Transcriptome analysis reveals the essential role of NK2 homeobox 1/thyroid transcription factor 1 (NKX2-1/TTF-1) in gastric adenocarcinoma of fundic-gland type.

BACKGROUND: Gastric adenocarcinoma of fundic-gland type (GA-FG) is a gastric malignancy with little relation to Helicobacter pylori. Clinical characteristics of GA-FG have been established, but molecular mechanisms leading to tumorigenesis have not yet been elucidated. METHODS: We subjected three GA-FG tumors-normal mucosa pairs to microarray analysis. Network analysis was performed for the top 30 up-regulated gene transcripts, followed by immunohistochemical staining to confirm the gene expression analysis results. AGS and NUGC4 cells were transfected with the gene-encoding NK2 homeobox 1/thyroid transcription factor 1 (NKX2-1/TTF-1) to evaluate transcriptional changes in its target genes. RESULTS: Comprehensive gene expression analysis identified 1410 up-regulated and 1395 down-regulated gene probes with ≥ two-fold difference in expression. Among the top 30 up-regulated genes in GA-FG, we identified transcription factor NKX2-1/TTF-1, a master regulator of lung/thyroid differentiation, together with surfactant protein B (SFTPB), SFTPC, and secretoglobin family 3A member 2(SCGB3A2), which are regulated by NKX2-1/TTF-1. Immunohistochemical analysis of 16 GA-FG specimens demonstrated significantly higher NKX2-1/TTF-1 and SFTPB levels, as compared to that in adjacent normal mucosa (P < 0.05), while SCGB3A2 levels did not differ (P = 0.341). Transduction of NKX2-1/TTF-1 into AGS and NUGC4 cells induced transactivation of SFTPB and SFTPC, indicating that NKX2-1/TTF-1 can function as normally in gastric cells as it can in the lung cells. CONCLUSIONS: Our first transcriptome analysis of GA-FG indicates significant expression of NKX2-1/TTF1 in GA-FG. Immunohistochemistry and cell biology show ectopic expression and normal transactivation ability of NKX2-1/TTF-1, suggesting that it plays an essential role in GA-FG development.

Artificial Restriction DNA Cutter Using Nuclease S1 for Site-Selective Scission of Genomic DNA.

By combining a pair of pseudo-complementary peptide nucleic acids (pcPNAs) with S1 nuclease, a novel tool to cut DNA at a predetermined site can be obtained. Complementary pcPNAs invade the DNA duplex and base pair to each strand of a target site, creating single-stranded regions that are cleaved by S1 nuclease. The scission site can be freely modulated by the design of pcPNAs. This method can be used to cleave a single site in the human genome. This protocol presents experimental details for site-selective scission using this versatile new tool. © 2019 by John Wiley & Sons, Inc.

Affinity Isolation of Defined Genomic Fragments Cleaved by Nuclease S1-based Artificial Restriction DNA Cutter.

The human genome is highly susceptible to various modifications, lesions, and damage. To analyze lesions and proteins bound to a defined region of the human genome, the genome should be fragmented at desired sites and the region of interest should be isolated. The few available methods for isolating a desired region of the human genome have serious drawbacks and can only be applied to specific sequences or require tedious experimental procedures. We have recently developed a novel method to isolate a desired fragment of the genome released by site-specific scission of DNA using a pair of pseudo-complementary peptide nucleic acids (pcPNAs) and S1 nuclease. When conjugated to biotin, one of the pcPNAs can be used to affinity purify the cleavage product. Here we report a detailed protocol to isolate defined kilobase-length DNA fragments that can be applied to plasmid or genomic DNA and is not limited by sequence. © 2019 by John Wiley & Sons, Inc.

One-Pot Isolation of a Desired Human Genome Fragment by Using a Biotinylated pcPNA/S1 Nuclease Combination.

Scission of the human genome at predetermined sites and isolation of a particular fragment are of great interest for the analysis of lesion/modification sites, in proteomics, and for gene therapy. However, methods for human genome scission and specific fragment isolation are limited. Here, we report a novel one-pot method for the site-specific scission of DNA by using a biotinylated pcPNA/S1 nuclease combination and isolation of a desired fragment by streptavidin-coated magnetic beads. The proof of concept was initially demonstrated for the clipping of plasmid DNA and isolation of the required fragment. Our method was then successfully applied for the isolation of a fragment from the cell-derived human genome.

