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PubMed2006

Requirement of nectin, but not cadherin, for formation of claudin-based tight junctions in annexin II-knockdown MDCK cells.

Adherens junctions (AJs) and tight junctions (TJs) comprise a junctional complex which plays key roles not only in cell adhesion and polarization but also in regulation of cell movement and proliferation in epithelial cells. E-Cadherin and nectin are major cell-cell adhesion molecules (CAMs) at AJs, whereas claudin is a major CAM at TJs. We have shown that the cadherin-based cell-cell adhesion is not formed in MDCK cells in which annexin II, a Ca(2+)- and phospholipid-binding protein, is knocked down. Here, we found that TJs and the nectin-based cell-cell adhesions were formed in annexin II-knockdown cells. The formation of TJs in annexin II-knockdown MDCK cells required the nectin-based cell-cell adhesion and afadin, a nectin- and actin-filament-binding protein. In addition, it required the activation of Cdc42 and Rac small G proteins and subsequent reorganization of the IQGAP1-dependent actin cytoskeleton which were induced by the nectin-based cell-cell adhesion. These results indicate that the nectin-based cell-cell adhesion and afadin, but not the cadherin-based cell-cell adhesion, are necessary for the formation of TJs and that the signaling by nectin and the subsequent reorganization of the actin cytoskeleton are also necessary for the formation of TJs under certain conditions.

Animals↗
PubMed2006

Knockdown of Sox4 expression by RNAi induces apoptosis in ACC3 cells.

Microarray RNA gene expression profiling analysis has shown that Sox4 (Sry-related high mobility group (HMG) box 4) is one of the most upregulated genes in adenoid cystic carcinoma (ACC), relative to non-neoplastic tissue of origin. Here, we show that Sox4 protein is similarly upregulated in ACC by immunohistochemistry of 28 primary cancers and 20 normal tissues. To elucidate the functional significance of these findings, RNA interference (RNAi)-mediated RNA silencing was used to downregulate Sox4 expression in the ACC-derived cell line, ACC3. With confirmed knockdown of Sox4 protein, cell viability was reduced by 51%, with a corresponding increase of apoptosis to 85% as compared to 12% in controls. Apoptosis was confirmed by cell morphology, DNA fragmentation and flow cytometry. Cells could be rescued from the proapoptotic effects of Sox4 RNAi by co-transfection with a construct expressing functional Sox4. Microarray gene expression profiling of RNAi knockdown experiments shows that downregulation of Sox4-modulated expression of critical genes involved in apoptosis and cell cycle control. Overall, our findings suggest that Sox4 contributes to the malignant phenotype of ACC cells by promoting cell survival.

Apoptosis↗
PubMed2004

Generation of single and double knockdowns in polarized epithelial cells by retrovirus-mediated RNA interference.

RNA interference (RNAi) is a ubiquitous mechanism of eukaryotic gene regulation that can be exploited for specific gene silencing. Retroviruses have been successfully used for stable expression of short hairpin RNAs in mammalian cells, leading to persistent inhibition of gene expression by RNAi. Here, we apply retrovirus-mediated RNAi to epithelial Madin-Darby canine kidney cells, whose properties limit the applicability of other RNAi methods. We demonstrate efficient suppression of a set of 13 target genes by retroviral coexpression of short hairpin RNAs and a selectable marker. We characterize the resulting knockdown cell populations with regard to composition and stability, and examine the usefulness of proposed guidelines for choosing RNAi target sequences. Finally, we show that this system can be used to simultaneously target two genes, giving rise to double knockdowns. Thus, retrovirus-mediated RNAi is a convenient method for gene silencing in Madin-Darby canine kidney cells, and is likely to be applicable to virtually any mammalian cell.

Cell Polarity↗
PubMed1997

A "knockdown" mutation created by cis-element gene targeting reveals the dependence of erythroid cell maturation on the level of transcription factor GATA-1.

The hematopoietic-restricted transcription factor GATA-1 is required for both mammalian erythroid cell and megakaryocyte differentiation. To define the mechanisms governing its transcriptional regulation, we replaced upstream sequences including a DNase I hypersensitive (HS) region with a neomycin-resistance cassette by homologous recombination in mouse embryonic stem cells and generated mice either harboring this mutation (neoDeltaHS) or lacking the selection cassette (DeltaneoDeltaHS). Studies of the consequences of these targeted mutations provide novel insights into GATA-1 function in erythroid cells. First, the neoDeltaHS mutation leads to a marked impairment in the rate or efficiency of erythroid cell maturation due to a modest (4- to 5-fold) decrease in GATA-1 expression. Hence, erythroid differentiation is dose-dependent with respect to GATA-1. Second, since expression of GATA-1 from the DeltaneoDeltaHS allele in erythroid cells is largely restored, transcription interference imposed by the introduced cassette must account for the "knockdown" effect of the mutation. Finally, despite the potency of the upstream sequences in conferring high-level, developmentally appropriate expression of transgenes in mice, other cis-regulatory elements within the GATA-1 compensate for its absence in erythroid cells. Our work illustrates the usefulness of targeted mutations to create knockdown mutations that may uncover important quantitative contributions of gene function not revealed by conventional knockouts.

