Inhibition Of Histone Acetylation Research Articles

Chromatin remodeling by curcumin alters endogenous aryl hydrocarbon receptor signaling

The aim of this study was to gain more information about the mechanisms that regulate expression of the aryl hydrocarbon receptor (AHR) target gene CYP1A1. Human hepatoma cells (HepG2 and Huh7) and human immortalized keratinocytes (HaCaT) were treated with different concentrations of the dietary polyphenolic compound curcumin (CUR) alone or in combination with the natural AHR agonist 6-formylindolo[3,2-b]carbazole (FICZ). In an earlier study, we described that CUR can activate the AHR indirectly by inhibiting metabolic clearance of FICZ. Here, we measured cell viability, activation of AHR signaling, oxidative stress and histone modifying activities in response to CUR at concentrations ranging from 0.1 to 50 μM. We observed apparent non-linear responses on cell viability and activation of AHR signaling. The CYP1A1 expression and the CYP1A1 enzyme activity in the presence of CUR reflected the histone acetylation efficiency observed in nuclear extracts. At the lowest concentration, CUR significantly decreased histone deacetylase activity and increased the FICZ-induced CYP1A1 activity. In contrast, at the highest concentration, CUR increased the formation of reactive oxygen species, significantly inhibited histone acetylation, and temporally decreased FICZ-induced CYP1A1 activity. The results suggest that CUR can both increase and decrease the accessibility of DNA and thereby influence transcriptional responses to the ligand-activated AHR. This suggestion was supported by the fact that chromatin remodeling treatments with trichostatin A, p300, or 5-aza-dC increased CYP1A1 transcription. We conclude that the AHR-dependent transcriptional efficiency is modified by factors that influence the cellular redox status and the chromatin structure.

Polycomb inhibits histone acetylation by CBP by binding directly to its catalytic domain

Drosophila Polycomb (PC), a subunit of Polycomb repressive complex 1 (PRC1), is well known for its role in maintaining repression of the homeotic genes and many others and for its binding to trimethylated histone H3 on Lys 27 (H3K27me3) via its chromodomain. Here, we identify a novel activity of PC: inhibition of the histone acetylation activity of CREB-binding protein (CBP). We show that PC and its mammalian CBX orthologs interact directly with the histone acetyltransferase (HAT) domain of CBP, binding to the previously identified autoregulatory loop, whose autoacetylation greatly enhances HAT activity. We identify a conserved PC motif adjacent to the chromodomain required for CBP binding and show that PC binding inhibits acetylation of histone H3. CBP autoacetylation impairs PC binding in vitro, and PC is preferentially associated with unacetylated CBP in vivo. PC knockdown elevates the acetylated H3K27 (H3K27ac) level globally and at promoter regions of some genes that are bound by both PC and CBP. Conversely, PC overexpression decreases the H3K27ac level in vivo and also suppresses CBP-dependent Polycomb phenotypes caused by overexpression of Trithorax, an antagonist of Polycomb silencing. We find that PC is physically associated with the initiating form of RNA polymerase II (Pol II) and that many promoters co-occupied by PC and CBP are associated with paused Pol II, suggesting that PC may play a role in Pol II pausing. These results suggest that PC/PRC1 inhibition of CBP HAT activity plays a role in regulating transcription of both repressed and active PC-regulated genes.

Platelet-Activating Factor Induces Epigenetic Modifications in Human Mast Cells

Ultraviolet (UV) radiation-induced systemic immune suppression is a major risk factor for skin cancer induction. The migration of dermal mast cells from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced keratinocyte-derived platelet-activating factor (PAF) activates mast cell migration, in part by up regulating the expression of CXCR4 on the surface of mast cells. Others have indicated that epigenetic mechanisms regulate CXCR4 expression, so we asked whether PAF activates epigenetic mechanisms in mast cells. Human mast cells were treated with PAF and the effect on DNA methylation and/or acetylation was measured. PAF suppressed the expression of DNA methyltransferase (DNMT) 1 and 3b. On the other hand, PAF increased p300 histone acetyltransferase expression, and the acetylation of histone H3, which coincided with a decreased expression of the histone deacetylase HDAC2. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the CXCR4 promoter. Finally, inhibiting histone acetylation blocked p300 up-regulation and suppressed PAF-induced surface expression of CXCR4. Our findings suggest a novel molecular mechanism for PAF, activation of epigenetic modifications. We suggest that PAF may serve as an endogenous molecular mediator that links the environment (UV radiation) with the epigenome.

Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model.

Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down-regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing.

Valproic acid-induced histone acetylation suppresses CYP19 gene expression and inhibits the growth and survival of endometrial stromal cells.

Endometriosis is a common type of estrogen‑dependent, gynecological and chronic inflammatory disease. Epigenetics refers to changes in gene expression that occur without altering the DNA sequence or DNA content. Histone modification dominates epigenetics, and histone acetylation is the most extensively studied type of histone modification. The CYP19 gene is the gene that encodes P450 aromatase, which regulates the synthesis of estrogen. Hence, we conducted this study to investigate whether histone acetylation has an effect on CYP19 expression and whether histone acetylation is related to endometrial stromal cells (ESCs). Reverse transcription-quantitative polymerase chain reaction (RT‑qPCR), western blot analysis and chromatin immunoprecipitation assays were performed. The results revealed that valproic acid (VPA) significantly promoted histone acetylation in the ESCs, which inhibited histone acetylation in the promoter region of the CYP19 gene, thus suppressing its expression. We also noted that VPA inhibited cell viability and proliferation, and induced the apoptosis, of ESCs. The findings of our study on histone acetylation, endometriosis and the CYP19 gene provide insight which may aid in the research of histone acetylation and suggest that the CYP19 gene may be a novel therapeutic target and method for the treatment of endometriosis.

Abstract 211: IL-35 Suppresses Endothelial Cell Activation by Inhibiting Histone H3K14 Acetylation and AP-1

Background: IL-35 is a newly identified anti-inflammatory cytokine, which inhibits inflammation and immune responses by inducing regulatory T cells and regulatory B cells and suppressing effector T cells and macrophages. We previously reported that IL-35 is a responsive cytokine that is not constitutively expressed in human tissues, but can be induced by proinflammatory stimuli in vascular endothelial cells (EC), smooth muscle cells and monocytes. However, the functions of IL-35 on non-lymphocytes such as vascular EC remain unknown. EC activation induced by proatherogenic stimuli including lysophosphatidylcholine (LPC) is considered as the initiation step of monocyte recruitment and atherosclerosis. In this study, we examined the expression of IL-35 during early atherosclerosis and the role of IL-35 in LPC-induced EC activation. Methods and Results: Using microarray method, we found that two IL-35 subunits and their receptor subunits are all significantly induced during early atherosclerosis in the aortas of atherosclerotic mouse model, apolipoprotein E (ApoE) knockout mice fed with 3 weeks of high fat diet. In addition, we found that IL-35 can suppress LPC-induced monocyte adhesion to human aortic ECs (HAEC). Mechanistic, our analysis using EC biology gene array shows that IL-35 selectively inhibits LPC-induced expressions of EC adhesion molecules ICAM-1, VCAM-1, and proinflammatory cytokine IL-1β in human aortic EC (HAEC). To further dissect the molecular mechanisms underlying the IL-35 inhibitory effects on LPC-induced EC activation gene upregulation, we performed mass spectrum analysis and electrophoretic mobility shift assay. We found that IL-35 blocked LPC-induced histone H3 lysine 14 (H3K14) acetylation as well as the binding of pro-inflammatory transcription factor AP-1 to the promoters of EC adhesion molecules in HAEC. Conclusions: IL-35 is induced in the aortas during early atherogenesis and suppresses human EC activation and monocyte adhesion by inhibiting histone acetylation and AP-1 nuclear translocation.

Curcumin-induced histone acetylation inhibition improves stress-induced gastric ulcer disease in rats.

