Recruitment Of P300 Research Articles

Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy.

Triple negative breast cancer (TNBC) cells express increased levels of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which promotes their proliferation and migration. Because TNBC patients are unresponsive to current targeted therapies, new therapeutic strategies are urgently needed. While proteasome inhibition by bortezomib (BZ) or carfilzomib (CZ) has been effective in treating hematological malignancies, it has been less effective in solid tumors, including TNBC, but the mechanisms are incompletely understood. Here we report that proteasome inhibition significantly increases expression of IL-8, and its receptors CXCR1 and CXCR2, in TNBC cells. Suppression or neutralization of the BZ-induced IL-8 potentiates the BZ cytotoxic and anti-proliferative effect in TNBC cells. The IL-8 expression induced by proteasome inhibition in TNBC cells is mediated by IκB kinase (IKK), increased nuclear accumulation of p65 NFκB, and by IKK-dependent p65 recruitment to IL-8 promoter. Importantly, inhibition of IKK activity significantly decreases proliferation, migration, and invasion of BZ-treated TNBC cells. These data provide the first evidence demonstrating that proteasome inhibition increases the IL-8 signaling in TNBC cells, and suggesting that IKK inhibitors may increase effectiveness of proteasome inhibitors in treating TNBC.

Inhibitor of CBP Histone Acetyltransferase Downregulates p53 Activation and Facilitates Methylation at Lysine 27 on Histone H3.

Tumor suppressor p53-directed apoptosis triggers loss of normal cells, which contributes to the side-effects from anticancer therapies. Thus, small molecules with potential to downregulate the activation of p53 could minimize pathology emerging from anticancer therapies. Acetylation of p53 by the histone acetyltransferase (HAT) domain is the hallmark of coactivator CREB-binding protein (CBP) epigenetic function. During genotoxic stress, CBP HAT-mediated acetylation is essential for the activation of p53 to transcriptionally govern target genes, which control cellular responses. Here, we present a small molecule, NiCur, which blocks CBP HAT activity and downregulates p53 activation upon genotoxic stress. Computational modeling reveals that NiCur docks into the active site of CBP HAT. On CDKN1A promoter, the recruitment of p53 as well as RNA Polymerase II and levels of acetylation on histone H3 were diminished by NiCur. Specifically, NiCur reduces the levels of acetylation at lysine 27 on histone H3, which concomitantly increases the levels of trimethylation at lysine 27. Finally, NiCur attenuates p53-directed apoptosis by inhibiting the Caspase 3 activity and cleavage of Poly (ADP-ribose) polymerase (PARP) in normal gastrointestinal epithelial cells. Collectively, NiCur demonstrates the potential to reprogram the chromatin landscape and modulate biological outcomes of CBP-mediated acetylation under normal and disease conditions.

Heme ameliorates dextran sodium sulfate-induced colitis through providing intestinal macrophages with noninflammatory profiles

The local environment is crucial for shaping the identities of tissue-resident macrophages (Mϕs). When hemorrhage occurs in damaged tissues, hemoglobin induces differentiation of anti-inflammatory Mϕs with reparative function. Mucosal bleeding is one of the pathological features of inflammatory bowel diseases. However, the heme-mediated mechanism modulating activation of intestinal innate immune cells remains poorly understood. Here, we show that heme regulates gut homeostasis through induction of Spi-C in intestinal CX3CR1high Mϕs. Intestinal CX3CR1high Mϕs highly expressed Spi-C in a heme-dependent manner, and myeloid lineage-specific Spic-deficient (Lyz2-cre; Spicflox/flox ) mice showed severe intestinal inflammation with an increased number of Th17 cells during dextran sodium sulfate-induced colitis. Spi-C down-regulated the expression of a subset of Toll-like receptor (TLR)-inducible genes in intestinal CX3CR1high Mϕs to prevent colitis. LPS-induced production of IL-6 and IL-1α, but not IL-10 and TNF-α, by large intestinal Mϕs from Lyz2-cre; Spicflox/flox mice was markedly enhanced. The interaction of Spi-C with IRF5 was linked to disruption of the IRF5-NF-κB p65 complex formation, thereby abrogating recruitment of IRF5 and NF-κB p65 to the Il6 and Il1a promoters. Collectively, these results demonstrate that heme-mediated Spi-C is a key molecule for the noninflammatory signature of intestinal Mϕs by suppressing the induction of a subset of TLR-inducible genes through binding to IRF5.

