Higher Reporter Activity Research Articles

WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

Analysis of the functional sequences in the promoter region of the human adhesion molecule close homolog of L1

Background Close Homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules. CHL1 gene is located on human chromosome 3 and has been linked to several pathologies, including 3p deletion syndrome, schizophrenia, and tumor growth and metastasis. Objective The goal of the present study was to determine which region of the CHL1 promoter is most competent in driving CHL1 gene expression. Methods: Five candidate DNA fragments in the promoter regions were selected by screening across six species for evolutionary conserved sequences. The activity of these five promoter regions was quantitatively evaluated using a GFP reporter gene in transfection experiments, performed in C6 glioma cells. Results Of the five promoter regions tested, three drove reporter GFP expression, with the conserved region 6 (CR6, Gene ID AC066595.5, 25851-26850) being the most active for transcription. Conclusion The identification of the CR6 activity provides a better understanding of the regulatory mechanisms underlying CHL1 expression. It may help future discovery of therapeutic strategies that involve influencing critical promoter regions to drive transcriptional regulation of the mammalian CHL1 gene. HIGHLIGHTS Conserved regions of CHL1 promoter sequences were identified by in-silico analysis. Five conserved regions were tested for gene regulatory activity using a reporter assay. Conserved regions CR5, CR6 and CR7 show gene regulatory function in a reporter assay. Co-transfection of CR5 and CR6 yielded the highest reporter activity. The core region of CR6 (CR6core) was identified as a cis-acting element. In-tandem promoter CR5core-CR6core was the best in a reporter assay.

ERG ENHANCER-BASED REPORTER IDENTIFIES LEUKEMIA CELLS WITH ELEVATED LEUKEMOGENIC POTENTIAL DRIVEN BY ERG-USP9X FEED-FORWARD REGULATION

Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to the insufficiently characterized subpopulations of Leukemia Stem Cells (LSCs) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter which enables stemness assessment to enrich and functionally characterize LSCs. We revealed heterogeneous activity of the ERG+85 enhancer based fluorescent reporter in human leukemias. Cells with high reporter activity (tagBFPHigh) exhibited elevated expression of stemness and chemo-resistance genes, demonstrated increased clonogenicity and resistance to chemo- and radio- therapy as compared to their tagBFPNeg counterparts. Moreover, tagBFPHigh enriched fraction was capable of regenerating the original cellular heterogeneity and demonstrated increased invasion ability. Most importantly, tagBFPHigh fraction was enriched for leukemia initiating cells in a xenograft assay. We also identified USP9X deubiquitinase enzyme as a novel ERG transcriptional target that sustains ERG+85 positive cells by controlling ERG ubiquitination. Therapeutic targeting of USP9X led to the preferential inhibition of the ERG-dependent leukemias. In summary, we have developed a new strategy to characterize LSCs and propose ERG targeting via USP9X inhibition as a potential anti-leukemia treatment.

An ERG Enhancer-Based Reporter Identifies Leukemia Cells with Elevated Leukemogenic Potential Driven by ERG-USP9X Feed-Forward Regulation.

Acute leukemia is a rapidly progressing blood cancer with low survival rates. Unfavorable prognosis is attributed to insufficiently characterized subpopulations of leukemia stem cells (LSC) that drive chemoresistance and leukemia relapse. Here we utilized a genetic reporter that assesses stemness to enrich and functionally characterize LSCs. We observed heterogeneous activity of the ERG+85 enhancer-based fluorescent reporter in human leukemias. Cells with high reporter activity (tagBFPHigh) exhibited elevated expression of stemness and chemoresistance genes and demonstrated increased clonogenicity and resistance to chemo- and radiotherapy as compared with their tagBFPNeg counterparts. The tagBFPHigh fraction was capable of regenerating the original cellular heterogeneity and demonstrated increased invasive ability. Moreover, the tagBFPHigh fraction was enriched for leukemia-initiating cells in a xenograft assay. We identified the ubiquitin hydrolase USP9X as a novel ERG transcriptional target that sustains ERG+85-positive cells by controlling ERG ubiquitination. Therapeutic targeting of USP9X led to preferential inhibition of the ERG-dependent leukemias. Collectively, these results characterize human leukemia cell functional heterogeneity and suggest that targeting ERG via USP9X inhibition may be a potential treatment strategy in patients with leukemia. SIGNIFICANCE: This study couples a novel experimental tool with state-of-the-art approaches to delineate molecular mechanisms underlying stem cell-related characteristics in leukemia cells.