Curcumin is known to possess anti‑inflammatory properties. Despite the fact that curcumin is known to be a strong inhibitor of H+, K+‑ATPase activity, the mechanism underlying the curcumin‑induced inhibition of the transcription of the H+, K+‑ATPase α subunit in gastric mucosal parietal cells remains unclear. The present study investigated the possible mechanism by which curcumin inhibits stomach H+, K+‑ATPase activity during the acute phase of gastric ulcer disease. A rat model of stress‑induced gastric ulcers was produced, in which the anti‑ulcer effects of curcumin were examined. Curcumin‑induced inhibition of the H+, K+‑ATPase promoter via histone acetylation, was verified using a chromatin immunoprecipitation assay. The results showed that curcumin improved stress‑induced gastric ulcer disease in rats, as demonstrated by increased pH values and reduced gastric mucosal hemorrhage and ulcer index. These effects were accompanied by a significant reduction in the level of histone H3 acetylation at the site of the H+, K+‑ATPase promoter and in the expression of the gastric H+,K+‑ATPase α subunit gene and protein. In conclusion, curcumin downregulated the acetylation of histone H3 at the site of the H+, K+‑ATPase promoter gene, thereby inhibiting the transcription and expression of the H+, K+‑ATPase gene. Curcumin was shown to have a preventive and therapeutic effect in gastric ulcer disease.

Epigenetic regulation of inflammatory gene expression in macrophages by selenium

Acetylation of histone and non-histone proteins by histone acetyltransferases plays a pivotal role in the expression of proinflammatory genes. Given the importance of dietary selenium in mitigating inflammation, we hypothesized that selenium supplementation may regulate inflammatory gene expression at the epigenetic level. The effect of selenium towards histone acetylation was examined in both in vitro and in vivo models of inflammation by chromatin immunoprecipitation assays and immunoblotting. Our results indicated that selenium supplementation, as selenite, decreased acetylation of histone H4 at K12 and K16 in COX-2 and TNFα promoters, and of the p65 subunit of the redox sensitive transcription factor NFκB in primary and immortalized macrophages. On the other hand, selenomethionine had a much weaker effect. Selenite treatment of HIV-1-infected human monocytes also significantly decreased the acetylation of H4 at K12 and K16 on the HIV-1 promoter, supporting the down-regulation of proviral expression by selenium. A similar decrease in histone acetylation was also seen in the colonic extracts of mice treated with dextran sodium sulfate that correlated well with the levels of selenium in the diet. Bone-marrow-derived macrophages from Trspfl/flCreLysM mice that lack expression of selenoproteins in macrophages confirmed the important role of selenoproteins in the inhibition of histone H4 acetylation. Our studies suggest that the ability of selenoproteins to skew the metabolism of arachidonic acid contributes, in part, to their ability to inhibit histone acetylation. In summary, our studies suggest a new role for selenoproteins in the epigenetic modulation of proinflammatory genes.

Novel INHAT repressor (NIR) is required for early lymphocyte development.

Novel inhibitor of histone acetyltransferase repressor (NIR) is a transcriptional corepressor with inhibitor of histone acetyltransferase activity and is a potent suppressor of p53. Although NIR deficiency in mice leads to early embryonic lethality, lymphoid-restricted deletion resulted in the absence of double-positive CD4(+)CD8(+) thymocytes, whereas bone-marrow-derived B cells were arrested at the B220(+)CD19(-) pro-B-cell stage. V(D)J recombination was preserved in NIR-deficient DN3 double-negative thymocytes, suggesting that NIR does not affect p53 function in response to physiologic DNA breaks. Nevertheless, the combined deficiency of NIR and p53 provided rescue of DN3L double-negative thymocytes and their further differentiation to double- and single-positive thymocytes, whereas B cells in the marrow further developed to the B220(+)CD19(+) pro-B-cell stage. Our results show that NIR cooperate with p53 to impose checkpoint for the generation of mature B and T lymphocytes.

