Action by CITED2 in the Nucleus B Κ

Negative Feedback Regulation of NF-κB Action by CITED2 in the Nucleus Xiwen Lou, Shaogang Sun, Wei Chen, Yi Zhou, Yuefeng Huang, Xing Liu, Yufei Shan and Chen Wang This information is current as of September 29, 2021. J Immunol 2011; 186:539-548; Prepublished online 22 November 2010; doi: 10.4049/jimmunol.1001650 http://www.jimmunol.org/content/186/1/539 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2010/11/22/jimmunol.100165 Material 0.DC1

References This article cites 45 articles, 19 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/186/1/539.full#ref-list-1

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Negative Feedback Regulation of NF-kB Action by CITED2 in the Nucleus

Xiwen Lou, Shaogang Sun, Wei Chen, Yi Zhou, Yuefeng Huang, Xing Liu, Yufei Shan, and Chen Wang

NF-kB is a family of important transcription factors that modulate immunity, development, inflammation, and cancer. The biological activity of NF-kB is subjected to various spatial and temporal regulations. Bioinformatics analysis predicts that CITED2 is topologically close to NF-kB in the protein interaction networks. In this study, we show that ectopic expression or knockdown of CITED2 attenuates or potentiates, respectively, the expression of NF-kB–responsive genes. Mechanistically, CITED2 constitutively localizes inside the nucleus and interacts specifically with the coactivator p300. This prevents p65 from binding to p300, impairs p65 acetylation, and attenuates p65 binding to its cognate promoters. Furthermore, LPS induces CITED2 expression via NF-kB in macrophages. CITED2 sensitizes cells to TNF-a–induced apoptosis. Collectively, this study identifies Downloaded from CITED2 as a novel regulator of NF-kB in the nucleus, which reveals a negative feedback mechanism for NF-kB signaling. The Journal of Immunology, 2011, 186: 539–548.

uclear factor kB is an important family of transcription modulation of NF-kB action inside the nucleus has recently

