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Long noncoding RNAs have been implicated in many of the hallmarks of cancer. Herein, we found that the expression of lncRNA152 (lnc152; a.k.a. DRAIC), which we annotated previously, is highly upregulated in luminal breast cancer (LBC) and downregulated in triple-negative breast cancer (TNBC). Knockdown of lnc152 promotes cell migration and invasion in LBC cell lines. In contrast, ectopic expression of lnc152 inhibits growth, migration, invasion, and angiogenesis in TNBC cell lines. In mice, lnc152 inhibited the growth of TNBC cell xenografts, as well as metastasis of TNBC cells in an intracardiac injection model. Transcriptome analysis of the xenografts indicated that lnc152 downregulates genes controlling angiogenesis. Using pull down assays followed by LC/MS-MS, we identified RBM47, a known tumor suppressor in breast cancer, as a lnc152-interacting protein. The effects of lnc152 in TNBC cells are mediated, in part, by regulating the expression of RBM47. Collectively, our results demonstrate that lnc152 is an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC. Implications: This study identifies lncRNA152 as an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC by upregulating the expression of the tumor suppressor RBM47. As such, lncRNA152 may serve as a biomarker to track aggressiveness of breast cancer, as well as therapeutic target for treating TNBC.

Supplementary Table from Functional Characterization of lncRNA152 as an Angiogenesis-Inhibiting Tumor Suppressor in Triple-Negative Breast Cancers

Supplementary Figure from Functional Characterization of lncRNA152 as an Angiogenesis-Inhibiting Tumor Suppressor in Triple-Negative Breast Cancers

Responses to developmental and environmental cues are dependent on precise spatiotemporal control of gene transcription. Enhancers, which comprise DNA elements bound by regulatory proteins, can activate target genes in response to these external signals. Recent studies have shown that enhancers are transcribed to produce enhancer RNAs (eRNAs). Do eRNAs play a functional role in activating gene expression or are they non-functional byproducts of nearby transcription machinery? The unstable nature of eRNAs and overreliance on knockdown approaches have made elucidating the possible functions of eRNAs challenging. Herein, we focus on studies that have cloned eRNAs to study their function as transcripts, revealing roles for eRNAs in enhancer-promoter looping, recruiting transcriptional machinery, and facilitating RNA polymerase pause release to regulate gene expression.

Complementary papers by Nguyen et al. (2021) and Baek et al. (2021) track the assembly of the pre-initiation complexes at gene promoters using single-molecule microscopy, revealing dynamic spatiotemporal regulation of transcription initiation.

Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor, making it an excellent target for endocrine therapy. When endogenous and exogenous ligands engage ERα, the receptor binds to regulatory regions in the genome known as enhancers, where it (1) associates with coregulatory proteins, (2) increases histone modifications associated with active enhancers, (3) promotes chromatin looping, (4) stimulates enhancer transcription and the production of enhancer RNAs (eRNAs), and (5) ultimately regulates target gene transcription. We have previously used global run-on sequencing (GRO-seq), a high-throughput method that identifies the location and orientation of all transcriptionally active RNA polymerases across the genome, to identify active enhancers based on enhancer transcription and enrichment of the aforementioned genomic features. In ERα-positive breast cancer cells, estrogen signaling promotes the synthesis of thousands of eRNAs that originate from ERα enhancers. We are interested in understanding the functions of these estrogen-regulated eRNAs and how they contribute to the etiology of breast cancer. Recent studies on the roles of eRNAs have suggested that they may (1) recruit regulatory proteins to enhancers; (2) promote RNA polymerase II transition; (3) regulate histone modification; and (4) increase chromosome looping. One challenge facing the field is the lack of accurate eRNA annotations and cloned full-length eRNAs; eRNAs have typically been studied as signals from genomic assays on browser tracks, not as physical entities (e.g., cDNAs) - in other words, “ghosts.” We have employed precision nuclear run-on of capped RNA (PRO-cap) to determine the transcription start sites of all expressed eRNAs. In addition, we have also used ultra-deep RNA-sequencing of polyA-depleted and polyA-enriched RNA fractions to detect eRNAs and examine for the potential splicing of eRNAs. Combining these genomic data, our annotations provide critical information for the large-scale cloning of eRNAs and testing them either by (1) tethering them to their cognate ERα enhancers by using CRISPR/dCas9 technology (i.e., cis) or (2) ectopically expressing them (i.e., trans) in MCF-7 breast cancer cells. When we tether eRNAs to their cognate ERα enhancers by using dCas9 and CRISPR guide RNAs, various aspects of ERα enhancer assembly and target gene expression are enhanced. Currently we are devising strategies to screen for functional eRNAs on a genome-wide basis to determine how they contribute to ERα enhancer assembly and function on a global scale, and ultimately identify the eRNAs that drive breast cancer initiation and growth. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K. Citation Format: Tim Y Hou, William L Kraus. Estrogen-regulated enhancer RNAs control enhancer assembly and function in breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-05-02.