The yeast Cyc8–Tup1 complex cooperates with Hda1p and Rpd3p histone deacetylases to robustly repress transcription of the subtelomeric FLO1 gene

We demonstrate that the yeast flocculation gene, FLO1, is representative of a distinct subset of subtelomeric genes that are robustly repressed by the Cyc8–Tup1 complex. We have examined Cyc8–Tup1 localisation, histone acetylation and long-range chromatin remodelling within the extensive FLO1 upstream region. We show that Cyc8–Tup1 is localised in a DNase I hypersensitive site within an ordered array of strongly positioned nucleosomes around −700 base pairs upstream of the transcription start site. In cyc8 deletion mutant strains, Tup1p localisation is absent, with concomitant histone hyperacetylation of adjacent regions at the FLO1 promoter. This is accompanied by extensive histone depletion across the upstream region and gene activation. The yeast histone deacetylases, Hda1p and Rpd3p, occupy the repressed FLO1 promoter region in a Cyc8–Tup1 dependent manner and coordinate histone deacetylation, nucleosome stabilisation and gene repression. Moreover, we show that the ATP-dependent chromatin remodelling complex Swi–Snf occupies the site vacated by Cyc8–Tup1 in a cyc8 mutant. These data suggest that distinctly bound Cyc8–Tup1 cooperates with Hda1p and Rpd3p to establish or maintain an extensive array of strongly positioned, deacetylated nucleosomes over the FLO1 promoter and upstream region which inhibit histone acetylation, block Swi–Snf binding and prevent transcription.

Anti-Inflammatory and Anti-Remodelling Effects of ISU201, a Modified Form of the Extracellular Domain of Human BST2, in Experimental Models of Asthma: Association with Inhibition of Histone Acetylation

There are few alternatives to glucocorticosteroids for treatment of asthma. We assessed the activity of a novel protein drug designated ISU201, the extracellular domain of the human cell surface protein BST2, stabilised by fusion with the Fc region of IgG, in mouse models of mild chronic asthma and an acute exacerbation of asthma. The ability of ISU201 to suppress airway inflammation and remodelling was compared with that of dexamethasone. Female BALB/c mice were systemically sensitised with ovalbumin, then received controlled low-level challenge with aerosolised ovalbumin for 6 weeks, which induced lesions of mild chronic asthma, and were treated with drugs during the final 2 weeks. Alternatively, sensitised mice received 4 weeks of chronic low-level challenge and were treated 24 and 2 hours before a final single moderate-level challenge, which triggered acute airway inflammation simulating an asthmatic exacerbation. Inflammation and remodelling were quantified, as was the expression of pro-inflammatory cytokines in bronchoalveolar lavage fluid and tissues. To identify cellular targets of ISU201, we assessed the effects of the drug on activated lymphocytes, macrophages and airway epithelial cells. In the model of mild chronic asthma, ISU201 was as effective as dexamethasone in suppressing airway inflammation and most changes of remodelling. In the model of an allergen-induced acute exacerbation of chronic asthma, ISU201 was also an effective anti-inflammatory agent, although it was less active than dexamethasone. The drug acted on multiple cellular targets, suppressing production of pro-inflammatory cytokines by lymphocytes and macrophages. ISU201 significantly reduced acetylation of histone H4 in airway epithelial cells, suggesting at least one potential mechanism of action. We conclude that in these models of asthma, ISU201 is a broad-spectrum inhibitor of both airway inflammation and remodelling. Thus, unlike drugs which target specific mediators, it could potentially be an alternative or an adjunct to glucocorticoids for the treatment of asthma.

Death Domain-associated Protein 6 (Daxx) Selectively Represses IL-6 Transcription through Histone Deacetylase 1 (HDAC1)-mediated Histone Deacetylation in Macrophages

As a multifunctional nuclear protein, death domain-associated protein 6 (Daxx) regulates a wide range of biological processes, including cell apoptosis and gene transcription. However, the function of Daxx in innate immunity remains unclear. In our study, we show that Daxx is highly expressed in macrophages and localized in nucleus of macrophages. The expression of Daxx is significantly up-regulated by stimulation with TLR ligands LPS and poly(I:C). Silence of Daxx selectively represses IL-6 expression at transcription level in LPS-activated macrophages. Upon stimulation of LPS, Daxx specifically binds to the promoter of IL-6 and inhibits histone acetylation at IL-6 promoter region. Further mechanism analyses show that histone deacetylase 1 (HDAC1) interacts with Daxx and binds to the promoter of IL-6. Daxx silencing decreases the association of HDAC1 to IL-6 promoter. Therefore, our data reveal that Daxx selectively represses IL-6 transcription through HDAC1-mediated histone deacetylation in LPS-induced macrophages, acting as a negative regulator of IL-6 during innate immunity and potentially preventing inflammatory response because of overproduction of IL-6.

