Gene 544 (2014) 19–24
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GCN5 is involved in regulation of immunoglobulin heavy chain gene expression in immature B cells Hidehiko Kikuchi a,b,⁎, Masami Nakayama a, Futoshi Kuribayashi b,c, Shinobu Imajoh-Ohmi b, Hideki Nishitoh a, Yasunari Takami a, Tatsuo Nakayama a a b c
Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan Laboratory Center for Proteomics Research, Graduate School of Frontier Sciences, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Tokyo 108-8639, Japan Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan
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Article history: Received 6 November 2013 Received in revised form 13 April 2014 Accepted 16 April 2014 Available online 18 April 2014 Keywords: GCN5 Gene targeting Histone acetyltransferase Immunoglobulin DT40
a b s t r a c t GCN5 is involved in the acetylation of core histones, which is an important epigenetic event for transcriptional regulation through alterations in the chromatin structure in eukaryotes. To investigate physiological roles of GCN5, we have systematically analyzed phenotypes of homozygous GCN5-deficient DT40 mutants. Here, we report participation of GCN5 in regulation of IgM heavy chain (H-chain) gene expression. GCN5-deficiency downregulates gene expressions of IgM H-chain (as whole, membrane-bound and secreted forms of its mRNA) but not light chain (L-chain), causing decreases in membrane-bound and secreted forms of IgM proteins. Chromatin immnoprecipitation assay revealed that GCN5 binds to the chicken IgM H-chain gene around its constant region but not L-chain gene, and acetylate Lys-9 residues of histone H3 within chromatin surrounding the constant region. These results suggest that GCN5 takes part in transcriptional regulation of the IgM H-chain gene via histone acetylation resulting in formation of relaxed chromatin arrangement around its coding region and plays a key role in epigenetic regulation of B cell functions. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Epigenetics can be defined as alterations in cellular phenotypes via control of gene expression without altering DNA sequences (Berger, 2007; Bernstein et al., 2007; Biel et al., 2005; Goldberg et al., 2007). Gene expression is regulated by several epigenetic mechanisms; histone modification, DNA methylation, small and non-coding RNAs and chromatin architecture (Berger, 2007; Bernstein et al., 2007; Biel et al., 2005; Espada and Esteller, 2007; Goldberg et al., 2007; Knowling and Morris, 2011). Histone modification is one of the most common and important epigenetic mechanisms (Berger, 2007; Bernstein et al., 2007; Biel et al., 2005; Butler et al., 2012; Goldberg et al., 2007; Jenuwein and Allis, 2001; Kouzarides, 2007). Core histones are subjected to various post-translational modifications including acetylation, methylation, phosphorylation, ubiquitination, sumoylation, etc. (Biel et al., 2005; Butler et al., 2012; Jenuwein and Allis, 2001; Kouzarides,
Abbreviations: ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde 3phosphate dehydrogenase; H-chain, heavy chain; H3K14, Lys-14 of histone H3; H3K9, Lys-9 of histone H3; HAT, histone acetyltransferase; HDAC, histone deacetylase; L-chain, light chain; RT-PCR, reverse transcription-polymerase chain reaction. ⁎ Corresponding author at: Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. E-mail address: [email protected] (H. Kikuchi).
http://dx.doi.org/10.1016/j.gene.2014.04.030 0378-1119/© 2014 Elsevier B.V. All rights reserved.
2007). In particular, histone acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays critical roles in the modulation of chromatin topology and thereby control of gene expressions (Biel et al., 2005; Jenuwein and Allis, 2001; Selvi and Kundu, 2009). Particular Lys residues at N-terminal tails of core histones have positive charges and tightly bind to DNA, resulting in the formation of heterochromatin. Acetylation catalyzed by HATs reduces positive charges of these Lys residues at the N-terminal histone tails and promotes euchromatin formation to allow easier access of transfactors to their target genes. In contrast, HDACs remove the acetyl groups from the N-terminal histone tails to silence gene expressions. Taken together, HATs and HDACs play essential roles in the regulation of gene expressions via collaboration of histone acetylation and deacetylation. We previously reported that in the chicken immature B cell line DT40, HDAC2 controls the amount of IgM heavy chain (H-chain) at the steps of transcription of its gene and alternative processing of its pre-mRNA (Takami et al., 1999) and down-regulates IgM light chain (L-chain) gene promoter activity (Takechi et al., 2002). As a result, DT40 cells continuously produce both membrane-bound and secreted forms of IgM proteins as complex of IgM H- and L-chains. In addition, HDAC2 regulates IgM H- and L-chain gene expressions via EBF1, Pax5, Ikaros, Aiolos and E2A gene expressions (Nakayama et al., 2007). These data have progressed studies on immunoglobulin gene regulations by HDACs as triggers. Studies using trichostatin A, a specific HDAC
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inhibitor, revealed that histone acetylation regulates transcription of genes controlling terminal B cell differentiation (Lee et al., 2003), and HDAC is involved in the immunoglobulin H-chain gene transcription via activation of 3′-IgH enhancers (Lu et al., 2005). It was also shown that there are distinct roles of HDAC1 and HDAC2 in transcription at the immunoglobulin loci in DT40 (Kurosawa et al., 2010). However, the understanding of physiological roles of HATs in immunoglobulin gene regulations is still poor in vertebrate cells. GCN5, a prototypical HAT, functions as a coactivator to promote transcriptional activation with enzymatic modification at chromatin (Brownell et al., 1996). To investigate physiological roles of GCN5, we generated homozygous GCN5-deficient DT40 mutants, GCN5−/−, by gene targeting techniques, which are excellent methods to study physiological roles of many genes involved in expressions of important cell functions of higher eukaryotes, including histone acetylation and deacetylation (Buerstedde and Takeda, 1991; Kikuchi et al., 2006). Our previous studies have revealed that GCN5-deficiency caused not only delayed growth rate, suppressed cell cycle progression at G1/S phase transition, and down- or up-regulated various G1/S phase transition-related genes (Kikuchi et al., 2005), but also drastic resistance against apoptosis induced by the B cell receptor-mediated stimulation through transcriptional regulation of various apoptosis-related genes (Kikuchi and Nakayama, 2008). In addition, GCN5 activated phosphatidylinositol 3-kinase/Akt survival pathway in cells exposed to oxidative stress via controlling gene expressions of Syk and Btk (Kikuchi et al., 2011a) and promoted the superoxide-generating system in leukocytes via controlling the gp91-phox gene expression (Kikuchi et al., 2011b). Very recently, we clarified that GCN5 is involved in UV–tolerance via controlling gene expression of DNA polymerase η (Kikuchi et al., 2012). In this study, we investigated the effects of GCN5-deficiency on amounts of IgM proteins and expressions of IgM H-chain and L-chain genes. GCN5-deficiency caused remarkable down-regulation of transcriptions of IgM H-chain gene but not L-chain gene. Results obtained in this study including chromatin immunoprecipitation (ChIP) assay revealed that GCN5 is involved in regulation of IgM H-chain gene expression through acetylation of Lys-9 residues of histone H3 within chromatin surrounding its constant region in immature B cells. 2. Materials and methods 2.1. Materials Bovine aprotinin (Sigma-Aldrich, St. Louis, MO, USA), PMSF (Wako, Osaka, Japan), monoclonal anti-GCN5 antibody (Chemicon, Temecula, CA, USA), Ex Taq DNA polymerase (Takara, Shiga, Japan), goat antichicken L-chain antisera and goat anti-chicken IgM H-chain antisera (Bethyl Laboratories, Montgomery, TX, USA), anti-acetylated histone antisera and protein G agarose/salmon sperm DNA (Millipore, Billerica, MA, USA), fluorescein isothiocyanate-conjugated tyramide-labeled anti-goat IgG (American Qualex, San Clemente, CA, USA), and horse radish peroxidase-conjugated anti-goat immunoglobulin (DAKO, Inc., Glostrup, Denmark) were used. 2.2. Cell cultures and flow cytometric analysis Generation of GCN5−/− was described in our previous report (Kikuchi et al., 2005). DT40 and GCN5−/− were cultured essentially as described (Kikuchi and Nakayama, 2008; Kikuchi et al., 2005, 2011a, 2011b, 2012). The cell surface membrane-bound IgM protein (as B cell receptor) was analyzed with a FACSCalibur (BD Biosciences, San Jose, CA, USA) as described (Takami et al., 1999).
