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Previous Article | Next Article 
Molecular and Cellular Biology, February 2008, p. 1171-1181, Vol. 28, No. 3
0270-7306/08/$08.00+0 doi:10.1128/MCB.01396-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Toru Suzuki,1,*
Kenichi Aizawa,1
Saku Miyamoto,1
Yasushi Imai,1
Takayoshi Matsumura,1
Masami Horikoshi,2 and
Ryozo Nagai1*
Department of Cardiovascular Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan,1 Laboratory of Developmental Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan2
Received 3 August 2007/ Returned for modification 3 September 2007/ Accepted 13 November 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One important question in chromatin transcription which remains poorly understood is the mechanism underlying region-specific (e.g., promoter-specific) nucleosomal regulation. As gene-specific transcription is likely dictated by the DNA-binding transcription factor whose actions are critically dictated through its cognate binding sequence on the promoter, investigations focused on understanding the role of the DNA-binding transcription factor in regulation of transcription at the nucleosomal level will likely be pivotal in helping our understanding of region-specific nucleosomal regulation in transcription.
To this aim, we have previously investigated the structural and functional basis of mechanisms of action of histone chaperones as well as addressing the role of DNA-binding transcription factor in chromatin transcription using the Sp/KLF (for Sp1- and Krüppel-like factor) family (27, 29-32, 44-47). The Sp/KLF family of transcription factors has received recent attention due to roles in various biological events, among which KLF5 (Krüppel-like factor 5) is involved in the cardiovascular remodeling response to stress and acts at the cellular level on cell proliferation and cell cycle regulation (46, 47). This family has been shown to functionally interact with all three classes of chromatin-related cofactors, which is a unique property for DNA-binding factors aside from histones (18, 27, 29, 44, 45, 47). Previously, we have addressed the pathways and mechanisms of transcriptional regulation mediated by Sp/KLF family members and chromatin-related cofactors, which include differential regulation through interaction and acetylation of Sp/KLF by acetylase (29, 44), inhibition of acetylation and DNA accessibility of Sp/KLF by histone chaperone (29, 45), and inhibition of acetylation and DNA accessibility of KLF5 by direct binding of deacetylase (27). Given our understanding of the actions and regulation of this family of factors on chromatin-related cofactors, they serve as an excellent target for further investigations on the role of DNA-binding transcription factor in transcription at the chromatin level, especially in regard to functional interaction with histone chaperones.
In the present study, we identify the novel histone chaperone acidic nuclear phosphoprotein 32B (ANP32B) as a factor interacting with KLF5 and through the analysis of their functional interaction show promoter region-specific histone incorporation and inhibition of histone acetylation as mediated by histone chaperone activity of ANP32B and KLF5-dependent recruitment of ANP32B onto the promoter.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation and identification of factors interacting with KLF5 ZF/DBD. Isolation of interactors with KLF5-ZF/DBD was done as previously described with modification by use of HeLa S3 nuclear extract (29). Protein identification was done using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and identifications were searched against the nonredundant National Center for Biotechnology Information database as previously described (29).
Construction of ANP32B expression vectors. Full-length human ANP32B (NM_006401.2) cDNA was amplified from a human aorta cDNA library (Clontech) by PCR and inserted into the expression vectors pcDNA3 (Invitrogen), pBICEP (SIGMA), pGEX4T-3 (GE Healthcare), and pET30b (Novagen).
Preparation of recombinant proteins. Glutathione S-transferase (GST)-tagged proteins (GST, GST-KLF5-ZF/DBD, and GST-ANP32B) were expressed and affinity purified as previously described (29). GST-KLF5 full length and activation domain were obtained from inclusion bodies and affinity purified. Hexahistidine-tagged proteins (6HIS-KLF5-ZF/DBD, 6HIS-ANP32B, and 6HIS-TAF-Iβ) were expressed and affinity purified as previously described (29). Affinity-purified 6HIS-ANP32B was further purified using Q-Sepharose (GE Healthcare).
Protein-protein interaction assay.
