Advertisement

Histone Modifications Are Associated with Δ9-Tetrahydrocannabinol-mediated Alterations in Antigen-specific T Cell Responses*

Open AccessPublished:May 19, 2014DOI:https://doi.org/10.1074/jbc.M113.545210
      Marijuana is one of the most abused drugs due to its psychotropic effects. Interestingly, it is also used for medicinal purposes. The main psychotropic component in marijuana, Δ9-tetrahydrocannabinol (THC), has also been shown to mediate potent anti-inflammatory properties. Whether the immunomodulatory activity of THC is mediated by epigenetic regulation has not been investigated previously. In this study, we employed ChIP-Seq technology to examine the in vivo effect of THC on global histone methylation in lymph node cells of mice immunized with a superantigen, staphylococcal enterotoxin B. We compared genome-wide histone H3 Lys-4, Lys-27, Lys-9, and Lys-36 trimethylation and histone H3 Lys-9 acetylation patterns in such cells exposed to THC or vehicle. Our results showed that THC treatment leads to the association of active histone modification signals to Th2 cytokine genes and suppressive modification signals to Th1 cytokine genes, indicating that such a mechanism may play a critical role in the THC-mediated switch from Th1 to Th2. At the global level, a significant portion of histone methylation and acetylation regions were altered by THC. However, the overall distribution of these histone methylation signals among the genomic features was not altered significantly by THC, suggesting that THC activates the expression of a subset of genes while suppressing the expression of another subset of genes through histone modification. Functional classification of these histone marker-associated genes showed that these differentially associated genes were involved in various cellular functions, from cell cycle regulation to metabolism, suggesting that THC had a pleiotropic effect on gene expression in immune cells. Altogether, the current study demonstrates for the first time that THC may modulate immune response through epigenetic regulation involving histone modifications.

      Introduction

      Marijuana is the most frequently used illicit substance in the United States (
      • Substance Abuse and Mental Health Services Administration
      ). In addition, many states in the United States have now legalized marijuana use, especially when authorized by a physician, for medical purposes, such as alleviation of nausea and vomiting from chemotherapy, wasting in AIDS patients, and chronic pain that is unresponsive to opioids (
      • Todaro B.
      Cannabinoids in the treatment of chemotherapy-induced nausea and vomiting.
      ,
      • Cinti S.
      Medical marijuana in HIV-positive patients: what do we know?.
      ). Moreover, two states in the United States have legalized marijuana for recreational use. Thus, studies evaluating the risks and benefits of marijuana use are critical.
      Δ9-Tetrahydrocannabinol (THC),
      The abbreviations used are:
      THC
      Δ9-tetrahydrocannabinol
      H3K4me3
      histone H3 lysine 4 trimethylation
      H3K27me3
      histone H3 lysine 27 trimethylation
      H3K36me3
      histone H3 lysine 36 trimethylation
      H3K9me3
      histone H3 lysine 9 trimethylation
      H3K9ac
      histone H3 lysine 9 acetylation
      TSS
      transcription start site
      SEB
      staphylococcal enterotoxin B
      LN
      lymph node(s)
      TTS
      transcription termination site
      miRNA
      microRNA
      ER
      estrogen receptor.
      the active psychotropic ingredient of marijuana, mediates its activity through cannabinoid receptors (CB1 and CB2). Cannabinoid receptors are typical transmembrane G protein-coupled receptors. Whereas CB1 is highly expressed in the brain, and to a lower extent in peripheral tissues (
      • Galiègue S.
      • Mary S.
      • Marchand J.
      • Dussossoy D.
      • Carrière D.
      • Carayon P.
      • Bouaboula M.
      • Shire D.
      • Le Fur G.
      • Casellas P.
      Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations.
      ), CB2 is predominant in immune cells (
      • Bouaboula M.
      • Rinaldi M.
      • Carayon P.
      • Carillon C.
      • Delpech B.
      • Shire D.
      • Le Fur G.
      • Casellas P.
      Cannabinoid-receptor expression in human leukocytes.
      ). Therefore, besides its psychoactive effects, THC can suppress inflammation through activation of cannabinoid receptors on immune cells, using multiple pathways (
      • Do Y.
      • McKallip R.J.
      • Nagarkatti M.
      • Nagarkatti P.S.
      Activation through cannabinoid receptors 1 and 2 on dendritic cells triggers NF-κB-dependent apoptosis: novel role for endogenous and exogenous cannabinoids in immunoregulation.
      ,
      • Rao G.K.
      • Zhang W.
      • Kaminski N.E.
      Cannabinoid receptor-mediated regulation of intracellular calcium by Δ9-tetrahydrocannabinol in resting T cells.
      ,
      • Newton C.A.
      • Klein T.W.
      • Friedman H.
      Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by Δ-9-tetrahydrocannabinol injection.
      ). THC has been shown to suppress Th1 while promoting Th2 cells (
      • Klein T.W.
      • Newton C.A.
      • Nakachi N.
      • Friedman H.
      Δ9-Tetrahydrocannabinol treatment suppresses immunity and early IFN-γ, IL-12, and IL-12 receptor β2 responses to Legionella pneumophila infection.
      ,
      • Yuan M.
      • Kiertscher S.M.
      • Cheng Q.
      • Zoumalan R.
      • Tashkin D.P.
      • Roth M.D.
      Δ9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells.
      ). In addition, THC induces CD11b+ Gr-1+ myeloid-derived suppressor cells (
      • Hegde V.L.
      • Nagarkatti M.
      • Nagarkatti P.S.
      Cannabinoid receptor activation leads to massive mobilization of myeloid-derived suppressor cells with potent immunosuppressive properties.
      ,
      • Kusmartsev S.A.
      • Li Y.
      • Chen S.H.
      Gr-1+ myeloid cells derived from tumor-bearing mice inhibit primary T cell activation induced through CD3/CD28 costimulation.
      ,
      • Bronte V.
      • Apolloni E.
      • Cabrelle A.
      • Ronca R.
      • Serafini P.
      • Zamboni P.
      • Restifo N.P.
      • Zanovello P.
      Identification of a CD11b+/Gr-1+/CD31+ myeloid progenitor capable of activating or suppressing CD8+ T cells.
      ) as well as Tregs (
      • Hegde V.L.
      • Hegde S.
      • Cravatt B.F.
      • Hofseth L.J.
      • Nagarkatti M.
      • Nagarkatti P.S.
      Attenuation of experimental autoimmune hepatitis by exogenous and endogenous cannabinoids: involvement of regulatory T cells.
      ), which have been shown to inhibit T cell proliferation. The induction of myeloid-derived suppressor cells by THC was associated with alterations in microRNA expression (
      • Hegde V.L.
      • Tomar S.
      • Jackson A.
      • Rao R.
      • Yang X.
      • Singh U.P.
      • Singh N.P.
      • Nagarkatti P.S.
      • Nagarkatti M.
      Distinct microRNA expression profile and targeted biological pathways in functional myeloid-derived suppressor cells induced by Δ9-tetrahydrocannabinol in vivo: regulation of CCAAT/enhancer binding protein α by microRNA-690.
      ). Moreover, we also noted that prenatal exposure to THC causes T cell dysfunction in the offspring (
      • Lombard C.
      • Hegde V.L.
      • Nagarkatti M.
      • Nagarkatti P.S.
      Perinatal exposure to Δ9-tetrahydrocannabinol triggers profound defects in T cell differentiation and function in fetal and postnatal stages of life, including decreased responsiveness to HIV antigens.