factors (1), including RelA (p65), RelB, C-Rel, p50 attracted much attention, revealing unexpected multilayers of tight http://www.jimmunol.org/ N (p105), and p52 (p100) (2). The p65/p50 heterodimer regulations (8, 9). For example, a dynamic equilibrium exists is the predominant form of NF-kB in most cells, and it controls a between promoter-bound and nucleoplasmic NF-kB, which ena- wide array of genes critical in the immune response and inflam- bles the cells to retune quickly its transcriptional programs mation (3). It might also play some potential roles in development according to external stimuli (10). In addition, the modification (4, 5). The aberrant action of NF-kB is causally associated with status of histones on a given promoter could define the accessi- cancer (6) and some autoimmune diseases (7). bility of the NF-kB dimers (11, 12). Furthermore, the modification The latent form of the NF-kB heterodimer is retained in the status of NF-kB per se markedly influences its transcriptional cytoplasm through binding to its inhibitor IkBa.IkBa is quickly activity (11, 13, 14). Nuclear p65 can be phosphorylated and phosphorylated, ubiquitinated, and degraded in response to a acetylated on distinct sites by different kinases and acetyl- by guest on September 29, 2021 myriad of extracellular and intracellular stimuli, such as TNF-a, transferases (15, 16). Promoter-bound NF-kB can also be ubiq- IL-1b, LPS, and so forth. This releases the NF-kB heterodimer, uitinated and degraded in certain conditions (17, 18). All of these which then translocates from cytoplasm into nucleus to mediate provide an array of delicate mechanisms for the rapid and re- the transcription of the corresponding target genes (2). The past versible integration of stress signals. two decades have witnessed the successful elucidation, at the Recently, a growing list of novel molecules has been implicated molecular and cellular levels, of the major signaling pathways in the modulation of nuclear NF-kB action via different mecha- leading to IkBa degradation and NF-kB nuclear translocation. nisms (19). For example, Akirin2 (20) and UXT (21) were re- As with any other pivotal molecules in various biological pro- cently demonstrated to function as essential coactivators in the cesses, the activity of NF-kB is controlled strictly in vivo. The NF-kB enhanceosome. Importantly, the transcriptional coactivator p300/CBP not only serves as a scaffold for the NF-kB enhanceosome but also directly acetylates both p65 and histones si- Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai multaneously (22). It is a great challenge to understand how these 200031, China proteins cooperate both dynamically and temporally to remodel Received for publication May 18, 2010. Accepted for publication October 20, 2010. the NF-kB enhanceosome. This work was supported by grants from the Ministry of Science and Technology of CITED2 (35 kDa), also known as MRG1 or p35srj, may function Shanghai (09XD1404800, 09ZR1436800), the National Natural Science Foundation in several biological processes. Knockout analysis demonstrated of China (30900762, 31030021), and the Ministry of Science and Technology of that CITED2 plays crucial roles in mouse embryo development. China (2007CB914504, 2010CB529703, 2011CB910900). The CITED22/2 embryos could not proceed beyond embryo- Address correspondence and reprint requests to Dr. Chen Wang and Dr. Yufei Shan, Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, genesis. Cardiac malformation, adrenal agenesis, neural crest Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue defects, and exencephaly were observed in the embryos (23–25). Yang Road, Shanghai 200031, China. E-mail addresses: [email protected] and Further studies revealed left–right patterning defects in these [email protected] embryos, especially in co-isogenic C57BL/6J backgrounds (26). The online version of this article contains supplemental material. Moreover, CITED2 could selectively maintain adult hemopoietic Abbreviations used in this paper: BD, binding domain; BMDM, bone marrow- derived macrophage; ChIP, chromatin immunoprecipitation; CHX, cycloheximide; stem cell functions, at least in part via Ink4a/Arf and Trp53 (27). IRF3, IFN regulatory factor 3; MOI, multiplicity of infection; PD, pull-down; PI, Mechanistically, CITED2 has been suggested as a multifunc- propidium iodide; SeV, Sendai virus; shRNA, short hairpin RNA; siRNA, small in- tional transcriptional cofactor. It can bind to the LIM domain of terfering RNA. Lhx2 and stimulate the expression of the glycoprotein hormone Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 a-subunit gene (28). It can also bind TFAP2 and potentiate its www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001650 540 CITED2 NEGATIVELY REGULATES NF-kB transcription activity, which is a possible explanation for the em- RNA. The amount of transcript of the genes of interest was measured by bryo lethality of CITED22/2 mice (23). Notably, CITED2 can be real-time RT-PCR method using the StepOne real-time PCR system (Ap- a repressor of the transcription of HIF-dependent genes (29, 30). plied Biosystems, Carlsbad, CA). The primers used were as follows: A20: sense 59-GCGTTCAGGACACAGACTTG-39, antisense 59-GCAAAGCC- To our knowledge, there is no investigation concerning the CCGTTTCAACAA-39; MCP-1: sense 59-TCGCCTCCAGCATGAAAG- function of CITED2 in immunity and inflammation. Using bioin- TC-39, antisense 59-GGCATTGATTGCATCTGGC-39; IL-8: sense 59-GG- formatics analysis, we noticed that CITED2 is topologically close to CAGCCTTCCTGATTTCTG-39, antisense 59-CTTGGCAAAACTGCAC- 9 9 9 NF-kB in the protein interaction networks. In this study, we dem- CTTCA-3 ; GRO-1: sense 5 -AGGAAGCTCACTGGTGGCTG-3 , antisense 59-TAGGCACAATCCAGGTGGC-39; IL-2: sense 59-GTCACAAA- onstrate that CITED2 is inducible by LPS. It prevents p65 from CAGTGCACCTAC-39, antisense 59-CCCTGGGTCTTAAGTGAAAG-39; binding to p300, impairs p65 acetylation, and weakens p65 binding IL-6 (mouse): sense 59-GAGAGGAGACTTCACAGAGGATAC-39, anti- to its cognate promoters, revealing that CITED2 is a novel repressor sense 59-GTACTCCAGAAGACCAGAGG-39; IL-12b (mouse): sense 59- of nuclear NF-kB. GCAACGTTGGAAAGGAAAGA-39, antisense 59-AAAGCCAACCAAG- CAGAAGA-39; b-actin (human): sense 59-AAAGACCTGTACGCCAA’- CAC-39, antisense 59-GTCATACTCCTGCTTGCTGAT-39; b-actin (mouse): Materials and Methods sense 59-AAAGACCTCTATGCCAACAC-39, antisense 59-ATCGTACTC- Cell culture and transfection CTGCTTGCTGAT-39. HEK293T, J774, Raw264.7, and MEF cells were cultured in DMEM medium Immunoprecipitation assay and immunoblot analysis (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT). Jurkat cells were cultured in RPMI 1640 medium (Invitrogen) sup- For immunoprecipitation, whole-cell lysates were obtained by lysing cul- plemented with 10% FBS. For the bone marrow-derived macrophages tured cells with lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM NaVO and protease (BMDMs), femora were aseptically removed from 8-wk-old male C57BL/6 3 Downloaded from mice. The bones were then cut off on both ends, and bone marrow cells were inhibitor mixture (Roche, Grenzacherstrasse, Basel); or nuclear extracts flushed out with a 27-gauge needle attached to a syringe containing 5 ml were obtained as previously described (32). The lysates were centrifuged, medium (RPMI 1640 supplemented with 20% FBS, 2 mM L-glutamine, 50 and the supernatant was collected and precleared with protein A/G agarose mM 2-mercaptoethanol, and 40 ng/ml macrophage colony stimulating beads (Santa Cruz Biotechnology) at 4˚C for 1 h. Then the precleared factor). The medium was changed every 2 d and the cells differentiated to supernatant was incubated with the indicated Abs at 4˚C overnight. Protein BMDMs after 1 wk. All cells were maintained in a humidified 5% CO2 A/G agarose beads were added later for another 1 h, and the immuno- incubator at 37˚C. Transient transfections were performed with Lipofect- precipitate was collected and washed extensively with wash buffer con- amine 2000 (Invitrogen) following the manufacturer’s instructions. The short taining 0.5% Triton X-100. Finally, each sample was diluted with 13 SDS http://www.jimmunol.org/ hairpin RNA (shRNA) plasmids were introduced into J774 cells and loading buffer and boiled for 3 min. For immunoblot analysis, all immu- BMDMs using recombinant adenovirus (Invitrogen). Adenovirus harboring noprecipitates and whole-cell lysates were subjected to SDS-PAGE. The the GFP gene was constructed via the AdEasy Adenoviral Vector Systems resolved proteins were then electrically transferred to a polyvinylidene (Stratagene, Lexington, MA), and the titer of all the adenoviruses was de- difluoride membrane (Bio-Rad). The membrane was blocked with skim- termined by the multiplicity of infection (MOI) test. Plasmids and small med milk and probed with indicated Abs. The protein bands were visu- interfering RNAs (siRNAs) were introduced into Jurkat cells using the Gene alized by using a SuperSignal West Pico chemiluminescence ECL kit Pulser Xcell Electroporation System (Bio-Rad, Hercules, CA). (Pierce, Rockford, IL). Plasmids and siRNAs Chromatin immunoprecipitation assay