The protein-protein interaction assay for experiments to investigate the binding of KLF5 and its deletion mutants to ANP32B was done as previously described (29). Commercially available recombinant proteins were used for p53, MyoD, and NF-
B (Santa Cruz). Briefly, GST fusion proteins, glutathione-Sepharose 4B resin, and hexahistidine-tagged proteins were incubated in a buffer containing 20 mM HEPES (pH 7.6 at 4°C), 20% glycerol, 0.2 mM EDTA, 0.1% Triton, 100 mM NaCl, and 100 µM ZnSO4 at 4°C for 2 h, and then the mixtures were washed two times in the same buffer. Bound proteins were analyzed by Western blotting using anti-His probe (G-18) antibody (Santa Cruz).
Preparation of anti-ANP32B antibody. Rabbits were immunized with 100 µg of full-length purified recombinant His6-human ANP32B for five times at 1-week intervals, after which serum was extracted. Antibody specificity was confirmed by Western blotting (see Fig. 1H).
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Experiments on endogenous ANP32B and KLF5 were performed as experiments on the effect of PMA stimulation on the interaction of ANP32B and KLF5 with slight modification. Briefly, transfection, serum starvation, and PMA stimulation were not done; extracts were incubated with 1 µg of anti-ANP32B antibody (Abcam) or normal rabbit IgG; and bound proteins were eluted using 0.5 M glycine (pH 2.5). Precipitate was analyzed by Western blotting using anti-FLAG-horseradish peroxidase (Sigma), rabbit polyclonal anti-ANP32B, and anti-KLF5 (KM1785) antibodies.
Gel shift DNA-binding assay. Gel shift DNA-binding assays were performed as previously described (27) with some modification. Briefly, recombinant proteins and 2.5 pmol of 5'-fluorescein isothiocyanate-labeled probes were incubated at room temperature for 30 min in BA150 (10 mM Tris HCl [pH 7.5], 10% glycerol, 150 mM NaCl, and 0.1% NP-40) containing 6 µM ZnSO4. Gels were analyzed using Typhoon 3700 (GE Healthcare).
Cotransfection reporter assay. The cotransfection reporter assay was done as previously described (3) with slight modification. Briefly, HeLa cells were transfected with the platelet-derived growth factor A (PDGF-A) chain (–900)-Luc promoter reporter (0.1 µg) and effector expression vectors. The total effector DNA amount in transfection reaction mixtures was corrected to 1 µg by addition of empty vector.
Histone binding assay. Core histones were purified from HeLa S3 cells as previously described (43). GST fusion proteins were immobilized onto glutathione-Sepharose 4B resin and incubated with 5 µg of core histones or calf thymus linker histones (Sigma) in BA150 (10 mM Tris HCl [pH 7.5], 10% glycerol, 150 mM NaCl, and 0.1% NP-40) at 4°C and then washed three times with BA150. Precipitated proteins were analyzed by Coomassie blue staining or Western blotting using anti-His antibody.
Plasmid supercoiling assay. Nucleosome assembly reactions were performed as previously described (30) with slight modification. Briefly, after preincubation for 30 min at 37°C, reaction mixtures and relaxed DNA were incubated at 37°C for 2 h.
MNase digestion assay. Nucleosome assembly reactions were done with twofold-increased reaction volumes and factors compared with those in the plasmid supercoiling assay, and then the reaction mixtures were incubated with micrococcal nuclease (MNase) (Takara) at each concentration in the presence of 6 mM CaCl2 for 7 min at room temperature followed by DNA extraction.
RNA interference analysis. The target sequences of small interfering RNA (siRNA) oligonucleotide probes were as follows: ANP32B, 5'-GAAGAGGAGUUUGAUGAAGAAGA-3' (34); KLF5, 5'-AACCCGGAUCUGGAGAAGCGA-3' (7); and secreted alkaline phosphatase (SEAP), 5'-AGGGCAACUUCCAGACCAUU-3' (3). siRNAs were constructed as previously described (3).