Comparison of effectiveness of 5'-regulatory sequences in transplastomic tobacco chloroplasts.

The development of tools which ensure the desired level of transgene expression in plastids is a prerequisite for the effective utilization of these plant organelles for the deployment of bioactive proteins. High-level accumulation of target proteins is considered as a positive feature of transplastomic plants, but excessive accumulation of foreign proteins may have deleterious effects on host plants. On the other hand, expression at low levels can result in ineffective phenotypes. We compared the effectiveness of different 5'-regulatory sequences in driving the expression of a reporter gene, β-glucuronidase (uidA), in tobacco chloroplasts. To achieve varying expression levels, we have chosen heterologous 5'-regulatory sequences which either differ significantly from their homologous counterparts or depend on specific nuclear encoded factors. The Medicago truncatula psbA promoter/5'-UTR supported the highest levels of protein accumulation, surpassing the other tested sequences by two to three orders of magnitude. The heterologous regulatory sequence of Phaseolus vulgaris rbcL gene was as efficient in tobacco chloroplasts as the corresponding homologous promoter/5'-UTR. The Arabidopsis thaliana ndhF promoter/5'-UTR supported as high reporter activity levels as the rbcL 5'-sequences, whereas the effectiveness of A. thaliana psbN promoter/5'-UTR was three fold lower. The characterized regulatory sequences can be utilized to establish transplastomic lines with desirable levels of target protein accumulation. The ability to control transgene expression should be useful for achieving appropriate levels of protein accumulation and thereby avoid their negative impacts on host plant physiology.

Abstract 2403: Molecular mechanisms orchestrating intratumoral heterogeneity in Kras-driven carcinomas

Abstract The considerable variability within tissue microenvironments as well as the multiclonality of cancers leads to intratumoral heterogeneity. This increases the probablility of cellular states that promote resistance to therapy and eventually lead to reconstitution of the tumor by treatment-resistant cancer cells, which in some cases have properties of normal tissue stem cells. Wnt signals are important in the maintenance of stem cells in various epithelial tissues, including in lung development and regeneration. We hypothesized that Wnt signals contribute to tumor heterogeneity in genetically engineered KrasG12D; Tp53Δ/Δ (“KP”) mouse lung adenocarcinomas (LUAD). We observed that a subpopulation of LUAD cells exhibited high Wnt reporter activity and had increased tumor forming ability, which could be suppressed by silencing of Wnt signaling pathway components or by small molecule Wnt inhibitors in vitro and in vivo. KP LUAD cells show hierarchical features with two distinct populations, one with increased Wnt reporter activity and another forming a niche that provides the Wnt signal. Lineage-tracing experiments in the autochthonous KP tumors demonstrated that Wnt responder cells have increased tumor propagation ability in vivo. Strikingly, selective ablation of the Wnt responder cells resulted in tumor stasis. CRISPR-based targeting or small molecules targeting Wnt signaling reduced tumor growth and prolonged survival in the autochthonous KP mouse lung cancer model. Interestingly, we found that a similar Wnt-driven mechanism operates to establish an intratumoral hierarchy also in other Kras-driven carcinomas. These results indicate that maintenance of heterogeneity within tumors may be advantageous for the tumor cell population collectively, which may be utilized as a therapeutic target to homogenize cancer cell phenotypes within tumors. Citation Format: Roxana Azimi. Molecular mechanisms orchestrating intratumoral heterogeneity in Kras-driven carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2403.

Polycistronic Expression of the Influenza A Virus RNA-Dependent RNA Polymerase by Using the Thosea asigna Virus 2A-Like Self-Processing Sequence.

