regulation of the ubiquitylation and deubiquitylation of ... · regulation of the ubiquitylation...

SC I ENCE S I GNAL ING | R E S EARCH ART I C L E

IMMUNOLOGY

1Department of Medicine, Acute Lung Injury Center of Excellence, Vascular MedicalInstitute, and Department of Cell Biology, University of Pittsburgh School of Medicine,Pittsburgh, PA 15213, USA. 2Department of Anesthesia, First Hospital of Jilin University,Changchun, China. 3Department of Critical Care Medicine, Xiangya Hospital, CentralSouth University, Changsha, Hunan, China.*These authors contributed equally to this work.†Corresponding author. Email: [email protected] (J.Z.); [email protected] (Y.Z.)

Wei et al., Sci. Signal. 10, eaak9660 (2017) 13 June 2017

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Regulation of the ubiquitylation and deubiquitylationof CREB-binding protein modulates histone acetylationand lung inflammationJianxin Wei,1 Su Dong,1,2 Rachel K. Bowser,1 Andrew Khoo,1 Lina Zhang,1,3 Anastasia M. Jacko,1

Yutong Zhao,1*† Jing Zhao1*†

Cyclic adenosine monophosphate (cAMP) response element–binding protein (CREB)–binding protein (CBP) is ahistone acetyltransferase that plays a pivotal role in the control of histone modification and the expression ofcytokine-encoding genes in inflammatory diseases, including sepsis and lung injury. We found that the E3 ubiquitinligase subunit FBXL19 targeted CBP for site-specific ubiquitylation and proteasomal degradation. The ubiquitylation-dependent degradation of CBP reduced the extent of lipopolysaccharide (LPS)–dependent histone acetylation andcytokine release in mouse lung epithelial cells and in a mouse model of sepsis. Furthermore, we demonstrated thatthe deubiquitylating enzyme USP14 (ubiquitin-specific peptidase 14) stabilized CBP by reducing its ubiquitylation.LPS increased the stability of CBP by reducing the association between CBP and FBXL19 and by activatingUSP14. Inhibition of USP14 reduced CBP protein abundance and attenuated LPS-stimulated histone acetylationand cytokine release. Together, our findings delineate the molecular mechanisms through which CBP stability isregulated by FBXL19 and USP14, which results in the modulation of chromatin remodeling and the expressionof cytokine-encoding genes.

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INTRODUCTIONCyclic adenosine monophosphate (cAMP) response element–bindingprotein (CREB)–binding protein (CBP), a member of the family ofhistone acetyltransferases (HATs), catalyzes histone acetylation, therebyswitching chromatin from the closed state to the open state. This changepromotes the binding of RNA polymerase II and basal transcriptionfactors to the open DNA to initiate transcription. The acetylation ofhistone H4 is correlated with increased expression of genes encodingproinflammatory factors, such as interleukin-8 (IL-8), IL-6, and tumornecrosis factor–a (TNF-a) (1–4). Multiple studies have attempted toinvestigate the regulation of CBP stability to better understand themolecular basis of its action. Ubiquitylation is a posttranslationalmodification that alters protein stability. Proteins conjugated to Lys48

(K48)–linked ubiquitin chains are degraded in the proteasome, whereasK63-linked polyubiquitylation triggers protein degradation in the lyso-some (5). Siah1 and Siah2 (collectively known as Siah1/2) are E3 ubi-quitin ligases that ubiquitylate CBP, leading to its degradation andthereby reducing the extent of CBP-mediated acetylation of p53 (6).However, much less is known about the effect of the Siah proteins onCBP-catalyzed histone acetylation. Sen and Snyder demonstrated thatSiah facilitates the acetylation of histone H3 and enhances the expres-sion of CREB-regulated genes (7). These studies suggest that Siah1/2specifically dampens the CBP that regulates p53 acetylation but notthe CBP that modulates histones. In addition, neither Siah1 nor Siah2is detectable in normal lung tissues (8). To reduce CBP activity in in-flammatory diseases, such as lung injury, there must be alternative E3ubiquitin ligases that target CBP for ubiquitylation and degradation toinhibit its effects on histone acetylation.

The Skp1–Cul1–F-box protein (SCF) ligase complex is one of thelargest among the family of E3 ubiquitin ligases. The F-box proteinconnects the ligase complex and specific substrates through its F-boxdomain and substrate-bindingmotif, respectively (9–11). A previouslyuncharacterized F-box protein, FBXL19, targets the IL-33 receptorST2L (12) and small guanosine triphosphatases (GTPases) (13–15)for ubiquitylation and proteasomal degradation.We previously showedthat FBXL19 reduces inflammatory responses, such as cytokine release,in a mouse model of acute lung injury (12), suggesting that FBXL19targets an unidentified substrate that plays a pivotal role in regulatingthe expression of cytokine-encoding genes. Protein ubiquitylationcan be reversed by deubiquitylating enzymes (DUBs), which enhanceprotein stability. Several DUBs [for example, USP3 (ubiquitin-specificpeptidase 3), USP22, and Mysm1] remove ubiquitin moieties fromubiquitylated histone H2A proteins (16–18). The HAT P/CAF phys-ically interacts with the DUB Mysm1 (19); however, whether DUBsplay a role in the regulation of HAT stability is unclear. We previouslyshowed that USP14 plays a proinflammatory role by enhancing theactivity of the transcription factor nuclear factor kB (NF-kB) (20).Xu and Guo showed that inhibition of USP14 by the small-moleculeinhibitor IU1 prevents ventilator-induced lung injury in rats (21).Together, these findings suggest that USP14 exhibits proinflammatoryproperties.

Here, we demonstrated that FBXL19 targets CBP for site-specificubiquitylation and degradation. FBXL19 functioned as an anti-inflammatory E3 ubiquitin ligase subunit by reducing the abundanceof CBP protein, which led to decreased histone acetylation and re-duced cytokine release. Together, these data suggest that an E3 ubiqui-tin ligase of the SCF family ubiquitylates aHAT, thereby leading to itsdegradation and the reduction of histone acetylation. Furthermore,we revealed that USP14 deubiquitylated CBP and promoted its sta-bility. Inhibition of USP14 decreased the amount of CBP protein,attenuated lipopolysaccharide (LPS)–stimulated histone acetylationand cytokine release, and lessened systemic and lung inflammationin mice.