Effect of RNA interference of ANP32B on expression levels of PDGF-A chain. Four micrograms of siRNA was transfected into 2 x 105 HeLa cells using Lipofectamine 2000. After 48 h of incubation using DMEM (10% FCS), cells were harvested and total RNA was obtained with the RNeasy preparation kit (Qiagen) and then reverse transcribed. cDNA was analyzed by quantitative PCR using gene-specific primer sets and Quantum RNA 18S internal standard primer set I (Ambion) as previously described (29). The relative intensity of each gene was calculated in reference to internal 18S rRNA. The gene-specific primer pairs were as follows: ANP32B, 5'-TGGATGGTGTGGATGAAGAGGA-3' and 5'-TGTTTCTGCAGGTCATCTGGGG-3'; KLF5, 5'-GGCTTGGCGCCCGTGTGCTTCC-3' and 5'-GGTTGCACAAAAGTTTATAC-3'; and PDGF-A chain, 5'-CAGCATCCGGGACCTCCAGCGACTC-3' and 5'-TCGTAAATGACCGTCCTGGTCTTGC-3'.
Chromatin immunoprecipitation (ChIP) assay. (i) Transfection and cell treatment. For experiments on RNA interference of ANP32B, 80 µg of siRNA was transfected into 2 x 106 HeLa cells. Cells were harvested after 48 h of incubation using DMEM (10% FCS). For experiments on localization of ANP32B, 24 µg of pBICEP-ANP32B was transfected into 2 x 106 HeLa cells. Cells were harvested after 48 h of incubation using DMEM (10% FCS). For experiments on RNA interference of both ANP32B and KLF5, 80 µg of siANP32B and/or siKLF5 was transfected into 6 x 106 HeLa cells. The total siRNA amount in transfection reaction mixtures was corrected to 160 µg by addition of siSEAP. Cells were harvested after 24 h of incubation using DMEM (10% FCS). For experiments on the effect of RNA interference of KLF5 on localization of ANP32B, 24 µg of pBICEP-ANP32B and 80 µg of siRNA were transfected into 2 x 106 HeLa cells. Cells were harvested after 48 h of incubation using DMEM (10% FCS). For experiments on the effect of PMA stimulation, 2 x 106 HeLa cells were treated with DMEM (10% FCS) for 24 h and then stimulated by 200 nM of PMA after treatment with DMEM for 24 h. For experiments on the effect of PMA stimulation on localization of ANP32B, 24 µg of pBICEP-ANP32B was transfected into 4 x 106 HeLa cells and then cells were treated with DMEM (10% FCS) for 24 h and further stimulated with 200 nM of PMA after treatment with DMEM for 24 h.
(ii) Anti-KLF5, FLAG-ANP32B, or acetylated histone H3 and H4 ChIP assays. Anti-KLF5 and anti-FLAG ChIP assays were performed as previously described (26) with slight modification. For anti-KLF5 ChIP analysis, fixed proteins were incubated with 2 µg of rabbit polyclonal anti-KLF5 (48) or normal rabbit IgG (Santa Cruz). For anti-acetylated histone H3 and H4 ChIP analyses, fixed proteins were incubated with 2 µg of rabbit polyclonal anti-acetylated histone H3 (K9 and K14) (Upstate Biotechnology), rabbit polyclonal anti-acetylated histone H4 (K5, K8, and K12) (Upstate Biotechnology), or normal rabbit IgG (Santa Cruz) in the presence of 50 mM sodium butyrate. For anti-FLAG ChIP analysis, fixed proteins were incubated with 2 µg of mouse monoclonal anti-FLAG (Sigma) or normal mouse IgG (Santa Cruz) antibodies. Proteins and antibodies were adsorbed to protein G Sepharose (GE Healthcare) and then washed. For anti-KLF5 and FLAG-ANP32B ChIP analyses, washing was done with wash buffer (10 mM Tris HCl [pH 8.0], 0.1% sodium dodecyl sulfate, 1% Triton, 1 mM EDTA, and 150 mM NaCl), LiCl buffer (10 mM Tris HCl [pH 8.0], 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, and 250 mM LiCl), and TE-200 (10 mM Tris HCl [pH 8.0], 1 mM EDTA, and 200 mM NaCl). For anti-acetylated histone H3 and H4 ChIP analyses, washing was done using the same buffer containing 50 mM sodium butyrate.