immunoblotting as described (Nakayama et al., 2007; Takami et al., 1999). The final culture media (1 ml) containing equal cell numbers of GCN5−/− or DT40 (1 × 106) were centrifuged at 1000 ×g at 4 °C for 5 min, and equal aliquots (2.5 μl) of the supernatants were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and electrotransferred onto polyvinyliden difluoride membranes. The protein-transferred membranes were immersed in a 5% skim milk solution in Tris–buffered saline and incubated with goat anti-chicken IgM H- or L-chain antiserum at 4 °C overnight. Antibody binding was detected using horse radish peroxidase-conjugated anti-goat immunoglobulin as a second antibody and SuperSignal west Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) as a substrate. Data analyses were carried out using a luminescent image analyzer LAS-1000plus (Fujifilm, Tokyo, Japan). 2.4. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) RT-PCR was carried out essentially as described (Kikuchi and Nakayama, 2008; Kikuchi et al., 2005, 2011a, 2011b, 2012). Total RNAs were isolated from DT40 and its mutant clones. RT was performed with a first strand DNA synthesis kit (Toyobo, Osaka, Japan) at 42 °C for 20 min, followed by heating at 99 °C for 5 min. PCRs were carried out using appropriate IgM sense and antisense primers (such as IgM Hc for whole IgM H-chain mRNA, IgM Hm for its membrane-bound form, IgM Hs for its secreted form and IgM L for IgM L-chain), which were synthesized according to the EST data deposited in GenBank and listed in previous reports (Kikuchi and Nakayama, 2008; Kikuchi et al., 2005, 2011a, 2011b, 2012; Nakayama et al., 2007). Chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as internal control. PCR products were subjected to 1.5% agarose gel electrophoresis. Data obtained by semiquantitative RT-PCR before reaching the plateau were analyzed by Image Gauge software Profile mode (densitometric analysis mode) using a luminescent image analyzer LAS-1000plus. Nucleotide sequences of all amplified RT-PCR products obtained were confirmed by the PCR sequencing method as described (Takami et al., 1999). 2.5. ChIP assay ChIP assay was performed using Chromatin Immunoprecipitation Assay Kit (Millipore) as described (Kikuchi et al., 2011b, 2012). Briefly, formaldehyde-fixed cells (1 × 106) were lysed in the presence of 1 mM PMSF and 100 μg/ml aprotinin. Cell lysates were sonicated with Biorupter UCD-250 (Cosmo Bio, Tokyo, Japan) to shear DNA to lengths between 200 and 1000 bp and centrifuged at 12,000 ×g at 4 °C for 10 min. Immunoprecipitation with 2 μg anti-GCN5 antibody (or 2 μg irrelevant IgG as negative control) or 2 μl anti-acetylated histone antisera (or 2 μl normal rabbit serum as negative control) was carried out. Immunoprecipitated and input DNAs were analyzed by PCR using appropriate primers (H-ChIP primers; sense 5′-TTCCCGTTGGTTCTGTGC TC-3′ and antisense 5′-GTTGGAATCGAACCACGTGA-3′, L-ChIP primers; sense 5′-TACACAGCCATACATACGCG-3′ and antisense 5′-TTCGTTCAGC TCCTCCTTTG-3′) corresponding to constant regions of the chicken IgM H- and L-chains, respectively. PCRs using Ex Taq DNA polymerase were carried out at 96 °C for 20 s, 55 °C for 20 s and 72 °C for 30 s for 31–34 cycles, and stopped before reaching the plateau. PCR products were subjected to 1.5% agarose gel electrophoresis and analyzed using a luminescent image analyzer LAS-1000plus. 2.6. Statistical analysis
2.3. Immunoblotting The amounts of secreted IgM protein consisting of secreted form of IgM H-chain and IgM L-chain in culture media were measured by
Results (PCR and immunoblotting) are expressed as the mean ± standard deviation. Statistical differences were calculated with Student's t test.
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3. Results 3.1. GCN5-deficiency causes decreases in membrane-bound and secreted forms of IgM proteins
% Control
100 IgM proteins, the first-produced antibody isotype in vertebrates, are present in the membrane-bound form to make up the B cell receptor (complex of membrane-bound form of IgM H-chain and L-chain) and secreted form to act as soluble antibody (complex of secreted form of IgM H-chain and L-chain) (Kikuchi and Nakayama, 2008; Nakayama et al., 2007; Takami et al., 1999). DT40, a chicken immature B cell line, continuously produces both membrane-bound and secreted forms of IgM proteins (Nakayama et al., 2007; Takami et al., 1999). To examine influences of GCN5-deficiency on amounts of the membrane-bound form of IgM proteins, we first carried out flow cytometric analysis on DT40 and GCN5−/−. As shown in Fig. 1, the amounts of the membranebound form of IgM proteins were remarkably down-regulated on the cell surface of GCN5−/−. Similar results were obtained in two other independent GCN5−/− clones (data not shown). Next, we examined influences of GCN5-deficiency on amounts of the secreted form of IgM proteins in culture media by immunoblotting. As shown in Fig. 2, the amounts of both secreted form of IgM H-chain and IgM L-chain were remarkably decreased (to ~35% and to ~25%, respectively) in three independent GCN5−/− clones. These results suggested that GCN5 is involved in regulations of the amounts of IgM proteins (both membrane-bound and secreted forms of IgM proteins).
75 50
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The IgM H-chain gene is first transcribed as pre-mRNA that is thereafter alternatively processed into membrane-bound (IgM Hm) and secreted (IgM Hs) forms of mRNA (Takami et al., 1999). To study influences of GCN5-deficiency on transcriptions of IgM H-chain and L-chain genes, we carried out semiquantitative RT-PCR using appropriate primers IgM Hc, IgM Hm, IgM Hs and IgM L (Nakayama et al., 2007) on total RNAs prepared from three independent GCN5−/− clones and DT40 (Fig. 3). As expected, GCN5-deficiency showed significant decreases in the amounts of all forms of IgM H-chain mRNAs; IgM Hc
L-chain
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Cell number
3.2. GCN5-deficiency down-regulates gene expressions of IgM H-chain but not L-chain
GCN5 -/--1
DT40
Fig. 2. Influences of GCN5-deficiency on the amount of the secreted form of IgM protein in culture media. The final culture media of DT40 and three independent GCN5−/− clones were used as samples for SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-chicken IgM H- or L-chain antiserum. (A) Quantitative data represent averages of three independent GCN5−/− clones (solid bars) and are indicated as percentages of control values (100%) obtained from DT40 cells (gray bars). Error bars indicate standard deviation. Statistical differences were calculated with Student's t test. **p b 0.01. (B) Typical immunoblotting profiles.