CITED2 was a gift from Dr. S. Bhattacharya (University of Oxford, Oxford, The chromatin immunoprecipitation (ChIP) assay kit (Upstate Bio- by guest on September 29, 2021 U.K.). CITED2(4m) (L243E/P244E/L246E/W247E) was constructed by technology, Billerica, MA) was used according to the manufacturer’s using the QuickChange XL Site-Directed Mutagenesis method (Stra- instructions with some modifications. Formaldehyde was added directly tagene). p300 was a gift from Dr. Warner C. Greene (University of Cal- into the culturing media to a final concentration of 1% at room temperature ifornia, San Francisco, CA). p65 tagged with or without FLAG, FLAG– for cross-linking. Ten minutes later, the cells were harvested and washed IkBaSR, FLAG–TRAF6, FLAG–MyD88, and the reporter gene 3*kB-luc and lysed with SDS lysis buffer. The lysates were sonicated until the have been described previously (21). The CITED2 (human) siRNA se- chromatin was sheared to an average size between 200 and 1000 bp. The quences were as follows: CITED2 (396), 59-CCACUACAUGCCGGA- sonicated lysates were centrifuged under 12,000 3 g for 10 min, and the UUUG-39; mCITED2 (396m), 59-CCACUACACACCGGAUUUG-39. The supernatant was diluted 10-fold by ChIP dilution buffer and precleared by nonspecific siRNA sequence (NC) was 59-UUCUCCGAACGUGUCA- protein A/G agarose beads that were saturated with salmon sperm DNA CGU-39. The CITED2 siRNA-resistant form was generated by introducing and BSA for 1 h. Then, the precleared supernatant was incubated with the two silent mutations (C396TC399T) into the siRNA CITED2 (396) target indicated Abs at 4˚C overnight. Protein A/G agarose was added later for sequence. The shRNA sequence targeting CITED2 (mouse) was as fol- another 1 h, and the immunoprecipitates were collected and washed se- lows: 59-CACCGCAGAAGCTCAACAACCAGTACGAATACTGGTTG- quentially and extensively with a series of wash buffer. Then, immuno- TTGAGCTTCTGC-39. The shRNA targeting LacZ was used as a negative precipitants were extracted with elusion buffer to release the immune control, and the sequence was 59-CACCGCTACACAAATCAGCGATT- complexes. The DNA and protein was de-cross-linked by heating at 65˚C TCGAAAAATCGCTGATTTGTGTAG-39. The siRNA sequence targeting under high salt conditions for more than 5 h. The RNA contaminants were p300 was previously reported (31). removed by adding RNase A at 37˚C for 1 h, and the protein contaminants were removed by incubating with protease K at 45˚C for 2 h. Then, DNA Reagents was purified by phenol/chloroform extraction and precipitated by anhy- drous ethanol and finally dissolved in sterile water. Then, PCR was per- CITED2 (JA22), p300 (N-15), p65 (C-20), p65 (F-6), IkBa, HA, cIAP1, formed to measure the amount of promoter DNA in each sample. and RNA polymerase II Abs were purchased from Santa Cruz Bio- technology (Santa Cruz, CA). FLAG and b-actin Abs were purchased from Immunofluorescence and microscopy Sigma-Aldrich (St. Louis, MO). Acetyl–NF-kB p65 (Lys310) Ab was obtained from Cell Signaling (Danvers, MA). rhTNF-a was obtained from Cells grown on coverslips were fixed with 4% paraformaldehyde, per- R&D Systems (Minneapolis, MN). PMA, ionomycin, LPS, PD98059, meabilized in 0.1% Triton X-100, blocked using 1% BSA, and stained with LY294002, and Bay11-7082 were purchased from Sigma-Aldrich. the indicated primary Abs followed by incubation with a Cy5-conjugated anti-mouse IgG (Millipore, Billerica, MA). Nuclei were counterstained Reporter assays with DAPI (Sigma-Aldrich). Slides were mounted using Aqua-Poly/Mount (Polysciences, Warrington, PA). Images were captured at room tempera- Reporter assays were performed as described previously (21). ture using a confocal microscope (TCS SP2 ACBS; Leica) with a 363 (numerical aperture 1.4) oil objective. The acquiring software was TCS Real-time RT-PCR (Leica, Solms, Germany). Total RNA was extracted from cultured cells using TRIzol reagent (Invi- Apoptosis assay trogen) according to the manufacturer’s instructions. Oligonucleotide primers and Moloney murine leukemia virus reverse transcriptase (Prom- The 293T cells were transfected with the indicated plasmids. Twenty-four ega, Madison, WI) were used for the reverse transcription of the purified hours later, cells were treated with 50 ng/ml TNF-a plus 5 mg/ml cy- The Journal of Immunology 541 cloheximide (CHX) or left untreated for 24 h. Suspended and ad- contrast, ectopic expression of CITED2 significantly attenuated the herent cells were collected, trypsinized, and washed twice with PBS. NF-kB–responsive luciferase reporter, which was stimulated by Then, the cells were analyzed with a FACSCalibur (BD Biosciences) flow TNF-a (Fig. 1B) or Sendai virus (SeV; Supplemental Fig. 1). cytometer using annexin V–FITC (BD Biosciences) and propidium iodide b (PI; Sigma-Aldrich) according to the manufacturer’s instructions. Consistently, this was also true for the IFN- and E-selectin luciferase reporters, which are tightly regulated by NF-kB(Fig.1C). To make it more physiologically relevant, we explored, via real- Results time RT-PCR, whether CITED2 influenced the expression of en- k Identification of CITED2 as a potential regulator of NF- B dogenous NF-kB target genes induced by TNF-a. Notably, ectopic signaling expression of CITED2 markedly impaired the expression of these Some proteins have been shown to play dual roles in development genes (A20, IL-8, MCP-1, and GRO-1) in 293T cells (Fig. 1D). and immunity (20, 33). As CITED2 is essential for development, We also constructed CITED2(4m) by introducing four amino acid we wondered if it could play a role in immunity. Notably, bio- changes (L243E/P244E/L246E/W247E) into the CITED2 se- informatics analysis with Cytoscape (http://www.cytoscape.org/) quence. In contrast, expression of CITED2(4m) could barely in- software predicts that CITED2 is topologically close to NF-kB, fluence the induction of these genes. Ectopic expression of IFN regulatory factor 3 (IRF3), and AP-1 (c-Jun/ATF3) in the CITED2 also decreased the induction of NF-kB–regulated genes protein interaction networks (Fig. 1A). (IL-2 and IL-8) in Jurkat T cells (Fig. 1E), suggesting that To explore the potential relevance of this prediction, the corre- CITED2 could be a negative regulator of NF-kB signaling. sponding specific luciferase reporters were used to test whether