(iii) Anti-histone H2B and H4 ChIP assays. Antihistone ChIP assays were performed as previously described (33) with modification of nucleus isolation according to previously described methods for nucleosome mapping with nuclease (21, 24). Cells were harvested and treated in cell lysis buffer (10 mM Tris HCl [pH 7.5], 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 0.15 mM spermine, 0.5 mM spermidine, and 50 mM sodium butyrate) and centrifuged at 8,000 rpm for 4 min at 4°C, and then precipitate was harvested as nuclei. Nuclei were suspended in nuclear buffer (10 mM Tris HCl [pH 7.5], 50 mM NaCl, 10 mM MgCl2, 0.2 mM EDTA, 0.2 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 1 mM β-mercaptoethanol, and 50 mM sodium butyrate). Nuclei were incubated with CaCl2 at a final concentration of 2 mM and 20 units of MNase (Takara) at 37°C for 5 min, and then reactions were stopped by addition of EDTA to a concentration of 10 mM. Soluble chromatin was harvested as previously described (33) and incubated with 8 µg of rabbit polyclonal anti-H4 (H-97; Santa Cruz), whose epitope corresponds to amino acids residues 7 to 103 of the N terminus of human histone H4; rabbit polyclonal anti-H2B (Abcam), whose epitope is derived from a peptide region containing from amino acid residue 100 to the C terminus of human histone H2B; or normal rabbit IgG (Santa Cruz) and then adsorbed to protein G Sepharose (GE Healthcare).
(iv) Analysis of immunoprecipitated DNA. Immunoprecipitated DNA was analyzed by quantitative PCR using a Light Cycler (Roche) and QuantiTect Sybr green PCR Kit (Qiagen). The region-specific primers pairs were as follows: 5'-(–3,000-bp) region, 5'-GCGAAAGTTTCTTCCCCTCT-3' and 5'-GAGTCAGGTGCCCCAAAATAG-3'; KLF5 site (promoter region), 5'-TGGCACTGGAGGGTGGGCAA-3' and 5'-GAAGCGCTAGGGGTTCGT-3'; and 3' region, 5'-GTGTTACATTCCTGAACCTACTATGTACGGTGC-3' and 5'-CTGCTTCACCGAGTGCTACAATACTTGCTTTGATG-3'.
Phorbol ester-induced expression of KLF5 and ANP32B. Cell lysate from HeLa cells stimulated with 200 nM of PMA following 24 h of treatment with DMEM was analyzed by Western blotting using anti-KLF5 (KM1785) or anti-ANP32B antibody. The relative intensity of KLF5 or ANP32B protein in reference to Coomassie blue staining was calculated by using National Institutes of Health Image software. Quantitative PCR using Quantum RNA 18S internal standard primer set I (Ambion) and calculation of the relative intensity of PDGF-A chain mRNA were done as previously described (29).
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RESULTS |
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We next investigated whether ANP32B directly and specifically interacts with KLF5-ZF/DBD by using deletion mutants (Fig. 1E) and other DNA-binding proteins (29) (Fig. 1F and G). A GST pull-down assay showed that ANP32B directly binds GST-KLF5 full length and GST-ZF/DBD (Fig. 1F, lanes 2 and 3) but not the activation domain, which excludes the ZF/DBD (Fig. 1F, lane 4). Further analysis showed that ANP32B binds KLF5 (Fig. 1G, lane 2) but not p53, MyoD, or NF-B (Fig. 1G, lanes 3 to 5). Therefore, ANP32B directly and specifically binds KLF5 through its ZF/DBD. Further, a coimmunoprecipitation assay using anti-ANP32B antibody, whose specificity was confirmed by Western blotting (Fig. 1H), showed that endogenous ANP32B and KLF5 proteins interact in the cell (Fig. 1I, lane 3). Thus, KLF5 and ANP32B specifically and directly bind in the cell.
