Regulation of neuronal traits by a novel transcriptional complex.

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Identification of the auxin-responsive element, AuxRE, in the primary indoleacetic acid-inducible gene, PS-IAA4/5, of pea (Pisum sativum).

The plant hormone auxin transcriptionally activates early genes in pea. We have developed a transient assay system using protoplasts of auxin-responsive pea seedling cells to define the auxin-responsive element, AuxRE, of the early auxin-induced PS-IAA4/5 gene. The auxin responsive protoplasts show an authentic hormonal response identical to that observed in intact pea tissue, with respect to rapidity, specificity and cycloheximide (CHX) inducibility of the PS-IAA4/5 transcript. The hormone also mediates rapid and specific induction of chloramphenicol acetyltransferase (CAT) activity in protoplasts transfected with a chimeric IAA4/5-CAT gene. The IAA-induced CAT activity is developmentally regulated and is observed only in protoplasts derived from auxin-responsive regions of the pea seedling. Extensive deletion analysis of the PS-IAA4/5 promoter defined a promoter region between -318 and -154 that confers auxin inducibility. This AuxRE mediates auxin-inducible CAT activity in pea cells driven by the non auxin-responsive CaMV 35S minimal promoter. The functionality of this promoter region as an AuxRE was further verified in tobacco plants using IAA4/5-GUS gene fusions. The AuxRE contains two domains: Domain A acts as an auxin switch; domain B has an enhancer-like activity. The A and B domains contain the highly conserved sequences found in various auxin-regulated genes (T/GGT-CCCAT (domain A) and C/AACATGGNC/AA/GTGTT/CT/CC/A (domain B)). DNase I footprinting reveals binding of nuclear proteins to the highly conserved sequence found in A and B domains. The sequence of the A domain does not correspond to any known regulatory elements found in other eukaryotic genes, and the data suggest that this conserved motif functions as an AuxRE. A model for the early transcriptional activation of the PS-IAA4/5 gene by IAA is discussed.

Transcriptional regulation of PS-IAA4/5 and PS-IAA6 early gene expression by indoleacetic acid and protein synthesis inhibitors in pea (Pisum sativum).

The transcription of two genes, PS-IAA4/5 and PS-IAA6, in pea is induced by indoleacetic acid (IAA) and protein synthesis inhibitors such as cycloheximide (CHX) and anisomycin (ANI). Induction by IAA is rapid, taking 5 and 7.5 minutes for PS-IAA4/5 and PS-IAA6, respectively, and is independent of IAA concentration and whether IAA has a free or esterified carboxyl group (ethyl-IAA). The rate of mRNA accumulation, however, is dependent on hormone concentration, and is greater with IAA than with ethyl-IAA. The turnover rates (t1/2) of the PS-IAA4/5 and PS-IAA6 mRNAs are 60 and 75 minutes, respectively, and are not affected by IAA. CHX or ANI induce the transcription of PS-IAA4/5 and PS-IAA6 more slowly than IAA (5 to 10 minutes for PS-IAA4/5 and 20 minutes for PS-IAA6). While protein synthesis inhibitors stabilize both mRNAs, the rapidity of induction by CHX and ANI cannot be accounted for solely by mRNA stabilization. The relationship between mRNA induction and protein synthesis inhibition does not obey Michaelis-Menten kinetics, but rather is best described by a hyperbolic curve, suggesting the release of transcriptional repression by the inhibition of protein synthesis. RNA expression experiments with transgenic tobacco seedlings or with transfected pea protoplasts using PS-IAA4/5 promoter GUS or CAT fusions reveal that CHX transcriptionally activates PS-IAA4/5 gene expression. Thus, protein synthesis inhibitors have a dual effect on PS-IAA4/5 and PS-IAA6. (1) They stabilize both mRNAs (possibly by a translational arrest-linked process or by preventing the synthesis of a labile nuclease(s)). (2) They activate transcription (possibly by preventing the synthesis or function of a repressor).

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein.

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5' end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAPalpha, specifically bound VirD2 in vivo and in vitro. VirD2-AtKAPalpha interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAPalpha was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin alpha family. Indeed, AtKAPalpha efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAPalpha specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.

Transient expression of the plasmid pCaMVCAT in plant protoplasts following transformation with polyethyleneglycol.

Petunia and carrot protoplasts have been transformed with the plasmid pCaMVCAT by the use of polyethyleneglycol (PEG) as a facilitator. Transformation was revealed by the appearance of the chloramphenicol-acetyl transferase (CAT) enzyme within the transformed cells. Maximal activity of the CAT enzyme was detected within 15 h following transformation, while after 60 h, its activity was significantly reduced, indicating transient expression of the CAT gene. The efficiency of transformation was highly dependent on the presence of CaCl2 in the transformation system, was stimulated by non-functional carrier DNA and was independent on the molecular weight (MW) of PEG used.

