Social isolation stress induces ATF-7 phosphorylation and impairs silencing of the 5-HT 5B receptor gene
2009; Springer Nature; Volume: 29; Issue: 1 Linguagem: Inglês
10.1038/emboj.2009.318
ISSN1460-2075
AutoresToshio Maekawa, Seungjoon Kim, Daisuke Nakai, Chieko Makino, Tsuyoshi Takagi, Hiroo Ogura, Kazuyuki Yamada, Bruno Chatton, Shunsuke Ishii,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoArticle5 November 2009free access Social isolation stress induces ATF-7 phosphorylation and impairs silencing of the 5-HT 5B receptor gene Toshio Maekawa Corresponding Author Toshio Maekawa Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan Search for more papers by this author Seungjoon Kim Seungjoon Kim Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, JapanPresent address: Laboratory of Veterinary Reproduction, Kyungpook National University, Sankyuk-dong, Buk-gu Daegu 702-701, South Korea Search for more papers by this author Daisuke Nakai Daisuke Nakai Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Ibaraki, Japan Search for more papers by this author Chieko Makino Chieko Makino Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Ibaraki, JapanPresent address: Division of Molecular Genetics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan Search for more papers by this author Tsuyoshi Takagi Tsuyoshi Takagi Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan Search for more papers by this author Hiroo Ogura Hiroo Ogura Tsukuba Research Laboratories, Eisai Co., Ltd, Ibaraki, Japan Search for more papers by this author Kazuyuki Yamada Kazuyuki Yamada Support Unit for Animal Experiment, Research Resources Center, Brain Science Institute (BSI), RIKEN, Wako, Saitama, Japan Search for more papers by this author Bruno Chatton Bruno Chatton Ecole Supérieure de Biotechnologie de Strasbourg, Université Louis Pasteur, Parc d'innovation, UMR7100 CNRS-ULP, Strasbourg, Illkirch Cedex, France Search for more papers by this author Shunsuke Ishii Corresponding Author Shunsuke Ishii Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Ibaraki, Japan Search for more papers by this author Toshio Maekawa Corresponding Author Toshio Maekawa Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan Search for more papers by this author Seungjoon Kim Seungjoon Kim Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, JapanPresent address: Laboratory of Veterinary Reproduction, Kyungpook National University, Sankyuk-dong, Buk-gu Daegu 702-701, South Korea Search for more papers by this author Daisuke Nakai Daisuke Nakai Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Ibaraki, Japan Search for more papers by this author Chieko Makino Chieko Makino Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Ibaraki, JapanPresent address: Division of Molecular Genetics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan Search for more papers by this author Tsuyoshi Takagi Tsuyoshi Takagi Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan Search for more papers by this author Hiroo Ogura Hiroo Ogura Tsukuba Research Laboratories, Eisai Co., Ltd, Ibaraki, Japan Search for more papers by this author Kazuyuki Yamada Kazuyuki Yamada Support Unit for Animal Experiment, Research Resources Center, Brain Science Institute (BSI), RIKEN, Wako, Saitama, Japan Search for more papers by this author Bruno Chatton Bruno Chatton Ecole Supérieure de Biotechnologie de Strasbourg, Université Louis Pasteur, Parc d'innovation, UMR7100 CNRS-ULP, Strasbourg, Illkirch Cedex, France Search for more papers by this author Shunsuke Ishii Corresponding Author Shunsuke Ishii Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Ibaraki, Japan Search for more papers by this author Author Information Toshio Maekawa 1,‡, Seungjoon Kim1,‡, Daisuke Nakai1,2, Chieko Makino1,2, Tsuyoshi Takagi1, Hiroo Ogura3, Kazuyuki Yamada4, Bruno Chatton5 and Shunsuke Ishii 1,2 1Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, Tsukuba, Ibaraki, Japan 2University of Tsukuba, Graduate School of Comprehensive Human Sciences, Tsukuba, Ibaraki, Japan 3Tsukuba Research Laboratories, Eisai Co., Ltd, Ibaraki, Japan 