Functional effects of interaction between ANP32B and KLF5. To next address the functional implications of interaction of ANP32B with KLF5, we first examined the effect of ANP32B on the DNA-binding activity of KLF5 by gel shift analysis using KLF5 and ANP32B recombinant proteins (Fig. 2A). Under conditions in which KLF5-ZF/DBD showed sequence-specific DNA binding (Fig. 2B) and ANP32B did not bind DNA (Fig. 2C, lanes 1 to 4), addition of GST-ANP32B inhibited DNA-binding activity of KLF5-ZF/DBD (Fig. 2C, lanes 5 to 7 versus 8 to 10). This indicates that ANP32B possesses the ability to negatively regulate DNA-binding activity of KLF5 or that KLF5 association with ANP32B and DNA is mutually exclusive.
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Characterization of the novel histone chaperone ANP32B. ANP32B is a member of a conserved superfamily which includes pp32/ANP32A, a factor that binds histones and inhibits their acetylation (19, 40). As the biochemical functions of ANP32B are unknown, we first examined whether ANP32B binds histones (Fig. 3A). A GST pull-down assay showed ANP32B to directly bind core histones, with a preference for core histones rather than linker histones (Fig. 3A, lanes 3 and 4).
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Effects of loss of function of ANP32B on KLF5-mediated chromatin transcription. As ANP32B possesses dual activities to stimulate nucleosome assembly and to inhibit DNA-binding activity of KLF5, we reasoned that ANP32B could regulate KLF5-mediated transcription by inhibiting binding of KLF5 to its cognate binding sites and by inducing incorporation of histones.
To investigate this, we first examined the effect of RNA interference of ANP32B on endogenous PDGF-A chain expression, as ANP32B represses KLF5-mediated transcription of this gene (Fig. 2D). RNA interference of ANP32B reduced levels of ANP32B (Fig. 4A, lanes 1 and 2) as previously described (34), which resulted in an increase in PDGF-A chain mRNA expression (Fig. 4A, lanes 3 and 4), while KLF5 expression was not affected (Fig. 4A, lanes 5 and 6). This indicates that ANP32B negatively regulates transcription of the KLF5-downstream gene PDGF-A chain.
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We further examined whether ANP32B participates in histone incorporation over the PDGF-A chain gene (Fig. 4C and D). Anti-histone H4 ChIP analysis showed that RNA interference of ANP32B showed decreased histone H4 levels over the KLF5 binding site on the PDGF-A chain gene promoter (Fig. 4C, lanes 3 and 4) while not affecting histone H4 levels over the 5'-(–3,000 bp) or the 3' regions of the gene (Fig. 4C, lanes 1, 2, 5, and 6). Anti-histone H2B ChIP analysis showed the same effect on histone H2B levels over the PDGF-A chain gene promoter (Fig. 4D, lanes 1 to 6). These findings indicate that ANP32B participates in incorporation of both histones H2A/H2B and histones H3/H4.
As ANP32B is a member of a conserved superfamily which includes pp32/ANP32A, a factor that binds histones and inhibits their acetylation (40), we reasoned that ANP32B inhibits acetylation of histones leading to transcriptional repression. To address the relationship between histone acetylation, histone eviction, and gene activation, we focused upon regulation of acetylation of K9 and K14 of H3 and K5, K8, and K12 of histone H4, which have been reported to be coupled with transcriptional activation and inhibition as well as recognition by INHAT (inhibition of histone acetyltransferase) subunits (37, 39). To address whether ANP32B participates in inhibition of histone acetylation leading to transcriptional activation, we examined the effect of RNA interference of ANP32B on acetylated histone levels (Fig. 4E and F). Anti-acetylated histone H3 and H4 ChIP assays showed that RNA interference of ANP32B increased levels of acetylated histones H3 and H4 over the KLF5 binding site on the PDGF-A chain gene promoter (Fig. 4E and F, lanes 3 and 4) while not affecting levels of acetylated histone H3 and H4 over the 5'-(–3,000 bp) or the 3' regions of the gene (Fig. 4E and F, lanes 1, 2, 5, and 6). This indicates that ANP32B participates in inhibition of histone acetylation in a promoter region-specific manner.
