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From the Department of Biochemistry andthe Department of Pediatric Surgery, Tohoku University Graduate School of Medicine, Seiryo-machi 1-1, Sendai 980-0874,
From the Department of Biochemistry andCenter for Regulatory Epigenome and Diseases,Tohoku University Graduate School of Medicine, Seiryo-machi 2-1, Sendai 980-8575,
To whom correspondence should be addressed: Dept. of Biochemistry, Tohoku University Graduate School of Medicine, Seiryo-machi 2-1, Sendai 980-8575, Japan. Tel.: 81-22-717-7595; Fax: 81-22-717-7598; E-mail: .
From the Department of Biochemistry andCREST, Japan Science and Technology Agency, Seiryo-machi 2-1, Sendai 980-8575,Center for Regulatory Epigenome and Diseases,Tohoku University Graduate School of Medicine, Seiryo-machi 2-1, Sendai 980-8575,
* This work was supported by Grants-in-aid 15H02506, 24390066, 21249014, 17054028, 25460352, and 24790271 from Japan Society for the Promotion of Science and the Network Medicine Global COE Program from the Ministry of Education, Culture, Sport, Science and Technology of Japan and The Takeda Foundation and Astellas Foundation for Research on Metabolic Disorders. Restoration of the laboratory from the damage due to the 2011 Tohoku earthquake was provided in part by the Astellas Foundation for Research on Metabolic Disorders, the Banyu Foundation, the Naito Foundation, A. Miyazaki, and A. Iida. The authors declare that they have no conflicts of interest with the contents of this article.
B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of plasma cell differentiation. The transcription factor Bach2 represses Blimp-1 expression in B cells to stall terminal differentiation, by which it supports reactions such as class switch recombination of the antibody genes. We found that histones H3 and H4 around the Prdm1 intron 5 Maf recognition element were acetylated at higher levels in X63/0 plasma cells expressing Blimp-1 than in BAL17 mature B cells lacking its expression. Conversely, methylation of H3-K9 was lower in X63/0 cells than BAL17 cells. Purification of the Bach2 complex in BAL17 cells revealed its interaction with histone deacetylase 3 (HDAC3), nuclear co-repressors NCoR1 and NCoR2, transducin β-like 1X-linked (Tbl1x), and RAP1-interacting factor homolog (Rif1). Chromatin immunoprecipitation confirmed the binding of HDAC3 and Rif1 to the Prdm1 locus. Reduction of HDAC3 or NCoR1 expression by RNA interference in B cells resulted in an increased Prdm1 mRNA expression. Bach2 is suggested to cooperate with HDAC3-containing co-repressor complexes in B cells to regulate the stage-specific expression of Prdm1 by writing epigenetic modifications at the Prdm1 locus.
B cells play important roles in humoral immunity. Upon stimulation by an antigen and additional stimulators, mature cells undergo clonal proliferation with concomitant somatic hypermutation and class switch recombination (CSR)
The abbreviations used are: CSR, class switch recombination; 2-ME, 2-mercaptoethanol; qPCR, quantitative PCR; MARE, Maf recognition element; IP, immunoprecipitation; HDAC, histone deacetylase; TSA, trichostatin A.
of immunoglobulin genes, followed by terminal differentiation into antibody-secreting plasma cells. These responses are coordinated by a network of transcription factor genes (
). Two DNA binding factors are known to repress transcription of Prdm1. One is Bcl6 (B cell lymphoma 6) that is essential for the formation of a germinal center and is required for somatic hypermutation (
). Another repressor is Bach2 (BTB and CNC homologue 2), which is a basic region-leucine zipper factor and forms heterodimers with small Maf proteins through their leucine zipper domain (
Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.
). Although Bach2 is central to the regulation of plasma cell differentiation, the mechanism for the regulation of Prdm1 by Bach2 remains to be elucidated. More specifically, little is known about the co-regulators of Bach2 or the epigenetic regulation of Prdm1 in B and plasma cells. Here, we compare changes in acetylation and methylation of histones at the Prdm1 locus before and after plasma cell differentiation, and we purified the Bach2 protein complex to identify proteins involved in this epigenetic regulation.
Experimental Procedures
Bach2 Complex Purification and Mass Spectrometry Analysis
The Bach2 complex was purified from nuclear extracts prepared from BAL17 cells stably expressing FLAG-hemagglutinin (HA) epitope-tagged Bach2 (eBach2) as described previously (
). The eBach2-expressing cells were collected by centrifugation for 8 min at 1,865 × g and were washed with phosphate-buffered saline. After centrifugation for 10 min at 1,190 × g, the pellets were suspended in 6 volumes of hypotonic buffer (10 mm Tris-HCl (pH 7.3), 10 mm KCl, 1.6 mm MgCl2, 2 mm 2-mercaptoethanol (2-ME), 40 μm phenylmethylsulfonyl fluoride (PMSF)) and were then collected by centrifugation for 5 min at 1,190 × g. Next, the cells were resuspended in the same volume of hypotonic buffer, and the suspensions were homogenized and centrifuged for 15 min at 2,330 × g to collect the nuclei. The obtained crude nuclei were then suspended in a half-volume of low salt buffer (0.02 m KCl, 20 mm Tris-HCl (pH 7.3), 25% glycerol, 1.5 mm MgCl2, 0.2 mm EDTA (pH 8.0)) for homogenization. The resulting suspension was dropped with a half-volume of high salt buffer (1.2 m KCl, 20 mm Tris-HCl (pH 7.3), 25% glycerol, 1.5 mm MgCl2, 0.2 mm EDTA) and then stirred gently for 60 min and centrifuged for 60 min at 48,384 × g. The supernatants were dialyzed against 50 volumes of BC-0 buffer (20 mm Tris-HCl (pH 7.3), 20% glycerol, 0.2 mm EDTA (pH 8.0), 2 mm 2-ME, 40 μm PMSF) until the conductivity reached 65–70 microsiemens/cm. The dialysate was centrifuged for 20 min at 23,700 × g, and the supernatant was used as a nuclear extract. eBach2 was immunoprecipitated from nuclear extracts by incubating with M2 anti-FLAG-agarose (Sigma) for 4 h with rotation. After an extensive wash with 0.1 m KCl-containing buffer B, the bound proteins were eluted from FLAG M2-agarose by incubating for 60 min with the FLAG peptide in the same buffer (0.5 mg/ml). The eluates were then incubated with anti-HA antibody-conjugated beads for 4 h with rotation. After extensive washing with 0.1 m KCl-containing buffer B, the bound proteins were eluted from anti-HA antibody-conjugated beads by incubating with 100 mm glycine-HCl (pH 2.5) and neutralized with 1 m Tris-HCl (pH 8.0). Purified proteins were separated by SDS-PAGE on a 4–20% gel, and the gel was subsequently stained with Coomassie Brilliant Blue. The stained bands were excised from the gel, and the proteins therein were subjected to in-gel reduction, S-carboxyamidomethylation, and digestion with trypsin (Promega). The molecular masses of the tryptic peptides were determined using LC-HCT plus (Bruker Daltonics), and protein identification was performed using the MASCOT search engine (Matrix Science).
