And Represses TNF Gene Expression Activating Transcription Factor-2

Histone Deacetylase 3, a Class I Histone Deacetylase, Suppresses MAPK11-Mediated Activating Transcription Factor-2 Activation and Represses TNF Gene Expression This information is current as of September 25, 2021. Ulrich Mahlknecht, Jutta Will, Audrey Varin, Dieter Hoelzer and Georges Herbein J Immunol 2004; 173:3979-3990; ; doi: 10.4049/jimmunol.173.6.3979 http://www.jimmunol.org/content/173/6/3979 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Histone Deacetylase 3, a Class I Histone Deacetylase, Suppresses MAPK11-Mediated Activating Transcription Factor-2 Activation and Represses TNF Gene Expression1

Ulrich Mahlknecht,2* Jutta Will,* Audrey Varin,† Dieter Hoelzer,* and Georges Herbein2†

During inflammatory events, the induction of immediate-early genes, such as TNF-␣, is regulated by signaling cascades including the JAK/STAT, NF-␬B, and the p38 MAPK pathways, which result in phosphorylation-dependent activation of transcription factors. We observed the direct interaction of histone deacetylase (HDAC) 3, a class I histone deacetylase, with MAPK11 (p38 ␤ isoform) by West-Western-based screening analysis, pull-down assay, and two-hybrid system analysis. Results further indicated that HDAC3 decreases the MAPK11 phosphorylation state and inhibits the activity of the MAPK11-dependent transcription factor, activating transcription factor-2 (ATF-2). LPS-mediated activation of ATF-2 was inhibited by HDAC3 in a time- and Downloaded from dose-dependent manner. Inhibition of HDAC3 expression by RNA interference resulted in increased ATF-2 activation in response to LPS stimulation. In agreement with decreased ATF-2 transcriptional activity by HDAC3, HDAC3-repressed TNF gene expression, and TNF protein production observed in response to LPS stimulation. Therefore, our results indicate that HDAC3 interacts directly and selectively with MAPK11, represses ATF-2 transcriptional activity, and acts as a regulator of TNF gene expression in LPS-stimulated cells, especially in mononuclear phagocytes. The Journal of Immunology, 2004, 173: 3979–3990. http://www.jimmunol.org/ echanisms of transcriptional activation have received recently been identified (6). The human histone deacetylase, considerable attention in recent years; however, it is HDAC3, is a 49 kDa protein that is expressed by many different M becoming evident that transcriptional repression, cell lines and multiple normal human tissues and is located both in which may be mediated by a number of different mechanisms, is the cell nucleus and in the cytoplasm (4, 7, 8). Although the C- just as important (1). Relaxation of the chromatin fiber facilitates terminal proportion of HDAC3 contributes to the protein’s local- transcription and is regulated by two competing enzymatic activ- ization in the nucleus via a nuclear localization sequence, which is ities, histone acetyltransferases (HATs)3 and histone deacetylases sufficient for the transport of a reporter fusion protein into the (HDACs), which modify the acetylation state of histone proteins nucleus and is required for both its deacetylase and transcriptional and other promoter-bound transcription factors (2). A still growing repression activities, the central portion of the HDAC3 protein by guest on September 25, 2021 number of HDACs has been identified in various systems and possesses a nuclear export signal (8). HDAC3 preferentially model organisms (3). On the basis of their similarity to corre- deacetylates histone lysines 5 and 12 of H4 and lysine 5 of H2A sponding yeast ancestor proteins, these enzymes have been clas- (9). HDAC3 forms oligomers in vitro and in vivo and gains its sified into three distinct families of which class I HDACs enzymatic activity in association with multiprotein repressor com- (HDAC1, 2, 3, and 8) are closely related to the yeast transcrip- plexes that contain silencing mediator for retinoid and thyroid hor- tional regulator, RPD3 (4). Class II HDACs (HDAC4, 5, 6, 7, 9, mone receptors and nuclear receptor corepressors (4, 10, 11). and 10) are similar to the yeast deacetylase, Hda1p (5). Finally, a Apart from deacetylating histone proteins and its capability to alter third class of HDACs, which contains the NAD-dependent sirtuin chromosome structure, which affects transcription factor access to proteins (SIRT1-SIRT7), which are homologous to yeast SIR2, has DNA, HDAC3 has also been described to directly associate with nonhistone proteins, such as the ATP-dependent chaperone protein, heat shock protein 70 (9) and with class II HDACs (7). In *Department of Hematology/Oncology, University of Frankfurt Medical Center, addition, HDAC3 has been linked with the direct suppression of Frankfurt am Main, Germany; and †Department of Virology, Franche-Comte« Uni- versity, Besançon, France the transcriptional potential of the transcription factors GATA-2, Received for publication February 17, 2004. Accepted for publication June 30, 2004. YY1, and TFII-I (12Ð14) and with the deacetylation of RelA, a subunit of NF-␬B, which has been reported to be exported from the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance nucleus in an IkB␣-dependent fashion (15). HDAC3 has also been with 18 U.S.C. Section 1734 solely to indicate this fact. demonstrated to inhibit the JNK pathway via G-protein pathway 1 This work was supported by the German National Science Foundation (Deutsche suppressor 2, which is a component of the nuclear receptor co- Forschungsgemeinschaft, MA 2057/2-3), the Heinrich and Fritz Riese Foundation, the August Scheidel Foundation, and the Franche-Comte University. repressor-HDAC3 complex (16). Four MAPK subgroups have been identified in humans: ERK, 2 Address correspondence and reprint requests to Dr. Georges Herbein, Department of Virology, Franche-Comte University, 2 Place Saint-Jacques, F-25030 Besançon, JNK/stress-activated protein kinases, the ERK5/big MAPK pro- France or Dr. Ulrich Mahlknecht at the current address: Medizinische Klinik und tein, and p38 MAPK. The p38 group of MAPK contains four iso- Poliklinic V, Universitatsklinicum Heidelberg, Hospitalstrasse 3, D-69115 Heidel- ␣ ␤ ␥ ␦ berg, Germany. E-mail addresses: [email protected] or Ulrich_Mahlknecht@ forms: p38 , p38 , p38 , and p38 (17). The p38 MAPK are med.uni-heidelberg.de strongly activated by cytokines, LPS, and environmental stress, but 3 Abbreviations used in this paper: HAT, histone acetyltransferase; HDAC, histone are poorly activated by growth factors and phorbol ester (17). They deacetylase; ATF-2, activating transcription factor-2; MKK, MAPK kinase; CRE, are widely expressed in many tissues and require dual phosphor- cAMP responsive element; pTNF-Luc, luciferase reporter plasmid driven by the human TNF promoter; M⌽, macrophage; TSA, trichostatin A; siRNA, small interfer- ylation on threonine and tyrosine residues to become activated. ence RNA. This phosphorylation is mediated by a protein kinase cascade,

