IL-9-triggered lncRNA Gm13568 regulates Notch1 in astrocytes through interaction with CBP/P300: contribute to the pathogenesis of experimental autoimmune encephalomyelitis.

BACKGROUND: Interleukin 9 (IL-9), produced mainly by T helper 9 (Th9) cells, has been recognized as an important regulator in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Astrocytes respond to IL-9 and reactive astrocytes always associate with blood-brain barrier damage, immune cell infiltration, and spinal injury in MS and EAE. Several long non-coding RNAs (lncRNAs) with aberrant expression have been identified in the pathogenesis of MS. Here, we examined the effects of lncRNA Gm13568 (a co-upregulated lncRNA both in EAE mice and in mouse primary astrocytes activated by IL-9) on the activation of astrocytes and the process of EAE. METHODS: In vitro, shRNA-recombinant lentivirus with glial fibrillary acidic protein (GFAP) promoter were performed to determine the relative gene expression and proinflammatory cytokines production in IL-9 treated-astrocytes using Western blot, real-time PCR, and Cytometric Bead Array, respectively. RIP and ChIP assays were analyzed for the mechanism of lncRNA Gm13568 regulating gene expression. Immunofluorescence assays was performed to measure the protein expression in astrocytes. In vivo, H&E staining and LFB staining were applied to detect the inflammatory cells infiltrations and the medullary sheath damage in spinal cords of EAE mice infected by the recombinant lentivirus. Results were analyzed by one-way ANOVA or Student's t test, as appropriate. RESULTS: Knockdown of the endogenous lncRNA Gm13568 remarkably inhibits the Notch1 expression, astrocytosis, and the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) as well as the production of inflammatory cytokines and chemokines (IL-6, TNF-α, IP-10) in IL-9-activated astrocytes, in which Gm13568 associates with the transcriptional co-activators CBP/P300 which are enriched in the promoter of Notch1 genes. More importantly, inhibiting Gm13568 with lentiviral vector in astrocytes ameliorates significantly inflammation and demyelination in EAE mice, therefore delaying the EAE process. CONCLUSIONS: These findings uncover that Gm13568 regulates the production of inflammatory cytokines in active astrocytes and affects the pathogenesis of EAE through the Notch1/STAT3 pathway. LncRNA Gm13568 may be a promising target for treating MS and demyelinating diseases.

TAF5L functions as transcriptional coactivator of MITF involved in the immune response of the clam Meretrix petechialis.

TAF5L is a component of the P300/CBP-associated factor (PCAF) histone acetylase complex, which serves as a coactivator and takes part in basal transcription such as promoter recognition, complex assembly and transcription initiation. In our study, the full-length sequence of MpTAF5L was identified and characterized in the clam M. petechialis. Sequence analysis showed that the predicted MpTAF5L protein had a N-terminal TAF5-NTD2 domain and a C-terminal WD40-repeats domain. The annotation and evolutionary analysis revealed MpTAF5L had close evolutionary relationship with other invertebrate species. Tissue distribution analysis of TAF5L claimed that it was highly expressed in the mantle, adductor muscle, foot and hepatopancreas. The mRNA expression of MpTAF5L was significantly up-regulated after Vibrio parahaemolyticus challenge, indicating its involvement in the immune response of clam. Yeast two-hybrid assays verified that MpTAF5L can interact with MpMITF (a critical immune-related transcription factor), and our further research clarified this interaction depended upon the N-terminal TAF5-NTD2 domain of MpTAF5L. Moreover, the mRNA expression of MpBcl-2 (a target gene of MITF) was significantly decreased but the mRNA expression of MpMITF was not significantly changed after knockdown of MpTAF5L, which indicated the reduction of MpMITF regulating activity at the same time. These results revealed that MpTAF5L interacted with MpMITF and enhanced the activation of MpMITF, which plays roles in the immune defense against V. parahaemolyticus.

Downregulation of the transcriptional co-activator PCAF inhibits the proliferation and migration of vascular smooth muscle cells and attenuates NF-κB-mediated inflammatory responses.