(to ~ 32%), IgM Hm (to ~ 30%) and IgM Hs (to ~ 35%). In contrast, the amount of IgM L-chain mRNA remained unchanged in GCN5−/−. These findings suggested that the remarkable decreases in the amounts of both IgM Hm and IgM Hs mRNAs (derived from IgM H-chain premRNA) but not IgM L mRNA resulted in decreased amounts of both membrane-bound and secreted forms of IgM proteins in GCN5−/−. In addition, treatment with CPTH2, a specific GCN5 inhibitor (Chimenti et al., 2009; Kikuchi et al., 2011b), inhibited transcription of IgM H-chain (IgM Hm, IgM Hs and IgM Hc) but not L-chain in DT40 (Fig. S1). These data, together, revealed that GCN5 would be involved in transcriptional regulation of IgM H-chain but not L-chain. 3.3. GCN5 binds to the constant region of chicken IgM H-chain gene and contributes to acetylation of H3K9 within chromatin surrounding the region
Fluorescence intensity Fig. 1. Influences of GCN5-deficiency on the amount of the membrane-bound form of IgM protein on cell surface. DT40 and GCN5−/− cells were incubated with anti-chicken IgM antibody followed by fluorescein isothiocyanate-conjugated tyramide-labeled anti-goat IgG, and analyzed by a FACSCalibur. Normal goat serum was used as negative control (dotted lines). Data were plotted on linear histograms as fluorescence intensity (x axis) against relative cell number (y axis).
To determine whether or not GCN5 interacts with the chicken IgM H-chain gene and acetylates Lys residues of histone H3 within chromatin surrounding the gene, we performed ChIP assay using anti-GCN5 antibody and anti-acetylated Lys-9 (H3K9) and Lys-14 (H3K14) residues of histone H3 antisera. These two Lys residues of histone H3 are typical acetylation sites catalyzed by GCN5 (Allis et al., 2007; Grant et al., 1999; Kikuchi et al., 2011b, 2012; Lee and Workman, 2007; Shimada et al., 2008; Suka et al., 2001). Cross-linked chromatins were co-precipitated from cell lysates of DT40 and GCN5−/− with each of these three
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Fig. 3. Influences of GCN5-deficiency on transcriptions of IgM H- and L-chain genes. Total RNAs were extracted from DT40 and three independent GCN5−/− clones, and mRNA levels of IgM H- and L-chains were determined by semiquantitative RT-PCR using appropriate primers (IgM Hc, whole IgM H-chain; IgM Hm, membrane-bound form of IgM H-chain; IgM Hs, secreted form of IgM H-chain; IgM L, IgM L-chain). Chicken GAPDH gene was used as internal control for calibration. (A) Quantitative data represent averages of three independent GCN5−/− clones (solid bars) and are indicated as percentages of control values (100%) obtained from DT40 cells (gray bars). Error bars indicate standard deviation. Statistical differences were calculated with Student's t test. **p b 0.01. (B) Typical semiquantitative RT-PCR profiles. Numbers below the panels indicate PCR cycle numbers.
antibody agents. Precipitated chromatins were amplified by PCR using specific H-ChIP or L-ChIP primers for the constant region of the chicken IgM H- or L-chain gene. Concerning GCN5, amplified DNA fragment
A
using H-ChIP primers could be detected in DT40 but not in GCN5−/−, but no signal using L-ChIP primers could be found in either DT40 or GCN5−/− (Fig. 4A). Regarding acetylated Lys-residues of histone H3, in
C irrelevant anti- irrelevant antiIgG GCN5 IgG GCN5
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normal anti- normal antiserum serum serum serum
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Fig. 4. GCN5 binds to constant region of IgM H-chain gene and acetylates Lys-9 residues of histone H3 within chromatin surrounding the region. (A) Interaction of GCN5 with the constant region of chicken IgM H-chain gene. The cross-linked chromatins from cell lysates of DT40 and GCN5−/− were co-precipitated by anti-GCN5 antibody. Irrelevant IgG was used as negative control. After de-cross-linking, co-precipitated chromatins (ChIP) and input samples (Input) were amplified by PCR using specific primers (H-ChIP and L-ChIP primers). PCR products were analyzed by 1.5% agarose gel electrophoresis. Typical patterns are shown. (B) Acetylation level of Lys residues of histone H3 within chromatin surrounding the constant region of the chicken IgM H-chain gene. ChIP assay was carried out as in (A) using antisera specific for acetylated H3K9 (Ac-H3K9) and H3K14 (Ac-H3K14). Rabbit normal serum was used as negative control. Typical patterns are shown. (C) Quantitative data for acetylation level of Lys residues of histone H3 within chromatin surrounding the constant region of the chicken IgM H-chain gene. The cross-linked chromatins from cell lysates of DT40 (open bars) and GCN5−/− (gray bars) were co-precipitated by antisera specific for acetylated H3K9 (Ac-H3K9) and H3K14 (Ac-H3K14). Data are indicated as percentages of control values (100%) obtained from DT40 and represent averages of three separate experiments including results shown in (B). Error bars indicate standard deviation. Statistical differences were calculated with Student's t test. **p b 0.01.
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GCN5−/− the acetylation level of H3K9 residues within chromatin surrounding the constant region of the IgM H-chain gene was remarkably decreased (to ~22% of control value obtained from DT40), but insignificant effect was observed on the acetylation level of H3K14 (Fig. 4B and C). The ChIP assay using antibodies of both GCN5 and acetylated Lysresidues of histone H3 showed similar results for two other GCN5−/− clones (data not shown). These results revealed that GCN5 binds near constant region of the chicken IgM H-chain gene and thereby acetylates H3K9 residues within chromatin surrounding the region in vivo, suggesting that GCN5 is certainly involved in the transcription regulation of the IgM H-chain gene in vertebrate immature B cells. 4. Discussion In vertebrate cells, tens of thousands of gene expressions are regulated by HATs, HDACs and others. However, less than 20 members of HATs have been found in vertebrates so far (Allis et al., 2007). Studies on specific and overlapped functions of HATs would be very important because these functions show redundancy. GCN5, one of the most important HATs, is well-known as the first-identified coactivator with transcription-related HAT activity (Brownell et al., 1996; Nagy and Tora, 2007). Importantly, mice lacking GCN5 die with mesodermal defects during embryogenesis (Xu et al., 2000; Yamauchi et al., 2000). These findings suggested that GCN5 has specialized functions necessary for life support systems in vertebrates. In fact, GCN5 plays specialized roles in transcriptional activation of various genes through chromatin structure changes (Grant et al., 1999; Kikuchi et al., 2011a, 2011b, 2012; Nagy and Tora, 2007; Shimada et al., 2008; Suka et al., 2001). Here, in addition to the above-mentioned functions, we revealed a new inherent function of GCN5 in B cells: transcriptional regulation of the IgM H-chain gene. GCN5-deficiency caused remarkable decreases in membrane-bound and secreted forms of IgM proteins (Figs. 1 and 2), based on significant decreases in amounts of all forms of IgM H-chain mRNA; IgM Hc, IgM Hm and IgM Hs (Fig. 3), indicating that GCN5 is involved in regulations of both mRNA and protein levels of IgM H-chain. By contrast, while transcription of the IgM L-chain gene remained unchanged (Fig. 3), the amount of IgM L-chain protein secreted into the culture media in GCN5−/− was remarkably decreased (Fig. 2). These data suggested that GCN5 is not directly involved in transcription regulation of the IgM L-chain gene, and the decreased protein level of IgM L-chain in the culture media may be due to limited formation of mature complex of IgM H- and L-chains (i.e. secreted form of IgM