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 3980 HDAC3 INHIBITS MAPK11/ATF-2-MEDIATED SIGNALING which consists of MAPK kinases (MKKs) that include MKK3 and cultivated in RPMI 1640 supplemented with 10% FBS. Cos7 cells were MKK6 (17). MAPK11 is known to activate downstream substrates cultured in DMEM (Invitrogen Life Technologies, Carlsbad, CA) supple- like the transcription factors serum response factor accessory pro- mented with 10% FCS, 1% glutamine, and 1% penicillin/streptomycin. Primary monocyte-derived macrophages (M⌽) were prepared from the pe- tein-1a and activating transcription factor-2 (ATF-2) through phos- ripheral blood of healthy donors and were cultured in RPMI 1640 medium phorylation (18). ATF-2 is a member of the ATF-CREB family of supplemented with 10% (v/v) pooled AB human serum (Sigma-Aldrich, transcription factors, which have been implicated in cell cycle pro- Munich, Germany), as previously described (29). gression, cell differentiation, transformation, and immune response Transfection and reporter assay (19). Being a leucine zipper transcription factor, ATF-2 binds an 8-bp response element (5Ј-TGACGTCA-3Ј), while as a homo-/ To carry out transient transfections, the DNA concentration was kept con- heterodimer it associates with other members of the ATF family or stant in the different samples by using the corresponding empty vector. A total of 5 ϫ 106 cells were transfected with 5Ð10 ␮g of total plasmid DNA, with members of the Jun/Fos family of transcription factors (20). which included the firefly luciferase-expressing vector using the DEAE- Most common is the ATF-2/c-Jun heterodimer, which recognizes dextran procedure (30). At 48 h posttransfection, luciferase activity was the AP-1/cAMP response element (CRE) target sequence (20). measured in cell lysates using a luminometer (TD-20/20; Promega) as pre- Upon its phosphorylation on Ser121, ATF-2 associates with p300/ viously described (30). Luciferase expression was normalized with respect CBP, which links it to the basal transcriptional complex (21). Like to protein concentrations using the Detergent-Compatible Protein Assay (Bio-Rad, Munich, Germany). For HDAC3 RNA interference experiments, p300, ATF-2 was also reported to elicit HAT activities that are 10 ␮g of pBS/U6/siHDAC3 (Upstate Biotechnology, Lake Placid, NY) increased upon its phosphorylation (18). were incubated with 106 cells in 300 ␮l of medium, 10% FCS, and elec- TNF-␣ exerts a variety of biological effects, including production troporated at 200 V. Cell lysates were prepared 3 days postelectroporation of inflammatory cytokines, up-regulation of adhesion molecules, pro- and analyzed by Western blotting using anti-HDAC3 (Upstate Biotechnol- Downloaded from ogy) and anti-tubulin Abs (Sigma-Aldrich). For reporter gene assays in liferation, differentiation, and cell death (22). Monocytic cells release U937 cells, 2 ␮g of pATF-2 were cotransfected with 2 ␮g of pBS/U6/ TNF in response to many stimuli, including the Gram-negative bac- SiHDAC3 and luciferase activity was measured 3 days after transfection. terial endotoxin, LPS, TNF itself, phorbol esters, superantigens, and To examine TNF promoter-driven gene expression, 107 U937 cells were viral agents (23, 24). Regulation is both transcriptional and posttran- cotransfected with 4 ␮g of pTNF-Luc and 2 ␮g of pHDAC3 vector or an scriptional, depending on the stimulus, cell type, and possibly differ- empty control vector, using the DEAE-dextran procedure (30). Twenty- four hours later, the cells were stimulated with LPS at 100 ng/ml (Sigma-

Ϫ http://www.jimmunol.org/ entiation (25, 26). Sequences in the proximal 172 bp and in the 627 Aldrich) and, at 48 h posttransfection, luciferase activity was measured. All to Ϫ487 bp region of the human TNF promoter, at least, contribute to transfections were done in triplicate. transcriptional control in monocytic cells (24, 26, 27). Each of the above stimuli modifies transcription factors interaction with the Ϫ116 EMSA to Ϫ88 bp region. This region includes putative CRE/ATF, NF-␬B, Nuclear extracts were prepared from U937 and THP-1 cells transfected CCAAT/enhancer-binding protein, and Ets binding sites (24). Factors with a HDAC3 expression vector or the empty control vector and left binding to the CRE/ATF site (Ϫ106 to Ϫ99 bp) and the NF-␬B site unstimulated or stimulated with 100 or 1000 ng/ml LPS during different Ϫ Ϫ periods of time. To measure AP-1 and NF-␬B activation, EMSA were ( 97 to 88 bp) cooperate functionally in both monocytic and lym- conducted as previously described (30, 31). To determine ATF-2 activa- phocytic cells (24, 25). tion, we used the Nufshift ATF-2 kit (Active Motif, Rixensart, Belgium). The purpose of this study was the identification and character- Nuclear extracts were incubated with 32P-end-labeled 23-mer double- by guest on September 25, 2021 ization of nonhistone interaction partners of the human histone stranded ATF-2 oligonucleotide, 5Ј-GATTCAATGACATCACGGCT Ј deacetylase, HDAC3. In this study, we show that HDAC3, a class GTG-3 (bold indicates ATF-2 binding sites). The specificity of the binding was examined by competition with a mutated unlabeled oligonucleotide I HDAC, interacts with MAPK11, inhibits the transcriptional ac- 5Ј-GATTCAAGAACATAGCGGCTGTG-3Ј. The DNA-protein complex tivity of the MAPK11-dependent transcription factor, ATF-2, and formed was analyzed on a 6% native polyacrylamide gel. The dried gels represses TNF gene expression. All together, our data indicate that were visualized and radioactive bands quantified by a PhosphorImager HDAC3 plays an important role in the regulation of TNF expres- (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software (Am- ersham Biosciences, Piscataway, NJ). The double-stranded oligonucleo- sion in monocytic cells. tides with AP-1 or NF-␬B-binding motifs were obtained from Eurobio (Paris, France). Materials and Methods Plasmids and construction of expression vectors Chromatin immunoprecipitation assays The pcDNA3.1-based expression vector for human HDAC3 has been de- U937 cells were transfected with a HDAC3 expressing vector (pHDAC3) scribed elsewhere (4). Deletion constructs were generated by standard PCR or the empty vector (mock), and treated 24 h later with 100 ng/ml LPS. and cloning procedures (28). HDAC3 constructs were verified by DNA se- After 0, 7, and 28 min of treatment, cells were treated with 1% parafor- quencing. MAPK11 cDNAs (GenBank U53442) were kindly provided by maldehyde at room temperature. After 10 min, the cells were harvested, Dr. J. Han (The Scripps Research Institute, La Jolla, CA) and the Reference and chromatin immunoprecipitation assays were performed in accordance Center of the German Human Genome Project (Deutsches Ressourcenzentrum with the manufacturer’s instructions (Upstate Biotechnology). Immunopre- cipitations were done overnight at 4¡C with 10 ␮g of anti-ATF-2 Ab or 10 fu¬r Genomforschung (RZPD), Berlin, Germany; GenBank AF031135). The ␮ luciferase reporter plasmid driven by the human TNF promoter (pTNF-Luc) g of anti-cFos Ab used as an irrelevant Ab (Active Motifs). Immune was kindly provided by Dr. D. Kwiatkowski (International Child Health complexes were collected with protein A-agarose preblocked with soni- Group, University Department of Pediatrics, John Radcliffe Hospital, Oxford, cated salmon sperm DNA and BSA (Upstate Biotechnology). Immunopre- U.K.; Ref. 26). In vivo detection of HDAC3/MAPK11 interactions was done cipitates were washed three times in washing buffer (immunoprecipitation by transient cotransfections of GAL4-HDAC3 and VP16-MAPK11 in the con- buffer supplemented with 0.1 mg/ml yeast tRNA) and three times in wash- text of a mammalian two-hybrid system (CheckMate; Promega, Mannheim, ing buffer containing 500 mM NaCl. Immune complexes were eluted with Germany). In vivo assessment of ATF-2 activation and the upstream p38 sig- 1% SDS and 100 mM NaHCO3. Formaldehyde cross-links were reversed nal transduction pathway was done with the PathDetect in vivo signal trans- by incubating the samples with 200 mM NaCl at 65¡C for 4 h. The im- duction pathway trans-reporting system from Stratagene (La Jolla, CA). Se- munoprecipitated DNA was purified by proteinase K treatment, phenol- quence authenticity was confirmed by cDNA cycle sequencing. chloroform extractions, and ethanol precipitation. Immunoprecipitated DNA and input (nonimmunoprecipitated) chromatin were analyzed by Cell culture PCR using the following primer pair for human TNF promoter detection (sense, 5Ј-GTC CCC AAC TTT CCA AAT CC-3Ј; and antisense, 5Ј-CAA Most of the study was performed with the promonocytic U937 cells and CCA GCG GAA AAC TTC CTT-3Ј). PCR was performed using the fol- with the Cos7 cells obtained from the American Type Culture Collection lowing primer pair for human ␤-globin gene detection (sense, 5Ј-GAA (Manassas, VA) and with human THP-1 promonocytes kindly provided by GAG CCA AGG ACA GGT AC-3Ј; and antisense, 5Ј-CAA CTT CAT Dr. G. Pancino (Institut Pasteur, Paris, France). U937 and THP-1 cells were CCA CGT TCA CC-3Ј). Amplification products were run on a 2% agarose The Journal of Immunology 3981 gel. Quantitative real-time PCR was performed according to manufacturer’s instructions using the ABI Prism 7000 (Applied Biosystems, Foster City, CA). West-Western identification of HDAC3 protein interactions Protein-protein interaction screening was performed on hEx1 high-density protein expression cDNA libraries with recombinant HDAC3 protein which was expressed from the prokaryotic expression vector pET-32 (No- vagen, Madison, WI) according to previously published protocols (32). Immunoprecipitation Cos7 cells, U937 cells, or primary M⌽ were left untreated or were treated with 100 ng/ml LPS for different periods of time. Cell lysates were precleared by adding 50 ␮l of Protein G Plus/Protein A-Agarose (Calbiochem-Novabio- chem, Bad Soden, Germany) during1hat4¡C. The cleared supernatants were removed, combined with 10 ␮g/ml anti-HDAC3 or anti-MAPK11 (Santa Cruz Biotechnology, Santa Cruz, CA) Abs and incubated overnight at 4¡C. Immune complexes were washed in the presence of protease inhibitors and the bound proteins were eluted with sample buffer and run on a 10% SDS-PAGE gels. SDS-PAGE and Western blot analysis were performed according to standard procedures (28). Western blots were developed with the ECL detection kit