Anti-inflammatory properties of cilostazol: Its interruption of DNA binding activity of NF-κB from the Toll-like receptor signaling pathways

Cilostazol, a selective inhibitor of phosphodiesterase type III with anti-platelet, anti-mitogenic, and vasodilating properties, is widely used to treat ischemic symptoms of peripheral vascular disease. Ample evidence has suggested that cilostazol also exhibits an anti-inflammatory effect, but its anti-inflammatory mechanism is not fully understood. Here, we showed that cilostazol specifically inhibited expression of cytokines, which are induced by nuclear factor-κB (NF-κB) activation, in RAW264.7 macrophage cells stimulated with different Toll-like receptor (TLR) ligands. Cilostazol was found to significantly reduce TLR-4 and TLR-3 ligands-stimulated NF-κB transcriptional activity, which was quantified by luciferase reporter assays. However, cilostazol was without effect on IκBα degradation and NF-κB p65 phosphorylation and nuclear translocation after challenge with the TLR-4 ligand lipopolysaccharide (LPS). Cilostazol did not also prevent the LPS-induced increase in phosphorylated levels of the mitogen-activated protein kinase (MAPK) family. On the other hand, using chromatin immunoprecipitation assays, we demonstrated that cilostazol reduced the LPS-induced transcriptional activities of interleukin-6 and tumor necrosis factor-α by preventing the recruitment of NF-κB p65 to these gene promoters. When cilostazol was given to mice by oral gavage daily for 7 days, LPS-induced aberrant pro-inflammatory cytokine production and end-organ tissue injury were significantly reduced. The results of this study suggest that cilostazol is capable of directly interrupting DNA binding activity of NF-κB proteins from the TLR signaling pathways. The therapy to specifically intervene in this pathway may be potentially beneficial for the prevention of different inflammatory disorders.

Abstract 1519: TonEBP promotes hepatocellular carcinogenesis, recurrence, and metastasis

Abstract Introduction: Hepatocellular carcinoma (HCC) is a common cancer with high rate of recurrence and mortality. Diverse etiological agents and wide heterogeneity in individual tumors impede effective and personalized treatment. Tonicity-responsive enhancer-binding protein (TonEBP) is a transcriptional cofactor for the expression of pro-inflammatory genes. Although inflammation is intimately associated with the pathogenesis of HCC, the role of TonEBP is unknown. We aimed to identify function of TonEBP in HCC. Methods: Tumors with surrounding hepatic tissues were obtained from 296 patients with HCC who received completion resection. TonEBP expression was analyzed by qRT-PCR and immunohistochemical analyses of tissue microarrays. Mice with TonEBP haplo-deficiency, and hepatocyte- and myeloid-specific TonEBP deletion were used along with HCC and hepatocyte cell lines. Results: TonEBP expression is higher in tumors than in adjacent non-tumor tissues in 92.6% of 296 patients with HCC regardless of etiology associated and DEN-induced mouse HCC. Hepatic induction of TonEBP is mediated by a fall in the miR-223 abundance. The TonEBP expression in tumors and adjacent non-tumor tissues predicts recurrence, metastasis, and death in multivariate analyses. Univariate analysis of two layers of patients showed that higher TonEBP expression was significantly associated with bigger tumor, advanced tumor grade, and vascular invasion. TonEBP promotes HCC initiation and growth via oxidative stress and inflammation in various mouse models of HCC. The association between TonEBP and inflammation was confirmed from analysis of the RNA-seq dataset from TCGA. TonEBP-dependent stimulation of tumor growth was dependent on COX-2. TonEBP drives the expression of COX-2 by stimulating the promoter in association with transcription factor YY1 and histone acetyltransferase p300. TonEBP is required for the recruitment of both YY1 and p300 to the promoter in situ. The interaction between TonEBP and YY1 is mediated by RHD and spacer domain. TonEBP deficiency resulted in reduced COX-2 expression leading to reduced production of prostaglandin E2 in various animal models of HCC and acute liver injury. In addition, self-renewal of hepatic cancer stem cells (hCSCs) contributing to recurrence was driven by TonEBP in association with ERCC1. In mouse models of HCC and acute liver injury, three common sites of TonEBP action in response to diverse etiological agents leading to tumorigenesis and tumor progression were found: cell injury and inflammation, induction by oxidative stress, and stimulation of the COX-2 promoter Conclusions: TonEBP is a key component of the common pathway in tumorigenesis and tumor progression of HCC in response to diverse etiological insults. TonEBP is involved in multiple steps along the pathway rendering it an attractive therapeutic target as well as a prognostic biomarker. Citation Format: Jun Ho Lee, Jae Hee Suh, Soo Youn Choi, Hyun Je Kang, Hwan Hee Lee, Whaseon Lee-Kwon, Jiyoung Park, Kyungjae Myung, Neung Hwa Park, Hyug Moo Kwon. TonEBP promotes hepatocellular carcinogenesis, recurrence, and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1519.