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Fig. 1. FBXL19mediates site-specific CBPubiquitylation anddegradation. (A andB)MLE12 cells were transfectedwith the cbp-v5or cbpK2103R-v5plasmids. Forty-eight hourslater, the cells were treated with CHX (20 mg/ml) for 0, 2, 4, or 6 hours. (A) Cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. (B)Determination of relative protein abundance by densitometric analysis of Western blots with ImageJ software. (B) Data aremeans ± SEM of three independent experiments. *P <0.01 by two-way analysis of variance (ANOVA) and post hoc Tukey’s test. (C and D) MLE12 cells were transfected with the indicated amounts of the Fbxl19-v5 plasmid. (C) Forty-eight hours later, cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. (D) The relative amounts of p300 and CBP proteins weredetermined by densitometric analysis. Data aremeans ± SEM of three independent experiments. *P < 0.01 by two-way ANOVA and post hoc Tukey’s test. Comparison wasmadeto the amount of CBP in cells that were not transfected with the Fbxl19-v5 plasmid. (E and F) MLE12 cells were transfectedwith the cbp-v5 and Fbxl19-v5 plasmids as indicated.(E) Forty-eight hours later, cell lysates were analyzed byWestern blotting with antibodies against V5 and b-actin. (F) The relative amounts of CBP-V5 protein were determinedby densitometric analysis. Data are means ± SEM of three independent experiments. *P < 0.01 by one-way ANOVA and post hoc Tukey’s test. Comparison wasmade to cells thatwere not transfectedwith the Fbxl19-v5 plasmid. (G andH) MLE12 cells were transfectedwith control (Cont) shRNA (−) or fbxl19-specific shRNA (+), and then cells were treated forthe indicated times with CHX (20 mg/ml). (G) Cell lysates were analyzed by Western blotting with antibodies against CBP, FBXL19, and b-actin. (H) The relative amounts of CBPproteinweredeterminedbydensitometric analysis. Dataaremeans±SEMof three independent experiments. *P<0.01by two-wayANOVAandpost hoc Tukey’s test. Comparisonwasmade to cells treatedwith control shRNA. (I and J) MLE12 cells were transfectedwith the indicated amounts of the Fbxl19-v5 plasmid. Forty-eight hours later, cell lysates weresubjected to immunoprecipitation (IP) with an anti-CBP antibody and Western blotting analysis with an anti-ubiquitin antibody. Input lysates were analyzed byWestern blottingwith antibodies against CBP, V5, and b-actin. (J) The relative amounts of ubiquitylatedCBPweredeterminedbydensitometric analysis. Data aremeans± SEMof three independentexperiments. P values were calculated by one-way ANOVA and post hoc Tukey’s test. Comparison was made to cells treated with control shRNA. IB, immunoblotting. Ubi(n),polyubiquitination. (K and L)MLE12 cellswere transfectedwith the Fbxl19-v5, cbp-v5, or cbpK2103R-v5plasmid, as indicated. (K) Cell lysateswere then analyzedbyWesternblottingwithantibodies against V5 and b-actin. (L) The relative amounts of CBP-V5were determined by densitometric analysis. Data aremeans ± SEMof three independent experiments.P values were determined by the unpaired Student’s t test. (M andN) Troponin T (TnT)–synthesized CBP-V5 or CBPK2103R-V5 were incubatedwith a full complement of ubiquitylationreaction componentswith (+) orwithout (−) purified Flag-tagged FBXL19. (M) Reactionmixtureswere subjected toWestern blotting analysiswith antibodies against ubiquitin andthe Flag tag. The relative amounts of ubiquitylated CBP-V5 and CBPK2103R-V5 were determined by densitometric analysis. Data are means ± SEM of three independentexperiments. P values were determined by one-way ANOVA and post hoc Tukey’s test. All Western blots are representative of at least three independent experiments.

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RESULTSFBXL19 targets CBP for ubiquitylation and degradationIt iswell known that CBP stability is regulated by the ubiquitin-proteasomesystem; however, the molecular control of CBP stability in the setting ofinflammatory responses is unclear, and the ubiquitin acceptor sitehas not been identified. Here, in experiments with the protein synthesisinhibitor cycloheximide (CHX), we confirmed that CBP was unstable,with a half-life of ~3.4 hours (Fig. 1, A and B). Lysine residues arecommonly the targets for ubiquitylation. We found that a lysine-to-argininemutant of CBP (CBPK2103R) had an extended half-life comparedto that of the wild-type (WT) protein (Fig. 1, A and B), suggesting that


Lys2103 was the potential ubiquitylation site within CBP. These data areconsistent with those from a previous proteomics analysis (22).

FBXL19 is a subunit of an SCF family E3 ubiquitin ligase, whichexhibits an anti-inflammatory property (23); however, its substratesrelated to cytokine gene expression have not been identified. We testedwhether FBXL19 regulated the protein stability of HATs. Ectopicallyexpressed FBXL19 reduced the abundances of endogenous and overex-pressed CBP proteins (Fig. 1, C to F, and fig. S1), without altering p300abundance (Fig. 1, C and D); however, knockdown of FBXL19 withFbxl19-specific short hairpin RNA (shRNA) enhanced the stability ofCBP (Fig. 1, G and H). In addition, overexpression of FBXL19 resulted



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ig. 2. FBXL19 dampens the LPS-stimulated increase in CBP protein abundance. (A and B) MLE12ells were treated with LPS (10 mg/ml) for 0, 3, 6, and 9 hours. (A) Cell lysates were analyzed by Westernlotting with antibodies against the indicated proteins. (B) Determination of relative CBP protein abunance by densitometric analysis of Western blots with ImageJ software. Data are means ± SEM of three

independent experiments. P valueswere calculatedbyone-wayANOVAandpost hoc Tukey’s test. (C andD)MLE12 cellswere treatedwith LPS (10 mg/ml) for 1, 5, or 20 hours. TotalRNAs were extracted and cbp (C) and kc (D) mRNA abundances were measured by reverse transcription polymerase chain reaction (RT-PCR). Data are means ± SEM of threeindependent experiments. P values were calculated by one-way ANOVA and post hoc Tukey’s test. gapdh, glyceraldehyde-3-phosphate dehydrogenase. (E and F) MLE12cells were treated with LPS (10 mg/ml, 3 hours). (E) Cell lysates were subjected to immunoprecipitation with an anti-CBP antibody and Western blotting with an anti-ubiquitinantibody. Input lysates were analyzed by Western blotting with an anti-CBP antibody. (F) The relative amounts of ubiquitylated CBP were determined by densitometric analysis.Data aremeans ± SEMof three independent experiments. P valueswere calculated by unpaired Student t test. (G andH) MLE12 cells were treatedwith LPS (10 mg/ml, 3 hours). (G)Cell lysates were subjected to immunoprecipitation with immunoglobulin G (IgG) or with antibody against FBXL19 or CBP, followed by Western blotting with antibodies againstCBP and FBXL19. (H) The relative amounts of CBP and FBXL19were determined bydensitometric analysis. Data aremeans± SEMof three independent experiments. P valueswerecalculated by unpaired Student t test. (I and J) MLE12 cells were transfectedwith the Fbxl19-v5 plasmid. Forty-eight hours later, the cells were treatedwith LPS (10 mg/ml, 6 hours).(I) Cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. (J) Determination of relative protein abundance by densitometric analysis ofWestern blots with ImageJ software. (J) Data are means ± SEM of three independent experiments. P values were determined by one-way ANOVA and post hoc Tukey’s test. AllWestern blots are representative of at least three independent experiments.



in a reduction in the amount of nuclearCBP, but not that of cytoplasmicCBP (fig. S2, A and B).