The RNA-dependent RNA polymerase (RdRp) of influenza A virus consists of three subunits, PB2, PB1, and PA, and catalyses both viral RNA genome replication and transcription. Cotransfection of four monocistronic expression vectors for these subunits and nucleoprotein with an expression vector for viral RNA reconstitutes functional viral ribonucleoprotein complex (vRNP). However, the specific activity of reconstituted RdRp is usually very low since the expression level and the ratio of the three subunits by transfection are uncontrollable at single-cell levels. For efficient reconstitution of RdRp and vRNP, their levels need to be at least comparable. We constructed polycistronic expression vectors in which the coding sequences of the three subunits were joined with the 2A-like self-processing sequence of Thosea asigna virus (TaV2A) in various orders. The level of PB1 protein, even when it was placed at the most downstream, was comparable with that expressed from the monocistronic PB1 vector. In contrast, the levels of PB2 and PA were very low, the latter of which was most likely due to proteasomal degradation caused by the TaV2A-derived sequences attached to the amino- and/or carboxyl-terminal ends in this expression system. Interestingly, two of the constructs, in which the PB1 coding sequence was placed at the most upstream, showed much higher reporter activity in a luciferase-based mini-genome assay than that observed by cotransfection of the monocistronic vectors. When the coding sequence of selective antibiotic marker was further placed at the most downstream of the PB1-PA-PB2 open reading frame, stable cells expressing RdRp were easily established, indicating that acquisition of antibiotic resistance assured the expression of upstream RdRp. The addition of an affinity tag to the carboxyl-terminal end of PB2 allowed us to isolate reconstituted vRNP. Taken together, the polycistronic expression system for influenza virus RdRp may be available for functional and structural studies on vRNP.

Abstract A64: β-catenin as a TIC therapeutic target in epithelial ovarian cancer.

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is the fifth leading cause of cancer deaths in women. The most common and lethal form of EOC which makes up greater than 75% of cases is high-grade serous cancer (HGSOC). Platinum-based drugs are used as first-line chemotherapeutics for all women diagnosed with HGSOC. Unfortunately, ~20% do not respond to this treatment and up to 80% of those patients which do respond recur within 5 years at which point the cancer is particularly difficult to eradicate. Emerging evidence supports the concept that platinum based therapies are able to eliminate the bulk of differentiated cancer cells but are unable to eliminate tumor-initiating cells (TICs) which are rare populations of tumor cells characterized by their ability to self-renew indefinitely and thus drive the expansion of drug-resistant tumors. Hence, there is immediate need of novel TIC targeted therapeutic approaches that can overcome chemo-resistance in EOC. Thus, identifying critical drivers of the stem-like phenotype that can be pharmacologically modulated in ovarian TICs is required. To date, no TIC targeting drugs have successfully completed clinical trials in EOC. Using primary HGSOC cells derived from HGSOC patient derived xenograft (PDX) models, we have recently identified β-catenin as a critical driver of HGSOC TIC phenotype in-vitro. β-catenin knockdown decreased stem-cell frequency and induced chemo-sensitivity in-vitro in platinum resistant HGSOC PDX derived primary cells. ALDH positive Ovarian TIC subpopulation with high Wnt reporter activity was platinum resistant and exhibited increased expression of β-catenin regulated stem-cell markers clearly identifying β-catenin regulated stem-cell transcriptional axis as a driver of chemo-resistance in Ovarian TICs. Accordingly, the β-catenin stem-cell transcriptional axis specific inhibitor iCG-001 (PRI-724) decreased stem-cell frequency and induced chemo-sensitivity in platinum resistant ovarian TICs, suggesting that pharmacologic inhibition of β-catenin could be a potential novel approach aimed at eradicating TICs in EOC. Furthermore, in-vivo tumor-initiation assays showed a robust decrease in tumor-initiation capacity upon β-catenin knockdown in platinum resistant HGSOC cells, thus establishing β-catenin as a driver of TIC phenotype in HGSOC. In sum, we have identified that pharmacologic inhibition of β-catenin can be a novel TIC targeted therapy approach to overcome chemo-resistance and improve patient outcome in in EOC. Our results suggest that the β-catenin specific inhibitor PRI-724, that has shown promising results in phase I clinical trials in solid tumors, can be a potential TIC targeting drug in EOC. These results will form a solid foundation for clinical trials with PRI-724 in patients with chemo-recurrent EOC. Citation Format: Anil Belur Nagaraj, Peronne Joseph, Olga Kovalenko, Sareena Singh, Amy Armstrong, Analisa DiFeo. β-catenin as a TIC therapeutic target in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A64.

Differential expression of CYB5A in Chinese and European pig breeds due to genetic variations in the promoter region.