protein). Moreover, ChIP assay revealed that GCN5 binds to the chicken IgM H-chain gene around its constant region and acetylates H3K9 residues but not H3K14 (Fig. 4). These data agreed with our previous data; GCN5-deficiency in DT40 cells led to decreased bulk acetylation level of H3K9 but not H3K14 (Kikuchi et al., 2005, 2011b, 2012). As is well-known, histone acetylation catalyzed by HATs reduces the binding affinity between histones and DNA, and thereby constructs euchromatin that allows access of various trans-acting factors to target genes (Biel et al., 2005; Jenuwein and Allis, 2001; Selvi and Kundu, 2009). Taken together, our results revealed that GCN5 is involved in acetylation of H3K9 residues within chromatin around the coding region of the IgM H-chain gene followed by activation of its transcription. Unfortunately, we could not perform ChIP assay using primers corresponding to nucleotide sequences around the 5′-flanking region of the IgM H-chain gene because of lack of information on them in chickens. However, our results obtained in this study strongly suggested that GCN5 plays a key role in transcription regulation of the IgM H-chain gene through euchromatin formation around its coding region based on histone acetylation. Needless to say, the behavior of GCN5 around the promoter region of the IgM H-chain gene should be elucidated in the future. Histone-modifying enzymes are well-known to be related to human diseases through various unusual epigenetic events (Butler et al., 2012). For instance, unbalance of histone acetylation level due to abnormalities of HATs and/or HDACs could lead to generation of cancer (Selvi and
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Kundu, 2009) and occurrence of autoimmune disease (Javierre et al., 2011). This report is the first case on a novel function of GCN5 as an effective transcription activator of IgM H-chain gene expression and may significantly help in the understanding of mechanisms specific for B cell normal and pathological functions. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.04.030. Conflict of interest The authors have no financial conflicts of interest. Acknowledgments We thank R. Masuya and N. Nagamatsu-Yamamoto for technical support and H. Madhyastha and R. Madhyastha for editorial reading of the manuscript. This work was supported in part by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 25430170, to HK). This work was also partly supported by the Grant for Joint Research Project of the Institute of Medical Science, The University of Tokyo (No. 2013230). References Allis, C.D., Berger, S.L., Cote, J., Dent, S., Jenuwien, T., Kouzarides, T., Pillus, L., Reinberg, D., Shi, Y., Shiekhatter, R., Shilatifard, A., Workman, J., Zhang, Y., 2007. New nomenclature for chromatin-modifiying enzymes. Cell 131, 633–636. Berger, S.L., 2007. The complex language of chromatin regulation during transcription. Nature 447, 407–412. Bernstein, B.E., Meissner, A., Lander, E.S., 2007. The mammalian epigenome. Cell 128, 669–681. Biel, M., Wascholowski, V., Giannis, A., 2005. Epigenetics—an epicenter of gene regulation: histones and histone-modifying enzymes. Angewandte Chemie International Edition in English 20, 3186–3216. Brownell, J.E., Zhou, J., Ranalli, T., Kobayashi, R., Edomondson, D.G., Roth, S.Y., Allis, C.D., 1996. Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell 84, 843–851. Buerstedde, J.M., Takeda, S., 1991. Increased ration of targeted to random integration after transfection of chicken B cell lines. Cell 67, 179–188. Butler, J.S., Koutelou, E., Schibler, A.C., Dent, S.Y., 2012. Histone-modifying enzymes: regulators of developmental decisions and drivers of human disease. Epigenomics 4, 163–177. Chimenti, F., Bizzarri, B., Maccioni, E., Secci, D., Bolasco, A., Chimenti, P., Fioravanti, R., Granese, A., Carradori, S., Tosi, F., Ballario, P., Vernarecci, S., Filetici, P., 2009. A novel histone acetyltransferase inhibitor modulating Gcn5 network: cyclopentylidene-[4(4′-chlorophenyl)thiazol-2-yl]hydrazone. Journal of Medicinal Chemistry 52, 530–536. Espada, J., Esteller, M., 2007. Epigenetic control of nuclear architecture. Cellular and Molecular Life Sciences 64, 449–457. Goldberg, A.D., Allis, C.D., Bernstein, E., 2007. Epigenetics: a landscape takes shape. Cell 128, 635–638. Grant, P.A., Eberharter, A., John, S., Cook, R.G., Turner, B.M., Workman, J.L., 1999. Expanded lysine acetylation specificity of Gcn5 in native complexes. Journal of Biological Chemistry 274, 5895–5900. Javierre, B.M., Hernando, H., Ballestar, E., 2011. Environmental triggers and epigenetic deregulation in autoimmune disease. Discovery Medicine 12, 535–545. Jenuwein, T., Allis, C.D., 2001. Translating the histone code. Science 293, 1074–1080. Kikuchi, H., Nakayama, T., 2008. GCN5 and BCR signalling collaborate to induce premature B cell apoptosis through depletion of ICAD and IAP2 and activation of caspase activities. Gene 419, 48–55. Kikuchi, H., Takami, Y., Nakayama, T., 2005. GCN5: a supervisor in all-inclusive control of vertebrate cell cycle progression through transcription regulation of various cell cycle-related genes. Gene 347, 83–97. Kikuchi, H., Barman, H.K., Nakayama, M., Takami, Y., Nakayama, T., 2006. Participation of histones, histone modifying enzymes and histone chaperons in vertebrate cell functions. In: Buerstedde, J.M., Takeda, S. (Eds.), Reviews and Protocols in DT40 Research. Springer-Verlag, Berlin, pp. 225–243. Kikuchi, H., Kuribayashi, F., Takami, Y., Imajoh-Ohmi, S., Nakayama, T., 2011a. GCN5 regulates the activation of PI3K/Akt survival pathway in B cells exposed to oxidative stress via controlling gene expressions of Syk and Btk. Biochemical and Biophysical Research Communications 405, 657–661. Kikuchi, H., Kuribayashi, F., Kiwaki, N., Takami, Y., Nakayama, T., 2011b. GCN5 regulates the superoxide-generating system in leukocytes via controlling gp91-phox gene expression. Journal of Immunology 186, 3015–3022. Kikuchi, H., Kuribayashi, F., Imajoh-Ohmi, S., Nishitoh, H., Takami, Y., Nakayama, T., 2012. GCN5 protects vertebrate cells against UV–irradiation via controlling gene expression of DNA polymerase η. Journal of Biological Chemistry 287, 39842–39849.
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Knowling, S., Morris, K.V., 2011. Non-coding RNA and antisense RNA. Nature's trash or treasure? Biochimie 93, 1922–1927. Kouzarides, T., 2007. Chromatin modifications and their function. Cell 128, 693–705. Kurosawa, K., Lin, W., Ohta, K., 2010. Distinct roles of HDAC1 and HDAC2 in transcription and recombination at the immunoglobulin loci in the chicken B cell line DT40. Journal of Biochemistry 148, 201–207. Lee, K.K., Workman, J.L., 2007. Histone acetyltransferase complexes: one size doesn't fit all. Nature Reviews Molecular Cell Biology 8, 284–295. Lee, S.C., Bottaro, A., Insel, R.A., 2003. Activation of terminal differentiation by inhibition of histone deacetylation. Molecular Immunology 39, 923–932. Lu, Z.P., Ju, Z.L., Shi, G.Y., Zhang, J.W., Sun, J., 2005. Histone deacetylase inhibitor Trichostatin A reduces anti-DNA autoantibody production and represses IgH gene transcription. Biochemical and Biophysical Research Communications 330, 204–209. Nagy, Z., Tora, L., 2007. Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation. Oncogene 26, 5341–5357. Nakayama, M., Suzuki, H., Yamamoto-Nagamatsu, N., Barman, H.K., Kikuchi, H., Takami, Y., Toyonaga, K., Yamashita, K., Nakayama, T., 2007. HDAC2 controls IgM H- and L-chain gene expressions via EBF1, Pax5, Ikaros, Aiolos and E2A gene expressions. Genes to Cells 12, 359–373. Selvi, R.B., Kundu, T.K., 2009. Reversible acetylation of chromatin: implication in regulation of gene expression, disease and therapeutics. Biotechnology Journal 4, 375–390.