Bach2 Complex Analysis by Reversible Cross-link Immunoprecipitation (ReCLIP)
The proteins interacting with Bach2 were isolated also by another method based on the one called “ReCLIP” (
). Approximately 1 × 108 BAL17 cells were fixed in PBS containing 0.5 mm dithiobis(succinimidyl propionate) and 0.5 mm dithiobismaleimidoethane, and then they were treated with Tris and l-cysteine in PBS for quenching. The fixed cells were suspended in RIPA buffer, and after supersonic treatment and centrifugation, the cleared lysate was incubated with anti-FLAG magnetic beads (Sigma M8823) for 1.5 h at 4 °C. The beads were then washed five times with RIPA buffer, and the proteins bound to the beads were eluted using 0.1 μg/μl FLAG peptide. The eluted proteins were treated with 50 mm DTT to dissolve the cross-link and were separated by SDS-PAGE using a 5–20% polyacrylamide gel (Oriental Instruments). After staining with 60 mg/liter Coomassie Brilliant Blue-G-250 and 35 mm HCl (
), each lane in the gel was divided into 12 sections, and the resulting gel blocks were treated with DTT for reduction and then with acrylamide for alkylation of the sulfhydryl groups. The proteins in every gel block were digested overnight by 10–20 ng of trypsin (Promega). The resulting peptides were extracted from the gel with 75% acetonitrile and 0.5% formic acid and were concentrated in a SpeedVac. One-half of each sample was analyzed using an LTQ Orbitrap Velos with ETD mass spectrometer (Thermo Fisher Scientific) equipped with a PAL HTC-xt autosampler (AMR) and an ADVANCE HPLC system (AMR, Tokyo, Japan). The peptides were separated on a PepSwift Monolithic Column (100 μm inner diameter × 25 cm, Thermo Fisher Scientific) at a flow rate of 300 nl/min with a 60-min linear gradient generated by aqueous solvent A (0.1% formic acid) and organic solvent B (100% acetonitrile) as follows: 2.5–22.5% B in 52 min, to 35% B in 56 min, to 95% B in 58 min, and they were directly electrosprayed into the mass spectrometer. The data acquisition of every sample was carried out for 70 min after the LC gradient was started, in which MS1 scans from m/z = 321 to 1,800 were carried out in the orbitrap with the resolution set at 100,000 with a lock mass at m/z = 445.120025, followed by sequential isolation of the 20 most intense precursor ions and MS2 acquisition by collision-induced dissociation in the ion trap in the normal resolution mode. The settings for MS2 scans were as follows: minimal signal intensity required = 500, isolation width = 2 m/z, AGC target = 10,000, maximum ion injection time = 200 ms, normal collision energy = 35, activation time = 10 ms, microscan = 1, and dynamic exclusion was enabled with a 60-s exclusion duration. The raw data files derived from samples in the same SDS-PAGE lane were converted together into a single MASCOT generic format file and used for the database search by MASCOT (version 2.5.1, Matrix Science) against the human proteins in SwissProt (Feb., 2015), and a custom database, including contaminant proteins and the FLAG-tagged mouse Bach2 protein. A maximum of three trypsin miscleavages was allowed. The peptide mass tolerance and MS/MS tolerance were set at 5 ppm and 0.5 Da, respectively. Protein N-terminal acetylation (+42.0106), oxidation of methionine (+15.9949), phosphorylation at serine/threonine (+79.9663), propionamidation at cysteine (+71.0371), propionamidated dithiobis(succinimidyl propionate) at lysine (+159.0354), and propionamidated dithiobismaleimidoethane at cysteine (+246.0674) were considered to be variable modifications. Peptides identified with MASCOT expectation values of <0.05 were selected as significant hits. The false discovery rates calculated by decoy database search were 2.05% in mock IP and 1.79% in FLAG-Bach2 IP.
