RNA: guiding gene silencing.

In diverse organisms, small RNAs derived from cleavage of double-stranded RNA can trigger epigenetic gene silencing in the cytoplasm and at the genome level. Small RNAs can guide posttranscriptional degradation of complementary messenger RNAs and, in plants, transcriptional gene silencing by methylation of homologous DNA sequences. RNA silencing is a potent means to counteract foreign sequences and could play an important role in plant and animal development.

Transcriptional and posttranscriptional gene silencing are mechanistically related.

Two distinct gene-silencing phenomena are observed in plants: transcriptional gene silencing (TGS), which involves decreased RNA synthesis because of promoter methylation, and posttranscriptional gene silencing (PTGS), which involves sequence-specific RNA degradation. PTGS is induced by deliberate [1-4] or fortuitous production (R.v.B., unpublished data) of double-stranded RNA (dsRNA). TGS could be the result of DNA pairing [5], but could also be the result of dsRNA, as was shown by the dsRNA-induced inactivation of a transgenic promoter [6]. Here, we show that when targeting flower pigmentation genes in Petunia, transgenes expressing dsRNA can induce PTGS when coding sequences are used and TGS when promoter sequences are taken. For both types of silencing, small RNA species are found, which are thought to be dsRNA decay products [7] and determine the sequence specificity of the silencing process [8, 9]. Furthermore, silencing is accompanied by the methylation of DNA sequences that are homologous to dsRNA. DNA methylation is assumed to be essential for regulating TGS and important for reinforcing PTGS [10]. Therefore, we conclude that TGS and PTGS are mechanistically related. In addition, we show that dsRNA-induced TGS provides an efficient tool to generate gene knockouts, because not only does the TGS of a PTGS-inducing transgene fully revert the PTGS phenotype, but also an endogenous gene can be transcriptionally silenced by dsRNA corresponding to its promoter.

Role of inverted DNA repeats in transcriptional and post-transcriptional gene silencing.

Transgenes and endogenous genes are sensitive to silencing, in particular when the genes are tandemly repeated. Their expression can be transcriptionally or post-transcriptionally repressed, or both. It is remarkable that very often, two or more genes or parts of the genes are arranged as inverted repeats (IR). Many of such IRs are dominant silencing loci. They can repress the expression of homologous genes elsewhere in the genome in trans which is usually associated with an increase in the level of DNA methylation. Trans-silencing has been explained by DNA-DNA pairing between a repetitive silencing locus and a homologous target locus. However, there is accumulating evidence that the trans effect might be mediated by dsRNA transcribed from the IR (trans)genes. Besides dsRNA-directed DNA methylation, dsRNA in plants as well as in other systems also induces the degradation of homologous RNAs and silence genes post-transcriptionally. These findings indicate that several features associated with gene silencing can be attributed to the activities of dsRNA, which would explain why inverted transgene repeats are such efficient silencing loci.

Post-transcriptional gene-silencing: RNAs on the attack or on the defense?

Post-transcriptional gene-silencing (PTGS) was first discovered in plants and results from the sequence-specific degradation of RNA. Degradation can be activated by introducing transgenes, RNA viruses or DNA sequences that are homologous to expressed genes. A similar RNA degradation mechanism which is inducible by double-stranded RNA (dsRNAs), has been discovered recently in vertebrates, invertebrates and protozoa. dsRNAs may also be potent activators of PTGS in plants. PTGS is not cell autonomous, suggesting the synthesis of sequence-specific silencing signals which are not only moving through the plant but are also amplified and an RNA-directed RNA Polymerase which has recently been cloned from various plant species is a candidate enzyme for amplifying silencing signals. The natural role of PTGS seems to be as a defence against plant viruses, so what first appeared to be RNAs on the attack may now be considered RNAs on the defense. BioEssays 22:520-531, 2000.

Distinct features of post-transcriptional gene silencing by antisense transgenes in single copy and inverted T-DNA repeat loci.