k Downloaded from CITED2 had any impact on these transcription factors. We observed Knockdown of CITED2 potentiates NF- B signaling that ectopic expression of CITED2 apparently had little effect on Alternatively, an RNA interference knockdown approach was used either AP-1 or IRF3 reporter (Fig. 1B and Supplemental Fig. 1). In to investigate whether the endogenous CITED2 regulated NF-kB http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Identification of CITED2 as a novel negative regulator of NF-kB activation. A, CITED2-associated proteins are determined from the known protein–protein interaction networks using Cytoscape. B, 293T cells were co-transfected with either empty vector or HA–CITED2 with AP-1, IRF3, and NF-kB reporter genes as indicated. Twenty-four hours after transfection, cells were treated with 10 ng/ml PMA or SeV (MOI, 0.5) or 10 ng/ml TNF-a for 10 h before the luciferase assays were performed. C, Empty vector or HA–CITED2 and the indicated reporter genes were transfected into 293T cells. After 24 h, the cells were treated with SeV (MOI, 0.5) or 10 ng/ml TNF-a for 10 h. The cells were assayed as described in B. D, 293T cells were transfected with CITED2 or its loss-of-function mutant CITED2(4m). Twenty-four hours later, the cells were treated with 10 ng/ml TNF-a for 1 h. The endogenous mRNA expression level of A20, IL-8, MCP-1, and GRO-1 were measured by real-time RT-PCR. E, Jurkat cells were transfected with CITED2 or its loss-of- function mutant CITED2(4m). Twenty-four hours later, the cells were treated with 50 ng/ml PMA plus 1.5 mM ionomycin for 3 h. The endogenous mRNA expression levels of IL-2 and IL-8 were measured by real-time RT-PCR. Data in B–E are represented as means 6 SD (error bars) of at least three independent experiments. *p , 0.05; **p , 0.01. 542 CITED2 NEGATIVELY REGULATES NF-kB signaling. The siRNA oligonucleotide was screened out and de- CITED2 potentiated the expression of NF-kB target genes, which termined to be efficient in reducing both the exogenous and en- was reversed by the ectopic expression of CITED2(FLr). dogenous CITED2 protein levels. At the same time, nonspecific We then went on to test the function of CITED2 in macrophages (NC) and mutant (mCITED2) siRNAs were used as negative con- (J774 and BMDM). The CITED2 siRNA oligonucleotide used above trols (Fig. 2A). Knockdown of endogenous CITED2 modestly po- did not knock down well in murine macrophages, so we screened out tentiated NF-kB target genes in HEK293T cells upon TNF-a another shRNA oligonucleotide and introduced it into macrophages treatment (Fig. 2B) and in Jurkat T cells after PMA/ionomycin via a recombinant adenovirus. An shRNA targeting the lacZ gene stimulation (Fig. 2C). was used as negative control (Fig. 2E). Adenovirus-GFP was used To rule out the potential off-target effect of the CITED2 siRNA, to confirm the transduction efficiency, which was ∼50–60% (Sup- an RNA interference-resistant construct of CITED2, CITED2 plemental Fig. 2). Consistently, a decrease in endogenous CITED2 (FLr), was generated, which contained silent mutations in the expression significantly boosted the induction of NF-kB target corresponding siRNA target region of CITED2 without changing genes stimulated by LPS (Fig. 2F,2G). Taken together, these data the protein sequence. HEK293T cells were first transfected with the indicated that CITED2 is a novel repressor of NF-kB signaling. control or CITED2 siRNA followed by transfection of the control k or CITED2(FLr) plasmids as indicated. Then, the induction of CITED2 modulates NF- B signaling inside the nucleus NF-kB target genes was measured by real-time RT-PCR after A key regulatory node in NF-kB signaling is the phosphorylation TNF-a treatment. As shown in Fig. 2D, knockdown of endogenous and ubiquitin-proteasome–mediated degradation of its inhibitor, Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Knockdown of endogenous CITED2 enhances NF-kB activation. A, The indicated siRNAs were transfected into 293T cells either with (left) or without (right) HA–CITED2. After 48 h, cells were collected and lysed, and the whole-cell lysate was analyzed by immunoblot with the indicated Abs. B, 293T cells were transfected with siRNAs as indicated. Forty-eight hours later, the cells were stimulated with 10 ng/ml TNF-a for 1 h. The total RNA was extracted, and real-time RT-PCR was performed to measure the level of IL-8 and GRO-1 mRNA in each sample. C, Jurkat cells were transfected with siRNAs as described in B. After 48 h, cells were treated with 50 ng/ml PMA plus 1.5 mM ionomycin for 3 h. Then, the total RNA was extracted, and the amount of IL-2 and IL-8 mRNA was measured with real-time RT-PCR in each sample. D, 293T cells were transfected with siRNAs as mentioned and then rescued with siRNA-resistant CITED2. Forty-eight hours later, the cells were treated with TNF-a for 1 h, and the expression of IL-8 and GRO-1 was measured by real-time RT-PCR (top). The expression of endogenous and exogenous CITED2 was analyzed by immunoblotting (bottom). E, J774 cells were transfected with the indicated shRNAs using adenovirus. Forty-eight hours later, the cells were harvested, and the whole-cell lysates were prepared and immunoblotted to measure the endogenous CITED2. F and G, J774 cells and BMDMs were transfected with the shRNA mentioned above and stimulated by LPS for 2 h. Then, the total RNA was extracted, and real-time RT-PCR was performed to determine the amount of IL-6 and IL-12b in each sample. Data in B–D, F, and G represent means 6 SD (error bars) of at least three independent experiments. *p , 0.05; **p , 0.01. The Journal of Immunology 543 Downloaded from