(Amersham Biosciences, Piscataway, NJ). Downloaded from Pull-down assays Fragments of cDNA encoding human HDAC3 were produced using con- venient restriction enzymes and PCR methods and then cloned into the GST fusion vector pGEX-4T-3 (Amersham Biosciences). The GST constructs were transformed into the Escherichia coli strain, BL21, (Strat-

agene) and the GST fusion proteins were puriﬁed according to manufac- http://www.jimmunol.org/ turer’s instructions. MAPK11 was transcribed and translated in vitro in the presence of [35S]methionine (Amersham Biosciences) with the SP6 or T7- coupled reticulocyte lysate systems for 90 min, 30¡C in accordance with the manufacturer’s instructions (Promega). For pull-down assays, 20 ␮gof the GST fusion proteins were incubated overnight at 4¡C with 5 ␮lofthe MAPK11 in vitro-transcribed and translated reaction mixture in PBS. The suspension was then washed three times in PBS, denaturized, and subsequently separated by SDS-PAGE before autoradiography. Kinase assay FIGURE 1. HDAC3 and MAPK11 interact both in vitro and in vivo. A,

Image analysis of a high-density protein filter array, which has been by guest on September 25, 2021 Kinase assays were then conducted with the TNT T7 (or SP6) Quick Cou- screened by nonradioactive West-Western blotting using human HDAC3 pled Transcription/Translation (Promega) in accordance with the manufacturer’s instructions. pcDNA3.1-HDAC3 and the pcDNA3.1 empty vectors protein as a probe. A magnified section of the blot is shown, indicating the were transcribed and translated in the presence of 1 mM methionine with- interacting double-spotted MAPK11 clones (black). Guide dots (white) on out biotinylated tRNA, while MAPK11 was transcribed and translated in the high-density filter membrane facilitate the identification of xy-coordi- vitro under the same conditions but in the presence of 2 ␮l of biotinylated nates of positive clones. B, Binding of MAPK11 to HDAC3 was measured tRNA (Promega) with the T7-coupled reticulocyte lysate system for 90 min in pull-down assays with in vitro translated 35S-labeled MAPK11 (left lane, 32 at 30¡C. After incubation for 5 min at room temperature, [ P]MAPK11 input; middle lane, pGEX-4T-3 empty vector; right lane, 428 aa full-length samples were washed three times (wash solution; Calbiochem-Novabio- HDAC3). The double bands represent phosphorylated and unphosphory- ␥ 32 chem) from the free [ - P]ATP and collected on a streptavidin membrane, lated MAPK11. Input corresponds to 10% of the material used for pull- which was followed by the addition of a liquid scintillation mixture (Roth, down. Results are representative of three independent experiments. C, To- Karlsruhe, Germany) and quantification on a beta counter (PerkinElmer, ⌽ Dreieich, Germany). tal cellular extracts from Cos7 cells, U937 cells, or primary M , were immunoprecipitated with antisera specific for HDAC3 (upper panel)or Results MAPK11 (lower panel), or isotype controls. Immunoprecipitated material HDAC3 interacts with MAPK11 via its N terminus was analyzed by Western blotting with antisera against MAPK11 (upper panel) or HDAC3 (lower panel). To identify potential protein/protein interactions of human HDAC3, we screened high-density protein expression filter membranes that contained 55,296 clones from a human fetal brain library by Far- Western analysis with recombinant HDAC3 as a bait (32). We iden- vitro, but also within several cell types, including primary M⌽. Ly- tified MAPK11 as a potential binding candidate (Fig. 1A). To further sates of Cos7 cells were immunoprecipitated with Abs directed test whether this interaction was direct or indirect, we expressed against other members of class I HDACs (HDAC1, HDAC2, and HDAC3 as a GST fusion protein in E. coli and tested its ability to HDAC8) and then detected by Western blot with the anti-MAPK11 interact with in vitro translated MAPK11. MAPK11 bound to HDAC3 (Fig. 1B). The two bands detected on the gel correspond to Ab. MAPK11 did not interact endogenously with other members of phosphorylated and unphosphorylated MAPK11, as demonstrated us- class I HDACs (data not shown), indicating that HDAC3/MAPK11 ing an anti-phospho-MAPK11 Ab in parallel (data not shown). En- interaction was specific. We further investigated the interaction be- dogenous MAPK11 coimmunoprecipitated with HDAC3 in Cos7 tween HDAC3 and MAPK11 using a mammalian two-hybrid assay. cells, promonocytic U937 cells, and primary M⌽, whereas the isotype We used HDAC3, which was fused to the GAL4 DNA-binding do- control immunoprecipitation showed no associated MAPK11 protein main (pBIND) as the bait vector and MAPK11, which was fused to (Fig. 1C). The interaction of HDAC3 and MAPK11 was also dem- the VP16-activation domain (pACT), as the prey vector (Fig. 2A). onstrated by transient transfection of Cos7 cells (data not shown). These constructs were transfected into mammalian cells along with These data indicate that HDAC3 interacts with MAPK11, not only in the pG5Luc vector, which contains five GAL4 binding sites upstream 3982 HDAC3 INHIBITS MAPK11/ATF-2-MEDIATED SIGNALING