4Support Unit for Animal Experiment, Research Resources Center, Brain Science Institute (BSI), RIKEN, Wako, Saitama, Japan 5Ecole Supérieure de Biotechnologie de Strasbourg, Université Louis Pasteur, Parc d'innovation, UMR7100 CNRS-ULP, Strasbourg, Illkirch Cedex, France ‡These authors contributed equally to this work *Corresponding authors: Laboratory of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Tel.: +81 29 836 9031; Fax: +81 29 836 9030; E-mail: [email protected] of Molecular Genetics, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. E-mail: [email protected] The EMBO Journal (2010)29:196-208https://doi.org/10.1038/emboj.2009.318 Present address: Laboratory of Veterinary Reproduction, Kyungpook National University, Sankyuk-dong, Buk-gu Daegu 702-701, South Korea 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 Many symptoms induced by isolation rearing of rodents may be relevant to neuropsychiatric disorders, including depression. However, identities of transcription factors that regulate gene expression in response to chronic social isolation stress remain elusive. The transcription factor ATF-7 is structurally related to ATF-2, which is activated by various stresses, including inflammatory cytokines. Here, we report that Atf-7-deficient mice exhibit abnormal behaviours and increased 5-HT receptor 5B (Htr5b) mRNA levels in the dorsal raphe nuclei. ATF-7 silences the transcription of Htr5B by directly binding to its 5′-regulatory region, and mediates histone H3-K9 trimethylation via interaction with the ESET histone methyltransferase. Isolation-reared wild-type (WT) mice exhibit abnormal behaviours that resemble those of Atf-7-deficient mice. Upon social isolation stress, ATF-7 in the dorsal raphe nucleus is phosphorylated via p38 and is released from the Htr5b promoter, leading to the upregulation of Htr5b. Thus, ATF-7 may have a critical role in gene expression induced by social isolation stress. Introduction ATF-7 (originally called ATFa) is structurally related to ATF-2 (Hai et al, 1989; Maekawa et al, 1989; Gaire et al, 1990), a member of the ATF–CREB family of transcription factors. ATF-2, ATF-7, and CRE-BPa (Nomura et al, 1993) form a subfamily in the ATF–CREB family. Each of these three factors contains a transcription-activation domain consisting of a metal-finger structure and stress-activated protein kinase (SAPK) phosphorylation sites, and a b-ZIP type DNA-binding domain. Various stresses, including inflammatory cytokines, activate SAPKs such as p38 and JNK (Davis, 2000), which then phosphorylate ATF-2 and activate its trans-activating capacity (Gupta et al, 1995; Livingstone et al, 1997; van Dam et al, 1997). ATF-7 is also phosphorylated by p38, but not by JNK (De Graeve et al, 1999). ATF-2 and ATF-7 can form homodimers or heterodimers with Jun and bind to cAMP response element (CRE) (5′-TGACGTCA-3′) (Chatton et al, 1994). ATF-7 binds to mouse ATFa-associated modulator (mAM) which is a component of the ESET complex (De Graeve et al, 2000; Wang et al, 2003). As ESET is a histone methyltransferase (HMTase) that converts lysine 9 of histone H3 (H3-K9) from the dimethyl to the trimethyl form, therefore ATF-7 is thought to support gene silencing by inducing histone H3-K9 trimethylation. Two reports have suggested a role for ATF-2 family transcription factors in epigenetic gene silencing. The yeast homologue of ATF-2, Atf1, contributes to heterochromatin formation independently of the RNAi machinery (Jia et al, 2004). Vertebrate ATF-2 also interacts with the histone variant macroH2A, which is enriched in the inactive X chromosome in female mammalian cells and functions to maintain gene silencing (Agelopoulos and Thanos, 2006). Both ATF-7 and ATF-2 are ubiquitously expressed in various tissues, including the brain (Takeda et al, 1991; Goetz et al, 1996). Atf-2 null mice die immediately after birth because of defects in respiration, which appear to be caused by impaired proliferation of cytotrophoblasts in the placenta (Maekawa et al, 1999). Atf-2 heterozygotes are highly prone to mammary tumours in which the expression