The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation

BackgroundThe concept of pioneer transcription factors is emerging as an essential part of the epigenetic regulation, taking place during cell development and differentiation. However, the precise molecular mechanism underlying pioneer factor activity remains poorly understood. We recently reported that the transcription factor c-Myb acts as a pioneer factor in haematopoiesis, and a point mutation in its DNA binding domain, D152V, is able to abrogate this function.ResultsHere, we show that specific histone modifications, including H3K27ac, prevent binding of c-Myb to histone tails, representing a novel effect of histone modifications, namely restricting binding of a pioneer factor to chromatin. Furthermore, we have taken advantage of the pioneer-defect D152V mutant to investigate mechanisms of c-Myb’s pioneer factor activity. We show that c-Myb-dependent transcriptional activation of a gene in inaccessible chromatin relies on c-Myb binding to histones, as well as on c-Myb interacting with the histone acetyltransferase p300. ChIP assays show that both wild type and the D152V mutant of c-Myb bind to a selected target gene at its promoter and enhancer, but only wild-type c-Myb causes opening and activation of the locus. Enhancement of histone acetylation restores activation of the same gene in the absence of c-Myb, suggesting that facilitating histone acetylation is a crucial part of the pioneer factor function of c-Myb.ConclusionsWe suggest a pioneer factor model in which c-Myb binds to regions of closed chromatin and then recruits histone acetyltransferases. By binding to histones, c-Myb facilitates histone acetylation, acting as a cofactor for p300 at c-Myb bound sites. The resulting H3K27ac leads to chromatin opening and detachment of c-Myb from the acetylated chromatin. We propose that the latter phenomenon, acetylation-induced chromatin dissociation, represents a mechanism for controlling the dynamics of pioneer factor binding to chromatin.

High-fat diet unveils an enhancer element at the Ped/Pea-15 gene responsible for epigenetic memory in skeletal muscle

BackgroundThe impact of nutrition on the evolution towards type 2 diabetes has recently received increasing attention because of the effect on chromatin structure and gene expression. PurposeEvaluate the effect of high-fat diet on chromatin remodelling and expression of Ped/Pea-15, a gene commonly overexpressed in individuals at risk of type 2 diabetes. MethodsWe used mouse and cell models to investigate Ped/Pea-15 transcriptional regulation by high-fat diet and glucose, respectively. Chromatin structure and histone modification marks were assessed by Micrococcal Nuclease Protection and Chromatin Immunoprecipitation assays. ResultsSixteen-week exposure of C57BL/6J mice to a high-fat diet impaired glucose tolerance and enhanced Ped/Pea-15 expression in their skeletal muscle tissue. This effect was associated with increased chromatin accessibility at specific regulatory sites at the Ped/Pea-15 gene. In particular, the region at −1900 to −1300 bp from Ped/Pea-15 transcription start site was revealed to feature enhancer activity as demonstrated by its function in the luciferase assay, increased p300 recruitment and H3K4me1 and H3K27Ac levels, all marks of functionally active enhancers. Returning mice to a standard chow diet was accompanied by rapid loss of acetylation of K27 on histone H3 and p300 recruitment at Ped/Pea-15. In contrast, the increased H3K4me1, which accompanied the high-fat diet exposure, remained stable. Incubation of muscle cells in culture medium supplemented with 25 mM glucose (HG) increased Ped/Pea-15 mRNA expression and H3K4me1 at the enhancer region. These effects became measurable upon 72 h of exposure to the HG medium and were not rescued upon returning the cells to the 5 mM glucose-containing medium. Interestingly, after 25 mM and sequential 5 mM glucose treatments, re-exposure of the same cells to HG medium further enhanced Ped/Pea-15 expression and increased H3K4me1 above the levels induced by the initial HG challenge already upon 24 h. ConclusionTransient exposure to HFD or HG unveiled the presence of an enhancer element at the Ped/Pea-15 gene. Epigenetic changes imposed at this region by diets, which impair glucose tolerance generate metabolic memory of the nutritional injury and leave Ped/Pea-15 induction in a poised state.

