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Advanced transgenic and gene-targeting approaches.

Over the past two decades the techniques associated with the manipulation of the mouse genome have provided a powerful approach toward the better understanding of gene function. Conventional transgenic and gene targeting approaches have been used extensively, and these techniques have been particularly rewarding for neuroscientists. Nevertheless, the traditional approaches toward genome manipulation have certain limitations that diminish their usefulness for studying more sophisticated biological processes. Therefore, variations to these techniques have recently been developed. The improvements are focused on two areas: one provides regulated control of transgene expression using an inducible expression system; and the other provides the opportunity to inactivate genes in specific cells and at predetermined developmental stages with a conditional gene targeting system. This review summarizes the advantages as well as some of the technical difficulties of these new approaches. The application of these advanced approaches in biomedical research, particularly neuroscience, are also discussed.

Animals↗

Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis.

Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited blistering skin disorder caused by point mutations in the suprabasal cytokeratins 1 or 10. Targeting the murine cytokeratin 10 gene in ES cells resulted in mice with different phenotypes in the homozygotes and heterozygotes; both of which exhibit similarities to specific clinical characteristics of BCIE. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes were apparently unaffected at birth, but developed hyperkeratosis with age. In both genotypes, aggregation of cytokeratin intermediate filaments, changes in cytokeratin expression, and alterations in the program of epidermal differentiation were observed. In addition we demonstrate, for the first time, the existence of the murine equivalent of human cytokeratin 16.

Animals↗

Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis.

The EWS/FLI-1 fusion gene results from the 11;22 chromosomal translocation in Ewing's sarcoma. The product of the gene is one of a growing number of structurally altered transcription factors implicated in oncogenesis. We have employed a subtractive cloning strategy of representational difference analysis in conjunction with a model transformation system to identify genes transcribed in response to EWS/FLI. We have characterized eight transcripts that are dependent on EWS/FLI for expression and two transcripts that are repressed in response to EWS/FLI. Three of the former were identified by sequence analysis as stromelysin 1, a murine homolog of cytochrome P-450 F1 and cytokeratin 15. Stromelysin 1 is induced rapidly after expression of EWS/FLI, suggesting that the stromelysin 1 gene may be a direct target gene of EWS/FLI. These results demonstrate that expression of EWS/FLI leads to significant changes in the transcription of specific genes and that these effects are at least partially distinct from those caused by expression of germ line FLI-1. The representational difference analysis technique can potentially be applied to investigate transformation pathways activated by a broad array of genes in different tumor systems.

Base Sequence↗

Identification of human myometrial target genes of the c-Jun NH2-terminal kinase (JNK) pathway: the role of activating transcription factor 2 (ATF2) and a novel spliced isoform ATF2-small.

Activating transcription factor 2 (ATF2), a ubiquitously expressed member of the basic region leucine zipper (bZIP) family of transcription factors activated by mitogen activated protein kinase (MAPK) pathways, is important in the mediation of cellular stress responses, development and transformation. We have previously reported the differential expression of active ATF2 in the human myometrium throughout pregnancy and labour, and identified and partially characterized a novel splice variant ATF2-small (ATF2-sm). To further understand the role of these factors in the myometrium, we have used gene microarrays to define the target genes in cultured myometrial cells stably-transfected with ATF2 and ATF2-sm cDNAs. Many of the genes identified appear to have potential roles in regulating myometrial function and include proteins involved in G-protein receptor signalling, cytokine signalling, transcriptional regulation, cell-cycle control, formation of the extracellular matrix and cytoskeletal architecture. ATF2 was found to affect the expression of 204 genes; 113 being up-regulated and 91 down-regulated whereas the novel ATF2-sm factor altered the expression of 55 genes; expression was increased in 29 cases and decreased in 26. A further 25 genes affected by ATF2-sm were identified by suppression subtractive hybridisation (SSH). Notably, the genes affected by ATF2 and ATF2-sm appear to belong to discrete groups: only two genes were affected by both factors.

Activating Transcription Factor 2↗

Phylogenetic footprinting and genome scanning identify vertebrate BMP response elements and new target genes.

