A gene expression control technology for cell‐free systems and synthetic cells via targeted gene silencing and transfection
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Wakana Sato↗
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Results for “gene silencing”
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Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation
Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.
Non-NASA Center↗
Comparative MicroRNA Expression Patterns in Fibroblasts after Low and High Doses of Low-LET Radiation Exposure
Exposure to ionizing radiation causes DNA damage to cells, and provokes a plethora of cellular responses controlled by unique gene-directed signaling pathways. MicroRNAs (miRNAs) are small (~22-nucleotide), non-coding RNAs which functionally silence gene expression by either degrading the messages or inhibiting translation. Here we investigate radiation-dependent changes in these negative regulators by comparing the expression patterns of all 462 known human miRNAs in fibroblasts, after exposure to low (0.1 Gy) or high (2 Gy) doses of X-rays at 30 min, 2, 6 and 24 hrs post-treatment. The expression patterns of microRNAs after low and high doses of radiation show a similar qualitative down-regulation trend at early (0.5 hr) and late (24 hr) time points, with a quantitatively steeper slope following the 2 Gy exposures. Interestingly, an interruption of this downward trend is observed after the 2 Gy exposure, i.e. a significant up-regulation of microRNAs at 2 hrs, then reverting to the downward trend by 6 hrs; this interruption at the intermediate time point was not observed with the 0.1 Gy exposure. At the early time point (0.5 hr), candidate gene targets of selected down-regulated microRNAs, common to both 0.1 and 2 Gy exposures, were those functioning in chromatin remodeling. Candidate target genes of unique up-regulated microRNAs seen at a 2 hr intermediate time point, after the 2 Gy exposure only, are those involved in cell death signaling. Finally, putative target genes of down-regulated microRNAs seen at the late (24 hr) time point after either doses of radiation are those involved in the up-regulation of DNA repair, cell signaling and homeostasis. Thus we hypothesize that after radiation exposure, microRNAs acting as hub negative regulators for unique signaling pathways needed to be down-regulated so as to de-repress their target genes for the proper cellular responses, including DNA repair and cell maintenance. The unique microRNAs up-regulated at 2 hr after 2 Gy suggest the cellular response to functionally suppress the apoptotic death signaling reflex after exposure to high dose radiation. Further analyses with transcriptome and global proteomic profiling will validate the reciprocal expression of signature microRNAs selected in our radiation-exposed cells, and their candidate target gene families, and test our hypothesis that unique radiation-specific microRNAs are keys in governing signaling responses for damage control of this environmental hazard.
Maes, Olivier C.↗
Delayed translational silencing of ceruloplasmin transcript in gamma interferon-activated U937 monocytic cells: role of the 3' untranslated region
Ceruloplasmin (Cp) is an acute-phase protein with ferroxidase, amine oxidase, and pro- and antioxidant activities. The primary site of Cp synthesis in human adults is the liver, but it is also synthesized by cells of monocytic origin. We have shown that gamma interferon (IFN-gamma) induces the synthesis of Cp mRNA and protein in monocytic cells. We now report that the induced synthesis of Cp is terminated by a mechanism involving transcript-specific translational repression. Cp protein synthesis in U937 cells ceased after 16 h even in the presence of abundant Cp mRNA. RNA isolated from cells treated with IFN-gamma for 24 h exhibited a high in vitro translation rate, suggesting that the transcript was not defective. Ribosomal association of Cp mRNA was examined by sucrose centrifugation. When Cp synthesis was high, i.e., after 8 h of IFN-gamma treatment, Cp mRNA was primarily associated with polyribosomes. However, after 24 h, when Cp synthesis was low, Cp mRNA was primarily in the nonpolyribosomal fraction. Cytosolic extracts from cells treated with IFN-gamma for 24 h, but not for 8 h, contained a factor which blocked in vitro Cp translation. Inhibitor expression was cell type specific and present in extracts of human cells of myeloid origin, but not in several nonmyeloid cells. The inhibitory factor bound to the 3' untranslated region (3'-UTR) of Cp mRNA, as shown by restoration of in vitro translation by synthetic 3'-UTR added as a "decoy" and detection of a binding complex by RNA gel shift analysis. Deletion mapping of the Cp 3'-UTR indicated an internal 100-nucleotide region of the Cp 3'-UTR that was required for complex formation as well as for silencing of translation. Although transcript-specific translational control is common during development and differentiation and global translational control occurs during responses to cytokines and stress, to our knowledge, this is the first report of translational silencing of a specific transcript following cytokine activation.