Curcumin-Mediated Cardiac Defects in Mouse is Associated with a Reduced Histone H3 Acetylation and Reduced Expression of Cardiac Transcription Factors

Histone acetylation plays an important role in heart development. However, the mechanism(s) remains unclear. This study was designed to evaluate the effect of curcumin-caused histone hypo-acetylation on the development of mouse embryonic heart and the expression of cardiac transcription factors in vivo. The results showed that curcumin treatment significantly decreased histone acetylase activity and histone acetylation level in mouse embryonic heart. In curcumin-treated mice, the hearts on E11.5 were smaller with thinner ventricular wall and a delayed development of trabeculae and ventricular septum compared with the controls. The ventricular septum was complete on E14.5; however, the ventricular wall and septum were thinner with fewer trabeculae than those in the controls. On E17.5, the cardiac structure appeared normal, but the ventricular wall and septum were thinner. The expression of GATA4, Nkx2.5 and Mef2c in the heart on E11.5 and E14.5 was decreased significantly as compared to the controls. There was no significant difference in Mef2c expression on E17.5 between curcumin-treated group and the controls, while GATA4 and Nkx2.5 expression remained significantly reduced. These results indicate that inhibition of histone acetylation by curcumin can reduce the expression of the cardiac transcription factors resulting in an abnormal heart development in mice.

LHX3 Interacts with Inhibitor of Histone Acetyltransferase Complex Subunits LANP and TAF-1β to Modulate Pituitary Gene Regulation

LIM-homeodomain 3 (LHX3) is a transcription factor required for mammalian pituitary gland and nervous system development. Human patients and animal models with LHX3 gene mutations present with severe pediatric syndromes that feature hormone deficiencies and symptoms associated with nervous system dysfunction. The carboxyl terminus of the LHX3 protein is required for pituitary gene regulation, but the mechanism by which this domain operates is unknown. In order to better understand LHX3-dependent pituitary hormone gene transcription, we used biochemical and mass spectrometry approaches to identify and characterize proteins that interact with the LHX3 carboxyl terminus. This approach identified the LANP/pp32 and TAF-1β/SET proteins, which are components of the inhibitor of histone acetyltransferase (INHAT) multi-subunit complex that serves as a multifunctional repressor to inhibit histone acetylation and modulate chromatin structure. The protein domains of LANP and TAF-1β that interact with LHX3 were mapped using biochemical techniques. Chromatin immunoprecipitation experiments demonstrated that LANP and TAF-1β are associated with LHX3 target genes in pituitary cells, and experimental alterations of LANP and TAF-1β levels affected LHX3-mediated pituitary gene regulation. Together, these data suggest that transcriptional regulation of pituitary genes by LHX3 involves regulated interactions with the INHAT complex.

Cigarette Smoke Component Acrolein Modulates Chromatin Assembly by Inhibiting Histone Acetylation

Chromatin structure and gene expression are both regulated by nucleosome assembly. How environmental factors influence histone nuclear import and the nucleosome assembly pathway, leading to changes in chromatin organization and transcription, remains unknown. Acrolein (Acr) is an α,β-unsaturated aldehyde, which is abundant in the environment, especially in cigarette smoke. It has recently been implicated as a potential major carcinogen of smoking-related lung cancer. Here we show that Acr forms adducts with histone proteins in vitro and in vivo and preferentially reacts with free histones rather than with nucleosomal histones. Cellular fractionation analyses reveal that Acr exposure specifically inhibits acetylations of N-terminal tails of cytosolic histones H3 and H4, modifications that are important for nuclear import and chromatin assembly. Notably, Acr exposure compromises the delivery of histone H3 into chromatin and increases chromatin accessibility. Moreover, changes in nucleosome occupancy at several genomic loci are correlated with transcriptional responses to Acr exposure. Our data provide new insights into mechanisms whereby environmental factors interact with the genome and influence genome function.