To investigate whether FBXL19 transferred ubiquitin chains toCBP, MLE12 cells were transfected with the Fbxl19-v5 plasmid,and then the ubiquitylation state of CBP was determined. We foundthat overexpression of FBXL19 led to the increased polyubiquitylationof CBP (Fig. 1, I and J). The FBXL19-mediated ubiquitylation of CBPwas K48-linked, but not K63-linked (fig. S3). The CBPK2103R mutantprotein was resistant to degradation in response to the overexpressionof FBXL19 (Fig. 1, K and L) and was not subjected to polyubiquityla-tion (Fig. 1, M and N). The mutation of specific lysine residues in CBPhad no effect on its cellular localization (fig. S2C). These data suggestthat FBXL19 induces the site-specific ubiquitylation of CBP and itssubsequent degradation.



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LPS increases CBP stabilitythrough the disassociation of theFBXL19-CBP complex and areduction in the abundanceof FBXL19CBP-mediated histone acetylation playsa crucial role in LPS-induced inflamma-tory responses (24–26). We examinedthe effect of LPS on CBP abundanceand found that the treatment of MLE12cells with LPS increased the amount ofCBP protein in a time-dependent manner(Fig. 2, A and B). The effect of LPS waslikely a result of an increase inCBP stabil-ity, because LPS did not substantially af-fect the abundance of Cbp mRNA (Fig.2C). As a positive control, LPS increasedthe abundance of kc mRNA in MLE12cells (Fig. 2D). To determine the molecu-lar mechanism by which LPS regulatedCBP stability, we examined the effect ofLPS on CBP ubiquitylation. LPS reducedboth the extent of CBP ubiquitylation(Fig. 2, E and F) and the association be-tweenCBPandFBXL19 at 3 hours (Fig. 2,G andH). Furthermore, the LPS-inducedincrease in CBP abundance was attenu-ated in cells transfected with plasmidexpressing FBXL19 (Fig. 2, I and J). To-gether, these data suggest that LPSincreased CBP abundance through disso-ciationofCBP fromFBXL19at early times,which led to a reduction in the extent ofCBP ubiquitylation. We found that LPSincreased the extent of serine phospho-rylation of CBP at 3 hours (fig. S4A). Wepreviously showed that the phosphoryl-ationof substrates promotes their bindingtoE3ubiquitin ligases (23).Thus, thephos-phorylation ofCBPmay inhibit its interac-tion with FBXL19.

To investigate whether FBXL19abundance was changed under inflam-matory conditions, wemeasured the rela-


tive amounts of FBXL19 in the lungs of mice 24 hours after they wereintratracheally challenged with Pseudomonas aeruginosa (strainPA103). We found that abundance of FBXL19 protein in the lungsof infectedmice was reduced compared to that in the lungs of uninfectedmice (Fig. 3, A and B). To confirm these data, we examined theamount of FBXL19 protein in LPS-treated MLE12 cells. We foundthat LPS decreased the abundance of FBXL19 in a time-dependentmanner (Fig. 3, C and D) and that these effects were attenuated by theproteasomal inhibitor MG132 (N-carbobenzyloxy-L-leucyl-L-leucyl-L-leucinal), but not the lysosomal inhibitor leupeptin (Fig. 3, C and D),which suggests that LPS stimulates the proteasomal degradationof FBXL19. Furthermore, we found that LPS increased the extent ofphosphorylation (fig. S4B) and K48-linked polyubiquitylation ofFBXL19 (Fig. 3, E and F), without substantially altering the abundance

Fig. 3. LPSdecreasesFBXL19 stability. (A and B) C57/BL6 mice were intratracheally administered with P. aeruginosastrain PA103 (104 colony-forming units per mouse). (A) Twenty-four hours later, lung tissues were analyzed by Westernblotting with antibodies against FBXL19 and b-actin. i.t., intratracheal. (B) The relative amounts of FBXL19 were determinedby densitometric analysis with ImageJ software. Data are means ± SEM of 6 (control) and 11 (PA103) mice. P values werecalculated by unpaired Student t test. (C andD) MLE12 cells were treated with DMSO (dimethyl sulfoxide), MG132 (20 mM),or leupeptin (100 mM) for 1 hour before theywere treatedwith LPS (10 mg/ml) for an additional 0, 6, or 9 hours. (C) Cell lysateswere analyzed by Western blotting with antibodies against FBXL19 and b-actin. (D) The relative amounts of FBXL19were determined by densitometric analysis with ImageJ software. Data are means ± SEM of three independentexperiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test. (E and F) HBEpCs were treatedwith LPS (10 mg/ml) for 6 hours. (E) Cell lysates were subjected to immunoprecipitation with an anti-FBXL19 antibodyand Western blotting with an anti–K48-ubiquitin antibody. Input lysates were analyzed by Western blotting with anti-bodies against CBP, FBXL19, and b-actin. (F) The relative amounts of FBXL19 were determined by densitometric anal-ysis with ImageJ software. Data are means ± SEM of three independent experiments. P values were calculated byunpaired Student t test. (G) MLE12 cells were treated with LPS (10 mg/ml) for 1, 5, or 20 hours. Total RNAs were extractedand fbxl19 mRNA abundance was measured by RT-PCR analysis. Data are means ± SEM of three independentexperiments. P values were calculated by one-way ANOVA and post hoc Tukey’s test. All Western blots are representativeof at least three independent experiments.

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of fbxl19mRNA (Fig. 3G), suggesting that LPS-induced FBXL19 deg-radation was mediated through ubiquitylation. It is well known that thephosphorylation of substrate can stimulate protein ubiquitylation (27).Thus, we hypothesized that phosphorylation may be essential forFBXL19 ubiquitylation. We found that overexpression of V5-taggedFBXL19 had no effect on the abundance of myc-tagged FBXL19 (fig.S5), which suggests that the degradation of FBXL19 was not a resultof auto-ubiquitylation and degradation by its own activity. Together,these data suggest that LPS increases CBP protein abundance by inhibitingthe FBXL19-CBP association at early times and reducing FBXL19 proteinabundance at later times, thereby increasing the stability of CBP.