Cytochrome b5 (CYB5A) is an important electron transfer protein with homologues in a number of different organisms. In pigs, CYB5A is related to boar taint because of its role in androstenone biosynthesis. To determine the variety of CYB5A expression in pig breeds, genetic variations in the porcine CYB5A promoter region in both Chinese and European pig breeds were examined. Three single nucleotide polymorphisms (NC_010443.4:g.165901487delG, g.165901767T>C and g.165902078C>T) were identified in the porcine CYB5A promoter region. These SNPs occurred in different frequencies in Chinese and European pigs. Chinese pigs were primarily haplotype B (denoted as delG-C-T: the position of nt 165901487 of the CYB5 gene is a G deletion, nt 165901767 is C and nt 165902078 is T), except for Licha black pigs, which were primarily haplotype A (denoted as G-T-C: nt 165901487 is G, nt 165901767 is T and nt 165902078 is C), similar to European pigs. Quantitative PCR data from liver tissues demonstrated that haplotype B individuals had higher CYB5A expression than did those with haplotype A. This was confirmed by in vitro cell transfection assays, in which haplotype B individuals had higher reporter activity than did those with haplotype A. In silico analysis predicted that Myc-associated zinc-finger protein (MAZ) is a potential transcription factor at position 165901767. Electrophoretic mobility shift assays showed this polymorphism affects the stable binding of transcription factors to the CYB5A promoter, which in turn affects the expression levels of this gene. Therefore, this variation of the porcine CYB5A promoter region may explain the differences in androstenone accumulation between Chinese and European pig breeds and may also prove useful as a genetic marker to distinguish the origin of different pig breeds.

P58. The biphasic function of Wnt signaling during neural differentiation of mouse embryonic stem cells

Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. However, contrary to these results, undifferentiated ES cells shows very low level of endogenous Wnt signaling and ectopic activation of Wnt signaling blocks neural differentiation. Therefore it is not clear whether or when endogenous Wnt/β-catenin signaling is necessary for self-renewal or neural differentiation of ES cells. When we examined the expression profile of Wnt signaling components, expressions of most Wnts are very low in undifferentiated ES cells while some of Wnts are increased during differentiation. We generated stable ES cell lines that probe Wnt signaling activity and these cells showed that Wnt signaling was very low in undifferentiated ES cells while increased during embryonic body formation or neural differentiation. However, conditioned media from differentiated ES cells did not induce higher reporter activity than that of undifferentiated ES cells, which suggests that ES cells may change the sensitivity to Wnt signaling during differentiation whereas the levels of secreted Wnts are same. Changes in the composition of Tcfs or Lef1 in ES cells at different conditions may be a possible mechanism. In addition, we observed that activation of Wnt/β-catenin signaling at different time periods resulted in differential effects on neural differentiation of 46C ES cells that show increased Sox1 promoter driven GFP during neural differentiation; activation of Wnt signaling by the treatment with BIO (a GSK3β inhibitor) in early or late period blocks or enhances neural differentiation, respectively. Consistent with these results, inactivation of Wnt/β-catenin signaling in late period by the treatment with endo-IWR1 (an Axin stabilizer) blocked neural differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays biphasic roles during neural differentiation of mouse embryonic stem cells. Supported by the National Research Foundation of Korea (2006-2004046) and Brain Korea 21 program.

Beta‐catenin/TCF complex regulates HNF4‐alpha expression in the liver

Hepatocyte nuclear factor‐alpha (HNF4α) is a liver‐enriched nuclear receptor of the steroid and thyroid hormone receptor superfamily. It is expressed at the highest levels in the liver, kidney, intestine, and pancreas, and plays an essential role in development, organogenesis, and maintenance of organ function. Previously, we have shown that loss of β‐catenin during prenatal liver development compromises hepatocyte maturation. We identified low HNF4α expression in the β‐catenin‐deficient hepatocytes. Also, livers from transgenic mice overexpressing stable β‐catenin and hepatocyte‐specific β‐catenin knockout mice show high and low HNFα expression, respectively. Here, we investigate the regulation of HNF4α expression by β‐catenin. Transfection of HepG2 cells, a human hepatoma cell line, which harbors constitutively active β‐catenin, with HNF4α‐ reporter plasmids demonstrates high luciferase activity, which was significantly reduced in response to β‐catenin siRNA transfection. Co‐transfection of Hep3B cells (contain wild‐type β‐catenin) with stable β‐catenin plasmid and HNF4α‐promoter luciferase expression plasmid also exhibited high reporter activity. ChIP assays utilizing β‐catenin and TCF antibodies clearly show binding to HNF4α promoter. These data indicate that β‐catenin can directly regulate HNF4α, a nuclear transcription receptor and plays a critical role hepatocyte homeostasis.