levels of Maspin, a tumour suppressor, and Gadd45α, which is induced by hypoxic stress, are decreased (Maekawa et al, 2007). Both these genes encode the regulators of apoptosis, suggesting that defects in the apoptotic machinery are linked to the occurrence of mammary tumours. In contrast, the physiological role of ATF-7 is unknown, although the Atf-2 and Atf-7 double mutant exhibits embryonic lethality with abnormalities in the developing liver and heart (Breitwieser et al, 2007). Human neuropsychiatric disorders, such as depression, have multiple-risk factors, including environmental and genetic factors. A loss of social contact is one environmental factor that appears to be linked to both the onset and relapse of depression (Paykel et al, 1980). Long-term social isolation of rodents after weaning provides a model to study the behavioural consequences of loss of social interactions. Many of the symptoms induced by isolation rearing may be relevant to neuropsychiatric disorders (Rodgers and Cole, 1993). Isolated animals are aggressive and exhibit anxiety-like behaviours and increased locomotor activity (Rodgers and Cole, 1993; Blanchard et al, 2001). One of the typical abnormal behaviour in isolation-reared mice is a deficit in pre-pulse inhibition (PPI) of the acoustic startle response (Wilkinson et al, 1994). In fact, isolation-induced disruption of PPI has been used as a disease model in screening antipsychotic drugs. In animal studies, isolation stress changes the activity of brain neurotransmitters (Blanc et al, 1980; Blanchard et al, 2001). In the case of acute stress, several transcription factors, including c-Fos and corticosteroid receptors, are activated and modulate multiple target genes (Kaufer et al, 1998). However, the transcription factors that are activated and the regulation of gene expression patterns in response to a chronic stress, such as social isolation stress, remain elusive. In addition, as the effect of social isolation stress on behaviour is long-lived, this stress may cause epigenetic changes. However, the mechanism by which epigenetic change is caused by isolation stress remains unknown. In this study, we have demonstrated that Atf-7-deficient (Atf-7−/−) mice exhibit abnormal behaviours reminiscent of isolation-reared wild-type (WT) mice. Social isolation stress induced the phosphorylation of ATF-7 and p38 in the dorsal raphe nuclei, as well as a release of ATF-7 from the promoter of the 5-HT receptor 5B (Htr5b) gene, leading to an impaired silencing of this gene. Results Abnormal behaviours of Atf-7−/− mice We generated Atf-7−/− mice (Supplementary Figure S1), and, under pathogen-free conditions, Atf-7−/− mice appeared healthy until at least 12 months of age. As Atf-7 mRNA is expressed at relatively high levels in parts of the brain (Goetz et al, 1996), we examined various behaviours originally using WT and Atf-7−/− littermate mice with a mixed CBA (25%) × C57BL/6 (75%) genetic background, and later using C57BL/6 congenic mice. In the marble-burying test, which is used to examine anxiety-related behaviours (Spooren et al, 2000), Atf-7−/− mice exhibited increased marble-burying behaviour compared with WT mice (Figure 1A and Supplementary Figure S2A). In other tests of anxiety-related behaviours, such as the amount of time spent in the centre of an open-field and the elevated plus-maze test (Spooren et al, 2000), there was no significant difference between Atf-7−/− and WT mice (Figure 1B and C and Supplementary Figure S2B). Atf-7−/− mice did exhibit a significant increase in the startle response to a pulse-alone stimulus (Figure 1D and Supplementary Figure S2C). PPI, in which the startle reflex response is attenuated by a pre-pulse, is an important measure of sensorimotor gating (Geyer et al, 1990). Atf-7−/− mice displayed lower levels of PPI of the acoustic startle response (Figure 1E and Supplementary Figure S2D). Although the association between the startle response and PPI is not currently clear, a negative correlation between the startle response and PPI in WT mice has been reported (Egashira et al, 2005). If these two