Long non-coding RNAs have been implicated in many of the hallmarks of cancer. Herein, we found that the expression oflncRNA152(lnc152; a.k.a.DRAIC), which we annotated previously, is highly upregulated in luminal breast cancer (LBC) and downregulated in triple-negative breast cancer (TNBC). Knockdown oflnc152promotes cell migration and invasion in LBC cell lines. In contrast, ectopic expression oflnc152inhibits growth, migration, invasion, and angiogenesis in TNBC cell lines. In mice,lnc152inhibited the growth of TNBC cell xenografts, as well as metastasis of TNBC cells in an intracardiac injection model. Transcriptome analysis of the xenografts indicated thatlnc152downregulates genes controlling angiogenesis. Using pull down assays coupled with LC-MS/MS, we identified RBM47, a known tumor suppressor in breast cancer, as alnc152-interacting protein. The effects oflnc152in TNBC cells are mediated, in part, by regulating the expression of RBM47. Collectively, our results demonstrate thatlnc152is an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC.Statement ofSignificanceThis study identifieslncRNA152as an angiogenesis-inhibiting tumor suppressor that attenuates the aggressive cancer-related phenotypes found in TNBC by upregulating the expression of the tumor suppressor RBM47. As such,lncRNA152may serve as a biomarker to track aggressiveness of breast cancer, as well as therapeutic target for treating TNBC.

Inhibitor of DNA binding (Id) genes comprise a family of four helix–loop–helix (HLH) transcriptional inhibitors. Our earlier studies revealed a role for ID2 within the circadian system, contributing to input, output, and core clock function through its interaction with CLOCK and BMAL1. Here, we explore the contribution of ID4 to the circadian system using a targeted disruption of the Id4 gene. Attributes of the circadian clock were assessed by monitoring the locomotor activity of Id4−/− mice, and they revealed disturbances in its operation. Id4-mutant mice expressed a shorter circadian period length, attenuated phase shifts in responses to continuous and discrete photic cues, and an advanced phase angle of entrainment under a 12:12 light:dark cycle and under short and long photoperiods. To understand the basis for these properties, suprachiasmatic nucleus (SCN) and retinal structures were examined. Anatomical analysis reveals a smaller Id4−/− SCN in the width dimension, which is a finding consistent with its smaller brain. As a result of this feature, anterograde tracing in Id4−/− mice revealed retinal afferents innovate a disproportionally larger SCN area. The Id4−/− photic entrainment responses are unlikely to be due to an impaired function of the retinal pathways since Id4−/− retinal anatomy and function tested by pupillometry were similar to wild-type mice. Furthermore, these circadian characteristics are opposite to those exhibited by the Id2−/− mouse, suggesting an opposing influence of the ID4 protein within the circadian system, or, the absence of ID4 results in changes in the expression or activity of other members of the Id gene family. Expression analysis of the Id genes within the Id4−/− SCN revealed a time-of-day specific elevated Id1. It is plausible that the increased Id1 and/or absence of ID4 result in changes in interactions with bHLH canonical clock components or with targets upstream and/or downstream of the clock, thereby resulting in abnormal properties of the circadian clock and its entrainment.

