Heat Shock Promoter Research Articles

Photoreceptor Cell Development Requires CDHR1a Protein Stability Controlled by Siah E3 Ubiquitin Ligase During Zebrafish Eye Development

E3 ubiquitin ligases mediate orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development. Our lab recently discovered that Siah family of E3 ligases plays a role in early ocular morphogenesis, in particular fusion of the optic fissure. Interestingly, Siah ligases are also expressed during photoreceptor development and are predicted to target CDHR1a, a cadherin superfamily of calcium‐dependent cell adhesion molecules and photoreceptor‐specific cadherin. Mutations in this cadherin are associated with inherited retinal dystrophies, such as cone‐rod dystrophy. Using whole mount in situ hybridization and Immuno‐histochemistry we detected Siah gene expression and protein localization in the outer nuclear layer (ONL) and in the retinal ganglion cell layer starting at 3 days‐post fertilization (dpf) until 7dpf. CDHR1a expression and protein localization overlaps with both Siah genes in the outer segment of the retina from 3 until 7dpf. We therefore hypothesized that Siah regulates CDHR1a during photoreceptor development. To test our hypothesis, I first confirmed siah‐mediated targeting of CDHR1a for degradation using a mammalian cell culture, co‐immunoprecipitation followed by western blotting analysis. Second, I created two transgenic zebrafish lines that express Siah1 or a dominant negative (Siah1DRING) under the control of the heat shock promoter. Using the heat shock line to overexpress Siah1 at 2dpf and 2.5dpf, we observed a decrease in the number of developing rods and cones in the retina at 3dpf. The number of retinal ganglion cells, amacrine, bipolar and horizontal cells, however, did not change. In addition, there was a significant reduction of proliferating (EdU+ and PH3+ cells in ONL) cells in the retina. Taken together, our results suggest that Siah ubiquitin ligases may control CDHR1a stability and therefore regulate the photoreceptor cell proliferation and differentiation.Support or Funding InformationThis work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq) under grant number 202970/2014‐0.

Identification and functional analysis of promoters of heat-shock genes from the fall armyworm, Spodoptera frugiperda

The functional information on heat-shock proteins (Hsp) and heat-shock promoters from an important agricultural insect pest, Spodoptera frugiperda, is still lacking. We conducted a genome-wide identification of Hsp genes and identified a total of 21 genes belonging to four major insect Hsp families (small heat-shock proteins, Hsp60, Hsp70, and Hsp90) in S. frugiperda. Expression of most of S. frugiperda (SfHsp) genes could be detected in Sf9 cells, embryos and larval tissues of S. frugiperda. The heat-inducible activity of heat-shock promoters from several SfHsp genes was tested in Sf9 cells and embryos. The promoter of SfHsp70D showed the high constitutive activity in cell line and embryos, while the activity of SfHsp20.15 and SfHsp20.71 promoters was most dramatically induced in Sf9 cells and embryos. In embryos, the heat-induced activity of SfHsp20.71 and SfHsp70D promoters outperformed commercially used ie1 and ie2 promoters. The heat-induced activity of SfHsp70D and SfHsp19.07 promoters were more robust than ie2 promoter in Sf9 cells. These SfHsp promoters with high basal activity or with heat-induced activity from low basal activity, could be used in S. frugiperda or other lepidopteran insects for many applications including transgenesis and genome editing.

Leucinostatin acts as a co-inducer for heat shock protein 70 in cultured canine retinal pigment epithelial cells

Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 co-inducer, we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin, which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line, also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that leucinostatin might be applied as a new co-inducer for Hsp70 expression.

Functional analysis of MTF-1 and MT promoters and their transcriptional response to zinc (Zn) and copper (Cu) in yellow catfish Pelteobagrus fulvidraco.