Applications of PNA-Based Artificial Restriction DNA Cutters.

More than ten years ago, artificial restriction DNA cutters were developed by combining two pseudo-complementary peptide nucleic acid (pcPNA) strands with either Ce(IV)/EDTA or S1 nuclease. They have remarkably high site-specificity and can cut only one predetermined site in the human genome. In this article, recent progress of these man-made tools have been reviewed. By cutting the human genome site-selectively, desired fragments can be clipped from either the termini of chromosomes (telomeres) or from the middle of genome. These fragments should provide important information on the biological functions of complicated genome system. DNA/RNA hybrid duplexes, which are formed in living cells, are also site-selectively hydrolyzed by these cutters. In order to further facilitate the applications of the artificial DNA cutters, various chemical modifications have been attempted. One of the most important successes is preparation of PNA derivatives which can form double-duplex invasion complex even under high salt conditions. This is important for in vivo applications, since the inside of living cells is abundant of metal ions. Furthermore, site-selective DNA cutters which require only one PNA strand, in place of a pair of pcPNA strands, are developed. This progress has opened a way to new fields of PNA-based biochemistry and biotechnology.

Suppression of Myogenic Differentiation of Mammalian Cells Caused by Fluidity of a Liquid-Liquid Interface.

There is growing evidence to suggest that the prevailing physical microenvironment and mechanical stress regulate cellular functions, including adhesion, proliferation, and differentiation. Moreover, the physical microenvironment determines the stem-cell lineage depending on stiffness of the substrate relative to biological tissues as well as the stress relaxation properties of the viscoelastic substrates used for cell culture. However, there is little known regarding the biological effects of a fluid substrate, where viscoelastic stress is essentially absent. Here, we demonstrate the regulation of myogenic differentiation on fluid substrates by using a liquid-liquid interface as a scaffold. C2C12 myoblast cells were cultured using water-perfluorocarbon (PFC) interfaces as the fluid microenvironment. We found that, for controlled in vitro culture at water-PFC interfaces, expression of myogenin, myogenic regulatory factors (MRF) family gene, is remarkably attenuated even when myogenic differentiation was induced by reducing levels of growth factors, although MyoD was expressed at the usual level (MyoD up-regulates myogenin under an elastic and/or viscoelastic environment). These results strongly suggest that this unique regulation of myogenic differentiation can be attributed to the fluid microenvironment of the interfacial culture medium. This interfacial culture system represents a powerful tool for investigation of the mechanisms by which physical properties regulate cellular adhesion and proliferation as well as their differentiation. Furthermore, we successfully transferred the cells cultured at such interfaces using Langmuir-Blodgett (LB) techniques. The combination of the interfacial culture system with the LB approach enables investigation of the effects of mechanical compression on cell functions.

Clipping of predetermined fragments from the human genome by S1 nuclease-PNA combinations.

By combining S1 nuclease with two strands of pseudo-complementary peptide nucleic acid (pcPNA), the whole human genome was selectively cut at targeted sites, and desired fragments were clipped from the genome.

An artificial restriction DNA cutter for site-selective gene insertion in human cells.

With the use of a chemistry-based artificial restriction DNA cutter (combination of Ce(IV)-EDTA and a pair of pcPNA), both an antibiotic-resistance gene and a fluorescent reporter protein gene were incorporated into the targeted site through homologous recombination in human cells.

Intracellular localization of PNA in human cells upon its introduction by electroporation.

Peptide nucleic acid (PNA) is one of the most useful DNA analogs in a wide variety of gene analysis in human cells. In order to exhibit its maximal functions, PNA must be localized to a desired place (e.g., nucleus, cytoplasm and other organelles). Here, we introduced PNAs into HeLa cells by electroporation and examined their localization at various time points. The PNA which binds to the mitochondrial COII gene was initially accumulated in the nucleus, and thereafter mostly transferred to cytoplasm. This time-dependent intracellular localization of PNA is ascribed to the breakdown of the nuclear envelope in the cell division. On the other hand, another PNA that binds to telomere repeat sequence mostly remained in the nucleus, even after the cell division occurred. The retention of this PNA in the nucleus was further enhanced when it was conjugated with Cy3.

Chemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter.

A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.

Site-selective scission of human genome by artificial restriction DNA cutter.