      ). Together, such data suggested that THC may trigger epigenetic modulations in immune cells.
      Epigenetic modification has been implicated in the establishment and maintenance of differential gene expression in T cells (
      • Araki Y.
      • Wang Z.
      • Zang C.
      • Wood 3rd, W.H.
      • Schones D.
      • Cui K.
      • Roh T.Y.
      • Lhotsky B.
      • Wersto R.P.
      • Peng W.
      • Becker K.G.
      • Zhao K.
      • Weng N.P.
      Genome-wide analysis of histone methylation reveals chromatin state-based regulation of gene transcription and function of memory CD8+ T cells.
      ). DNA methylation and histone modifications are common epigenetic pathways leading to alterations in gene expression. Epigenetic modifications have been shown to regulate T cell differentiation by modifying the chromatin at the related genes, such as Ifn-γ, Foxp3, and IL-4 (
      • Morinobu A.
      • Kanno Y.
      • O'Shea J.J.
      Discrete roles for histone acetylation in human T helper 1 cell-specific gene expression.
      ). Genome-wide histone modification studies using the ChIP-Seq method in human T cells have linked histone methylation patterns to the specific gene activity in different T cell subtypes (
      • Araki Y.
      • Wang Z.
      • Zang C.
      • Wood 3rd, W.H.
      • Schones D.
      • Cui K.
      • Roh T.Y.
      • Lhotsky B.
      • Wersto R.P.
      • Peng W.
      • Becker K.G.
      • Zhao K.
      • Weng N.P.
      Genome-wide analysis of histone methylation reveals chromatin state-based regulation of gene transcription and function of memory CD8+ T cells.
      ,
      • Barski A.
      • Cuddapah S.
      • Cui K.
      • Roh T.Y.
      • Schones D.E.
      • Wang Z.
      • Wei G.
      • Chepelev I.
      • Zhao K.
      High-resolution profiling of histone methylations in the human genome.
      ,
      • Roh T.Y.
      • Cuddapah S.
      • Cui K.
      • Zhao K.
      The genomic landscape of histone modifications in human T cells.
      ,
      • Lim P.S.
      • Hardy K.
      • Bunting K.L.
      • Ma L.
      • Peng K.
      • Chen X.
      • Shannon M.F.
      Defining the chromatin signature of inducible genes in T cells.
      ). Histone methylation mainly occurs on the lysine and arginine residues, and lysines can be mono-, di-, or trimethylated. Histone H3 methylation on lysine 4, lysine 9, lysine 27, and lysine 36 are among the most extensively studied histone methylations (
      • Greer E.L.
      • Shi Y.
      Histone methylation: a dynamic mark in health, disease and inheritance.
      ). In general, histone H3 lysine 4 trimethylation (H3K4me3) in the promoter region is associated with transcription activation, whereas histone H3 lysine 27 trimethylation (H3K27me3) within the promoter region is associated with transcription repression. However, H3K4me3 and H3K27me3 that seem to be associated with opposite functions can co-exist in the same regions. This so-called “bivalent domain” has been found in embryonic stem cells and T cells and are proposed to lead to activation or suppression (
      • Bernstein B.E.
      • Mikkelsen T.S.
      • Xie X.
      • Kamal M.
      • Huebert D.J.
      • Cuff J.
      • Fry B.
      • Meissner A.
      • Wernig M.
      • Plath K.
      • Jaenisch R.
      • Wagschal A.
      • Feil R.
      • Schreiber S.L.
      • Lander E.S.
      A bivalent chromatin structure marks key developmental genes in embryonic stem cells.
      ,
      • Roh T.Y.
      • Wei G.
      • Farrell C.M.
      • Zhao K.
      Genome-wide prediction of conserved and nonconserved enhancers by histone acetylation patterns.
      ,
      • Wei G.
      • Wei L.
      • Zhu J.
      • Zang C.
      • Hu-Li J.
      • Yao Z.
      • Cui K.
      • Kanno Y.
      • Roh T.Y.
      • Watford W.T.
      • Schones D.E.
      • Peng W.
      • Sun H.W.
      • Paul W.E.
      • O'Shea J.J.
      • Zhao K.
      Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells.
      ). Histone lysine 36 trimethylation (H3K36me3) has been linked to transcription elongation and is enriched in the body of active transcripts (
      • Bannister A.J.
      • Schneider R.
      • Myers F.A.
      • Thorne A.W.
      • Crane-Robinson C.
      • Kouzarides T.
      Spatial distribution of di- and trimethyl lysine 36 of histone H3 at active genes.
      ,
      • Mikkelsen T.S.
      • Ku M.
      • Jaffe D.B.
      • Issac B.
      • Lieberman E.
      • Giannoukos G.
      • Alvarez P.
      • Brockman W.
      • Kim T.K.
      • Koche R.P.
      • Lee W.
      • Mendenhall E.
      • O'Donovan A.
      • Presser A.
      • Russ C.
      • Xie X.
      • Meissner A.
      • Wernig M.
      • Jaenisch R.
      • Nusbaum C.
      • Lander E.S.
      • Bernstein B.E.
      Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.
      ). Histone lysine 9 trimethylation (H3K9me3) has been linked to the silencing of the gene. This mark is enriched in the telomeric region and terminal repeats (
      • Barski A.
      • Cuddapah S.
      • Cui K.
      • Roh T.Y.
      • Schones D.E.
      • Wang Z.
      • Wei G.
      • Chepelev I.
      • Zhao K.
      High-resolution profiling of histone methylations in the human genome.
      ,
      • Mikkelsen T.S.
      • Ku M.
      • Jaffe D.B.
      • Issac B.
      • Lieberman E.
      • Giannoukos G.
      • Alvarez P.
      • Brockman W.
      • Kim T.K.
      • Koche R.P.
      • Lee W.
      • Mendenhall E.
      • O'Donovan A.
      • Presser A.
      • Russ C.
      • Xie X.
      • Meissner A.
      • Wernig M.
      • Jaenisch R.
      • Nusbaum C.
      • Lander E.S.
      • Bernstein B.E.
      Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.
      ,
      • Vakoc C.R.
      • Mandat S.A.
      • Olenchock B.A.
      • Blobel G.A.
      Histone H3 lysine 9 methylation and HP1γ are associated with transcription elongation through mammalian chromatin.
      ,
      • Wang Z.
      • Zang C.
      • Rosenfeld J.A.
      • Schones D.E.
      • Barski A.
      • Cuddapah S.
      • Cui K.
      • Roh T.Y.
      • Peng W.
      • Zhang M.Q.
      • Zhao K.
      Combinatorial patterns of histone acetylations and methylations in the human genome.
      ). However, it has been shown that H3K9me3 is also enriched in many promoters (
      • Squazzo S.L.
      • O'Geen H.
      • Komashko V.M.
      • Krig S.R.
      • Jin V.X.
      • Jang S.W.
      • Margueron R.
      • Reinberg D.
      • Green R.
      • Farnham P.J.
      Suz12 binds to silenced regions of the genome in a cell-type-specific manner.
      ). Histone acetylation in general is associated with gene activation. One of the most well studied histone acetylation markers is histone H3 acetylation at lysine 9 (H3K9ac), which is enriched near the transcription start site (TSS) of highly expressed genes (
      • Karmodiya K.
      • Krebs A.R.
      • Oulad-Abdelghani M.
      • Kimura H.
      • Tora L.
      H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells.