A Trichostatin A (TSA)/Sp1-mediated mechanism for the regulation of SALL2 tumor suppressor in Jurkat T cells

SALL2 is a transcription factor involved in development and disease. Deregulation of SALL2 has been associated with cancer, suggesting that it plays a role in the disease. However, how SALL2 is regulated and why is deregulated in cancer remain poorly understood. We previously showed that the p53 tumor suppressor represses SALL2 under acute genotoxic stress. Here, we investigated the effect of Histone Deacetylase Inhibitor (HDACi) Trichostatin A (TSA), and involvement of Sp1 on expression and function of SALL2 in Jurkat T cells. We show that SALL2 mRNA and protein levels were enhanced under TSA treatment. Both, TSA and ectopic expression of Sp1 transactivated the SALL2 P2 promoter. This transactivation effect was blocked by the Sp1-binding inhibitor mithramycin A. Sp1 bound in vitro and in vivo to the proximal region of the P2 promoter. TSA induced Sp1 binding to the P2 promoter, which correlated with dynamic changes on H4 acetylation and concomitant recruitment of p300 or HDAC1 in a mutually exclusive manner. Our results suggest that TSA-induced Sp1-Lys703 acetylation contributes to the transcriptional activation of the P2 promoter. Finally, using a CRISPR/Cas9 SALL2-KO Jurkat-T cell model and gain of function experiments, we demonstrated that SALL2 upregulation is required for TSA-mediated cell death. Thus, our study identified Sp1 as a novel transcriptional regulator of SALL2, and proposes a novel epigenetic mechanism for SALL2 regulation in Jurkat-T cells. Altogether, our data support SALL2 function as a tumor suppressor, and SALL2 involvement in cell death response to HDACi.

The distal upstream region of insulin-like growth factor–binding protein-1 enhances its expression in endometrial stromal cells during decidualization

We have previously shown that decidualization of human endometrial stromal cells (ESCs) causes a genome-wide increase in the levels of acetylation of histone-H3 Lys-27 (H3K27ac). We also reported that the distal gene regions, more than 3 kb up- or downstream of gene transcription start sites have increased H3K27ac levels. Insulin-like growth factor-binding protein-1 (IGFBP-1) is a specific decidualization marker and has increased H3K27ac levels in its distal upstream region (-4701 to -7501 bp). Here, using a luciferase reporter gene construct containing this IGFBP-1 upstream region, we tested the hypothesis that it is an IGFBP-1 enhancer. To induce decidualization, we incubated ESCs with cAMP and found that cAMP increased luciferase expression, indicating that decidualization increased the transcriptional activity from the IGFBP-1 upstream region. Furthermore, CRISPR/Cas9-mediated deletion of this region in HepG2 cells significantly reduced IGFBP-1 expression, confirming its role as an IGFBP-1 enhancer. A ChIP assay revealed that cAMP increased the recruitment of the transcriptional regulators CCAAT enhancer-binding protein β (C/EBPβ), forkhead box O1 (FOXO1), and p300 to the IGFBP-1 enhancer in ESCs. Of note, C/EBPβ knockdown inhibited the stimulatory effects of cAMP on the levels of H3K27ac, chromatin opening, and p300 recruitment at the IGFBP-1 enhancer. These results indicate that the region -4701 to -7501 bp upstream of IGFBP-1 functions as an enhancer for IGFBP-1 expression in ESCs undergoing decidualization, that C/EBPβ and FOXO1 bind to the enhancer region to up-regulate IGFBP-1 expression, and that C/EBPβ induces H3K27ac by recruiting p300 to the IGFBP-1 enhancer.