The complex gene regulatory networks governed by growth factor signaling are still poorly understood. In order to accelerate the rate of progress in uncovering these networks, we explored the usefulness of interspecies sequence comparison (phylogenetic footprinting) to identify conserved growth factor response elements. The promoter regions of two direct target genes of Bone Morphogenetic Protein (BMP) signaling in Xenopus, Xvent2 and XId3, were compared with the corresponding human and/or mouse counterparts to identify conserved sequences. A comparison between the Xenopus and human Vent2 promoter sequences revealed a highly conserved 21 bp sequence that overlaps the previously reported Xvent2 BMP response element (BRE). Reporter gene assays using Xenopus animal pole ectodermal explants (animal caps) revealed that this conserved 21 bp BRE is both necessary and sufficient for BMP responsiveness. We combine the same phylogenetic footprinting approach with luciferase assays to identify a highly conserved 49 bp BMP responsive region in the Xenopus Id3 promoter. GFP reporters containing multimers of either the Xvent2 or XId3 BREs appear to recapitulate endogenous BMP signaling activity in transgenic Xenopus embryos. Comparison of the Xvent2 and the XId3 BRE revealed core sequence features that are both necessary and sufficient for BMP responsiveness: a Smad binding element (SBE) and a GC-rich element resembling an OAZ binding site. Based on these findings, we have implemented genome scanning to identify over 100 additional putative target genes containing 2 or more BRE-like sequences which are conserved between human and mouse. RT-PCR and in situ analyses revealed that this in silico approach can effectively be used to identify potential BMP target genes.

Animals↗

B cell antigen receptor signaling enhances IFN-gamma-induced Stat1 target gene expression through calcium mobilization and activation of multiple serine kinase pathways.

The signal transducers and activators of transcription 1 (Stat1) are essential for the majority of interferon-gamma (IFN-gamma)-regulated gene expression. Phosphorylation of serine 727 in the transcription activation domain of Stat1 is induced in response to IFN-gamma for maximal transcription activity. In this report, we show that crosslinking of B cell antigen receptor (BCR) or T cell antigen receptor (TCR) can enhance S727 phosphorylation in Stat1 and result in increased expression of Stat1 target genes. We further demonstrate that this enhancement by BCR cross-linking involves the widely used secondary messenger Ca2+ and simultaneous activation of multiple serine kinase pathways. When cells are exposed to both IFN-gamma and a Ca2+ fluxing reagent, the level of S727 phosphorylation is enhanced, resulting in increased transcription activation of Stat1 target genes. We directly demonstrate that the biochemical function of phospho-Ser-727 is to enhance the recruitment of transcription coactivator CBP/p300 to the promoters of Stat1 target genes. Furthermore, we show that both the p38 mitogen-activated protein kinase (MAPK) and the Ca(2+)/calmodulin-dependent kinase (CaMKII) are activated in response to BCR signaling to converge on Stat1 S727 for maximal gene expression. These studies demonstrate that a wide variety of noncytokine signaling pathways can modulate cytokine signaling through modulation of Stat1 serine phosphorylation.

Animals↗

Identification of circulating miRNA alterations in diabetes patients excluding periodontitis effects: insights into target gene downregulation in diabetic complications.

BACKGROUND: Diabetes mellitus (DM) induces systemic complications through chronic metabolic dysregulation. Circulating exosomal microRNAs (miRNAs) are emerging as key regulators of post-transcriptional gene expression and may drive diabetes-associated pathologies. Although miRNAs have been widely studied in diabetes, the characterization of PD-independent miRNA signatures across tissues remains limited. This study aimed to identify DM-specific miRNA alterations and their contribution to systemic metabolic dysfunction independent of PD. METHODS: Exosomes were isolated from plasma samples, and small RNA sequencing was performed to identify differentially expressed miRNAs (DE-miRs) using the limma R package. Predicted target genes were identified using TargetScan and validated through bulk RNA sequencing datasets from four tissues-foot, kidney, pancreas, and retina. Differentially expressed genes (DEGs) were analyzed, followed by Gene Ontology Biological Process (GOBP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment to elucidate diabetes-related mechanisms. RESULTS: We identified 9 upregulated and 6 downregulated DE-miRs specific to the diabetic group. TargetScan predicted 216 upregulated and 64 downregulated target genes. Functional validation revealed that these genes were enriched in pathways related to glucose metabolism, cellular stress response, and tissue repair. Notably, SREK1 and GLIPR1 were commonly detected across all four tissues, suggesting potential systemic regulators of diabetes-related complications. CONCLUSION: This study suggests that circulating exosomal miRNAs, independent of periodontitis, may function as systemic regulators in diabetes. Unlike previous studies, which did not distinguish co-morbid periodontitis, we specifically defined PD-independent miRNA signatures and validated their cross-organ regulatory effects on target genes. Our results revealed a cross-organ miRNA-mRNA regulatory network and identified common regulatory targets. These findings provide insights into both systemic and organ-specific mechanisms underlying diabetic complications and highlight the potential of miRNAs as biomarkers and therapeutic targets.