      ).
      Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that triggers a massive Th1-cytokine storm leading to lethal toxic shock syndrome (
      • Fraser J.D.
      • Proft T.
      The bacterial superantigen and superantigen-like proteins.
      ). In this study, we investigated the effect of THC on SEB-induced T cell activation in vivo and determined whether THC modifies global histone methylation in activated immune cells. Using a ChIP-Seq approach, we compared genome-wide H3K4me3, H3K27me3, H3K36me3, H3K9me3, and H3K9ac patterns in SEB-activated popliteal lymph node (LN) cells in mice with or without THC pretreatment. Our data showed that a significant portion of histone methylation and acetylation regions are altered by THC treatment at the genomic level. However, the associated methylation markers, not the H3K9ac marker, in key Th1/Th2 cytokine genes are altered by THC treatment, which is consistent with the ability of THC to induce a shift in Th1-Th2 balance. Moreover, we identified many other genes whose expression may be regulated by THC through histone modification.

      DISCUSSION

      The immune response and the establishment of functionally specialized immune cell lineages are controlled by multiple transcription factors as well as epigenetic modifications, and these epigenetic modifications can be altered by various environmental factors or bioactive drug components. In this study, we examined the effect of THC on four histone methylation markers and one histone acetylation marker across the whole genome in SEB superantigen-activated lymph node cells in vivo. A significant amount of histone modification clusters were found to be unique to THC treatment. These results suggested that THC could specifically activate or suppress the expression of genes.
      THC has been shown to have anti-inflammatory and immunosuppression properties and to induce apoptosis of immune cells (
      • Nagarkatti P.
      • Pandey R.
      • Rieder S.A.
      • Hegde V.L.
      • Nagarkatti M.
      Cannabinoids as novel anti-inflammatory drugs.
      ). Indeed, the size of the popliteal lymph node was smaller, and the cell number was lower in SEB + THC-treated mice than that in the SEB + vehicle-treated mice. The histone methylation pattern in several proinflammatory and anti-inflammatory cytokines was consistent with data that indicated that THC suppressed proinflammatory cells such as Th1. H3K27me3, the suppression marker, was the only signal present in the promoter of Ifn-γ in the SEB + THC-treated sample in this study, and the expression of Ifn-γ was suppressed, although SEB is a potent agent to induce inflammation. In contrast, the Ifn-γ promoter in the SEB + vehicle-activated lymphocytes had both H3K4me3 and H3K27me3. The bivalent modification of H3K4me3 and H3K27me3 in the promoter of Ifn-γ suggested that the expression of Ifn-γ can be quickly modulated according to the external signal. Similarly, TBX21, a Th1-specific transcription factor that controls the expression of Ifn-γ, also had this bivalent modification in the SEB + vehicle-treated sample. This kind of modification might be critical for a balanced immune response because prolonged expression of proinflammatory cytokines can have adverse effects on the host. Despite a significant difference in overall H3K9ac pattern in vehicle- and THC-treated cells, we did not find a difference in the association of H3K9ac in these genes. The unexpected decrease of H3K9ac signal near the TSS of SEB + vehicle-treated cells may indicate that SEB affects the function of enzymes that regulate histone acetylation and deacetylation, and THC may partially relieve that effect. In the future, we will use other antigens to activate the immune cells to determine whether the H3K9ac pattern in this experiment is unique to SEB stimulation.
      In this study, we identified many genes with bivalent modification. H3K4me3 and H3K27me3 bivalent modification has been proposed to explain the plasticity of T cell differentiation, and genes with bivalent modification can be either expressed or silenced (
      • Wei G.
      • Wei L.
      • Zhu J.
      • Zang C.
      • Hu-Li J.
      • Yao Z.
      • Cui K.
      • Kanno Y.
      • Roh T.Y.
      • Watford W.T.
      • Schones D.E.
      • Peng W.
      • Sun H.W.
      • Paul W.E.
      • O'Shea J.J.
      • Zhao K.
      Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells.
      ). However, our study also demonstrated that some genes are oppositely modified in SEB + vehicle and SEB + THC samples. For example, the promoter of Brca2 had active H3K4me3 marker in the SEB + vehicle-treated sample but had suppressive H3K27me3 marker in the SEB + THC-treated sample. Whereas Cbx-1 had H3K27me3 in the SEB + vehicle-treated sample, it had H3K4me3 in the SEB + THC-treated sample. This suggested that the expression of these genes could be permanently altered by THC. Determination of whether this is the case, however, requires further investigation.
      It is known that many histone modifications can independently regulate gene expression. For example, in human CD8+ T cells, some active genes are associated with high levels of H3K4me3, whereas others are associated with H3K9ac (
      • Araki Y.
      • Wang Z.
      • Zang C.
      • Wood 3rd, W.H.
      • Schones D.
      • Cui K.
      • Roh T.Y.
      • Lhotsky B.
      • Wersto R.P.
      • Peng W.
      • Becker K.G.
      • Zhao K.
      • Weng N.P.
      Genome-wide analysis of histone methylation reveals chromatin state-based regulation of gene transcription and function of memory CD8+ T cells.
      ). That may explain the lack of histone methylation markers in Rorc while its expression is down-regulated by THC. It is possible that it is associated with other epigenetic modifications, such as other histone acetylation markers and DNA methylation.
      Long noncoding RNAs and miRNAs are parts of the epigenetic regulation mechanism. Bic is a long noncoding RNA whose expression is elevated in the activated T cells (
      • Tam W.
      Identification and characterization of human BIC, a gene on chromosome 21 that encodes a noncoding RNA.
      ,
      • Haasch D.
      • Chen Y.W.
      • Reilly R.M.
      • Chiou X.G.
      • Koterski S.
      • Smith M.L.
      • Kroeger P.
      • McWeeny K.
      • Halbert D.N.
      • Mollison K.W.
      • Djuric S.W.
      • Trevillyan J.M.
      T cell activation induces a noncoding RNA transcript sensitive to inhibition by immunosuppressant drugs and encoded by the proto-oncogene, BIC.
      ). Bic can be further processed into miR-155. It has been shown that Bic/miR-155 is essential for immune function, and mice with deficiency in Bic/miR-155 are immunodeficient (
      • Rodriguez A.
      • Vigorito E.
      • Clare S.
      • Warren M.V.
      • Couttet P.
      • Soond D.R.
      • van Dongen S.
      • Grocock R.J.
      • Das P.P.
      • Miska E.A.
      • Vetrie D.
      • Okkenhaug K.
      • Enright A.J.
      • Dougan G.
      • Turner M.
      • Bradley A.
      Requirement of bic/microRNA-155 for normal immune function.
      ). In a study of vulvar lichen sclerosus and lichen planus, autoimmune disorders that are characterized by a strong Th1 response, the expression of Bic/miR-155 was profoundly elevated (
      • Terlou A.
      • Santegoets L.A.
      • van der Meijden W.I.
      • Heijmans-Antonissen C.
      • Swagemakers S.M.
      • van der Spek P.J.
      • Ewing P.C.
      • van Beurden M.
      • Helmerhorst T.J.
      • Blok L.J.
      An autoimmune phenotype in vulvar lichen sclerosus and lichen planus: a Th1 response and high levels of microRNA-155.
      ). miR-155 has also been shown to be overexpressed in other autoimmune diseases and to enhance inflammatory T cell development (
      • O'Connell R.M.
      • Kahn D.
      • Gibson W.S.
      • Round J.L.
