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JmjC enzyme KDM2A is a regulator of rRNA transcription in response to starvation

2010; Springer Nature; Volume: 29; Issue: 9 Linguagem: Inglês

10.1038/emboj.2010.56

1460-2075

Yuji Tanaka, Kengo Okamoto, Kwesi Teye, Toshiyuki Umata, Noriyuki Yamagiwa, Yutaka Suto, Yi Zhang, Makoto Tsuneoka,

Genomics and Chromatin Dynamics

Article8 April 2010free access JmjC enzyme KDM2A is a regulator of rRNA transcription in response to starvation Yuji Tanaka Yuji Tanaka Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Kengo Okamoto Kengo Okamoto Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Kwesi Teye Kwesi Teye Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Toshiyuki Umata Toshiyuki Umata Radioisotope Research Center, Research Facility for Occupational and Environmental Health, University of Occupational and Environmental Health, Kitakyushu, Japan Search for more papers by this author Noriyuki Yamagiwa Noriyuki Yamagiwa Department of Organic Chemistry, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Yutaka Suto Yutaka Suto Department of Organic Chemistry, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Yi Zhang Yi Zhang Department of Biochemistry and Biophysics, Linberger Comprehensive Cancer Center, and Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Makoto Tsuneoka Corresponding Author Makoto Tsuneoka Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Yuji Tanaka Yuji Tanaka Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Kengo Okamoto Kengo Okamoto Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Kwesi Teye Kwesi Teye Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Toshiyuki Umata Toshiyuki Umata Radioisotope Research Center, Research Facility for Occupational and Environmental Health, University of Occupational and Environmental Health, Kitakyushu, Japan Search for more papers by this author Noriyuki Yamagiwa Noriyuki Yamagiwa Department of Organic Chemistry, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Yutaka Suto Yutaka Suto Department of Organic Chemistry, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Yi Zhang Yi Zhang Department of Biochemistry and Biophysics, Linberger Comprehensive Cancer Center, and Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Search for more papers by this author Makoto Tsuneoka Corresponding Author Makoto Tsuneoka Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan Search for more papers by this author Author Information Yuji Tanaka1, Kengo Okamoto1, Kwesi Teye1, Toshiyuki Umata2, Noriyuki Yamagiwa3, Yutaka Suto3, Yi Zhang4 and Makoto Tsuneoka 1 1Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan 2Radioisotope Research Center, Research Facility for Occupational and Environmental Health, University of Occupational and Environmental Health, Kitakyushu, Japan 3Department of Organic Chemistry, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki, Japan 4Department of Biochemistry and Biophysics, Linberger Comprehensive Cancer Center, and Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA *Corresponding author. Department of Molecular Pharmacy, Faculty of Pharmacy, Takasaki University of Health and Welfare, Takasaki 370-0033, Japan. Tel.: +81 27 352 1180; Fax: +81 27 352 1118; E-mail: [email protected] The EMBO Journal (2010)29:1510-1522https://doi.org/10.1038/emboj.2010.56 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The rate-limiting step in ribosome biogenesis is the transcription of ribosomal RNA, which is controlled by environmental conditions. The JmjC enzyme KDM2A/JHDM1A/FbxL11 demethylates mono- and dimethylated Lys 36 of histone H3, but its function is unclear. Here, we show that KDM2A represses the transcription of ribosomal RNA. KDM2A was localized in nucleoli and bound to the ribosomal RNA gene promoter. Overexpression of KDM2A repressed the transcription of ribosomal RNA in a demethylase activity-dependent manner. When ribosomal RNA transcription was reduced under starvation, a cell-permeable succinate that inhibited the demethylase activity of