vivo, we protein 1), that associates with Set9 and is required for all three generated strains expressing functional Flag-Pdp1 and Set9- states of H4K20me. We demonstrated that Pdp1 interacts with myc at their native chromosomal loci (Figure S1). When protein histones and regulates the association of Set9 with chromatin. extracts from cells expressing both Set9-myc and Flag-Pdp1 The PWWP domain belongs to the Royal family (Maurer-Stroh were used to perform immunoprecipitation analysis, Set9-myc et al., 2003). Members of this family also include Tudor, chromo, coimmunoprecipitated with Flag-Pdp1 (Figure 2A). In addition, and MBT domains, which use aromatic residues to form hydro- we could immunoprecipitate endogenous Set9 with Flag-Pdp1 phobic cavities to interact with methylated histones or chromatin (Figure 2B). proteins (Maurer-Stroh et al., 2003; Ruthenburg et al., 2007). We Because Pdp1 is the only protein present in stoichiometric discovered that the PWWP domain of Pdp1 binds to H4K20me amount with Set9 as indicated by Coomassie staining and Mud- in vitro and in vivo. Furthermore, mutations of two conserved PIT analysis (Figures 1B and 1D), we examined whether these aromatic residues in a hydrophobic cavity of the PWWP domain two proteins associate with each other directly using a GST abolished this interaction and resulted in the selective loss of pull-down assay.
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