Metal-responsive transcription factor-1 (MTF-1) and metallothionein (MT) expression are involved in metal homeostasis and detoxification. Here, we characterized the structure and functions of mtf-1 and mt promoters in yellow catfish Pelteobagrus fulvidraco. Many important binding sites of transcriptional factors, such as heat shock promoter element (HSE) and metal responsive element (MRE), were predicted on their promoter regions. Cu did not significantly influence the activity of mtf-1 promoter, but Zn increased its promoter activity. Cu and Zn induced the increase of mt promoter activity. HSE site of mtf-1 promoter was the functional binding locus responsible for Zn-induced mtf-1 transcriptional activation. Zn and Cu induced transcriptional activation of mt gene through the MTF-1- and MRE-dependent pathway. Using primary hepatocytes of yellow catfish, we found that Cu and Zn induced the mt expression; Cu did not significantly influence the mRNA and total protein levels of MTF-1, but Zn up-regulated its mRNA and total protein expression. Both Zn and Cu treatment also up-regulated MTF-1 nuclear protein expression, which in turn increased the mt expression. Taken together, these findings delineated the transcriptional regulation of MT and MTF-1 under Zn or Cu treatments, and provided some mechanisms for the regulation of Cu and Zn homeostasis in vertebrates.

Transcription Factor Induction of Ectopic Vascular Blood Stem Cell Niches In Vivo

The hematopoietic stem and progenitor cell (HSPC) niche is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial cells (ECs) that directly interact with HSPCs and promote stem cell function. Utilizing spatial transcriptomics, in combination with tissue-specific RNA-seq, we identified 29 genes selectively enriched in ECs of the zebrafish fetal hematopoietic niche. Using upstream regulatory sequences for two of these genes, mrc1a and selectin E (sele), we generated GFP reporter lines that allowed us to selectively isolate niche ECs for ATAC-seq. This analysis identified 6,848 regions of chromatin that were accessible in niche ECs but not ECs from other tissues. Several of these regions were associated with the 29 genes. To evaluate whether these regions might be enhancers we coupled them to GFP and injected them into embryos. 12/15 sequences drove GFP expression in niche ECs. Upon closer examination of the mrc1a and sele genes, we identified enhancer sequences as short as 125 bp and 158 bp, respectively, which drove niche EC-specific expression. A genome-wide motif enrichment analysis of the 6,848 uniquely open chromatin regions revealed that Ets, SoxF and Nuclear Hormone Receptor (RXRA/NR2F2, specifically) sites were most enriched. In contrast, 4,522 pan-EC elements were enriched for Ets sites but not SoxF or NHR motifs. Using mutant variants of the 125 bp and 158 bp enhancer sequences, we demonstrated that Ets, SoxF and RXRA/NR2F2 sites were independently required for specific transgene expression. Gel shift experiments demonstrated that NR2F2 could bind the 125 bp and 158 bp zebrafish enhancers and this binding was disrupted upon mutation of the NR2F2 binding sites. Knockdown of the endogenous zebrafish nr2f2 gene resulted in a loss of expression of the 125 bp mrc1a enhancer-GFP construct and a significantly reduced number of HSPCs in the fetal niche. We next injected pools of human transcription factors, including at least one member from each of the three families, under the control of a ubiquitous promoter. Strikingly, we found that a combination of ETV2 or ETS1 with SOX7 and NR2F2 generated ectopic patches of mrc1a+ niche ECs that recruited runx1+ HSPCs outside of the endogenous niche. Using high-resolution live cell imaging we could observe HSPCs initially arriving at the ectopic sites, lodging for several hours and then eventually dividing and migrating away from the site through circulation. HSPCs localized to the ectopic regions were found in both intravascular and extravascular spaces, and were often enwrapped by ECs and in contact with cxcl12a+ stromal cells, similar to what is observed in the endogenous niche. Ectopic regions of niche EC gene expression were similarly observed when alternative regulatory elements were used for transcription factor overexpression, including a pan-EC enhancer (nrp1b), a muscle promoter (mylz2) and a heat shock promoter (hsp70). These results suggest the three-factor combinations are sufficient to reprogram niche EC identify in vivo. Lastly, we evaluated by RNA-seq the expression of our niche EC signature in the zebrafish kidney marrow (the site of adult hematopoiesis) and in ECs from multiple organs of the mouse, including the heart, kidney, liver, lung and bone marrow, at multiple stages of development (E11-13, E14-15, E16-17, P2-P4 and adult). Strikingly, 23/29 genes were highly expressed in ECs of the zebrafish kidney and 21/29 genes were enriched in the ECs of a mammalian hematopoietic organ - the fetal liver and/or adult bone marrow - relative to their expression in ECs from non-hematopoietic organs at the same stage. Notably, for a subset of the genes the expression patterns mirrored the temporal dynamics of HSPC ontogeny in the mouse, showing robust expression in fetal liver ECs and then later in adult bone marrow ECs with a concomitant reduction in liver ECs. An analysis of transcription factor expression within these EC populations revealed that Ets1, the SoxF factor Sox18, and Nr2f2 were the most highly expressed members of the Ets, Sox and NHR families. Collectively our work has uncovered a conserved gene expression signature and transcriptional regulatory program unique to the vascular niche of hematopoietic organs. These findings have important implications for designing a synthetic vascular niche for blood stem cells or for modulating the niche in a therapeutic context. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

