Targeted gene expression by the Gal4-UAS system in zebrafish

Targeted gene expression by the Gal4-UAS system is a powerful methodology for analyzing function of genes and cells in vivo and has been extensively used in genetic studies in Drosophila . On the other hand, the Gal4-UAS system had not been applied effectively to vertebrate systems for a long time mainly due to the lack of an efficient transgenesis method. Recently, a highly efficient transgenesis method using the medaka fish Tol2 transposable element was developed in zebrafish. Taking advantage of the Tol2 transposon system, we and other groups developed the Gal4 gene trap and enhancer trap methods and established various transgenic fish expressing Gal4 in specific cells. By crossing such Gal4 lines with transgenic fish lines harboring various reporter genes and effector genes downstream of UAS (upstream activating sequence), specific cells can be visualized and manipulated in vivo by targeted gene expression. Thus, the Gal4 gene trap and enhancer trap approaches together with various UAS lines should be important tools for investigating roles of genes and cells in vertebrates.     

Multiple GUS expression patterns of a single Arabidopsis gene

Ten independent transposant lines with gene or enhancer traps (ET) inserted into the same gene (At2g01170) were identified in Arabidopsis thaliana . Transposon insertions were confirmed for each line. Only three of five ET lines and only one of the five gene trap (GT) lines displayed uidA (GUS) staining. The GUS (β-glucuronidase) expression patterns of the ET lines were different in all three lines. In the GT line, the GUS expression was restricted to the vascular tissue under all conditions examined. The variation in ET GUS expression suggests that each ET was controlled by different enhancer elements or the different elements of the trapped locus may give rise to different GUS expression patterns. Of five GT lines, three have the GUS gene in the same orientation as the At2g01170 open reading frame, yet only one yielded GUS staining. Regardless of the insertion construct, only those transposants with an insertion at the 3' end of the gene yielded GUS staining. Some transposants displayed a longer root phenotype in the presence of kanamycin that was also observed in 3' insertion sites in At2g01170. Taken together, these data show that insertions in the 5' end of the gene disrupted expression and emphasise the complexity encountered with ET and GT constructs to characterise the expression patterns of genes of interest based solely on GUS expression patterns.     

Identification of a root-specific glycosyltransferase from Arabidopsis and characterization of its promoter

A set of Ds-element enhancer trap lines of Arabidopsis thaliana was generated and screened for expression patterns leading to the identification of a line that showed root-specific expression of the bacterial uidA reporter gene encoding beta-glucuronidase (GUS). The insertion of the Ds element was found to be immediately downstream to a glycosyltransferase gene At1g73160. Analysis of At1g73160 expression showed that it is highly root-specific. Isolation and characterization of the upstream region of the At1g73160 gene led to the definition of a 218 bp fragment that is sufficient to confer root-specific expression. Sequence analysis revealed that several regulatory elements were implicated in expression in root tissue. The promoter identified and characterized in this study has the potential to be applied in crop biotechnology for directing the root-specific expression of transgenes.     

Sox9 transactivation and testicular expression of a novel human gene,KIAA0800

The Sry and Sox9 sex-determination factors initiate and promote testis differentiation by gene transactivation through similar promoter elements. However, knowledge is limited concerning what genes are regulated by Sry/Sox9 in the testis. Identification and characterization of Sry/Sox9-regulated genes are critical for understanding sexual differentiation. We now demonstrate that a novel human gene, KIAA0800, is preferentially expressed in the testis and is transactivated by Sox9. The KIAA0800 promoter is repressed by an upstream element involving a polyT track and two Alu repeats. Two specific Sox9-bindings sites have been identified in the KIAA0800 promoter by using DNaseI footprinting assays and gel electrophoretic mobility shift assays. Sox9 transactivation of the KIAA0800 promoter appears to be exerted mainly through the relief of promoter repression. Genes homologous to the human KIAA0800 exist in organisms with differentiated sex tissues including mouse, Drosophila, and C. elegans, but not in unicellular organisms, including yeast and bacteria. Further, our recent sequence analysis shows that KIAA0800 protein is 97% identical between human and mouse. Thus, KIAA0800 is a novel Sox9-activated gene that is evolutionarily conserved and potentially involved in sexual differentiation.     

AtHVA22 gene family in Arabidopsis: phylogenetic relationship,ABA and stress regulation,and tissue-specific expression