Humans↗

The crystal structure of the gene targeting homing endonuclease I-SceI reveals the origins of its target site specificity.

The I-SceI homing endonuclease enhances gene targeting by introducing double-strand breaks at specific chromosomal loci, thereby increasing the recombination frequency. Here, we report the crystal structure of the enzyme complexed to its DNA substrate and Ca(2+) determined at 2.25A resolution. The structure shows the prototypical beta-saddle of LAGLIDADG homing endonucleases that is contributed by two pseudo-symmetric domains. The high specificity of I-SceI is explained by the large number of protein-DNA contacts, many that are made by a long beta-hairpin loop that reaches into the major groove of the DNA. The DNA minor groove is compressed at the catalytic center, bringing the two scissile phosphodiester bonds into close proximity. The protein-Ca(2+)-DNA structure shows the protein bound to its DNA substrate in a pre-reactive state that is defined by the presence of two asymmetric active sites, one of which appears poised to first cleave the DNA bottom strand.

Base Sequence↗

Plasmid-mediated muscle-targeted gene therapy for circulating therapeutic protein replacement: a tale of the tortoise and the hare?

There is now conclusive evidence that gene therapy can lead to real clinical benefit. Initial enthusiasm has been muted by set-backs related to viral vectors including retroviral oncogenesis and adenoviral inflammatory response. Plasmid-mediated muscle-targeted gene transfer offers the potential of a cost-effective pharmaceutical grade therapy delivered by simple intramuscular injection without the need for anaesthetic, cell culture, transplantation or immunosuppression. This approach is particularly appropriate for long-term circulating therapeutic protein replacement currently requiring repeated injection therapy. Wide-ranging clinical applications include haemophilia, chronic anaemia, growth hormone deficiency and diabetes. Inadequate transgene expression, unregulated protein delivery and immune response have been major limiting factors. Recent innovations including in situ electroporation enabling sustained systemic protein delivery within the therapeutic range are reviewed. Pharmacological and physiological approaches to regulation are discussed in addition to the role of innate and humoral immunity. Translation of advances in all of these areas to clinical success will enable muscle-targeted gene therapy to capitalise on its inherent strengths and realise its long-standing promise.

Animals↗

[Expression of NFkappaB p65 and its target genes in gastric cancer and precancerous lesions].

OBJECTIVE: To study the expression of NFkappaB p65 and its target genes in intestinal metaplasia (IM), dysplasia (Dys), gastric cancer (GC) infected with Helicobacter pylori (Hp) and explore the mechanism of infection by cytotoxin-associated antigen A expressing Hp (CagA(+)Hp) in the development of gastric cancer. METHODS: CagA antibody in blood sample of 289 patients was determined by ELISA. Hp was detected by rapid urease test and Warthin starry staining. Expression of NFkappaB p65 and its target genes in IM, Dys and GC was examined by immunohistochemistry. RESULTS: In IMI approximately II, IMIII, DysI, DysII approximately III and GC, the expression of NFkappaB p65 was significantly higher in patients with CagA(+)Hp infection than those without CagA Hp infection. In IMIII and DysII approximately III, the expression of NFkappaB p65, c-myc, CyclinD(1) and bcl-xl was significantly higher in patients with CagA Hp infection than those without CagA Hp infection. In gastric cancer infected with CagA(+)Hp, the expression of NFkappaB p65, c-myc, CyclinD(1) and bcl-xl was significantly higher in intestinal type than in diffuse type. CONCLUSION: There are different mechanisms in intestinal type and diffuse type in the development of gastric cancer. The occurrence of intestinal type gastric cancer is associated with CagA(+)Hp infection which by NFkappaB p65 upregulating the expression of c-myc, CyclinD(1),bcl-xl in patients with IMIII, DysII approximately III. It may be an effective method to prevent gastric cancer by inhibiting NFkappaB p65.

Adult↗

Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity.

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics.

Base Sequence↗

Recruitment of distinct chromatin-modifying complexes by tamoxifen-complexed estrogen receptor at natural target gene promoters in vivo.

