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PubMed2004

FGF signaling functions in the hypodermis to regulate fluid balance in C. elegans.

Signaling by the Caenorhabditis elegans fibroblast growth factor receptor EGL-15 is activated by LET-756, a fibroblast growth factor, and attenuated by CLR-1, a receptor tyrosine phosphatase. Hyperactive EGL-15 signaling results in a dramatic Clr phenotype characterized by the accumulation of clear fluid within the pseudocoelomic space, suggesting that regulated EGL-15 signaling is essential for fluid homeostasis in C. elegans. To determine the cellular focus of EGL-15 signaling, we identified an enhancer element (e15) within the egl-15 promoter, which is both necessary for the promoter activity and sufficient when duplicated to drive either egl-15 or clr-1 rescue activity. This enhancer drives GFP expression in hypodermal cells. Consistent with this finding, immunofluorescence studies of EGL-15 indicate that EGL-15 is expressed in hypodermal cells, and hypodermal promoters can drive full clr-1 and egl-15 rescue activity. Moreover, a mosaic analysis of mpk-1, which acts downstream of egl-15, suggests that its suppression of Clr (Soc) function is required in the hypodermis. These results suggest that EGL-15 and CLR-1 act in the hypodermis to regulate fluid homeostasis in worms.

Animals↗
PubMedDate not supplied

Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40.

Genetic analysis of eukaryotic transcriptional promoters has revealed that protein-coding genes often contain a complex array of cis-control elements consisting of upstream activator sequences and enhancer elements. The metallothionein genes provide a useful example for dissecting the action of multiple interspersed control elements that govern both basal level and regulated expression in animal cells. The human metallothionein (hMTIIA) promoter has been analysed in detail and found to contain no less than five distinct control elements in the 5' flanking regions of the gene that mediate specificity and regulation of transcription. These different control elements can be functionally subdivided into two categories: basal and induced elements. There are several distinct basal recognition sequences, which include a TATA-box, a GC-box, and at least two basal level enhancer (BLE) sequences, that function like classical enhancer elements. The hMTIIA gene also responds to induction by heavy metals and by steroid hormones through the action of metal regulatory elements (MRE) and glucocorticoid responsive elements (GRE). Here we report the identification of two cellular DNA-binding proteins that interact selectively with sequences governing the basal level expression of hMTIIA. One of these factors is a novel activator protein (AP1) that interacts with sequences in the BLE of hMTIIA and also binds to a site within the 72-base pair (bp) repeats of the simian virus 40 (SV40) enhancer region. The second protein has been purified to homogeneity and shown to be transcription factor Sp1 which recognizes and binds to a single GC-box element within the hMTIIA promoter.

Base Sequence↗
PubMed1989

Repression of polyoma virus DNA replication by 5'-flanking region of mouse DNA polymerase beta gene containing transcriptional silencer elements.

Dual cis-acting silencer elements are located upstream of the mouse DNA polymerase beta gene (Yamaguchi, M., Hayashi, Y., and Matsukage, A. (1989) J. Biochem. (Tokyo) 105, 79-83). In order to examine possible involvement of transcriptional silencer elements in the regulation of DNA replication, we have utilized a transient replication system of the plasmid DNA carrying replication origin of polyoma virus DNA in mouse MOP8 cells, which is constitutively producing polyoma virus large T-antigen. The polyoma virus origin of DNA replication is composed of three cis-acting genetic elements called alpha, beta, and core, in which alpha and beta elements correspond to enhancer domains. When the 5'-flanking regions of the DNA polymerase beta gene containing silencer elements were placed at the late gene border of alpha element, they effectively repressed the DNA replication. However, when placed at the early gene border of core element, it only marginally repressed the DNA replication. These results suggest that the silencer elements at cis position repress polyoma virus DNA replication by impeding the enhancer function that activates the DNA replication.

Animals↗
PubMed2001

Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis.

Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.

Animals↗
PubMed1998

Chondrocyte-specific enhancer elements in the Col11a2 gene resemble the Col2a1 tissue-specific enhancer.