FBXL19 inhibits histone acetylation and cytokine productionin lung epithelial cells and a mouse model of sepsis



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Histone acetylation is an essen-tial step to control the expressionof cytokine-encoding genes inresponse to LPS (28–30). Toinvestigate whether FBXL19affected histone acetylation,FBXL19 and CBP were over-expressed in lung epithelial cells.Overexpression of CBP aloneslightly increased the extent ofacetylation of histone H4 at Lys8

(H4K8ac), whereas the effectwas attenuated in cells in whichFBXL19 was overexpressed(Fig. 4, A and B). LPS stimu-lated the acetylation of histoneH4K8 in lung epithelial cells(Fig. 4, C and D); however, theeffects were attenuated by theexpression of FBXL19, where-as H4K8 acetylation was pro-moted by a reduction in theabundance of FBXL19 (Fig. 4,E and F). Furthermore, we foundthat FBXL19 reduced the extentof acetylation of histone H4K12(fig. S6, A and B) and thebinding of acetylated H4K8 tothe Il8 promoter (fig. S7) in re-sponse to LPS. These data suggestthat FBXL19 reduced histoneacetylation through the desta-bilization of CBP. We then ex-amined the effects of FBXL19 oncytokine release by lung epi-thelial cells. The knockdownof FBXL19 with fbxl19-specificshRNA promoted the LPS-stimulated release of the cytokineKC (keratinocyte-derived cyto-kine) by murine lung epithelialcells (Fig. 4G). Overexpressionof FBXL19 attenuated the LPS-induced production of IL-8 byhuman lung epithelial cells


overexpressing WT CBP, but not CBPK2103R (Fig. 4H), suggestingthat the FBXL19 exerted its anti-inflammatory properties by reduc-ing CBP stability and the extent of histone acetylation. These datareveal previously uncharacterized biological functions for FBXL19and suggest that FBXL19 limits LPS-induced histone acetylation andcytokine release.

We previously showed that overexpression of FBXL19 in mouselungs attenuates intratracheally instilled, LPS-induced cytokine releaseinto the bronchoalveolar lavage (BAL) fluid (BALF) (12). To investigatewhether FBXL19 lessened systemic inflammation, we intravenouslyinjected a lentiviral vector expressing Fbxl19 into mice before they weresubjected to cecal ligation and puncture (CLP), a well-established mu-rine model of sepsis. CLP increased the acetylation of histone H4K8 inmouse lungs in a time-dependent manner (Fig. 5, A and B), whereas

Fig. 4. The reduction of CBP stability by FBXL19 impairs histone acetylation and cytokine release. (A and B) MLE12 cellswere transfected with cbp-v5 and Fbxl19-v5 plasmids. (A) Forty-eight hours later, isolated histone lysates were analyzed byWestern blotting with antibodies against histone H4K8ac and histone H4. Cell lysates were analyzed by Western blotting withantibodies against V5 and b-actin. (B) Determination of relative H4K8ac protein abundance by densitometric analysis of West-ern blots with ImageJ software. Data are means ± SEM of three independent experiments. P values were calculated by two-wayANOVA and post hoc Tukey’s test. (C and D) HBEpCs were transfected with the Fbxl19-v5 plasmid, and then cells were treatedwith LPS (10 mg/ml, 3 hours). (C) Isolated histone lysates were analyzed by Western blotting with antibodies against H4K8ac andH4. (D) Determination of relative H4K8ac protein abundance by densitometric analysis of Western blots with ImageJ software.Data are means ± SEM of three independent experiments. P values were calculated by two-way ANOVA and post hoc Tukey’stest. (E and F) MLE12 cells transfected with fbxl19 shRNA (shfbxl19) plasmid. Seventy-two hours later, cells were treated withLPS (10 mg/ml) for 3 hours. (E) Isolated histone lysates were analyzed by Western blotting with antibodies against H4K8ac andH4. (F) Determination of relative H4K8ac protein abundance by densitometric analysis of Western blots with ImageJ software.Data are means ± SEM of three independent experiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test.(G) MLE12 cells transfected with or without the fbxl19 shRNA plasmid. Seventy-two hours later, cells were treated with LPS (10 mg/ml)for 3 hours. KC release was measured by enzyme-linked immunosorbent assay (ELISA). Data are means ± SEM of threeindependent experiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test. (H) HBEpCs were transfectedwith the Fbxl19-v5, cbp-v5, or cbpK2103R-V5 plasmid as indicated. Forty-eight hours later, cells were treated with LPS (10 mg/ml, 3 hours).The amount of IL-8 secreted into the medium was measured by ELISA. Data are means ± SEM of three independentexperiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test. All Western blots are representative ofat least three independent experiments. n.s., not significant.

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these effects were reduced in the lungs of mice overexpressing FBXL19(Fig. 5, C andD). CLP-inducedKC release in plasma and BALFs andmy-eloperoxidase activity (a marker of lung inflammation) in the lungs werereduced inmice infected with lentivirus expressing Fbxl19 (Fig. 5, E to G).These data suggest that FBXL19 exhibits an anti-inflammatory effect inmurine models of both local and systemic inflammatory diseases.

USP14 deubiquitylates and stabilizes CBPDeubiquitylation, the reverse process of ubiquitylation, is enacted byDUBs, which control protein stability, localization, and enzyme activity.We previously showed that USP14 decreases the stability of inhibitor ofNF-kB (IkB) protein (20), indicating that USP14 promotes inflamma-tory responses. Here, we tested whether USP14 regulated CBP stability,because CBP plays a critical role in proinflammatory responses. Wefound that overexpression of USP14 resulted in the increased abun-dance of CBP, without altering the abundance of p300 (Fig. 6, A and B).Knockdown of USP14 with usp14-specific shRNA (Fig. 6, C and D)or inhibition of USP14 activity with IU1 (Fig. 6, E and F) resulted inreduced CBP abundance. Furthermore, pretreatment of MLE12 cellswith IU1 attenuated the LPS-dependent increase in CBP protein


abundance (Fig. 6, G and H). To investigate whether USP14 deubi-quitylated CBP, we transfectedMLE12 cells to overexpress USP14 ortreated them with IU1, and then we examined the ubiquitylation ofCBP. We found that overexpression of USP14 reduced CBP ubiqui-tylation, whereas IU1 had the opposite effect (Fig. 6, I and J). Further-more, coimmunoprecipitation studies showed an association betweenUSP14 andCBP (Fig. 6K), whichwas unaffected by LPS (Fig. 6L). Thesedata suggest that USP14 stabilizes CBP through the deubiquitylationof CBP.