FIGURE 3. CITED2 is a nuclear regulator of NF-kB. A, 293T cells were transfected with an empty vector, CITED2, or CITED2(4m). Twenty-four hours later, the cells were treated with 10 ng/ml TNF-a for the indicated time, and the cell lysates were prepared to analyze the degradation of IkBa. B, 293T cells were transfected with the indicated siRNAs and were treated and analyzed as described in A. C, CITED2 or CITED2(4m) was transfected into 293T cells with NF-kB–luc as a reporter gene and MyD88, TRAF6, IKKb, or p65 as an activator. Twenty-four hours after transfection, the cells were collected and applied to a luciferase assay. The data represent means 6 SD (error bars) of at least three independent experiments. *p , 0.05; **p , 0.01. http://www.jimmunol.org/

IkBa. We observed that neither ectopic expression nor knockdown indicated that p65 bound p300 specifically upon stimulation, and of CITED2 could affect the degradation of IkBa (Fig. 3A,3B). this interaction is essential for NF-kB enhanceosome assembly In addition, ectopic expression of MyD88, TRAF6, IKKb, and (34). We confirmed that CITED2 could interact with p300 directly p65 respectively could activate NF-kB signaling, and all of which and tightly, via immunoprecipitation and GST pull-down (Fig. were attenuated by the coexpression of CITED2 (Fig. 3C). Given 5A). Notably, the CH1 domain of p300 has been implicated, via the hierarchical relations of these molecules in NF-kB pathway, a yeast two-hybrid approach, to bind to CITED2, p65, Hif-1a, and we reasoned that CITED2 modulates NF-kB inside the nucleus. so forth (35). So we speculated whether CITED2 could influence