Alleles↗
PubMed2003

Epigenomic stress response. Knockdown of DNA methyltransferase 1 triggers an intra-S-phase arrest of DNA replication and induction of stress response genes.

The DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase 1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2'-deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novo synthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such as NF-kappaB, JunB, ATF-3, and GADD45beta (growth arrest DNA damage 45beta gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information.

Cell Division↗
PubMed2005

TRPC4 knockdown suppresses epidermal growth factor-induced store-operated channel activation and growth in human corneal epithelial cells.

Epidermal growth factor (EGF) in corneal epithelial cells stimulates proliferation by inducing capacitative calcium entry (CCE). However, neither the identity nor the mechanism of activation of the plasma membrane influx pathway that mediates CCE is known. Accordingly, we determined, in human corneal epithelial cells, whether or not (i) CCE is dependent upon stimulation of storeoperated channel (SOC) activity, (ii) the canonical transient receptor potential (TRP) protein isoform TRPC4 is a component of such channels, and (iii) suppression of TRPC4 protein expression decreases EGF-induced stimulation of SOC activity and proliferation. The whole cell patch-clamp technique was used to monitor TRPC4-mediated stimulation of SOC activity following intracellular calcium store depletion and induction of CCE. TRPC4 small interfering RNA transfection suppressed TRPC4 protein expression. Reverse transcription-PCR and Western blot analysis were used to assess knockdown efficiency of mRNA and protein expression. [(3)H]Thymidine incorporation was used to evaluate EGF-in-duced mitogenesis. Ca(2+) transients were measured by single-cell fluorescence imaging. TRPC4 knockdown decreased mRNA and protein expression by 89 and 87%, respectively. In these cells, EGF-induced SOC activation elicited by intracellular calcium store depletion was obviated; 2) EGF-induced CCE fell by 76%; 3) EGF-induced stimulation of SOC activity was eliminated; and 4) EGF-induced increases in proliferation fell by 54%. Thus, TRPC4 is a component of SOC in human corneal epithelial cells whose activation by EGF is requisite for an optimum mitogenic response to this growth factor.

Blotting, Western↗
PubMed2005

Knockdown of glutamate-cysteine ligase by small hairpin RNA reveals that both catalytic and modulatory subunits are essential for the survival of primary neurons.

Glutathione deficiency is an early biochemical feature that occurs during apoptotic neuronal death associated with certain neurological disorders such as Parkinson disease. However, whether specific targeting of glutathione biosynthesis in neurons is sufficient to trigger neurodegeneration remains undetermined. To address this issue, we used a vector-based small hairpin RNA (shRNA) strategy to knock down each subunit of glutamate-cysteine ligase (GCL; gamma-glutamylcysteine synthetase), the heterodimeric enzyme that catalyzes the rate-limiting step of glutathione biosynthesis. Independent targeting of the catalytic and modulatory subunits by shRNA caused disruption of GCL as assessed by Northern and Western blotting, enzyme activity, and glutathione concentrations. Silencing each subunit in primary cortical neurons spontaneously elicited time-dependent apoptotic death, an effect that was synergistic with glutamate or nitric oxide treatment. Moreover, neuronal apoptosis by GCL knockdown was rescued by expressing the corresponding subunit full-length cDNA carrying silent mutations within the shRNA target cDNA sequence and by incubating neurons with gamma-glutamylcysteine or glutathione ethyl ester. In contrast, supplying glutathione precursors to neurons from co-cultured astrocytes did not prevent the apoptotic death triggered by GCL knockdown. Finally, overexpressing the catalytic (but not modulatory) GCL subunit full-length cDNA increased enzyme activity and glutathione concentrations, yielding neurons more resistant to glutamate- or nitric oxide-mediated apoptosis. Thus, specific and independent disruption of each subunit of GCL in neurons can be said to cause a primary decrease in glutathione that is sufficient to promote neurodegeneration.

Amino Acid Sequence↗
PubMed2006

Knockdown of MBP-1 in human prostate cancer cells delays cell cycle progression.

