KIAA1718 is a histone demethylase that erases repressive histone methyl marks

KIAA1718 is a histone demethylase that erases repressive histone methyl marks Atsushi Yokoyama, Yosuke Okuno, Toshihiro Chikanishi, Waka Hashiba, Hiroki Sekine, Ryoji Fujiki and Shigeaki Kato* Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan The methylation states of histone lysine residues are regarded as significant epigenetic marks governing transcriptional regulation. A number of histone demethylases containing a jumonji C (JmjC) domain have been recognized; however, their properties remain to be investigated. Here, we show that KIAA1718, a PHF2/PHF8 subfamily member, possesses histone demethylase activity specific for H3K9 and H3K27, transcriptionally repressive histone marks. Biochemical purification of the KIAA1718 interactants reveals that KIAA1718 forms complexes with several factors including KAP1, a transcriptional co-activator. Consistent with these findings, KIAA1718 shows a transcriptional activation function in the chromatin context. Thus, our study identifies KIAA1718 as a histone demethylase for repressive methyl marks and shows that it is involved in transcriptional activation. Introduction In eukaryotic cells, DNA is wrapped around histone octamers, forming nucleosomes, the primary units of chromatin structure (Luger 2002). Both histone N-terminal tails and globular domains are subject to multiple covalent post-translational modifications such as acetylation, methylation, ubiquitination and phosphorylation, which constitute a ‘histone code’ capable of modulating diverse nuclear processes including transcription (Kouzarides 2007; Ruthenburg et al. 2007; Cairns 2009; Campos & Reinberg 2009). Of these histone modifications, methylation of lysine residues is generally regarded as one of the most significant histone modification, as it triggers alterations in chromatin structure (Garcia-Bassets et al. 2007). Methylation of H3K9 and H3K27 leads to chromatin silencing, whereas H3K4 methylation enhances chromatin activity (Kouzarides 2007; Li et al. 2007; Campos & Reinberg 2009). Similar to other post-translational modifications that take place on histone tails, histone methylation is enzymatically reversible by a number of histone demethylases (Klose & Zhang 2007; Shi & Whetstine 2007; Cloos et al. 2008). LSD1, the first histone demethylase identified, can demethylase both Communicated by : Kohei Miyazono *Correspondence: uskato@mail.ecc.u-tokyo.ac.jp H3K4me1 ⁄ 2 and H3K9me1 ⁄ 2 (Shi et al. 2004; Metzger et al. 2005; Yokoyama et al. 2008). Subsequently, studies recognized Jumonji C (JmjC) domain-containing proteins that possess histone demethylase activity in the presence of a-ketoglutarate and Fe (II) cofactors (Tsukada et al. 2006; Yamane et al. 2006; Christensen et al. 2007; Iwase et al. 2007). JmjC domain-containing proteins can be divided into seven groups based on alignment of the JmjC domain (Klose et al. 2006). The PHF2 ⁄ PHF8 subfamily includes PHF2, PHF8 and KIAA1718; they have a plant homeo domain (PHD)-type zinc finger motif in addition to a JmjC domain. However, little is known about the enzymatic properties of the PHF2 ⁄ PHF8 subfamily members. Biochemical characterization of histone modifying enzymes has revealed that these enzymes often form multiprotein complexes in the nucleus. Complex formation appears to be indispensable for the proteins to be enzymatically active in chromatin (Kitagawa et al. 2003; Ohtake et al. 2007; Takada et al. 2007; Fujiki et al. 2009; Sawatsubashi et al. 2010). However, the possibility of complex formation by PHF2 ⁄ PHF8 family proteins has not been biochemically tested. Here, we show that KIAA1718 has histone demethylase activity for H3K9 and H3K27 in vitro and in vivo and acts as a transcriptional activator. Furthermore, proteomic analysis revealed complex formation of KIAA1718 with several factors such as KAP1, supporting its transcriptional activation function. DOI: 10.1111/j.1365-2443.2010.01424.x  2010 The Authors Journal compilation  2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. Genes to Cells (2010) 15, 867–873 867 A Yokoyama et al. Results KIAA1718 is a putative histone demethylase which is enriched in brain tissue KIAA1718 is a member of the PHF2 ⁄ PHF8 family which is characterized by the presence of a PHD-type zinc finger motif in addition to the JmjC domain (Fig. 1A). To examine the tissue distribution of KIAA1718 mRNA, we carried out real-time quantitative PCR (qPCR) analysis using an adult mouse cDNA library. As shown in Fig. 1B, KIAA1718 mRNA was ubiquitously expressed, with relatively high expression levels in the brain, indicating that KIAA1718 might function in general transcriptional