This was further substantiated by the observation that CITED2 the specific interaction between p65 and p300. by guest on September 29, 2021 is constitutively expressed inside the nucleus (Fig. 4A). To test this possibility, we mapped the C terminus of CITED2 CITED2 prevents p65 from binding to its cognate promoters (aa 221–270) to explore the binding site of p300 (Fig. 5B). A series of mutagenesis analyses uncovered four amino acid residues It was apparent that CITED2 did not interact directly with either NF- of CITED2 to be critical for this interaction. Therefore, CITED2 k k a B p65 or p50, although I B co-immunoprecipitated strongly (L243E/P244E/L246E/W247E), named as CITED2(4m), could with them (Fig. 4B). Besides, UXT was recently uncovered as an barely interact with p300 (Fig. 5B), whereas CITED2(L243E/ k integral component of NF- B enhanceosome (21). Neither could L246E) could partially do so (X. Lou and C. Wang, unpublished k CITED2 promote the export of NF- Bfromthenucleustothe data). Notably, ectopic expression of CITED2(4m) was unable to k k a cytoplasm or actively retain NF- B in the nucleus, whereas I B attenuate the activation of the NF-kB reporter gene (Fig. 3C)or (SR) (Fig. 4C)orUXT(Fig.4D) could do so, respectively. the induction of NF-kB responsive genes (Fig. 1D,1E). Neither Consequently, ChIP assay was used to investigate the status of could CITED2(4m) expression impair p65 to bind its cognate k NF- B on its cognate promoters. Notably, ectopic expression of promoters (Fig. 4E,4G). We noticed that expression of CITED2 CITED2 could apparently decrease the amount of p65 on the (4m) could, to a lesser extent, influence the binding of RNA promoters of A20 and IL-8. This was also true for RNA poly- polymerase II to its cognate promoters (Fig. 4E). These data in- merase II on these promoters (Fig. 4E,4G). In contrast, an indicated that the CITED2 and p300 interaction is functionally crease of CITED2 expression had no obvious effect on the important for modulating NF-kB activity inside the nucleus. k GAPDH transcription complex, which is not an NF- B target gene Furthermore, we carried out a mammalian two-hybrid assay to (Fig. 4E,4G). Furthermore, ectopic expression of CITED2 dis- explore whether CITED2 competed against p65 for binding to p300. played no observable effect on IRF3 in terms of its corresponding The N terminus (aa 1–800) of p300 was fused with the yeast GAL4 promoters (X. Lou and C. Wang, unpublished data). Consistently, binding domain (BD). As expected, ectopic expression of this fu- knockdown of the endogenous CITED2 protein level led to a mod- sion protein and p65 induced the expression of a luciferase reporter erate increase in the amount of p65 and RNA polymerase II on the harboring five tandem repeats of the GAL4 binding site. Notably, promoters of A20 or IL-8 (Fig. 4F,4H). Knockdown of CITED2 had introduction of CITED2 into this system significantly reduced the no effect on the GAPDH promoter. Taken together, these data in- induction of the GAL4-reporter. In contrast, ectopic expression of dicate that CITED2 regulates the affinity of NF-kB toward its cognate promoters but does not directly interact with NF-kB. CITED2(4m) slightly potentiated the induction of the GAL4- reporter by p300 N800-BD and p65, possibly due to the dominant CITED2 attenuates p65 acetylation by preventing its access to negative effect of CITED2(4m) (Fig. 5C). These data indicate that coactivator p300 CITED2 disrupted the interaction between p300 and p65. There exists research suggesting that CITED2 interacted with the Alternatively, p65 was co-transfected respectively with CITED2, transcriptional coactivator p300/CBP (29). Also, previous studies CITED2(4m), or empty vector. Then, the cell extracts were 544 CITED2 NEGATIVELY REGULATES NF-kB Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. CITED2 reduces the amount of p65 on its cognate promoters. A, Subcellular localization of endogenous and exogenous CITED2 in 293T cells. 293T cells were transfected with (bottom) or without (top) HA–CITED2. Immunofluorescence was performed with the indicated Abs. Scale bar, 20 mm. Original magnification 363. B, 293T cells were transfected with FLAG–p65 (left) or FLAG–p50 (right) together with empty vector, HA–CITED2, or HA–IkBa. Twenty-four hours after transfection, the whole-cell lysate was prepared, immunoprecipitated, and immunoblotted as indicated. C, 293T cells grown on coverslips were transfected with plasmids as mentioned. Twenty-four hours later, the cells were treated with 10 ng/ml TNF-a for 30 min or left untreated. Then, the cells were stained with an anti-p65 primary Ab and a Cy5-conjugated secondary Ab. The nucleus was counterstained with DAPI. Scale bar, 20 mm. Original magnification 363. D, 293T cells grown on coverslips were transfected with siRNAs as mentioned and then stimulated and analyzed as in C. Scale bar, 20 mm. Original magnification 363. Stains in A, C, and D are Cy5 (red) and DAPI (blue). E and F, 293T cells were transfected with plasmids or siRNAs as indicated. Twenty-four hours or 48 h later, the cells were treated with 10 ng/ml TNF-a for 30 min or left untreated. Then, the cells were immediately cross-linked with formaldehyde and applied to ChIP assay in terms of A20, IL-8, and GAPDH promoters using the corresponding Abs and primers. G and H, Cells were applied to ChIP assay as in E and F and analyzed using real-time RT-PCR. The Journal of Immunology 545 Downloaded from http://www.jimmunol.org/

FIGURE 5. CITED2 blocks the interaction of p65 and p300, attenuating p65 acetylation. A, The nuclear extracts of 293T cells were applied to immunoprecipitation with either an anti-CITED2 or anti-p300 Ab (left). 293T cells were transfected with the indicated plasmids, and then the cells were harvested and applied to immunoprecipitation with an anti-FLAG Ab (middle) or GST pull-down with GST or the GST–CH1 protein (right). Finally, they were analyzed by guest on September 29, 2021 by immunoblot analysis. PD, pull-down. B, Schematic of CITED2 and its mutants (top). CITED2 or its mutants as mentioned earlier were overexpressed in 293T cells. Then, cell lysate was prepared and used for GST pull-down assay with the GST–CH1 protein (bottom). (CITED2 and CITED2[1–221] were tagged with HA, and CITED2(4m) was tagged with FLAG.) C, The mammalian two-hybrid reporter gene pFR–luc was transfected into 293T cells with the plasmids as indicated. Twenty-four hours after transfection, the cells were collected and analyzed by a luciferase assay. Data represent means 6 SD (error bars) of at least three independent experiments. D, The plasmids as indicated were transfected into 293T cells. The cells were harvested and used for GST pull-down assay with the GST–CH1 protein and then analyzed by immunoblotting. E and F, 293T cells were transfected with either plasmids as indicated in E or siRNAs as indicated in F. Twenty-four hours or 48 h later, the cells were treated with TNF-a for 1 h, and the nuclear extracts were obtained for immunoprecipitation with an anti-p65 Ab and then analyzed by immunoblotting. G, 293T cells were transfected with plasmids as indicated. After 24 h, the whole-cell lysates were prepared and immunoblotted with the indicated Abs, including an Ab that could detect acetyl lysine 310 of p65. subjected to a GST pull-down assay with recombinant GST–p300 experiments (Supplemental Fig. 3). We observed that knockdown CH1. Again, in the absence of CITED2, p65 was pulled down by of p300 markedly decreased the amount of p65 and RNA poly- GST–p300 CH1. Notably, in the presence of CITED2, much less merase II on the A20 and IL-8 promoters upon TNF-a stimulation. amount of p65 was pulled down, whereas CITED2(4m) did not This finding is consistent with the above results (Fig. 4E–H) that influence p65 pull-down (Fig. 5D). Importantly, co-immunopre- CITED2 could block the binding of p65 to its cognate promoters. cipitation assays indicated that overexpression of CITED2 could a impair the endogenous interaction between p300 and p65, whereas CITED2 sensitizes cells to TNF- –induced apoptosis knockdown of CITED2 could potentiate the interaction between TNF-a with a low concentration of CHX (5 mg/ml) is often used p300 and p65 upon TNFa stimulation (Fig. 5E,5F). to induce cell apoptosis (36, 37). NF-kB signaling antagonizes this p65 is reported to be acetylated by p300 on the enhanceosome. In apoptosis by inducing the transcription of antiapoptotic genes, particular, acetylation of lysine 310 is critical for NF-kB transcrip- such as c-FLIP, cIAP-1, and cIAP-2 (38, 39). Blockade of NF-kB tional activity (16). Therefore, p65 and p300 were co-transfected activation can amplify the indicated apoptosis. Consequently, ec- into 293T cells along with CITED2 or its mutants as indicated. topic expression of CITED2 apparently sensitized cells to apo- Expectedly, p300 could catalyze the acetylation of p65. This mod- ptosis induced by TNF-a plus CHX, as evidenced by a differential ification was sharply reduced in the presence of CITED2, whereas interference contrast microscope (Fig. 6A) and flow cytometry CITED2mutantsfailedtodoso(Fig.5G). This further substantiated (Fig. 6B,6C). the finding that CITED2 prevents p65 from binding to p300. At the concentration of #10 mg/ml, CHX only reduces but does p300 is a coactivator for p65, and we hypothesized that it may not block de novo protein synthesis (37). We verified this result by promote p65 binding to its cognate promoters. We confirmed this showing the expression of CITED2 and its mutant in the presence by transfecting cells with siRNA targeting p300, followed by ChIP of a low dose of CHX (5 mg/ml) (Fig. 6E). We also demonstrated 546 CITED2 NEGATIVELY REGULATES NF-kB Downloaded from