Cell Culture
BAL17 mature B cells and X63/0 plasma cells were maintained in Iscove's modified Dulbecco's medium (Gibco) supplemented with 10% FBS (Nichirei Bioscience), 50 μm 2-ME (Wako), 100 μg/ml streptomycin (Gibco), and 100 units/ml penicillin (Gibco). Splenic B cells were isolated from 8- to 11-week-old wild-type or Bach2−/− C57BL/6 mice, purified by MACS magnetic cell sorting with CD45R (B220) microbeads or B cell isolation kit, mouse (Miltenyi Biotec), cultured in RPMI 1640 medium (Sigma) supplemented with 10% FBS, 10 mm HEPES (Gibco), 1 mm sodium pyruvate (Gibco), 0.1 mm non-essential amino acids (Gibco), 50 μm 2-ME (Gibco), 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco), and stimulated with 20 μg/ml LPS (0111: B4; Sigma), as described previously (
). For experiments using the HDAC inhibitor trichostatin A (TSA), 1.0 × 106 BAL17 cells were cultured for 12 h with 7.5 μg/ml TSA (Wako). For selective inhibition of HDAC3, 1.0 × 107 BAL17 cells were cultured for 12 h with 10 or 50 μm RGFP966 (Abcam). Details of retroviral infection were previously described (
). Briefly, purified splenic B cells were preactivated with 20 μg/ml LPS for 24 h before infection. Infected splenic B cells were then cultured with 20 μg/ml LPS for a further 48 h and were analyzed. 293T human embryonic kidney cells were cultured with DMEM (Sigma) supplemented with 10% FBS (Nichirei Bioscience), 100 μg/ml streptomycin (Gibco), and 100 units/ml penicillin (Gibco). B1-8Hi mice were obtained from Dr. M. C. Nussenzweig (Rockefeller University) and Dr. T. Kurosaki (Osaka University) (
). Purified B cells from B1-8Hi mice were cultured in RPMI 1640 medium, with supplemental reagents as above, and stimulated with IL-2, IL-4, IL-5, CD40 ligand, and NP-Ficoll. Retroviral infection was performed as described (
). Infected cells were sorted on the basis of GFP expression after 72 h and were analyzed for mRNA transcripts or differentiation efficiency with FACS.
Immunoblot Analysis
Whole cell extracts were prepared from BAL17 cells as described previously (
). Each sample was resolved on SDS-polyacrylamide gels and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked for 1 h in blocking buffer (3% (w/v) skim milk, 0.05% (v/v) Tween 20 in TBS) and were subsequently incubated with primary antibodies in the blocking buffer overnight and secondary antibodies in the TBS with 0.05% (v/v) Tween 20 for 0.5 h. The primary antibodies used were anti-Bach2 antiserum (F69-1 (
Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.
) or Bach2N-2 (see below)), anti-Tbl1x antibody (H-367; Santa Cruz Biotechnology), anti-Rif1 antibody (600-401-871; Rockland), and anti-HDAC3 antibody (06-890; Upstate Biotechnology). ECL Western blotting detection reagents (Thermo Scientific Pierce) were used to detect antibody-antigen complexes.
Immunoprecipitation
His-tagged mouse Bach2 protein (amino acid residues 119–318) was overexpressed in Escherichia coli and purified using nickel resin. The recombinant Bach2 protein was used to immunize a rabbit, resulting in anti-Bach2 antisera (Bach2N-1 and N-2). These antisera were found useful for the immunoblot analysis and immunoprecipitation assays. Whole cell extracts of BAL17 cells were pre-cleared with protein G-Sepharose beads at 4 °C for 2 h and immunoprecipitated with the anti-Bach2 antiserum (Bach2N-2) or anti-HDAC3 antibody (NB500-126; Novus) for 2 h to analyze the interaction of endogenous proteins. Immunoprecipitates were recovered with protein G-Sepharose beads and were washed seven times. Samples were analyzed by immunoblot analysis as described above (
Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.
)) and anti-HDAC3 antibody (06-890; Upstate Biotechnology). The secondary antibody was HRP-conjugated anti-rabbit IgG (GE Healthcare). In some experiments, rabbit IgG TrueBlot (eBioscience) was used to eliminate the interference of signal detection by the immunoglobulin heavy and light chains. For primary B cell immunoprecipitation, ReCLIP was carried by using control IgG or anti-Bach2 antibodies (Bach2N-1) that were conjugated with beads, followed by immunoblot analysis. Anti-NCoR (ab24552; Abcam) was used as the primary antibody in addition to the above antibodies. Intensities of data images on the films were measured by ImageJ software.
TABLE 1The list of proteins identified as Bach2-interacting proteins using ReCLIP
). Mouse Rif1 expression plasmid was generated by N. Y. and H. M. and will be described elsewhere.
RNA Interference
Stealth RNAi duplexes against HDAC3 were designed using the BLOCK-iT RNAi Designer (Invitrogen). We also used Stealth RNAi siRNA negative control (Invitrogen) that is not homologous to any sequence of the vertebrate transcriptome. For delivering RNAi duplexes, 3.0 × 106 mouse splenic B cells were transfected with 6 μl of stock Stealth RNAi duplexes (20 μm) using a basic nucleofection solution for mouse primary B cell (VPA-1010; Lonza) with the nucleofector program (Z-001; Lonza). Retroviral plasmids encoding short hairpin RNA oligonucleotides were constructed and transduced as described previously (
). The oligonucleotide sequences of short hairpin RNA directed against Rif1 and NCoR1 mRNA were described as follows: Rif1 1162, 5′-CGTGAACCGTATTCAGAATCAA-3′; Rif1 #2993, 5′-CCAGAGTACAATAAGTGTTGAT-3′; NCoR1 #196, 5′-CACCGCTCTTCTCATATTGAAG-3′; NCoR1 #3138, 5′-CCCGCATCAAGTGATAACTAAC-3′; and Bach2 #7678, 5′-GGGTGCTAAACTTCTACCAAAC-3′.