Efficient functioning of plant promoters and poly(A) sites in Xenopus oocytes.

Mature Xenopus oocytes were challenged with DNA constructs including plant regulatory elements, namely, the Cauliflower mosaic virus (CaMV) 35S promoter as well as the nopaline synthase (NOS) promoter and polyadenylation signal. The bacterial chloramphenicol acetyl transferase (CAT) was used as a reporter gene. When microinjected into these cells, the plant-derived DNA constructs effectively promoted CAT synthesis in a manner dependent on the presence of the plant promoters and probably also on the polyadenylation signals. Structural studies revealed that the supercoiled structures of the above DNA plasmids were much more active in supporting CAT synthesis in microinjected oocytes than their linear forms, with clear correlation between efficient gene expression and DNA topology. In contrast, the linear forms of these plasmids were considerably more active than the supercoiled ones in transfected plant protoplasts. These findings demonstrate, for the first time, the activity of regulatory elements from plant genes in Xenopus oocytes and shed new light on the specific rules applicable for gene expression in plant and animal cells.

Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts.

Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)(+) RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.

Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-IAA4/5.

The plant growth hormone indole-3-acetic acid (IAA) transcriptionally activates expression of several genes in plants. We have previously identified a 164-bp promoter region (-318 to -154) in the PS-IAA4/5 gene that confers IAA inducibility. Linker-scanning mutagenesis across the region has identified two positive domains: domain A (48 bp; -203 to -156) and domain B (44 bp; -299 to -256), responsible for transcriptional activation of PS-IAA4/5 by IAA. Domain A contains the highly conserved sequence 5'-TGTCCCAT-3' found among various IAA-inducible genes and behaves as the major auxin-responsive element. Domain B functions as an enhancer element which may also contain a less efficient auxin-responsive element. The two domains act cooperatively to stimulate transcription; however, tetramerization of domain A or B compensates for the loss of A or B function. The two domains can also mediate IAA-induced transcription from the heterologous cauliflower mosaic virus 35S promoter (-73 to +1). In vivo competition experiments with icosamers of domain A or B show that the domains interact specifically and with different affinities to low abundance, positive transcription factor(s). A model for transcriptional activation of PS-IAA4/5 by IAA is discussed.

DNA elements responsive to auxin.

Genes induced by the plant hormone auxin are probably involved in the execution of vital cellular functions and developmental processes. Experimental approaches designed to elucidate the molecular mechanisms of auxin action have focused on auxin perception, genetic dissection of the signaling apparatus and specific gene activation. Auxin-responsive promoter elements of early genes provide molecular tools for probing auxin signaling in reverse. Functional analysis of several auxin-specific promoters of unrelated early genes suggests combinatorial utilization of both conserved and variable elements. These elements are arranged into autonomous domains and the combination of such modules generates uniquely composed promoters. Modular promoters allow for auxin-mediated transcriptional responses to be revealed in a tissue- and development-specific manner.

Transient gene expression of foreign genes in preheated protoplasts: stimulation of expression of transfected genes lacking heat shock elements.

Transfection of preheated petunia protoplasts with several biologically active DNA constructs resulted in a significantly higher gene expression than that observed in transfected unheated protoplasts. It was observed with supercoiled, linearized and single-stranded DNA structures that stimulation of transient gene expression in preheated protoplasts was neither dependent on the reporter gene nor on the regulatory elements used. Heat treatment at 42 degrees C also increased expression in protoplasts transfected with a plasmid bearing the tobacco mosaic virus (TMV) translational enhancer, omega. Northern blot analysis revealed that heat treatment of protoplasts before the transfection event greatly increased the amount of the newly synthesized transcripts. Preheating of protoplasts did not affect the transfection efficiency, namely the number of transfected cells in the population, nor the amount of DNA in transfected nuclei, as was inferred from histochemical staining and Southern blot analysis, respectively. The possible mechanism by which heat treatment stimulates transient gene expression of genes lacking obvious heat shock elements is offered. The relevance of the present findings to transient gene expression in plants in general and to viral gene expression in particular is discussed.

CoREST: a functional corepressor required for regulation of neural-specific gene expression.

Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for CoREST interaction. Mutations of the motif that disrupt binding also abrogate repression. When fused to a Gal4 DNA-binding domain, CoREST functions as a repressor. CoREST is present in cell lines that express REST, and the proteins are found in the same immunocomplex. CoREST contains two SANT (SW13/ADA2/NCoR/TFIIIB B) domains, a structural feature of the nuclear receptor and silencing mediator for retinoid and thyroid human receptors (SMRT)-extended corepressors that mediate inducible repression by steroid hormone receptors. Together, REST and CoREST mediate repression of the type II sodium channel promoter in nonneural cells, and the REST/CoREST complex may mediate long-term repression essential to maintenance of cell identity.