Target Gene Expression In Cells Research Articles

Exosome Determinants of Physiological Aging and Age-Related Neurodegenerative Diseases.

Aging is consistently reported as the most important independent risk factor for neurodegenerative diseases. As life expectancy has significantly increased during the last decades, neurodegenerative diseases became one of the most critical public health problem in our society. The most investigated neurodegenerative diseases during aging are Alzheimer disease (AD), Frontotemporal Dementia (FTD) and Parkinson disease (PD). The search for biomarkers has been focused so far on cerebrospinal fluid (CSF) and blood. Recently, exosomes emerged as novel biological source with increasing interest for age-related neurodegenerative disease biomarkers. Exosomes are tiny Extracellular vesicles (EVs; 30–100 nm in size) released by all cell types which originate from the endosomal compartment. They constitute important vesicles for the release and transfer of multiple (signaling, toxic, and regulatory) molecules among cells. Initially considered with merely waste disposal function, instead exosomes have been recently recognized as fundamental mediators of intercellular communication. They can move from the site of release by diffusion and be retrieved in several body fluids, where they may dynamically reflect pathological changes of cells present in inaccessible sites such as the brain. Multiple evidence has implicated exosomes in age-associated neurodegenerative processes, which lead to cognitive impairment in later life. Critically, consolidated evidence indicates that pathological protein aggregates, including Aβ, tau, and α-synuclein are released from brain cells in association with exosomes. Importantly, exosomes act as vehicles between cells not only of proteins but also of nucleic acids [DNA, mRNA transcripts, miRNA, and non-coding RNAs (ncRNAs)] thus potentially influencing gene expression in target cells. In this framework, exosomes could contribute to elucidate the molecular mechanisms underneath neurodegenerative diseases and could represent a promising source of biomarkers. Despite the involvement of exosomes in age-associated neurodegeneration, the study of exosomes and their genetic cargo in physiological aging and in neurodegenerative diseases is still in its infancy. Here, we review, the current knowledge on protein and ncRNAs cargo of exosomes in normal aging and in age-related neurodegenerative diseases.

Maternal circulating syncytiotrophoblast-derived extracellular vesicles contain biologically active 5’-tRNA halves

The placenta releases syncytiotrophoblast-derived extracellular vesicles (STB-EV) into the maternal circulation throughout gestation. STB-EV dependent signalling is believed to contribute to the widespread maternal adaptive physiological changes seen in pregnancy. Transfer RNA (tRNA) halves have been identified in vesicles released from other human and murine organ systems, which alter gene expression in target cells. Here, we characterise tRNA-half expression in STB-EV and demonstrate biological activity of a highly abundant tRNA-half. Short RNA from ex-vivo, dual-lobe placental perfusion STB-EV was sequenced, showing that most (>95%) comprised tRNA species. Whole placental tissue contained <50% tRNA species, suggesting selective packaging and export of tRNA into STB-EV. Most tRNA within STB-EV were 5′-tRNA halves cleaved at 30-32 nucleotides. The pattern of tRNA expression differed depending on the size/origin of the STB-EV; this was confirmed by qPCR. Protein synthesis was suppressed in human fibroblasts when they were cultured with a 5′-tRNA half identified from STB-EV sequencing. This study is the first to evaluate tRNA species in STB-EV. The presence of biologically active 5′-tRNA halves, specific to a vesicular origin, suggests a novel mechanism for maternal-fetal signalling in normal pregnancy.

New insights into the immunomodulatory role of exosomes in cardiovascular disease

Exosomes, nanosized lipid bilayer membranous vesicles, are secreted by a variety of cells and contain protein, lipids, mRNA, miRNA, and signaling molecules that participate in intercellular material transfer and information exchange through binding, fusion or endocytosis. Exosomes mediate the gene expression of target cells and regulate pathological and physiological processes, thereby playing a key role in the occurrence and development of various diseases. Accumulated studies has shown that exosomes hold therapeutic potential though their anti-apoptotic and anti-fibrotic roles. They also have been shown to promote angiogenesis, inhibit ventricular remodeling and improve cardiac function, as well as inhibiting local inflammation and regulating the immune response. As such, exosomes represent a new target for the treatment of cardiovascular diseases. This review summarizes the literature in this field to date, including the basic biological characteristics of exosomes, and new progress in the understanding of the mechanisms of their involvement in immune regulation in cardiovascular diseases. In this way, it servrs as a basis for future research and the development of therapeutic exosomes.