NASA Discipline Regulatory Physiology↗
Uncovering Unique Molecular Adaptations in the Arabidopsis Thaliana Cvi-0 Ecotype
This research proposal aims to investigate the unique molecular adaptations exhibited by Arabidopsis Thaliana, specifically focusing on the Cape Verde Islands (Cvi-0) ecotype, in response to microgravity conditions. The study examines data from NASA’s Open Science Data Repository and applies a multifaceted RNAseq analysis pipeline using tools in the UseGalaxy.org open platform. Through transcriptomic analysis, differential gene expression patterns were identified in Cvi-0, revealing an absence of heat shock protein (HSP) upregulation and an upregulation of Rubisco Activase (RCA) and chloroplast-related pathways. To test the hypothesis that these adaptations may contribute to Cvi-0’s increased adaptability in microgravity, a three-fold experimental design is proposed. Four experimental groups will be cultivated under simulated microgravity and ground control conditions, including Cvi-0, Col-0, and genetically modified Col-0 with silenced HSP genes, and genetically modified Col-0 with upregulated RCA gene. Growth parameters will be measured to assess plant resilience, and RNA sequencing will provide transcriptomic data for pathway analysis. Anticipated outcomes include improved markers of plant health (mass, growth, etc.) of Cvi-0 in simulated microgravity and enhanced resilience in genetically altered Col-0 variants, providing insights into potential mechanisms of adaptation. This research would bear significance for space agriculture, nutrition for extended space missions, and sustainable terrestrial crop enhancement. Moreover, the insights gained could reshape crop engineering on Earth, enhancing robustness to climate induced stresses and bolstering global food security. The proposal’s trajectory blends scientific curiosity with practical applicability, forging a path towards sustainable food production and improving human exploration beyond our planet.
GL4HS↗
A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification
A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.
NASA Program Fundamental Space Biology↗
T-Cell Shenanigans: The Impact of MHC Pathway & Lipid Metabolism Genes on T-Cell Differentiation in the Thymus
Astronaut health and proper immune function are key to the success and sustainability of long term missions in space. While previous studies have observed adaptive immune dysfunction such as diminished WBC counts and cytokine production during spaceflight, the underlying mechanisms behind why immune function worsens are poorly understood. Using transcriptomic data analyzed from mouse thymus tissues available from the GLDS-421 experiment on the Rodent Research-9 mission, we used the GeneLab standardized RNA-Seq pipeline and identified four genes that were largely dysregulated (p<0.05): lipid metabolic genes Hpgd and Pgr and cell cycle regulator genes Cenpe and Kif11. We analyzed how these genes regulate the MHC-TCR interaction, and used this understanding to propose a novel mechanism for alterations to T-cell differentiation. In our experimental methodology, we propose to use 36 transgenic CD4-CD8 mice and 12 wild-type mice from the Jackson Lab to create a microgravity-simulating hindlimb unloading model and test our hypothesis. By silencing the aforementioned genes using custom siRNA based primers, we will use flow cytometry and RNA-Seq to quantify Treg counts in comparison to naive CD4+ cells, Alamar Blue assay for observing the metabolic activity in T-cells, and Enzyme-Linked Immunosorbent Assay (ELISA) to observe the general immune response in terms of T-cell differentiation. We hope this methodology will improve our knowledge of MHC-TCR interaction, contribute to a better understanding of the mechanisms of autoimmune diseases such as lupus, and improve the success rate of newer cancer treatments such as CAR-T cell therapy.
GL4HS↗
Radio-sensitization of Prostate Cancer Cells by Monensin Treatment and its associated Gene Expression Profiling Changes
Radio-resistant or recurrent prostate cancer represents a serious health risk for approximately 20%-30% of patients treated with primary radiation therapy for clinically localized prostate cancer. Here, we investigated the effect of monensin on sensitizing radiation mediated cell killing of two radio-resistant prostate cell lines Lncap (P53+ and AR+) and PC3 (P53- and AR-). Treatment with monensin alone (5 micromoles-20 micromoles) showed a significant direct cell killing of Lncap (10-30%), but not PC3 cells. Monensin was also shown to successfully sensitize Lncap cells to X-ray radiation (2Gy-10Gy) mediated cell death, up to 50% of killing with the combined treatment. To better understand the mechanisms of radio-resistance of these two cell lines and their different response to monensin, the apoptosis related gene expression profiles in both cell lines were analyzed using cDNA PCR array. Without any treatment, PC3 showed a much higher expression level of antiapoptosis genes than Lncap in the BCL2 family, the caspase/card family and the TNF ligand/receptor family. At 2 hr after 20 micormolar monensin treatment alone, only the TRAF and CIDE family showed a greater induction in Lncap cells than in PC3. Exposures to 10 Gy X-rays alone of Lncap cells significantly induced gene expression levels in the death and death receptor domain family, the TNF ligand and receptor family, and apoptotic group of BCL2 family; whereas exposures of PC3 induced only the expression of genes in the anti-apoptosis group of CASP and CARD family. Furthermore, we selectively suppressed the expression of several anti-apoptosis genes (BCL-xl, Bcl2A1, BIRC2, BIRC3 and CASP2) in PC3 cells by using the siRNA treatment. Exposure to 10Gy X-rays alone showed an enhanced cell killing (about 15%) in BCL-x1 silenced cells, but not in cells with siRNA treatment targeting other anti-apoptosis genes. We also exposed PC3 cells to protons in the Bragg peak region to compare the effectiveness of cell killing of X-rays. Interestingly, in comparison to X-rays, protons significantly reduced the gene expression in the anti-apoptosis family, suggesting that proton treatment may be more effective for PC3 cells. As a conclusion, monensin was found to sensitize Lncap cells, but not PC3, and over-expression of Bcl-xl cells may be responsible for the radio- or chemo-resistance characteristics of PC3 cells.