FIGURE 2. HDAC3 and MAPK11 interact in vitro in a mammalian two hybrid assay. A, Schematic representation of expression constructs, which have been used for cotransfection experiments in this mammalian two hybrid setting. B, Mammalian two hybrid analysis with HDAC3 being fused to Downloaded from the GAL4 DNA-binding domain and MAPK11 being fused to the VP16 activator domain. Luciferase assays were conducted on total extracts from Cos 7 cells that have been transfected with the luciferase expressing construct pG5-Luc, pBIND-HDAC3, pACTMAPK11, or control plasmids. Results http://www.jimmunol.org/ represent the average of a triplicate experi- ment where luciferase activity was normalized to protein expression. As a positive control, two plasmids, pACT-MyoD and pBIND-Id, were cotransfected while cotransfection of their empty vectors was used as a negative control. Results represent mean values of three independent experiments. by guest on September 25, 2021

of a minimal TATA box upstream of the luciferase gene. The inter- HDAC3 decreases both MAPK11 and ATF-2 phosphorylation states action between the GAL4-HDAC3 and VP16-MAPK11 fusion con- Using in vitro translated and transcribed MAPK11, human HDAC3 structs resulted in a 4-fold increase of relative firefly luciferase ex- decreased significantly the MAPK11 phosphorylation state, as mea- pression over the negative control (20,345 vs 5,259 relative light units sured by an in vitro kinase assay (Fig. 4A). Because HDAC3 reduced (rlu); Fig. 2B). Expression of pGAL4-HDAC3 alone or pVP16- the MAPK11 phosphorylation state in vitro, we further assessed MAPK11 alone did not result in increased luciferase activity vs neg- HDAC3 effect on the MAPK11 phosphorylation state in vivo. Be- ative controls (5305 and 378 rlu, respectively; Fig. 2B). To determine the MAPK11 binding domain(s), we generated a se- cause LPS has been reported to increase the MAPK11 phosphoryla- ries of plasmids expressing sequential C-terminal truncations of the tion state (33), we assessed the effect of HDAC3 on the LPS-induced 180 GST-tagged HDAC3 protein at amino acid positions 3, 31, 49, 59, 66, MAPK11 phosphorylation state using an anti-phospho-p38 (Thr / 182 137, 198, 266, and 331 (Fig. 3). We then analyzed their MAPK11 Tyr ) polyclonal Ab. LPS treatment increased the MAPK11 phos- binding capacity by GST pull-down assays (Fig. 3). MAPK11 binding phorylation state which was significantly inhibited in U937 cells tran- capacity was totally annihilated in GST pull-down assays using GST- siently transfected with an HDAC3 expressing vector construct, but tagged HDAC3 constructs with truncations upstream of aa 49 (Fig. 3). not with an empty control vector (Fig. 4B). Because activation of These results are consistent with the hypothesis that HDAC3 interacts MAPK11 results in increased ATF-2 activation (34), we assessed the with MAPK11 via its N terminus. state of ATF-2 phosphorylation following LPS stimulation, in the The Journal of Immunology 3983

FIGURE 3. HDAC3 interacts with MAPK11 via its N terminus. A, MAPK11 binds to HDAC3 via its N terminus as demonstrated using wild- type or truncated HDAC3 constructs. Binding of MAPK11 to the C or N terminus of HDAC3 was measured in pull-down assays with in vitro translated 35S-labeled MAPK11. Input Downloaded from corresponds to 10% of the material used for pulldown. Results are representative of two independent experiments. B, Schematic representation of wild-type (428 aa) and truncated HDAC3 constructs. HDAC3 func-

tional domains contain the MAPK11 http://www.jimmunol.org/ binding site (upstream aa 49), the oli- gomerization domain (aa 1Ð122), the nuclear export signal (aa 180Ð313), the nuclear localization signal (aa 313Ð428), and the HDAC domain (aa 401Ð428; Ref. 8). by guest on September 25, 2021

presence or absence of HDAC3 overexpression. Increased ATF-2 hibited by 80% in the presence of HDAC3 overexpression (Fig. phosphorylation was observed following LPS treatment of U937 cells 5A). MKK3-mediated ATF-2 activation was inhibited following and was significantly inhibited upon transient transfection of cells HDAC3 expression (Fig. 5A). The MAPK11-mediated ATF-2 with a HDAC3-expressing vector construct but not with the empty activation was almost completely blocked by the p38 kinase vector (Fig. 4C). All together, these data indicate that both MAPK11 inhibitor, SB202190 (Fig. 5A). To determine whether HDAC3 and ATF-2 phosphorylation states are negatively regulated by had a direct or indirect negative effect on ATF-2-mediated tran- HDAC3 in LPS-treated cells. Following LPS treatment, the amount of scription, we cotransfected pcDNA3.1-HDAC3 together with endogenous MAPK11/HDAC3 interacting complexes decreased tran- pFA-ATF-2 in the presence of the luciferase reporter gene pFR- siently in primary M⌽ (Fig. 4D) and in U937 cells (data not shown), Luc. We found that HDAC3 had no direct inhibitory effect on indicating that LPS relieves HDAC3/MAPK11 interaction. ATF-2 activity (Fig. 5A), indicating that HDAC3 does not directly repress ATF-2 transcriptional activity, but probably does HDAC3 represses MAPK11-mediated ATF-2 activation it through its interaction with MAPK11. To directly examine To determine whether HDAC3 can modulate MAPK11-depen- the contribution of endogenous HDAC3 to ATF-2 repression, dent transcriptional activity, we conducted transient cotransfec- we performed loss-of-function studies. To decrease endogenous tions of pcDNA3.1-HDAC3 and pcDNA3.1-MAPK11 in the HDAC3 protein levels, we used a vector directing the expres- presence of the luciferase reporter gene pFR-Luc and the down- sion of a small interference RNA (siRNA) designed to target the stream MAPK11-dependent transcription factor pFA-ATF-2. cellular HDAC3-encoding mRNA (35). Transient transfection The activity of ATF-2 is known to be induced by phosphory- of this vector led to an efficient depletion of HDAC3 levels in lated MAPK11 (18). We confirmed that overexpression of Cos7 cells and U937 cells (Fig. 5B). Concomitantly, expression MAPK11 up-regulated transcriptional activity of ATF-2 (Fig. of HDAC3 siRNA completely abrogated transcriptional repres- 5A). Furthermore, MAPK11-mediated ATF-2 activation was in- sion of ATF-2 following transient expression of HDAC3 in 3984 HDAC3 INHIBITS MAPK11/ATF-2-MEDIATED SIGNALING

FIGURE 4. HDAC3 represses both MAPK11 and ATF-2 phosphorylation. A, The level of MAPK11 phosphorylation was assessed, in the absence and presence of human HDAC3 overexpression, using an in vitro kinase assay. Results represent means of three independent experiments. B, LPS-induced MAPK11 phosphorylation is inhibited by HDAC3. Total cellular extracts from U937 cells transiently transfected with an HDAC3-expressing vector construct or with an empty control

vector, left untreated or stimulated Downloaded from with 1 ␮g/ml LPS for different periods of time, were analyzed by West- ern blotting with an antiserum directed against phospho-MAPK11 or against tubulin as a control (data not shown). Results are representative of two independent experiments. C, http://www.jimmunol.org/ LPS-induced ATF-2 phosphorylation is inhibited by HDAC3. Total cellular extracts from U937 cells transiently transfected with an HDAC3- expressing vector construct or with an empty vector, left untreated or stimulated with 1 ␮g/ml LPS for different periods of time, were analyzed by Western blotting with an antiserum directed against phospho- by guest on September 25, 2021 ATF-2 or against tubulin as a control. Results are representative of two independent experiments. D, Total cellular extracts from primary M⌽ left untreated or treated with 100 ng/ml LPS for different periods of time were immunoprecipitated with an antiserum speciﬁc for MAPK11 or an isotype control (data not shown). Im- munoprecipitated material was analyzed by Western blotting with antisera against HDAC3. Results are representative of two independent experiments.