Inhibition of dicer activity in lepidopteran and dipteran cells by baculovirus-mediated expression of Flock House virus B2

Prior studies have suggested that insect DNA viruses are negatively affected by dicer-2-mediated RNA interference (RNAi). To examine this further, we utilized an in vitro assay to measure dicer activity in lepidopteran and dipteran cells, combined with baculoviruses expressing the RNAi suppressor B2 from Flock House virus or Aedes aegypti dicer-2 (Aedicer-2) using a constitutive heat shock promoter. Addition of cell lysates containing baculovirus-expressed B2 to lysates from dipteran (S2, Aag2) or lepidopteran (Sf9) cells inhibited endogenous dicer activity in a dose-dependent manner, while expression of Aedicer-2 restored siRNA production in Ae. albopictus C6/36 cells, which are dicer-2 defective. However, B2 expression from the constitutive heat shock promoter had no impact on baculovirus replication or virulence in cell lines or larvae that were either highly permissive (Trichoplusia ni) or less susceptible (Spodoptera frugiperda) to infection. We determined that this constitutive level of B2 expression had little to no ability to suppress dicer activity in cell lysates, but higher expression of B2, following heat shock treatment, inhibited dicer activity in all cells tested. Thus, we cannot rule out the possibility that optimized expression of B2 or other RNAi suppressors may increase baculovirus replication and expression of heterologous proteins by baculoviruses.

Petal abscission in roses is associated with the activation of a truncated version of the animal PDCD4 homologue, RbPCD1

Abscission is a developmental process that leads to shedding of organs not needed by the plant. Apart from wall hydrolysis, the cells of the abscission zone (AZ) are also believed to undergo programmed cell death (PCD). We show that ethylene-induced petal abscission in Rosa bourboniana is accompanied with the activation of RbPCD1 (PROGRAMMED CELL DEATH LIKE 1) encoding a protein of 78 amino acids. Its expression increases during natural and ethylene-induced petal abscission. Its transcription in most tissues is up-regulated by ethylene. RbPCD1 shows similarity to the N-terminal domain of animal PDCD4 (PROGRAMMED CELL DEATH PROTEIN 4) proteins that are activated during apoptosis and function as transcriptional and translational repressors. RbPCD1 resides in the nucleus and cytoplasm and acts as a transcriptional repressor. Constitutive expression of RbPCD1 in transgenic Arabidopsis is seedling lethal. Heat-induced expression of RbPCD1 under the soybean heat-shock promoter affects leaf function, inflorescence development, silique formation, seed yield and reduces survival. Nuclear localization of RbPCD1 is necessary for manifestation of its effects. RbPCD1 may be necessary to mediate some of the ethylene-induced changes during abscission and senescence in specific tissues.