Zhang Ye↗
Cellular Bases of Light-regulated Gravity Responses
This report summarizes the most significant research accomplished in our NAG2-1347 project on the cellular bases of light-regulated gravity responses, It elaborates mainly on our discovery of the role of calcium currents in gravity-directed polar development in single germinating spore cells of the fern Ceratopteris, our development of RNA silencing as a viable method of suppressing the expression of specific genes in Ceratopteris, and on the structure, expression and distribution of members of the annexin family in flowering plants, especially Arabidopsis.
Roux, Stanley J.↗
NRSF causes cAMP-sensitive suppression of sodium current in cultured hippocampal neurons
The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na(+) channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1-2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na(+) channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K(+) currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic "silencer" and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.
Hippocampus/cytology/metabolism↗
WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis
Wild-type Arabidopsis roots develop a wavy pattern of growth on tilted agar surfaces. For many Arabidopsis ecotypes, roots also grow askew on such surfaces, typically slanting to the right of the gravity vector. We identified a mutant, wvd2-1, that displays suppressed root waving and leftward root slanting under these conditions. These phenotypes arise from transcriptional activation of the novel WAVE-DAMPENED2 (WVD2) gene by the cauliflower mosaic virus 35S promoter in mutant plants. Seedlings overexpressing WVD2 exhibit constitutive right-handed helical growth in both roots and etiolated hypocotyls, whereas the petioles of WVD2-overexpressing rosette leaves exhibit left-handed twisting. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs. In roots, the phenotype is accompanied by a change in the arrangement of cortical microtubules within peripheral cap cells and cells at the basal end of the elongation zone. WVD2 transcripts are detectable by reverse transcriptase-polymerase chain reaction in multiple organs of wild-type plants. Its predicted gene product contains a conserved region named "KLEEK," which is found only in plant proteins. The Arabidopsis genome possesses seven other genes predicted to encode KLEEK-containing products. Overexpression of one of these genes, WVD2-LIKE 1, which encodes a protein with regions of similarity to WVD2 extending beyond the KLEEK domain, results in phenotypes that are highly similar to wvd2-1. Silencing of WVD2 and its paralogs results in enhanced root skewing in the wild-type direction. Our observations suggest that at least two members of this gene family may modulate both rotational polarity and anisotropic cell expansion during organ growth.
NASA Discipline Plant Biology↗
Investigating the Relationship between the Cell Wall Integrity Pathway and Unfolded Protein Response
Plants have made significant contributions to astronaut health in spaceflight missions. To further spaceflight research in optimizing plant viability, this study aims to understand the factors involved in maintaining cell wall integrity, which is vital to plant morphology and structural stability. Spaceflight can negatively impact the cell wall; thus, it is crucial to investigate how to mitigate spaceflight stressors to maintain the integrity of the cell wall. The structural integrity of plants’ cell walls depends on secondary cell wall biogenesis, which enables the repair and architectural support of plants like A. thaliana. This biogenesis is triggered by a signal transduction cascade: first initiated by cell wall stress, the CWI (cell wall integrity) pathway is activated, followed by the UPR (unfolded protein response), then the cell wall’s integrity is maintained through secondary cell wall biogenesis. Through a re-analysis of GeneLab Dataset 321 (GLDS-321), a study from NASA’s Open Science Data Repository that investigates the effects of spaceflight on the UPR, several genes were found to be associated with the cell wall. This proposal postulates a relationship between the UPR and the CWI pathway and their direct effect on secondary cell wall biogenesis by investigating IRX7, a gene associated with secondary cell wall biogenesis. The predicted outcome of overexpressing IRX7 is increased resilience of the cell wall by upregulating both the UPR and the CWI pathway, while silencing IRX7 is predicted to compromise the cell wall integrity by downregulating the UPR and the CWI pathway. This study will give insight into the needed measures to increase cell wall resilience in stressful environments: As spaceflight durations increase and uncertain climate change events progress on Earth, understanding how to optimize cell wall resilience – a fundamental pillar of plant health – can effectively enhance mass crop production and quality and ensure the physical and psychological health of astronauts in long-term space missions.