Shimada, M., Niida, H., Zineldeen, D.H., Tagami, H., Tanaka, M., Saito, H., Nakanishi, M., 2008. Chk1 is a histone H3 threonine 11 kinase that regulates DNA damageinduced transcriptional repression. Cell 132, 221–232. Suka, N., Suka, Y., Carmen, A.A., Wu, J., Grunstein, M., 2001. Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin. Molecular Cell 8, 473–479. Takami, Y., Kikuchi, H., Nakayama, T., 1999. Chicken histone deacetylase-2 controls the amount of the IgM H-chain at the steps of both transcription of its gene and alternative processing of its pre-mRNA in the DT40 cell line. Journal of Biological Chemistry 274, 23977–23990. Takechi, S., Adachi, M., Nakayama, T., 2002. Chicken HDAC2 down-regulates IgM light chain gene promoter activity. Biochemical and Biophysical Research Communications 299, 263–267. Xu, W., Edmondson, D.G., Evrard, Y.A., Wakamiya, M., Behringer, R.R., Roth, S.Y., 2000. Loss of Gcn5l2 leads to increased apoptosis and mesodermal defects during mouse development. Nature Genetics 26, 229–232. Yamauchi, T., Yamauchi, J., Kuwata, T., Tamura, T., Yamashita, T., Bae, N., Westphal, H., Ozato, K., Nakatani, Y., 2000. Distinct but overlapping roles of histone acetylase PCAF and of the closely related PCAF-B/GCN5 in mouse embryogenesis. Proceedings of the National Academy of Sciences of the United States of America 97, 11303–11306.
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Gene journal homepage: www.elsevier.com/locate/gene
GCN5 is involved in regulation of immunoglobulin heavy chain gene expression in immature B cells Hidehiko Kikuchi a,b,⁎, Masami Nakayama a, Futoshi Kuribayashi b,c, Shinobu Imajoh-Ohmi b, Hideki Nishitoh a, Yasunari Takami a, Tatsuo Nakayama a a b c
Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan Laboratory Center for Proteomics Research, Graduate School of Frontier Sciences, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Tokyo 108-8639, Japan Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan
a r t i c l e
i n f o
Article history: Received 6 November 2013 Received in revised form 13 April 2014 Accepted 16 April 2014 Available online 18 April 2014 Keywords: GCN5 Gene targeting Histone acetyltransferase Immunoglobulin DT40
a b s t r a c t GCN5 is involved in the acetylation of core histones, which is an important epigenetic event for transcriptional regulation through alterations in the chromatin structure in eukaryotes. To investigate physiological roles of GCN5, we have systematically analyzed phenotypes of homozygous GCN5-deficient DT40 mutants. Here, we report participation of GCN5 in regulation of IgM heavy chain (H-chain) gene expression. GCN5-deficiency downregulates gene expressions of IgM H-chain (as whole, membrane-bound and secreted forms of its mRNA) but not light chain (L-chain), causing decreases in membrane-bound and secreted forms of IgM proteins. Chromatin immnoprecipitation assay revealed that GCN5 binds to the chicken IgM H-chain gene around its constant region but not L-chain gene, and acetylate Lys-9 residues of histone H3 within chromatin surrounding the constant region. These results suggest that GCN5 takes part in transcriptional regulation of the IgM H-chain gene via histone acetylation resulting in formation of relaxed chromatin arrangement around its coding region and plays a key role in epigenetic regulation of B cell functions. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Epigenetics can be defined as alterations in cellular phenotypes via control of gene expression without altering DNA sequences (Berger, 2007; Bernstein et al., 2007; Biel et al., 2005; Goldberg et al., 2007). Gene expression is regulated by several epigenetic mechanisms; histone modification, DNA methylation, small and non-coding RNAs and chromatin architecture (Berger, 2007; Bernstein et al., 2007; Biel et al., 2005; Espada and Esteller, 2007; Goldberg et al., 2007; Knowling and Morris, 2011). Histone modification is one of the most common and important epigenetic mechanisms (Berger, 2007; Bernstein et al., 2007; Biel et al., 2005; Butler et al., 2012; Goldberg et al., 2007; Jenuwein and Allis, 2001; Kouzarides, 2007). Core histones are subjected to various post-translational modifications including acetylation, methylation, phosphorylation, ubiquitination, sumoylation, etc. (Biel et al., 2005; Butler et al., 2012; Jenuwein and Allis, 2001; Kouzarides,
Abbreviations: ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde 3phosphate dehydrogenase; H-chain, heavy chain; H3K14, Lys-14 of histone H3; H3K9, Lys-9 of histone H3; HAT, histone acetyltransferase; HDAC, histone deacetylase; L-chain, light chain; RT-PCR, reverse transcription-polymerase chain reaction. ⁎ Corresponding author at: Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. E-mail address: [email protected] (H. Kikuchi).
http://dx.doi.org/10.1016/j.gene.2014.04.030 0378-1119/© 2014 Elsevier B.V. All rights reserved.
2007). In particular, histone acetylation regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) plays critical roles in the modulation of chromatin topology and thereby control of gene expressions (Biel et al., 2005; Jenuwein and Allis, 2001; Selvi and Kundu, 2009). Particular Lys residues at N-terminal tails of core histones have positive charges and tightly bind to DNA, resulting in the formation of heterochromatin. Acetylation catalyzed by HATs reduces positive charges of these Lys residues at the N-terminal histone tails and promotes euchromatin formation to allow easier access of transfactors to their target genes. In contrast, HDACs remove the acetyl groups from the N-terminal histone tails to silence gene expressions. Taken together, HATs and HDACs play essential roles in the regulation of gene expressions via collaboration of histone acetylation and deacetylation. We previously reported that in the chicken immature B cell line DT40, HDAC2 controls the amount of IgM heavy chain (H-chain) at the steps of transcription of its gene and alternative processing of its pre-mRNA (Takami et al., 1999) and down-regulates IgM light chain (L-chain) gene promoter activity (Takechi et al., 2002). As a result, DT40 cells continuously produce both membrane-bound and secreted forms of IgM proteins as complex of IgM H- and L-chains. In addition, HDAC2 regulates IgM H- and L-chain gene expressions via EBF1, Pax5, Ikaros, Aiolos and E2A gene expressions (Nakayama et al., 2007). These data have progressed studies on immunoglobulin gene regulations by HDACs as triggers. Studies using trichostatin A, a specific HDAC
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inhibitor, revealed that histone acetylation regulates transcription of genes controlling terminal B cell differentiation (Lee et al., 2003), and HDAC is involved in the immunoglobulin H-chain gene transcription via activation of 3′-IgH enhancers (Lu et al., 2005). It was also shown that there are distinct roles of HDAC1 and HDAC2 in transcription at the immunoglobulin loci in DT40 (Kurosawa et al., 2010). However, the understanding of physiological roles of HATs in immunoglobulin gene regulations is still poor in vertebrate cells. GCN5, a prototypical HAT, functions as a coactivator to promote transcriptional activation with enzymatic modification at chromatin (Brownell et al., 1996). To investigate physiological roles of GCN5, we generated homozygous GCN5-deficient DT40 mutants, GCN5−/−, by gene targeting techniques, which are excellent methods to study physiological roles of many genes involved in expressions of important cell functions of higher eukaryotes, including histone acetylation and deacetylation (Buerstedde and Takeda, 1991; Kikuchi et al., 2006). Our previous studies have revealed that GCN5-deficiency caused not only delayed growth rate, suppressed cell cycle progression at G1/S phase transition, and down- or up-regulated various G1/S phase transition-related genes (Kikuchi et al., 2005), but also drastic resistance against apoptosis induced by the B cell receptor-mediated stimulation through transcriptional regulation of various apoptosis-related genes (Kikuchi and Nakayama, 2008). In addition, GCN5 activated phosphatidylinositol 3-kinase/Akt survival pathway in cells exposed to oxidative stress via controlling gene expressions of Syk and Btk (Kikuchi et al., 2011a) and promoted the superoxide-generating system in leukocytes via controlling the gp91-phox gene expression (Kikuchi et al., 2011b). Very recently, we clarified that GCN5 is involved in UV–tolerance via controlling gene expression of DNA polymerase η (Kikuchi et al., 2012). In this study, we investigated the effects of GCN5-deficiency on amounts of IgM proteins and expressions of IgM H-chain and L-chain genes. GCN5-deficiency caused remarkable down-regulation of transcriptions of IgM H-chain gene but not L-chain gene. Results obtained in this study including chromatin immunoprecipitation (ChIP) assay revealed that GCN5 is involved in regulation of IgM H-chain gene expression through acetylation of Lys-9 residues of histone H3 within chromatin surrounding its constant region in immature B cells. 2. Materials and methods 2.1. Materials Bovine aprotinin (Sigma-Aldrich, St. Louis, MO, USA), PMSF (Wako, Osaka, Japan), monoclonal anti-GCN5 antibody (Chemicon, Temecula, CA, USA), Ex Taq DNA polymerase (Takara, Shiga, Japan), goat antichicken L-chain antisera and goat anti-chicken IgM H-chain antisera (Bethyl Laboratories, Montgomery, TX, USA), anti-acetylated histone antisera and protein G agarose/salmon sperm DNA (Millipore, Billerica, MA, USA), fluorescein isothiocyanate-conjugated tyramide-labeled anti-goat IgG (American Qualex, San Clemente, CA, USA), and horse radish peroxidase-conjugated anti-goat immunoglobulin (DAKO, Inc., Glostrup, Denmark) were used. 2.2. Cell cultures and flow cytometric analysis Generation of GCN5−/− was described in our previous report (Kikuchi et al., 2005). DT40 and GCN5−/− were cultured essentially as described (Kikuchi and Nakayama, 2008; Kikuchi et al., 2005, 2011a, 2011b, 2012). The cell surface membrane-bound IgM protein (as B cell receptor) was analyzed with a FACSCalibur (BD Biosciences, San Jose, CA, USA) as described (Takami et al., 1999).