Tributyrin Administration Targets Alcohol‐induced Pathogenic Mechanisms in the Gut‐liver Axis: Relevance to the Development of Therapeutic Strategies in Alcoholic Liver Disease

IntroductionEmerging evidence has established the important role of the “gut‐liver” axis in the development of alcoholic liver disease (ALD). Alcohol‐induced enteric dysbiosis plays an essential role in the pathogenesis of alcoholic liver disease on the gut liver axis. Tributyrin (Tb) is a triglyceride that is rapidly absorbed and metabolized to butyrate by pancreatic lipases. Butyrate is the primary fuel source for colonocytes and improves gut barrier function. Moreover, butyrate is a known HDAC inhibitor.ObjectiveTributyrin modulates alcohol‐induced gut microbiome and barrier function and prevents hepatic steatosis via HDAC inhibition at CPT‐1 promoterMethodsMice were fed liquid Lieber‐DeCarli diet without or with alcohol (5% v/v) for a duration 7 weeks. A subset of mice were administered tributyrin (orally gavaged). Metagenomic analysis of the gut microbiome was performed by analyzing the fecal DNA by amplification of the V3–V5 regions of the 16S rRNA gene and large‐scale parallel sequencing on the Illumina MiSeq platform. Primary rat hepatocytes were exposed to alcohol (50 mM) with/without butyrate pre‐treatment for 24 hrs. Isolation of RNA was carried out using Trizol. Chromatin immunoprecipitation (ChIP) analysis was performed to examine the epigenetic modifications occurring in response to alcohol and butyrate at CPT‐1 promoter.ResultsEffect of tributyrin on alcohol‐induced intestinal dysbiosis and hepatic injury was evaluated. Tributyrin co‐administration prevented chronic alcohol‐induced i) microbial dysbiosis as documented by decrease in the diversity of the gut microbiome, ii) increase in systemic endotoxemia iii) hepatic steatosis and injury. Additionally, pharmacokinetic studies showed increase in plasma butyrate concentration in the hepato‐portal vein after tributyrin gavage. Notably, tributyrin prevented the ethanol‐induced down‐regulation of hepatic CPT‐1, a key enzyme in free fatty acid β‐oxidation. Specifically the effect of butyrate as HDAC inhibitor was evaluated in primary rat hepatocytes by examining CPT‐1 promoter region. Hepatocytes exposed to alcohol showed an increase in HDAC1 recruitment at the CPT‐1 promoter, which was prevented by butyrate. Correspondently, butyrate led to an increase in p300 recruitment with a subsequent increase in transcriptionally permissive histone acetylation at the proximal region of CPT‐1 promoter. This butyrate induced open chromatin configuration further allowed recruitment of RNA‐Pol II leading to CPT‐1 transcription.ConclusionsIn conclusion, tributyrin administration increases the diversity of the bacterial communities and prevents alcohol induced gut dysbiosis. Moreover, Tb attenuates hepatic steatosis by affecting alcohol‐induced, HDAC‐mediated epigenetic mechanisms the down regulate CPT‐1 gene expression. Taken together, these data also support that Tb supplementation is a potential therapeutic strategy in the treatment of ALD.Support or Funding InformationThis work is supported by NIH/NIAAA grants.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhancer epigenomic regulation in differentiation, development and cancer

Enhancers control cell type‐specific gene expression and are marked by H3K4me1. Active enhancers are further marked by H3K27ac. We previously identified CBP/p300 as major H3K27 acetyltransferases and MLL3/MLL4 as major enhancer H3K4me1 methyltransferases [1, 2]. We show that during differentiation of adipocytes, myocytes and ES cells, MLL3/MLL4 are required for enhancer activation, cell type‐specific gene expression, and cell differentiation [2, 3]. Further, we show that while enhancer‐priming by MLL3/MLL4 is dispensable for cell identity maintenance, it controls cell fate transition by orchestrating CBP/p300‐mediated enhancer activation. MLL4 protein, rather than MLL4‐mediated H3K4me1, controls p300 recruitment to enhancers [3, 4].Our data suggest a model of sequential actions of epigenomic regulators on enhancers: 1) pioneer TFs and lineage‐determining TFs recruit MLL3/MLL4 to prime enhancer regions and label them with H3K4me1; 2) MLL3/MLL4 facilitate the binding of CBP/p300, which activate enhancers and label them with H3K27ac; 3) H3K27ac and acetylated TFs are recognized by the epigenomic reader Brd4, which recruits Mediator and RNA Pol II to establish enhancer‐promoter interactions and activate cell type‐specific genes (Nat Comm, in press).MLL3/MLL4 are tumor suppressors frequently mutated in many types of cancers as well as developmental diseases. We show that MLL3/MLL4 are required for the development of adipose, muscle, mammary gland, B cells, T cells, and heart and that depletion of MLL3/MLL4 in mice promotes tumorigenesis [2, 5–9]. Our findings suggest that mutations in MLL3/MLL4 would lead to defects in enhancer activation, cell type‐specific gene expression and cell differentiation. Such a mechanism may contribute to the pathogenesis of these developmental diseases and cancers.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIDDK, NIH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ferritin heavy chain controls the HIF-driven hypoxic response by activating the asparaginyl hydroxylase FIH