Cos7 cells (Fig. 5A). These results conﬁrm that HDAC3 is a HDAC3 represses LPS-induced ATF-2 activation crucial functional component that represses MAPK11-mediated In addition to MAPK11 and ATF-2 activation, LPS activates the tran- ATF-2 activation. To assess directly the role of histone acety- scription factors, NF-␬B and AP-1 (19, 36, 37). Therefore, we investi- lation in ATF-2 transcriptional activity, we overexpressed tran- gated the ability of LPS to activate ATF-2, NF-␬B, and AP-1 in mono- siently in Cos7 cells a HDAC3 mutant lacking the deacetylase cytic cells U937 and THP-1, in the presence and absence of HDAC3 enzymatic activity (HDAC⌬DEAC; Ref. 4). MAPK11-medi- expression, as measured by EMSA. LPS activated ATF-2 in a time- and ated ATF-2 activation was signiﬁcantly increased in the pres- dose-dependent manner (Fig. 6A, and data not shown). ATF-2 activation ence of HDAC⌬DEAC overexpression (Fig. 5C). was time-dependent, with optimal activation occurring at ϳ21 min The Journal of Immunology 3985

FIGURE 5. HDAC3 represses MAPK11- mediated ATF-2 activation. A, Overexpres- sion of HDAC3 inhibits MAPK11-mediated ATF-2 transcription. Upper panel, Overview of expression constructs, which have been used for cotransfection experiments of Cos7 cells in this experimental setting. Lower left panel, Cotransfection experiments of the luciferase-expressing construct pFR-Luc with

pFA-ATF-2, which was fused to the GAL4 Downloaded from DNA-binding domain and pMAPK11, in the presence or absence of HDAC3, indicated that HDAC3 represses ATF-2 transcriptional activity mediated via MAPK11. Transient transfection of pFC-MKK3 in conjunction with ATF-2 was used as positive controls. Results represent the average of triplicate experiments where lu- http://www.jimmunol.org/ ciferase activity was normalized to protein expression. Lower right panel, Loss of HDAC3 function releases ATF-2 from repression. Ex- pression constructs coding for ATF-2 were cotransfected with empty (siRNA) or HDAC3 siRNA expression vectors. Reporter activation was measured 2 days after transfection. Results represent the average of triplicate experiments where luciferase activity was normalized to pro- by guest on September 25, 2021 tein expression. B, Expression of HDAC3 siRNA efﬁciently depletes endogenous HDAC3 protein levels in both U937 cells and Cos7 cells. Lysates of transfected cells were analyzed 3 days after transfection by immunoblotting using an anti-HDAC3 Ab and an anti-tubulin Ab as a control. C, MAPK11-mediated ATF-2 activation is signiﬁcantly increased in the presence of a HDAC3 mutant lacking its deacetylase enzymatic activity (pHDAC⌬DEAC). Cotrans- fection experiments of the luciferase expressing construct pFR-Luc with pFA-ATF-2 and pMAPK11 in the presence or absence of pHDAC⌬DEAC, indicated that, besides the MAPK11 pathway, HDAC3 represses ATF-2 transcriptional activity via its deacetylase activity. Results represent the average of triplicate experiments where luciferase activity was normalized to protein expression. 3986 HDAC3 INHIBITS MAPK11/ATF-2-MEDIATED SIGNALING

(Fig. 6A). Disappearance of the ATF-2 band by competition with unlabeled oligonucleotide indicates that the interaction was speciﬁc (Fig. 6B). ATF-2 activation observed in response to LPS treatment disappeared following transient transfection with a HDAC3-expressing vector construct but not with the empty control vector (Fig. 6C). To directly examine the contribution of endogenous HDAC3 to ATF-2 repression, we performed loss-of-function studies. Expression of HDAC3 siRNA resulted in increased ATF-2 DNA binding following LPS stimulation (Fig. 6D), further indicating HDAC3-mediated transcriptional repression of ATF-2. Following LPS stimulation, activation of NF-␬B and AP-1 has been reported in monocytic cells (19, 37). Therefore, we assessed NF-␬B and AP-1 activation in LPS-treated U937 cells expressing HDAC3 siRNA. In LPS-treated U937 cells, expression of HDAC3 siRNA resulted in increased NF-␬B activation (Fig. 6E), but had no effect on AP-1 induction (data not shown). These results indicate that HDAC3 inhibits LPS-mediated ATF-2 activation, but also LPS-mediated NF-␬B activation, in monocytic cells. Downloaded from HDAC3 inhibits LPS-mediated TNF gene expression Because LPS triggers the production of proinﬂammatory cytokines, we assessed the role of HDAC3 in the modulation of LPS- mediated TNF production. To determine whether HDAC3 can suppress the transcriptional activity of the human TNF promoter