By using an artificial restriction DNA cutter which is composed of Ce(iv)/EDTA and two pseudo-complementary peptide nucleic acid strands (pcPNAs), only one target site in the whole genome of human beings (one site in the X chromosome) was selectively hydrolyzed.

Homologous recombination in human cells using artificial restriction DNA cutter.

The double strand break induced by an artificial restriction DNA cutter (ARCUT) was successfully repaired in human cells with high frequencies through homologous recombination.

The R336Q mutation in human mitochondrial EFTu prevents the formation of an active mt-EFTu.GTP.aa-tRNA ternary complex.

The mitochondrial translational machinery allows the genes encoded by mitochondrial DNA (mtDNA) to be translated in situ. Mitochondrial translation requires a number of nucleus-encoded protein factors, some of which have been found to carry mutations in patients affected by mitochondrial encephalomyopathies. We have previously described the first, and so far only, mutation in the mitochondrial elongation factor Tu, mt-EFTu, in a baby girl with polycystic encephalopathy, micropolygyria, and leukodystrophic changes. Despite that the mutant mt-EFTu was present in normal amount in the patient's tissues, mitochondrial translation was severely reduced, determining multiple defects in the amount and activity of mtDNA-dependent respiratory chain complexes. By an in-vitro reconstructed translational system, we here provide evidence that the mutant mt-EFTu variant fails to bind to aminoacylated mitochondrial tRNAs, thus explaining the observed impairment of mitochondrial translation. This is the first analysis on the molecular mechanism of a mtDNA translation defect due to a nuclear gene mutation.

Direct preparation of sticky-ended duplexes within PCR by using caged primers.

Control of the terminal structures of PCR products is crucially important to facilitate molecular biology and biotechnology. Here, we report a new method to prepare PCR products having desired sticky ends directly after thermal cycles. When a pair of caged primers is used, polymerase reaction is site-selectively terminated in front of the caged nucleotide, and the 5'- portion of the primer remains single-stranded throughout the reaction. Successive removal of the photo-cleavable protecting group gives restriction-enzyme free sticky ends on the product. We succeeded in applying this technique to make a recombinant vector bearing GFP gene.

Site-specific gene manipulation of fluorescent proteins using artificial restriction DNA cutter.

Two of three amino acid residues, which compose the chromophore of the enhanced green fluorescent protein (EGFP), were converted to others by using artificial restriction DNA cutter (ARCUT). The vector prepared by ARCUT was easily connected with the insert by using oligonucleotide additive and resultant fluorescent protein such as blue fluorescent protein (BFP) was successfully expressed in cells.

Site-selective blocking of PCR by a caged nucleotide leading to direct creation of desired sticky ends in the products.

In order to terminate the polymerase reaction at a desired position, a caged thymine derivative--4-O-[2-(2-nitrophenyl)propyl]thymine--was incorporated into PCR primers. In the PCR cycles, the elongation of the nascent strand (5'-->3' direction) by polymerase was site-selectively terminated at the 3'-side of T(NPP). Accordingly, predetermined protruding ends were obtained after the removal of the protecting group by short UVA irradiation. Recombinant vectors coding the GFP gene were successfully prepared by direct ligation of these light-assisted cohesive-ending PCR (LACE-PCR) products with scission fragments obtained by use either of restriction enzymes or of artificial restriction DNA cutters and were used for transformation of E. coli.

Measuring mRNA decay in human mitochondria.

Human mitochondria contain a genome encoding 13 proteins, all of which are components of respiratory chain complexes. Mutations in human mitochondrial DNA often have pathological consequences. Although 12 of the mitochondrial mRNAs are generated from the same polycistronic transcript, the steady-state level of each mRNA differs. The stability of each mitochondrial mRNA is post-transcriptionally controlled by polyadenylation and deadenylation. However, the molecular mechanism by which each mRNA attains a unique stability is not fully understood. In this report, we describe a practical method for measuring the half-lives of human mitochondrial mRNAs using quantitative real-time reverse transcription PCR.

Gene manipulation of adenovirus vector by artificial restriction DNA cutter.

Adenovirus vector is a useful tool for research in gene expression and regulation as well as gene therapy. However, the size of this vector is too large to construct it by a simple method. In this study, we successfully cleaved specifically at one target site of this large vector by artificial restriction DNA cutter (ARCUT). This result suggests that ARCUT will be the powerful tool for construction of large sized virus vectors.