Tamoxifen, a breast cancer therapeutic, is a tissue-selective estrogen receptor modulator (SERM), which acts as an antiestrogen in the mammary tissue and displays estrogenic activity in other tissues such as bone and uterus. In order to understand the mechanisms underlying the antiestrogenic effect of this prototype SERM, we performed an analysis of the cofactors that interact with ER complexed with 4-hydroxytamoxifen (OHT) at natural target genes in a human breast tumor cell line MCF-7. Employing chromatin immunoprecipitation (ChIP), we observed that treatment with OHT rapidly induces the binding of ERalpha to the E-responsive promoter regions of pS2 and c-myc genes. Promoter-bound OHT-complexed ERa coordinately recruited the components of a multiprotein complex containing the corepressor NCoR, histone deacetylase 3 (HDAC3), and a WD40-repeat protein TBL1. Surprisingly, the OHT-complexed ERalpha also recruited a chromatin-remodeling NuRD complex in which histone deacetylase 1 (HDAC1) is associated with several polypeptides including metastasis-associated protein 1/2 (MTA1/2), and SWI2/SNF2-related ATPase Mi2. Kinetic studies revealed that following OHT addition the recruitment of these HDAC complexes to pS2 or the c-myc promoter occurs in a sequential manner; the NCoR-HDAC3 complex is recruited earlier than the NuRD complex. Serial ChIP experiments indicated that the ER-NCoR-HDAC3 and ER-NuRD complexes are distinct, and they do not occupy the target gene promoter simultaneously. We also established a close temporal link between the appearance of the HDAC complexes at the E-responsive regions of pS2 and c-myc promoters, local hypoacetylation of specific lysine residues in N-terminal tails of histones H3 and H4, and disappearance of RNA polymerase II from the target gene loci. Collectively, our studies indicated that transcriptional repression by tamoxifen-bound ER at E-regulated gene promoters involves a dynamic interplay of multiple distinct chromatin-modifying/remodeling complexes.

Cell Line, Tumor↗

Identification of target genes for the Xenopus Hes-related protein XHR1, a prepattern factor specifying the midbrain-hindbrain boundary.

The midbrain-hindbrain boundary (MHB) acts as a local organizer in the development of the CNS in vertebrates. Previously, we identified an MHB-specific bHLH-WRPW transcriptional repressor gene, Xenopus Hes-related 1 (XHR1), which is initially expressed in the presumptive MHB (pre-MHB) region at the early gastrula stage. To better understand the gene cascades involved in MHB formation, we investigated the genes downstream from XHR1 by differential screening using a Xenopus cDNA macroarray and a dexamethasone (DEX)-inducible, dominant-negative transcriptional activator construct of XHR1 (XHR1-VP16-GR). Among the newly identified candidate target genes of XHR1 were Enhancer of split-related genes (ESR1, ESR3/7, and ESR9) and Xenopus laevis cleavage 2 (XLCL2). XHR1-VP16-GR induced the expression of the ESR genes and XLCL2 as well as Xdelta1, Xngnr1, and XHR1 itself in the presence of DEX even after pretreatment with the protein synthesis inhibitor, cycloheximide. This suggests that these genes are direct targets of XHR1. XHR1-knockdown experiments with antisense morpholino oligos and ectopic expression of wild-type XHR1 revealed that XHR1 is necessary and sufficient to repress ESR genes in the pre-MHB region. Misexpression of the ESR genes in the pre-MHB region repressed the MHB marker gene, Pax2, suggesting that the repression of the ESR genes by XHR1 is at least partly required for the early development of the pre-MHB. Our data also show that XHR1 is not activated by Notch signaling, differing from ESR genes. Taken together, we propose a model in which XHR1 defines the pre-MHB region as a prepattern gene by repressing those possible direct target genes.

Animals↗

Altered skeletal muscle phenotypes in calcineurin Aalpha and Abeta gene-targeted mice.