which contains ATF-2-binding domains, we assessed directly the http://www.jimmunol.org/ levels of ATF-2 binding in the human TNF promoter during LPS stimulation. Therefore, we performed chromatin immunoprecipitation experiments. U937 cells were stimulated with LPS, in the presence and absence of HDAC3 expression. ATF-2 was immunoprecipitated from the chromatin solution, and the presence of TNF promoter sequences was analyzed by PCR. This analysis re- vealed that the TNF promoter became relatively enriched in ATF-2 during the first 28 min after LPS stimulation (Fig. 7A). Enrichment of the TNF promoter in ATF-2 almost totally disappeared follow- by guest on September 25, 2021 ing transient transfection with a HDAC3-expressing vector construct but not with the empty control vector (Fig. 7A). Following LPS stimulation, ATF-2 associates specifically (a 2.5-fold enrichment) with the promoter region of TNF, whereas no increase in binding was observed in the ␤-globin region used as a negative control (Fig. 7A). Then, we examined the effect of HDAC3 on the transcriptional activity of the isolated TNF promoter in a reporter assay (26). LPS treatment increased the activity of the transfected TNF promoter construct by ϳ2-fold in THP-1 cells (Fig. 7B). In- terestingly, expression of siRNA HDAC3 further increased LPS- induced TNF-promoter activity (Fig. 7B; p Ͻ 0.05), indicating that endogenous HDAC3 represses TNF promoter activity in THP-1 cells. TNF mRNA levels were decreased by 60% in LPS-treated THP-1 cells transfected with a HDAC3-expressing vector vs LPS- FIGURE 6. HDAC3 represses LPS-mediated ATF-2 activation. A, Time course of ATF-2 activation following LPS treatment. U937 cells (1 ϫ treated cells transfected with an empty vector, as measured by 106/ml) were treated with 1 ␮g/ml LPS for different periods of time at 37¡C RT-PCR assay (Fig. 7C), further indicating that the inhibition of and then ATF-2 activation was measured. B, Specificity of ATF-2 activa- TNF production by HDAC3 occurred at the transcriptional level. tion by LPS. Nuclear extracts from U937 cells treated with 100 ng/ml LPS LPS-treated THP-1 cells were treated with an HDAC inhibitor, for 21 min were incubated 20 min with an unlabeled ATF-2 probe or trichostatin A (TSA), and expression of TNF mRNA was assessed mutated ATF-2 probe, and then ATF-2 activity was measured as described by RT-PCR analysis. LPS-mediated TNF mRNA up-regulation under Materials and Methods. C, ATF-2 activation following LPS treat- was repressed by HDAC3 and was relieved after the addition of 6 ment is inhibited by HDAC3 overexpression. THP-1 cells (1 ϫ 10 /ml) TSA to the culture medium (Fig. 7D). These data further indicate were transfected with a HDAC3-expressing vector (pHDAC3) or the that the TNF promoter is suppressed by the activity of an HDAC. empty vector (mock), and treated 24 h later with 100 ng/ml LPS for different periods of time at 37¡C, and then ATF-2 activation was measured, as described under Materials and Methods. D, ATF-2 activation following LPS treatment is repressed by endogenous HDAC3. U937 cells (1 ϫ 106/ releases NF-␬B from repression. THP-1 cells (1 ϫ 106/ml) were trans- ml) were transfected with empty (siRNA) or HDAC3 siRNA expression fected with empty (siRNA) or HDAC3 siRNA expression vectors and vectors and treated 24 h later with 100 ng/ml LPS for different periods of treated 24 h later with 100 ng/ml LPS for different periods of time at 37¡C, time at 37¡C, and ATF-2 activation was measured as described under Ma- and NF-␬B activation was measured as described under Materials and terials and Methods. E, Following LPS treatment, loss of HDAC3 function Methods. The Journal of Immunology 3987 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 7. HDAC3 represses LPS-mediated TNF expression. A, TNF-promoter enrichment in ATF-2 following LPS treatment is inhibited by HDAC3 overexpression. U937 cells (1 ϫ 106/ml) were transfected with a HDAC3-expressing vector (pHDAC3) or the empty control vector (mock), and treated 24 h later with 100 ng/ml LPS for different periods of time at 37¡C, and then enrichment of the TNF promoter in ATF-2 was measured using chromatin immunoprecipitation as described under Materials and Methods. Briefly, cross-linked cell extracts from mock or HDAC3-transfected cells expressing chromosomal ATF-2 were immunoprecipitated by anti-ATF-2 Abs and assayed for the presence of the TNF promoter or ␤-globin regions. Left panel, Amplification products were run on a 2% agarose gel. As a negative control, DNA was amplified from extracts tagged with c-Fos. Input DNA (control) is shown to demonstrate equal DNA amount. Results are representative of two independent experiments. Right panel, Time course of the recruitment of ATF-2 to the TNF promoter. ATF-2 binding to the TNF promoter was measured using quantitative real-time PCR amplification. Results are represented as a histogram of the relative enrichment (immunoprecipitated/input) in DNA associated with ATF-2 relative to untreated cells. Results are representative of two independent experiments. B, Endogenous HDAC3 represses human TNF promoter activity. U937 cells were cotransfected with pTNF-Luc and with empty (siRNA) or HDAC3 siRNA expression vectors, and treated 24 h later with 100 ng/ml LPS for 20 min. Luciferase activity was measured in cell lysates. The results represent means of three independent experiments. C, LPS-mediated TNF gene expression is repressed by HDAC3. THP-1 cells were transiently transfected with an HDAC3-expressing vector construct or with an empty control vector (mock). Twenty-four hours posttransfection, the cells were left untreated or were treated with SB202190 100 ␮M for 1 h before treatment with 500 ng/ml LPS for 2 h. Total cellular RNA extracts and real-time RT-PCR quantification of human TNF mRNA were performed as described under Materials and Methods. Results represent mean values of three independent experiments. D, LPS-mediated TNF gene expression depends on histone acetylation. U937 cells were transiently transfected with an HDAC3- expressing vector construct or with an empty control vector and treated with 500 ng/ml LPS for 2 h, in the presence or absence of TSA at 1 ␮M. Total cellular RNA extracts and real-time RT-PCR quantification of human TNF mRNA were performed as described under Materials and Methods. Results are representative of two independent experiments. E, LPS-mediated TNF protein production is repressed by HDAC3. THP-1 cells were transiently transfected with an HDAC3-expressing vector or an empty control vector and treated with 10 ng/ml LPS for different periods of time. TNF protein amounts were quantified in culture supernatants as described under Materials and Methods. Results are representative of two independent experiments. 3988 HDAC3 INHIBITS MAPK11/ATF-2-MEDIATED SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 8. Model for the regulation of TNF gene expression by HDAC3. This scheme indicates the potential effect of the HDAC3/MAPK11 interaction on ATF-2 activity and TNF gene expression both under basal conditions and following LPS stimulation. In contrast to basal conditions, following LPS stimulation HDAC3 repression is suppressed, MAPK11 which is activated via the p38 signaling cascade, is phosphorylated and subsequently phosphor- ylates the transcription factor ATF-2. Phosphorylated ATF-2 binds to the human TNF promoter and via the formation of a p300/CREB-binding protein/ ATF-2 activator complex stimulates TNF gene expression and subsequently TNF protein production. HDAC3 interaction with NF-␬B pathway is not represented. The Journal of Immunology 3989

The production of the TNF protein was almost totally abolished at responsive loci for the binding of determined regulatory factors. 8 h after LPS treatment in THP-1 cells expressing transiently Very recently, it has been reported that such changes may be in- pHDAC3 vs LPS-treated control cells transfected with the empty voked by p38-dependent histone modification, where inflamma- vector (Fig. 7E). All together, these data indicate that HDAC3 tory stimuli selectively induce p38 MAPK-dependent phosphory- represses TNF expression in LPS-treated monocytic cells. lation and phosphoacetylation of the histone, H3, on the promoters of a subset of cytokine and chemokine genes where p38 activity is Discussion required to enhance the accessibility of cryptic NF-␬B binding The events leading from MAPK11 phosphorylation to enhanced sites (41). In agreement with these data, we observed that HDAC3 target gene transcription are poorly understood. Our results indi- represses both LPS-mediated ATF-2 and NF-␬B activation in cate that p38 signaling is regulated by the human histone deacety- monocytic cells. HDAC3, by interfering with p38 activity, could lase, HDAC3, at the level of the MAPK11 protein. Transient trans- both decrease ATF-2 transcriptional activity via dephosphoryla- fections of truncated HDAC3 mutants indicated the HDAC3 N tion and decrease NF-␬B activation by diminishing the accessibil- terminus to be associated with MAPK11. HDAC3 inhibited the ity of cryptic NF-␬B binding sites present in the promoter of TNF MAPK11-dependent transcriptional activity of ATF-2, a transcrip- gene. In fact, ATF-2 and NF-␬B complexes bind sites between tion factor binding to the promoter of human TNF gene. In agree- Ϫ106 and Ϫ88 bp of the human TNF promoter (19). Proximate ment with this observation, HDAC3 inhibited TNF gene expres- cooperating ATF-2 and NF-␬B sites are also present in the pro- sion and TNF protein production by monocytic cells following moters of the E-selectin and IFN-␤ genes (44). After LPS expo- LPS stimulation. Together our data indicate that HDAC3 partici- sure, glucocorticoids suppress TNF transcription in monocytic pates to the control of proinflammatory cytokine gene expression, cells by preventing transactivation of ATF and NF-␬B sites be- Downloaded from such as TNF, via repressing MAPK11/ATF-2 signaling. tween Ϫ106 and Ϫ88 bp of the promoter and both ATF and Transcription activation by de-repression, as described here for NF-␬B sites contribute independently but additively to glucocor- ATF-2, is not uncommon in eukaryotic gene regulation. Similar ticoid response (19). In agreement with these data, we observed principles are well described in the cases of NF-␬B, nuclear re- that HDAC3 inhibits the binding of both ATF-2 and NF-␬B com- ceptors, and numerous other transcription factors. Repression as a plexes to the TNF promoter following LPS stimulation of mono-