KDM2A prevented the reduction of ribosomal RNA transcription. Starvation reduced the levels of mono- and dimethylated Lys 36 of histone H3 marks on the rDNA promoter, and treatment with the cell-permeable succinate suppressed the reduction of the marks during starvation. The knockdown of KDM2A increased mono- and dimethylated Lys 36 of histone H3 marks, and suppressed the reduction of ribosomal RNA transcription under starvation. These results show a novel mechanism by which KDM2A activity is stimulated by starvation to reduce ribosomal RNA transcription. Introduction Regulation of cell growth ultimately depends on the control of new ribosome synthesis (Grummt, 2003). Although the supply of ribosomal components involves the activities of three forms of nuclear RNA polymerase (pol I, pol II, and pol III) in eukaryotic cells, pol I has a central role in the regulation of ribosome biogenesis (Laferte et al, 2006; Chedin et al, 2007; Grewal et al, 2007). Pol I transcribes the eukaryotic ribosomal RNA genes (rDNA) in nucleoli. rDNA code 18S, 5.8S, and 28S ribosomal RNA, which are three of the four structured RNA molecules constituting the ribosome. These three RNA result from the processing of one precursor transcript, pre-ribosomal RNA (pre-rRNA). Many discoveries about the relationship between chromatin structures and transcription have been made during the past decade, and several chemical modifications of chromatin components, including DNA methylation and histone acetylation, have been identified (Berger, 2007). One key component of chromatin structures in biological regulation is the methylation of lysine residues in histone proteins, which has been vigorously studied over the past several years. Highly specific enzymes catalysing the synthesis of methyl marks, as well as proteins recognizing distinct methylated lysine residues, have been identified. Recently, an increasing number of histone demethylases, JmjC domain-containing enzymes, has been discovered and has highlighted the dynamic nature of the regulation of histone methylation (Kustatscher and Ladurner, 2007; Stavropoulos and Hoelz, 2007). In addition, the activities of JmjC enzymes require small molecules, including molecular oxygen, Fe(II), and α-ketoglutarate (α-KG), as co-substrates (Klose et al, 2006a). Therefore, these substrates and their cognate products, such as the succinate for α-KG, can affect the activities of JmjC enzymes and the transcription regulated by them. However, the roles of JmjC enzymes on rDNA chromatin have not been well studied. Previously, we identified the JmjC protein Mina53 (Tsuneoka et al, 2002), which is involved in mammalian cell proliferation. The expression of the mina53 gene is directly controlled by the oncogene myc (Tsuneoka et al, 2002) and elevated in some types of cancer (Teye et al, 2004, 2007; Tsuneoka et al, 2004; Fukahori et al, 2007; Ishizaki et al, 2007; Zhang et al, 2008; Komiya et al, 2009). Mina53 exists in nucleoli (Tsuneoka et al, 2002) and binds to nucleolar proteins (Eilbracht et al, 2005). However, the substrate of this putative enzyme has not yet been identified, and the role of JmjC proteins including Mina53 in ribosome biogenesis is still unclear. To determine whether the dynamics of histone methylation mediated by demethylases affect ribosome biogenesis, we first attempted to identify a histone demethylase containing a JmjC domain that regulates rDNA transcription. For this, we searched the database of the ∼700 human nucleolar proteins that have been identified using high sensitivity mass spectrometry (Andersen et al, 2005), and found a candidate JmjC enzyme, KDM2A. It had been reported that KDM2A had histone demethylase activity in vivo on the dimethylated Lys 36 of histone H3 (H3K36me2) and in vitro on the monomethylated Lys 36 of histone H3 (H3K36me1) in addition to H3K36me2 (H3K36me1/2) (Tsukada et al, 2006). The amino acid sequence of human KDM2A is 97.4 and 87.0% identical to those of Mus musculus and Gallus gallus, respectively. These suggest that KDM2A has an essential role in