P300/CBP-associated factor (PCAF) regulates vascular inflammation. This study was to explore the effect of PCAF on the proliferation and migrationof vascular smooth muscle cells (VSMCs) and neointimal hyperplasia in balloon-injured rat carotid artery. Downregulation of PCAF remarkably suppressed VSMCs proliferation and migration induced by lipopolysaccharide, and also significantly inhibit the nuclear translocation of nuclear factor-kappaB p65. Meanwhile, downregulation of PCAF inhibited the mRNA expression of tumor necrosis factor-α and interleukin-6, and also the levels in culture supernatants. Moreover, downregulation of PCAF profoundly reduced the intima area and the ratio of intima area to media area in balloon-injured rat carotid artery. In addition, the expression of PCNA and NF-κB p65 in intima were decreased by downregulation of PCAF. These results highlight that PCAF may be a potential target for prevention and treatment of neointimal hyperplasia and restenosis after angioplasty.

Inhibition of p300/CBP-Associated Factor Attenuates Renal Tubulointerstitial Fibrosis through Modulation of NF-kB and Nrf2.

p300/CBP-associated factor (PCAF), a histone acetyltransferase, is involved in many cellular processes such as differentiation, proliferation, apoptosis, and reaction to cell damage by modulating the activities of several genes and proteins through the acetylation of either the histones or transcription factors. Here, we examined a pathogenic role of PCAF and its potential as a novel therapeutic target in the progression of renal tubulointerstitial fibrosis induced by non-diabetic unilateral ureteral obstruction (UUO) in male C57BL/6 mice. Administration of garcinol, a PCAF inhibitor, reversed a UUO-induced increase in the renal expression of total PCAF and histone 3 lysine 9 acetylation and reduced positive areas of trichrome and α-smooth muscle actin and collagen content. Treatment with garcinol also decreased mRNA levels of transforming growth factor-ß, matrix metalloproteinase (MMP)-2, MMP-9, and fibronectin. Furthermore, garcinol suppressed nuclear factor-κB (NF-κB) and pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6, whereas it preserved the nuclear expression of nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and levels of Nrf2-dependent antioxidants including heme oxygense-1, catalase, superoxide dismutase 1, and NAD(P)H:quinone oxidoreductase 1. These results suggest that the inhibition of inordinately enhanced PCAF could mitigate renal fibrosis by redressing aberrant balance between inflammatory signaling and antioxidant response through the modulation of NF-κB and Nrf2.

[Influence of TNF-α on the immunomodulatory property of laryngeal mucosa mesenchymal stromal cells'].

Objective: To investigate the effect of tumor necrosis factor-alpha(TNF-α)on the immunoregulatory capacity of laryngeal mucosal mesenchymal stromal cells (LM-MSCs) and its potential molecular mechanism, and provide a theoretical basis for the study of chronic laryngitis. Methods: LM-MSCs were separated from epiglottal mucosa. The LM-MSCs cells were directly co-cultured with T cells in vitro to detect the immunomodulatory property of LM-MSCs. After long-term stimulation with inflammatory factors TNF-α in vitro, the differences were compared in the immunomodulatory ability of LM-MSCs between normal LM-MSCs and TNF-α stimulated LM-MSCs. The expression of general control non-repressed protein5(GCN5), FAS, FASL in normal LM-MSCs and TNF-α stimulated LM-MSCs was detected by Western blot and quantitative real-time RT-PCR(RT-qPCR). Results: After chronic stimulation of TNF-α, the RNA relative expression of GCN5 was 0.31±0.03 (3 days) and 0.53±0.06 (7 days) compared with control group, showing significant difference (F=13.45, P<0.05). The percentage of LM-MSC-induced T cell apoptosis was 6.27%±0.81% (3 days) and 4.99%±0.52% (7 days) in chronic stimulation group compared with control group 10.02%±1.02%. There is a significant difference among these groups (F=11.13, P<0.05). Moreover, the ability of LM-MSCs to induce T cell apoptosis is regulated by GCN5. Conclusion: With the chronic stimulation of TNF-α, the expression of GCN5 in LM-MSCs is decreased, thus impairing its immunoregulatory capacity.

Grainyhead-like-2 confers NK-sensitivity through interactions with epigenetic modifiers.

Natural Killer (NK) cells suppress tumor initiation and metastasis. Most carcinomas are heterogeneous mixtures of epithelial, mesenchymal and hybrid tumor cells, but the relationships of these phenotypes to NK susceptibility are understood incompletely. Grainyhead-like-2 (GRHL2) is a master programmer of the epithelial phenotype, that is obligatorily down-regulated during experimentally induced Epithelial-Mesenchymal Transition (EMT). Here, we utilize GRHL2 re-expression to discover unifying molecular mechanisms that link the epithelial phenotype with NK-sensitivity. GRHL2 enhanced the expression of ICAM-1, augmenting NK-target cell synaptogenesis and NK killing of target cells. The expression of multiple interferon response genes, including ICAM1, anti-correlated with EMT. We identified two novel GRHL2-interacting proteins, the histone methyltransferases KMT2C and KMT2D. Mesenchymal-epithelial transition, NK-sensitization and ICAM-1 expression were promoted by GRHL2-KMT2C/D interactions and by GRHL2 inhibition of p300, revealing novel and potentially targetable epigenetic mechanisms connecting the epithelial phenotype with target cell susceptibility to NK killing.