We have previously shown that MBP-1 acts as a general transcriptional repressor, and forced expression of MBP-1 exerts an anti-proliferative effect on a number of human cancer cells. In this report, we have investigated the role of endogenous MBP-1 in cell growth regulation. For this, we generated human prostate cancer cells (PC3) stably transfected with short hairpin RNA targeting MBP-1. We have observed retarded growth and longer doubling time of MBP-1 knockdown PC3 cells as compared with control mock-transfected PC3 cells. Fluorescence-activated cell sorter analysis suggested that PC3 cells expressing MBP-1-specific small interfering RNA accumulated during G2/M phase of the cell cycle. Further analysis suggested that depletion of MBP-1 was associated with reduction of cyclin A and cyclin B1 expression when compared with that of the control cells. A delayed induction of cyclin A and B1 expression was observed in MBP-1-depleted PC3 cells (PC3-4.2) upon serum stimulation, although the level of expression was much lower than that of control PC3 cells. Supplementation of MBP-1 in PC3-4.2 cells restored cyclin A and cyclin B1 expression. Together, these results suggest that knockdown of MBP-1 in prostate cancer cells perturbs cell proliferation by inhibiting cyclin A and cyclin B1 expression.

Cell Cycle↗
PubMed2006

RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts.

U2AF is a heterodimeric splicing factor composed of a large (U2AF65) and a small (U2AF35) subunit. In humans, alternative splicing generates two U2AF35 variants, U2AF35a and U2AF35b. Here, we used RNA interference to specifically ablate the expression of each isoform in HeLa cells. Our results show that knockdown of the major U2AF35a isoform reduced cell viability and impaired mitotic progression, leading to accumulation of cells in prometaphase. Microarray analysis revealed that knockdown of U2AF35a affected the expression level of approximately 500 mRNAs, from which >90% were underrepresented relative to the control. Among mRNAs underrepresented in U2AF35a-depleted cells we identified an essential cell cycle gene, Cdc27, for which there was an increase in the ratio between unspliced and spliced RNA and a significant reduction in protein level. Furthermore, we show that depletion of either U2AF35a or U2AF35b altered the ratios of alternatively spliced isoforms of Cdc25B and Cdc25C transcripts. Taken together our results demonstrate that U2AF35a is essential for HeLa cell division and suggest a novel role for both U2AF35 protein isoforms as regulators of alternative splicing of a specific subset of genes.

Alternative Splicing↗
PubMed1986

Genetic divergence under uniform selection. II. Different responses to selection for knockdown resistance to ethanol among Drosophila melanogaster populations and their replicate lines.

We have tested the hypothesis that genetic differences among conspecific populations may result in diverse responses to selection, using natural populations of Drosophila melanogaster. Selection for ethanol tolerance in a tube measuring knockdown resistance was imposed on five West Coast populations. In 24 generations the selected lines increased their mean knockdown times, on average, by a factor of 2.7. An initially weak latitudinal cline was steepened by selection. The two southernmost populations showed the same increases in the selected character, but differed consistently in their correlated responses in characters related to ethanol tolerance. This result indicates that the populations responded to selection by different genetic changes. Selection decreased female body weight and increased resistance to acetone, suggesting components of the response unrelated to ethanol metabolism. The Adhs allele was favored by selection in all populations at the onset, but increased in frequency only in the selected lines of the southernmost population. There was a correlation between latitude and Adh frequency changes, suggesting that fitnesses of the Adh alleles were dependent on the genetic background. Genetic background also had a large effect on the loss of fitness due to selection. Genetic drift between replicate lines caused more variation in selection response than initial genetic differences between populations. This result demonstrates the importance of genetic drift in divergence among natural populations undergoing uniform selection, since the effective population sizes approached those of small natural populations. Drift caused greater divergence between selected replicates than control replicates. Implications of this result for the genetic model of selection response are discussed.

Alcohol Dehydrogenase↗
PubMed1996

Response of two heat shock genes to selection for knockdown heat resistance in Drosophila melanogaster.