Chromatin Immunoprecipitation (ChIP)
Chromatin fixation and immunoprecipitation were carried out using BAL17 cells and X63/0 cells as described previously (
). The antibodies used were as follows: anti-acetylated histone H3 (Lys-9 and Lys-14) (06-599; Upstate Biotechnology); anti-acetylated histone H4 (Lys-5, Lys-8, Lys-12, and/or Lys-16) (06-866; Upstate Biotechnology); anti-acetylated histone H3-K9 (07-352; Upstate Biotechnology); anti-acetylated histone H3-K18 (07-354; Upstate Biotechnology); anti-acetylated histone H3-K27 (07-360; Upstate Biotechnology); anti-mono-methylated histone H3-K4 (ab8895; Abcam); anti-di-methylated histone H3-K4 (ab7766; Abcam); anti-tri-methylated histone H3-K4 (ab8580; Abcam); anti-di-methylated histone H3-K9 (ab1220; Abcam); anti-tri-methylated histone H3-K9 (ab8898; Abcam); Bach2N-2, anti-Rif1 antibody (600-401-871; Rockland); and anti-HDAC3 antibody (06-890; Upstate Biotechnology). Normal rabbit serum (011-000-120; Jackson ImmunoResearch) or purified rabbit IgG (02-6120; Invitrogen) was used in negative control experiments. Quantitative PCR was carried out by using FastStart Essential DNA Green Master with a LightCycler nano system class switch recombination and by using SYBR Green Real Time PCR Master Mix with a 7500 Real Time PCR system (Applied Biosystems and Life Technologies, Inc.). Primers used to detect promoter 1,800 MARE and intron 5 MARE were previously described (
). Primers used to detect promoter region of Mcm5 were forward primer 5′-GCGAAAGTCGGCTTCCTCTA-3′ and reverse primer 5′-CAATTCCCTCACCTCACAGC-3′.
Reverse Transcription and Quantitative PCR
Total RNA was isolated from various cells using a total RNA isolation minikit (Agilent Technologies) or RNeasy Plus minikit (Qiagen). cDNA was synthesized by Omniscript reverse transcriptase (Qiagen) using the random priming method. Quantitative PCR was carried out using LightCycler Fast Start DNA Master SYBR Green I (Roche Applied Science) and LightCycler 1.5 or LightCycler Nano (Roche Applied Science) with mouse β-actin (Roche Applied Science) expression as the normalization control. The primers were as follows: Blimp-1 forward primer 5′-CCCTCATCGGTGAAGTCTA-3′ and reverse primer 5′-ACGTAGCGCATCCAGTTG-3′; HDAC3 forward primer 5′-CCTCGGGTGCTCTACATTGATA-3′ and reverse primer 5′-CCACTCTCTGCTCCAACTTCAT-3′; Rif1 forward primer 5′-GGAGAGATACATTCTGCTGTTGT-3′ and reverse primer 5′-AGCCTACAATGAAGAAACCAATG-3′; and NCoR1 forward primer 5′-GTCGTGAGTCACAGCCCATT-3′ and reverse primer 5′-TCTCTGTAACAGGTAAGCAGCA-3′.
Statistical Analysis
The statistical analysis was performed using Student's t test or Wilcoxon test, as appropriate. p value < 0.05 was considered to be significant.
Results
Histone Acetylation at the Prdm1 Locus Increases upon Plasma Cell Differentiation
Prdm1 contains two MAREs in the promoter upstream region and intron 5 (Fig. 1A) (
). The pattern of histone modifications around these MAREs was compared in BAL17 mature B cells and X63/0 plasma cells, in which Prdm1 mRNA expression is low and high, respectively (
). Quantitative PCR-based ChIP assays with anti-acetylated histone H3 (Lys-9 and Lys-14, Fig. 1B) and anti-acetylated H4 (Lys-5, Lys-8, Lys-12, and/or Lys-16, Fig. 1C) antibodies revealed that the levels of acetylation of histone H3 and H4 at the intron 5 MARE of the Prdm1 gene were higher in X63/0 cells than in BAL17 cells. There was no significant difference between BAL17 and X63/0 in the amount of acetylation of histone H3 on the promoter MARE region, although the amount of acetylated histone H4 on the promoter region was low in X63/0 cells. We also compared individual acetylation of Lys-9, Lys-18, or Lys-27 of histone H3 using their respective antibodies. The levels of acetylation of H3-K9, -K18, and -K27 at the Prdm1 promoter MARE was slightly decreased in X63/0 cells compared with BAL17 (Fig. 1, D and F) or did not differ in these cells (Fig. 1E). In contrast, the levels of H3-K9, -K18, or -K27 acetylation at the Prdm1 intron 5 MARE were higher in X63/0 plasma cells than in BAL17 cells (Fig. 1D). Taken together, these results suggested that hyperacetylation of Lys-9, Lys-18, and Lys-27 of histone H3 at the Prdm1 intron 5 MARE may be involved in the repression of Prdm1 in mature B cells.
FIGURE 1Histone acetylation at the Prdm1 promoter and intron 5 MARE regions in BAL17 and X63/0 cells.A, schematic representation of mouse Prdm1 is shown. The small lines denote MARE. B and C, ChIP assays of BAL17 (mature B cell) and X63/0 (plasma cell) using antibodies against acetylated histone H3 (Lys-9 and Lys-14) (B) or acetylated histone H4 (Lys-5, Lys-8, Lys-12, and/or Lys-16) (C) at the Prdm1 promoter 1,800 MARE (left panel) and intron 5 MARE (right panel). D–F, ChIP assays of BAL17 and X63/0 cells with antibodies against indicated acetylated residues of H3K9ac (D), K18ac (E), and K27ac (F). The relative levels of enrichment in B–F are shown as mean values of two independent experiments and standard error of the mean with p values (Student's t test) for differences between BAL17 and X63/0 cells. NRS, normal rabbit serum.