Sumoylation of RORγt regulates TH17 differentiation and thymocyte development.

RORγt controls the differentiation of TH17 cells, which are mediators of autoimmune conditions such as experimental autoimmune encephalomyelitis (EAE). RORγt also regulates thymocyte development and lymph node genesis. Here we show that the function of RORγt is regulated by its sumoylation. Loss of Sumo3, but not Sumo1, dampens TH17 differentiation and delays the progression of thymic CD8+ immature single-positive cells (ISPs). RORγt is SUMO3-modified by E3 ligase PIAS4 at lysine 31 (K31), and the mutation of K31 to arginine in mice prevents RORγt sumoylation, leading to impaired TH17 differentiation, resistance to TH17-mediated EAE, accumulation of thymic ISPs, and a lack of Peyer's patches. Mechanistically, sumoylation of RORγt-K31 recruits histone acetyltransferase KAT2A, which stabilizes the binding of SRC1 to enhance RORγt transcription factor activity. This study thus demonstrates that sumoylation is a critical mechanism for regulating RORγt function, and reveals new drug targets for preventing TH17-mediated autoimmunity.

Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.

A key event required for effective resolution of inflammation is efferocytosis, which is defined as phagocytic removal of apoptotic cells mostly by macrophages acquiring an alternatively activated phenotype (M2). c-Myc has been reported to play a role in alternative activation of human macrophages and is proposed as one of the M2 macrophage markers. We found that M2-like peritoneal macrophages from zymosan A-treated mice exhibited a marked accumulation of Myc-nick, a truncated protein generated by a Calpain-mediated proteolytic cleavage of full-length c-Myc. Further, ectopic expression of Myc-nick in murine bone marrow-derived macrophages promoted the M2 polarization and, consequently, enhanced their efferocytic capability. Notably, Myc-nick-induced efferocytosis was found to be tightly associated with α-tubulin acetylation by K acetyltransferase 2a (Kat2a/Gcn5) activity. These findings suggest Myc-nick as a novel proresolving mediator that has a fundamental function in maintaining homeostasis under inflammatory conditions.-Zhong, X., Lee, H.-N., Kim, S. H., Park, S.-A., Kim, W., Cha, Y.-N., Surh, Y.-J. Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.

Bioenergetic state regulates innate inflammatory responses through the transcriptional co-repressor CtBP.

The innate inflammatory response contributes to secondary injury in brain trauma and other disorders. Metabolic factors such as caloric restriction, ketogenic diet, and hyperglycemia influence the inflammatory response, but how this occurs is unclear. Here, we show that glucose metabolism regulates pro-inflammatory NF-κB transcriptional activity through effects on the cytosolic NADH:NAD+ ratio and the NAD(H) sensitive transcriptional co-repressor CtBP. Reduced glucose availability reduces the NADH:NAD+ ratio, NF-κB transcriptional activity, and pro-inflammatory gene expression in macrophages and microglia. These effects are inhibited by forced elevation of NADH, reduced expression of CtBP, or transfection with an NAD(H) insensitive CtBP, and are replicated by a synthetic peptide that inhibits CtBP dimerization. Changes in the NADH:NAD+ ratio regulate CtBP binding to the acetyltransferase p300, and regulate binding of p300 and the transcription factor NF-κB to pro-inflammatory gene promoters. These findings identify a mechanism by which alterations in cellular glucose metabolism can influence cellular inflammatory responses.Several metabolic factors affect cellular glucose metabolism as well as the innate inflammatory response. Here, the authors show that glucose metabolism regulates pro-inflammatory responses through effects on the cytosolic NADH:NAD+ ratio and the NAD(H)-sensitive transcription co-repressor CtBP.

The Lysine Acetyltransferase GCN5 Is Required for iNKT Cell Development through EGR2 Acetylation.