higher animals, but it is not clear how KDM2A functions and on which genes. Here, we show that KDM2A represses the transcription of ribosomal RNA by binding to the rDNA promoter and demethylating H3K36me1/2, and that its activity is controlled by succinate. Results KDM2A gene encodes two proteins An antibody was produced against the recombinant polypeptide from Leu 763 to Gly 855 of the human KDM2A protein (Figure 1A). When a human cell lysate was analysed by western blotting using this KDM2A-specific antibody (Figure 1A, anti-pan-KDM2A antibody), two bands were recognized (Figure 1B). The protein with the lower mobility migrated to the same spot as the polypeptide exogenously expressed by the human KDM2A cDNA (GenBank Accession No. NM_012308) (Figure 1C, arrowhead), indicating that this protein was KDM2A. The protein with the higher mobility may be a degradation product of KDM2A or an mRNA product with a shorter ORF coded in the KDM2A gene. Figure 1.Proteins encoded by the KDM2A gene. (A) Diagrams of human KDM2A proteins. KDM2A (upper bar) has 1162 amino acids and contains the JmjC domain (AA148–316, shown by the white box) (GenBank Accession No. NM_012308). The numbers with AA in parentheses show amino acid numbers of KDM2A. SF-KDM2A (lower bar) has 620 amino acids and corresponds to the polypeptide from Met 543 to the end of KDM2A (AA 543–1162). The first Met for SF-KDM2A occurs in exon 14 of the KDM2A gene. The anti-pan-KDM2A antibody was produced against the polypeptide from Leu 763 to Gly 855 of KDM2A, and recognized both KDM2A and SF-KDM2A. An anti-KDM2A antibody was produced against the polypeptide from Ser 360 to Val 451 of KDM2A, and recognized only KDM2A. KDM2A-specific siRNA is a stealth RNA cognated to a partial nucleotide sequence for only KDM2A mRNA. The numbers with nn in parentheses show nucleotide numbers from the A of the first Met of KDM2A mRNA. (B) Western blot analysis to detect KDM2A proteins. Breast adenocarcinoma cell line MCF-7 cells were transfected with KDM2A-specific siRNA (KDM2A siRNA) or control siRNA. After 48 h culture, cells were lysed, and the extracts were subjected to western blotting using anti-pan-KDM2A antibody or anti-KDM2A antibody. The positions of KDM2A and SF-KDM2A are indicated by an arrowhead and arrow, respectively. The positions of protein markers with defined molecular weights are indicated on the right side of the figure. (C) The expression vector for KDM2A, SF-KDM2A, or the empty control vector was introduced into MCF-7 cells and analysed by western blotting as in (B) using anti-pan-KDM2A antibody. Download figure Download PowerPoint Using high-resolution maps of histone lysine methylations and pol II across the human KDM2A genome (Barski et al, 2007) and previously deposited sequences of EST clones, we identified a new mRNA whose transcription started from part of intron 12 of the KDM2A gene. Finally, we found that in addition to KDM2A protein, a smaller protein was expressed by the KDM2A gene (Figure 1A; a detailed description is included in the Supplementary text and Figure S1 of the Supplementary data). The protein with higher mobility had the same mobility as the polypeptide exogenously expressed from the cDNA encoding the smaller protein (Figure 1C). The cognate siRNA duplex specific for KDM2A reduced the band for KDM2A but not the band with the higher mobility (Figure 1B). These results indicate that the protein with the higher mobility was produced by mRNA with a shorter ORF coded in the KDM2A gene. We named the polypeptide SF-KDM2A (short-form KDM2A), and deposited the sequence in the GenBank (Accession No. AB490246). SF-KDM2A does not have a JmjC domain. Although KDM2A possessed demethylase activity for dimethylated Lys36 histone H3 (H3K36me2) as reported before, SF-KDM2A did not (Supplementary Figure S2). These results suggest that SF-KDM2A has a different function from KDM2A. To investigate the specific role of histone lysine methylation on the rDNA chromatin, we focused this study on KDM2A. KDM2A is localized in nucleoli and binds to ribosomal