ground state might help to avoid inadvertent or spurious activation cytic cells, suggesting that HDAC3 might mimic the effect of glu- http://www.jimmunol.org/ of target genes, which, in the case of ATF-2, could cause inap- cocorticoids in regard to the control of proinflammatory cytokines propriate TNF production and activation of the immune system. in mononuclear phagocytes. Our results indicate that HDAC3 re- Activation by de-repression has also been described for other tran- presses TNF expression primarily at a transcriptional level, al- scription factors such as c-Jun (38). Interaction assays, as well as though we cannot rule out a posttranscriptional control of TNF gain- and loss-function studies, identified HDAC3 as a repressor of expression by HDAC3 (45). We observed that HDAC3 blocks MAPK11-mediated ATF-2 activation. The function of HDAC3 as early rather than late LPS-induced TNF secretion, suggesting a a negative transcriptional cofactor has been described in the con- temporary and partial effect. In agreement with our data, deacety- text of nuclear receptors such as the thyroid hormone or retinoic lation of the RelA subunit of NF-␬B by HDAC3 has been shown acid receptors (39). ATF-2 can be relieved of repression either by to act as an intranuclear molecular switch that controls the duration by guest on September 25, 2021 signal-dependent MAPK11-mediated phosphorylation, such as of the NF-␬B transcriptional response (15). We also observed that following LPS stimulation, or signal independently, by titrating HDAC3 represses the IL-1␤ gene expression and the IL-1␤ protein out one or more limiting components of the repressor complex production in LPS-treated THP-1 cells (data not shown), indicating through increased ATF-2 protein levels. Both mechanisms are not that HDAC3 represses the secretion of proinflammatory cytokines, mutually exclusive and might in principle operate in conjunction to such as TNF and IL-1␤, in activated monocytic cells (46). increase transcription activation by ATF-2. Consistently, Based on our observations, we speculate that HDAC3 plays a MAPK11 signaling might not only increase phosphorylation but critical role in determining the threshold level at which a cell, also the protein levels of ATF-2 thus counteracting repression in especially mononuclear phagocytes, produces TNF in response to two ways. LPS stimulation. HDAC3 could allow some level of LPS stimu- We observed that a HDAC3 mutant, deleted for its deacetylase lation without a concomitant increase in TNF expression, a situa- domain, strongly activates ATF-2. These data suggest that ATF-2 tion that might allow for controlled immune activation. When the activation of TNF transcription could be accompanied by local LPS stimulus is sufficiently strong, the suppressive effect of hyperacetylation of histones in agreement with HAT activity elic- HDAC3 on TNF expression would be overwhelmed, resulting in ited by ATF-2 (18). Furthermore, treatment of cells with the his- TNF expression and enhanced immune response against the patho- tone deacetylase inhibitor, TSA, activates TNF gene expression. gen. Therefore, according to this model, HDAC 3 might serve as These observations suggest that the TNF promoter belongs to a a rheostat modulating the TNF production in response to LPS stim- small fraction of cellular promoters that are under the control of a ulation (Fig. 8). Our observations that overexpression of HDAC 3 repressive HDAC and can be activated by the inhibition of HDAC inhibits TNF production and that inhibition of HDAC 3 expression activity (40). Because the induction of several immediate early via siRNA enhances TNF production support this model. Future genes is controlled through phosphorylation of transcription fac- experiments in transgenic mice will directly test this hypothesis tors such as ATF-2 (41), our data suggest that HDAC3 could con- and further define the role of HDAC 3 in the regulation of the trol ATF-2-mediated transcription via both decreased ATF-2 phos- immune response. phorylation and decreased acetylation of histone proteins (42). A number of regulatory cascades such as JAK/STAT, NF-␬B, Acknowledgments and MAPK pathways are known to regulate inflammatory gene The MAPK11 cDNA has been kindly provided by Dr. J. Han (Department expression. This process goes along with the phosphorylation-de- of Immunology, The Scripps Research Institute), and the RZPD. We thank pendent activation of transcription factors while chromatin may A. Bourgeois for help with the chromatin immunoprecipitation assay. selectively be altered by posttranslational acetylation and phos- References phorylation of histone tails or by direct remodeling of nucleosomes 1. McKenna, N. J., and B. W. O’Malley. 2002. Combinatorial control of gene ex- involving ATP-dependent complexes (43), to specifically prepare pression by nuclear receptors and coregulators. Cell 108:465. 3990 HDAC3 INHIBITS MAPK11/ATF-2-MEDIATED SIGNALING

2. Kouzarides, T. 2000. Acetylation: a regulatory modification to rival phosphory- 25. Tsai, E. Y., J. Yie, D. Thanos, and A. E. Goldfeld. 1996. Cell-type-specific lation? EMBO J. 19:1176. regulation of the human tumor necrosis factor-␣ gene in B cells and T cells by 3. Ng, H. H., and A. Bird. 2000. Histone deacetylases: silencers for hire. Trends NFATp and ATF-2/Jun. Mol. Cell. Biol. 16:5232. Biochem. Sci. 25:121. 26. Udalova, I. A., J. C. Knight, V. Vidal, S. A. Nedospasov, and D. Kwiatkowski. 4. Emiliani, S., W. Fischle, C. Van Lint, Y. Al-Abed, and E. Verdin. 1998. Char- 1998. Complex NF-␬B interactions at the distal tumor necrosis factor promoter acterization of a human RPD3 ortholog, HDAC3. Proc. Natl. Acad. Sci. USA region in human monocytes. J. Biol. Chem. 273:21178. 95:2795. 27. Tang, X., M. J. Fenton, and S. Amar. 2003. Identification and functional char- 5. Grozinger, C. M., C. A. Hassig, and S. L. Schreiber. 1999. Three proteins define acterization of a novel binding site on TNF-␣ promoter. Proc. Natl. Acad. Sci. a class of human histone deacetylases related to yeast Hda1p. Proc. Natl. Acad. USA 100:4096. Sci. USA 96:4868. 28. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Lab- 6. Grozinger, C. M., E. D. Chao, H. E. Blackwell, D. Moazed, and S. L. Schreiber. oratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring 2001. Identification of a class of small molecule inhibitors of the sirtuin family of Harbor. NAD-dependent deacetylases by phenotypic screening. J. Biol. Chem. 29. Mahlknecht, U., C. Deng, M. C. Lu, T. C. Greenough, J. L. Sullivan, W. A. 276:38837. O’Brien, and G. Herbein. 2000. Resistance to apoptosis in HIV-infected CD4ϩ T 7. Fischle, W., F. Dequiedt, M. Fillion, M. J. Hendzel, W. Voelter, and E. Verdin. lymphocytes is mediated by macrophages: role for Nef and immune activation in 2001. Human HDAC7 histone deacetylase activity is associated with HDAC3 in viral persistence. J. Immunol. 165:6437. vivo. J. Biol. Chem. 276:35826. 30. Varin, A., S. K. Manna, V. Quivy, A. Z. Decrion, C. Van Lint, G. Herbein, and 8. Yang, W. M., S. C. Tsai, Y. D. Wen, G. Fejer, and E. Seto. 2002. Functional B. B. Aggarwal. 2003. Exogenous Nef protein activates NF-␬B, AP-1, and c-Jun domains of histone deacetylase-3. J. Biol. Chem. 277:9447. N-terminal kinase and stimulates HIV transcription in promonocytic cells: role in 9. Johnson, C. A., D. A. White, J. S. Lavender, L. P. O’Neill, and B. M. Turner. AIDS pathogenesis. J. Biol. Chem. 278:2219. 2002. Human class I histone deacetylase complexes show enhanced catalytic 31. Manna, S. K., and B. B. Aggarwal. 1999. Immunosuppressive leflunomide me- activity in the presence of ATP and co-immunoprecipitate with the ATP-depen- tabolite (A77 1726) blocks TNF-dependent nuclear factor-␬B activation and gene dent chaperone protein Hsp70. J. Biol. Chem. 277:9590. expression. J. Immunol. 162:2095. 10. Guenther, M. G., O. Barak, and M. A. Lazar. 2001. The SMRT and N-CoR 32. Mahlknecht, U., O. G. Ottmann, and D. Hoelzer. 2001. Far-Western based pro- corepressors are activating cofactors for histone deacetylase 3. Mol. Cell. Biol. tein-protein interaction screening of high-density protein filter arrays. J. Biotech- 21:6091. nol. 88:89. Downloaded from 11. Li, J., J. Wang, Z. Nawaz, J. M. Liu, J. Qin, and J. Wong. 2000. Both corepressor 33. Goh, K. C., M. J. deVeer, and B. R. Williams. 2000. The protein kinase PKR is proteins SMRT and N-CoR exist in large protein complexes containing HDAC3. required for p38 MAPK activation and the innate immune response to bacterial EMBO J. 19:4342. endotoxin. EMBO J. 19:4292. 12. Ozawa, Y., M. Towatari, S. Tsuzuki, F. Hayakawa, T. Maeda, Y. Miyata, 34. Ouwens, D. M., N. D. de Ruiter, G. C. van der Zon, A. P. Carter, J. Schouten, M. Tanimoto, and H. Saito. 2001. Histone deacetylase 3 associates with and C. van der Burgt, K. Kooistra, J. L. Bos, J. A. Maassen, and H. van Dam. 2002. represses the transcription factor GATA-2. Blood 98:2116. Growth factors can activate ATF-2 via a two-step mechanism: phosphorylation of 13. Wen, Y. D., W. D. Cress, A. L. Roy, and E. Seto. 2003. Histone deacetylase 3 Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-