phenomena are correlated in Atf7−/− mice, an increase in startle reactivity may lead to decreased PPI. However, we cannot exclude the possibility that ATF-7 is independently involved in the modulation of startle response and its PPI. Figure 1.Abnormal behaviours in Atf-7−/− mice. Wild-type (WT; +/+) and Atf-7−/− C57BL/6 congenic mice were used for all assays. Data are mean±s.e.m. (A) Marble-burying test. **P<0.01 (n=10–12 for each group). (B) Center of the open-field test. Time spent in the center of the test apparatus is expressed as a percent of total time (10 min). NS, no significant difference (n=13–16 for each group). (C) Elevated plus-maze test. Mice were observed in an elevated plus-maze for 5 min. Percentage of entries into open arms (left) and the time spent in open arms (right) are shown (n=13–16 for each group). (D) Acoustic startle response. Amplitude of the startle response to a 120 dB acoustic stimulus is shown (n=13–16 for each group). (E) Pre-pulse inhibition of the acoustic startle response. The response to a white noise stimulus of 120 dB after a 20 ms pre-pulse warning stimulus (70 or 80 dB) is shown (n=13–16 for each group). Download figure Download PowerPoint The Atf-7−/− and WT mice responses were indistinguishable in other behavioural tests. We examined spontaneous locomotor activity in a new environment by placing mice in an open-field chamber and monitoring their behaviour. There was no significant difference in the locomotor activity of Atf-7−/− and WT mice on the first and second day of the trials (Supplementary Figure S3A and B). We also examined motor coordination using a rotating rod treadmill. Overall, the amount of time mice spent on the rotarod increased with training (Supplementary Figure S3C). The retention time of Atf-7−/− mice on the rod was not significantly different from that of WT mice at 0 (stationary), 5, or 10 r.p.m. In the footprint test, there was no significant difference in the stride length and the step width between mutant and WT mice (Supplementary Figure S3D). In the forced swimming test, there was also no difference between Atf-7−/− and WT mice (Supplementary Figure S4A). We also examined spatial learning ability using the Morris Water Maze task. WT and Atf-7−/− mice took similar lengths of time to reach the visual platform to escape from the water (Supplementary Figure S4B), thus indicating that Atf-7−/− mice have normal vision, motor function, and escape behaviour in the water maze task. Mice were then trained in a hidden platform task, in which mice search for a submerged platform to escape from the water. Atf-7−/− and WT mice took similar lengths of time over the 7 days of testing to locate the hidden platform (Supplementary Figure S4C). Thus, Atf-7−/− and WT mice were able to learn the location of a hidden platform during the course of the trials. We then carried out a probe test, in which the platform is removed from the pool after completion of the hidden platform task, and the trained mice are allowed to swim freely for 60 s. The time spent in the target quadrant by Atf-7−/− mice was similar to that of WT mice (Supplementary Figure S4D). These results indicate that a normal spatial learning ability is present in Atf-7−/− mice. The number of crossings of the hidden platform and the swimming distance of Atf-7−/− and WT mice was also similar during the probe trial (Supplementary Figure S4E and F). Thus, the performance of Atf-7−/− mice in the hidden platform task was indistinguishable from WT mice. Upregulation of the Htr5b gene in the dorsal raphe nucleus of Atf-7−/− mice Atf-7 mRNA was detected in the cortex, the cerebellum, the hippocampus, and the brainstem, including the medulla, the pons, and the midbrain of WT mice (Figure 2A), but not of Atf-7−/− mice (Supplementary Figure S5). No obvious morphological abnormalities were found in these tissues in Atf-7−/− mice (data not shown). Western blotting indicated that ATF-7 expression levels varied in these tissues (Figure 2B). Several studies have linked abnormal marble-burying behaviour to 5-HT function (Jenck et al, 1998). Further, disruptions