The potential role of miRNAs and exosomes in chemotherapy in ovarian cancer.

Chemoresistance is one of the major obstacles in the treatment of cancer patients. It poses a fundamental challenge to the effectiveness of chemotherapy and is often linked to relapse in patients. Chemoresistant cells can be identified in different types of cancers; however, ovarian cancer has one of the highest rates of chemoresistance-related relapse (50% of patients within 5 years). Resistance in cells can either develop through prolonged cycles of treatment or through intrinsic pathways. Mechanistically, the problem of drug resistance is complex mainly because numerous factors are involved, such as overexpression of drug efflux pumps, drug inactivation, DNA repair mechanisms and alterations to and/or mutations in the drug target. Additionally, there is strong evidence that circulating miRNAs participate in the development of chemoresistance. Recently, miRNAs have been identified in exosomes, where they are encapsulated and hence protected from degradation. These miRNAs within exosomes (exo-miRNAs) can regulate the gene expression of target cells both locally and systemically. Exo-miRNAs play an important role in disease progression and can potentially facilitate chemoresistance in cancer cells. In addition, and from a diagnostic perspective, exo-miRNAs profiles may contribute to the development of predictive models to identify responder and non-responder chemotherapy. Such model may also be used for monitoring treatment response and disease progression. Exo-miRNAs may ultimately serve as both a predictive biomarker for cancer response to therapy and as a prognostic marker for the development of chemotherapy resistance. Therefore, this review examines the potential role of exo-miRNAs in chemotherapy in ovarian cancer.

Rapid colorimetric detection of p53 protein function using DNA-gold nanoconjugates with applications for drug discovery and cancer diagnostics.

The tumor suppressor protein p53 plays a central role in preventing cancer through interaction with DNA response elements (REs) to regulate target gene expression in cells. Due to its significance in cancer biology, relentless efforts have been directed toward understanding p53-DNA interactions for the development of cancer therapeutics and diagnostics. In this paper, we report a rapid, label-free and versatile colorimetric assay to detect wildtype p53 DNA-binding function in complex solutions. The assay design is based on a concept that alters interparticle-distances between RE-AuNPs from a crosslinking effect induced through tetramerization of wildtype p53 protein (p53-WT) upon binding to canonical DNA motifs modified on gold nanoparticles (RE-AuNPs). This leads to a visible solution color change from red to blue, which is quantifiable by the UV- visible absorption spectra with a detection limit of 5 nM. Contrastingly, no color change was observed for the binding-deficient p53 mutants and non-specific proteins due to their inability to crosslink RE-AuNPs. Based on this sensing principle, we further demonstrate its utility for fast detection of drug-induced DNA binding function to cancer-associated Y220C mutant p53 protein using well-established reactivating compounds. By exploiting the dominant-negative property of mutant p53 over p53-WT and interactions with RE-AuNPs, this assay is configurable to detect low numbers of mutant p53 expressing cells in miniscule sample fractions obtained from typical core needle biopsy-sized tissues without signal attrition, alluding to the potential for biopsy sampling in cancer diagnostics or for defining cancer margins. This nanogold enabled colorimetric assay provides a facile yet robust method for studying important parameters influencing p53-DNA interactions with great promises for clinically pertinent applications.

Platelet Microparticles and miRNA Transfer in Cancer Progression: Many Targets, Modes of Action, and Effects Across Cancer Stages.