RNA gene promoter To investigate the subcellular localization of KDM2A, an antibody specific to KDM2A was produced against a recombinant polypeptide whose amino acid sequence was found in KDM2A but not in SF-KDM2A (Figure 1A). Western blot analysis showed that the antibody recognized the band that was reduced by the siRNA for KDM2A (Figure 1B). These results indicate that this antibody specifically recognized KDM2A. Immunostaining of human cells with the antibody produced signals localized in the nucleoli (Figure 2A), and the siRNA for KDM2A clearly reduced the nucleolar signals. Most of the signals for KDM2A overlapped with those for the nucleolar protein nucleolin (Figure 2A). It was reported that exogenously expressed KDM2A localized throughout the nucleoplasm as a heterochromatin-associated protein (Frescas et al, 2008). However, we observed that when KDM2A was moderately expressed exogenously but not highly overexpressed, the protein was located in nucleoli (Figure 2B). These results show that KDM2A exists in nucleoli, although some part of it may exist outside of nucleoli. Figure 2.KDM2A with JmjC domain was localized in nucleoli and bound to rDNA. (A) MCF-7 cells were transfected with control or KDM2A siRNA and double-stained with anti-KDM2A (red) and mouse anti-nucleolin (green) antibodies. The specimen was observed through a fluorescence and differential interference contrast (DIC) microscope, and representative images are shown. The anti-KDM2A antibody produced signals localized in the nuclei (control siRNA). KDM2A siRNA reduced them. Most of the signals for KDM2A overlapped with those for nucleolin (merge). (B) HeLa cells were transfected with an expression vector encoding Flag-tagged KDM2A, and double-stained with anti-Flag (green) and rabbit anti-nucleolin antibodies (red). Representative images observed using a fluorescence microscope are shown. Most of the signals for KDM2A are co-localized with those for nucleolin when KDM2A was moderately expressed. (C) MCF7 cells were transfected with control or KDM2A siRNA and analysed by chromatin immunoprecipitation (ChIP) analysis using the anti-KDM2A antibody. The specific signals for the binding of KDM2A to DNA (white bars) were detected in all regions of rDNA genes including the promoter region, and the signals were reduced when cells were treated with KDM2A siRNA before ChIP analysis (black bars). The experiments were performed three times, and mean values with standard deviations are indicated. *P<0.05. A diagram of human rDNA and the positions of PCR primers used in this experiment are shown at the bottom of this figure. The numbers in parentheses show nucleotide numbers in a human ribosomal DNA complete repeating unit (GenBank Accession No. U13369). Download figure Download PowerPoint Next, the binding of KDM2A to rDNA was investigated. First, the distribution of histone H3 through rDNA was examined. Chromatin immunoprecipitation (ChIP) analysis using anti-H3 antibody indicated that histone H3 was almost evenly distributed to all regions of rDNA (Supplementary Figure S3A). The anti-KDM2A antibody collected the fragment of the rDNA promoter (H0 region) (Supplementary Figure S3B). Enrichment of the fragment is dependent on KDM2A binding to the rDNA promoter, because the siRNA for KDM2A abolished recovery of the rDNA fragment by the anti-KDM2A antibody (Supplementary Figures S3B; Figure 2C). Additionally, the KDM2A binding detected here is specific, because the anti-KDM2A antibody hardly enriched the promoter and exon 5 genomic DNA fragments of the TATA-binding protein (TBP) gene (Supplementary Figure S3B). ChIP analysis also showed that the anti-KDM2A antibody collected DNA fragments from all regions of rDNA (Figure 2C). The siRNA for KDM2A abolished recovery of these DNA fragments, confirming the specific binding of KDM2A to the rDNA (Figure 2C). Overexpression of KDM2A represses transcription of rDNA in demethylase activity-dependent manner To investigate whether KDM2A regulates rDNA transcription, KDM2A was exogenously expressed, and the