Inhibition of USP14 attenuates LPS-induced histoneacetylation and cytokine release and lessens systemic andlung inflammationWe next found that LPS increased the deubiquitylating activity ofUSP14 and confirmed the inhibitory effect of IU1 on USP14 activity(Fig. 7A). Because USP14 stabilized CBP, we investigated whetherUSP14 promoted CBP-mediated histone acetylation. The LPS-inducedacetylation of histone H4K8 was enhanced in MLE12 cells overexpress-ing USP14 (Fig. 7, B and C), whereas knockdown of USP14 withusp14-specific shRNA attenuated the acetylation of histone H4K8

Fig. 5. FBXL19 mitigates histone acetylation and inflammatory responses in a CLP-induced murine model of sepsis. (A and B) C57/BL6 mice were subjected toCLP for 4, 24, or 48 hours. (A) Isolated histone lysates of lung tissues were analyzed by Western blotting with antibodies against histone H4K8ac and histone H4. (B)Determination of relative H4K8ac protein abundance by densitometric analysis of Western blots with ImageJ software. Data are means ± SEM of six to eight samples pergroup. P values were calculated by one-way ANOVA and post hoc Tukey’s test. (C and D) C57/BL6 mice were intravenously infected with control lentivirus (Lenti-cont) orlentivirus encoding Fbxl19 (Lenti-Fbxl19) before they were subjected to CLP. (C) Twenty-four hours later, isolated histone lysates of lung tissues were analyzed byWestern blotting with antibodies against histone H4K8ac and histone H4. (D) Determination of relative H4K8ac protein abundance by densitometric analysis of Westernblots with ImageJ software. Data are means ± SEM of 11 samples per group. P values were calculated by unpaired Student t test. (E and F) Plasma and BAL KCconcentrations were measured by ELISA. Data are means ± SEM of five to eight samples per group. P values were calculated by two-way ANOVA and post hoc Tukey’stest. (G) Myeloperoxidase (MPO) activity in lung tissues was measured by ELISA. Data are means ± SEM of five to eight samples per group. P values were calculated bytwo-way ANOVA and post hoc Tukey’s test.

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(Fig. 7, D and E). Furthermore, transfection ofMLE12 cells with usp14-specific shRNA (Fig. 8A) or treatment with IU1 (Fig. 8B and fig. S8, Aand B) attenuated the LPS-induced release of KC and IL-6 by MLE12cells and RAW264.7 cells (a mouse macrophage cell line). The anti-inflammatory effect of IU1 was also confirmed in experiments withLPS-treated peripheral blood mononuclear cells (fig. S8C).

Todetermine the anti-inflammatory effects of IU1 inmurinemodelsof inflammatory diseases, such as sepsis and lung injury, IU1 was intra-


peritoneally injected into mice after CLP. IU1 increased the survival rateof these mice (Fig. 8C) and reduced the concentrations of IL-6 and KC inboth plasma and BALF (Fig. 8, D to G). Intratracheally administeredIU1 reduced the LPS-induced increase in IL-6 abundance in the BAL(Fig. 8H) and inhibited the influx of neutrophils into the alveolar spaces(Fig. 8I). These data indicate that the inhibition of USP14 by IU1 re-duces inflammatory responses by reducing CBP stability and histoneacetylation.

Fig. 6. USP14 deubiquitylates and stabilizes CBP. (A and B) MLE12 cells were transfected with the indicated amounts of Usp14-ha plasmid. (A) Forty-eight hourslater, cell lysates were analyzed by Western blotting with antibodies against indicated proteins. (B) Determination of relative CBP and p300 protein abundance bydensitometric analysis of Western blots with ImageJ software. Data are means ± SEM of three independent experiments. P values were calculated by two-way ANOVAand post hoc Tukey’s test. (C and D) MLE12 cells were transfected with Cont shRNA or usp14 shRNA. (C) Seventy-two hours later, cell lysates were analyzed by Westernblotting with antibodies against the indicated proteins. (D) Determination of relative CBP protein abundance by densitometric analysis of Western blots with ImageJsoftware. Data are means ± SEM of three independent experiments. P values were calculated by unpaired Student’s t test. (E and F) MLE12 cells were treated with IU1 (0,10, 20, or 50 mM). (E) Twenty-four hours later, cell lysates were analyzed by Western blotting with antibodies against CBP and b-actin. (F) Determination of relative CBPprotein abundance by densitometric analysis of Western blots with ImageJ software. Data are means ± SEM of three independent experiments. P values were calculatedby one-way ANOVA and post hoc Tukey’s test. (G and H) MLE12 cells were treated with increasing concentrations of IU1 for 16 hours before they were treated with LPSfor a further 9 hours. (G) Cell lysates were analyzed by Western blotting with antibodies against CBP and b-actin. (H) Determination of relative CBP protein abundanceby densitometric analysis of Western blots with ImageJ software. Data are means ± SEM of three independent experiments. P values were calculated by one-wayANOVA and post hoc Tukey’s test. (I and J) MLE12 cells were transfected with Usp14-ha plasmid for 48 hours or were pretreated with IU1 (50 mM) for 4 hours. (I) Cellswere then subjected to immunoprecipitation with an anti-CBP antibody and Western blotting analysis with antibodies against ubiquitin and CBP. Veh, vehicle. (J) Therelative amounts of ubiquitylated CBP were determined by densitometric analysis with ImageJ software. Data are means ± SEM of three independent experiments.P values were calculated by one-way ANOVA and post hoc Tukey’s test. (K) Top: MLE12 cell lysates were subjected to immunoprecipitation with control IgG or an anti-CBP antibody, followed by Western blotting with antibodies against USP14 and CBP. Middle: MLE12 cells were subjected to immunoprecipitation with control IgG or ananti-USP14 antibody, followed by Western blotting with antibodies against CBP and USP14. Bottom: Input lysates were analyzed by Western blotting with antibodiesagainst CBP, USP14, and b-actin. (L) HBEpCs were treated with LPS (10 mg/ml, 6 hours), and cell lysates were subjected to immunoprecipitation with an anti-CBPantibody, followed by Western blotting with antibodies against CBP and USP14. Input lysates were analyzed by Western blotting with antibodies against CBP,USP14, and b-actin. All Western blots are representative of at least three independent experiments.

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DISCUSSIONE3 ubiquitin ligase complexes of the SCF family regulate diversecellular functions, including inflammatory responses (11–13, 31, 32).Among the subunits of the SCF E3 ligases, the F-box protein linkssubstrates to the E3 ligase complex.We previously showed that FBXL19regulates the stability of the IL-33R (12) and of small GTPases, such asRhoA (14), Rac1 (13), and Rac3 (15). Here, we demonstrated that CBP isa previously uncharacterized substrate of FBXL19. We found thatFBXL19mediated the site-specific ubiquitylation of CBP, thus reducingits stability and inhibiting histone acetylation and cytokine release. Fur-thermore, our study revealed that LPS increased the stability of CBP byinterfering with the FBXL19-CBP interaction and reducing the abun-dance of FBXL19. Ubiquitylation is reversed by DUBs. We found thatthe DUBUSP14 targeted CBP for deubiquitylation, resulting in stabiliza-tion of CBP. Understanding the regulation of CBP stability in the settingof inflammatory responses is essential to identifying previously unchar-acterized molecular targets that could mediate the expression of genesencoding proinflammatory cytokines. We also reported that the USP14inhibitor IU1 reduced the stability of CBP, thereby mitigating against in-flammatorypathogen-inducedhistonemodification and cytokine release.