Platelet-derived microparticles (PMPs) have long been known to increase in circulation in the presence of cancer, and have been considered to be cancer promoting by multiple mechanisms including shrouding of circulating tumor cells allowing immune evasion, inducing a procoagulant state associated with increased risk for venous thromboembolic events in cancer patients, and supporting metastatic dissemination by establishment of niches for anchorage of circulating tumor cells. These modes of PMP-enhanced progression of late stage cancer are generally based on the adhesive and procoagulant surfaces of PMPs. However, it is now clear that PMPs can also act as intercellular signaling vesicles, by fusion with target cells and transfer of a broad array of platelet-derived molecular contents including growth factors, angiogenic modulators, second messengers, lipids, and nucleic acids. It is also now well established that PMPs are major repositories of microRNAs (miRNAs). In recent years, new roles of PMPs in cancer have begun emerging, primarily reflecting their ability to transfer miRNA contents and modulate gene expression in target cells, allowing PMPs to affect cancer development at many stages. PMPs have been shown to interact with and transfer miRNAs to various blood vascular cells including endothelium, macrophages and neutrophils. As each of these contributes to cancer progression, PMP-mediated miRNA transfer can affect immune response, NETosis, tumor angiogenesis, and likely other cancer-associated processes. Recently, PMP miRNA transfer was found to suppress primary tumor growth, via PMP infiltration in solid tumors, anchorage to tumor cells and direct miRNA transfer, resulting in tumor cell gene suppression and inhibition of tumor growth. This mini-review will summarize current knowledge of PMP-miRNA interactions with cancer-associated cells and effects in cancer progression, and will indicate new research directions for understanding platelet-cancer interactions.

CD63, MHC class 1, and CD47 identify subsets of extracellular vesicles containing distinct populations of noncoding RNAs

Extracellular vesicles (EVs) mediate the intercellular transfer of RNAs, which alter gene expression in target cells. EV heterogeneity has limited progress towards defining their physiological functions and utility as disease-specific biomarkers. CD63 and MHC1 are widely used as markers to purify EVs. CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis was addressed by comparing Jurkat T cell EVs captured using CD47, CD63, and MHC1 antibodies. These EV subsets have similar sizes but divergent RNA contents. Apart from differences in numbers of nonannotated transcripts, CD63+, MHC1+, and CD47+ EVs have similar overall contents of most noncoding RNA classes, but the relative enrichment of specific RNAs differs. The enrichment of micro-RNAs is highly divergent, and some including miR320a are selectively concentrated in CD47+ EVs. Small nucleolar RNAs including SNORD116@ and SNHG10 are also selectively enriched in CD47+ EVs, whereas no small nuclear RNAs are enriched in CD47+ EVs. Conversely, MHC1+ EVs are selectively enriched in a subset of tRNAs including TRE-CTC and TRR-CCG. This heterogeneity in RNA composition suggests multiple sorting mechanisms that direct specific RNAs into subsets of EVs that express specific surface markers.

Emerging Evidence of Epigenetic Modifications in Vascular Complication of Diabetes.

Genes, dietary, and lifestyle factors have been shown to be important in the pathophysiology of diabetes and associated microvascular complications. Epigenetic modifications, such as DNA methylation, histone acetylation, and post-transcriptional RNA regulation, are being increasingly recognized as important mediators of the complex interplay between genes and the environment. Recent studies suggest that diabetes-induced dysregulation of epigenetic mechanisms resulting in altered gene expression in target cells can lead to diabetes-associated complications, such as diabetic cardiomyopathy, diabetic nephropathy, retinopathy, and so on, which are the major contributors to diabetes-associated morbidity and mortality. Thus, knowledge of dysregulated epigenetic pathways involved in diabetes can provide much needed new drug targets for these diseases. In this review, we constructed our search strategy to highlight the role of DNA methylation, modifications of histones and role of non-coding RNAs (microRNAs and long non-coding RNAs) in vascular complications of diabetes, including cardiomyopathy, nephropathy, and retinopathy.

Impact of opioids on oxidative status and related signaling pathways: An integrated view.

Opioids produce reactive oxygen species (ROS) which are highly reactive molecules that damage cells and tissues, and are suggested to contribute to the opioid use disorders. Thus, antioxidant supplementation might improve the disturbance in redox (oxidation-reduction) homeostasis. However, randomized trials on antioxidant therapy have not shown beneficial effects. The purpose of this review is to shed lights on the oxidative changes resulting from opioid use and to highlight the unanswered questions regarding oxidative profile in an effort to provide a comprehensive view of different aspects of an efficient antioxidant therapy in clinical settings. The studies were identified and gathered from the PubMed database over the past 16 years (2000-2016). Our search results were limited to articles in English, both animals and human and in vitro and in vivo studies. A total of 50 full text articles were reviewed and summarized. Opioids elevate the level of ROS and decrease the function of enzymatic antioxidants such as superoxide dismutase, catalase, and glutathione peroxidase. They increase the risk of vitamin deficiency and modify gene expression of target cells through ROS production. The effects of opioids on their target cells are exerted through different way and various mechanisms. Opioids modulate the redox homeostasis; therefore, understanding the profile of oxidative changes in individuals with opioid use disorder could be of significant benefits in the clinical setting, to help with selection of an efficient antioxidant therapy and diminishing oxidative damage.