FIGURE 6. CITED2 sensitizes cells to apoptosis induced by TNF-a. A, 293T cells were transfected with plasmids as mentioned. Twenty-four hours after transfection, the cells were stimulated with 50 ng/ml TNF-a and 5 mg/ml CHX or left untreated for another 24 h. Representative microscopic images are shown. Scale bar, 50 mm. B and C, The cells were prepared as in A, harvested, and analyzed by annexin Vor PI to measure the percentage of apoptotic cells in each sample. Data represents means 6 SD (error bars) of at least three independent experiments. D, 293T cells were transfected with plasmids as

indicated. Twenty-four hours later, cells were either treated with TNF-a (50 ng/ml) alone or TNF-a plus CHX (5 mg/ml) for the indicated time or left http://www.jimmunol.org/ untreated. Then, lysates were analyzed by immunoblotting with the indicated Abs. E, 293T cells were transfected with the indicated plasmids. One day after transfection, cells were treated with TNF-a (50 ng/ml) plus CHX (5 mg/ml) for 24 or 4 h or left untreated. Then, lysates were analyzed by immunoblotting. Original magniﬁcation 320.

that the protein level of cIAP-1 was lower when CITED2 was kB to be subjected to meticulous checks and balances. Recently, overexpressed, compared with CITED2(4m) or vector, under much attention has been focused on the regulation of NF-kB in- TNF-a/CHX treatment (Fig. 6D). side the nucleus. Several nuclear coactivators have been demonstrated as critical in the regulation of NF-kB activity and the by guest on September 29, 2021 LPS robustly induces CITED2 expression via NF-kB corresponding enhanceosome, via specific mechanisms. For ex- LPS, also known as endotoxin, is a potent inducer of NF-kBacti- ample, p300/CBP is an essential coactivator for NF-kB, which vation. The NF-kB activation is quickly downregulated to avoid the simultaneously serves as a scaffold protein for the NF-kB en- detrimental side effects toward the host such as chronic in- hanceosome and catalyzes the acetylation of p65 (22). In addition, flammation. To address the potential role of CITED2 in this nega- the nuclear proteins UXT (21), CARM1 (40), protein kinase C bΙΙ tive regulation, we challenged the murine macrophage cell lines (41), Akirin2 (20), and so on are essential for NF-kB to induce J774, Raw 264.7, and BMDM with LPS. Notably, the CITED2 a subset of target genes. mRNA was quickly induced by LPS in these cells (Fig. 7A). The Notably, our understanding of the mechanisms that decrease protein level of CITED2 was also elevated (Fig. 7B). nuclear NF-kB activity has been more rudimentary than of those Given that LPS activates several signaling pathways, we used that lead to its activation. A few corepressors have been suggested specific inhibitors to probe how CITED2 was induced in the to impair NF-kB activity in the nucleus. For example, PIAS1 can murine macrophage. We observed that a MAPK kinase inhibitor interact with p65 and block its interaction with the promoters (PD98059) and a PI3K inhibitor (LY294002) could not impair the of NF-kB–responsive genes induced by TNF-a and LPS (42). induction of CITED2 upon LPS treatment. In contrast, an NF-kB PDLIM2 interacts directly with p65 in certain conditions, which signaling inhibitor (Bay11-7082) significantly attenuated the promotes the relocalization of p65 to insoluble nuclear bodies and inductionofCITED2byLPS(Fig.7C). Furthermore, wild-type its subsequent ubiquitin-mediated degradation (43). These obser- 2/2 MEF and p65 MEF cells were respectively challenged with vations nicely indicate that the strength and duration of NF-kB 2/2 LPS. Induction of CITED2 was scarcely observed in p65 response inside the nucleus is modulated through more compli- MEF cells compared with that in the wild-type MEF cells cated mechanisms than previously expected. (Fig. 7D). Using bioinformatics analysis, we noticed that CITED2 is topo- Consistently, bioinformatics analysis identified an authentic kB logically close to NF-kB in the protein interaction networks. In this site on the promoter of CITED2. ChIP assay revealed that p65 study, we characterized CITED2 as a novel corepressor for the NF- bound to this particular site after LPS stimulation, indicating that kB enhanceosome. Several lines of observation support this argu- NF-kB regulates the expression of CITED2 per se (Fig. 7E). ment (1). Ectopic expression of CITED2 attenuated NF-kB reporter Taken together, these data uncovered a novel negative feedback genes in response to multiple stimuli. Epistatic analysis supported mechanism for regulating NF-kB inside the nucleus. that CITED2 functioned in the NF-kB enhanceosome, which was consistent with its nuclear localization. Furthermore, ectopic ex- Discussion pression of CITED2 reduced the transcription of NF-kB target Given the physiological and pathological roles of NF-kB in im- genes (2). Conversely, knockdown of endogenous CITED2 resulted munity, cancer, and autoimmune diseases, it is necessary for NF- in an apparent increase of the expression of NF-kB–responsive The Journal of Immunology 547