To confirm that ANP32B mediates histone incorporation over the KLF5 binding sites, we examined whether ANP32B localizes to the PDGF-A chain gene promoter (Fig. 4G). The ChIP assay showed that relative levels of FLAG-ANP32B over the 5'-(–3,000 bp) or the 3' regions of the gene were markedly lower than that over the KLF5 sites of the promoter (Fig. 4G, lanes 1 to 3). This indicates that ANP32B localizes to the PDGF-A chain gene in a promoter-specific manner.
These findings collectively suggest that ANP32B represses KLF5-mediated transcription by dual pathways of inhibition of DNA-binding activity of KLF5 by protein-protein interaction and inhibition of promoter access of KLF5 by histone incorporation over its cognate binding sites.
Recruitment of ANP32B by KLF5. As KLF5 and ANP32B directly interact and ANP32B localizes on the PDGF-A chain gene promoter in a region-specific manner, we reasoned that KLF5 recruits ANP32B onto the promoter region (Fig. 5A). To address whether KLF5 possesses the ability to recruit ANP32B onto the promoter region, we examined effects of RNA interference of KLF5 on ANP32B levels over the promoter region. Under the conditions in which the ChIP assay showed promoter region-specific localization of ANP32B, RNA interference of KLF5 decreased ANP32B levels over the promoter region (Fig. 5A, lanes 3 and 4). This indicates that ANP32B is recruited onto the promoter region of the PDGF-A chain gene in a KLF5-dependent manner.
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Dynamics of ANP32B, KLF5, and histones over the PDGF-A chain gene in response to transactivational stimulus. To examine the cellular implications of functional interaction of KLF5 and ANP32B in chromatin transcription, we investigated how ANP32B, KLF5, and histones act over the PDGF-A chain gene promoter in response to a KLF5-activating stimulus. For this purpose, we examined effects of phorbol ester (as represented by PMA) on PDGF-A chain gene expression as mediated by KLF5. PMA is a model agonist of inducible extracellular pathophysiological stimulation known to transcriptionally activate KLF5, which in turn upregulates its endogenous downstream gene, PDGF-A chain (3, 42).
We first assessed expression levels of ANP32B and KLF5 proteins and PDGF-A chain mRNA under PMA stimulation (Fig. 6A). ANP32B protein expression was not affected under the condition in which KLF5 protein expression and PDGF-A chain mRNA expression were induced by PMA stimulation as previously described (29). ANP32B is thus a constitutive factor under this condition. Next, we assessed whether interaction of KLF5 and ANP32B is regulated by PMA stimulation by using a coimmunoprecipitation assay (Fig. 6B). As KLF5 is an inducible factor, experiments were done with cotransfection of ANP32B and KLF5 under the condition in which expression levels of KLF5 and ANP32B were not affected by PMA stimulation (Fig. 6B, lanes 7 to 12). The coimmunoprecipitation assay showed that interaction of KLF5 and ANP32B was attenuated by PMA stimulation (Fig. 6B, lanes 4 to 6). Thus, while ANP32B is not an inducible protein, its interaction with KLF5 is attenuated by PMA stimulation.
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To address the functional implications of regulation of histone acetylation and transcriptional activation by PMA stimulation, we examined the effect of PMA stimulation on levels of acetylated histones H3 and H4 (Fig. 7D). ChIP assays of anti-acetylated histones H3 and H4 showed that PMA stimulation increased acetylated histone H3 and H4 levels over the promoter region of the PDGF-A chain gene (Fig. 7D, lanes 3 and 4) while not affecting histone H4 levels over the 5'-(–3,000 bp) or the 3' regions of the gene (Fig. 7D, lanes 1, 2, 5, and 6). These findings indicate that transcriptional activation mediated by PMA stimulation is coupled with promoter access of KLF5 as well as promoter region-specific histone eviction and histone acetylation.