Therapeutic effects of Schisandra chinensis on the hyperprolactinemia in rat.

Prolactin (PRL) is secreted from the pituitary gland in response to eating, mating, and ovulation. Increased serum concentration of PRL during pregnancy contributes to enlargement of the mammary glands of the breasts and prepares for production of milk. However, high PRL levels derived from prolactinoma and hyperprolactinemia induce physiological disorders such as infertility and early menopause. Natural compounds isolated from S.chinensis have been known to possess anti-oxidative, anti-inflammatory and anti-diabetic effects. In the present study, we examined the therapeutic effect of S.chinensis and its single compounds on hyperprolactinemia in the pituitary gland. In rat pituitary cells, PRL expression levels were examined using real-time PCR and western blot assay. Crude S.chinensis extract and its single compound, gomisin N, reduced mRNA and protein levels of PRL in GH3 cells. In addition, cell proliferation and PRL target gene expression in cells were modulated by S.chinensis. Similar to the invitro experiments, crude S.chinensis extract and gomisin N reduced PRL levels in the pituitary and serum of immature female rats. These results show that S.chinensis and its single compound, gomisin N, are regulators of PRL production and may be candidates for treatment of hyperprolactinemia and prolactinoma.

Sulfide-responsive transcriptional repressor SqrR functions as a master regulator of sulfide-dependent photosynthesis

Sulfide was used as an electron donor early in the evolution of photosynthesis, with many extant photosynthetic bacteria still capable of using sulfur compounds such as hydrogen sulfide (H2S) as a photosynthetic electron donor. Although enzymes involved in H2S oxidation have been characterized, mechanisms of regulation of sulfide-dependent photosynthesis have not been elucidated. In this study, we have identified a sulfide-responsive transcriptional repressor, SqrR, that functions as a master regulator of sulfide-dependent gene expression in the purple photosynthetic bacterium Rhodobacter capsulatus SqrR has three cysteine residues, two of which, C41 and C107, are conserved in SqrR homologs from other bacteria. Analysis with liquid chromatography coupled with an electrospray-interface tandem-mass spectrometer reveals that SqrR forms an intramolecular tetrasulfide bond between C41 and C107 when incubated with the sulfur donor glutathione persulfide. SqrR is oxidized in sulfide-stressed cells, and tetrasulfide-cross-linked SqrR binds more weakly to a target promoter relative to unmodified SqrR. C41S and C107S R. capsulatus SqrRs lack the ability to respond to sulfide, and constitutively repress target gene expression in cells. These results establish that SqrR is a sensor of H2S-derived reactive sulfur species that maintain sulfide homeostasis in this photosynthetic bacterium and reveal the mechanism of sulfide-dependent transcriptional derepression of genes involved in sulfide metabolism.

Characterization of alendronic- and undecylenic acid coated magnetic nanoparticles for the targeted delivery of rosiglitazone to subcutaneous adipose tissue

Obesity is a state of positive energy balance where excess white adipose tissue accumulates to the detriment of metabolic health. Improving adipocyte function with systemic administration of thiazolidinediones (TZDs) improves metabolic outcomes in obesity, however TZD use is limited clinically due to undesirable side effects. Here we evaluate magnetic nanoparticles (MNPs) as a tool to target rosiglitazone (Rosi) specifically to adipose tissue. Results show Rosi can be adsorbed to MNPs (Rosi-MNPs) with hydrophobic coatings for which we present binding and release kinetics. Rosi adsorbed to MNPs retained the ability to induce PPARγ target gene expression in cells. Biodistribution analysis of radiolabeled Rosi-MNPs revealed a fat-implanted magnet significantly enhanced localization of Rosi to the targeted adipose tissue when administered by subcutaneous injection to obese mice. We propose MNPs for targeted delivery of anti-diabetic agents to superficially located subcutaneous adipose tissue.

MiRNA in Macrophage Development and Function.