Dynamic Changes of Histone Methylation Pattern at the Prdm1 Locus between Mature B Cells and PCs
Histone H3-K4 methylation is associated with regulatory regions of actively transcribed genes, whereas histone H3-K9 methylation is associated with inactive genes. We therefore assessed histone methylation at the promoter and intron 5 MARE regions of the Prdm1 locus. The levels of H3-K4 mono-, di-, and tri-methylation (me1, me2, and me3) at the promoter MARE were similar between BAL17 and X63/0 cells (Fig. 2, A–C). In contrast, these modifications at the intron 5 MARE were higher in X63/0 than in BAL17 cells (Fig. 2, A–C). In particular, H3K4me2 showed a profound difference. Next, we investigated the levels of histone H3-K9 methylation. The levels of H3K9me2 and H3K9me3 at the intron 5 MARE were significantly reduced in X63/0 cells (Fig. 2, D and E). These modifications at the promoter MARE tended to be lower in X63/0 cells than in BAL17 cells (Fig. 2, D and E). Collectively, these results suggest that histone modifications involving acetylation and methylation may act together to regulate the plasma cell-specific Prdm1 expression. A competition between acetylation and methylation of H3-K9 may operate at the intron MARE region.
FIGURE 2Histone methylation at the Prdm1 promoter and intron 5 MARE regions in BAL17 and X63/0 cells.A–C, ChIP assays of mono-, di-, and tri-methylation of histone H3-K4 as in Fig. 1. D and E, ChIP assays of di- and tri-methylation of histone H3-K9. The relative levels of enrichment are shown as mean values of three experiments and standard error of the mean with p values (Student's t test) for differences between BAL17 and X63/0 cells. NRS, normal rabbit serum.
Bach2 Promotes Histone Deacetylation of Prdm1 Chromatin in B Cells
To examine whether Bach2 is involved in the epigenetic modifications of the Prdm1 locus in B cells, we carried out ChIP analysis of the locus using splenic B cells isolated from wild-type and Bach2−/− mice and stimulated with LPS in vitro. The levels of H3K9ac at the promoter and intron 5 MAREs were significantly higher in Bach2−/− splenic B cells than in wild-type B cells (Fig. 3A). To ascertain whether the elevated levels of H3K9ac directly resulted from the absence of Bach2, we reconstituted Bach2 expression in Bach2−/− splenic B cells by a retroviral transduction, and we determined the levels of acetylation by ChIP analysis (Fig. 3B). The levels of H3K9ac at both the promoter and intron 5 MAREs were lower in the B cells reconstituted with Bach2 than those infected with the control virus. Taken together, these results suggested that Bach2 reduced H3-K9 acetylation at Prdm1 in B cells.
FIGURE 3Bach2 is required to maintain Prdm1 H3-K9 acetylation at a lower level in B cells.A, ChIP assays of histone H3K9ac. Splenic B cells from wild-type or Bach2−/− mice were compared as in Fig. 1. The results from two independent ChIP assays evaluated by qPCR in triplicates are shown as mean values and standard error of the mean with p values (Student's t test) for differences between wild-type and Bach2−/− B cells. NRS, normal rabbit serum. B, 2 days after infection of Bach2−/− B cells with control enhanced GFP or Bach2 retroviruses, the levels of histone H3K9ac were compared with ChIP assays. The relative levels of enrichment are shown as mean values of two independent experiments and standard error of the mean with p values (Student's t test) for differences between enhanced GFP-expressing and Bach2-expressing Bach2−/− B cells. C, ChIP assays of endogenous Bach2 and MafK using primary wild-type splenic B cells. The promoter region of the Mcm5 gene was also compared. The relative levels of enrichment from three independent ChIP assays are shown as mean values and standard error of the mean with p values (Student's t test) for differences between anti-Bach2 (left panel) or anti-MafK (right panel) antiserum and control IgG.
We examined whether Bach2 bound to the two regulatory regions of Prdm1 in B cells. We generated a new anti-Bach2 antiserum (Bach2N-2), which recognized endogenous Bach2 in B cells in immunoblot analysis (see under “Materials and Methods”). Using this antiserum, we carried out ChIP assays of primary B cells stimulated with LPS. As shown in Fig. 3C, both Bach2 and MafK were found to bind to the promoter and intron 5 MAREs. Bach2 did not bind to the promoter region of the Mcm5 gene, confirming specific binding of Bach2 of the Prdm1 MAREs. These results together indicate that Bach2 promotes histone deacetylation, including H3-K9 of the Prdm1 locus by directly binding to the regulatory regions in B cells.
Characterization of Bach2 Complex in B Cells
To elucidate the factors involved in Prdm1 repression by Bach2, Bach2 was purified from BAL17 cells. FLAG-HA epitope-tagged Bach2 (eBach2) was stably expressed in BAL17 cells (Fig. 4A). The expression level of epitope-tagged Bach2 was seven times higher than that of endogenous Bach2 according to a densitometry analysis. The purification of eBach2 was performed by sequential steps of affinity chromatography using anti-FLAG antibody-conjugated agarose followed by anti-HA antibody-conjugated beads. The associated proteins were analyzed by SDS-PAGE along with samples that were purified with the same procedures from control cells (mock; Fig. 4B). Several proteins were found to associate with eBach2. These proteins were specific components of the Bach2 complex inasmuch as they were not enriched in the mock purification. Protein bands were excised from the gels, and their identities were determined by mass spectrometry analysis. Rif1, Tbl1x, HDAC3, MafG, and MafK were identified among these proteins (Fig. 4, C–G). In another set of purification, using the ReCLIP method (see “Experimental Procedures”), the lanes of the gels were excised as slices, and proteins in the slices were determined by mass spectrometry analysis. We focused on proteins that had been identified in the FLAG-Bach2 sample but absent from the mock purification. The list of these proteins included nuclear co-repressors NCoR1, NCoR2 (also know as SMRT) in addition to Rif1, HDAC3, Tbl1x, MafG, MafK and other proteins (Table 1). Bach2 was found to interact with several DNA binding transcription factors, chromatin-binding factors, RNA binding factors, and several proteins involved in ubiquitination such as Huwe1 (Fig. 5A). By integrating these results with the known protein interaction database, some of these proteins appear to interact with Bach2 by forming distinct complexes (Fig. 5B). The list of Bach2-interacting proteins will aid future analysis into the function and regulation of Bach2 in B cells.