Moreover, to confirm that ANP32B participates in regulation of promoter region-specific histone eviction and histone acetylation as mediated by PMA stimulation, we examined the effect of PMA stimulation on localization of ANP32B over the PDGF-A chain gene promoter (Fig. 7E). Anti-ANP32B ChIP analysis showed that PMA stimulation decreased ANP32B levels over the promoter region (Fig. 7E, lanes 3 and 4), while not affecting ANP32B levels over the 5'-(–3,000 bp) and 3' regions of the gene (Fig. 7E, lanes 1, 2, 5, and 6). This indicates that ANP32B localizes over the PDGF-A chain promoter during transcriptional repression while not during transcriptional activation.
These findings collectively indicate that a transactivating stimulus regulates interaction of ANP32B and KLF5 from an associative to a dissociative state, thus leading to transcriptional activation in a manner correlated with region-specific histone eviction. Thus, transcriptional activation of the PDGF-A chain gene is mediated by histone eviction over the KLF5 binding site and binding of KLF5 to its cognate binding site.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We showed that ANP32B harbors multiple activities which include nucleosome assembly in vitro (Fig. 3), histone incorporation in vivo (Fig. 4), inhibition of histone acetylation (Fig. 4), inhibition of DNA-binding activity of KLF5 by protein-protein interaction (Fig. 2), and repression of transcription of PDGF-A chain, which is a downstream gene of KLF5 (Fig. 2 and 4).
Importantly, functional interaction of histone ANP32B and KLF5 resulted in promoter region-specific histone incorporation and inhibition of histone acetylation on the KLF5-downstream PDGF-A chain gene which was coupled with transcriptional repression (Fig. 4 and 5). An extracellular stimulus (e.g., phorbol ester) further regulated this mechanism in the cell, which supports the biological relevance of this interaction (Fig. 6 and 7). RNA interference of KLF5 resulted in decreased ANP32B and histone levels over the PDGF-A chain gene promoter (Fig. 5), which indicates that recruitment of ANP32B onto the PDGF-A chain gene promoter leading to histone incorporation by ANP32B requires KLF5. PMA stimulation showed an increase in KLF5 levels and a decrease in ANP32B and histone levels (Fig. 7), which indicates that recruitment of ANP32B or KLF5 onto the PDGF-A chain gene promoter results in eviction of ANP32B or KLF5. Further, as ANP32B inhibited the DNA-binding activity of KLF5 (Fig. 2), it would seem that, while KLF5 and ANP32B cooperatively interact during recruitment, occupancy on the promoter is mutually exclusive.
In perspective, the coupling of transcriptional regulation and histone eviction/incorporation has been best studied on the Saccharomyces cerevisiae PHO5 promoter. The transcription factors Pho2 and Pho4 activate PHO5 by promoter access (10, 49). A recent study showed that the histone chaperone Spt6 incorporates histones during transcriptional repression of PHO5 and that histone incorporation by Spt6 and promoter access to Pho2/Pho4 are independent (2). Importantly, as protein-protein interaction and/or coregulation of Spt6 and Pho2/Pho4 has not been addressed (as Spt6 and Pho2/Pho4 are not regulated by protein-protein interaction and/or coregulation), regulation of histone incorporation/eviction and DNA-binding activity of transcription factors are mutually independent in the Spt6-Pho2/Pho4 system. In contrast to the Spt6-Pho2/Pho4 system, histone incorporation/eviction and DNA-binding activity of transcription factor are coupled in regulation of transcription as mediated by ANP32B and KLF5.
A recent study showed that the histone chaperone JDP2, an interactor with the transcription factor Jun (6, 15), inhibits DNA-binding activity of Jun, assembles nucleosomes, and represses Jun-mediated transcription (16). We have shown that the histone chaperone TAF-Iβ inhibits KLF5-mediated transcription by protein-protein interaction with KLF5 ZF/DBD (29). These histone chaperones, JDP-2 and TAF-I, directly bind transcription factors and negatively regulate transcription; however, involvement of these histone chaperones in histone incorporation in a promoter region-specific manner was not addressed.
Collectively, we have described cooperative regulation of promoter region-specific histone incorporation in transcriptional repression by DNA-binding transcription factor and histone chaperone.
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ACKNOWLEDGMENTS |
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We declare that we have no competing financial interest.
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FOOTNOTES |
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Published ahead of print on 26 November 2007.
These authors contributed equally.
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