FIGURE 7. CITED2 is induced by LPS through NF-kB. A, J774 cells, Raw264.7 cells, and BMDMs were treated with LPS and harvested at different time points. Then, the total RNA was extracted and assayed using real-time RT-PCR for the mRNA level of CITED2. B, J774 and Raw264.7 cells were given a time-course treatment with LPS, and the whole-cell lysates were collected and immunoblotted to detect the protein level of CITED2. C, J774 cells were pretreated with the indicated inhibitors and stimulated with LPS for 1 h. The total RNA was extracted, and real-time RT-PCR was performed. D, MEF (WT) and MEF (p652/2) cells were Downloaded from stimulated with LPS for the indicated time periods, and then the cells were lysed and the lysates used for immunoblot analysis. E, J774 cells were stimulated by LPS for either 30 or 60 min or left untreated; ChIP assay was performed to detect the amount of p65 on the http://www.jimmunol.org/ CITED2 promoter. *p , 0.05; **p , 0.01. by guest on September 29, 2021 genes, stimulated by TNF-a or PMA/ionomycin or LPS. Notably, CITED2 on other signaling pathways important in innate immu- this increase could be dampened by an siRNA-resistant exogenous nity such as IRF3 or AP-1. CITED2 (3). CITED2 did not influence the molecular events The apparent multifunctionality of CITED2 can be understood leading to NF-kB translocation into the nucleus or the amount of by the observation that CITED2 interacts strongly with p300/CBP. nuclear NF-kB. However, CITED2 markedly impaired the ability of A large number of transcription factors bind to p300/CBP on NF-kB to bind its cognate promoters (4). CITED2 is unable to their different regions; therefore, depending on the topological interact directly with NF-kB. Notably, CITED2 could tightly bind orientations of p300/CBP, CITED2, and the corresponding tran- to the p300 CH1 domain, which was also recognized by p65. The scription factors, CITED2 and the relevant transcription factor antagonism between CITED2 and p65 was substantiated by mam- could antagonize or cooperate in terms of binding to p300/CBP, malian two-hybrid, GST pull-down, and co-immunoprecipitation thus attenuating or promoting the transcriptional activity, respec- assays (5). Disruption of the p65 and p300 interaction led to a re- tively. For instance, Bhattacharya et al. (29) proposed a model in duction in p65 acetylation catalyzed by p300, a strong correlation which CITED2 competes against HIF-1a for the CH1 domain of with the decreased presence of NF-kB on its cognate promoters (6). p300, thus CITED2 serves as a negative regulator for HIF-1a. The expression of CITED2 sensitized cells to TNF-a–induced ap- Also, Bhattacharya et al. reported that the C terminus of CITED2 optosis. This result is consistent with the antiapoptotic role of NF- (aa 224–255) is sufficient for binding of CITED2 with p300 CH1. kB. Taken together, a tentative model is proposed in which CITED2 In this study, we refined the model and showed that the amino seems to compete against NF-kB for binding to p300, thus atten- acids L243, P244, L246, and W247 are all important residues for uating p65 acetylation and its binding to the cognate promoters. CITED2 to bind to p300 CH1. This mechanism also can occur CITED2 is a member of the CREB/p300-interacting trans- between other transcription factors. For example, STAT2 and NF- activator with Glu/Asp-rich C-terminal domain (CITED) protein kB cross-inhibit each other by competing for p300/CBP at the family, which has four members (CITED1–4). CITED1, CITED2, CH1 binding site (45), indicating that the amount of nuclear p300/ and CITED4 have been found in human and mouse. CITED2 CBP is very limited. This finding is consistent with our observa- localizes exclusively in the nucleus and functions as a transcription that ectopic expression of p300/CBP was sufficient to reverse tion coactivator or corepressor depending on the context. For the inhibitory effects of CITED2 on NF-kB (data not shown). It is example, CITED2 is a coactivator for HNF4a in the development possible that CITED2 serves as a versatile valve to control dy- of the liver (44) and for TFAP2A in the neural tube formation and namically the access of important transcription factors to p300/ cardiac development (23). In contrast, CITED2 is an inducible and CBP, given that CITED2 is inducible under stress conditions. This negative regulator of HIF-1a under hypoxia conditions. In this hypothesis is substantiated by the observation that CITED2 per se study, we demonstrated that CITED2 is a negative regulator of was significantly induced by LPS, and the induction was NF-kB NF-kB. Besides, we did not observe any regulatory effects of dependent. 548 CITED2 NEGATIVELY REGULATES NF-kB

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