The development of CD1d-restricted invariant natural killer T (iNKT) cells, a population that is critical for both innate and adaptive immunity, is regulated by multiple transcription factors, but the molecular mechanisms underlying how the transcriptional activation of these factors are regulated during iNKT development remain largely unknown. We found that the histone acetyltransferase general control non-derepressible 5 (GCN5) is essential for iNKT cell development during the maturation stage. GCN5 deficiency blocked iNKT cell development in a cell-intrinsic manner. At the molecular level, GCN5 is a specific lysine acetyltransferase of early growth responsive gene 2 (EGR2), a transcription factor required for iNKT cell development. GCN5-mediated acetylation positively regulated EGR2 transcriptional activity, and both genetic and pharmacological GCN5 suppression specifically inhibited the transcription of EGR2 target genes in iNKT cells, including Runx1, promyelocytic leukemia zinc finger protein (PLZF), interleukin (IL)-2Rb, and T-bet. Therefore, our study revealed GCN5-mediated EGR2 acetylation as a molecular mechanism that regulates iNKT development.

LPS depletes PHLPP levels in macrophages through the inhibition of SP1 dependent transcriptional regulation.

We have previously reported that bacterial endotoxin LPS attenuates expression of PHLPP, a ser/thr phosphatase, at both transcript and protein levels in different immune cells, however the underlying molecular mechanism is unknown and is of significant interest. Here, in line with the decreased transcript levels upon LPS treatment, we observed that LPS caused significant reduction in PHLPP promoter activity. We observed that SP1, a transcription factor frequently associated with inflammation, was recruited to the PHLPP promoter region. Ectopic expression of SP1 enhanced both transcript and protein levels of PHLPP while knockdown of SP1 or pharmacological inhibition of SP1 DNA binding by mithramycin reduced PHLPP expression. Moreover, over-expression of SP1 co-activators CBP/p300 augmented SP1 driven PHLPP promoter activity. Of note, LPS treatment depleted SP1 and CBP protein levels due to which recruitment of SP1 to PHLPP promoter was reduced. Further, we found that re-introduction of SP1 restored promoter activity and transcript levels of PHLPP in LPS stimulated cells. Collectively, our data revealed the molecular mechanism underlying the regulation of PHLPP expression during LPS induced macrophage inflammatory response.

Ubiquitin-mediated NFκB degradation pathway.

The nuclear factor κB (NFκB) transcription factor plays critical roles in inflammation and immunity. The dysregulation of NFκB is associated with inflammatory and autoimmune diseases and cancer. NFκB activation is negatively regulated by the ubiquitin-dependent proteasomal degradation pathway. In the present review, we discuss recent advances in our understanding of how ubiquitin ligases regulate the NFκB degradation pathway.

Coordinated histone H3 methylation and acetylation regulate physiologic and pathologic fas ligand gene expression in human CD4+ T cells.

Activation-induced Fas ligand (FasL) mRNA expression in CD4+ T cells is mainly controlled at transcriptional initiation. To elucidate the epigenetic mechanisms regulating physiologic and pathologic FasL transcription, TCR stimulation-responsive promoter histone modifications in normal and alcohol-exposed primary human CD4+ T cells were examined. TCR stimulation of normal and alcohol-exposed cells led to discernible changes in promoter histone H3 lysine trimethylation, as documented by an increase in the levels of transcriptionally permissive histone 3 lysine 4 trimethylation and a concomitant decrease in the repressive histone 3 lysine 9 trimethylation. Moreover, acetylation of histone 3 lysine 9 (H3K9), a critical feature of the active promoter state that is opposed by histone 3 lysine 9 trimethylation, was significantly increased and was essentially mediated by the p300-histone acetyltransferase. Notably, the degree of these coordinated histone modifications and subsequent recruitment of transcription factors and RNA polymerase II were significantly enhanced in alcohol-exposed CD4+ T cells and were commensurate with the pathologic increase in the levels of FasL mRNA. The clinical relevance of these findings is further supported by CD4+ T cells obtained from individuals with a history of heavy alcohol consumption, which demonstrate significantly greater p300-dependent H3K9 acetylation and FasL expression. Overall, these data show that, in human CD4+ T cells, TCR stimulation induces a distinct promoter histone profile involving a coordinated cross-talk between histone 3 lysine 4 and H3K9 methylation and acetylation that dictates the transcriptional activation of FasL under physiologic, as well as pathologic, conditions of alcohol exposure.

Nrf2 expression and activity in human T lymphocytes: stimulation by T cell receptor activation and priming by inorganic arsenic and tert-butylhydroquinone.