To better understand the functions of non-coding enhancer RNAs (eRNAs), we annotated the estrogen-regulated eRNA transcriptome in estrogen receptor α (ERα)-positive breast cancer cells using PRO-cap and RNA sequencing. We then cloned a subset of the eRNAs identified, fused them to single guide RNAs, and targeted them to their ERα enhancers of origin using CRISPR/dCas9. Some of the eRNAs tested modulated the expression of cognate, but not heterologous, target genes after estrogen treatment by increasing ERα recruitment and stimulating p300-catalyzed H3K27 acetylation at the enhancer. We identified a ∼40 nucleotide functional eRNA regulatory motif (FERM) present in many eRNAs that was necessary and sufficient to modulate gene expression, but not the specificity of activation, after estrogen treatment. The FERM interacted with BCAS2, an RNA-binding protein amplified in breast cancers. The ectopic expression of a targeted eRNA controlling the expression of an oncogene resulted in increased cell proliferation, demonstrating the regulatory potential of eRNAs in breast cancer.

Approximately 70% of breast cancers express estrogen receptor alpha (ERα), a hormone-regulated transcription factor, making it an excellent target for endocrine therapy. When endogenous and exogenous ligands engage ERα, the receptor binds to regulatory regions in the genome known as enhancers, where it associates with coregulatory proteins, increases histone modifications associated with active enhancers, promotes chromatin looping, stimulates enhancer transcription and the production of enhancer RNAs (eRNAs), and ultimately regulates target gene transcription. We have previously used global run-on sequencing (GRO-seq), a high-throughput method that identifies the location and orientation of all transcriptionally active RNA polymerases across the genome, to identify active enhancers based on enhancer transcription and enrichment of the aforementioned genomic features. Current studies on the roles of eRNAs have suggested that eRNAs may 1) recruit regulatory proteins to enhancers; 2) promote RNA polymerase II transition; 3) regulate histone modification; and 4) increase chromosome looping. One challenge facing the field is the lack of accurate eRNA annotations and cloned full-length eRNAs; eRNAs have typically been studied as signals from genomic assays on ‘browser tracks,’ not as physical entities (e.g., cDNAs). We have employed precision nuclear run-on of capped RNA (PRO-cap) to determine the transcription start sites of all expressed eRNAs. In addition, we have also used ultra-deep RNA-sequencing of polyA-depleted and polyA-enriched RNA fractions to detect eRNAs and examine for the potential splicing of eRNAs. Combining these genomic data, our annotations provide critical information for the large-scale cloning of eRNAs. We are now testing whether eRNAs originating from ERα enhancers may regulate target gene expression, either in cis or in trans. We cloning specific eRNAs based on our genomic annotations and testing them either by (1) tethering them to their cognate ERα enhancers by using CRISPR/dCas9 technology (i.e., cis) or (2) ectopically expressing them (i.e., trans) in MCF-7 breast cancer cells. Ultimately, our genomic annotation and biochemical tools will allow us to determine the mechanisms by which eRNA contribute to ERα enhancer assembly and function. This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K.