To identify genes involved in stress resistance and heat hardening, replicate lines of Drosophila melanogaster were selected for increased resistance to knockdown by a 39 degrees heat stress. Two selective regimes were used, one with and one without prior hardening. Mean knockdown times were increased from approximately 5 min to > 20 min after 18 generations. Initial realized heritabilities were as high as 10% for lines selected without hardening, and crosses between lines indicated simple additive gene effects for the selected phenotypes. To survey allelic variation and correlated selection responses in two candidate stress genes, hsr-omega and hsp68, we applied denaturing gradient gel electrophoresis to amplified DNA sequences from small regions of these genes. After eight generations of selection, allele frequencies at both loci showed correlated responses for selection following hardening, but not without hardening. The hardening process itself was associated with a hsp68 frequency change in the opposite direction to that associated with selection that followed hardening. These stress loci are closely linked on chromosome III, and the hardening selection established a disequilibrium, suggesting an epistatic effect on resistance. The data indicate that molecular variation in both hsr-omega and.hsp68 contribute to natural heritable variation for hardened heat resistance.

Alcohol Dehydrogenase↗
PubMed2004

Establishment of stable hFis1 knockdown cells with an siRNA expression vector.

Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.

Animals↗
PubMed2003

GluR2(B) knockdown accelerates CA3 injury after kainate seizures.

Ca2+ currents are thought to enhance glutamate excitotoxicity. To investigate whether reduced expression of the Ca2+ limiting GluR2(B) subunit enhances seizure-induced vulnerability to either CA1 or CA3 neurons, we delivered GluR2(B) oligodeoxynucleotides (AS-ODNs) to the dorsal hippocampus of adult rats before inducing kainate (KA) seizures. After knockdown, no changes in behavior, electrographic activity, or histology were observed. In contrast, GluR2(B) knockdown and KA-induced status epilepticus produced accelerated histological injury to the ipsilateral CA3a-b and hilar subregions. At 8 to 12 h, the CA3a was preferentially labeled by both silver and TUNEL methods. TUNEL staining revealed 2 types of nuclei. They were round with uniform label, features of necrosis, or had DNA clumping or speckled chromatin deposits within surrounding cytosol, features of apoptosis. At 16 to 24 h, many CA3a-c neurons were shrunken, eosinophilic, argyrophilic, or completely absent. Immunohistochemistry revealed marked decreases in GluR2(B) subunits throughout the hippocampus, NR1 immunoreactivity was also reduced but to a lesser extent. In contrast, GluR1 and NR2A/B immunohistochemistry was relatively uniform except in regions of cell loss or within close proximity to the CA1 infusion site. At 144 h, the CA3 was still preferentially injured although bilateral CA1 injury was also observed in some AS-ODN-, S-ODN-, and KA-only-treated animals. Glutamate receptor antibodies revealed generalized decreases in the CA3 with all probes tested at this delayed time. In contrast, GluR2(B) expression was increased within CA1 irregularly shaped, injured neurons. Therefore, hippocampal deprivation of GluR2(B) subunits is insufficient to induce cell death in mature animals but may accelerate the already known CA3/hilar lesion, possibly by triggering apoptosis within CA3 neurons. CA1 and DG survive the first week despite their loss of GluR2(B) subunits, suggesting that other intrinsic properties such as increased Na+ conductance and reduced ability of the GluR2(B) subunit to interact with certain cytoplasmic proteins may be responsible for the augmented cell death rather than changes in AMPA receptor-mediated Ca2+ permeability. Alternatively, changes in allosteric interactions that affect other receptor classes of high density at the mossy fiber synapse (e.g. KA receptors) may augment KA neurotoxicity. Latent GluR2(B) increases in CA1 injured neurons support a role for AMPA receptor subunit alterations in seizure-induced tolerance.

Animals↗
PubMed2005

Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference.

RNA interference (RNAi)-mediated gene knockdown is a potent approach for studying gene function. We have previously reported a plasmid-based, tamoxifen-inducible gene knockdown system in cultured cells using a combined RNAi and Cre-LoxP system. Here, we validate this system in mouse and show that it can be used to suppress the expression of an endogenous gene (Fgfr2) with high efficiency. We show that transgenic mice carrying the U6-ploxPneo-Fgfr2 RNAi construct are normal, displaying Fgfr2 transcripts equivalent to those of wild-type controls, indicating that the U6 promoter is inactive in vivo due to the presence of the neo in the promoter. After excision of the neo by crossing with transgenic mice that express Cre in the mouse germline, the U6 promoter is activated, leading to over 95% reduction of Fgfr2 transcripts, and consequently, embryonic lethality. On the other hand, activation of the U6 promoter using transgenic mice that express Cre in the progress zone of the limb results in live mice with malformation of digits of both the forelimbs and hindlimbs. This method provides a fast, yet efficient way to decipher gene functions in vivo in a tissue-specific manner.