amount of pre-rRNA was measured by quantitative reverse transcription-mediated polymerase chain reaction (qRT–PCR). When the wild-type KDM2A was expressed, the amount of pre-rRNA was reduced (Figure 3A). KDM2A that had His 212 in the JmjC domain replaced with Ala (H212A mutant) did not show the demethylase activity (Tsukada et al, 2006) (Supplementary Figure S2). The H212A mutant also did not show a capacity to decrease the amount of pre-rRNA in these experimental conditions (Figure 3A). It was confirmed that comparable amounts of the wild-type KDM2A and H212A mutant were expressed on both RNA and protein levels (Figure 3A). Ongoing ribosomal RNA synthesis was also assessed by fluorouridine (FUrd) incorporation in in situ run-on assays (Kruhlak et al, 2007). Although high FUrd incorporation at nucleolar sites was observed in cells, ectopic expression of KDM2A led to a pronounced decrease in nucleolar FUrd incorporation (Figure 3B and C). This effect was not observed when Escherichia coli β-galactosidase targeted to the nucleus (Tsuneoka and Mekada, 1992) was expressed. Furthermore, the H212A mutant and SF-KDM2A did not show reduced FUrd incorporation (Figures 3B and C). These results indicate that the JmjC domain of KDM2A has a crucial role in the reduction. Together, these results show that KDM2A represses the transcription of rDNA in a demethylase activity-dependent manner. Figure 3.KDM2A reduced rDNA transcription. (A) MCF-7 cells were transfected with the KDM2A- or H212A mutant-expressing vector or the empty vector by electroporation and cultured for 2 days. Total RNA was isolated and analysed by quantitative real-time PCR (qRT–PCR) using specific primers for pre-rRNA, KDM2A, and RNA polymerase II subunit a (Polr2a). The values were normalized using the amounts of mRNA for Polr2a. The experiments were performed three times, and mean values with standard deviations are indicated. *P<0.05. KDM2A protein was also detected by western blotting. β-actin was detected as a loading control. The positions of the molecular weight markers are indicated on the right side of the figure. (B) MCF-7 cells transfected with vector encoding KDM2A, the H212A mutant, SF-KDM2A, or nuclear-localizing E. coli β-galactosidase. Two days later, cells were cultured with 2 mM FUrd for 15 min, fixed, and stained for FUrd and Flag-tagged KDM2A or β-galactosidase for assessment by FUrd incorporation assays. The incorporated FUrd (green) and exogenously expressed protein (red) were observed by a fluorescence microscopy. Representative images are shown. One of the cells with positive signals for exogenous proteins in one filed is indicated by an arrowhead. (C) Percentages of FUrd-positive cells were calculated in cells that exogenously expressed KDM2A, the H212A mutant, SF-KDM2A, or nuclear-localizing E. coli β-galactosidase (black bars), or in cells that did not exogenously express each protein in the same specimen (white bars). The experiments were performed three times, and mean values with standard deviations are indicated. *P 0.1 (no significant difference). Download figure Download PowerPoint JmjC enzyme is involved in reduction of rDNA transcription under starvation Cells of the human breast adenocarcinoma cell line MCF-7 retain a good ability to change the levels of rDNA transcription in response to environmental conditions. When MCF-7 cells were cultured in starvation conditions, it was observed that starvation reduced the amount of pre-rRNA (Figure 4A). The effects of starvation on ongoing ribosomal RNA synthesis were also assessed by the FUrd incorporation assays. Although high FUrd incorporation at nucleolar sites was observed in cells in growth conditions, starvation clearly reduced the incorporation (Figure 4B). Figure 4.Levels of rDNA transcription under starvation conditions in the presence or absence of cell-permeable succinate (DMS). (A) MCF-7 cells were cultured with or without starvation for 9 h. Total RNA was isolated from cells, and analysed by qRT–PCR with specific primers for pre-rRNA and Polr2a mRNA, as described