Hypoxia-inducible factor 1 (HIF-1) is a key player in cellular response to hypoxia. The stability and transcriptional activity of this protein are oxygen-dependently regulated by the prolyl hydroxylases PHD1-3 and the asparaginyl hydroxylase FIH. Recently, ferritin heavy chain (FTH1) has been characterized to reinforce the HIF-1 signaling pathway in an indirect way through the inhibition of PHD activity by depleting the free iron pool in the cytoplasm. In the present study, we addressed the role of FTH1 in the FIH control of HIF-1 activity. Unexpectedly, immunoprecipitation analyses revealed that FTH1 directly interacted with FIH. In an in vitro hydroxylation assay, FTH1 was found to facilitate the FIH-mediated Asn803 hydroxylation in HIF-1α. As expected, FTH1 prevented the recruitment of p300 to HIF-1α through the Asn803 hydroxylation. In luciferase reporter analyses, FTH1 was found to repress the transcriptional activity of HIF-1α in HCT116 cells under either normoxic or hypoxic conditions. Consequently, FTH1 downregulated the expression of the HIF-1 target genes, such as VEGF, CA9 and GLUT1. Our results suggest a new role of FTH1 as a co-regulator for the FIH-mediated oxygen sensing pathway. Since HIF-1α is involved in pathogenesis of diverse hypoxia-associated diseases, we propose that FTH1 be a potential target in developing new therapeutic strategies against such diseases.

Upregulation of CXCR4 through promoter demethylation contributes to inflammatory hyperalgesia in rats.

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms yet are incompletely defined. DNA methylation has been implicated in the pathogenesis of chronic pain. However, the specific genes regulated by DNA methylation under inflammatory pain condition remain largely unknown. Here, we investigated how chemokine receptor CXCR4 expression is regulated by DNA methylation and how it contributes to inflammatory pain induced by complete Freund's adjuvant (CFA) in rats. Intraplantar injection of CFA could not only induce significant hyperalgesia in rats, but also significantly increase the expression of CXCR4 mRNA and protein in the dorsal root ganglion (DRG). Intrathecal injection of CXCR4 antagonist AMD3100 significantly relieved hyperalgesia in inflammatory rats in a time- and dose-dependent manner. Bisulfite sequencing and methylation-specific PCR demonstrate that CFA injection led to a significant demethylation of CpG island at CXCR4 gene promoter. Consistently, the expression of DNMT3b was significantly downregulated after CFA injection. Online software prediction reveals three binding sites of p65 in the CpG island of CXCR4 gene promoter, which has confirmed by the chromatin immunoprecipitation assay, CFA treatment significantly increases the recruitment of p65 to CXCR4 gene promoter. Inhibition of NF-kB signaling using p65 inhibitor pyrrolidine dithiocarbamate significantly prevented the increases of the CXCR4 expression. Upregulation of CXCR4 expression due to promoter demethylation followed by increased recruitment of p65 to promoter of CXCR4 gene contributes to inflammatory hyperalgesia. These findings provide a theoretical basis for the treatment of chronic pain from an epigenetic perspective.