FIGURE 4Purification of Bach2 complex in BAL17 mature B cells.A, expression of FLAG-HA epitope-tagged Bach2 (eBach2). Immunoblotting analysis was carried out with anti-Bach2 antiserum using nuclear extracts from non-transduced (Mock) and eBach2-expressing BAL17 cells. IB, immunoblot. B, Bach2 complex was purified from nuclear extracts prepared from eBach2-expressing BAL17 cells. Mock purification from nuclear extracts prepared from nontransduced BAL17 cells was performed as a control. IP, immunoprecipitation. C–G, identification of Rif1, Tbl1x, HDAC3, MafG, and MafK by mass spectrometry. Amino acid sequences of peptides were determined by a mass spectrometry/mass spectrometry analysis.
FIGURE 5Proteins interacting with Bach2.A, schematic representation of candidate proteins interacting with Bach2. The mass spectrometry data from independent purifications were compiled. Proteins were selected based on two criteria, protein score of more than 130 and being absent in the mock samples. The protein score of HDAC3 was lower than 130 in this experiment but is indicated here to give its context. The thickness of each edge represents protein scores. Proteins are colored based on their representative functions as in the color key. B, interaction of Bach2 with known protein complexes. Known interactions are integrated with the results in A.
The presence of HDAC3, Tbl1x, and Rif1 in the Bach2 complex was verified by immunoblotting analyses using HDAC3-, Tbl1x-, or Rif1-specific antibodies (Fig. 6A). The possible interaction between endogenous Bach2 and HDAC3 in BAL17 cells was examined using an immunoprecipitation assay. Not only endogenous Bach2 but also endogenous HDAC3 were precipitated by the anti-Bach2 antibodies (Fig. 6B). In a reciprocal assay, endogenous HDAC3 and Bach2 were precipitated by the anti-HDAC3 antibodies (Fig. 6C). The enriched Bach2 migrated faster than the major form of Bach2. This band may represent an alternative splicing form of Bach2 (
Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.
) or a post-translationally modified Bach2. These results showed that endogenous Bach2 and HDAC3 interacted with each other.
FIGURE 6Bach2-HDAC3-Rif1 interactions.A, immunoblot analysis of the affinity-purified samples (derived from Fig. 4B) using anti-Rif1 anti-Bach2, anti-Tbl1x, or anti-HDAC3 antibodies. B, lysates from BAL17 cells were immunoprecipitated (IP) with anti-Bach2 antiserum (Bach2N-2) or control antibodies (IgG) and analyzed by immunoblotting (IB) using anti-Bach2 or anti-HDAC3 antibodies. Inputs were also analyzed. C, lysates were immunoprecipitated with anti-HDAC3 or control antibodies and analyzed by immunoblotting using anti-Bach2 or anti-HDAC3 antibodies. Inputs were also analyzed. The dots indicate the bands that reacted with the anti-Bach2 antiserum. D, interaction of Rif1 with Bach2. FLAG-Rif1 and Bach2 were overexpressed in 293T cells in the indicated combinations. FLAG-Rif1 was immunoprecipitated with anti-FLAG antibody. The resulting samples were analyzed by immunoblotting using anti-FLAG or anti-Bach2 antibodies. An image of immunoblotting analysis of input samples (1% amount of loading for immunoprecipitation) is also shown to clarify the levels of Bach2 expression. E, interaction of Rif1 with HDAC3. FLAG-Rif1 and HA-HDAC3 were overexpressed in 293T cells in the indicated combinations, and their interaction was analyzed as in D. An image of shorter exposure is also shown for the HDAC3 immunoblotting (bottom) to clarify the levels of HDAC3 expression. IP, immunoprecipitate. IB, immunoblot. The dot and asterisk on the left indicate specific and nonspecific bands, respectively.
Originally, Rif1 has been implicated in genome stability such as telomere DNA synthesis in yeast and regulation of DNA replication timing and DNA repair in mammalian cells (
). However, its role in transcription remains less clear. To confirm the interaction between Rif1 and Bach2, we performed co-immunoprecipitation analysis of FLAG-Rif1 and Bach2 overexpressed in 293T human embryonic kidney cells. We detected their co-immunoprecipitation (Fig. 6D), confirming that Rif1 and Bach2 form a protein complex. We next examined whether HDAC3 and Rif1 interact with each other. When overexpressed in 293T cells, HA-HDAC3 was co-precipitated with FLAG-Rif1 (Fig. 6E).
Bach2-interacting Proteins Are Involved in the Repression of Prdm1
To examine whether the Bach2-mediated repression of Prdm1 involves HDACs, we first treated BAL17 cells with the HDAC inhibitor trichostatin A (TSA) for 12 h. The amount of Prdm1 mRNA increased following TSA treatment (Fig. 7A), suggesting that Prdm1 is repressed in mature B cells by TSA-sensitive HDACs. To investigate whether HDAC3 is involved in the Prdm1 repression, we treated BAL17 cells with a HDAC3 selective inhibitor, RGFP966, at different concentrations for 12 h and assessed the Prdm1 expression (Fig. 7B). Quantitative RT-PCR analysis clearly demonstrated that inhibition of HDAC3 resulted in a significant up-regulation of Prdm1 expression, suggesting that HDAC3 is required for Prdm1 repression. To confirm this observation, an RNAi strategy was used to reduce the expression of HDAC3 mRNA in mouse splenic B220-expressing B cells to investigate whether HDAC3 was required for Prdm1 repression. Mouse splenic B cells were pre-activated with LPS for 12 h to induce plasma cell differentiation and then electroporated with HDAC3 siRNA or control siRNA. The expression levels of HDAC3 and Blimp-1 mRNAs were determined after 36 and 48 h by RT-qPCR. Although the knockdown of HDAC3 was transient, it resulted in a mild but reproducible derepression of Prdm1 (Fig. 7C). Furthermore, ChIP experiments clearly demonstrated that HDAC3 binds to the MAREs at the Prdm1 locus (Fig. 7D). Twelve hours after treatment of BAL17 cells with RGFP966, the levels of H3K27ac increased dramatically at both the promoter and intron 5 MAREs (Fig. 7E). Collectively, these results suggested that HDAC3 was involved in the repression of Prdm1 in B cells through altering epigenetic modification.