The acetylation of core histones plays amajor role in the pathogenesisof various diseases, including inflammatory diseases (1, 2, 4, 33). CBP, oneof the major members of the HAT family, facilitates chromatin remod-eling, recruitment of the transcriptional machinery to the promoterregion, and initiation of the expression of cytokine-encoding genes


(33–35). Studies showed thatthe E3 ubiquitin ligases Siah1and Siah2mediate the ubiquityl-ation and degradation of CBP,thereby limiting p53 biologicalfunction(6).Polekhinaetal. dem-onstrated that Siah1 potentiatesthe TNF-a–induced activationof NF-kB (36), suggesting thatSiah proteins play a proinflam-matory role. These data are con-sistent with the finding of Senand Snyder that showed thatSiah increases histoneH3 acety-lation and the expression ofCREB-regulated genes (7). To-gether, these studies suggestthat Siah specifically targetsp53-associatedCBP for its degra-dation, without regulating theCBP-mediated acetylation ofhistones and subsequent cyto-kine release. Here, we identifiedFBXL19 as the E3 ubiquitin li-gase subunit responsible forthe ubiquitylation and degra-dation of CBP. The FBXL19-mediated ubiquitylation of CBPwas K48-linked, but not K63-linked.

It is common for multipleE3 ubiquitin ligases to targetthe same substrate under differ-ent biological conditions. Our

study revealed that FBXL19 is an anti-inflammatory E3 ubiquitin ligasesubunit, because it reduced the extents of histone acetylation and cyto-kine release in vitro and in vivo. We identified Lys2013 within CBP as aubiquitin acceptor site for FBXL19, whereas the ubiquitin acceptor sitefrom Siah1/2 has not been identified. It is possible that Siah1 and Siah2target a distinct lysine residue in CBP, which may lead to a biologicalrole distinct from that caused bymodification by FBXL19. For example,the stability and activity of p53 are regulated by multiple E3 ubiquitinligases, including Mdm2, makorin-1, RING finger protein MSL2, andE4F transcription factor 1. The ubiquitylation of p53 at different lysinesby these enzymes stimulates distinct biological processes, such as deg-radation, nuclear export, and translocation to chromatin (37). Ourdata are consistent with the following pathway: LPS reduces the extentof the association between FBXL19 and CBP and reduces the abun-dance of FBXL19 protein, which results in increased CBP stability, his-tone acetylation, and cytokine release. Our study suggests that FBXL19is a potential target for dampening CBP-modulated inflammatory dis-eases. To our knowledge, there is no single-nucleotide polymorphism(SNP) that affects CBP Lys2103. Future studies will focus on identifyingpreviously uncharacterized SNPs associated with CBP stability. Weshowed that that lentiviral expression of FBXL19 reduced lung inflam-mation in amousemodel; however, the effect of FBXL19 on lung repairand remodeling has not yet been revealed. The generation of lung-specific FBXL19-transgenic mice will be useful to determine the path-ogenic role of FBXL19 in lung inflammatory diseases in future studies.

Fig. 7. Inhibition of USP14 impairs histone acetylation. (A) MLE12 cells were transfected with Usp14-ha plasmid. Forty-eighthours later, cells were treated with LPS (10 mg/ml, 1 hour). USP14-HA (hemagglutinin) was immunoprecipitated with an anti-HAantibody, and then the immunoprecipitated complex was incubated with or without IU1. A deubiquitylation assay was per-formed according to the manufacturer’s instructions. Deubiquitylating activity was normalized by the control. Data are means ±SEM of three independent experiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test. (B and C) MLE12cells were transfected with Usp14-ha plasmid. (B) Forty-eight hours later, cells were treated with LPS (10 mg/ml, 3 hours). Isolatedhistone lysates were analyzed by Western blotting with antibodies against histone H4K8ac and histone H4. (C) Determination ofrelative H4K8ac protein abundance by densitometric analysis of Western blots with ImageJ software. Data are means ± SEM of threeindependent experiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test. (D and E) MLE12 cells weretransfected with usp14 shRNA plasmid. (D) Seventy-two hours later, cells were treated with LPS (10 mg/ml, 3 hours). Isolated histonelysates were analyzed by Western blotting with antibodies against histone H4K8ac and histone H4. (E) Determination of relativeH4K8ac protein abundance by densitometric analysis of Western blots with ImageJ software. Data are means ± SEM of threeindependent experiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test. All Western blots are represen-tative of at least three independent experiments.

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USP14, one of the ubiquitin-specific peptidases, disassembles andrecycles the ubiquitin chain from the distal tip of its substrates (38, 39).We previously showed that USP14 decreases the stability of IkB byinteracting with theNF-kB subunit RelA (20). This finding is consistentwith another study demonstrating that the inhibition ofUSP14 increasesIkB stability in rat lungs andprevents ventilator-induced lung injury (21).Other studies also suggest that USP14 stabilizes its substrates through itsdeubiquitylating activity (40, 41). Here, we showed that USP14 targetsand stabilizes a previously uncharacterized substrate, CBP. Furthermore,we revealed a previously uncharacterized biological function of USP14:Inhibition of USP14 reduced the stability of CBP, thereby lessening theseverity of pathogen-induced lung inflammation. Previously,we showedthat LPS stimulates the serine phosphorylation of USP14 (20). Here, weprovided further evidence showing that LPS increases USP14 activity.Thus, it will be important to determine the role of serine phosphoryl-ation in the regulation of USP14 activity in future studies. USP14 is amajor regulator of the proteasome. Inhibition of USP14 by IU1 may


cause unexpected cellular responses. This raises concerns regardingthe translational potential of IU1 in treating lung inflammatory diseases.

Our study revealed the molecular mechanism of regulation of CBPstability by FBXL19 and USP14. We showed that LPS reduced theinteraction betweenCBP and its degrader (FBXL19) aswell as the abun-dance of FBXL19 and activated USP14, thus increasing CBP stability,enhancing histone acetylation, and promoting the expression of genesencoding proinflammatory cytokines. This study provides a previouslyuncharacterized therapeutic strategy to treat inflammatory disorders byinhibiting the activity of USP14 with IU1.