in PPI of the startle response are correlated not only with D2 dopamine and N-methyl-D-aspartate signalling systems, but also with 5-HT (Geyer et al, 2001). Therefore, we focused our attention on the dorsal raphe nuclei of the brainstem, where much of the 5-HT in the brain is localized and relatively high levels of ATF-7 are expressed. Figure 2.Increased levels of 5-HT receptor 5B (Htr5b) mRNA in the Atf-7−/− brainstem. (A) Atf-7 mRNA expression in various regions of the brain was examined by in situ hybridization with anti-sense and sense probes. Bar, 100 μm. (B) Extracts (20 μg of protein) from the indicated regions of wild-type (WT) or Atf-7−/− brains were used for western blotting with anti-ATF-7. The bands indicated by arrows are the ATF-7 signals. (C) Real-time RT–PCR analysis of Htr5b mRNA levels using total RNA from the brainstem (n=4). ***P<0.001. (D) Htr5b mRNA expression in the dorsal raphe nuclei was examined by in situ hybridization using probes for Htr5b (green) and the serotonin transporter (5-HTT, red). Cell nuclei were identified by DNA staining using TOTO-3 (blue). The sections were examined by laser confocal microscopy, and representative images are presented. The panels at the right show the merged images. Bar, 100 μm. The white box indicates a subregion of each image that is presented at higher magnification below. (E, F) A 5-HT 5B receptor antagonist reduced the abnormal behaviour of Atf-7−/− mice. Marble-burying behaviour (E) and pre-pulse inhibition (PPI) (F) of WT and Atf-7−/− C57BL/6 congenic mice was examined after administration of either vehicle or methiothepin (n=10–12 for each group in E, and n=7 for each group in F). *P<0.05. Download figure Download PowerPoint To identify the ATF-7 target genes in the brainstem that may have a role in the abnormal behaviour in Atf-7−/− mice, we performed a DNA microarray analysis using RNA from the brainstem of Atf-7−/− and WT mice. The results indicate that 25 genes were upregulated and 38 genes downregulated by more than two-fold by the loss of Atf-7. Of these ATF-7 target genes, the functions of 11 of the upregulated genes are known, whereas the functions of only 7 of the downregulated genes have been reported. Among these genes, only the Htr5b and the ciliary neurotrophic factor receptor (Cntfr) genes have been associated with neuronal function. As the 5-HT system appeared to be associated with the abnormal behaviour of Atf-7−/− mice as described above, upregulation of the Htr5b gene may be associated with the phenotype of Atf-7−/− mice. CNTF is a cytokine that has neurotrophic and differentiating effects on cells in the central nervous system, and the CNTF–CNTF receptor system affects motor neurons (Vergara and Ramirez, 2004). However, there has been no report demonstrating a connection between the CNTF system and anxiety-related behaviours. Therefore, we focused our attention on the Htr5b gene. As Htr5b is thought to act as an autoreceptor (Serrats et al, 2004), its upregulation may lead to a decrease in the extracellular concentration of serotonin (5-HT). There is abundant evidence for the role of decreased 5-HT in depression and anxiety disorders (Artigasa et al, 1996). Selective serotonin re-uptake inhibitors (SSRIs), which increase the extracellular concentration of 5-HT in the dorsal raphe nuclei, are widely used as anti-depressant drugs. Htr5b mRNA levels in the Atf-7−/− brainstem were approximately 12-fold higher than in WT (Figure 2C). In situ hybridization showed higher levels of Htr5b mRNA expression in the Atf-7−/− dorsal raphe nuclei, which also expressed a serotonin transporter mRNA, than in the WT (Figure 2D and Supplementary Figure S6). There appeared to be no obvious difference in the levels of Htr5b mRNA between WT and Atf-7−/− mice in other regions, including the hippocampus, the habenular nucleus, and the inferior olivary nucleus (Supplementary Figure S7). Among the many reported antagonists of 5-HT receptors, methiothepin has a relatively high affinity for the 5-HT 5B receptor, although it also binds to other 5HT receptors, including the 5-HT 1A receptor (Boess and Martin, 1994). Injection of methiothepin into Atf-7−/− mice suppressed the increased marble-burying behaviour, whereas saline-treated control Atf-7−/− mice still exhibited increased marble-burying behaviour (Figure 2E). Furthermore, methiothepin also alleviated the lower levels of PPI in Atf-7−/− mice (Figure 2F). These results suggest that increased expression of Htr5b mRNA in the Atf-7−/− brainstem may, at least partly, contribute to their abnormal behaviour, although loss of ATF-7 could cause changes in the expression of other genes in various regions of the brain and also contribute to abnormal behaviours. Silencing of the Htr5b gene by ATF-7 via direct binding to its 5′-region Analysis of the DNA sequence in the 5′ region of the mouse Htr5b gene identified three CRE-like sites at nucleotides −3374, −3340, and −2325 (where +1 is the major transcriptional start site), all of which have only a 1 or 2 bp difference from the consensus CRE sequence (Figure 3A). Gel mobility-shift assays were carried out using nine DNA probes, which cover approximately 4.4 kbp of the 5′-region of Htr5b, and nuclear extracts from 293T cells that were transfected with a Flag-tagged ATF-7 expression plasmid or control empty vector. When the #2 or #4 probes were used, a retarded band was detected in extracts containing Flag-ATF-7 (Figure 3B). These specific retarded bands were further shifted when an anti-Flag antibody was added, indicating that the bands contained Flag-ATF-7. In contrast, no retarded bands were observed with the other probes (Supplementary Figure S8). We then used 54 bp and 18 bp oligonucleotides containing the CRE-like sites derived from the #2 and #4 probes, respectively. The retarded bands generated using either probe were competed out by excess amounts of unlabelled competitor oligonucleotide, but not by competitors, which contained mutated CRE-like sites (Figure 3C). These results indicate that ATF-7 binds directly to the CRE-like sites in the 5′-region of the mouse Htr5b gene. Figure 3.Binding of ATF-7 to the 5-HT receptor 5B (Htr5b) promoter region leads to silencing. (A) Presence of cAMP response element (CRE)-like sites in the 5′ region of the mouse Htr5b gene. The CRE-like sites in the 5′-region of mouse Htr5b, and nine DNA probes used for gel mobility-shift assays are shown. (B, C) Gel mobility-shift assays were performed using nuclear extracts prepared from 293T cells transfected with a Flag-ATF-7 expression vector or control empty vector. The #2 and #4 DNA probes were used as the probes in (B). In some lanes, anti-Flag or control IgG was added. In (C), oligonucleotides containing the two CRE-like sites derived from probe #2 or the one CRE-like site from probe #4 were used as probes. In some lanes, a 50-fold excess of competitor containing the same sequence as the probe (wild-type (WT)), or a mutated CRE-like site, was added. Free probe is indicated by a closed arrowhead, whereas ATF-7-bound DNA is shown by an open arrowhead. The ATF-7-DNA complex, which was super-shifted by the anti-Flag antibody, is indicated by the arrow. (D) ATF-7 represses Htr5b gene transcription. RN46A cells were transfected with the indicated Htr5b promoter-luciferase construct together with 1 (+), 2 (++), or 3 (+++) μg of the ATF-7 expression plasmid, or the control empty vector (−), and luciferase activity was measured. Values indicate mean±s.d. (n=3). *P<0.05, **P<0.01. Download figure Download PowerPoint When a Htr5b promoter-luciferase reporter containing the 4.4 kb 5′-region of the Htr5b gene was cotransfected into RN46A cells, which are derived from rat medullary raphe nucleus cells, ATF-7 inhibited luciferase expression by approximately 70% (Figure 3D). In contrast, mutation of the three CRE-like sites in this reporter relieved the ATF-7-dependent silencing. These results suggest that ATF-7 suppresses the transcription of Htr5b through interaction with CRE-like sites. The results of chromatin immunoprecipitation (ChIP) assays using the brainstem chromatin and an anti-ATF-7 antibody indicated that ATF-7 bound to the region containing CRE-like sites of