FIGURE 7Involvement of HDAC3 and NCoR1 in Prdm1 repression.A, HDAC activity is required for Prdm1 repression. BAL17 cells were treated with or without 7.5 μg/ml TSA for 12 h. The mRNA levels of Prdm1 were determined by RT-qPCR. The results are shown as mean values of three independent experiments and standard error of the mean with a p value (Student's t test). B, BAL17 cells were treated with 10 or 50 μm of RGFP966 for 12 h. The mRNA levels of Prdm1 were determined by RT-qPCR. The results are shown as mean values of two independent experiments and standard error of the mean with p values (Student's t test) for differences between RGFP966 and control DMSO treatments. C, expression of HDAC3 and Blimp-1 mRNA in mouse primary B cells expressing RNAi duplex targeting HDAC3. The expression levels of HDAC3 and Blimp-1 mRNA in HDAC3 knockdown LPS-stimulated splenic B cells were evaluated by RT-qPCR. The values represent the means and standard error of the mean with p values (Student's t test) at 36 or 48 h of LPS stimulation. n = 7 (36 h) or 4 (48 h). D, HDAC3 binds to the Prdm1 locus. ChIP assays of BAL17 cells were performed with antibody against HDAC3. The relative levels of enrichment of the indicated genomic DNA regions are shown as mean values of two independent ChIP assays and standard error of the mean with p values (Student's t test) for differences between anti-HDAC3 antibody and normal IgG. E, 12 h after treatment of BAL17 cells with control DMSO or 10 mm RGFP966, the levels of histone H3K27ac at indicated regions of Prdm1 were compared with ChIP assays. The relative levels of enrichment are shown as mean values of two independent experiments and standard error of the mean with p values (Student's t test) for differences between DMSO and RGFP966. F, effect of NCoR1 knockdown upon Prdm1 expression. The expression levels of NCoR1 (left panel) and Blimp-1 (right panel) mRNA were determined by RT-qPCR in BAL17 cells expressing shRNA targeting the NCoR1 mRNA or a control shRNA. The results are shown as mean values of two independent experiments and standard error of the mean with p values (Student's t test).
HDAC3 is known to associate with co-repressors such as NCoR1 or its related factor NCoR2. Their complexes are recruited to target genes by DNA-binding transcription factors (
). To investigate whether NCoR1 is also involved in the repression of transcription of the Prdm1 gene, we knocked down the expression of NCoR1 by short hairpin RNA (shRNA). The NCoR1 mRNA expression levels were reduced to nearly 50% by two independent shRNAs (Fig. 7F, left panel). The expression levels of Blimp-1 mRNA were significantly increased (Fig. 7F, right panel). Together with the previously reported interaction between Bach2 and NCoR2 (
), these results suggest that HDAC3 forms co-repressor complexes with NCoR1 and/or NCoR2 at the Prdm1 locus.
To assess the putative function of Rif1 in the regulation of Prdm1, we performed ChIP assays of Rif1. We observed a recruitment of Rif1 to both of the Prdm1 regions examined (Fig. 8A). These experiments also revealed that Rif1 was recruited to the Mcm5 promoter region, suggesting that Rif1 may possess a widespread function. To explore the involvement of Rif1 in Prdm1 repression, we carried out knockdown of Rif1 by two independent short hairpin RNA (shRNA) clones in BAL17 cells. Although we confirmed a reduction of the Rif1 expression by RT-qPCR, the amount of Prdm1 transcript was virtually unaffected (Fig. 8B). The role of Rif1 for the Bach2-mediated transcription repression therefore remains to be clarified.
FIGURE 8Binding of Rif1 to the Prdm1 locus.A, Rif1 binds to the Prdm1 locus. ChIP assays of BAL17 cells were performed with antibody against Rif1. The relative levels of enrichment of the indicated genomic DNA regions are shown as mean values of two independent ChIP assays and standard error of the mean with p values (Student's t test) for differences between anti-Rif1 antibody and normal rabbit serum (NRS). B, effect of Rif1 knockdown on Prdm1 expression. The expression levels of Rif1 (left panel) and Blimp-1 (right panel) mRNA were determined by RT-qPCR in BAL17 cells expressing shRNA targeting Rif1 mRNA and control shRNA. The results are shown as mean values of two independent experiments and standard error of the mean with p values (Student's t test).
Bach2 Interacts with HDAC3 and Mediates the Repression of Prdm1 in Primary B Cells
To further confirm that Bach2 was involved in the repression of Prdm1 together with HDAC3, we utilized mature B cells isolated from B1-8hi knock-in mice carrying immunoglobulin heavy chain gene that can produce an antibody to a pre-specified antigen (
). The mature B cells isolated from these mice show rather uniform reactions upon activation in vitro. The B1-8hi mature B cells were stimulated with an antigen, and nuclear extracts were prepared. Endogenous Bach2 was immunoprecipitated. Immunoblot analyses of the precipitates revealed that endogenous MafK and HDAC3 were both co-precipitated along with Bach2 (Fig. 9A). The signal for NcoR1 was also detected, although it was not clear due to its large molecular weight. We confirmed their presence using mass spectrometry analysis of the immunoprecipitates. In contrast, we failed to detect an interaction of Bach2 with Rif1 in these experiments. Reducing Bach2 by an shRNA-mediated interference in B1-8hi mature B cells resulted in increases in the expression of Prdm1 and plasma cell differentiation (Fig. 9, B and C). Therefore, these results established that Bach2 interacts with HDAC3 in primary B cells and suggest that this interaction is important for the repression of Prdm1 by Bach2.