MicroRNAs (miRNAs) control cellular gene expression via primarily binding to 3' or 5' untranslated region of the target transcript leading to translational repression or mRNA degradation. In most cases, miRNAs have been observed to fine-tune the cellular responses and, therefore, act as a rheostat rather than an on/off switch. Transcription factor PU.1 is a master switch that controls monocyte/macrophage development from hematopoietic stem cells. Recent Advances: PU.1 induces a specific set of miRNAs while suppressing the miR17-92 cluster to regulate monocyte/macrophage development. In addition to development, miRNAs tightly control the macrophage polarization continuum from proinflammatory M1 or proreparative M2 by regulating expression of key transcription factors involved in the process of polarization. miRNAs are intricately involved with fine-tuning fundamental macrophage functions such as phagocytosis, efferocytosis, inflammation, tissue repair, and tumor promotion. Macrophages are secretory cells that participate in intercellular communication by releasing regulatory molecules and microvesicles (MVs). MVs are bilayered lipid membranes packaging a hydrophilic cargo, including proteins and nucleic acids. Macrophage-derived MVs carry functionally active miRNAs that suppress gene expression in target cells via post-transcriptional gene silencing, thus regulating cell function. In summary, miRNAs fine-tune several major facets of macrophage development and function. Such fine-tuning is critical in preventing exaggerated macrophage response to endogenous or exogenous stimuli. A critical role of miRNAs in the regulation of innate immune response and macrophage biology, including development, differentiation, and activation, has emerged. A clear understanding of such regulation on macrophage function remains to be elucidated. Antioxid. Redox Signal. 25, 795-804.

728. Tissue-Directed Transgene Engineering for AAV and Lentivector Gene Therapy Approaches

Clinical gene therapy frequently is encumbered by low transgene product biosynthesis at predictably safe vector doses. It has been hypothesized that the presence of rare codons may regulate transgene product expression through depletion of the available cognate tRNA pool. Codon optimization is the prominent strategy utilized to overcome this hypothesized limitation and involves replacing rare, presumably translation rate-limiting, codons with the most frequent ones. Typical algorithms attempt to match the codon usage frequency of the transgene coding sequence to that of target organism's total mRNA pool, which has been shown to approximate the overall available tRNA concentrations. Upon closer examination, it appears that both codon frequency and tRNA content vary between tissue types. Therefore, we hypothesize that codon-optimization can be improved by tailoring to the codon-frequency of the most highly expressed genes present in target cell types. Our tissue-directed codon optimization algorithm utilizes novel codon usage indices generated from target cell gene expression data. As proof of concept, we developed tissue-codon optimized variants of coagulation factor VIII (FVIII) to be utilized in lenti- and adeno-associated viral (LV and AAV, respectively) vectors. These two vectors are the leading platforms for clinical gene therapy of hemophilia A. LV is utilized to target autologous hematopoietic cell types ex vivo while AAV is delivered intravenously to genetically-modify hepatocytes. However to date, development-stage LV and AAV gene therapy products for hemophilia A have been characterized by low-level FVIII transgene product biosynthesis. Initially, we designed human hepatocyte-, monocyte- and standard overall human-optimized FVIII constructs (LCO, MCO and HCO, respectively) to be compared to wild-type FVIII. Upon initial examination, expression of LCO-FVIII was shown to be 3-fold greater than either MCO- or wild-type FVIII from the human hepatoma cell line, HepG2, following transient transfection. In contrast, LCO-FVIII and MCO-FVIII expression was diminished 12 and 4-fold, respectively, compared to wild-type in the ‘neutral’ human embryonic kidney 293T cell line. Furthermore, following hydrodynamic injection of naked plasmid DNA into hemophilia A mice, LCO-FVIII exhibited a sustained 10-fold increase in FVIII expression relative to the HCO-FVIII comparator. In attempt to generate a lead candidate for clinical translation, we utilized several of our most promising vector components to construct a liver-optimized AAV8 vector consisting of LCO-ET3, our previously described high-expression bioengineered FVIII variant, transcriptionally driven by a novel 146bp liver-directed promoter and adjacent MVM intron. Following intravenous delivery into hemophilia A mice, vector doses of 1e12 and 1e11 vector genomes per kg achieved sustained, predictable curative plasma FVIII levels of 200% and 20% of normal human levels, respectively. These initial results support the utility of our novel approach of clinical tissue-directed transgene optimization.