regulation, especially in the nervous system. KIAA1718 possesses a histone demethylase activity which can be stimulated by H3K4me3 marks To determine whether KIAA1718 has histone demethylase activity, FLAG-tagged wild-type mouse KIAA1718 was transiently expressed in 293F cells and purified to near homogeneity (Fig. 2A). In the presence (A) of a-ketoglutarate and Fe (II) cofactors, the protein was subjected to in vitro histone demethylase assays using calf thymus histone as substrates and immunoblotting with a series of methylation-specific antibodies to screen for its substrates. This analysis revealed that KIAA1718 substantially reduced the levels of H3K9me2 and H3K27me2 without affecting the level of H3K4me2 (Fig. 2B). These results suggest that KIAA1718 is a histone demethylase that can specifically remove methyl groups from H3K9me2 and H3K27me2 in vitro. Recent studies showed the presence of histone modification cross-talk, i.e., one histone modification recruits histone modifying factors to modulate a second histone modification (Suganuma & Workman 2008). KIAA1718’s PHD-type zinc finger motif can recognize trimethylated H3K4 (H3K4me3) (Li et al. 2006; Pena et al. 2006; Shi et al. 2006; Wysocka et al. 2006). To test the idea that H3K4me3 marks affect the demethylation activity of KIAA1718, we carried out an in vitro histone demethylase assay in the presence or absence of H3K4me3 peptides. The addition of unmodified peptides did not significantly affect the enzymatic activity of KIAA1718. However, H3K4me3 peptides enhanced its activity (Fig. 2C), implying that recognition of H3K4me3 leads to allosteric activation of the demethylation activity of the KIAA1718. Biochemical purification of KIAA1718-interacting proteins (B) Figure 1 KIAA1718 is a member of the PHF2 ⁄ PHF8 protein family. (A) Schematic representation of mouse KIAA1718 and the PHF2 ⁄ PHF8 protein family. The percentage identity is indicated in the aligned sequences for the PHD finger and jumonji C domains. (B) Tissue distribution of KIAA1718 mRNA was assessed by qPCR using a mouse cDNA library from adult C57 ⁄ BL6 mouse (30 w; N = 3). The expression levels of the KIAA1718 gene were normalized to the endogenous expression of the 36B4 gene. The error bars indicate standard deviations. 868 Genes to Cells (2010) 15, 867–873 To better understand the molecular function of KIAA1718, we took a biochemical approach to identify partners associating with KIAA1718. First, we established 293F cell lines stably expressing FLAGtagged KIAA1718 by retrovirus infection. Nuclear extracts were prepared from the 293F-KIAA1718 cell lines and subjected to affinity purification using FLAG M2 antibody resin (Fig. 3A). Interacting proteins were eluted by FLAG peptide and subjected to silver staining (Fig. 3B). The KIAA1718-interacting proteins were excised from the silver-stained gel, and their tryptic digestion products were subjected to liquid chromatographytandem mass spectrometry (LC-MS ⁄ MS) analysis. In addition, proteins eluted from the resin were also directly digested with trypsin protease for protein identification using LC-MS ⁄ MS. A total of 13 factors were identified, as shown in Fig. 3C. Some of the identified proteins were transcriptional co-activators such as KAP-1 which recruits histone acetyl transferase SRC-2 (Rambaud  2010 The Authors Journal compilation  2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. KIAA1718 as a histone demethylase (A) (B) (C) Figure 2 KIAA1718 possesses histone demethylase activity. (A) Purified FLAG-tagged KIAA1718 from transiently transfected 293F cells was separated by SDS-PAGE and stained with Coomassie blue. (B) Calf thymus histones were incubated with purified KIAA1718 protein. Baculovirus-derived LSD1 was used as a positive control for demethylation assay. (C) In vitro demethylation assays using KIAA1718 protein were carried out in the presence of H3K4 or H3K4me3 peptide. (A) (B) (C) Figure 3 Proteomic analysis of KIAA1718 protein. (A) Purification scheme for KIAA1718-associated proteins from 293F stable transformants expressing FLAG-KIAA1718. (B) KIAA1718-associated proteins were visualized by silver staining for subsequent LC-MS ⁄ MS analysis. Identified proteins are indicated on the right side. (C) Total identified peptides are listed. Coverage means the percent sequence coverage identified from MS ⁄ MS results. MW, molecular weight; Accession no., accession number in NCBI; Peptides identified, number of the identified peptides by LC-MS ⁄ MS.  2010 The Authors Journal compilation  2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. Genes to Cells (2010) 15, 867–873 869 A Yokoyama et al. et al. 2009). DBC1 is also known for its co-activator function for the androgen