Characterisation of γ-interferon responsive promoters in fish

Reporter constructs of three interferon (IFN)-γ-induced rainbow trout genes were generated to examine specificity to type I or type II IFN. Constructs included γIP-10, LMP2 and TAP2 and were used to transfect trout fibroblast cells (RTG-2) which were then exposed to rainbow trout rIFNs. The γIP-10 construct showed high reporter activity even in the absence of rIFNs. The LMP2 promoter contained one GAS element and two double ISRE elements, of four constructs made, only those with ISRE elements showed significant reporter activity following rIFN-γ stimulation. The TAP2 regulatory region contained two GAS, two ISRE and one C/EBP element from which four constructs were made. Reporter expression for the construct containing all five elements showed an 11- and 2-fold increase in response to rIFN-γ and type I rIFN, respectively. Constructs containing only the GAS elements did not respond to rIFNs. The TAP2 construct with two ISRE and the C/EBP gave the greatest dose-dependent reporter response to rIFN-γ, with no significant response to type I rIFN. These data suggest that the ISRE elements, or elements nearby, are essential for the induction of type II IFN responsive genes in trout. The TAP2 construct is a candidate to develop a IFN-γ reporter stable cell line.

Sulfiredoxin is Regulated by NRF2 During Oxidative Stress in Lungs

Mammalian sulfiredoxin (SRX) plays a role in the regulation of oxidative stress. However, the transcriptional regulation of SRX in response to oxidative stress is not clear. Here we demonstrated that NRF2 transcription factor regulates SRX expression following oxidative stress in lungs. In response to cigarette smoke exposure, lungs from wild‐type mice showed significant increase in the mRNA and protein expression of SRX, while no induction was observed in lungs from NRF2‐/‐ mice. Similarly, diquat‐induced oxidative stress resulted in increased expression of SRX in wild‐type mouse embryonic fibroblasts (MEF), but not in the NRF2‐/‐ MEF cells. Silencing SRX by siRNA induced greater generation of ROS in Raw 264.7 macrophages. Promoter analysis revealed two antioxidant response elements (ARE) in the mouse SRX promoter. Transient transfection studies, using reporter constructs containing the SRX promoter, in NRF2‐/‐, wild‐type, and KEAP1‐/‐ MEF cells demonstrated low, modest and high reporter activity, respectively, suggesting NRF2‐dependent transcriptional regulation. Chromatin immunoprecipitation assays revealed binding of NRF2 to the ARE element upon hydrogen peroxide treatment. Taken together, our data demonstrates that NRF2 regulates basal and inducible expression of SRX in response to oxidative stress, which may have an important role in cell signaling and protection.

Evidence that a polymorphism within the 3′UTR of glutathione peroxidase 4 is functional and is associated with susceptibility to colorectal cancer

Low selenium (Se) status has been associated with increased risk of colorectal cancer (CRC). Se is present as the amino acid selenocysteine in selenoproteins, such as the glutathione peroxidases. Se incorporation requires specific RNA structures in the 3' untranslated region (3'UTR) of the selenoprotein mRNAs. A single nucleotide polymorphism (SNP) occurs at nucleotide 718 (within the 3'UTR) in the glutathione peroxidase 4 gene. In the present study, Caco-2 cells were transfected with constructs in which type 1 iodothyronine deiodinase coding region was linked to the GPx4 3'UTR with either C or T variant at position 718. Higher reporter activity was observed in cells expressing the C variant compared to those expressing the T variant, under either Se-adequate or Se-deficient conditions. In addition, a disease association study was carried out in cohorts of patients with either adenomatous polyps, colorectal adenocarcinomas and in healthy controls. A higher proportion of individuals with CC genotype at the GPx4 T/C 718 SNP was present in the cancer group, but not in the polyp group, compared with the control group (P < 0.05). The present data demonstrate the functionality of the GPx4 T/C 718 SNP and suggest that T genotype is associated with lower risk of CRC.

Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

The survival of motor neuron (SMN) protein, responsible for the neurodegenerative disease spinal muscular atrophy (SMA), oligomerizes and forms a stable complex with seven other major components, the Gemin proteins. Besides the SMN protein, Gemin2 is a core protein that is essential for the formation of the SMN complex, although the mechanism by which it drives formation is unclear. We have found a novel interaction, a Gemin2 self-association, using the mammalian two-hybrid system and the in vitro pull-down assays. Using in vitro dissociation assays, we also found that the self-interaction of the amino-terminal SMN protein, which was confirmed in this study, became stable in the presence of Gemin2. In addition, Gemin2 knockdown using small interference RNA treatment revealed a drastic decrease in SMN oligomer formation and in the assembly activity of spliceosomal small nuclear ribonucleoprotein (snRNP). Taken together, these results indicate that Gemin2 plays an important role in snRNP assembly through the stabilization of the SMN oligomer/complex via novel self-interaction. Applying the results/techniques to amino-terminal SMN missense mutants that were recently identified from SMA patients, we successfully showed that amino-terminal self-association, Gemin2 binding, the stabilization effect of Gemin2, and snRNP assembly activity were all lowered in the mutant SMN(D44V), suggesting that instability of the amino-terminal SMN self-association may cause SMA in patients carrying this allele.

Potential Role of cAMP Response Element-Binding Protein in Ethanol-InducedN-Methyl-d-aspartate Receptor 2B Subunit Gene Transcription in Fetal Mouse Cortical Cells

We have shown previously that long-term ethanol treatment causes an up-regulation of N-methyl-D-aspartate (NMDA) receptor 2B subunit (NR2B) number and function in cultured fetal mouse cortical neurons. To examine the intracellular signaling pathways involved in this NR2B gene transcription, we have subjected fetal cortical neurons to long-term treatment with ethanol and studied its effect on cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) levels by Western blot and enzyme-linked immunosorbent assay. We find a significant increase in phosphorylated CREB, without change in total CREB protein, in cells treated with ethanol for 5 days. Long-term ethanol treatment did not increase levels of both total and phospho-ERK in serum-free medium, whereas it did increase ERK phosphorylation in medium containing serum, without affecting total ERK levels. CREB phosphorylation was increased by ethanol treatment in both media, irrespective of the presence of serum. Electrophoretic mobility shift assay, using a 25-base pair (bp) double-stranded DNA fragment containing the cyclic AMP response element (CRE)-like sequence of the NR2B promoter as (32)P-labeled probe, showed an increase in specific CRE binding to nuclear proteins isolated from cells undergoing long-term ethanol treatment. A 467-bp DNA fragment of the NR2B promoter containing the CRE sequence cloned into the luciferase vector exhibited high reporter activity in transient cotransfection assay of mouse cortical neurons, and ethanol treatment increased this activity. Introducing site-directed mutation in the CRE sequence significantly reduced the reporter activity relative to the wild-type construct, and it also abolished the stimulatory effect by ethanol. Our results indicate that CREB is probably involved in mediating ethanol-induced up-regulation of NR2B gene.

Spontaneous switch from Aγ- to β-globin promoter activity in a stable transfected dual reporter vector

Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for Aγ- and β-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 1–2 years from transfection. The transfected GM979 cells rapidly (within 1 month) evolved into a stable population which expresses constant levels of reporters for more than a year of continuous bulk culture. No silencing of the inserted construct was observed over time. In contrast, after 1 month, the reporter activity (both from Aγ- and β-promoter) expressed per cell increased over time. The analysis of the Luciferase contained in single cell clones indicated that the higher reporter activity was due to increased gene expression per cell rather than to clonal selection of the most expressing clones. Since the activity driven by the β-promoter increased 10-fold more than that driven by the Aγ one, the ratio between Aγ-driven/( Aγ-driven + β-driven) reporter activity in the cells decreased after 1 month and became similar to the γ/(γ + β) globin mRNA ratio expressed by adult erythroid cells. Moreover, although both cells from early and late bulk culture responded to incubation with butyric acid, a known inducer of fetal globin gene expression, by increasing the reporter activity driven by the Aγ-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the β-promoter. These results suggest that the rapid changes in activity driven by the Aγ- and β-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the Aγ- and β-promoter during the fetal to adult hemoglobin switch and confirm GM979 cells stably transfected with the dual reporter construct as a reliable assay for automated screening of chemical inducers of fetal globin gene activation.