Type XI collagen and type II collagen are coexpressed in all cartilage, and both are essential for normal cartilage differentiation and skeletal morphogenesis. This laboratory has recently identified a 48-base pair (bp) enhancer element in the type II collagen gene Col2a1 that contains several HMG-type protein-binding sites and that can direct chondrocyte-specific expression in transient transfection and in transgenic mice. The present study has identified two short chondrocyte-specific enhancer elements within a region in the 5' portion of the type XI collagen gene Col11a2 that has previously been shown to influence chondrocyte-specific expression in transgenic mice. These Col11a2 enhancer elements, like the Col2a1 enhancer, contain several sites with homology to the high mobility group (HMG) protein-binding consensus sequence. In electrophoretic mobility shift assays, the Col11a2 elements formed a DNA-protein complex that was dependent on the presence of the HMG-like sites. It had the same mobility as the complex formed with the Col2a1 48-bp enhancer and appeared to contain the same or similar proteins, including SOX9. The Col11a2 elements directed gene expression in transient transfections of chondrocytes but not fibroblasts, and their activity was abolished by mutation of the HMG-like sites. Ectopically expressed SOX9 activated these enhancers in non-chondrocytic cells, as it also activates the Col2a1 enhancer. Finally, the Col11a2 enhancer elements both directed transgene expression to cartilage in developing mouse embryos. Overall, our results indicate that the two Col11a2 chondrocyte-specific enhancer elements share many similarities with the Col2a1 48-bp enhancer. These similarities suggest the existence of a genetic program designed to coordinately regulate the expression of these and perhaps other genes involved in the chondrocyte differentiation pathway.

Animals↗
PubMed1995

Determination of a necdin cis-acting element required for neuron specific expression by using zebra fish.

To determine cis-acting elements required for neuron specific expression of a necdin gene, we tried to use zebra fish assay system in vivo instead of cell lines in vitro. Various expression vectors carrying upstream sequences of necdin gene fused to MEKA (lacZ) gene as a reporter were injected into fertilized zebra fish embryos and then the expression of the reporter gene was analyzed by the whole mount immunochemical method. No promoter activity was obtained with a construct carrying sequence from -63 to +63 of the necdin gene, while promoter activity with preferential skin expression was obtained with a construct having sequence from -86 to +28. Further upstream sequence from -173 to +28 exhibited neuron specific expression as well as that from -845 to +63. These results indicate that a cis-acting element responsible for neuron specific expression is located in an 87bp sequence from -173 to -87 of necdin gene.

Animals↗
PubMed2004

Multiple regulatory elements with spatially and temporally distinct activities control the expression of the epithelial differentiation gene lin-26 in C. elegans.

Epithelial differentiation is a very early event during development of most species. The nematode Caenorhabditis elegans, with its well-defined and invariant lineage, offers the possibility to link cell lineage, cell fate specification and gene regulation during epithelial differentiation. Here, we focus on the regulation of the gene lin-26, which is required for proper differentiation of epithelial cells in the ectoderm and mesoderm (somatic gonad). lin-26 expression starts in early embryos and remains on throughout development, in many cell types originating from different sublineages. Using GFP reporters and mutant rescue assays, we performed a molecular dissection of the lin-26 promoter and could identify almost all elements required to establish its complex spatial and temporal expression. Most of these elements act redundantly, or synergistically once combined, to drive expression in cells related by function. We also show that lin-26 promoter elements mediate activation in the epidermis (hypodermis) by the GATA factor ELT-1, or repression in the foregut (pharynx) by the FoxA protein PHA-4. Taken together, our data indicate that lin-26 regulation is achieved to a large extent through tissue-specific cis-regulatory elements.

Animals↗
PubMed1994

Enhancer traps in the Drosophila bithorax complex mark parasegmental domains.

Eight P elements carrying a beta-galactosidase (lacZ) reporter have been mapped to sites within the Drosophila bithorax complex. The bithorax complex contains three homeotic genes, and at least nine regulatory regions which control their expression in successive parasegments of the fly. The enhancer traps inserted at the promoter of one of the genes, Ultrabithorax, express lacZ in patterns which mimic the Ultrabithorax protein pattern. Enhancer traps in the regulatory regions do not mimic the endogenous genes, but express lacZ globally in the relevant parasegments. Some P elements carry large DNA fragments upstream of the lacZ promoter but internal to the P element. In cases where these internal sequences specify a lacZ pattern, that pattern is generally suppressed when the element is inserted in the bithorax complex. In embryos mutant for genes of the Polycomb group, the lacZ expression from the enhancer traps spreads to all segments. Thus, the enhancer traps reveal parasegmental domains that are maintained by Polycomb-mediated repression. Such domains may be realized by parasegmental differences in chromatin structure.

Animals↗
PubMed1996

Analysis of human CD59 tissue expression directed by the CMV-IE-1 promoter in transgenic rats.