receptor (Fu et al. 2009). Interestingly, some virus-related proteins such as E1B 55K and PSIP 1 (PC4 and SFRS1 interacting protein) were also identified (Yew & Berk 1992; Llano et al. 2006). KIAA1718 is a transcriptional activator in the chromatin context Finally, to directly assess the role of KIAA1718 in transcription, we tethered KIAA1718 to the GAL4 DNA-binding domain and tested its transcriptional activity using 293F cells stably integrating a pGL4.31 reporter gene containing five GAL4 upstream activation sequence (UAS) sites in the luciferase gene promoter [293F-pGL4.31: chromatin Luc. assay (Yokoyama et al. 2008)]. Figure 4A shows that the GAL4-fused KIAA1718 transcriptional activation function was approximately 4.5-times greater than the GAL4 control, indicating that KIAA1718 acts as a transcriptional activator in the chromatin context. However, the KIAA1718 H282A mutant lacking the demethylation activity (Huang et al. 2010) lost their transcriptional activation function, suggesting that (A) (B) Figure 4 KIAA1718 acts as a transcriptional activator in chromatin. (A) A chromatin luc. assay was carried out using 293F-pGL4.31 cells. Cells were transiently transfected with pM or pM KIAA1718 vector (400 ng each) and pGL4.75 vector for control (0.1 ng). The error bars indicate standard deviations. (B) Chromatin immunoprecipitation analysis of histone modification at the GAL4 upstream activation sequence site. 293F-pGL4.31 cells were transiently transfected with pM or pM KIAA1718. After 48- h incubation, DNA fragments were precipitated with anti-H3K9me2 and antiAcH3. IgG was used as a negative control. 870 Genes to Cells (2010) 15, 867–873 KIAA1718 exerts the transcriptional activity through its histone demethylase activity. Next, we conducted a chromatin immunoprecipitation (ChIP) assay using 293F-pGL4.31 cells to determine whether recruited KIAA1718 modified histones in the promoter. Using antibodies shown in Fig. 4B, the immunoprecipitated chromatins were subjected to PCR using primers corresponding to the GAL4 UAS site. When GAL4-KIAA1718 was expressed, the level of H3K9me2 at the GAL4 UAS site was significantly reduced, corresponding to GAL4-KIAA1718 expression. Furthermore, the acetyl histone H3 (AcH3) level was increased, consistent with transcriptional activation. However, these changes in histone modification were not observed with KIAA1718 H282A (Fig. 4B), demonstrating that H3K9 demethylation by KIAA1718 triggers the histone acetylation, resulting in transcriptional activation. Together, these results suggest that KIAA1718 erases repressive histone marks such as H3K9me2 and recruits transcriptional activators to the targeted promoters, thereby activating transcription in vivo. Discussion In this study, we identified KIAA1718 as a histone demethylase that removed repressive histone marks such as H3K9me2 and H3K27me2. We also found that its recognition of H3K4me3 marks (presumably through the PHD finger motif) stimulates histone demethylase activity in vitro. Furthermore, we showed that KIAA1718 acts as a transcriptional activator in chromatin when targeted to promoters. Finally, using a biochemical approach, we identified proteins which interact with KIAA1718, and these results support its transcriptional activation function. Methylation of both H3K9 and H3K27 leads to chromatin inactivation (Li et al. 2007). Among the JmjC domain-containing proteins, JMJD1A and JMJD1B were previously reported as demethylases specific for H3K9 (Loh et al. 2007), whereas UTX and JMJD3 demethylate H3K27 (Agger et al. 2007). Therefore, in this respect, KIAA1718 is a unique enzyme that demethylates both H3K9 and H3K27, repressive histone methyl marks. Therefore, we suggest that KIAA1718 serves as a transcriptional switch on its targeted genes, changing silenced states to active chromatin states, an idea supported by the chromatin luciferase assay (Fig. 4A). Recently, several groups reported that KIAA1718 possesses a histone demethylase activity specific for H3K9 and H3K27 (Horton et al. 2010; Huang et al.  2010 The Authors Journal compilation  2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. KIAA1718 as a histone demethylase 2010; Kleine-Kohlbrecher et al. 2010), consistent with our present results. However, Horton et al. reported that coexistence of H3K4me3 and H3K9me2 on the same peptides inhibits enzymatic activity of KIAA1718 toward H3K9me2 caused by the structural problem (Horton et al. 2010). Intriguingly, our result shows that the enzymatic activity was stimulated when these modifications are present in trans, using core histones as substrates. Although both results could not be compared simply because of the difference in the experimental systems, these