The Role of Nesprin in the Facial Branchiomotor Neuron (FBMN) Migration of Danio rerio (zebrafish) development

Nuclear envelope spectrin‐repeat proteins (Nesprins) are part of the LINC (LInker of Nucleoskeleton and Cytoskeleton) complex, which plays a vital role in the binding of F‐actin cytoskeleton to the nuclear envelope. Nesprins are crucial in maintaining the integrity of the nuclear membrane. Nuclear membrane movement is a fundamental aspect in cell migration and muscle development. Previous work has demonstrated that Nesprins may be associated with myosin‐dependent tension, which has implications in muscular dystrophy due to the role that nesprins play in muscle cells. We hypothesize that expressing dominant‐negative nesprin in Danio rerio (zebrafish) embryos would result in defects in the migration of facial branchiomotor neurons and also cause disruptions in nuclear positioning in multinucleated muscle cells. To test this hypothesis, we use transient transgenic expression of dominant Nesprin in cranial motor neurons using the islet1 promoter. We quantify the extent of migration in neurons with inactive Nesprin. In addition, we express dominant‐negative Nesprin in muscle cells using a heat shock promoter and explore the positioning of nuclei in developing skeletal muscle myofibrils. Our preliminary data shows that transgenic zebrafish embryos with expression of dominant‐negative nesprin have disruptions and defects in tangential FBMN migration. However, with further experimentation and data collection in progress, we hope to analyze our findings for statistical significance. Our goal is to gain insight into the function of Nesprins in embryo development to better devise therapeutic strategies for patients with mutations in Nesprins.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Study on Possible Gene Therapy on Diabetics Type I Using Insulin Gene under Control of Heat Shock Promoter in Laboratory Animals

Background: Gene therapy is one of the treatment method for diabetes mellitus. An insulin gene, under control of heat shock inducer promoter was used in the study.Materials and Methods: Six mice used in this study. Streptozotocin (STZ) induced diabetes mellitus in BALB/C mice and rats. Recombinant plasmid was injected to each animal. Animal’s blood sugar (BS) was measured. Warming was done at injection site with a hair dryer to induced gene expression.Results: Immediately after warming blood sugar was increased in mouse 1 and decreased after one hour. However, blood sugar increased again in mice 2 and 4. Blood sugar increased for two hours after warming in mouse 3. Blood sugar increased to 150 mg/dl after STZ injection and immediately after warming reached to 160 mg/dl, after one hour BS dropped to 116 mg/dl and on the second hour was 126 mg/dl, in mouse 5 BS had an increasing trend, BS in mouse 6 had a similar pattern to mouse 2.Conclusion: With fixing the defects of the project will be used in gene therapy in future.

Transcriptomic Analysis of Zebrafish TDP-43 Transgenic Lines.

Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disorder that affects both upper and lower motor neurons, leading to muscle atrophy with spasticity and eventual death in 3–5 years after the disease onset. More than 50 mutations linked to ALS have been found in the gene TARDBP, encoding the protein TDP-43 that is the predominant component of neuronal inclusions in ALS. TDP-43 is an RNA binding protein with glycine-rich domains that binds to more than 6,000 RNAs in the human brain. However, ALS-related mutations do not appear to affect the function of these genes, indicating that a toxic gain-of-function may occur. We generated transgenic zebrafish lines expressing human TDP-43, either the wild-type form or the ALS-causative G348C mutation identified in a subset of ALS patients, with the transgene expression driven by an inducible heat shock promoter in order to bypass a potential early mortality. The expression of the mutant but not the wild-type human TDP-43 in zebrafish embryos induced a reduction of the locomotor activity in response to touch compared to controls and moderate axonopathy of the motor neurons of the spinal cord, with premature branching of the main axonal branch, recapitulating previous results obtained by mRNA injections. We used these lines to investigate transcriptomic changes due to the presence of mutant TDP-43 using RNA sequencing and have found 159 genes that are differentially expressed compared to control, with 67 genes up-regulated and 92 genes down-regulated. These transcriptomic changes are in line with recent transcriptomic data obtained in mouse models, indicating that these zebrafish transgenic lines are adequate to further study TDP-43-related ALS.