HVA22 is an ABA- and stress-inducible gene first isolated from barley (Hordeum vulgare L.). Homologues of HVA22 have been found in plants, animals, fungi and protozoa, but not in prokaryotes, suggesting that HVA22 plays a unique role in eukaryotes. Five HVA22 homologues, designated AtHVA22a, b, c, d and e, have been identified in Arabidopsis. These five AtHVA22 homologues can be separated into two subfamilies, with AtHVA22a, b and c grouped in one subfamily and AtHVA22d and e in the other. Phylogenetic analyses show that AtHVA22d and e are closer to barley HVA22 than to AtHVA22a, bandc, suggesting that the two subfamilies had diverged before the divergence of monocots and dicots. The distribution and size of exons of AtHVA22 homologues and barley HVA22 are similar, suggesting that these genes are descendents of a common ancestor. AtHVA22 homologues are differentially regulated by ABA, cold, dehydration and salt stresses. These four treatments enhance AtHVA22a, d and e expression, but have little or even suppressive effect on AtHVA22c expression. ABA and salt stress induce AtHVA22b expression, but cold stress suppresses ABA induction of this gene. Expression of AtHVA22d is the most tightly regulated by these four treatments among the five homologues. In general, AtHVA22 homologues are expressed at a higher level in flower buds and inflorescence stems than in rosette and cauline leaves. The expression level of these homologues in immature siliques is the lowest among all tissues analyzed. It is suggested that some of these AtHVA22 family members may play a role in stress tolerance, and others are involved in plant reproductive development.     

Arabidopsis thaliana vegetative storage protein (VSP) genes: gene organization and tissue-specific expression

We have previously identified two cDNAs encoding vegetative storage proteins (VSPs) in Arabidopsis thaliana. Unlike soybean in which VSPs accumulate at high levels in leaves, A. thaliana VSP mRNAs are abundant in flowers. To understand tissue-specific expression and possible roles of VSPs on reproductive organ development, genes corresponding to VSPs (Vsp1 and Vsp2) and their putative promoters were characterized in this study. Genomic sequence analysis revealed that Vsp1 and Vsp2 resemble each other except in their introns, and that these two genes were organized in a tandem array with an interval of 6 kb in a region. The expression patterns of Vsp1 and Vsp2 were examined using transgenic A. thaliana plants carrying a promoter from Vsp1 or Vsp2 fused to a bacterial -glucuronidase (GUS) reporter gene. The promoter from Vsp1 expressed its effect in gynoecia, especially in styles, the basal and distal ends of ovaries and in siliques, whereas the promoter from Vsp2 showed its activity in vegetative shoots, petioles, peduncles and receptacles of floral organs. These results suggest that expression of Vsp1 and Vsp2 may be developmentally regulated in A. thaliana. In the transgenic plants, the GUS activity was induced by wounding in an area around the mid-rib of leaves. Therefore, Vsp1 and Vsp2 promoters appear to have elements required for both tissue specificity and wounding.     

Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis

The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and β-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY™ vector conversion system. The method can be extended to other species when enhancer trap lines become available.     

Involvement of the INK4a/Arf gene locus in senescence

The INK4a/ARF locus encodes two proteins whose expression limits cellular proliferation. Whilst the biochemical activities of the two proteins appear very different, they both converge on regulating the retinoblastoma and p53 tumour suppressor pathways. Neither protein is required for normal development, but lack of either predisposes to the development of malignancy. Both proteins have also been implicated in the establishment of senescence states in response to a variety of stresses, signalling imbalances and telomere shortening. The INK4a/Arf regulatory circuits appear to be partially redundant and show evidence of rapid evolution. Especially intriguing are the large number of biological differences documented between mice and man. We review here the brief history of INK4a/Arf and explore possible links with organismal aging and the evolution of longevity.     

Efficient activation of gene expression using a heat-shock inducible Gal4/Vp16-UAS system in medaka

Background

Genetic interference by DNA, mRNA or morpholino injection is a widely used approach to study gene function in developmental biology. However, the lack of temporal control over the activity of interfering molecules often hampers investigation of gene function required during later stages of embryogenesis. To elucidate the roles of genes during embryogenesis a precise temporal control of transgene expression levels in the developing organism is on demand.

Results

We have generated a transgenic Gal4/Vp16 activator line that is heat-shock inducible, thereby providing a tool to drive the expression of specific effector genes via Gal4/Vp16. Merging the Gal4/Vp16-UAS system with the I-SceI meganuclease and the Sleeping Beauty transposon system allows inducible gene expression in an entirely uniform manner without the need to generate transgenic effector lines. Combination of this system with fluorescent protein reporters furthermore facilitates the direct visualization of transgene expressing cells in live embryos.

Conclusion

The combinatorial properties of this expression system provide a powerful tool for the analysis of gene function during embryonic and larval development in fish by ectopic expression of gene products.

     

An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus

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Crosstalk between histone modifications maintains the developmental pattern of gene expression on a tissue-specific locus

Genome wide studies have provided a wealth of information related to histone modifications. Particular modifications, which can encompass both broad and discrete regions, are associated with certain genomic elements and gene expression status. Here we focus on how studies on the ß-globin gene cluster can complement the genome wide effort through the thorough dissection of histone modifying protein crosstalk. The ß-globin locus serves as a model system to study both regulation of gene expression driven at a distance by enhancers and mechanisms of developmental switching of clustered genes. We investigate recent studies, which uncover that histone methyltransferases, recruited at the ß-globin enhancer, control gene expression by long range propagation on chromatin. Specifically, we focus on how seemingly antagonistic complexes, such as those including MLL2, G9a and UTX, can cooperate to functionally regulate developmentally controlled gene expression. Finally, we speculate on the mechanisms of chromatin modifying complex propagation on genomic domains.