n-3 polyunsaturated fatty acids (PUFA) have been shown in many clinical studies to attenuate inflammatory responses. Although inflammatory responses are orchestrated by a wide spectrum of cells, CD4(+) T cells play an important role in the etiology of many chronic inflammatory diseases such as inflammatory bowel disease and obesity. In light of recent concerns over the safety profiles of non-steroidal anti-inflammatory drugs (NSAIDs), alternatives such as bioactive nutraceuticals are becoming more attractive. In order for these agents to be accepted into mainstream medicine, however, the mechanisms by which nutraceuticals such as n-3 PUFA exert their anti-inflammatory effects must be fully elucidated. Lipid rafts are nanoscale, dynamic domains in the plasma membrane that are formed through favorable lipid-lipid (cholesterol, sphingolipids, and saturated fatty acids) and lipid-protein (membrane-actin cytoskeleton) interactions. These domains optimize the clustering of signaling proteins at the membrane to facilitate efficient cell signaling which is required for CD4(+) T cell activation and differentiation. This review summarizes novel emerging data documenting the ability of n-3 PUFA to perturb membrane-cytoskeletal structure and function in CD4(+) T cells. An understanding of these underlying mechanisms will provide a rationale for the use of n-3 PUFA in the treatment of chronic inflammation.

The International Agency for Research on Cancer recently released an assessment classifying red and processed meat as “carcinogenic to humans” on the basis of the positive association between increased consumption and risk for colorectal cancer. Diet, however, can also decrease the risk for colorectal cancer and be used as a chemopreventive strategy. Bioactive dietary molecules, such as n-3 polyunsaturated fatty acids, curcumin, and fermentable fiber, have been proposed to exert chemoprotective effects, and their molecular mechanisms have been the focus of research in the dietary/chemoprevention field. Using these bioactives as examples, this review surveys the proposed mechanisms by which they exert their effects, from the nucleus to the cellular membrane. In addition, we discuss emerging technologies involving the culturing of colonic organoids to study the physiological effects of dietary bioactives. Finally, we address future challenges to the field regarding the identification of additional molecular mechanisms and other bioactive dietary molecules that can be utilized in our fight to reduce the incidence of colorectal cancer.

The mechanisms by which n-3 polyunsaturated fatty acids (n-3 PUFA), abundant in fish oil, exert their anti-inflammatory effects have not been rigorously defined. We have previously demonstrated that n-3 PUFA decrease the amount of phosphatidylinositol-(4,5)-bisphosphate, [PI(4,5)P2], in CD4(+) T cells, leading to suppressed actin remodeling upon activation. Since discrete pools of PI(4,5)P2 exist in the plasma membrane, we determined whether n-3 PUFA modulate spatial organization of PI(4,5)P2 relative to raft and non-raft domains. We used Förster resonance energy transfer (FRET) to demonstrate that lipid raft mesodomains in the plasma membrane of CD4(+) T cells enriched in n-3 PUFA display increased co-clustering of Lck(N10) and LAT(ΔCP), markers of lipid rafts. CD4(+) T cells enriched in n-3 PUFA also exhibited a depleted plasma membrane non-raft PI(4,5)P2 pool as detected by decreased co-clustering of Src(N15), a non-raft marker, and PH(PLC-δ), a PI(4,5)P2 reporter. Incubation with exogenous PI(4,5)P2 rescued the effects on the non-raft PI(4,5)P2 pool, and reversed the suppression of T cell proliferation in CD4(+) T cells enriched with n-3 PUFA. Furthermore, CD4(+) T cells isolated from mice fed a 4% docosahexaenoic acid (DHA)-enriched diet exhibited a decrease in the non-raft pool of PI(4,5)P2, and exogenous PI(4,5)P2 reversed the suppression of T cell proliferation. Finally, these effects were not due to changes to post-translational lipidation, since n-3 PUFA did not alter the palmitoylation status of signaling proteins. These data demonstrate that n-3 PUFA suppress T cell proliferation by altering plasma membrane topography and the spatial organization of PI(4,5)P2.