Role of Arsenic Trioxide in EVI-1 Apoptosis in Patients with Acute Myeloid Leukaemia

Background:High expression of the ecotropic viral integration site-1 (EVI-1) is an independent negative prognostic indicator of survival in leukaemia patients. Zebrafish (Danio rerio) is a vertebrate animal model commonly used to examine haematopoiesis and myeloid malignancies. To clarify the molecular mechanisms of EVI-1, we previously introduced the human EVI-1 gene into embryonic zebrafish through a heat-shock promoter and established the stable germ-line Tg(EVI-1: HSE: EGFP) zebrafish (Shen et al, 2013). Arsenic trioxide (As2O3, ATO) is one of the effective anticancer drugs, especially for patients with leukaemia (Udupa et al, 2017). We thus aimed to explore the anticancer effects of ATO and the underlying functions associated with EVI-1 in an in vivo zebrafish model and in AML cells in vitro.Results:We determined EVI-1 expression in mononuclear cells isolated from the bone marrow and peripheral blood of AML patients and healthy donors by RT-qPCR and Western blot analysis. EVI-1 was highly expressed in primary AML (Fig 1A). Then, EVI-1 expression was detected in five leukaemia cell lines (K562, HL-60, U937, THP-1 and MV4-11) and normal PBMCs. Among these five leukaemia cell lines, THP-1 has the highest EVI-1 expression (Fig 1B and Fig1C).Primary acute monocytic leukaemia cells from one patient with high expression of EVI-1 were treated with ATO. We found ATO could significantly decrease EVI-1 mRNA (Fig 2A). Between the ATO-treated groups and the control group, the expression of EVI-1 were significantly reduced in the THP-1 cell line (Fig 2B). Next, we evaluated the EVI-1 expression in Tg(EVI-1: HSE; EGFP) transgenic zebrafish embryos over dose courses of ATO exposure (Fig 2C). Consistent with the results of our in vitro study, ATO decreased EVI-1 expression in a dose-dependent manner after 72 h (Fig 2C). Taken together, these results indicate that ATO is an inhibitor of EVI-1 expression both in vivo and in vitro.We investigate whether the reduction of THP-1 cells viability is due to apoptosis, THP-1 cells were incubated with 3 µM of ATO for 24 h, 48 h or 72 h. In the light microscopy images, THP-1 cells exhibited typical apoptotic characteristics (Fig 3A). The proportion of apoptotic cells was represented as early apoptotic cells (annexin V+/PI- staining, the lower right quadrant) plus late apoptotic cells (annexin V+/PI+ staining, the upper right quadrant) (Fig 3B). In cytometric analysis, ATO increased the percentage of apoptotic THP-1 cells in a dose- and time-dependent manner. We found that ATO increased the expressions of JNK, p-JNK, p-P53, PUMA, Bax, caspase-9 and caspase-3 (including cleaved caspase-9 and -3) but decreased the expressions of Bcl-2 and Bcl-xl (Fig 3C).To further verify the role of the JNK pathway in ATO-mediated THP-1 cell apoptosis, we examined if the inhibitor of JNK (SP600125) could reverse ATO-induced apoptosis in THP-1 cells. We found SP600125 not only decreased the pro-apoptotic effect of ATO in the THP-1 cell line (Fig 4A and Fig 4B) but also decreased the activation of the JNK-mediated apoptotic signalling pathway (Fig 4C). SP600125 silenced the activation of JNK by completely inhibiting the phosphorylation of JNK but had little effect on EVI-1 expression (Fig 4C).To test whether EVI-1 modulates apoptosis via the JNK signalling pathway, we transiently transfected THP-1 cells with EVI-1 siRNA which significantly reduced EVI-1 expression (Fig 5A). Silencing EVI-1 had a significant effect on the activation of the JNK pathway and the induction of THP-1 cell apoptosis (Fig 5B and Fig 5C).Conclusion:Our study demonstrated that the apoptotic pathway in THP-1 cells induced by ATO is closely associated with the oncogene EVI-1, the pro-apoptotic protein JNK, p-JNK, p-P53, PUMA, Bax, caspase-9 and caspase-3 (including cleaved caspase-9 and cleaved caspase-3), and the anti-apoptotic proteins Bcl-2 and Bcl-xL. ATO can downregulate EVI-1 mRNA and oncoprotein and block the repression of EVI-1 in the JNK pathway. Furthermore, the activated JNK signalling pathway regulated the expression level of apoptosis-associated proteins, including p-P53, PUMA, Bax, Bcl‐xL, Bcl‐2, Bax, caspase-9 and caspase-3(Fig 6). These findings may provide a novel theoretical basis for the development of personalized medical strategies for the treatment of EVI-1 positive AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Stable transgenic C9orf72 zebrafish model key aspects of the ALS/FTD phenotype and reveal novel pathological features

