Overexpression of Hdac6 enhances resistance to virus infection in embryonic stem cells and in mice.
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ABSTRACT:
SUBMITTER: Wang D
PROVIDER: S-EPMC4312767 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence.
| S-EPMC3541792 | biostudies-literature
Project description:The importance of epigenetic regulation for maintenance of embryonic stem cell (ESC) pluripotency or for initiation of differentiation is widely accepted. However, the molecular mechanisms are poorly understood. We recently reported that a hypoxic microenvironment induces ESC differentiation. In the present study, we found that hypoxia-responsive histone deacetylase 6 (HDAC6) performs an essential signaling function for myogenic differentiation of ESCs. HDAC6 was downregulated in hypoxic ESCs or during differentiation. A knock-down of HDAC6 in ESCs resulted in induction of myogenic markers, including Pax7. Suppression of HDAC6 increased acetylation of core histones H3 and H4, leading to enhanced binding of RNA polymerase II to the Pax7 promoter. Transplantation of HDAC6 knock-down cells facilitated muscle regeneration in vivo. Importantly, the downregulation of HDAC6 by hypoxia was not mediated by HIF1? or HIF2?, master transcription regulators under hypoxia, but by induction of microRNA-26a that directly targeted the 3'-untranslated region (3'-UTR) of HDAC6. A point mutation of the microRNA-26a-binding sequence in the HDAC6 3'-UTR diminished the luciferase reporter activity. Taken together, these results suggest that environmental cues of differentiation modulate the epigenetic machinery and guide stem cells to commit to a specific lineage.
| S-EPMC4344521 | biostudies-literature
Project description:The promise of pluripotent stem cells as a research and therapeutic tool is partly undermined by the technical challenges of generating and maintaining these cells in culture. Human embryonic stem cells (hESCs) are exquisitely sensitive to culture conditions, and require constant signaling by growth factors and cell-cell and cell-matrix interactions to prevent apoptosis, senescence, and differentiation. Previous work from our laboratory demonstrated that overexpression of the prosurvival gene BCL2 in mouse embryonic stem cells overrode the requirement of serum factors and feeder cells to maintain mESCs in culture. To determine whether this prosurvival gene could similarly protect hESCs, we generated hESC lines that constitutively or inducibly express BCL2. We find that BCL2 overexpression significantly decreases dissociation-induced apoptosis, resulting in enhanced colony formation from sorted single cells, and enhanced embryoid body formation. In addition, BCL2-hESCs exhibit normal growth in the absence of serum, but require basic fibroblast growth factor to remain undifferentiated. Furthermore, they maintain their pluripotency markers, form teratomas in vivo, and differentiate into all three germ layers. Our data suggest that the BCL2 signaling pathway plays an important role in inhibiting hESC apoptosis, such that its overexpression in hESCs offers both a survival benefit in conditions of stress by resisting apoptosis and obviates the requirement for serum or a feeder layer for maintenance.
| S-EPMC3044421 | biostudies-literature
Project description:BackgroundSoybean mosaic virus (SMV) is the most prevalent viral disease in many soybean production areas. Due to a large number of SMV resistant loci and alleles, SMV strains and the rapid evolution in avirulence/effector genes, traditional breeding for SMV resistance is complex. Genetic engineering is an effective alternative method for improving SMV resistance in soybean. Potassium (K+) is the most abundant inorganic solute in plant cells, and is involved in plant responses to abiotic and biotic stresses. Studies have shown that altering the level of K+ status can reduce the spread of the viral diseases. Thus K+ transporters are putative candidates to target for soybean virus resistance.ResultsThe addition of K+ fertilizer significantly reduced SMV incidence. Analysis of K+ channel gene expression indicated that GmAKT2, the ortholog of Arabidopsis K+ weak channel encoding gene AKT2, was significantly induced by SMV inoculation in the SMV highly-resistant genotype Rsmv1, but not in the susceptible genotype Ssmv1. Transgenic soybean plants overexpressing GmAKT2 were produced and verified by Southern blot and RT-PCR analysis. Analysis of K+ concentrations on different leaves of both the transgenic and the wildtype (Williams 82) plants revealed that overexpression of GmAKT2 significantly increased K+ concentrations in young leaves of plants. In contrast, K+ concentrations in the old leaves of the GmAKT2-Oe plants were significantly lower than those in WT plants. These results indicated that GmAKT2 acted as a K+ transporter and affected the distribution of K+ in soybean plants. Starting from 14 days after inoculation (DAI) of SMV G7, severe mosaic symptoms were observed on the WT leaves. In contrast, the GmAKT2-Oe plants showed no symptom of SMV infection. At 14 and 28 DAI, the amount of SMV RNA in WT plants increased 200- and 260- fold relative to GmAKT2-Oe plants at each time point. Thus, SMV development was significantly retarded in GmAKT2-overexpressing transgenic soybean plants.ConclusionsOverexpression of GmAKT2 significantly enhanced SMV resistance in transgenic soybean. Thus, alteration of K+ transporter expression is a novel molecular approach for enhancing SMV resistance in soybean.