      • Scholz R.L.
      • Chaudhuri A.A.
      • Kahn M.E.
      • Rao D.S.
      • Baltimore D.
      MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development.
      ). The altered histone methylation signal found in this study suggested that THC may also exert its function by regulating the expression of non-coding regulatory RNAs. Another example of histone methylation-mediated miRNA expression is miR-212 and miR-132. These miRNAs play important roles in immune response, apoptosis, and neuronal function. Expression of miR-212 enhances TRAIL-induced apoptosis, whereas inhibition of miR-212 renders cells resistant to TRAIL treatment (
      • Incoronato M.
      • Garofalo M.
      • Urso L.
      • Romano G.
      • Quintavalle C.
      • Zanca C.
      • Iaboni M.
      • Nuovo G.
      • Croce C.M.
      • Condorelli G.
      miR-212 increases tumor necrosis factor-related apoptosis-inducing ligand sensitivity in non-small cell lung cancer by targeting the antiapoptotic protein PED.
      ). miR-132 has been indicated as an early response miRNA after viral infection and has been suggested as an innate immunity regulation miRNA (
      • Lagos D.
      • Pollara G.
      • Henderson S.
      • Gratrix F.
      • Fabani M.
      • Milne R.S.
      • Gotch F.
      • Boshoff C.
      miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator.
      ). It has also been shown to potentiate anti-inflammatory signaling (
      • Shaked I.
      • Meerson A.
      • Wolf Y.
      • Avni R.
      • Greenberg D.
      • Gilboa-Geffen A.
      • Soreq H.
      MicroRNA-132 potentiates cholinergic anti-inflammatory signaling by targeting acetylcholinesterase.
      ). Results from miR-212 and miR-132 knock-out mice indicated that these miRNAs regulate synaptic transmission and plasticity (
      • Remenyi J.
      • van den Bosch M.W.
      • Palygin O.
      • Mistry R.B.
      • McKenzie C.
      • Macdonald A.
      • Hutvagner G.
      • Arthur J.S.
      • Frenguelli B.G.
      • Pankratov Y.
      miR-132/212 knockout mice reveal roles for these miRNAs in regulating cortical synaptic transmission and plasticity.
      ). Altered histone methylation signal in their transcription start sites after THC treatment suggested that THC could exert a broad biological effect by modulating miRNA expression.
      In this study, we found that some genes have all four histone H3 methylations, whereas others only have one type of methylation signal. It is unclear whether the regulation of genes with more epigenetic modifications has greater complexity than those with fewer modification signals. It is also not clear whether genes with two active markers, such as H3K4me3 and H3K36me3 are more active than those with only one marker. It is also possible that the multiple modification signals may come from different types of cells found in the lymph node.
      In summary, we demonstrate the association between THC-mediated histone modifications and a switch from Th1 to Th2 response against bacterial superantigen. The precise mechanisms through which THC regulates histone methylation remain to be further addressed. In the current study, we examined the expression of some major histone methyltranferase, demethylase, acetyltransferase, and deacetylase that are known to control these histone modifications (
      • Shahbazian M.D.
      • Grunstein M.
      Functions of site-specific histone acetylation and deacetylation.
      ,
      • Klose R.J.
      • Zhang Y.
      Regulation of histone methylation by demethylimination and demethylation.
      ) and found that THC treatment failed to alter the expression of these enzymes, as determined by real-time PCR. However, it is possible that the expression of other enzymes might be altered by THC. In addition, THC could modulate the functional activity of these enzymes. Some studies suggested that THC could act directly on the epigenetic modification machinery. For example, anandamide, an endocannabinoid, has been shown to increase the DNA methylation level in human keratinocytes through p38 (
      • Paradisi A.
      • Pasquariello N.
      • Barcaroli D.
      • Maccarrone M.
      Anandamide regulates keratinocyte differentiation by inducing DNA methylation in a CB1 receptor-dependent manner.
      ). As for histone modification, it has been shown that agonists of cannabinoid receptors can increase the number of H3K9me3-positive glioma stem-like cells, and this effect is blocked by CB antagonists (
      • Aguado T.
      • Carracedo A.
      • Julien B.
      • Velasco G.
      • Milman G.
      • Mechoulam R.
      • Alvarez L.
      • Guzmán M.
      • Galve-Roperh I.
      Cannabinoids induce glioma stem-like cell differentiation and inhibit gliomagenesis.
      ). Interestingly, in four histone markers examined in this study, THC had the most profound effect on H3K9me3. Another example for the role of cannabinoids in histone modification is the association of increased overall histone H3 acetylation and decreased level of CB1 in Huntington disease (
      • Sadri-Vakili G.
      • Bouzou B.
      • Benn C.L.
      • Kim M.O.
      • Chawla P.
      • Overland R.P.
      • Glajch K.E.
      • Xia E.
      • Qiu Z.
      • Hersch S.M.
      • Clark T.W.
      • Yohrling G.J.
      • Cha J.H.
      Histones associated with downregulated genes are hypo-acetylated in Huntington's disease models.
      ), suggesting that cannabinoid signaling could affect histone acetylation enzymes. Furthermore, THC has been shown to alter histone deacetylase 3 in a dose-dependent manner (
      • Khare M.
      • Taylor A.H.
      • Konje J.C.
      • Bell S.C.
      Δ9-Tetrahydrocannabinol inhibits cytotrophoblast cell proliferation and modulates gene transcription.
      ). Histone deacetylase 3 is a member of the histone deacetylase family and, along with other histone deacetylases, is responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (
      • Guenther M.G.
      • Lazar M.A.
      Biochemical isolation and analysis of a nuclear receptor corepressor complex.
      ). Although we did not identify a significant change in the expression of Sirt1, the major deacetylase responsible for H3K9ac deacetylation in this study, we did observe a significant change in overall H3K9ac pattern after THC treatment (Fig. 4, b and c). Determination of whether the expression and activity of other histone acetylation enzymes are altered by THC requires further investigation. Another piece of evidence that suggests that cannabinoids may directly regulate epigenetic modification comes from cannabinoid receptor knock-out mice. In CB1 knock-out mice, it has been shown that CB1 regulates chromatin remodeling during spermiogenesis (
      • Chioccarelli T.
      • Cacciola G.
      • Altucci L.
      • Lewis S.E.
      • Simon L.
      • Ricci G.
      • Ledent C.
      • Meccariello R.
      • Fasano S.
      • Pierantoni R.
      • Cobellis G.
      Cannabinoid receptor 1 influences chromatin remodeling in mouse spermatids by affecting content of transition protein 2 mRNA and histone displacement.
      ).
      As for THC-mediated alteration in histone methylation, currently there is no study that indicates that THC directly regulates the expression or activity of histone methyltransferases or demethylases. However, THC could indirectly regulate the activity of enzymes involved in histone methylation. For example, cannabinoids have been shown to down-regulate the PI3K/AKT signaling pathway (
      • Ellert-Miklaszewska A.
      • Kaminska B.
      • Konarska L.
      Cannabinoids down-regulate PI3K/Akt and Erk signalling pathways and activate proapoptotic function of Bad protein.
      ,
      • Greenhough A.
      • Patsos H.A.
      • Williams A.C.
      • Paraskeva C.
      The cannabinoid Δ9-tetrahydrocannabinol inhibits RAS-MAPK and PI3K-AKT survival signalling and induces BAD-mediated apoptosis in colorectal cancer cells.