The transcription factor nuclear factor-erythroid 2-related-2 (Nrf2) controls cellular redox homeostasis and displays immunomodulatory properties. Nrf2 alters cytokine expression in murine T cells, but its effects in human T lymphocytes are unknown. This study investigated the expression and activity of Nrf2 in human activated CD4(+) T helper lymphocytes (Th cells) that mediate the adaptive immune response. Th cells were isolated from peripheral blood mononuclear cells and activated with antibodies against CD3 and CD28, mimicking physiologic Th cell stimulation by dendritic cells. Nrf2 is hardly detectable in unstimulated Th cells. Activation of Th cells rapidly and strongly increases the levels of Nrf2 protein by increasing NRF2 gene transcription. Th cell activation also enhances mRNA and protein levels of Nrf2 target genes encoding antioxidant enzymes. Blocking Nrf2 expression using chemical inhibitors or siRNAs prevents these gene inductions. Pretreatment with inorganic arsenic, a Nrf2 inducer that does not alter NRF2 gene expression, increases protein level and transcriptional activity of Nrf2 induced by Th cell stimulation. Inorganic arsenic enhances nuclear translocation of Nrf2, its interaction with the coactivator protein p300, and its DNA binding activity. Inhibition of Nrf2 expression abrogates the effects of inorganic arsenic on mRNA levels of antioxidant genes, but does not alter the expression of IL-2, TNF-α, interferon-γ, or IL-17 in Th cells activated in the absence or presence of the metalloid. In conclusion, this study demonstrates for the first time that stimulation of human Th cells increases transcription of the NRF2 gene and activity of the Nrf2 protein. However, modulation of Nrf2 levels does not modify the secretion of inflammatory cytokines from these T lymphocytes.

Set7/9 impacts COL2A1 expression through binding and repression of SirT1 histone deacetylation.

Type II collagen is a key cartilaginous extracellular protein required for normal endochondral development and cartilage homeostasis. COL2A1 gene expression is positively regulated by the NAD-dependent protein deacetylase Sirtuin 1 (SirT1), through its ability to bind chromatin regions of the COL2A1 promoter and enhancer. Although SirT1/Sox9 binding on the enhancer site of COL2A1 was previously demonstrated, little is known about its functional role on the gene promoter site. Here, we examined the mechanism by which promoter-associated SirT1 governs COL2A1 expression. Human chondrocytes were encapsulated in three-dimensional (3D) alginate beads where they exhibited upregulated COL2A1 mRNA expression and increased levels of SirT1 occupancy on the promoter and enhancer regions, when compared to monolayer controls. Chromatin immunoprecipitation (ChIP) analyses of 3D cultures showed augmented levels of the DNA-binding transcription factor SP1, and the histone methyltransferase Set7/9, on the COL2A1 promoter site. ChIP reChIP assays revealed that SirT1 and Set7/9 form a protein complex on the COL2A1 promoter region of 3D-cultured chondrocytes, which also demonstrated elevated trimethylated lysine 4 on histone 3 (3MeH3K4), a hallmark of Set7/9 methyltransferase activity. Advanced passaging of chondrocytes yielded a decrease in 3MeH3K4 and Set7/9 levels on the COL2A1 promoter and reduced COL2A1 expression, suggesting that the SirT1/Set7/9 complex is preferentially formed on the COL2A1 promoter and required for gene activation. Interestingly, despite SirT1 occupancy, its deacetylation targets (ie, H3K9/14 and H4K16) were found acetylated on the COL2A1 promoter of 3D-cultured chondrocytes. A possible explanation for this phenotype is the enrichment of the histone acetyltransferases P300 and GCN5 on the COL2A1 promoter of3 D-cultured chondrocytes. Our study indicates that Set7/9 prevents the histone deacetylase activity of SirT1, potentiating euchromatin formation on the promoter site of COL2A1 and resulting in morphology-dependent COL2A1 gene transactivation.

Lysine acetyltransferase PCAF is a key regulator of arteriogenesis.