Nicotine primes the effect of cocaine on the induction of LTP in the amygdala

In human populations, there is a well-defined sequence of involvement in drugs of abuse, in which the use of nicotine or alcohol precedes the use of marijuana, which in turn, precedes the use of cocaine. The term “Gateway Hypothesis” describes this developmental sequence of drug involvement. In prior work, we have developed a mouse model to study the underlying metaplastic behavioral, cellular and molecular mechanisms by which exposure to one drug, namely nicotine, affects the response to another drug, namely cocaine. We found that nicotine enhances significantly the changes in synaptic plasticity in the striatum induced by cocaine (Levine et al., 2011). Here we ask: does the metaplastic effect of nicotine on cocaine also apply in the amygdala, a brain region that is involved in the orchestration of emotions and in drug addiction? We find that pretreatment with nicotine enhances long-term synaptic potentiation (LTP) in response to cocaine in the amygdala. Both short-term (1 day) and long-term (7 days) pre-exposure to nicotine facilitate the induction of LTP by cocaine. The effect of nicotine on LTP is unidirectional; exposure to nicotine following treatment with cocaine is ineffective. This metaplastic effect of nicotine on cocaine is long lasting but reversible. The facilitation of LTP can be obtained for 24 but not 40 days after cessation of nicotine. As is the case in the striatum, pretreatment with Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, simulates the priming effect of nicotine. These results provide further evidence that the priming effect of nicotine may be achieved, at least partially, by the inhibition of histone acetylation and indicate that the amygdala appears to be an important brain structure for the processing of the metaplastic effect of nicotine on cocaine.This article is part of the Special Issue entitled ‘Glutamate Receptor-Dependent Synaptic Plasticity’.

The Transcription Factor Jdp2 Controls Bone Homeostasis and Antibacterial Immunity by Regulating Osteoclast and Neutrophil Differentiation

Jdp2 is an AP-1 family transcription factor that regulates the epigenetic status of histones. Previous invitro studies revealed that Jdp2 is involved in osteoclastogenesis. However, the roles of Jdp2 invivo and its pleiotropic functions are largely unknown. Here we generated Jdp2(-/-) mice and discovered itscrucial roles not only in bone metabolism but also in differentiation of neutrophils. Jdp2(-/-) mice exhibited osteopetrosis resulting from impaired osteoclastogenesis. Jdp2(-/-) neutrophils were morphologically normal but had impaired surface expression of Ly6G, bactericidal function, and apoptosis. We also found that ATF3 was an inhibitor of neutrophil differentiation and that Jdp2 directly suppresses itsexpression via inhibition of histone acetylation. Strikingly, Jdp2(-/-) mice were highly susceptible to Staphylococcus aureus and Candida albicans infection. Thus, Jdp2 plays pivotal roles in invivo bone homeostasis and host defense by regulating osteoclast and neutrophil differentiation.

Epigenetic imprinting by commensal probiotics inhibits the IL-23/IL-17 axis in an in vitro model of the intestinal mucosal immune system

The pathophysiology of IBD is characterized by a complex interaction between genes and the environment. Genetic and environmental differences are attributed to the heterogeneity of the disease pathway and to the epigenetic modifications that lead to altered gene expression in the diseased tissues. The epigenetic machinery consists of short interfering RNA, histone modifications, and DNA methylation. We evaluated the effects of Bifidobacterium breve (DSMZ 20213) and LGG (ATCC 53103), as representatives of commensal probiotics on the expression of IL-17 and IL-23, which play an important role in IBD, and on the epigenetic machinery in a 3D coculture model composed of human intestinal HT-29/B6 or T84 cells and PBMCs. The cells were treated with LPS in the presence or absence of bacteria for 48 h, and the expression of IL-17, IL-23, and CD40 at the mRNA and protein levels was assessed using TaqMan qRT-PCR and ELISA, respectively. Western blotting was used to assess the expression of the MyD88, the degradation of IRAK-1 and IκBα, the expression of the NF-κB p50/p65 subunits, the p-p38 MAPK and p-MEK1, as well as histone modifications. NF-κB activity was assessed by NF-κB-dependent luciferase reporter gene assays. The accumulation of Ac-H4 and DNA methylation was quantitatively assessed using colorimetric assays. B. breve and LGG diminished the LPS-induced expression of IL-17, IL-23, CD40, and histone acetylation, while slightly enhancing DNA methylation. These effects were paralleled by a decrease in the nuclear translocation of NF-κB, as demonstrated by a decrease in the expression of MyD88, degradation of IRAK-1 and IκBα expression of the nuclear NF-κB p50/p65 subunits, p-p38 MAPK and p-MEK1, and NF-κB-dependent luciferase reporter gene activity in LPS-stimulated cells. B. breve and LGG may exert their anti-inflammatory effects in the gut by down-regulating the expression of the IBD-causing factors (IL-23/IL-17/CD40) associated with epigenetic processes involving the inhibition of histone acetylation and the optimal enhancement of DNA methylation, reflected in the limited access of NF-κB to gene promoters and reduced NF-κB-mediated transcriptional activation. We describe a new regulatory mechanism in which commensal probiotics inhibit the NF-κB-mediated transcriptional activation of IBD-causing factors (IL-23/IL-17/CD40), thereby simultaneously reducing histone acetylation and enhancing DNA methylation.