in Figure 3A. A cell-permeable succinate, DMS, was added at a final concentration of 50 mM during starvation in the indicated experiments. The experiments were performed three times, and mean values with standard deviations are indicated. **P<0.01. (B) MCF-7 cells were cultured with or without starvation for 9 h. In the indicated experiments, DMS was added during starvation. Cells were incubated with 2 mM FUrd for 15 min, fixed, and stained for FUrd. Representative images observed through a fluorescence (FUrd) and differential interference contrast (DIC) microscopy are shown. (C) KDM2A produces succinate during demethylation. When JmjC-domain enzymes execute demethylation reactions, they catalyse α-ketoglutarate (α-KG) as a co-substrate to succinate (Tsukada et al, 2006; Klose et al, 2006a). Thus, excess amounts of succinate may inhibit the activity of JmjC-domain enzymes. (D) MCF-7 cells were transfected with a Flag-KDM2A expression vector in serum-free DMEM, cultured for 1 day, and further cultured in serum-free DMEM for another day in the presence or absence of a cell-permeable succinate, dimethyl succinate (DMS) (50 mM). Cells were stained with anti-H3K36me2 (red)- and anti-Flag (green)-specific antibodies. Representative images observed by a fluorescence and differential interference contrast (DIC) microscopy are shown. Cells with positive signals for KDM2A are indicated by arrowheads. DMS inhibited the reduction of H3K36me2 marks by the ectopic expression of KDM2A. Cells with decreased H3K36me2 levels were 59 and 28% of cells in the absence and presence of DMS, respectively. These results provide in vivo evidence that succinate inhibits the demethylase activity of KDM2A. Download figure Download PowerPoint To test whether JmjC-domain enzymes are involved in the reduction of rDNA transcription by starvation, MCF-7 cells were treated with a compound to inhibit the demethylase activity of the enzymes. Succinate is produced from α-KG by the JmjC-domain enzymes during its demethylation reaction (Figure 4C). As an enzymatic activity depends on the product/substrate equilibrium, succinate may inhibit the activity of the enzymes. Indeed, we observed in vivo inhibition of the demethylase activity of KDM2A by a cell-permeable succinate, dimethyl succinate (DMS) (Figure 4D). As shown in Figure 4A, DMS suppressed the reduction in the amount of pre-rRNA by starvation. DMS also suppressed the reduction of FUrd incorporation at nucleolar sites by starvation (Figure 4B). Together these results suggest that a JmjC-domain enzyme regulates rDNA transcription during starvation. Levels of KDM2A substrates, H3K36me1/2 marks, are changed on the rDNA promoter during starvation The effects of starvation and DMS on the levels of substrates of KDM2A, H3K36me2 marks (Tsukada et al, 2006), were investigated. As shown in Figure 5A, starvation decreased the level of H3K36me2 marks on the rDNA promoter, and treatment with DMS increased it during starvation (Figure 5B). As starvation and DMS treatment hardly affected the amount of KDM2A protein (Supplementary Figure S4A) and DMS inhibited the demethylase activity of KDM2A (Figure 4D), these results are consistent with the possibility that KDM2A reduces rDNA transcription in an enzyme activity-dependent manner during starvation. DMS did not increase the level of H3K36me3 marks in starvation. Interestingly, although H3K36me1 has not been identified as a substrate of KDM2A in vivo, starvation decreased the level of H3K36me1 marks on the rDNA promoter (Figure 5A), and treatment with DMS increased it (Figure 5B). These results suggest the possibility that H3K36me1 on the rDNA promoter may also be recognized by KDM2A as a substrate. Figure 5.Levels of methyl histone marks under starvation conditions in the presence or absence of cell-permeable succinate (DMS). (A) MCF-7 cells were cultured with or without starvation for 9 h, and the histone methylation status on rDNA promoters was investigated by ChIP analyses using specific antibodies to H3K36me1, H3K36me2, H3K36me3, H3K4me3, and