Estrogen-responsive breast cancer cells exhibit both basal and estrogen-regulated transcriptional programs, which lead to the transcription of many different transcription units (i.e., genes), including those that produce coding and non-coding sense (e.g., mRNA, lncRNA) and antisense (i.e., asRNA) transcripts. We have previously characterized the global basal and estrogen-regulated transcriptomes estrogen receptor alpha (ERα)-positive MCF-7 breast cancer cells. Herein, we have mined genomic data to define three classes of antisense transcription in MCF-7 cells based on where their antisense transcription termination sites reside relative to their cognate sense mRNA and lncRNA genes. These three classes differ in their response to estrogen treatment, the enrichment of a number of genomic features associated with active promoters (H3K4me3, RNA polymerase II, open chromatin architecture), and the biological functions of their cognate sense genes as analyzed by DAVID gene ontology. We further characterized two estrogen-regulated antisense transcripts arising from the MYC gene in MCF-7 cells, showing that these antisense transcripts are five-prime capped, three-prime polyadenylated, and localized to different compartments of the cell. Together, our analyses have revealed distinct classes of antisense transcription correlated to different biological processes and response to estrogen stimulation, uncovering another layer of hormone-regulated gene regulation.

Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietaryn-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conductedin vitroandin vivoexperiments to examine the effects ofn-3 PUFA on CD4+T cell membrane order, mitochondrial bioenergetics and lymphoproliferation.n-3 PUFA were incorporated into human primary CD4+T cells phospholipidsin vitroin a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses,n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 gn-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+T cell plasma membrane organisation byn-3 PUFA may modulate down-stream clonal expansion.

Cluster of differentiation 4(+) (CD4(+)) effector T-cell subsets [e.g., T-helper (Th) 1 and Th17] are implicated in autoimmune and inflammatory disorders such as multiple sclerosis, psoriasis, and rheumatoid arthritis. Interleukin (IL)-6 is a pleiotropic cytokine that induces Th17 polarization via signaling through the membrane-bound transducer glycoprotein 130 (GP130). Previously, we demonstrated that n-3 (ω-3) polyunsaturated fatty acids (PUFAs) reduce CD4(+) T-cell activation and differentiation into pathogenic Th17 cells by 25-30%. Here we report that n-3 PUFAs alter the response of CD4(+) T cells to IL-6 in a lipid raft membrane-dependent manner. Naive splenic CD4(+) T cells from fat-1 transgenic mice exhibited 30% lower surface expression of the IL-6 receptor. This membrane-bound receptor is known to be shed during cellular activation, but the release of soluble IL-6 receptor after treatment with anti-CD3 and anti-CD28 was not changed in the CD4(+) T cells from fat-1 mice, suggesting that the decrease in surface expression was not due to ectodomain release. We observed a significant 20% decrease in the association of GP130 with lipid rafts in activated fat-1 CD4(+) T cells and a 35% reduction in GP130 homodimerization, an obligate requirement for downstream signaling. The phosphorylation of signal transducer and activator of transcription 3 (STAT3), a downstream target of IL-6-dependent signaling, was also decreased by 30% in response to exogenous IL-6 in fat-1 CD4(+) T cells. Our results suggest that n-3 PUFAs suppress Th17 cell differentiation in part by reducing membrane raft-dependent responsiveness to IL-6, an essential polarizing cytokine.

Little is known about the impact of n3 (ω3) PUFAs on polarization of CD4(+) T cells into effector subsets other than Th1 and Th2. We assessed the effects of dietary fat [corn oil (CO) vs. fish oil (FO)] and fermentable fiber [cellulose (C) vs. pectin (P)] (2 × 2 design) in male C57BL/6 mice fed CO-C, CO-P, FO-C, or FO-P diets for 3 wk on the ex vivo polarization of purified splenic CD4(+) T cells (using magnetic microbeads) into regulatory T cells [Tregs; forkhead box P3 (Foxp3(+)) cells] or Th17 cells [interleukin (IL)-17A(+) and retinoic acid receptor-related orphan receptor (ROR) γτ(+) cells] by flow cytometry. Treg polarization was unaffected by diet; however, FO independently reduced the percentage of both CD4(+) IL-17A(+) (P < 0.05) and CD4(+) RORγτ(+) cells (P < 0.05). Moreover, expression of another critical Th17-cell-related transcription factor, signal transducer and activator of transcription 3, was reduced by FO. Dietary FO reduced the surface expression of both IL-6R and IL-23R on polarized Th17 cells (P ≤ 0.05), thus interfering with the promotive effects of these critical cytokines on Th17 polarization. Additionally, C57BL/6 mice fed diets enriched in eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or DHA + EPA similarly reduced Th17-cell polarization in comparison to CO by reducing expression of the Th17-cell signature cytokine (IL-17A; P = 0.0015) and transcription factor (RORγτ P = 0.02), whereas Treg polarization was unaffected. Collectively, these data show that n3 PUFAs exert a direct effect on the development of Th17 cells in healthy mice, implicating a novel n3 PUFA-dependent, anti-inflammatory mechanism of action via the suppression of the initial development of this inflammatory T-cell subset.