The investigation of human complement (C) inhibitors with a view to overcoming C-mediated tissue injury stands to benefit from the production of anatomically suitable transgenic animals. In this study, we used the CMV-IE1 enhancer/promoter to control the expression in vivo in transgenic rats of the human terminal C protein inhibitor CD59. Five transgenic rats were identified, of which four possessed at least one complete copy of the transgene. The presence of human CD59 transcripts and protein was demonstrated in two transgenic rat lines. A widespread tissue distribution of cells expressing human CD59, similar in the two lines, was observed-principally in pancreas, brain, heart, kidney, intestine and striated muscle. Whereas expression in pancreas and brain was uniform, mosaicism of CD59 expression was observed in some tissues such as heart and kidney, a proportion of cells within the tissue not expressing the transgene. Immunohistological analysis revealed surface expression of human CD59 in a variety of cells, including fibroblasts, epithelial cells and muscle cells, but not in endothelial cells. In conclusion, this paper analyses at the cellular level human CD59 expression directed by the CMV promoter in transgenic rats, amd discusses how they could be used to investigate in vivo the role of C in a variety of pathologies.

Animals↗
PubMed1991

The gene encoding human TFE3, a transcription factor that binds the immunoglobulin heavy-chain enhancer, maps to Xp11.22.

TFE3, a member of the helix-loop-helix family of transcription factors, binds to the microE3 motif of the immunoglobulin heavy-chain enhancer and is expressed in many cell types. We have localized human TFE3 to the proximal short arm of the X chromosome using a somatic cell hybrid panel. A frequent RsaI RFLP detected by the TFE3 cDNA was found and used to confirm this location by linkage analysis in 20 pedigrees. Two-point and multipoint lod scores place TFE3 near markers in Xp11.22 with the most likely order DXS7-DXS255-TFE3-DXS146-DXS14.

Animals↗
PubMed2003

Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster.

Octopamine is an important neuroactive substance that modulates several physiological functions and behaviors of invertebrate species. Its biosynthesis involves two steps, one of which is catalyzed by Tyramine beta-hydroxylase enzyme (TBH). The Tbetah gene has been previously cloned from Drosophila melanogaster, and null mutations have been generated resulting in octopamine-less flies that show profound female sterility. Here, I show that ovulation process is defective in the mutant females resulting in blockage of mature oocytes within the ovaries. The phenotype is conditionally rescued by expressing a Tbetah cDNA under the control of a hsp70 promoter in adult females. Fertility of the mutant females is also restored when TBH is expressed, via the GAL4-UAS system, in cells of the CNS abdominal ganglion that express TBH and produce octopamine. This neuronal population differs from the dopamine- and serotonin-expressing cells indicating distinct patterns of expression and function of the three substances in the region. Finally, I demonstrate that these TBH-expressing cells project to the periphery where they innervate the ovaries and the oviducts of the reproductive system. The above results point to a neuronal focus that can synthesize and release octopamine in specific sites of the female reproductive system where the amine is required to trigger ovulation.

Adrenergic alpha-Agonists↗
PubMed1999

Use of the Gal4-UAS technique for targeted gene expression in the zebrafish.

The most common way to analyze the function of cloned genes in zebrafish is to misexpress the gene product or an altered variant of it by mRNA injection. However, mRNA injection has several disadvantages. The GAL4-UAS system for targeted gene expression allows one to overcome some of these disadvantages. To test the GAL4-UAS system in zebrafish, we generated two different kinds of stable transgenic lines, carrying activator and effector constructs, respectively. In the activator lines the gene for the yeast transcriptional activator GAL4 is under the control of a given promoter, while in the effectors the gene of interest is fused to the sequence of the DNA-binding motif of GAL4 (UAS). Crosses of animals from the activator and effector lines show that effector genes are transcribed with the spatial pattern of the activators. This work smoothes the way for a novel method of misexpression of gene products in zebrafish in order to analyze the function of genes in developmental processes.

Actins↗
PubMed2003

Molecular pathways needed for regeneration of spinal cord and muscle in a vertebrate.

The tail of the frog tadpole, comprising spinal cord, muscle, and notochord, regenerates following partial amputation. We show that, in Xenopus, this occurs throughout development, except for a "refractory period" between stages 45 and 47, when tails heal over without regeneration. Regeneration can be enabled during this refractory period by activation of either the BMP or Notch signaling pathways. Conversely, regeneration can be prevented during the later, regenerative, stages by inhibition of either pathway. BMP signaling will cause regeneration of all tissues, whereas Notch signaling activates regeneration of spinal cord and notochord, but not muscle. An activated form of Msx1 can promote regeneration in the same way as BMP signaling. Epistasis experiments suggest that BMP signaling is upstream of Notch signaling but exerts an independent effect on muscle regeneration. The results demonstrate that regenerative capability can be enabled by genetic modifications that reactivate specific components of the developmental program.