A hexanucleotide repeat expansion (HRE) within the chromosome 9 open reading frame 72 (C9orf72) gene is the most prevalent cause of amyotrophic lateral sclerosis/fronto-temporal dementia (ALS/FTD). Current evidence suggests HREs induce neurodegeneration through accumulation of RNA foci and/or dipeptide repeat proteins (DPR). C9orf72 patients are known to have transactive response DNA binding protein 43 kDa (TDP-43) proteinopathy, but whether there is further cross over between C9orf72 pathology and the pathology of other ALS sub-types has yet to be revealed.To address this, we generated and characterised two zebrafish lines expressing C9orf72 HREs. We also characterised pathology in human C9orf72-ALS cases. In addition, we utilised a reporter construct that expresses DsRed under the control of a heat shock promoter, to screen for potential therapeutic compounds.Both zebrafish lines showed accumulation of RNA foci and DPR. Our C9-ALS/FTD zebrafish model is the first to recapitulate the motor deficits, cognitive impairment, muscle atrophy, motor neuron loss and mortality in early adulthood observed in human C9orf72-ALS/FTD. Furthermore, we identified that in zebrafish, human cell lines and human post-mortem tissue, C9orf72 expansions activate the heat shock response (HSR). Additionally, HSR activation correlated with disease progression in our C9-ALS/FTD zebrafish model. Lastly, we identified that the compound ivermectin, as well as riluzole, reduced HSR activation in both C9-ALS/FTD and SOD1 zebrafish models.Thus, our C9-ALS/FTD zebrafish model is a stable transgenic model which recapitulates key features of human C9orf72-ALS/FTD, and represents a powerful drug-discovery tool.

Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

BackgroundMedaka (Oryzias latipes) is a popular animal model used in vertebrate genetic analysis. Recently, an efficient (~ 30%) knock-in system via non-homologous end joining (NHEJ) was established in zebrafish using the CRISPR/Cas9 system. If the same technique were applicable in medaka, it would greatly expand the usefulness of this model organism. The question of the applicability of CRISPR/Cas9 in medaka, however, has yet to be addressed.ResultsWe report the highly efficient generation of knock-in transgenic medaka via non-homologous end joining (NHEJ). Donor plasmid containing a heat-shock promoter and a reporter gene was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, an sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. Broad transgene expression in the expression domain of a target gene was observed in approximately 25% of injected embryos. By raising these animals, we established stable knock-in transgenic fish with several different constructs for five genetic loci, obtaining transgenic founders at efficiencies of > 50% for all five loci. Further, we show that the method is useful for obtaining mutant alleles. In the experiments where transgene integrations were targeted between the transcription start site and the initiation methionine, the resultant transgenic fish became mutant alleles.ConclusionWith its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in via NHEJ will become a standard method for the generation of transgenic and mutant medaka.

Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters

SummaryTranscription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters—an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.