immunoblotting as described (Nakayama et al., 2007; Takami et al., 1999). The final culture media (1 ml) containing equal cell numbers of GCN5−/− or DT40 (1 × 106) were centrifuged at 1000 ×g at 4 °C for 5 min, and equal aliquots (2.5 μl) of the supernatants were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and electrotransferred onto polyvinyliden difluoride membranes. The protein-transferred membranes were immersed in a 5% skim milk solution in Tris–buffered saline and incubated with goat anti-chicken IgM H- or L-chain antiserum at 4 °C overnight. Antibody binding was detected using horse radish peroxidase-conjugated anti-goat immunoglobulin as a second antibody and SuperSignal west Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) as a substrate. Data analyses were carried out using a luminescent image analyzer LAS-1000plus (Fujifilm, Tokyo, Japan). 2.4. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) RT-PCR was carried out essentially as described (Kikuchi and Nakayama, 2008; Kikuchi et al., 2005, 2011a, 2011b, 2012). Total RNAs were isolated from DT40 and its mutant clones. RT was performed with a first strand DNA synthesis kit (Toyobo, Osaka, Japan) at 42 °C for 20 min, followed by heating at 99 °C for 5 min. PCRs were carried out using appropriate IgM sense and antisense primers (such as IgM Hc for whole IgM H-chain mRNA, IgM Hm for its membrane-bound form, IgM Hs for its secreted form and IgM L for IgM L-chain), which were synthesized according to the EST data deposited in GenBank and listed in previous reports (Kikuchi and Nakayama, 2008; Kikuchi et al., 2005, 2011a, 2011b, 2012; Nakayama et al., 2007). Chicken glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as internal control. PCR products were subjected to 1.5% agarose gel electrophoresis. Data obtained by semiquantitative RT-PCR before reaching the plateau were analyzed by Image Gauge software Profile mode (densitometric analysis mode) using a luminescent image analyzer LAS-1000plus. Nucleotide sequences of all amplified RT-PCR products obtained were confirmed by the PCR sequencing method as described (Takami et al., 1999). 2.5. ChIP assay ChIP assay was performed using Chromatin Immunoprecipitation Assay Kit (Millipore) as described (Kikuchi et al., 2011b, 2012). Briefly, formaldehyde-fixed cells (1 × 106) were lysed in the presence of 1 mM PMSF and 100 μg/ml aprotinin. Cell lysates were sonicated with Biorupter UCD-250 (Cosmo Bio, Tokyo, Japan) to shear DNA to lengths between 200 and 1000 bp and centrifuged at 12,000 ×g at 4 °C for 10 min. Immunoprecipitation with 2 μg anti-GCN5 antibody (or 2 μg irrelevant IgG as negative control) or 2 μl anti-acetylated histone antisera (or 2 μl normal rabbit serum as negative control) was carried out. Immunoprecipitated and input DNAs were analyzed by PCR using appropriate primers (H-ChIP primers; sense 5′-TTCCCGTTGGTTCTGTGC TC-3′ and antisense 5′-GTTGGAATCGAACCACGTGA-3′, L-ChIP primers; sense 5′-TACACAGCCATACATACGCG-3′ and antisense 5′-TTCGTTCAGC TCCTCCTTTG-3′) corresponding to constant regions of the chicken IgM H- and L-chains, respectively. PCRs using Ex Taq DNA polymerase were carried out at 96 °C for 20 s, 55 °C for 20 s and 72 °C for 30 s for 31–34 cycles, and stopped before reaching the plateau. PCR products were subjected to 1.5% agarose gel electrophoresis and analyzed using a luminescent image analyzer LAS-1000plus. 2.6. Statistical analysis
2.3. Immunoblotting The amounts of secreted IgM protein consisting of secreted form of IgM H-chain and IgM L-chain in culture media were measured by
Results (PCR and immunoblotting) are expressed as the mean ± standard deviation. Statistical differences were calculated with Student's t test.
H. Kikuchi et al. / Gene 544 (2014) 19–24
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A
3. Results 3.1. GCN5-deficiency causes decreases in membrane-bound and secreted forms of IgM proteins
% Control
100 IgM proteins, the first-produced antibody isotype in vertebrates, are present in the membrane-bound form to make up the B cell receptor (complex of membrane-bound form of IgM H-chain and L-chain) and secreted form to act as soluble antibody (complex of secreted form of IgM H-chain and L-chain) (Kikuchi and Nakayama, 2008; Nakayama et al., 2007; Takami et al., 1999). DT40, a chicken immature B cell line, continuously produces both membrane-bound and secreted forms of IgM proteins (Nakayama et al., 2007; Takami et al., 1999). To examine influences of GCN5-deficiency on amounts of the membrane-bound form of IgM proteins, we first carried out flow cytometric analysis on DT40 and GCN5−/−. As shown in Fig. 1, the amounts of the membranebound form of IgM proteins were remarkably down-regulated on the cell surface of GCN5−/−. Similar results were obtained in two other independent GCN5−/− clones (data not shown). Next, we examined influences of GCN5-deficiency on amounts of the secreted form of IgM proteins in culture media by immunoblotting. As shown in Fig. 2, the amounts of both secreted form of IgM H-chain and IgM L-chain were remarkably decreased (to ~35% and to ~25%, respectively) in three independent GCN5−/− clones. These results suggested that GCN5 is involved in regulations of the amounts of IgM proteins (both membrane-bound and secreted forms of IgM proteins).