GL4HS↗
Dual Genes of Muscle Cell Senescence: Unraveling the Radiation-Induced Roles of HDAC4 and MORC2a
As the human race ventures further into space and plans missions to explore the Moon and the expanse that lies ahead, addressing health challenges that may hinder exploration becomes an imperative task. However, limited research exists on the molecular mechanisms within skeletal muscle cells in response to long-term radiation-induced oxidative stress. Upon analyzing GLDS-426 from the GeneLab Repository, upregulation of the DNA damage response gene HDAC4 in space radiation conditions with 1G was observed, where radiation-induced oxidative stress increased DNA damage. DNA damage response gene MORC2a was found to be downregulated in space radiation conditions in 1G with a culture treatment of nanoceria, an antioxidant that alleviates the effects of oxidative stress and causes less DNA damage. Literature review further indicated that MORC2a recruits HDAC4 at the promoter to prevent the transcription of p21. Upregulation of p21 has been linked to cell senescence and muscle atrophy on Earth, but no research has studied its changes in radiation conditions. To address the knowledge gap regarding the HDAC4/MORC2a pathway impact on p21 expression in radiation, our experiment aims to silence HDAC4 and MORC2a in the Mus musculus C2C12 cell culture line. These cultures will be exposed to radiation to assess the effects on cellular senescence (Aim 1). Additionally, we will silence p21 and subject the cell cultures to radiation to measure muscle cell proliferation (Aim 2). Muscle atrophy in future long-term space missions can contribute to the risk of degenerative diseases and hinder the completion of tasks in space. Furthermore, exploring the impact of these genes on muscle atrophy may provide therapeutic insights for individuals experiencing skeletal muscle degeneration and elderly individuals affected by sarcopenia.
GL4HS↗
The Role of Gravity Mechanotransduction in Regulating Stem Cell Tissue Regenerative Potential at the Single Cell Expressome Level
Gravity is an omnipresent force on Earth, and all living organisms have evolved under the influence of constant gravity. Mechanical forces generated by gravity are potent modulators of stem cell based tissue regenerative mechanisms, inducing cell fate decisions and tissue specific commitment. A novel mechanical unloading investigation assessed the formation, morphology, and gene expression of embryoid bodies (EB), a transitory cell model of early differentiation. After 15 days of spaceflight, the mechanotransduction-null EB cells showed upregulated proliferative mechanisms while differentiation cues were silenced.
Juran, Cassandra M.↗
Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts
Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.
Non-NASA Center↗
Rapid and efficient suppression of gene expression in a single-cell model system, Ceratopteris richardii
No abstract available
NASA Discipline Plant Biology↗
High LET Radiation Can Enhance TGF(Beta) Induced EMT and Cross-Talk with ATM Pathways
The TGF(Beta) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation in mammary epithelial cells. We investigated possible interactions between the TGF(Beta) and ATM pathways following simulated space radiation using hTERT immortalized human esophageal epithelial cells (EPC-hTERT), mink lung epithelial cells (Mv1lu), and several human fibroblast cell lines. TGF(Beta) is a key modulator of the Epithelial-Mesenchymal Transition (EMT), important in cancer progression and metastasis. The implication of EMT by radiation also has several lines of developing evidence, however is poorly understood. The identification of TGF(Beta) induced EMT can be shown in changes to morphology, related gene over expression or down regulation, which can be detected by RT-PCR, and immunostaining and western blotting. In this study, we have observed morphologic and molecular alternations consistent with EMT after Mv1lu cells were treated with TGF(Beta) High LET radiation enhanced TGF(Beta) mediated EMT with a dose as low as 0.1Gy. In order to consider the TGF(Beta) interaction with ATM we used a potent ATM inhibitor Ku55933 and investigated gene expression changes and Smad signaling kinetics. Ku559933 was observed to reverse TGF(Beta) induced EMT, while this was not observed in dual treated cells (radiation+TGF(Beta)). In EPC-hTERT cells, TGF(Beta) alone was not able to induce EMT after 3 days of application. A combined treatment with high LET, however, significantly caused the alteration of EMT markers. To study the function of p53 in the process of EMT, we knocked down P53 through RNA interference. Morphology changes associated with EMT were observed in epithelial cells with silenced p53. Our study indicates: high LET radiation can enhance TGF(Beta) induced EMT; while ATM is triggering the process of TGF(Beta)-induced EMT, p53 might be an essential repressor for EMT phenotypes.
Wang, Minli↗