| S-EPMC4074861 | biostudies-literature
Project description:GATA2 is essential for the endothelial-to-hematopoietic transition (EHT) and generation of hematopoietic stem cells (HSCs). It is poorly understood how GATA2 controls the development of human pluripotent stem cell (hPSC)-derived HS-like cells. Here, using human embryonic stem cells (hESCs) in which GATA2 overexpression was induced by doxycycline (Dox), we elucidated the dual functions of GATA2 in definitive hematopoiesis before and after the emergence of CD34+CD45+CD90+CD38- HS-like cells. Specifically, GATA2 promoted expansion of hemogenic precursors via the EHT and then helped to maintain HS-like cells in a quiescent state by regulating cell cycle. RNA sequencing showed that hPSC-derived HS-like cells were very similar to human fetal liver-derived HSCs. Our findings will help to elucidate the mechanism that controls the early stages of human definitive hematopoiesis and may help to develop a strategy to generate hPSC-derived HSCs.
| S-EPMC6626852 | biostudies-literature
Project description:BACKGROUND:Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of beta-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages. METHODS AND FINDINGS:Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a beta-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca(2+) microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca(2+) homeostasis of beta-cells developing in embryoid bodies produced from such HESC. Cells expressing key beta-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green. CONCLUSION:Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative beta-cells. Our findings provide a novel foundation to study the mechanism of pancreatic beta-cells differentiation during early human development and to help evaluate strategies for the generation of purified beta-cells for future clinical applications.
| S-EPMC2262135 | biostudies-literature
Project description:Deciphering the clues of a regenerative mechanism for the mammalian adult heart would save millions of lives in the near future. Heart failure due to cardiomyocyte loss is still one of the significant health burdens worldwide. Here, we show the potential of a single molecule, DAND5, in mouse pluripotent stem cell-derived cardiomyocytes specification and proliferation. Dand5 loss-of-function generated the double of cardiac beating foci compared to the wild-type cells. The early formation of cardiac progenitor cells and the increased proliferative capacity of Dand5 KO mESC-derived cardiomyocytes contribute to the observed higher number of derived cardiac cells. Transcriptional profiling sequencing and quantitative RT-PCR assays showed an upregulation of early cardiac gene networks governing cardiomyocyte differentiation, cell cycling, and cardiac regenerative pathways but reduced levels of genes involved in cardiomyocyte maturation. These findings prompt DAND5 as a key driver for the generation and expansion of pluripotent stem cell-derived cardiomyocytes systems with further clinical application purposes.
| S-EPMC8078107 | biostudies-literature
Project description:Intracellular potassium (K+ ) transported by plants under the action of a number of transport proteins is crucial for plant survival under distinct abiotic and biotic stresses. A correlation between K+ status and disease incidence has been found in many studies, but the roles of K+ in regulating disease resistance to viral diseases remain elusive. Here, we report that HIGH-AFFINITY K+ TRANSPORTER 5 (OsHAK5) regulates the infection of rice grassy stunt virus (RGSV), a negative-sense single-stranded bunyavirus, in rice (Oryza sativa). We found the K+ content in rice plants was significantly inhibited on RGSV infection. Meanwhile, a dramatic induction of OsHAK5 transcripts was observed in RGSV-infected rice plants and in rice plants with K+ deficiency. Genetic analysis indicated that disruption of OsHAK5 facilitated viral pathogenicity. In contrast, overexpression of OsHAK5 enhanced resistance to RGSV infection. Our analysis of reactive oxygen species (ROS) including H2 O2 and O2- , by DAB and NBT staining, respectively, indicated that RGSV infection as well as OsHAK5 overexpression increased ROS accumulation in rice leaves. The accumulation of ROS is perhaps involved in the induction of host resistance against RGSV infection in OsHAK5 transgenic overexpression rice plants. Furthermore, RGSV-encoded P3 induced OsHAK5 promoter activity, suggesting that RGSV P3 is probably an elicitor for the induction of OsHAK5 transcripts during RGSV infection. These findings indicate the crucial role of OsHAK5 in host resistance to virus infection. Our results may be exploited in the future to increase crop yield as well as improve host resistance via genetic manipulations.
| S-EPMC9276945 | biostudies-literature
Project description:Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo-expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with ?-globin production. Moreover, HPCs generated from RUNX1a EBs possess ?9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.
| S-EPMC3624936 | biostudies-literature
Project description:Heat-stressed crops suffer dehydration, depressed growth, and a consequent decline in water productivity, which is the yield of harvestable product as a function of lifetime water consumption and is a trait associated with plant growth and development. Heat shock transcription factor (HSF) genes have been implicated not only in thermotolerance but also in plant growth and development, and therefore could influence water productivity. Here it is demonstrated that Arabidopsis thaliana plants with increased HSFA1b expression showed increased water productivity and harvest index under water-replete and water-limiting conditions. In non-stressed HSFA1b-overexpressing (HSFA1bOx) plants, 509 genes showed altered expression, and these genes were not over-represented for development-associated genes but were for response to biotic stress. This confirmed an additional role for HSFA1b in maintaining basal disease resistance, which was stress hormone independent but involved H?O? signalling. Fifty-five of the 509 genes harbour a variant of the heat shock element (HSE) in their promoters, here named HSE1b. Chromatin immunoprecipitation-PCR confirmed binding of HSFA1b to HSE1b in vivo, including in seven transcription factor genes. One of these is MULTIPROTEIN BRIDGING FACTOR1c (MBF1c). Plants overexpressing MBF1c showed enhanced basal resistance but not water productivity, thus partially phenocopying HSFA1bOx plants. A comparison of genes responsive to HSFA1b and MBF1c overexpression revealed a common group, none of which harbours a HSE1b motif. From this example, it is suggested that HSFA1b directly regulates 55 HSE1b-containing genes, which control the remaining 454 genes, collectively accounting for the stress defence and developmental phenotypes of HSFA1bOx.
| S-EPMC3733161 | biostudies-other