Wingless is a positive regulator of eyespot color patterns in Bicyclus anynana butterflies

Eyespot patterns of nymphalid butterflies are an example of a novel trait yet, the developmental origin of eyespots is still not well understood. Several genes have been associated with eyespot development but few have been tested for function. One of these genes is the signaling ligand, wingless, which is expressed in the eyespot centers during early pupation and may function in eyespot signaling and color ring differentiation. Here we tested the function of wingless in wing and eyespot development by down-regulating it in transgenic Bicyclus anynana butterflies via RNAi driven by an inducible heat-shock promoter. Heat-shocks applied during larval and early pupal development led to significant decreases in wingless mRNA levels and to decreases in eyespot size and wing size in adult butterflies. We conclude that wingless is a positive regulator of eyespot and wing development in B. anynana butterflies.

Functional characterization of Aedes aegypti alkaline phosphatase ALP1 involved in the toxicity of Cry toxins from Bacillus thuringiensis subsp. israelensis and jegathesan

Presently three major groups of proteins from Aedes aegypti, cadherin, alkaline phosphatases (ALP) and aminopeptidases N (APN), have been identified as Cry11Aa toxin receptors. To further characterize their role on toxicity, transgenic mosquitoes with silenced Aedes cadherin expression were previously generated and the role of cadherin in mediating the toxicity of four different mosquitocidal toxins (Cry11Aa, Cry11Ba, Cry4Aa and Cry4Ba) was demonstrated. Here, we investigated the role of another reported Cry11Aa receptor, ALP1. As with Aedes cadherin, this protein is localized in the apical cell membrane of distal and proximal gastric caecae and the posterior midgut. We also successfully generated transgenic mosquitoes that knockdowned ALP1 transcript levels using an inducible Aedes heat shock promoter, Hsp70A driving dsALP1RNA. Four different mosquitocidal toxins were used for larval bioassays against this transgenic mosquito. Bioassay results show thatCry11Aa toxicity to these transgenic larvae following a heat shock decreased (4.4 fold) and Cry11Ba toxicity is slightly attenuated. But Cry4Aa and Cry4Ba toxicity to ALP1 silenced larvae is unchanged. Without heat shock, toxicity of all four toxins does not change, suggesting this heat shock promoter is heat-inducible. Notably, transgenic mosquitoes with ALP1 knockdown are about 3.7 times less resistant to Cry11Aa toxin than those with Aedes cadherin knockdown. These results demonstrate that the ALP1 is an important secondary receptor for Cry11Aa and Cry11Ba, but it might not be involved in Cry4Aa and Cry4Ba toxicity.

Hydroxycinnamic acids and curcumin production in engineered Escherichia coli using heat shock promoters

Hydroxycinnamic acids and curcumin are compounds with great therapeutic potential, including anticancer properties. In this study, p-coumaric acid, caffeic acid and curcumin were produced in Escherichia coli. Their production was induced by heat using the dnaK and ibpA heat shock promoters. The ribosome binding site (RBS) used was tested and further optimized for each gene to assure an efficient translation. p-Coumaric acid was successfully produced from tyrosine and caffeic acid was produced either from tyrosine or p-coumaric acid using tyrosine ammonia lyase (TAL) from Rhodotorula glutinis, 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris. The highest p-coumaric acid production obtained was 2.5mM; caffeic acid production reached 370μM. Regarding curcumin, 17μM was produced using 4-coumarate-CoA ligase (4CL1) from Arabidopsis thaliana, diketide-CoA synthase (DCS) and curcumin synthase 1 (CURS1) from Curcuma longa. Stronger RBSs and/or different induction conditions should be further evaluated to optimize those production levels. Herein it was demonstrated that the biosynthetic pathway of p-coumaric acid, caffeic acid and curcumin in E. coli can be triggered by using heat shock promoters, suggesting its potential for the development of new industrial bioprocesses or even new bacterial therapies.