MATERIALS AND METHODSCells and reagentsMurine lung epithelial (MLE12) cells were obtained from the AmericanType Culture Collection and cultured with HITES (hydrocortisone,insulin, transferrin, estradiol, and selenium)mediumcontaining10% fetal

Fig. 8. Inhibition of USP14 reduces cytokine release in vitro and in vivo. (A) MLE12 cells were transfected with Cont shRNA or usp14 shRNA before they weretreated with LPS (10 mg/ml) for 6 hours. KC release was measured by ELISA. Data are means ± SEM of three independent experiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test. Cell lysates were analyzed by Western blotting with antibodies against USP14 and b-actin. (B) Raw264.7 cells were treated withIU1 (50 mM) for 16 hours before they were treated with LPS (200 ng/ml) for 3 hours. The relative abundance of kcmRNA was analyzed by real-time PCR. Data are means ± SEMof three independent experiments. P values were calculated by two-way ANOVA and post hoc Tukey’s test. (C) C57/BL6 mice were subjected to CLP, which was followed byintraperitoneal injection of IU1 (10 mg/kg body weight). Survival rates were determined for up to 100 hours (n = 13 mice per group), and P values were calculated byMantel-Cox log-rank test. (D to G) C57/BL6 mice were subjected to CLP, which was followed by intraperitoneal (i.p.) injection of IU1 (10 mg/kg body weight). Twenty-fourhours later, the concentrations of plasma IL-6 (D), BAL IL-6 (E), plasma KC (F), and BAL KC (G) were analyzed by ELISA. Data are means ± SEM of five to seven samples pergroup. P values were calculated by two-way ANOVA and post hoc Tukey’s test. (H) C57/BL6micewere subjected to intratracheal injection of LPS (5mg/kg bodyweight), followedby intratracheal injection of IU1 (10mg/kgbodyweight). Twenty-four hours later, the concentration of IL-6 in the BALwasmeasured by ELISA.Data aremeans ± SEMof six to eightsamples per group. P values were calculated by two-way ANOVA and post hoc Tukey’s test. (I) Lung tissues from the experiments depicted in (H) were stained with hematoxylinand eosin. Scale bars, 20 mm. Images are representative of lung tissues from six to eight mice.

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bovine serum (FBS) and antibiotics at 37°C in an incubator containing 5%CO2. RAW264.7 cells were cultured in minimum essential mediumcontaining 10% FBS and antibiotics. Human bronchial epithelial cells(HBEpCs, Lonza) were cultured in bronchial epithelial basal medium(BEBM) with growth factors (Lonza). The anti-V5 antibody (catalog#461157), anti-ubiquitin antibody (catalog #131600), the plasmidpcDNA3.1/His-V5-topo, andEscherichia coliTop10competent cellswereobtained from Life Technologies. Antibodies against H4K8ac (catalog#A4028) and H4K12ac (catalog #A4029) were from EpiGentek. Theanti-FBXL19antibody (catalog #AP18447b)waspurchased fromAbgent.CHX, leupeptin, LPS, the anti–b-actin antibody (catalog #A2228), theanti-HA tag antibody (catalog #H6908), Usp14-specific shRNA, andcontrol shRNA were obtained from Sigma. MG132 and antibodiesagainstH4 (catalog #05-858), p300 (catalog #05-257), andphosphoserine(catalog #05-1000) were fromMillipore. The Fbxl19-specific shRNAwasobtained fromOpenBiosystems. ImmunobilizedproteinA/Gbeads, con-trol IgG, anti-CBP (catalog #sc-369), and anti-USP14 (catalog #sc-393872)antibodies were from Santa Cruz Biotechnology. All materials used inthe experiments were of the highest grades commercially available.

Transfection of cells with plasmids and shRNAsComplementary DNAs (cDNAs) encodingWT andmutantmouse CBPwere inserted into pCDNA3.1/V5-His-Topo vector (Invitrogen). Primersused to amplify cbp cDNA were as follows: forward, CACCATGGCC-GAGAACTTGCTG; reverse, CAAACCCTCCACAAACTTTTCT. Brief-ly, cbp-encoding cDNAwas synthesized by PCR using the mouse cDNAas a template. ThePCRproductwas purified and ligated into pCDNA3.1/V5-His-Topo vector according to the manufacturer’s instructions. Site-directed mutagenesis was performed to generate cbpmutants accordingto the manufacturer’s instructions (Agilent Technologies). MLE12 cellswere transfected with plasmids or shRNAs with the Amaxa Nucleofectorsystem(Lonza). Lipofectamine2000 (LifeTechnologies)wasused to trans-fect HBEpCs with plasmids according to the manufacturer’s instructions.

Immunoprecipitations and Western blotting analysisCells grown on 35-, 60-, or 100-mm dishes were washed with coldphosphate-buffered saline (PBS), collected in cell lysis buffer, and soni-cated. An equal amount of cell lysates (20 mg) was subjected to SDS–polyacrylamide gel electrophoresis, electrotransferred to membranes,and analyzed by Western blotting according to standard protocols.For immunoprecipitations, equal amounts of cell lysates (1 mg) wereincubated with the appropriate antibody overnight at 4°C, which wasfollowed by the addition of 40 ml of protein A/G agarose and incubationfor an additional 2 hours at 4°C. The immunoprecipitated complex waswashed three times with 0.1% Triton X-100 in ice-cold PBS and thenanalyzed by Western blotting.

ImmunostainingMLE12 cells were cultured in glass-bottom dishes and fixed with 3.7%paraformaldehyde for 20 min, which was followed by permeabilizationin 0.1% Triton X-100 for 1min. Cells were exposed to primary antibody,which was followed by incubation with a fluorescently labeled secondaryantibody. Immunofluorescent cell imagingwas performedwith aNikonA1 confocal microscope according to standard protocols.

In vitro translation of cDNAs for Cbp and WT andmutant Fbxl19In vitro transcription and translation were performed with a TnT invitro quick-coupled transcription-translation system (Promega) according


to the manufacturer’s instructions. This mammalian-based systemexpresses soluble, functional proteins that are posttranslationally mod-ified. Translated CBP protein and WT and mutant FBXL19 proteinswere confirmed by Western blotting analysis.

In vitro ubiquitin conjugation assayThe ubiquitylation of CBP was performed in a reaction mixturecontaining synthesized WT or mutant CBP protein in 50 mM tris(pH 7.6), 5 mMMgCl2, 0.6 mMdithiothreitol (DTT), 2 mM adenosinetriphosphate, E1 enzyme (1.5 ng/ml, Boston Biochem), Ubc5 (10 ng/ml),Ubc7 (10 ng/ml), ubiquitin (1 mg/ml, Boston Biochem), 1 mM ubiquitinaldehyde, and His-purified recombinant Cullin 1, Skp1, Rbx1, andsynthesizedFBXL19.The reactionmixturewas then subjected toWesternblotting analysis with anti-ubiquitin antibody.

Assay of DUB activityMLE12 cells transfected with plasmid encoding USP14-HA or the ap-propriate controls were treated with LPS and then subjected to immu-noprecipitation of USP14-HA with an anti-HA antibody. The DUBactivity in the immunoprecipitateswas analyzedwith theDUB-Detectordeubiquitylation assay kit (Active Motif). Briefly, the immunoprecipi-tated complex was incubated for 20 min in the presence or absence of100nMIU1.After the incubation, fluorescentubiquitin substrate (100nM)was added, and the fluorescence intensity of the sample was measuredafter 30 min with an excitation wavelength of 485 nm and an emissionwavelength of 535 nm.