      ), a pathway also known to cause global alterations of H3K27me3 (
      • Zuo T.
      • Liu T.M.
      • Lan X.
      • Weng Y.I.
      • Shen R.
      • Gu F.
      • Huang Y.W.
      • Liyanarachchi S.
      • Deatherage D.E.
      • Hsu P.Y.
      • Taslim C.
      • Ramaswamy B.
      • Shapiro C.L.
      • Lin H.J.
      • Cheng A.S.
      • Jin V.X.
      • Huang T.H.
      Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer.
      ). On the other hand, some studies showed that administration of THC increases phosphorylation of AKT in the mouse brain through CB1 (
      • Ozaita A.
      • Puighermanal E.
      • Maldonado R.
      Regulation of PI3K/Akt/GSK-3 pathway by cannabinoids in the brain.
      ). The discrepancy regarding the role of THC in AKT signaling may be due to the difference in cell type. Nonetheless, the effect of THC on AKT pathways may lead to regulation of histone methylation. AKT can phosphorylate EZH2 and suppress its methyltranferase activity, which results in a decrease of H3K27me3 (
      • Cha T.L.
      • Zhou B.P.
      • Xia W.
      • Wu Y.
      • Yang C.C.
      • Chen C.T.
      • Ping B.
      • Otte A.P.
      • Hung M.C.
      Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3.
      ). AKT also targets the association of histone with CBP, which regulates histone H3 acetylation (
      • Liu Y.
      • Xing Z.B.
      • Zhang J.H.
      • Fang Y.
      Akt kinase targets the association of CBP with histone H3 to regulate the acetylation of lysine K18.
      ). Additional studies are necessary to investigate whether the activity of EZH2 is altered by THC through the AKT pathway.
      THC may also indirectly regulate histone methylation through other pathways, such as the estrogen receptor (ER) pathway. It has been shown that histone demethylases LSD1 and KDM2A are required for the induction of ER signaling after E2 stimulation (
      • Garcia-Bassets I.
      • Kwon Y.S.
      • Telese F.
      • Prefontaine G.G.
      • Hutt K.R.
      • Cheng C.S.
      • Ju B.G.
      • Ohgi K.A.
      • Wang J.
      • Escoubet-Lozach L.
      • Rose D.W.
      • Glass C.K.
      • Fu X.D.
      • Rosenfeld M.G.
      Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors.
      ). On the other hand, histone demethylase, KDM4B, is induced in an ER-α-dependent manner after E2 stimulation (
      • Kawazu M.
      • Saso K.
      • Tong K.I.
      • McQuire T.
      • Goto K.
      • Son D.O.
      • Wakeham A.
      • Miyagishi M.
      • Mak T.W.
      • Okada H.
      Histone demethylase JMJD2B functions as a co-factor of estrogen receptor in breast cancer proliferation and mammary gland development.
      ), indicating that activation of the ER pathway modulates histone methylation status. Many studies have shown that cannabinoid and estrogen pathways regulate each other. For example, some studies have suggested that both crude cannabis extract and THC inhibit the binding of estradiol to estradiol receptors in vivo (
      • Sauer M.A.
      • Rifka S.M.
      • Hawks R.L.
      • Cutler Jr., G.B.
      • Loriaux D.L.
      Marijuana: interaction with the estrogen receptor.
      ,
      • von Bueren A.O.
      • Schlumpf M.
      • Lichtensteiger W.
      Δ9-Tetrahydrocannabinol inhibits 17β-estradiol-induced proliferation and fails to activate androgen and estrogen receptors in MCF7 human breast cancer cells.
      ). Recent studies showed that some estrogen receptor modulators can bind to cannabinoid receptors (
      • Kumar P.
      • Song Z.H.
      CB2 cannabinoid receptor is a novel target for third-generation selective estrogen receptor modulators bazedoxifene and lasofoxifene.
      ,
      • Prather P.L.
      • FrancisDevaraj F.
      • Dates C.R.
      • Greer A.K.
      • Bratton S.M.
      • Ford B.M.
      • Franks L.N.
      • Radominska-Pandya A.
      CB1 and CB2 receptors are novel molecular targets for tamoxifen and 4OH-tamoxifen.
      ). These results have raised the possibility that THC could regulate histone methylation through ER signaling.
      Thus, the current study opens new avenues to investigate the epigenetic pathways through which THC regulates the immune response. Because histone modifications can occur at many sites and at different levels, additional studies are necessary to address this because the current study focused on only certain histone markers. Second, the regulation of enzymes involved in histone modifications is very complex; thus, further investigations are necessary.

      REFERENCES

        • Substance Abuse and Mental Health Services Administration
        Results from the 2009 National Survey on Drug Use and Heath: Summary of National Findings. United States Department of Health and Human Services, Washington, D. C2010
        • Todaro B.
        Cannabinoids in the treatment of chemotherapy-induced nausea and vomiting.
        J. Natl. Compr. Canc. Netw. 2012; 10: 487-492
        • Cinti S.
        Medical marijuana in HIV-positive patients: what do we know?.
        J. Int. Assoc. Physicians AIDS Care (Chic.). 2009; 8: 342-346
        • Galiègue S.
        • Mary S.
        • Marchand J.
        • Dussossoy D.
        • Carrière D.
        • Carayon P.
        • Bouaboula M.
        • Shire D.
        • Le Fur G.
        • Casellas P.
        Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations.
        Eur. J. Biochem. 1995; 232: 54-61
        • Bouaboula M.
        • Rinaldi M.
        • Carayon P.
        • Carillon C.
        • Delpech B.
        • Shire D.
        • Le Fur G.
        • Casellas P.
        Cannabinoid-receptor expression in human leukocytes.
        Eur. J. Biochem. 1993; 214: 173-180
        • Do Y.
        • McKallip R.J.
        • Nagarkatti M.
        • Nagarkatti P.S.
        Activation through cannabinoid receptors 1 and 2 on dendritic cells triggers NF-κB-dependent apoptosis: novel role for endogenous and exogenous cannabinoids in immunoregulation.
        J. Immunol. 2004; 173: 2373-2382
        • Rao G.K.
        • Zhang W.
        • Kaminski N.E.
        Cannabinoid receptor-mediated regulation of intracellular calcium by Δ9-tetrahydrocannabinol in resting T cells.
        J. Leukoc. Biol. 2004; 75: 884-892
        • Newton C.A.
        • Klein T.W.
        • Friedman H.
        Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by Δ-9-tetrahydrocannabinol injection.
        Infect. Immun. 1994; 62: 4015-4020
        • Klein T.W.
        • Newton C.A.
        • Nakachi N.
        • Friedman H.
        Δ9-Tetrahydrocannabinol treatment suppresses immunity and early IFN-γ, IL-12, and IL-12 receptor β2 responses to Legionella pneumophila infection.
        J. Immunol. 2000; 164: 6461-6466
        • Yuan M.
        • Kiertscher S.M.
        • Cheng Q.
        • Zoumalan R.
        • Tashkin D.P.
        • Roth M.D.
        Δ9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells.
        J. Neuroimmunol. 2002; 133: 124-131
        • Hegde V.L.
        • Nagarkatti M.
        • Nagarkatti P.S.
        Cannabinoid receptor activation leads to massive mobilization of myeloid-derived suppressor cells with potent immunosuppressive properties.
        Eur. J. Immunol. 2010; 40: 3358-3371
        • Kusmartsev S.A.
        • Li Y.
        • Chen S.H.