Establishment of a tetracycline-off and heat shock-on gene expression system in tobacco

The tetracycline (Tet)-off gene expression regulation system based on the TetR-VP16/Top10 construct has not been widely utilized in plants, for its highly expressed TetR-VP16 activator is toxic to some plants and repeatedly replenishing tetracycline to turn off the constitutively active system is a tedious process. To solve these problems, a Tet-off and heat shock (HS)-on gene expression regulation system was constructed in this study. This system is composed of a chimeric transactivator gene TetR-HSF that is derived from a Tet repressor (TetR) and a HS transcription factor (HSF) controlled by a HS promoter HSP70m, and a Tet operator containing hybrid promoter, Om35S, that drives expression of the β-glucuronidase (GUS) gene. The resultant system yields a GUS expression pattern similar to that of the HSP70m promoter under inducing temperatures and at 35 and 40°C drives GUS expression to a similar level as the Cauliflower mosaic virus (CaMV) 35S promoter. Further examination revealed that the TetR-HSF and GUS genes were induced by HS, reaching peak expression after 1 and 6 h treatment, respectively, and the HS induction of the expression system could be inhibited by Tet. This system will provide a useful tool for transgenic studies of plants in the laboratory and in the field, including transgene function analysis, agronomic trait improvement, biopharmaceutical protein production and others.

Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase (NuRD) complex.

Depending on its stringency, exposure to heat in early life leads to either resilience or vulnerability to heat stress later in life. We hypothesized that epigenetic alterations in genes belonging to the cell proteostasis pathways are attributed to long-term responses to heat stress. Epigenetic regulation of the mRNA expression of the molecular chaperone heat-shock protein (HSP) 70 (HSPA2) was evaluated in the chick hypothalamus during the critical period of thermal-control establishment on day 3 post-hatch and during heat challenge on day 10. Both the level and duration of HSP70 expression during heat challenge a week after heat conditioning were more pronounced in chicks conditioned under harsh versus mild temperature. Analyzing different segments of the promoter invitro indicated that methylation of a distal part altered its transcriptional activity. In parallel, DNA-methylation level of this segment invivo was higher in harsh- compared to mild-heat-conditioned chicks. Hypermethylation of the HSP70 promoter in high-temperature-conditioned chicks was accompanied by a reduction in both POU Class 2 Homeobox 1 (POU2F1) binding and recruitment of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex. As a result, histone H3 acetylation levels at the HSP70 promoter were higher in harsh-temperature-conditioned chicks than in their mild-heat-conditioned counterparts. These results suggest that methylation level of a distal part of the HSP70 promoter and POU2F1 recruitment may reflect heat-stress-related epigenetic memory and may be useful in differentiating between individuals that are resilient or vulnerable to stress.

Cerberus–Nodal–Lefty–Pitx signaling cascade controls left–right asymmetry in amphioxus

Many bilaterally symmetrical animals develop genetically programmed left-right asymmetries. In vertebrates, this process is under the control of Nodal signaling, which is restricted to the left side by Nodal antagonists Cerberus and Lefty. Amphioxus, the earliest diverging chordate lineage, has profound left-right asymmetry as a larva. We show that Cerberus, Nodal, Lefty, and their target transcription factor Pitx are sequentially activated in amphioxus embryos. We then address their function by transcription activator-like effector nucleases (TALEN)-based knockout and heat-shock promoter (HSP)-driven overexpression. Knockout of Cerberus leads to ectopic right-sided expression of Nodal, Lefty, and Pitx, whereas overexpression of Cerberus represses their left-sided expression. Overexpression of Nodal in turn represses Cerberus and activates Lefty and Pitx ectopically on the right side. We also show Lefty represses Nodal, whereas Pitx activates Nodal These data combine in a model in which Cerberus determines whether the left-sided gene expression cassette is activated or repressed. These regulatory steps are essential for normal left-right asymmetry to develop, as when they are disrupted embryos may instead form two phenotypic left sides or two phenotypic right sides. Our study shows the regulatory cassette controlling left-right asymmetry was in place in the ancestor of amphioxus and vertebrates. This includes the Nodal inhibitors Cerberus and Lefty, both of which operate in feedback loops with Nodal and combine to establish asymmetric Pitx expression. Cerberus and Lefty are missing from most invertebrate lineages, marking this mechanism as an innovation in the lineage leading to modern chordates.