Age-dependent regulation of antioxidant genes by p38α MAPK in the liver

p38α is a redox sensitive MAPK activated by pro-inflammatory cytokines and environmental, genotoxic and endoplasmic reticulum stresses. The aim of this work was to assess whether p38α controls the antioxidant defense in the liver, and if so, to elucidate the mechanism(s) involved and the age-related changes. For this purpose, we used liver-specific p38α-deficient mice at two different ages: young-mice (4 months-old) and old-mice (24 months-old). The liver of young p38α knock-out mice exhibited a decrease in GSH levels and an increase in GSSG/GSH ratio and malondialdehyde levels. However, old mice deficient in p38α had higher hepatic GSH levels and lower GSSG/GSH ratio than young p38α knock-out mice. Liver-specific p38α deficiency triggered a dramatic down-regulation of the mRNAs of the key antioxidant enzymes glutamate cysteine ligase, superoxide dismutase 1, superoxide dismutase 2, and catalase in young mice, which seems mediated by the lack of p65 recruitment to their promoters. Nrf-2 nuclear levels did not change significantly in the liver of young mice upon p38α deficiency, but nuclear levels of phospho-p65 and PGC-1α decreased in these mice. p38α-dependent activation of NF-κB seems to occur through classical IκB Kinase and via ribosomal S6 kinase1 and AKT in young mice. However, unexpectedly the long-term deficiency in p38α triggers a compensatory up-regulation of antioxidant enzymes via NF-κB activation and recruitment of p65 to their promoters. In conclusion, p38α MAPK maintains the expression of antioxidant genes in liver of young animals via NF-κΒ under basal conditions, whereas its long-term deficiency triggers compensatory up-regulation of antioxidant enzymes through NF-κΒ.

Gene-specific mechanisms direct glucocorticoid-receptor-driven repression of inflammatory response genes in macrophages.

The glucocorticoid receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis in mouse macrophages reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB-binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class; however, it effects repression by targeting distinct temporal events and components of transcriptional machinery.

MUC1-C Induces PD-L1 and Immune Evasion in Triple-Negative Breast Cancer.

The immune checkpoint ligand PD-L1 and the transmembrane mucin MUC1 are upregulated in triple-negative breast cancer (TNBC), where they contribute to its aggressive pathogenesis. Here, we report that genetic or pharmacological targeting of the oncogenic MUC1 subunit MUC1-C is sufficient to suppress PD-L1 expression in TNBC cells. Mechanistic investigations showed that MUC1-C acted to elevate PD-L1 transcription by recruitment of MYC and NF-κB p65 to the PD-L1 promoter. In an immunocompetent model of TNBC in which Eo771/MUC1-C cells were engrafted into MUC1 transgenic mice, we showed that targeting MUC1-C associated with PD-L1 suppression, increases in tumor-infiltrating CD8+ T cells and tumor cell killing. MUC1 expression in TNBCs also correlated inversely with CD8, CD69, and GZMB, and downregulation of these markers associated with decreased survival. Taken together, our findings show how MUC1 contributes to immune escape in TNBC, and they offer a rationale to target MUC1-C as a novel immunotherapeutic approach for TNBC treatment.Significance: These findings show how upregulation of the transmembrane mucin MUC1 contributes to immune escape in an aggressive form of breast cancer, with potential implications for a novel immunotherapeutic approach. Cancer Res; 78(1); 205-15. ©2017 AACR.

Periodic variation in bile acids controls circadian changes in uric acid via regulation of xanthine oxidase by the orphan nuclear receptor PPARα

Xanthine oxidase (XOD), also known as xanthine dehydrogenase, is a rate-limiting enzyme in purine nucleotide degradation, which produces uric acid. Uric acid concentrations in the blood and liver exhibit circadian oscillations in both humans and rodents; however, the underlying mechanisms remain unclear. Here, we demonstrate that XOD expression and enzymatic activity exhibit circadian oscillations in the mouse liver. We found that the orphan nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) transcriptionally activated the mouse XOD gene and that bile acids suppressed XOD transactivation. The synthesis of bile acids is known to be under the control of the circadian clock, and we observed that the time-dependent accumulation of bile acids in hepatic cells interfered with the recruitment of the co-transcriptional activator p300 to PPARα, thereby repressing XOD expression. This time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the hepatic expression of XOD, which, in turn, led to circadian alterations in uric acid production. Finally, we also demonstrated that the anti-hyperuricemic effect of the XOD inhibitor febuxostat was enhanced by administering it at the time of day before hepatic XOD activity increased. These results suggest an underlying mechanism for the circadian alterations in uric acid production and also underscore the importance of selecting an appropriate time of day for administering XOD inhibitors.