FIGURE 9Bach2 interacts with HDAC3 and inhibits Prdm1 expression in primary B cells.A, whole cell extracts from B1-8hi B cells activated in vitro for 12 h were immunoprecipitated with anti-Bach2 antibody. Immunoblot analyses were carried out with the indicated antibodies. The arrowhead indicates NCoR1. The results are representative of two independent experiments. B, expression of indicated genes in B1-8hi B cells with or without knockdown of Bach2 mRNA. The results are shown as mean values of three independent experiments and S.E. with p values. C, plasma cell differentiation of B1-8hi B cells was monitored by the expression of syndecan 1 with or without knockdown of Bach2 mRNA. The data are representative of three independent experiments.
There has so far been no report on histone modification at the Prdm1 locus in B cells and plasma cells. By comparing histone modifications at the Prdm1 regulatory regions in BAL17 and X63/0 cells, we found that acetylation and methylation of H3-K9 at the intron MARE region correlated well with the Prdm1 expression in these cells. In addition, acetylation of H3-K18 and Lys-27 in this region was higher in X63/0 than in BAL17 cells. Therefore, we suggest that H3-K9 acetylation inhibits Lys-9 methylation, promoting Prdm1 expression in plasma cells. An important finding in this study is that Bach2 decreased the level of H3-K9 acetylation in primary B cells activated with LPS. Because the kinetics of transition between activated B cell and plasma cell is governed by Bach2 (
), our present observations suggest that Bach2 inhibits the process of plasma cell differentiation by promoting epigenetic modifications (low H3K9ac and high H3K9me) at the intron MARE region. Because H3-K9 acetylation at the promoter MARE region was also increased in Bach2-deficient primary B cells, Bach2 appears to regulate histone modification at the promoter region as well.
The analysis of Bach2-interacting proteins in BAL17 cells revealed several interesting candidate molecules for the epigenetic regulation by Bach2. Regarding the histone deacetylation, the presence of HDAC3, NCoR1, and NCoR2 is important. Because we have reported previously that Bach2 interacts directly with NCoR2 (
Co-repressor SMRT and class II histone deacetylases promote Bach2 nuclear retention and formation of nuclear foci that are responsible for local transcriptional repression.
), Bach2 may also directly interact with NCoR1. Both NCoR1 and NCoR2 are known to form co-repressor complexes with HDAC3 and other histone deacetylases (
). Therefore, Bach2 appears to recruit NCoR1 and NCoR2 complexes to its target genes to promote histone deacetylation. Consistent with this model, Prdm1 expression was increased in primary B cells upon knockdown of HDAC3 or NCoR1 and in response to the specific HDAC3 inhibitor RGFP966. It should be noted that Bcl6, with which Bach2 cooperates to repress the expression of Prdm1 in B cells, interacts with both NCoR1 and NCoR2 (
). Importantly, the binding sites of Bach2 and Bcl6 in the intron 5 are juxtaposed, and Bcl6 fails to repress Prdm1 reporter expression with a mutation at the MARE (
), suggesting that Bach2 is required for proper epigenetic regulation at the region by cooperating with Bcl6 in recruiting these co-repressor complexes. Recently, HDAC3 has been reported as a new therapeutic target of multiple myeloma (
). Considering that Prdm1 is essential for the terminal differentiation of plasma cells, an inhibition of HDAC3 in multiple myeloma may confer a therapeutic effect by promoting Prdm1 expression.
This study also identified other molecules that may be involved in the transcription repression by Bach2. Interestingly, Rif1 is known to interact with Sirt3, Sirt7 (
). Sirt3 and Sirt7 deacetylate histones, whereas Setdb1 methylates histone at H3-K9. Therefore, Rif1 may recruit additional histone-modifying enzymes such as those to the Prdm1 locus. Another interesting category of molecules includes Arid1a (BAF250) and Smarcd2 (BAF60B), which are the components of SWI/SNF chromatin remodelers (
). The presence of these molecules in the Bach2 complex suggests that chromatin remodeling may also be involved in the repression of Prdm1 and other target genes in B cells. Because Bach2 regulates the expression of diverse sets of genes important not only for B cells but also T cells (
), further studies of the Bach2-interacting proteins will allow mechanistic insights into the regulation of the immune system in the light of a protein network for gene expression.
Author Contributions
H. T., A. M., K. O., and K. I. conceived and coordinated the study and wrote the paper. H. T. and A. M. designed, performed, and analyzed the experiments shown in Fig. 1. A. M. designed, performed, and analyzed the experiments shown in FIGURE 2, FIGURE 3. H. T., K. O., H. S., Y. K., N. S., S. T., T. I., Y. H., and T. N. designed, performed, and analyzed the experiments shown in Fig. 4. H. S. carried out mass spectrometry analysis, and H. S. and K. I. analyzed the data shown in Fig. 5. H. T., N. S., and A. B. designed, performed, and analyzed the experiments shown in Fig. 6, and N. Y. and H. M. provided unpublished reagents. H. T., A. M., N. S., and S. T. designed, performed, and analyzed the experiments shown in Fig. 7. A. M., N. S., and S. T. designed, performed, and analyzed the experiments shown in Fig. 8. K. O. and N. S. designed, performed, and analyzed the experiments shown in Fig. 9. K. I. and M. N. reviewed data. All authors reviewed the results and approved the final version of the manuscript.
Acknowledgments
We thank Dr. Rahul Roychoudhuri for advice on chromatin immunoprecipitation and members of our laboratories for discussion.
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Orchestration of plasma cell differentiation by Bach2 and its gene regulatory network.
Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.
Co-repressor SMRT and class II histone deacetylases promote Bach2 nuclear retention and formation of nuclear foci that are responsible for local transcriptional repression.
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