        Gr-1+ myeloid cells derived from tumor-bearing mice inhibit primary T cell activation induced through CD3/CD28 costimulation.
        J. Immunol. 2000; 165: 779-785
        • Bronte V.
        • Apolloni E.
        • Cabrelle A.
        • Ronca R.
        • Serafini P.
        • Zamboni P.
        • Restifo N.P.
        • Zanovello P.
        Identification of a CD11b+/Gr-1+/CD31+ myeloid progenitor capable of activating or suppressing CD8+ T cells.
        Blood. 2000; 96: 3838-3846
        • Hegde V.L.
        • Hegde S.
        • Cravatt B.F.
        • Hofseth L.J.
        • Nagarkatti M.
        • Nagarkatti P.S.
        Attenuation of experimental autoimmune hepatitis by exogenous and endogenous cannabinoids: involvement of regulatory T cells.
        Mol. Pharmacol. 2008; 74: 20-33
        • Hegde V.L.
        • Tomar S.
        • Jackson A.
        • Rao R.
        • Yang X.
        • Singh U.P.
        • Singh N.P.
        • Nagarkatti P.S.
        • Nagarkatti M.
        Distinct microRNA expression profile and targeted biological pathways in functional myeloid-derived suppressor cells induced by Δ9-tetrahydrocannabinol in vivo: regulation of CCAAT/enhancer binding protein α by microRNA-690.
        J. Biol. Chem. 2013; 288: 36810-36826
        • Lombard C.
        • Hegde V.L.
        • Nagarkatti M.
        • Nagarkatti P.S.
        Perinatal exposure to Δ9-tetrahydrocannabinol triggers profound defects in T cell differentiation and function in fetal and postnatal stages of life, including decreased responsiveness to HIV antigens.
        J. Pharmacol. Exp. Ther. 2011; 339: 607-617
        • Araki Y.
        • Wang Z.
        • Zang C.
        • Wood 3rd, W.H.
        • Schones D.
        • Cui K.
        • Roh T.Y.
        • Lhotsky B.
        • Wersto R.P.
        • Peng W.
        • Becker K.G.
        • Zhao K.
        • Weng N.P.
        Genome-wide analysis of histone methylation reveals chromatin state-based regulation of gene transcription and function of memory CD8+ T cells.
        Immunity. 2009; 30: 912-925
        • Morinobu A.
        • Kanno Y.
        • O'Shea J.J.
        Discrete roles for histone acetylation in human T helper 1 cell-specific gene expression.
        J. Biol. Chem. 2004; 279: 40640-40646
        • Barski A.
        • Cuddapah S.
        • Cui K.
        • Roh T.Y.
        • Schones D.E.
        • Wang Z.
        • Wei G.
        • Chepelev I.
        • Zhao K.
        High-resolution profiling of histone methylations in the human genome.
        Cell. 2007; 129: 823-837
        • Roh T.Y.
        • Cuddapah S.
        • Cui K.
        • Zhao K.
        The genomic landscape of histone modifications in human T cells.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 15782-15787
        • Lim P.S.
        • Hardy K.
        • Bunting K.L.
        • Ma L.
        • Peng K.
        • Chen X.
        • Shannon M.F.
        Defining the chromatin signature of inducible genes in T cells.
        Genome Biol. 2009; 10: R107
        • Greer E.L.
        • Shi Y.
        Histone methylation: a dynamic mark in health, disease and inheritance.
        Nat. Rev. Genet. 2012; 13: 343-357
        • Bernstein B.E.
        • Mikkelsen T.S.
        • Xie X.
        • Kamal M.
        • Huebert D.J.
        • Cuff J.
        • Fry B.
        • Meissner A.
        • Wernig M.
        • Plath K.
        • Jaenisch R.
        • Wagschal A.
        • Feil R.
        • Schreiber S.L.
        • Lander E.S.
        A bivalent chromatin structure marks key developmental genes in embryonic stem cells.
        Cell. 2006; 125: 315-326
        • Roh T.Y.
        • Wei G.
        • Farrell C.M.
        • Zhao K.
        Genome-wide prediction of conserved and nonconserved enhancers by histone acetylation patterns.
        Genome Res. 2007; 17: 74-81
        • Wei G.
        • Wei L.
        • Zhu J.
        • Zang C.
        • Hu-Li J.
        • Yao Z.
        • Cui K.
        • Kanno Y.
        • Roh T.Y.
        • Watford W.T.
        • Schones D.E.
        • Peng W.
        • Sun H.W.
        • Paul W.E.
        • O'Shea J.J.
        • Zhao K.
        Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells.
        Immunity. 2009; 30: 155-167
        • Bannister A.J.
        • Schneider R.
        • Myers F.A.
        • Thorne A.W.
        • Crane-Robinson C.
        • Kouzarides T.
        Spatial distribution of di- and trimethyl lysine 36 of histone H3 at active genes.
        J. Biol. Chem. 2005; 280: 17732-17736
        • Mikkelsen T.S.
        • Ku M.
        • Jaffe D.B.
        • Issac B.
        • Lieberman E.
        • Giannoukos G.
        • Alvarez P.
        • Brockman W.
        • Kim T.K.
        • Koche R.P.
        • Lee W.
        • Mendenhall E.
        • O'Donovan A.
        • Presser A.
        • Russ C.
        • Xie X.
        • Meissner A.
        • Wernig M.
        • Jaenisch R.
        • Nusbaum C.
        • Lander E.S.
        • Bernstein B.E.
        Genome-wide maps of chromatin state in pluripotent and lineage-committed cells.
        Nature. 2007; 448: 553-560
        • Vakoc C.R.
        • Mandat S.A.
        • Olenchock B.A.
        • Blobel G.A.
        Histone H3 lysine 9 methylation and HP1γ are associated with transcription elongation through mammalian chromatin.
        Mol. Cell. 2005; 19: 381-391
        • Wang Z.
        • Zang C.
        • Rosenfeld J.A.
        • Schones D.E.
        • Barski A.
        • Cuddapah S.
        • Cui K.
        • Roh T.Y.
        • Peng W.
        • Zhang M.Q.
        • Zhao K.
        Combinatorial patterns of histone acetylations and methylations in the human genome.
        Nat. Genet. 2008; 40: 897-903
        • Squazzo S.L.
        • O'Geen H.
        • Komashko V.M.
        • Krig S.R.
        • Jin V.X.
        • Jang S.W.
        • Margueron R.
        • Reinberg D.
        • Green R.
        • Farnham P.J.
        Suz12 binds to silenced regions of the genome in a cell-type-specific manner.
        Genome Res. 2006; 16: 890-900
        • Karmodiya K.
        • Krebs A.R.
        • Oulad-Abdelghani M.
        • Kimura H.
        • Tora L.
        H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells.
        BMC Genomics. 2012; 13: 424
        • Fraser J.D.
        • Proft T.
        The bacterial superantigen and superantigen-like proteins.
        Immunol. Rev. 2008; 225: 226-243
        • Pandey R.
        • Hegde V.L.
        • Nagarkatti M.
        • Nagarkatti P.S.
        Targeting cannabinoid receptors as a novel approach in the treatment of graft-versus-host disease: evidence from an experimental murine model.
        J. Pharmacol. Exp. Ther. 2011; 338: 819-828
        • Langmead B.
        • Trapnell C.
        • Pop M.
        • Salzberg S.L.
        Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.
        Genome Biol. 2009; 10: R25
        • Zang C.
        • Schones D.E.