Serum-resistant CpG-STAT3 decoy for targeting survival and immune checkpoint signaling in acute myeloid leukemia

AbstractTargeting oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) in acute myeloid leukemia (AML) can reduce blast survival and tumor immune evasion. Decoy oligodeoxynucleotides (dODNs), which comprise STAT3-specific DNA sequences are competitive inhibition of STAT3 transcriptional activity. To deliver STAT3dODN specifically to myeloid cells, we linked STAT3dODN to the Toll-like receptor 9 (TLR9) ligand, cytosine guanine dinucleotide (CpG). The CpG-STAT3dODN conjugates are quickly internalized by human and mouse TLR9+ immune cells (dendritic cells, B cells) and the majority of patients' derived AML blasts, including leukemia stem/progenitor cells. Following uptake, CpG-STAT3dODNs are released from endosomes, and bind and sequester cytoplasmic STAT3, thereby inhibiting downstream gene expression in target cells. STAT3 inhibition in patients' AML cells limits their immunosuppressive potential by reduced arginase expression, thereby partly restoring T-cell proliferation. Partly chemically modified CpG-STAT3dODNs have >60 hours serum half-life which allows for IV administration to leukemia-bearing mice (50% effective dose ∼ 2.5 mg/kg). Repeated administration of CpG-STAT3dODN resulted in regression of human MV4-11 AML in mice. The antitumor efficacy of this strategy is further enhanced in immunocompetent mice by combining direct leukemia-specific cytotoxicity with immunogenic effects of STAT3 blocking/TLR9 triggering. CpG-STAT3dODN effectively reduced Cbfb/MYH11/Mpl AML burden in various organs and eliminated leukemia stem/progenitor cells, mainly through CD8/CD4 T-cell–mediated immune responses. In contrast, small-molecule Janus kinase 2/STAT3 inhibitor failed to reproduce therapeutic effects of cell-selective CpG-STAT3dODN strategy. These results demonstrate therapeutic potential of CpG-STAT3dODN inhibitors with broad implications for treatement of AML and potentially other hematologic malignancies.

HIV-1 Infection Dysregulates Cell Cycle Regulatory Protein p21 in CD4+ T Cells Through miR-20a and miR-106b Regulation.

Both CD4+ T lymphocytes and macrophages are the major targets of human immunodeficiency virus type 1 (HIV-1); however, they respond differently to HIV-1 infection. We hypothesized that HIV-1 infection alters gene expression in CD4+ T cells and monocyte-derived macrophages (MDMs) in a cell specific manner and microRNAs (miRNAs) in part play a role in cell-specific gene expression. Results indicate that 183 and 31 genes were differentially regulated in HIV-1 infected CD4+ T cells and MDMs, respectively, compared to their mock-infected counterparts. Among the differentially expressed genes, cell cycle regulatory gene, p21 (CDKN1A) was upregulated in virus infected CD4+ T cells both at the mRNA and protein level in CD4+ T cells, whereas no consistent change was observed in MDMs. Productively infected CD4+ T cells express higher amount of p21 compared to bystander cells. In determining the mechanism(s) of cell type specific regulation of p21, we found that the miRNAs miR-106b and miR-20a that target p21 were specifically downregulated in HIV-1 infected CD4+ T cells. Overexpression of these two miRNAs reduced p21 expression significantly in HIV-1 infected CD4+ T cells. These findings provide a potential mechanism, by which, HIV-1 could exploit host cellular machineries to regulate selective gene expression in target cells. J. Cell. Biochem. 117: 1902-1912, 2016. © 2016 Wiley Periodicals, Inc.

Epigenetic Modifications the Development of Different Heart Failure Phenotypes

Heart failure (HF) remains a leading cause of death in patient population with known cardiovascular disease. Within last two decades there are evidences regarding decline to determine newel cases with HF with reduced ejection fraction (HFrEF) in developed countries, whereas the frequency of newly-diagnosed HF with preserved ejection fraction (HFpEF) exhibits dramatically rise. Epigenetic modification is considered a modification of the non- DNA sequences related heritable changes in gene expression of target cells. Epigenetic modifications affect several molecular mechanisms, i.e., DNA methylation and deactylation, ATP-dependent chromatin remodeling, histone modifications, and microRNA regulation. The short commentary is clarified the implication of epigenetic modifications in development of different HF phenotypes.