facts might imply that demethylation activity of KIAA1718 is inhibited or stimulated when these modifications are present in cis or trans, respectively. More detailed biochemical data is, thus, required for the precise characterization of enzymatic activity of this protein. Biochemical purification of KIAA1718-interacting proteins revealed that KIAA1718 interacts with KAP1. This factor is reportedly a transcriptional coactivator for Nur77 (Rambaud et al. 2009), a nuclear receptor that is constitutively expressed in brain (Watson & Milbrandt 1990). Given that KIAA1718 is enriched in brain tissue (Tsukada et al. 2010), it would be intriguing to test whether KIAA1718 coregulates the transcriptional function of Nur77 and whether KIAA1718 is recruited to the Nur77targeted promoters in the nervous system. Additionally, some virus-related proteins such as PSIP1 were identified as KIAA1718 interactants. PSIP1 is known as the cofactor of viral integration to the chromosome (Llano et al. 2006). Recently, a couple of groups reported that KAP1 also regulates the endogenous retroviral genes (Matsui et al. 2010; Rowe et al. 2010). Therefore, KIAA1718 might have a role in the regulation of the endogenous retroviral genes expression, although it remains to be tested. More detailed analysis of KIAA1718 complex formation remains to be carried out. It is unclear at this stage whether KIAA1718 is a subunit stably integrated in multiprotein complexes or whether it transiently associates with those complexes. From this point of view, further biochemical characterization of KIAA1718 and other PHF2 ⁄ PHF8 family proteins would be of great interest. Experimental procedures KIAA1718 was also inserted into pM vector. Anti-FLAG and anti-FLAG M2 agarose were from Sigma (St Louis, MO, USA). Anti-H3 and anti-H3K9me2 were from Abcam (Cambridge, UK). Anti-H3K4me2 and anti-H3K27me2 were from Millipore. Unmodified histone H3 and H3K4me3 peptides were from Millipore (Billerica, MA, USA). Cell culture and transfection We maintained 293F cells in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum and antibiotics. For culture of 293F-pGL4.31 cells, cells were grown in DMEM supplemented with 200 lg ⁄ mL hygromycin (Invitrogen (Carlsbad, CA, USA)). For establishment of 293F FLAGKIAA1718 stable transformants, 293F cells were infected with retrovirus carrying the FLAG-KIAA1718 gene. For transfection, we used Lipofectamine2000 (Invitrogen) according to the manufacturer’s guidance. Preparation of nuclear extracts and KIAA1718 complex purification For purification of the KIAA1718-containing complex from 293F cells, cells were cultured in 30 500 cm2 TC-treated culture dishes (Corning, Corning, NY, USA). Nuclear extracts were prepared by previously described methods (Yokoyama et al. 2008). Briefly, collected cells were swollen in hypotonic buffer [10 mM Hepes (pH 7.6), 10 mM KCl and 1.5 mM MgCl2] and homogenized. Isolated nuclei were collected and suspended in 0.5 nuclear pellet volume (npv) of low salt buffer (50 mM KCl). Finally, nuclear proteins were extracted by adding 0.5 npv of high salt buffer (1.0 M KCl) dropwise and dialyzed against BC100 buffer [20 mM Hepes (pH 7.6), 100 mM KCl, 0.2 mM EDTA, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol]. To purify the complex, nuclear extracts were incubated with FLAG M2 resin (Sigma) in BC100. The interactants were eluted with FLAG peptide (Sigma), separated by SDS-PAGE and subjected to silver staining. Visible interactants were excised from the gel and analyzed by LC-MS ⁄ MS. Eluted proteins were also precipitated by the methanol-chloroform method, trypsinized and then directly subjected to the LC-MS ⁄ MS analysis as previously described (Fujiyama-Nakamura et al. 2009). Histone demethylase assay The histone demethylation assay was carried out as previously described (Lee et al. 2006; Tsukada & Zhang 2006). Calf thymus histones were purchased from Sigma. Plasmids and antibodies Full-length mouse KIAA1718 with N-terminal FLAG tag was amplified by PCR from the Neuro2a cell cDNA library and was cloned in-frame into pQCXIN vector. Full-length mouse ChIP assay Soluble chromatin from 293F-pGL4.31 cells was immunoprecipitated using the Acetyl-Histone H4 Immunoprecipitation  2010 The Authors Journal compilation  2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd. Genes to Cells (2010) 15, 867–873 871 A Yokoyama et al. assay kit (Millipore) with antibodies against the indicated proteins. Specific primer pairs were designed to amplify the GAL4 UAS sites of the pGL4.31 reporter gene (5¢-ggccggtac cgagtttcta-3¢ and 5¢-cccccacccccttttatag-3¢). PCR products were visualized on 1.5% agarose ⁄ TAE gels. 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