75 50
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The IgM H-chain gene is first transcribed as pre-mRNA that is thereafter alternatively processed into membrane-bound (IgM Hm) and secreted (IgM Hs) forms of mRNA (Takami et al., 1999). To study influences of GCN5-deficiency on transcriptions of IgM H-chain and L-chain genes, we carried out semiquantitative RT-PCR using appropriate primers IgM Hc, IgM Hm, IgM Hs and IgM L (Nakayama et al., 2007) on total RNAs prepared from three independent GCN5−/− clones and DT40 (Fig. 3). As expected, GCN5-deficiency showed significant decreases in the amounts of all forms of IgM H-chain mRNAs; IgM Hc
L-chain
25K
Cell number
3.2. GCN5-deficiency down-regulates gene expressions of IgM H-chain but not L-chain
GCN5 -/--1
DT40
Fig. 2. Influences of GCN5-deficiency on the amount of the secreted form of IgM protein in culture media. The final culture media of DT40 and three independent GCN5−/− clones were used as samples for SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-chicken IgM H- or L-chain antiserum. (A) Quantitative data represent averages of three independent GCN5−/− clones (solid bars) and are indicated as percentages of control values (100%) obtained from DT40 cells (gray bars). Error bars indicate standard deviation. Statistical differences were calculated with Student's t test. **p b 0.01. (B) Typical immunoblotting profiles.
(to ~ 32%), IgM Hm (to ~ 30%) and IgM Hs (to ~ 35%). In contrast, the amount of IgM L-chain mRNA remained unchanged in GCN5−/−. These findings suggested that the remarkable decreases in the amounts of both IgM Hm and IgM Hs mRNAs (derived from IgM H-chain premRNA) but not IgM L mRNA resulted in decreased amounts of both membrane-bound and secreted forms of IgM proteins in GCN5−/−. In addition, treatment with CPTH2, a specific GCN5 inhibitor (Chimenti et al., 2009; Kikuchi et al., 2011b), inhibited transcription of IgM H-chain (IgM Hm, IgM Hs and IgM Hc) but not L-chain in DT40 (Fig. S1). These data, together, revealed that GCN5 would be involved in transcriptional regulation of IgM H-chain but not L-chain. 3.3. GCN5 binds to the constant region of chicken IgM H-chain gene and contributes to acetylation of H3K9 within chromatin surrounding the region
Fluorescence intensity Fig. 1. Influences of GCN5-deficiency on the amount of the membrane-bound form of IgM protein on cell surface. DT40 and GCN5−/− cells were incubated with anti-chicken IgM antibody followed by fluorescein isothiocyanate-conjugated tyramide-labeled anti-goat IgG, and analyzed by a FACSCalibur. Normal goat serum was used as negative control (dotted lines). Data were plotted on linear histograms as fluorescence intensity (x axis) against relative cell number (y axis).
To determine whether or not GCN5 interacts with the chicken IgM H-chain gene and acetylates Lys residues of histone H3 within chromatin surrounding the gene, we performed ChIP assay using anti-GCN5 antibody and anti-acetylated Lys-9 (H3K9) and Lys-14 (H3K14) residues of histone H3 antisera. These two Lys residues of histone H3 are typical acetylation sites catalyzed by GCN5 (Allis et al., 2007; Grant et al., 1999; Kikuchi et al., 2011b, 2012; Lee and Workman, 2007; Shimada et al., 2008; Suka et al., 2001). Cross-linked chromatins were co-precipitated from cell lysates of DT40 and GCN5−/− with each of these three
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A
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Fig. 3. Influences of GCN5-deficiency on transcriptions of IgM H- and L-chain genes. Total RNAs were extracted from DT40 and three independent GCN5−/− clones, and mRNA levels of IgM H- and L-chains were determined by semiquantitative RT-PCR using appropriate primers (IgM Hc, whole IgM H-chain; IgM Hm, membrane-bound form of IgM H-chain; IgM Hs, secreted form of IgM H-chain; IgM L, IgM L-chain). Chicken GAPDH gene was used as internal control for calibration. (A) Quantitative data represent averages of three independent GCN5−/− clones (solid bars) and are indicated as percentages of control values (100%) obtained from DT40 cells (gray bars). Error bars indicate standard deviation. Statistical differences were calculated with Student's t test. **p b 0.01. (B) Typical semiquantitative RT-PCR profiles. Numbers below the panels indicate PCR cycle numbers.
antibody agents. Precipitated chromatins were amplified by PCR using specific H-ChIP or L-ChIP primers for the constant region of the chicken IgM H- or L-chain gene. Concerning GCN5, amplified DNA fragment
A
using H-ChIP primers could be detected in DT40 but not in GCN5−/−, but no signal using L-ChIP primers could be found in either DT40 or GCN5−/− (Fig. 4A). Regarding acetylated Lys-residues of histone H3, in
C irrelevant anti- irrelevant antiIgG GCN5 IgG GCN5
H-chain
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normal anti- normal antiserum serum serum serum
Ac-H3K9
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Fig. 4. GCN5 binds to constant region of IgM H-chain gene and acetylates Lys-9 residues of histone H3 within chromatin surrounding the region. (A) Interaction of GCN5 with the constant region of chicken IgM H-chain gene. The cross-linked chromatins from cell lysates of DT40 and GCN5−/− were co-precipitated by anti-GCN5 antibody. Irrelevant IgG was used as negative control. After de-cross-linking, co-precipitated chromatins (ChIP) and input samples (Input) were amplified by PCR using specific primers (H-ChIP and L-ChIP primers). PCR products were analyzed by 1.5% agarose gel electrophoresis. Typical patterns are shown. (B) Acetylation level of Lys residues of histone H3 within chromatin surrounding the constant region of the chicken IgM H-chain gene. ChIP assay was carried out as in (A) using antisera specific for acetylated H3K9 (Ac-H3K9) and H3K14 (Ac-H3K14). Rabbit normal serum was used as negative control. Typical patterns are shown. (C) Quantitative data for acetylation level of Lys residues of histone H3 within chromatin surrounding the constant region of the chicken IgM H-chain gene. The cross-linked chromatins from cell lysates of DT40 (open bars) and GCN5−/− (gray bars) were co-precipitated by antisera specific for acetylated H3K9 (Ac-H3K9) and H3K14 (Ac-H3K14). Data are indicated as percentages of control values (100%) obtained from DT40 and represent averages of three separate experiments including results shown in (B). Error bars indicate standard deviation. Statistical differences were calculated with Student's t test. **p b 0.01.