Calcineurin is a calcium-regulated serine-threonine protein phosphatase that controls developmental and inducible biological responses in diverse cell types, in part through activation of the transcription factor nuclear factor of activated T cells (NFAT). In skeletal muscle, calcineurin has been implicated in the regulation of myoblast differentiation, hypertrophy of mature myofibers, and fiber type switching in response to alterations in intracellular calcium concentration. However, considerable disagreement persists about the functional role of calcineurin signaling in each of these processes. Here we evaluated the molecular phenotypes of skeletal muscle from both calcineurin Aalpha and calcineurin Abeta gene-targeted mice. Calcineurin Aalpha was observed to be the predominant catalytic isoform expressed in nearly all skeletal muscles examined. Neither calcineurin Aalpha or Abeta null mice showed any gross growth-related alterations in skeletal muscle, nor was fiber size or number altered in glycolytic/fast muscle types. In contrast, both calcineurin Aalpha and Abeta gene-targeted mice demonstrated an alteration in myofiber number in the soleus, an oxidative/slow-type muscle. More significantly, calcineurin Aalpha and Abeta gene-targeted mice showed a dramatic down-regulation in the oxidative/slow fiber type program in multiple muscles (both slow and fast). Associated with this observation, NFAT-luciferase reporter transgenic mice showed significantly greater activity in slow fiber-containing muscles than in fast. However, only calcineurin Aalpha null mice showed a defect in NFAT nuclear occupancy or NFAT-luciferase transgene activity in vivo. Collectively, our results suggest that calcineurin signaling plays a critical role in regulating skeletal muscle fiber type switching but not hypertrophy. Our results also suggest that fiber type switching occurs through an NFAT-independent mechanism.

Animals↗

Curbing the nuclear activities of beta-catenin. Control over Wnt target gene expression.

Wnt molecules control numerous developmental processes by altering specific gene expression patterns, and deregulation of Wnt signaling can lead to cancer. Many Wnt factors employ beta-catenin as a nuclear effector. Upon Wnt stimulation, beta-catenin heterodimerizes with T-cell factor (TCF) DNA-binding proteins to form a transcriptional activator complex. As the activating subunit of this complex, beta-catenin performs dual tasks: it alleviates repression of target gene promoters and subsequently it activates them. Beta-catenin orchestrates these effects by recruiting chromatin modifying cofactors and contacting components of the basal transcription machinery. Although beta-catenin and TCFs are universal activators in Wnt signaling, their target genes display distinct temporal and spatial expression patterns. Apparently, post-translational modifications modulate the interactions between TCFs and beta-catenin or DNA, and certain transcription factors can sequester beta-catenin from TCFs while others synergize with beta-catenin-TCF complexes in a promoter-specific manner. These mechanisms provide points of intersection with other signaling pathways, and contribute to the complexity and specificity of Wnt target gene regulation.

Animals↗

A large-scale Agrobacterium-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.).

A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10(-3) to 10(-5) compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 10(3) stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10(-2) using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.

Diphtheria Toxin↗

Efficient gene targeting in the filamentous fungus Alternaria alternata.

To characterize homologous recombination of transforming DNA in the filamentous fungus Alternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. The A. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus. BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internal BRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Mel-) transformants accounted for 30 to 80% of hygromycin B-resistant (HyR) transformants, correlating closely with the size of the BRM1 segment in the transforming DNA. Restriction enzyme digestion within the BRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of the BRM1 segments. Plasmids carrying both BRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Mel- transformants accounted for only 5% of HyR transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within the BRM1 segment was used, almost all transformants were Mel-. These results indicate that homologous integration of circular molecules in A. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.

Alternaria↗

Identification of potential target genes for RFX4_v3, a transcription factor critical for brain development.

Regulatory factor X4 variant transcript 3 (Rfx4_v3) gene disruption in mice demonstrated that interruption of a single allele (heterozygous, +/-) prevented formation of the subcommissural organ, resulting in congenital hydrocephalus, while interruption of two alleles (homozygous, -/-) caused fatal failure of dorsal midline brain structure formation. To identify potential target genes for RFX4_v3, we used microarray analysis to identify differentially expressed genes in Rfx4_v3-deficient mouse brains at embryonic day 10.5, before gross structural changes were apparent. Of 109 differentially expressed transcripts, 24 were chosen for validation and 22 were confirmed by real-time PCR. Many validated genes encoded critical proteins involved in brain morphogenesis, such as the signaling components in the Wnt, bone morphogenetic protein (BMP) and retinoic acid (RA) pathways. Cx3cl1, a CX3C-type chemokine gene that is highly expressed in brain, was down-regulated in the Rfx4_v3-null mice. Both human and mouse Cx3cl1 proximal promoters contained highly conserved X-boxes, known cis-acting elements for RFX protein binding. Using the Cx3cl1 promoter as an example of a target gene, we demonstrated direct binding of RFX4_v3 to the Cx3cl1 promoter, and trans-acting activity of RFX4_v3 protein to stimulate gene expression. These data suggest that RFX4_v3 may act upstream of critical signaling pathways in the process of brain development.

Animals↗