Histone isolationThe EpiQuik total histone extraction kit (EpiGentek) was used to isolatecore histones from the cells or tissue samples indicated in the figurelegends. Cell pellets or lung tissue were lysed or disaggregated in prelysisbuffer containing 400 nM trichostatin A (Cayman Chemical), which wasfollowed by centrifugation of the samples at 10,000g for 1min at 4°C. Thesupernatant was discarded and the pellet was resuspended in lysis buffer(3× volume) and incubated on ice for 30 min. After centrifugation of thesamples at 12,000g for 5 min at 4°C, the supernatant was collected andneutralized by a balance buffer containing DTT (EpiGentek). Aliquotswere taken for the determination of protein concentration and were ana-lyzed by Western blotting to detect acetylated H4K8, H4K12, and H4.

Chromatin immunoprecipitation assaysCells were cross-linked for 5 min with 0.3% formaldehyde, and the re-action was terminated by the addition of glycine. The EZ NucleosomalDNA Prep Kit (Zymo Research) was used to isolate nuclei and preparenucleosomal DNA. Immunoprecipitation was performed with an anti-H4K8ac antibody or with IgG as a control. Chromatin DNA wasextractedwith the Zymo-SpinChIPKit. The Il8 proximal promoter regionwas detected by real-timePCRwith primers specific for a regionwithin theIl8promoter. The sequences of thePCRprimersused for the analysis of thehuman Il8 proximal promoter were as follows: 5′-GTGTGATGACT-CAGGTTTGCCC-3′ and 5′-AGTGCTCCGGTGGCTTTTTATATC-3′.

MiceHuman Fbxl19 cDNA was inserted into the pLVX-IRES-tdTomatovector (Clontech). Lentivirus expressing Fbxl19 and control virus weregenerated andconcentratedwitha lentiviruspackaging system(Clontech).C57/BL6 mice were housed in a specific pathogen–free barrier facil-ity maintained by the University of Pittsburgh Animal ResourcesCenter. C57/BL6 mice were intravenously administered Lenti-control

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or Lenti-Fbxl19 (109 plaque-forming units per mouse) for 7 daysbefore they were subjected to CLP to induce sepsis. Briefly, the cecumwith the adjoining intestine was ligated at 0.5 cm from its end. Theligated cecum was then punctured with an 18-gauge needle, whichenabled entrapped fecal material to leak into the normally sterileperitoneal cavity. The cecum was then repositioned in the peritonealcavity, and the abdomen was closed. Sham-operated animals receivedlaparotomy only. After the times indicated in the figure legends, plasma,BALF, and lung tissues were collected. Core histones in the lung tissuewere isolated and analyzed by Western blotting to detect H4 andH4K8ac.

Cytokine measurementCell culture medium, BALF, or mouse plasma was centrifuged at 500gfor 10 min to remove cell debris. The amounts of KC, IL-6, and IL-8 inthe samples were then measured with specific ELISA kits according tothe manufacturer’s instructions (Invitrogen).

Statistical analysisStatistical analysis was performed by one-way or two-way ANOVAwith Tukey’s test for the post hoc test or by unpaired Student’s t testto compare continuousmeasurements orwith theMantel-Cox log-ranktest to compare survival curves. P < 0.05 was considered to be statisti-cally significant.

on Fttp://stke.sciencem

ag.org/

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/483/eaak9660/DC1Fig. S1. FBXL19 induces CBP degradation.Fig. S2. FBXL19 mediates CBP degradation in the nucleus.Fig. S3. FBXL19 induces the K48-linked polyubiquitylation of CBP.Fig. S4. LPS stimulates the phosphorylation of CBP and FBXL19.Fig. S5. FBXL19 is not auto-ubiquitylated.Fig. S6. The reduction in CBP stability attenuates the LPS-induced acetylation of histoneH4on Lys12.Fig. S7. FBXL19 attenuates histone H4K8ac–mediated Il8 promoter activity.Fig. S8. IU1 attenuates LPS-stimulated cytokine release.

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Acknowledgments: We thank J. Franz for English editing of the manuscript. We thankJ. G. Yabes for help with the statistical analysis. Funding: This work was supported by theNIH (R01GM115389 to J.Z. and R01HL112791 and R01HL131665 to Y.Z.), the American HeartAssociation (16GRNT30660001 to Y.Z.), and American Lung Association BiomedicalResearch Grant RG350146 (to J.Z.). Author contributions: J.Z. and Y.Z. jointly designed andperformed experiments, analyzed the data, and wrote the manuscript; J.W., R.K.B., S.D., A.K.,A.M.J., and L.Z. performed experiments and analyzed the data; and J.Z. and Y.Z. oversawand directed the study. Competing interests: The authors declare that they have nocompeting interests.

Submitted 28 October 2015Resubmitted 21 September 2016Accepted 30 May 2017Published 13 June 201710.1126/scisignal.aak9660

Citation: J. Wei, S. Dong, R. K. Bowser, A. Khoo, L. Zhang, A. M. Jacko, Y. Zhao, J. Zhao,Regulation of the ubiquitylation and deubiquitylation of CREB-binding protein modulateshistone acetylation and lung inflammation. Sci. Signal. 10, eaak9660 (2017).

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histone acetylation and lung inflammationRegulation of the ubiquitylation and deubiquitylation of CREB-binding protein modulates

Jianxin Wei, Su Dong, Rachel K. Bowser, Andrew Khoo, Lina Zhang, Anastasia M. Jacko, Yutong Zhao and Jing Zhao

DOI: 10.1126/scisignal.aak9660 (483), eaak9660.10Sci. Signal.

manipulation of CBP stability may provide a means to control inflammatory responses to infections.Pharmacological inhibition of USP14 led to reduced CBP abundance and decreased cytokine production, suggesting thatthe FBXL19-CBP interaction and enhanced USP14 activity, thus promoting inflammatory cytokine production. USP14 reversed this process, enhancing CBP stability. Furthermore, signaling by the bacterial product LPS preventedFBXL19 ubiquitylated CBP, targeting it for proteasomal degradation. The authors also showed that the deubiquitylase

showed that the E3 ubiquitin ligase subunitet al.stability could help in the treatment of inflammatory diseases. Wei which enables the expression of genes encoding proinflammatory cytokines. Understanding the regulation of CBP

Acetylation of histone H4 by the histone acetyltransferase CBP during sepsis switches chromatin to an open state,Targeting CBP stability

ARTICLE TOOLS http://stke.sciencemag.org/content/10/483/eaak9660

MATERIALSSUPPLEMENTARY http://stke.sciencemag.org/content/suppl/2017/06/12/10.483.eaak9660.DC1

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http://stke.sciencemag.org/content/sigtrans/11/536/eaao2387.fullhttp://science.sciencemag.org/content/sci/351/6268/aac6633.fullhttp://stke.sciencemag.org/content/sigtrans/10/498/eaaq0150.fullhttp://stm.sciencemag.org/content/scitransmed/7/295/295ra109.fullhttp://stke.sciencemag.org/content/sigtrans/9/443/ra86.fullhttp://stke.sciencemag.org/content/sigtrans/8/391/ra85.fullhttp://stke.sciencemag.org/content/sigtrans/9/448/ec231.abstract

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