Amputation, Surgical↗
PubMed2001

Green fluorescent protein (GFP) transgenic fish and their applications.

The coupling of the GFP reporter system with the optical clarity of embryogenesis in model fish such as zebrafish and medaka is beginning to change the picture of transgenic fish study. Since the advent of first GFP transgenic fish in 1995, GFP transgenic fish technology have been quickly employed in many areas such as analyses of gene expression patterns and tissue/organ development, dissection of promoters/enhancers, cell lineage and axonal pathfinding, cellular localization of protein products, chimeric embryo and nuclear transplantation, cell sorting, etc. The GFP transgenic fish also have the potentials in analysis of upstream regulatory factors, mutagenesis screening and characterization, and promoter/enhancer trap. Our own studies indicate that GFP transgenic fish may become a new source of novel variety of ornamental fish. Efforts are also being made in our laboratory to turn GFP transgenic fish into biomonitoring organisms for surveillance of environmental pollution.

Animals↗
PubMed1997

Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1.

LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) alpha enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCR alpha enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCR alpha enhancer.

DNA, Superhelical↗
PubMed2005

Differential use of functional domains by coiled-coil coactivator in its synergistic coactivator function with beta-catenin or GRIP1.

beta-Catenin, a pivotal component of the Wnt-signaling pathway, binds to and serves as a transcriptional coactivator for the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcriptional activator proteins and for the androgen receptor (AR), a nuclear receptor. Three components of the p160 nuclear receptor coactivator complex, including CARM1, p300/CBP, and GRIP1 (one of the p160 coactivators), bind to and cooperate with beta-catenin to enhance transcriptional activation by TCF/LEF and AR. Here we report that another component of the p160 nuclear receptor coactivator complex, the coiled-coil coactivator (CoCoA), directly binds to and cooperates synergistically with beta-catenin as a coactivator for AR and TCF/LEF. CoCoA uses different domains to bind GRIP1 and beta-catenin, and it uses different domains to transmit the activating signal to the transcription machinery, depending on whether it is bound to GRIP1 or beta-catenin. CoCoA associated specifically with the promoters of transiently transfected and endogenous target genes of TCF/LEF, and reduction of the endogenous CoCoA level decreased the ability of TCF/LEF and beta-catenin to activate transcription of transient and endogenous target genes. Thus, CoCoA uses different combinations of functional domains to serve as a physiologically relevant component of the Wnt/beta-catenin signaling pathway and the androgen signaling pathway.

Animals↗
PubMed1993

The hLEF/TCF-1 alpha HMG protein contains a context-dependent transcriptional activation domain that induces the TCR alpha enhancer in T cells.

hLEF/TCF-1 alpha is a lymphoid cell-specific HMG protein that activates the distal enhancer of the gene encoding the alpha-subunit of the T-cell receptor (TCR alpha). We have shown previously that transcriptional activation by hLEF is highly dependent on the context of its binding site within the TCR alpha enhancer. Here, we demonstrate that hLEF contains a potent transcriptional activation domain that is separate from the HMG motif and is preferentially active in T cells. We find that hLEF/GAL4 fusion proteins can activate a GAL4-substituted TCR alpha enhancer up to 50-fold in T-cell lines and are as active as GAL4/VP16 in this context. Unlike GAL4/VP16, however, hLEF/GAL4 could not activate heterologous promoters bearing only GAL4 DNA-binding sites. Thus, activation by hLEF/GAL4, like that noted previously for the native hLEF activator, was strongly influenced by the context of its DNA-binding site within the TCR alpha enhancer. Inspection of enhancer mutants suggests that trans-activation by hLEF/GAL4 is especially dependent on TCF-2, a distinct T-cell-enriched protein that binds to sequences flanking the hLEF-binding site in the enhancer. Analysis of small deletion or clustered amino acid substitution mutants in the hLEF-coding sequences identified a minimal activation region between amino acids 80 and 256 that appears to be bipartite in structure. The hLEF activation domain is not notably acid or glutamine-rich but is proline-rich and includes a motif rich in tyrosine and serine residues. We conclude that sequences outside of the hLEF HMG box mediate cell- and context-specific activation of the TCR alpha enhancer and may facilitate interactions between hLEF and other T-cell-specific factors recruited to the enhancer.

Amino Acid Sequence↗