binds to and regulates the multifunctional transcription factor TFII-I. J. Biol. p38. EMBO J. 21:3782. http://www.jimmunol.org/ Chem. 278:1841. 35. Sui, G., C. Soohoo, B. Affar el, F. Gay, Y. Shi, and W. C. Forrester. 2002. A 14. Yao, Y. L., W. M. Yang, and E. Seto. 2001. Regulation of transcription factor DNA vector-based RNAi technology to suppress gene expression in mammalian YY1 by acetylation and deacetylation. Mol. Cell. Biol. 21:5979. cells. Proc. Natl. Acad. Sci. USA 99:5515. 15. Chen, L., W. Fischle, E. Verdin, and W. C. Greene. 2001. Duration of nuclear 36. Han, J., Y. Jiang, Z. Li, V. V. Kravchenko, and R. J. Ulevitch. 1997. Activation NF-␬B action regulated by reversible acetylation. Science 293:1653. of the transcription factor MEF2C by the MAP kinase p38 in inﬂammation. 16. Zhang, J., M. Kalkum, B. T. Chait, and R. G. Roeder. 2002. The N-CoR-HDAC3 Nature 386:296. nuclear receptor corepressor complex inhibits the JNK pathway through the in- 37. Karin, M., and A. Lin. 2002. NF-␬B at the crossroads of life and death. Nat. tegral subunit GPS2. Mol. Cell. 9:611. Immunol. 3:221. 17. Dong, C., R. J. Davis, and R. A. Flavell. 2002. MAP kinases in the immune 38. Weiss, C., S. Schneider, E. F. Wagner, X. Zhang, E. Seto, and D. Bohmann. response. Annu. Rev. Immunol. 20:55. 2003. JNK phosphorylation relieves HDAC3-dependent suppression of the tran- 18. Kawasaki, H., L. Schiltz, R. Chiu, K. Itakura, K. Taira, Y. Nakatani, and scriptional activity of c-Jun. EMBO J. 22:3686.

K. K. Yokoyama. 2000. ATF-2 has intrinsic histone acetyltransferase activity 39. Glass, C. K., and M. G. Rosenfeld. 2000. The coregulator exchange in transcrip- by guest on September 25, 2021 which is modulated by phosphorylation. Nature 405:195. tional functions of nuclear receptors. Genes Dev. 14:121. 19. Steer, J. H., K. M. Kroeger, L. J. Abraham, and D. A. Joyce. 2000. Glucocorti- 40. Van Lint, C., S. Emiliani, and E. Verdin. 1996. The expression of a small fraction coids suppress tumor necrosis factor-␣ expression by human monocytic THP-1 of cellular genes is changed in response to histone hyperacetylation. Gene Expr. cells by suppressing transactivation through adjacent NF-␬B and c-Jun-activating 5:245. transcription factor-2 binding sites in the promoter. J. Biol. Chem. 275:18432. 41. Saccani, S., S. Pantano, and G. Natoli. 2002. p38-dependent marking of inflam- 20. van Dam, H., D. Wilhelm, I. Herr, A. Steffen, P. Herrlich, and P. Angel. 1995. matory genes for increased NF-␬B recruitment. Nat. Immunol. 3:69. ATF-2 is preferentially activated by stress-activated protein kinases to mediate 42. Saccani, S., and G. Natoli. 2002. Dynamic changes in histone H3 Lys 9 meth- c-jun induction in response to genotoxic agents. EMBO J. 14:1798. ylation occurring at tightly regulated inducible inflammatory genes. Genes Dev. 21. Kawasaki, H., J. Song, R. Eckner, H. Ugai, R. Chiu, K. Taira, Y. Shi, N. Jones, 16:2219. and K. K. Yokoyama. 1998. p300 and ATF-2 are components of the DRF com- 43. Strahl, B. D., and C. D. Allis. 2000. The language of covalent histone modifi- plex, which regulates retinoic acid- and E1A-mediated transcription of the c-jun cations. Nature 403:41. gene in F9 cells. Genes Dev. 12:233. 44. Whitley, M. Z., D. Thanos, M. A. Read, T. Maniatis, and T. Collins. 1994. A 22. Tracey, K. J., and A. Cerami. 1994. Tumor necrosis factor: a pleiotropic cytokine striking similarity in the organization of the E-selectin and ␤ interferon gene and therapeutic target. Annu. Rev. Med. 45:491. promoters. Mol. Cell. Biol. 14:6464. 23. Herbein, G., and S. Gordon. 1997. 55- and 75-kilodalton tumor necrosis factor 45. Dumitru, C. D., J. D. Ceci, C. Tsatsanis, D. Kontoyiannis, K. Stamatakis, receptors mediate distinct actions in regard to human immunodeficiency virus J. H. Lin, C. Patriotis, N. A. Jenkins, N. G. Copeland, G. Kollias, and type 1 replication in primary human macrophages. J. Virol. 71:4150. P. N. Tsichlis. 2000. TNF-␣ induction by LPS is regulated posttranscriptionally 24. Yao, J., N. Mackman, T. S. Edgington, and S. T. Fan. 1997. Lipopolysaccharide via a Tpl2/ERK-dependent pathway. Cell 103:1071. induction of the tumor necrosis factor-␣ promoter in human monocytic cells: 46. Hsu, H. Y., and M. H. Wen. 2002. Lipopolysaccharide-mediated reactive oxygen regulation by Egr-1, c-Jun, and NF-␬B transcription factors. J. Biol. Chem. species and signal transduction in the regulation of interleukin-1 gene expression. 272:17795. J. Biol. Chem. 277:22131.