Arachidonic acid (20:4(Δ5,8,11,14), AA)-derived eicosanoids regulate inflammation and promote cancer development. Previous studies have targeted prostaglandin enzymes in an attempt to modulate AA metabolism. However, due to safety concerns surrounding the use of pharmaceutical agents designed to target Ptgs2 (cyclooxygenase 2) and its downstream targets, it is important to identify new targets upstream of Ptgs2. Therefore, we determined the utility of antagonizing tissue AA levels as a novel approach to suppressing AA-derived eicosanoids. Systemic disruption of the Fads1 (Δ5 desaturase) gene reciprocally altered the levels of dihomo-γ-linolenic acid (20:3(Δ8,11,14), DGLA) and AA in mouse tissues, resulting in a profound increase in 1-series-derived and a concurrent decrease in 2-series-derived prostaglandins. The lack of AA-derived eicosanoids, e.g., PGE₂ was associated with perturbed intestinal crypt proliferation, immune cell homeostasis, and a heightened sensitivity to acute inflammatory challenge. In addition, null mice failed to thrive, dying off by 12 weeks of age. Dietary supplementation with AA extended the longevity of null mice to levels comparable to wild-type mice. We propose that this new mouse model will expand our understanding of how AA and its metabolites mediate inflammation and promote malignant transformation, with the eventual goal of identifying new drug targets upstream of Ptgs2.

n−3 PUFA (polyunsaturated fatty acids), i.e. DHA (docosahexaenoic acid), found in fish oil, exhibit anti-inflammatory properties; however, the molecular mechanisms remain unclear. Since PtdIns(4,5)P2 resides in raft domains and DHA can alter the size of rafts, we hypothesized that PtdIns(4,5)P2 and downstream actin remodelling are perturbed by the incorporation of n−3 PUFA into membranes, resulting in suppressed T-cell activation. CD4+ T-cells isolated from Fat-1 transgenic mice (membranes enriched in n−3 PUFA) exhibited a 50% decrease in PtdIns(4,5)P2. Upon activation by plate-bound anti-CD3/anti-CD28 or PMA/ionomycin, Fat-1 CD4+ T-cells failed to metabolize PtdIns(4,5)P2. Furthermore, actin remodelling failed to initiate in Fat-1 CD4+ T-cells upon stimulation; however, the defect was reversed by incubation with exogenous PtdIns(4,5)P2. When Fat-1 CD4+ T-cells were stimulated with anti-CD3/anti-CD28-coated beads, WASP (Wiskott–Aldrich syndrome protein) failed to translocate to the immunological synapse. The suppressive phenotype, consisting of defects in PtdIns(4,5)P2 metabolism and actin remodelling, were recapitulated in CD4+ T-cells isolated from mice fed on a 4% DHA triacylglycerol-enriched diet. Collectively, these data demonstrate that n−3 PUFA, such as DHA, alter PtdIns(4,5)P2 in CD4+ T-cells, thereby suppressing the recruitment of WASP to the immunological synapse, and impairing actin remodelling in CD4+ T-cells.