histone H3. The results were expressed as fold changes to the values without starvation. (B) MCF-7 cells were cultured with starvation in the presence or absence of 50 mM DMS for 9 h, and the histone methylation status on rDNA promoters was detected as in (A). The results are expressed as fold changes to the values without DMS. For (A) and (B), the experiments were performed at least three times, and mean values with standard deviations are indicated. *P<0.05; **P 0.1 (no significant difference). Download figure Download PowerPoint An H3K4me3 mark was recently reported to be an active mark for rDNA transcription and demethylated by a JmjC-domain demethylase KDM2B/JHDM1B/FbxL10 (Frescas et al, 2007). However, neither starvation nor DMS affected the level of H3K4me3 marks in the rDNA promoter region (Figure 5A and B), suggesting that a JmjC-domain enzyme demethylating H3K4me3 on the rDNA promoter did not work during starvation. Next, we investigated the effects of starvation and DMS on H3K36 methylation in the other genomic regions. In the TBP gene, the levels of H3K36me1, H3K36me2, and H3K36me3 (H3K36me1/2/3) marks were higher in a transcribed region than in the promoter region (Supplementary Figure S4B). The levels of H3K36me1/2/3 in the two regions of the TBP gene did not change in response to starvation and DMS (Supplementary Figure S4B). In rDNA, the levels of H3K36me1/2/3 in the transcribed regions (H1, H4, and H13) were not higher than those in the promoter region (H0) (Supplementary Figure S5). Interestingly, starvation and DMS did not significantly change the levels of H3K36me1/2/3 marks in the transcribed and untranscribed regions (H1, H4, H13, and H27) of rDNA (Supplementary Figure S5). These results together with the results shown in Figure 5 suggest that the starvation signal is specifically transduced to an H3K36me1/2 demethylase located in the rDNA promoter region. KDM2A regulates levels of H3K36me1/2 and rDNA transcription in response to starvation To directly clarify the involvement of KDM2A in the regulation of H3K36me1/2 marks in the rDNA promoter region and rDNA transcription under starvation conditions, the expression of KDM2A was reduced using siRNA for KDM2A. The KDM2A knockdown reduced the amount of KDM2A protein in cells and on the rDNA promoter, and increased the levels of not only H3K36me2 but also H3K36me1 marks there (Figure 6A). Importantly, the KDM2A knockdown inhibited the decrease of H3K36me1/2 levels during starvation, indicating the involvement of KDM2A in the reduction of the marks under starvation. Figure 6.KDM2A was involved in the reduction of rDNA transcription by starvation, and its activity was controlled by succinate. (A) MCF-7 cells were transfected with control or KDM2A siRNA. Forty-eight hours after transfection, cells were further cultured 2 h with or without starvation. ChIP analyses using H3K36me1, H3K36me2, histone H3, and KDM2A antibodies were performed on the rDNA promoter as shown in Figure 2C. The experiments were performed three times, and mean values with standard deviations are indicated. *P<0.05; **P<0.01. The knockdown of KDM2A was confirmed by western blotting (lower panel). The positions of the molecular weight markers are indicated on the right side of the figure. (B) MCF-7 cells were transfected with control or KDM2A siRNA. Forty-eight hours after transfection, cells were further cultured 9 h with or without starvation. The amounts of pre-rRNA and KDM2A mRNA were measured by qRT–PCR as described in Figure 3A. The results are expressed as amounts relative to the values of cells treated with control siRNA and without starvation. For (B), (C), and (D), the experiments were performed three times, and mean values with standard deviations are indicated. *P<0.05; **P<0.01. (C) Increased levels of pre-rRNA transcript by KDM2A knockdown in (B) were expressed against the values with control siRNA. The increase of rDNA transcription with starvation was higher than that without starvation. *P<0.05. (D) Succinate functions through KDM2A to regulate rDNA transc