Cloning and characterization of metallothionein gene in ayu Plecoglossus altivelis

Metallothionein (MT) has been used widely as a potential molecular marker to detect the deleterious effects of heavy metals in aquatic ecosystem. Here we exposed ayu, Plecoglossus altivelis, to zinc (Zn) and tested the distribution as well as the induction of MT in various tissues such as liver, kidney, intestine and stomach. MT induction was significant in liver tissue, followed by kidney and intestine, whereas no induction was detected in stomach. The gene encoding ayu MT was successfully cloned and characterized. Complete nucleotide sequencing and analysis of the 4.5 kb DNA fragment containing the ayu MT gene revealed that the gene has three exons interrupted by two introns, a 5′-flanking region of about 2.5 kb and about 1.6 kb of 3′-flanking region. In grouper heart and kidney cells, the 2.5 kb promoter containing eight metal responsive elements (MREs), two hepatic nuclear factor 5 responsive elements (HNF5REs) and one cAMP responsive element (CRE) had the highest reporter activity.

In vivo-targeted gene delivery using antibody-based nonviral vector.

Tissue-specific gene transfer remains one of the main challenges to deliver genes into designated and/or disseminated cells. We have previously shown successful gene transfer with a nonviral gene delivery system based on the simple chemical conjugation of plasmid DNA with antibody. However, this approach was hampered by low efficiency due to the poor translocation rate of DNA to the nucleus. To improve this approach, we have modified our vector by introducing noncovalent binding between the antibody and DNA, allowing the possibility to introduce different important molecules. The noncovalent association was achieved with neutravidin and biotinylated components: (1) biotinylated antibodies; (2) a biotinylated hemagglutinin fusogenic peptide of influenza virus to favor endosomal escape; and (3) biotinylated histone H1 to compact, protect, and associate DNA to the complex. We report here that this delivery system can be internalized by tumor cells targeted by a specific monoclonal antibody, permits the protection of the transfected DNA, and allows its subsequent transfer into the nucleus after escape from the endosomal compartment. We also demonstrate that, in vitro, gene transfer with this vector showed much higher reporter activity in cells (15 vs. 0.5%) and a stronger production of murine interleukin 2 as compared with our previous vector. In vivo, a single intravenous injection of the vector containing an antibody directed to the G250 renal cell carcinoma-associated antigen led to beta-galactosidase expression in engrafted tumor bearing G250 but not in G250-negative tumor or in other tissues. Altogether, these results indicate that our antibody-based vector is suitable to promote gene delivery in vitro and in vivo in tumor cells.

The Upstream Region of the Rpe65 Gene Confers Retinal Pigment Epithelium-specific Expression in Vivo and in Vitro and Contains Critical Octamer and E-box Binding Sites

RPE65 is essential for all-trans- to 11-cis-retinoid isomerization, the hallmark reaction of the retinal pigment epithelium (RPE). Here, we identify regulatory elements in the Rpe65 gene and demonstrate their functional relevance to Rpe65 gene expression. We show that the 5' flanking region of the mouse Rpe65 gene, like the human gene, lacks a canonical TATA box and consensus GC and CAAT boxes. The mouse and human genes do share several cis-acting elements, including an octamer, a nuclear factor one (NFI) site, and two E-box sites, suggesting a conserved mode of regulation. A mouse Rpe65 promoter/beta-galactosidase transgene containing bases -655 to +52 (TR4) of the mouse 5' flanking region was sufficient to direct high RPE-specific expression in transgenic mice, whereas shorter fragments (-297 to +52 or -188 to +52) generated only background activity. Furthermore, transient transfection of analogous TR4/luciferase constructs also directed high reporter activity in the human RPE cell line D407 but weak activity in the non-RPE cell lines HeLa, HepG2, and HS27. Functional binding of potential transcription factors to the octamer sequence, AP-4, and NFI sites was demonstrated by directed mutagenesis, electrophoretic mobility shift assay, and cross-linking. Mutations of these sites abolished binding and corresponding transcriptional activity and indicated that octamer and E-box transcription factors synergistically regulate the RPE65 promoter function. Thus, we have identified the regulatory region in the Rpe65 gene that accounts for tissue-specific expression in the RPE and found that octamer and E-box transcription factors play a critical role in the transcriptional regulation of the Rpe65 gene.