RIG-I is a cytosolic sensor of viral RNA that plays crucial roles in the induction of type I interferons. The C-terminal domain (CTD) of RIG-I is responsible for the recognition of viral RNA with 5′ triphosphate (5′ ppp). However, the mechanism of viral RNA recognition by RIG-I is still not fully understood. Here we show that RIG-I CTD binds 5′ ppp dsRNA or ssRNA, as well as blunt-ended dsRNA, and exhibits the highest affinity for 5′ ppp dsRNA. Crystal structures of RIG-I CTD bound to 5′ ppp dsRNA with GC- and AU- rich sequences revealed that RIG-I recognizes the termini of the dsRNA and interacts with the 5′ triphosphate through extensive electrostatic interactions. Mutagenesis and RNA binding studies demonstrated that similar binding surfaces are involved in the recognition of different forms of RNA. Mutations of key residues at the RNA binding surface affected RIG-I signaling in cells.

CD4+ effector T cell subsets (e.g., Th1, Th17) are implicated in autoimmune and inflammatory disorders such as multiple sclerosis, psoriasis and rheumatoid arthritis. We have demonstrated t...

Long-chain n-3 polyunsaturated fatty acids (PUFA) have been shown to provide health benefits in a number of diseases including several forms of cancer. In this chapter we will discuss in detail some of the prominent mechanisms through which n-3 PUFA and its metabolites are believed to function in the prevention of colon tumorigenesis. At the plasma membrane, n-3 PUFA antagonize the production of inflammatory and procarcinogenic n-6 PUFA (i.e., arachidonic acid)-derived metabolites. Additionally, the highly unsaturated nature of n-3 PUFA impacts cell membrane properties and dynamics thereby altering numerous cellular functions, including intracellular signaling, cell growth, survival, and proliferation. Due to the sterically incompatible relationship between docosahexaenoic acid, sphingolipids, and cholesterol, the major constituents of lipid rafts, n-3 PUFA modulate these crucial membrane microdomains and perturb efficient signal transduction thereby eliciting the same biological effects exploited by some anti-cancer therapies. Moreover, we discuss how alterations in lipid rafts and downstream signaling impact both epithelial cells and the activation of immune cells. This is noteworthy, because chronic inflammation plays a critical role in tumorigenesis. Therefore, we also present an overview regarding the anti-inflammatory and immunomodulatory mechanisms through which n-3 PUFA perturb the tumor microenvironment and downregulate the activation of critical transcription factors and target genes with an established role in cancer development. Finally, we discuss recent evidence suggesting that n-3 PUFA in combination with other dietary bioactive nutrients, such as soluble fiber and curcumin, could be beneficial in cancer prevention. Collectively, we demonstrate that dietary n-3 PUFA have utility in the prevention of cancer development through mechanisms centered at both the molecular, cellular (plasma membrane), and tissue level.

Every 4 years, researchers in the cross-disciplinary field of nutritional immunology convene for a FASEB-sponsored meeting entitled, “Nutritional Immunology: Role in Health and Disease”, which was held this summer in Carefree, AZ, USA. The scope of the conference encompassed a diverse list of research topics, including, but not restricted to, obesity and immune dysfunction, nutrient–gene interactions, mucosal immunity and a discussion of future directions for the field. Here, we summarize some of the findings shared at the conference, specifically focusing on obesity, immunological function of dietary components (n-3 polyunsaturated fatty acids and flavanoids), gut immunity and the microbiota, and relevant emerging technologies and databases.