OBJECTIVE: Therapeutic arteriogenesis, that is, expansive remodeling of preexisting collaterals, using single-action factor therapies has not been as successful as anticipated. Modulation of factors that act as a master switch for relevant gene programs may prove more effective. Transcriptional coactivator p300-CBP-associated factor (PCAF) has histone acetylating activity and promotes transcription of multiple inflammatory genes. Because arteriogenesis is an inflammation-driven process, we hypothesized that PCAF acts as multifactorial regulator of arteriogenesis. APPROACH AND RESULTS: After induction of hindlimb ischemia, blood flow recovery was impaired in both PCAF(-/-) mice and healthy wild-type mice treated with the pharmacological PCAF inhibitor Garcinol, demonstrating an important role for PCAF in arteriogenesis. PCAF deficiency reduced the in vitro inflammatory response in leukocytes and vascular cells involved in arteriogenesis. In vivo gene expression profiling revealed that PCAF deficiency results in differential expression of 3505 genes during arteriogenesis and, more specifically, in impaired induction of multiple proinflammatory genes. Additionally, recruitment from the bone marrow of inflammatory cells, in particular proinflammatory Ly6C(hi) monocytes, was severely impaired in PCAF(-/-) mice. CONCLUSIONS: These findings indicate that PCAF acts as master switch in the inflammatory processes required for effective arteriogenesis.

Polycomb antagonizes p300/CREB-binding protein-associated factor to silence FOXP3 in a Kruppel-like factor-dependent manner.

Inducible gene expression underlies the epigenetically inherited differentiation program of most immune cells. We report that the promoter of the FOXP3 gene possesses two distinct functional states: an "off state" mediated by the polycomb histone methyltransferase complex and a histone acetyltransferase-dependent "on state." Regulating these states is the presence of a Kruppel-like factor (KLF)-containing Polycomb response element. In the KLF10(-/-) mouse, the FOXP3 promoter is epigenetically silenced by EZH2 (Enhancer of Zeste 2)-mediated trimethylation of Histone 3 K27; thus, impaired FOXP3 induction and inappropriate adaptive T regulatory cell differentiation results in vitro and in vivo. The epigenetic transmittance of adaptive T regulatory cell deficiency is demonstrated throughout more than 40 generations of mice. These results provide insight into chromatin remodeling events key to phenotypic features of distinct T cell populations.

Endothelial cell activation by antiphospholipid antibodies is modulated by Kruppel-like transcription factors.

Antiphospholipid syndrome is characterized by thrombosis and/or recurrent pregnancy loss in the presence of antiphospholipid antibodies (APLAs). The majority of APLAs are directed against phospholipid-binding proteins, particularly ß2-glycoprotein I (ß2GPI). Anti-ß2GPI antibodies activate endothelial cells in a ß2GPI-dependent manner through a pathway that involves NF-κB. Krüppel-like factors (KLFs) play a critical role in regulating the endothelial response to inflammatory stimuli. We hypothesized that activation of endothelial cells by APLA/anti-ß2GPI antibodies might be associated with decreased expression of KLFs, which in turn might facilitate cellular activation mediated through NF-κB. Our experimental results confirmed this hypothesis, demonstrating markedly decreased expression of KLF2 and KLF4 after incubation of cells with APLA/anti-ß2GPI antibodies. Restoration of KLF2 or KLF4 levels inhibited NF-κB transcriptional activity and blocked APLA/anti-ß2GPI-mediated endothelial activation despite NF-κB p65 phosphorylation. Chromatin immunoprecipitation analysis demonstrated that inhibition of NF-κB transcriptional activity by KLFs reflects sequestration of the cotranscriptional activator CBP/p300, making this cofactor unavailable to NF-κB. These findings suggest that the endothelial response to APLA/anti-ß2GPI antibodies reflects competition between KLFs and NF-κB for their common cofactor, CBP/p300. Taken together, these observations are the first to implicate the KLFs as novel participants in the endothelial proinflammatory response to APLA/anti-ß2GPI antibodies.

p300-mediated acetylation stabilizes the Th-inducing POK factor.

The lineage-specifying factor Th-inducing POK (ThPOK) directs the intrathymic differentiation of CD4 T cells. Although the regulation of ThPOK at the transcription level has been extensively studied, specific posttranslational modifications regulating the activity of ThPOK have not been addressed. In this paper, we show that ThPOK is an unstable protein that is more readily degraded in CD8 T cells compared with CD4 T cells. Among the various proteins that bind ThPOK, acetyltransferase p300 specifically promotes the acetylation of ThPOK at K210, K216, and K339, outcompeting ubiquitination, thereby stabilizing the protein. In CD4 T cells, attenuation of p300-mediated acetylation promotes the degradation of ThPOK. In contrast, mutation of lysines 210, 216, and 339 to arginines stabilizes ThPOK and enhances its ability to suppress the expression of CD8 molecule and cytotoxic effectors in CD8 T cells. Our results reveal an essential role of p300-mediated acetylation in regulating the stability of ThPOK and suggest that such regulation may play a part in CD4/CD8 lineage differentiation.