        • Zeng C.
        • Cui K.
        • Zhao K.
        • Peng W.
        A clustering approach for identification of enriched domains from histone modification ChIP-Seq data.
        Bioinformatics. 2009; 25: 1952-1958
        • Blankenberg D.
        • Von Kuster G.
        • Coraor N.
        • Ananda G.
        • Lazarus R.
        • Mangan M.
        • Nekrutenko A.
        • Taylor J.
        Galaxy: a web-based genome analysis tool for experimentalists.
        Curr. Protoc. Mol. Biol. 2010; (Chapter 19, Unit 19.10): 11-21
        • Ross-Innes C.S.
        • Stark R.
        • Teschendorff A.E.
        • Holmes K.A.
        • Ali H.R.
        • Dunning M.J.
        • Brown G.D.
        • Gojis O.
        • Ellis I.O.
        • Green A.R.
        • Ali S.
        • Chin S.F.
        • Palmieri C.
        • Caldas C.
        • Carroll J.S.
        Differential oestrogen receptor binding is associated with clinical outcome in breast cancer.
        Nature. 2012; 481: 389-393
        • Shin H.
        • Liu T.
        • Manrai A.K.
        • Liu X.S.
        CEAS: cis-regulatory element annotation system.
        Bioinformatics. 2009; 25: 2605-2606
        • Liu T.
        • Ortiz J.A.
        • Taing L.
        • Meyer C.A.
        • Lee B.
        • Zhang Y.
        • Shin H.
        • Wong S.S.
        • Ma J.
        • Lei Y.
        • Pape U.J.
        • Poidinger M.
        • Chen Y.
        • Yeung K.
        • Brown M.
        • Turpaz Y.
        • Liu X.S.
        Cistrome: an integrative platform for transcriptional regulation studies.
        Genome Biol. 2011; 12: R83
        • Nagarkatti P.
        • Pandey R.
        • Rieder S.A.
        • Hegde V.L.
        • Nagarkatti M.
        Cannabinoids as novel anti-inflammatory drugs.
        Future Med. Chem. 2009; 1: 1333-1349
        • Zhu L.X.
        • Sharma S.
        • Stolina M.
        • Gardner B.
        • Roth M.D.
        • Tashkin D.P.
        • Dubinett S.M.
        Δ-9-Tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway.
        J. Immunol. 2000; 165: 373-380
        • Shahbazian M.D.
        • Grunstein M.
        Functions of site-specific histone acetylation and deacetylation.
        Annu. Rev. Biochem. 2007; 76: 75-100
        • Klose R.J.
        • Zhang Y.
        Regulation of histone methylation by demethylimination and demethylation.
        Nat. Rev. Mol. Cell Biol. 2007; 8: 307-318
        • Boyer L.A.
        • Plath K.
        • Zeitlinger J.
        • Brambrink T.
        • Medeiros L.A.
        • Lee T.I.
        • Levine S.S.
        • Wernig M.
        • Tajonar A.
        • Ray M.K.
        • Bell G.W.
        • Otte A.P.
        • Vidal M.
        • Gifford D.K.
        • Young R.A.
        • Jaenisch R.
        Polycomb complexes repress developmental regulators in murine embryonic stem cells.
        Nature. 2006; 441: 349-353
        • Lee T.I.
        • Jenner R.G.
        • Boyer L.A.
        • Guenther M.G.
        • Levine S.S.
        • Kumar R.M.
        • Chevalier B.
        • Johnstone S.E.
        • Cole M.F.
        • Isono K.
        • Koseki H.
        • Fuchikami T.
        • Abe K.
        • Murray H.L.
        • Zucker J.P.
        • Yuan B.
        • Bell G.W.
        • Herbolsheimer E.
        • Hannett N.M.
        • Sun K.
        • Odom D.T.
        • Otte A.P.
        • Volkert T.L.
        • Bartel D.P.
        • Melton D.A.
        • Gifford D.K.
        • Jaenisch R.
        • Young R.A.
        Control of developmental regulators by Polycomb in human embryonic stem cells.
        Cell. 2006; 125: 301-313
        • Vastenhouw N.L.
        • Schier A.F.
        Bivalent histone modifications in early embryogenesis.
        Curr. Opin. Cell Biol. 2012; 24: 374-386
        • Karmaus P.W.
        • Chen W.
        • Crawford R.
        • Kaplan B.L.
        • Kaminski N.E.
        Δ9-Tetrahydrocannabinol impairs the inflammatory response to influenza infection: role of antigen-presenting cells and the cannabinoid receptors 1 and 2.
        Toxicol. Sci. 2013; 131: 419-433
        • Zhu W.
        • Friedman H.
        • Klein T.W.
        Δ9-Tetrahydrocannabinol induces apoptosis in macrophages and lymphocytes: involvement of Bcl-2 and caspase-1.
        J. Pharmacol. Exp. Ther. 1998; 286: 1103-1109
        • Kozela E.
        • Juknat A.
        • Kaushansky N.
        • Rimmerman N.
        • Ben-Nun A.
        • Vogel Z.
        Cannabinoids decrease the th17 inflammatory autoimmune phenotype.
        J. Neuroimmune Pharmacol. 2013; 8: 1265-1276
        • Mercer T.R.
        • Mattick J.S.
        Structure and function of long noncoding RNAs in epigenetic regulation.
        Nat. Struct. Mol. Biol. 2013; 20: 300-307
        • Tam W.
        Identification and characterization of human BIC, a gene on chromosome 21 that encodes a noncoding RNA.
        Gene. 2001; 274: 157-167
        • Haasch D.
        • Chen Y.W.
        • Reilly R.M.
        • Chiou X.G.
        • Koterski S.
        • Smith M.L.
        • Kroeger P.
        • McWeeny K.
        • Halbert D.N.
        • Mollison K.W.
        • Djuric S.W.
        • Trevillyan J.M.
        T cell activation induces a noncoding RNA transcript sensitive to inhibition by immunosuppressant drugs and encoded by the proto-oncogene, BIC.
        Cell Immunol. 2002; 217: 78-86
        • Rodriguez A.
        • Vigorito E.
        • Clare S.
        • Warren M.V.
        • Couttet P.
        • Soond D.R.
        • van Dongen S.
        • Grocock R.J.
        • Das P.P.
        • Miska E.A.
        • Vetrie D.
        • Okkenhaug K.
        • Enright A.J.
        • Dougan G.
        • Turner M.
        • Bradley A.
        Requirement of bic/microRNA-155 for normal immune function.
        Science. 2007; 316: 608-611
        • Terlou A.
        • Santegoets L.A.
        • van der Meijden W.I.
        • Heijmans-Antonissen C.
        • Swagemakers S.M.
        • van der Spek P.J.
        • Ewing P.C.
        • van Beurden M.
        • Helmerhorst T.J.
        • Blok L.J.
        An autoimmune phenotype in vulvar lichen sclerosus and lichen planus: a Th1 response and high levels of microRNA-155.
        J. Invest. Dermatol. 2012; 132: 658-666
        • O'Connell R.M.
        • Kahn D.
        • Gibson W.S.
        • Round J.L.
        • Scholz R.L.
        • Chaudhuri A.A.
        • Kahn M.E.
        • Rao D.S.
        • Baltimore D.
        MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development.
        Immunity. 2010; 33: 607-619
        • Incoronato M.
        • Garofalo M.
        • Urso L.
        • Romano G.
        • Quintavalle C.
        • Zanca C.