VHL Deficiency Drives Enhancer Activation of Oncogenes in Clear Cell Renal Cell Carcinoma

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Roles for noncoding cis-regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profiles, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specific aspects of malignancy. Superenhancer profiling identified ZNF395 as a ccRCC-specific and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivoVHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2α-HIF1β heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter-enhancer interactions. Subtype-specific driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression.Significance: Comprehensive epigenomic profiling of ccRCC establishes a compendium of somatically altered cis-regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL, a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2α and recruitment of histone acetyltransferase p300 at preexisting lineage-specific promoter-enhancer complexes. Cancer Discov; 7(11); 1284-305. ©2017 AACR.See related commentary by Ricketts and Linehan, p. 1221This article is highlighted in the In This Issue feature, p. 1201.

Pyruvate Kinase M2 Is Required for the Expression of the Immune Checkpoint PD-L1 in Immune Cells and Tumors.

Blocking interaction of the immune checkpoint receptor PD-1 with its ligand PD-L1 is associated with good clinical outcomes in a broad variety of malignancies. High levels of PD-L1 promote tumor growth by restraining CD8+ T-cell responses against tumors. Limiting PD-L1 expression and function is therefore critical for allowing the development of antitumor immune responses and effective tumor clearance. Pyruvate kinase isoform M2 (PKM2) is also a key player in regulating cancer as well as immune responses. PKM2 catalyzes the final rate-limiting step of glycolysis. Furthermore, PKM2 as a dimer translocates to the nucleus, where it stimulates hypoxia-inducible factor 1α (Hif-1α) transactivation domain function and recruitment of p300 to the hypoxia response elements (HRE) of Hif-1α target genes. Here, we provide the first evidence of a role for PKM2 in regulating the expression of PD-L1 on macrophages, dendritic cells (DCs), T cells, and tumor cells. LPS-induced expression of PD-L1 in primary macrophages was inhibited by the PKM2 targeting compound TEPP-46. Furthermore, RNA silencing of PKM2 inhibited LPS-induced PD-L1 expression. This regulation occurs through direct binding of PKM2 and Hif-1α to HRE sites on the PD-L1 promoter. Moreover, TEPP-46 inhibited expression of PD-L1 on macrophages, DCs, and T cells as well as tumor cells in a mouse CT26 cancer model. These findings broaden our understanding of how PKM2 may contribute to tumor progression and may explain the upregulation of PD-L1 in the tumor microenvironment.

Epigenetic regulation of interleukin-8 expression by class I HDAC and CBP in ovarian cancer cells.

Although inhibitors of epigenetic regulators have been effective in the treatment of cutaneous T cell lymphoma (CTCL) and other hematopoietic malignancies, they have been less effective in solid tumors, including ovarian cancer (OC). We have previously shown that inhibition of histone deacetylase (HDAC) activity induces expression of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (CXCL8, IL-8) in OC cells, resulting in their increased survival and proliferation. Here, we show that in addition to ovarian cancer SKOV3, OVCAR3, and CAOV3 cells, HDAC inhibition induces the CXCL8 expression in HeLa cells, but not in CTCL Hut-78 cells. In OC cells, the CXCL8 expression is induced by pharmacological inhibition of class I HDACs. Interestingly, while an individual suppression of HDAC1, HDAC2, or HDAC3 by corresponding siRNAs inhibits the CXCL8 expression, their simultaneous suppression induces the CXCL8 expression. The induced CXCL8 expression in OC cells is dependent on histone acetyltransferase (HAT) activity of CREB-binding protein (CBP), but not p300, and is associated with HAT-dependent p65 recruitment to CXCL8 promoter. Together, our results show that the CXCL8 expression in OC cells is induced by combined inhibition of HDAC1, -2, and -3, and silenced by suppression of HAT activity of CBP. In addition, our data indicate that the induced CXCL8 expression may be responsible for the limited effectiveness of HDAC inhibitors in OC and perhaps other solid cancers characterized by CXCL8 overexpression, and suggest that targeting class I HDACs and CBP may provide novel combination strategies by limiting the induced CXCL8 expression.