Elevated histone acetylations in Müller cell contribute to inflammation: A novel inhibitory effect of minocycline

Inflammation plays important roles in the development of diabetic retinopathy (DR). How Müller cells contribute to DR-related inflammation remains unclear. We hypothesized that under diabetic conditions, elevated histone acetylations in Müller cells contribute to the inflammatory response. In this study, significantly increased histone acetylations, elevated histone acetyltranferases levels, and decreased histone deacetylases levels were found in the retinas of diabetic rats. Elevated AcH3K9 and AcH3K18 were partially co-stained with Müller cells on retinal sections by immunofluorescence staining. Consistently, high-glucose (HG) treated rMC-1 cells, a Müller cell line, also showed upregulation of acetylated histones, accompanied with the overexpression of GFAP, p-STAT3, and NFκB-p65, and two inflammatory genes, TNFα and MCP-1. Meanwhile, sodium butyrate (NaB)-induced upregulation of acetylated histones is also accompanied with transcription of inflammatory genes. Minocycline, a drug with beneficial effects on DR, was found to downregulate HG-induced Müller cell activation, inflammation, and acetylated H3K18 bound to the promoters of GFAP and inflammatory genes by chromatin immunoprecipitation assay. Furthermore, the effects of minocycline on HG-induced elevation in histone acetylations were also demonstrated in isolated primary rat Müller cells. These findings suggest the elevation of histone acetylations in Müller cells plays important regulating roles in the inflammatory response during diabetic conditions. Inhibition of histone acetylation by minocycline is a novel function that may contribute to its beneficial effects on DR.

Helicobacter pylori decreases p27 expression through the delta opioid receptor-mediated inhibition of histone acetylation within the p27 promoter

Chronic Helicobacter pylori infection is associated with the decreased expression of the gastric tumour suppressor protein p27. Because transcription of the gene p27 may be regulated epigenetically through histone acetylation, which is mediated by G-protein coupled delta opioid receptor (DOR) stimulation, we examined whether H. pylori regulates the DOR/histone acetylation/p27 promoter pathway. The levels of acetylated histone and p300, a gene-specific histone acetyltransferase within the p27 promoter, were measured using ChIP assays. The expression of phospho-DOR was evaluated by Western blot and immunohistochemical analyses. Growth curves were constructed, and cell proliferation was assessed after BrdU incorporation. Low p27 expression in acutely H. pylori-infected AGS gastric epithelial cells and in chronically H. pylori-infected AGS-derived HS3C cells was associated with approximate 20% and 40% decreases in p27 mRNA expression, respectively, when compared to p27 mRNA levels in uninfected AGS parental cells. The low p27 mRNA levels following H. pylori infection were associated with a 15–60% reduction in p27 promoter histone H4 acetylation. The recruitment of p300 to the p27 promoter was also markedly decreased by H. pylori infection. The expression of phospho-DOR was decreased by H. pylori infection in cell lines in vitro and in H. pylori-infected human gastric mucosa in vivo. The level of cellular p27 inversely correlated with cell proliferation in HS3C cells. These results demonstrate that H. pylori decreases p27 expression by modulating the DOR and thereby inhibiting histone acetylation of the p27 promoter. These findings link low gastric p27 expression levels with increased instances of gastric carcinogenesis associated with H. pylori infection.