        • Iaboni M.
        • Nuovo G.
        • Croce C.M.
        • Condorelli G.
        miR-212 increases tumor necrosis factor-related apoptosis-inducing ligand sensitivity in non-small cell lung cancer by targeting the antiapoptotic protein PED.
        Cancer Res. 2010; 70: 3638-3646
        • Lagos D.
        • Pollara G.
        • Henderson S.
        • Gratrix F.
        • Fabani M.
        • Milne R.S.
        • Gotch F.
        • Boshoff C.
        miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator.
        Nat. Cell Biol. 2010; 12: 513-519
        • Shaked I.
        • Meerson A.
        • Wolf Y.
        • Avni R.
        • Greenberg D.
        • Gilboa-Geffen A.
        • Soreq H.
        MicroRNA-132 potentiates cholinergic anti-inflammatory signaling by targeting acetylcholinesterase.
        Immunity. 2009; 31: 965-973
        • Remenyi J.
        • van den Bosch M.W.
        • Palygin O.
        • Mistry R.B.
        • McKenzie C.
        • Macdonald A.
        • Hutvagner G.
        • Arthur J.S.
        • Frenguelli B.G.
        • Pankratov Y.
        miR-132/212 knockout mice reveal roles for these miRNAs in regulating cortical synaptic transmission and plasticity.
        PLoS One. 2013; 8: e62509
        • Paradisi A.
        • Pasquariello N.
        • Barcaroli D.
        • Maccarrone M.
        Anandamide regulates keratinocyte differentiation by inducing DNA methylation in a CB1 receptor-dependent manner.
        J. Biol. Chem. 2008; 283: 6005-6012
        • Aguado T.
        • Carracedo A.
        • Julien B.
        • Velasco G.
        • Milman G.
        • Mechoulam R.
        • Alvarez L.
        • Guzmán M.
        • Galve-Roperh I.
        Cannabinoids induce glioma stem-like cell differentiation and inhibit gliomagenesis.
        J. Biol. Chem. 2007; 282: 6854-6862
        • Sadri-Vakili G.
        • Bouzou B.
        • Benn C.L.
        • Kim M.O.
        • Chawla P.
        • Overland R.P.
        • Glajch K.E.
        • Xia E.
        • Qiu Z.
        • Hersch S.M.
        • Clark T.W.
        • Yohrling G.J.
        • Cha J.H.
        Histones associated with downregulated genes are hypo-acetylated in Huntington's disease models.
        Hum. Mol. Genet. 2007; 16: 1293-1306
        • Khare M.
        • Taylor A.H.
        • Konje J.C.
        • Bell S.C.
        Δ9-Tetrahydrocannabinol inhibits cytotrophoblast cell proliferation and modulates gene transcription.
        Mol. Hum. Reprod. 2006; 12: 321-333
        • Guenther M.G.
        • Lazar M.A.
        Biochemical isolation and analysis of a nuclear receptor corepressor complex.
        Methods Enzymol. 2003; 364: 246-257
        • Chioccarelli T.
        • Cacciola G.
        • Altucci L.
        • Lewis S.E.
        • Simon L.
        • Ricci G.
        • Ledent C.
        • Meccariello R.
        • Fasano S.
        • Pierantoni R.
        • Cobellis G.
        Cannabinoid receptor 1 influences chromatin remodeling in mouse spermatids by affecting content of transition protein 2 mRNA and histone displacement.
        Endocrinology. 2010; 151: 5017-5029
        • Ellert-Miklaszewska A.
        • Kaminska B.
        • Konarska L.
        Cannabinoids down-regulate PI3K/Akt and Erk signalling pathways and activate proapoptotic function of Bad protein.
        Cell. Signal. 2005; 17: 25-37
        • Greenhough A.
        • Patsos H.A.
        • Williams A.C.
        • Paraskeva C.
        The cannabinoid Δ9-tetrahydrocannabinol inhibits RAS-MAPK and PI3K-AKT survival signalling and induces BAD-mediated apoptosis in colorectal cancer cells.
        Int. J. Cancer. 2007; 121: 2172-2180
        • Zuo T.
        • Liu T.M.
        • Lan X.
        • Weng Y.I.
        • Shen R.
        • Gu F.
        • Huang Y.W.
        • Liyanarachchi S.
        • Deatherage D.E.
        • Hsu P.Y.
        • Taslim C.
        • Ramaswamy B.
        • Shapiro C.L.
        • Lin H.J.
        • Cheng A.S.
        • Jin V.X.
        • Huang T.H.
        Epigenetic silencing mediated through activated PI3K/AKT signaling in breast cancer.
        Cancer Res. 2011; 71: 1752-1762
        • Ozaita A.
        • Puighermanal E.
        • Maldonado R.
        Regulation of PI3K/Akt/GSK-3 pathway by cannabinoids in the brain.
        J. Neurochem. 2007; 102: 1105-1114
        • Cha T.L.
        • Zhou B.P.
        • Xia W.
        • Wu Y.
        • Yang C.C.
        • Chen C.T.
        • Ping B.
        • Otte A.P.
        • Hung M.C.
        Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3.
        Science. 2005; 310: 306-310
        • Liu Y.
        • Xing Z.B.
        • Zhang J.H.
        • Fang Y.
        Akt kinase targets the association of CBP with histone H3 to regulate the acetylation of lysine K18.
        FEBS Lett. 2013; 587: 847-853
        • Garcia-Bassets I.
        • Kwon Y.S.
        • Telese F.
        • Prefontaine G.G.
        • Hutt K.R.
        • Cheng C.S.
        • Ju B.G.
        • Ohgi K.A.
        • Wang J.
        • Escoubet-Lozach L.
        • Rose D.W.
        • Glass C.K.
        • Fu X.D.
        • Rosenfeld M.G.
        Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors.
        Cell. 2007; 128: 505-518
        • Kawazu M.
        • Saso K.
        • Tong K.I.
        • McQuire T.
        • Goto K.
        • Son D.O.
        • Wakeham A.
        • Miyagishi M.
        • Mak T.W.
        • Okada H.
        Histone demethylase JMJD2B functions as a co-factor of estrogen receptor in breast cancer proliferation and mammary gland development.
        PLoS One. 2011; 6: e17830
        • Sauer M.A.
        • Rifka S.M.
        • Hawks R.L.
        • Cutler Jr., G.B.
        • Loriaux D.L.
        Marijuana: interaction with the estrogen receptor.
        J. Pharmacol. Exp. Ther. 1983; 224: 404-407
        • von Bueren A.O.
        • Schlumpf M.
        • Lichtensteiger W.
        Δ9-Tetrahydrocannabinol inhibits 17β-estradiol-induced proliferation and fails to activate androgen and estrogen receptors in MCF7 human breast cancer cells.
        Anticancer Res. 2008; 28: 85-89
        • Kumar P.
        • Song Z.H.
        CB2 cannabinoid receptor is a novel target for third-generation selective estrogen receptor modulators bazedoxifene and lasofoxifene.
        Biochem. Biophys. Res. Commun. 2014; 443: 144-149
        • Prather P.L.
        • FrancisDevaraj F.
        • Dates C.R.
        • Greer A.K.
        • Bratton S.M.
        • Ford B.M.
        • Franks L.N.
        • Radominska-Pandya A.
        CB1 and CB2 receptors are novel molecular targets for tamoxifen and 4OH-tamoxifen.
        Biochem. Biophys. Res. Commun. 2013; 441: 339-343