H. Kikuchi et al. / Gene 544 (2014) 19–24
GCN5−/− the acetylation level of H3K9 residues within chromatin surrounding the constant region of the IgM H-chain gene was remarkably decreased (to ~22% of control value obtained from DT40), but insignificant effect was observed on the acetylation level of H3K14 (Fig. 4B and C). The ChIP assay using antibodies of both GCN5 and acetylated Lysresidues of histone H3 showed similar results for two other GCN5−/− clones (data not shown). These results revealed that GCN5 binds near constant region of the chicken IgM H-chain gene and thereby acetylates H3K9 residues within chromatin surrounding the region in vivo, suggesting that GCN5 is certainly involved in the transcription regulation of the IgM H-chain gene in vertebrate immature B cells. 4. Discussion In vertebrate cells, tens of thousands of gene expressions are regulated by HATs, HDACs and others. However, less than 20 members of HATs have been found in vertebrates so far (Allis et al., 2007). Studies on specific and overlapped functions of HATs would be very important because these functions show redundancy. GCN5, one of the most important HATs, is well-known as the first-identified coactivator with transcription-related HAT activity (Brownell et al., 1996; Nagy and Tora, 2007). Importantly, mice lacking GCN5 die with mesodermal defects during embryogenesis (Xu et al., 2000; Yamauchi et al., 2000). These findings suggested that GCN5 has specialized functions necessary for life support systems in vertebrates. In fact, GCN5 plays specialized roles in transcriptional activation of various genes through chromatin structure changes (Grant et al., 1999; Kikuchi et al., 2011a, 2011b, 2012; Nagy and Tora, 2007; Shimada et al., 2008; Suka et al., 2001). Here, in addition to the above-mentioned functions, we revealed a new inherent function of GCN5 in B cells: transcriptional regulation of the IgM H-chain gene. GCN5-deficiency caused remarkable decreases in membrane-bound and secreted forms of IgM proteins (Figs. 1 and 2), based on significant decreases in amounts of all forms of IgM H-chain mRNA; IgM Hc, IgM Hm and IgM Hs (Fig. 3), indicating that GCN5 is involved in regulations of both mRNA and protein levels of IgM H-chain. By contrast, while transcription of the IgM L-chain gene remained unchanged (Fig. 3), the amount of IgM L-chain protein secreted into the culture media in GCN5−/− was remarkably decreased (Fig. 2). These data suggested that GCN5 is not directly involved in transcription regulation of the IgM L-chain gene, and the decreased protein level of IgM L-chain in the culture media may be due to limited formation of mature complex of IgM H- and L-chains (i.e. secreted form of IgM protein). Moreover, ChIP assay revealed that GCN5 binds to the chicken IgM H-chain gene around its constant region and acetylates H3K9 residues but not H3K14 (Fig. 4). These data agreed with our previous data; GCN5-deficiency in DT40 cells led to decreased bulk acetylation level of H3K9 but not H3K14 (Kikuchi et al., 2005, 2011b, 2012). As is well-known, histone acetylation catalyzed by HATs reduces the binding affinity between histones and DNA, and thereby constructs euchromatin that allows access of various trans-acting factors to target genes (Biel et al., 2005; Jenuwein and Allis, 2001; Selvi and Kundu, 2009). Taken together, our results revealed that GCN5 is involved in acetylation of H3K9 residues within chromatin around the coding region of the IgM H-chain gene followed by activation of its transcription. Unfortunately, we could not perform ChIP assay using primers corresponding to nucleotide sequences around the 5′-flanking region of the IgM H-chain gene because of lack of information on them in chickens. However, our results obtained in this study strongly suggested that GCN5 plays a key role in transcription regulation of the IgM H-chain gene through euchromatin formation around its coding region based on histone acetylation. Needless to say, the behavior of GCN5 around the promoter region of the IgM H-chain gene should be elucidated in the future. Histone-modifying enzymes are well-known to be related to human diseases through various unusual epigenetic events (Butler et al., 2012). For instance, unbalance of histone acetylation level due to abnormalities of HATs and/or HDACs could lead to generation of cancer (Selvi and
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Kundu, 2009) and occurrence of autoimmune disease (Javierre et al., 2011). This report is the first case on a novel function of GCN5 as an effective transcription activator of IgM H-chain gene expression and may significantly help in the understanding of mechanisms specific for B cell normal and pathological functions. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.04.030. Conflict of interest The authors have no financial conflicts of interest. Acknowledgments We thank R. Masuya and N. Nagamatsu-Yamamoto for technical support and H. Madhyastha and R. Madhyastha for editorial reading of the manuscript. This work was supported in part by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 25430170, to HK). This work was also partly supported by the Grant for Joint Research Project of the Institute of Medical Science, The University of Tokyo (No. 2013230). References Allis, C.D., Berger, S.L., Cote, J., Dent, S., Jenuwien, T., Kouzarides, T., Pillus, L., Reinberg, D., Shi, Y., Shiekhatter, R., Shilatifard, A., Workman, J., Zhang, Y., 2007. New nomenclature for chromatin-modifiying enzymes. Cell 131, 633–636. Berger, S.L., 2007. The complex language of chromatin regulation during transcription. Nature 447, 407–412. Bernstein, B.E., Meissner, A., Lander, E.S., 2007. The mammalian epigenome. Cell 128, 669–681. Biel, M., Wascholowski, V., Giannis, A., 2005. Epigenetics—an epicenter of gene regulation: histones and histone-modifying enzymes. Angewandte Chemie International Edition in English 20, 3186–3216. Brownell, J.E., Zhou, J., Ranalli, T., Kobayashi, R., Edomondson, D.G., Roth, S.Y., Allis, C.D., 1996. Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell 84, 843–851. Buerstedde, J.M., Takeda, S., 1991. Increased ration of targeted to random integration after transfection of chicken B cell lines. Cell 67, 179–188. Butler, J.S., Koutelou, E., Schibler, A.C., Dent, S.Y., 2012. Histone-modifying enzymes: regulators of developmental decisions and drivers of human disease. Epigenomics 4, 163–177. Chimenti, F., Bizzarri, B., Maccioni, E., Secci, D., Bolasco, A., Chimenti, P., Fioravanti, R., Granese, A., Carradori, S., Tosi, F., Ballario, P., Vernarecci, S., Filetici, P., 2009. A novel histone acetyltransferase inhibitor modulating Gcn5 network: cyclopentylidene-[4(4′-chlorophenyl)thiazol-2-yl]hydrazone. Journal of Medicinal Chemistry 52, 530–536. Espada, J., Esteller, M., 2007. Epigenetic control of nuclear architecture. Cellular and Molecular Life Sciences 64, 449–457. Goldberg, A.D., Allis, C.D., Bernstein, E., 2007. Epigenetics: a landscape takes shape. Cell 128, 635–638. Grant, P.A., Eberharter, A., John, S., Cook, R.G., Turner, B.M., Workman, J.L., 1999. Expanded lysine acetylation specificity of Gcn5 in native complexes. Journal of Biological Chemistry 274, 5895–5900. Javierre, B.M., Hernando, H., Ballestar, E., 2011. Environmental triggers and epigenetic deregulation in autoimmune disease. Discovery Medicine 12, 535–545. Jenuwein, T., Allis, C.D., 2001. Translating the histone code. Science 293, 1074–1080. Kikuchi, H., Nakayama, T., 2008. GCN5 and BCR signalling collaborate to induce premature B cell apoptosis through depletion of ICAD and IAP2 and activation of caspase activities. Gene 419, 48–55. Kikuchi, H., Takami, Y., Nakayama, T., 2005. GCN5: a supervisor in all-inclusive control of vertebrate cell cycle progression through transcription regulation of various cell cycle-related genes. Gene 347, 83–97. Kikuchi, H., Barman, H.K., Nakayama, M., Takami, Y., Nakayama, T., 2006. Participation of histones, histone modifying enzymes and histone chaperons in vertebrate cell functions. In: Buerstedde, J.M., Takeda, S. (Eds.), Reviews and Protocols in DT40 Research. Springer-Verlag, Berlin, pp. 225–243. Kikuchi, H., Kuribayashi, F., Takami, Y., Imajoh-Ohmi, S., Nakayama, T., 2011a. GCN5 regulates the activation of PI3K/Akt survival pathway in B cells exposed to oxidative stress via controlling gene expressions of Syk and Btk. Biochemical and Biophysical Research Communications 405, 657–661. Kikuchi, H., Kuribayashi, F., Kiwaki, N., Takami, Y., Nakayama, T., 2011b. GCN5 regulates the superoxide-generating system in leukocytes via controlling gp91-phox gene expression. Journal of Immunology 186, 3015–3022. Kikuchi, H., Kuribayashi, F., Imajoh-Ohmi, S., Nishitoh, H., Takami, Y., Nakayama, T., 2012. GCN5 protects vertebrate cells against UV–irradiation via controlling gene expression of DNA polymerase η. Journal of Biological Chemistry 287, 39842–39849.
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