the Htr5b gene, but not to the RNA start site or the 2nd exon (Figure 4A). Further, the binding of ATF-7 to this region was not detected using the Atf-7−/− brainstem chromatin. Binding of ATF-2 to this region was also not detected (Figure 4B). Figure 4.Binding of ATF-7 to the 5-HT receptor 5B (Htr5b) promoter is correlated with histone H3-K9 trimethylation. (A, B) Chromatin immunoprecipitation (ChIP) assays were carried out using the brainstem of wild-type (WT) and Atf-7−/− mice, and anti-ATF-7 (A), anti-ATF-2 (B), or control IgG (A, B). Extracted DNA was amplified by real-time PCR using primers that cover the cAMP response element (CRE)-like sites, the transcription start site, or the 2nd exon of the Htr5b gene. The relative densities of bands are indicated, and each bar represents the mean±s.d. (n=3). (C) Generation of an RN46A cell line in which ATF-7 levels are downregulated (ATF-7 kd-RN46A) by expression of a small hairpin-type double-stranded RNA. Nuclear extracts of the parental RN46A cells and the ATF-7 kd-RN46A cells were used for western blotting to detect ATF-7. (D) Real-time RT–PCR analysis of Htr5b mRNA levels using RNAs from the parental RN46A cells and ATF-7 kd-RN46A cells. Values are mean±s.d. (n=3). (E) ChIP assays were carried out using anti-histone H3-K9m3 and parental RN46A cells or ATF-7 kd-RN46A cells. Extracted DNA was amplified by real-time PCR using primers that cover the CRE-like sites, the transcription start site, or the 2nd exon of the Htr5b gene. The relative densities of the bands are indicated, and each bar represents the mean±s.d. (n=3). *P<0.05, **P<0.01, ***P<0.001. Download figure Download PowerPoint ATF-7 mediates histone H3-K9 trimethylation of the Htr5b promoter region by recruiting the ESET HMTase The results of ChIP assays using the WT brainstem chromatin and anti-H3-K9m3 antibodies indicated that histone H3 in the region containing the CRE-like sites, the RNA start site, and the 2nd exon of Htr5b is trimethylated at K9 (Supplementary Figure S9A). When the Atf-7−/− brainstem chromatin was used, similar levels of histone H3-K9 trimethylation were detected. This result may indicate that Htr5b gene transcription is repressed by histone methylation in most cells of the brainstem with the exception of the dorsal raphe nucleus in Atf-7−/− mice. To assess the role of ATF-7 in histone H3-K9 methylation, we generated an ATF-7 knock-down (kd) RN46A cell line by expressing a small hairpin-type double-strand RNA. The ATF-7 level was approximately one-eighth that of the parental cell line (Figure 4C). In kd-RN46A cells, Htr5b mRNA levels increased approximately 2.3-fold compared with the parental cell line (Figure 4D). In ChIP assays, binding of ATF-7 to the region containing the CRE-like sites of the Htr5b gene was detected in parental RN46A cells, but not in the ATF-7 kd-RN46A cells (Supplementary Figure S9B). Binding of ATF-2 to the same region was not detected (Supplementary Figure S9C). The degree of histone H3-K9 trimethylation in the region containing the CRE-like sites or the 2nd exon of Htr5b gene was lower in the kd-RN46A cells than in parental cells (Figure 4E). Thus, ATF-7 contributes to H3-K9 trimethylation at the Htr5b gene promoter. To investigate whether the ESET HMTase is involved in the silencing of Htr5b by ATF-7, we examined ATF-7–ESET interactions by co-immunoprecipitation. Immunocomplexes prepared from RN46A cell lysates using anti-ATF-7 contained ESET, whereas immunocomplexes prepared with control IgG did not contain ESET (Figure 5A). The results of ChIP assays using the WT brainstem chromatin and anti-ESET antibodies indicated that ESET bound to the region containing CRE-like sites of Htr5b, but not to the region containing the RNA start site or the 2nd exon (Figure 5B). Further, binding of ESET to this region was not detected using the Atf-7−/− brainstem chromatin. Similar results were also obtained in ChIP assays using the parental RN46A and ATF-7 kd-RN46A cells (Figure 5C). Figure 5.ATF-7 recruits the ESET HMTase to the 5-HT receptor 5B (Htr5b) g
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