Animals↗
PubMed2006

Knockdown of an arbuscular mycorrhiza-inducible phosphate transporter gene of Lotus japonicus suppresses mutualistic symbiosis.

cDNA for a major arbuscular mycorrhiza (AM)-inducible phosphate (Pi) transporter of Lotus japonicus, LjPT3, was isolated from Glomus mosseae-colonized roots. The LjPT3 transcript was expressed in arbuscule-containing cells of the inner cortex. The transport activity of the gene product was confirmed by the complementation of a yeast mutant that lacks high-affinity Pi transporters. In contrast to most AM-inducible Pi transporters thus far reported, LjPT3 has an amino acid sequence that has much in common with those of other members of the Pht1 family of plant Pi transporters, such as StPT3 of potato. To understand better the physiological role of this AM-inducible Pi transporter, knockdown transformants of the gene were prepared through hairy root transformation and RNA interference. Under Pi-limiting conditions, the transformants showed a reduction of Pi uptake via AM and growth retardation. The transformants also exhibited a decrease in G. mosseae arbuscules. Additionally, when Mesorhizobium loti was inoculated into the knockdown transformants in combination with G. mosseae, necrotic root nodules were observed. Based on these findings, we consider that the genetically engineered host plants had monitored insufficient Pi uptake via AM or low expression of LjPT3, excluding the existing fungi and rhizobia and/or preventing further development of the fungal and nodule structures.

Alphaproteobacteria↗
PubMed2004

Quantitative trait loci affecting knockdown resistance to high temperature in Drosophila melanogaster.

Knockdown resistance to high temperature is an ecologically important trait in small insects. A composite interval mapping was performed on the two major autosomes of Drosophila melanogaster to search for quantitative trait loci (QTL) affecting knockdown resistance to high temperature (KRHT). Two dramatically divergent lines from geographically different thermal environments were artificially selected on KRHT. These lines were crossed to produce two backcross (BC) populations. Each BC was analysed for 200 males with 18 marker loci on chromosomes 2 and 3. Three X-linked markers were used to test for X-linked QTL in an exploratory way. The largest estimate of autosome additive effects was found in the pericentromeric region of chromosome 2, accounting for 19.26% (BC to the low line) and 29.15% (BC to the high line) of the phenotypic variance in BC populations, but it could represent multiple closely linked QTL. Complete dominance was apparent for three QTL on chromosome 3, where heat-shock genes are concentrated. Exploratory analysis of chromosome X indicated a substantial contribution of this chromosome to KRHT. The results show that a large-effect QTL with dominant gene action maps on the right arm of chromosome 3. Further, the results confirm that QTL for heat resistance are not limited to chromosome 3.

Animals↗
PubMed2006

Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth.

ALR (MLL2) is a member of the human MLL family, which belongs to a larger SET1 family of histone methyltransferases. We found that ALR is present within a stable multiprotein complex containing a cohort of proteins shared with other SET1 family complexes and several unique components, such as PTIP and the jumonji family member UTX. Like other complexes formed by SET1 family members, the ALR complex exhibited strong H3K4 methyltransferase activity, conferred by the ALR SET domain. By generating ALR knockdown cell lines and comparing their expression profiles to that of control cells, we identified a set of genes whose expression is activated by ALR. Some of these genes were identified by chromatin immunoprecipitation as direct ALR targets. The ALR complex was found to associate in an ALR-dependent fashion with promoters and transcription initiation sites of target genes and to induce H3K4 trimethylation. The most characteristic features of the ALR knockdown cells were changes in the dynamics and mode of cell spreading/polarization, reduced migration capacity, impaired anchorage-dependent and -independent growth, and decreased tumorigenicity in mice. Taken together, our results suggest that ALR is a transcriptional activator that induces the transcription of target genes by covalent histone modification. ALR appears to be involved in the regulation of adhesion-related cytoskeletal events, which might affect cell growth and survival.

Animals↗
PubMed2002

Effect of DMT1 knockdown on iron, cadmium, and lead uptake in Caco-2 cells.

DMT1 (divalent metal transporter 1) is a hydrogen-coupled divalent metal transporter with a substrate preference for iron, although the protein when expressed in frog oocytes transports a broad range of metals, including the toxic metals cadmium and lead. Wild-type Caco-2 cells displayed saturable transport of lead and iron that was stimulated by acid. Cadmium and manganese inhibited transport of iron, but zinc and lead did not. The involvement of DMT1 in the transport of toxic metals was examined by establishing clonal DMT1 knockdown and control Caco-2 cell lines. Knockdown cell lines displayed much lower levels of DMT1 mRNA and a smaller V(max) for iron uptake compared with control cell lines. One clone was further characterized and found to display an approximately 50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, but uptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters these cells through an independent hydrogen-driven mechanism.

Base Sequence↗