Protein kinase C modulates transcriptional activation by the juvenile hormone receptor methoprene-tolerant

Juvenile hormone (JH) controls many biological events in insects by triggering dramatic changes in gene expression in target cells. The Methoprene-tolerant (MET) protein, an intracellular JH receptor, acts as a transcriptional regulator and binds to the promoters of tissue- and stage-specific JH target genes when JH is present. Our recent study has demonstrated that the transcriptional activation by MET is modulated by a membrane-initiated JH signaling pathway, involving phospholipase C (PLC) and calcium/calmodulin-dependent protein kinase II (CaMKII). Here we report that protein kinase C (PKC) is another essential intermediate of this pathway. PKC was activated by JH and this action was PLC-dependent. Inhibition of the PKC activity substantially weakened the JH-induced gene expression in mosquito cells. RNAi experiments indicated that several PKC isoforms were involved in the JH action during the post-emergence development of adult female mosquitoes. JH treatment considerably increased the binding of MET to the promoters of JH response genes in cultured mosquito abdomens that were collected from newly emerged female adults. The JH-induced DNA binding of MET was hindered when the abdomens were treated with a PKC inhibitor and JH. Therefore, the results suggest that PKC modulates the transactivation activity of MET by enhancing the binding of MET to JH response elements in the JH target genes. This mechanism may allow for variable and stage- and tissue-specific genomic responses to JH.

Mechanisms of Enhancer-mediated Hormonal Control of Vitamin D Receptor Gene Expression in Target Cells

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), whose expression in bone cells is regulated positively by 1,25(OH)2D3, retinoic acid, and parathyroid hormone through both intergenic and intronic enhancers. In this report, we used ChIP-sequencing analysis to confirm the presence of these Vdr gene enhancers in mesenchyme-derived bone cells and to describe the epigenetic histone landscape that spans the Vdr locus. Using bacterial artificial chromosome-minigene stable cell lines, CRISPR/Cas9 enhancer-deleted daughter cell lines, transient transfection/mutagenesis analyses, and transgenic mice, we confirmed the functionality of these bone cell enhancers in vivo as well as in vitro. We also identified VDR-binding sites across the Vdr gene locus in kidney and intestine using ChIP-sequencing analysis, revealing that only one of the bone cell-type enhancers bound VDR in kidney tissue, and none were occupied by the VDR in the intestine, consistent with weak or absent regulation by the 1,25(OH)2D3 hormone in these tissues, respectively. However, a number of additional sites of VDR binding unique to either kidney or intestine were present further upstream of the Vdr gene, suggesting the potential for alternative regulatory loci. Importantly, virtually all of these regions retained histone signatures consistent with those of enhancers and exhibited unique DNase I hypersensitivity profiles that reflected the potential for chromatin access. These studies define mechanisms associated with hormonal regulation of the Vdr and hint at the differential nature of VDR binding activity at the Vdr gene in different primary target tissues in vivo.

Microguards and micromessengers of the genome.

The regulation of gene expression is of fundamental importance to maintain organismal function and integrity and requires a multifaceted and highly ordered sequence of events. The cyclic nature of gene expression is known as ‘transcription dynamics'. Disruption or perturbation of these dynamics can result in significant fitness costs arising from genome instability, accelerated ageing and disease. We review recent research that supports the idea that an important new role for small RNAs, particularly microRNAs (miRNAs), is in protecting the genome against short-term transcriptional fluctuations, in a process we term ‘microguarding'. An additional emerging role for miRNAs is as ‘micromessengers'—through alteration of gene expression in target cells to which they are trafficked within microvesicles. We describe the scant but emerging evidence that miRNAs can be moved between different cells, individuals and even species, to exert biologically significant responses. With these two new roles, miRNAs have the potential to protect against deleterious gene expression variation from perturbation and to themselves perturb the expression of genes in target cells. These interactions between cells will frequently be subject to conflicts of interest when they occur between unrelated cells that lack a coincidence of fitness interests. Hence, there is the potential for miRNAs to represent both a means to resolve conflicts of interest, as well as instigate them. We conclude by exploring this conflict hypothesis, by describing some of the initial evidence consistent with it and proposing new ideas for future research into this exciting topic.