Anopheles gambiae , the primary African vector of malaria parasites, exhibits numerous rhythmic behaviors including flight activity, swarming, mating, host seeking, egg laying, and sugar feeding. However, little work has been performed to elucidate the molecular basis for these daily rhythms. To study how gene expression is regulated globally by diel and circadian mechanisms, we have undertaken a DNA microarray analysis of An. gambiae under light/dark cycle (LD) and constant dark (DD) conditions. Adult mated, non–blood-fed female mosquitoes were collected every 4 h for 48 h, and samples were processed with DNA microarrays. Using a cosine wave-fitting algorithm, we identified 1,293 and 600 rhythmic genes with a period length of 20–28 h in the head and body, respectively, under LD conditions, representing 9.7 and 4.5% of the An. gambiae gene set. A majority of these genes was specific to heads or bodies. Examination of mosquitoes under DD conditions revealed that rhythmic programming of the transcriptome is dependent on an interaction between the endogenous clock and extrinsic regulation by the LD cycle. A subset of genes, including the canonical clock components, was expressed rhythmically under both environmental conditions. A majority of genes had peak expression clustered around the day/night transitions, anticipating dawn and dusk. Genes cover diverse biological processes such as transcription/translation, metabolism, detoxification, olfaction, vision, cuticle regulation, and immunity, and include rate-limiting steps in the pathways. This study highlights the fundamental roles that both the circadian clock and light play in the physiology of this important insect vector and suggests targets for intervention.

ID2 is a rhythmically expressed HLH transcriptional repressor. Deletion of Id2 in mice results in circadian phenotypes, highlighted by disrupted locomotor activity rhythms and an enhanced photoentrainment response. ID2 can suppress the transactivation potential of the positive elements of the clock, CLOCK-BMAL1, on mPer1 and clock-controlled gene (CCG) activity. Misregulation of CCGs is observed in Id2(-/-) liver, and mutant mice exhibit associated alterations in lipid homeostasis. These data suggest that ID2 contributes to both input and output components of the clock and that this may be via interaction with the bHLH clock proteins CLOCK and BMAL1. The aim of the present study was to explore this potential interaction. Coimmunoprecipitation analysis revealed the capability of ID2 to complex with both CLOCK and BMAL1, and mammalian two-hybrid analysis revealed direct interactions of ID2, ID1 and ID3 with CLOCK and BMAL1. Deletion of the ID2 HLH domain rendered ID2 ineffective at inhibiting CLOCK-BMAL1 transactivation, suggesting that interaction between the proteins is via the HLH region. Immunofluorescence analysis revealed overlapping localization of ID2 with CLOCK and BMAL1 in the cytoplasm. Overexpression of CLOCK and BMAL1 in the presence of ID2 resulted in a significant reduction in their nuclear localization, revealing that ID2 can sequester CLOCK and BMAL1 to the cytoplasm. Serum stimulation of Id2(-/-) mouse embryonic fibroblasts resulted in an enhanced induction of mPer1 expression. These data provide the basis for a molecular mechanism through which ID2 could regulate aspects of both clock input and output through a time-of-day specific interaction with CLOCK and BMAL1.

Id2 is a helix-loop-helix transcription factor gene expressed in a circadian manner in multiple tissues with a phase-locked relationship with canonical clock genes (1). Our previous studies have identified circadian phenotypes in Id2 null mice, including enhanced photo-entrainment and disruption of activity rhythms, and have demonstrated a potent inhibitory effect of ID proteins upon CLOCK-BMAL1 transactivation of clock gene and clock-controlled gene activity (1). We have now begun to explore the potential role that ID2 may play in specifically regulating clock output. Here we show that ID2 protein is rhythmically expressed in mouse liver. Time-of-day-specific liver gene expression in Id2+/+ and Id2−/− mice under circadian conditions was studied using DNA microarray analysis, identifying 651 differentially expressed genes, including a subset of 318 genes deemed rhythmically expressed in other studies. Examination of individual time courses reveals that these genes are dysregulated in a highly time-specific manner. A cohort of different functional groups were identified, including genes associated with glucose and lipid metabolism, e.g. serum protein Igfbp1 and lipoprotein lipase. We also reveal that the Id2−/− mice show a reduction in lipid storage in the liver and white adipose tissue, suggesting that disruption of normal circadian activity of components of lipid metabolism can result in overt physiological alterations. These data reveal a role for the transcriptional repressor ID2 as a circadian output regulator in the periphery.