The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as-RNA) are expected to result in strong silencing but often there is no clear correlation between as-RNA levels and the degree of silencing. To obtain insight into these puzzling observations, we have analyzed several petunia transformants of which the pigmentation gene chalcone synthase (Chs) is post-transcriptionally silenced in corollas by antisense (as) Chs transgenes. The transformants were examined with respect to the steady-state as-RNA level, transcription level of the as-transgenes, the repetitiveness and structure of the integrated T-DNAs, and the methylation status of the transgenes. This revealed that the transformants can be divided in two classes: the first class contains a single copy (S) T-DNA of which the as-Chs gene is transcribed, although several-fold lower than the endogenous Chs genes. As there are not sufficient as-RNAs to degrade every mRNA, we speculate that silencing is induced by double-stranded RNA. The second class contains two T-DNAs which are arranged as inverted repeats (IRs). These IR loci are severely methylated and the as-Chs transgenes transcriptionally barely active. The strongest silencing was observed with IR loci in which the as-Chs transgenes were proximal to the centre of the IR. Similar features have been described for co-suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisense IRs also occurs by co-suppression, either via ectopic DNA pairing or via dsRNA.

Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants.

Posttranscriptional silencing of chalcone synthase (Chs) genes in petunia transformants occurs by introducing T-DNAs that contain a promoter-driven or promoterless Chs transgene. With the constructs we used, silencing occurs only by T-DNA loci which are composed of two or more T-DNA copies that are arranged as inverted repeats (IRs). Since we are interested in the mechanism by which these IR loci induce silencing, we have analyzed different IR loci and nonsilencing single-copy (S) T-DNA loci with respect to the expression and methylation of the transgenes residing in these loci. We show that in an IR locus, the transgenes located proximal to the IR center are much more highly methylated than are the distal genes. A strong silencing locus composed of three inverted T-DNAs bearing promoterless Chs transgenes was methylated across the entire locus. The host Chs genes in untransformed plants were moderately methylated, and no change in methylation was detected when the genes were silenced. Run-on transcription assays showed that promoter-driven transgenes located proximal to the center of a particular IR are transcriptionally more repressed than are the distal genes of the same IR locus. Transcription of the promoterless Chs transgenes could not be detected. In the primary transformant, some of the IR loci were detected together with an unlinked S locus. We observed that the methylation and expression characteristics of the transgenes of these S loci were comparable to those of the partner IR loci, suggesting that there has been cross talk between the two types of loci. Despite the similar features, S loci are unable to induce silencing, indicating that the palindromic arrangement of the Chs transgenes in the IR loci is critical for silencing. Since transcriptionally silenced transgenes in IRs can trigger posttranscriptional silencing of the host genes, our data are most consistent with a model of silencing in which the transgenes physically interact with the homologous host gene(s). The interaction may alter epigenetic features other than methylation, thereby impairing the regular production of mRNA.

Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression.

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of beta-D-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)7CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.

Identification of Endogenous Gibberellins in Petunia Flowers (Induction of Anthocyanin Biosynthetic Gene Expression and the Antagonistic Effect of Abscisic Acid).

The elongation and pigmentation of corollas of Petunia hybrida requires the presence of anthers. The ability of exogenous gibberellic acid (GA3) to substitute for the anthers suggests a role for endogenous GAs. Here we report the identification of endogenous GAs in corollas and in anthers and show that both tissues contain detectable levels of GA1, GA4, and GA9, of which GA4 is the most abundant. These GAs stimulate corolla pigmentation, chalcone synthase (chs) mRNA accumulation, and chs transcription in an in vitro flower bud culture system. Methyl ester derivatives of GA3 and GA4 were not active but did not inhibit the bioactive GAs. Even though it is unknown whether abscisic acid (ABA) is involved in corolla maturation, ABA inhibited pigmentation of intact flowers, overruling the effect of the anthers. In detached flower buds it was shown that ABA prevented activation of the chs promoter by GA3. The synthesis of anthocyanin pigments requires the coordinate expression of at least 15 structural genes. Expression of early biosynthetic genes and of late biosynthetic genes are regulated by different transcriptional activators. GA induces both classes of genes with similar kinetics, indicating that GA acts relatively early in the signaling pathway.

Conditional inhibition of beta-glucuronidase expression by antisense gene fragments in petunia protoplasts.

Antisense RNA-mediated inhibition of gene expression is a valuable tool to induce mutant phenotypes. We are interested in the application of antisense gene fragments with the aim to improve the efficiency of inhibition and to be able to selectively suppress gene family members in plants. Protoplasts may provide a rapid system to screen the efficiency of antisense gene segments. As a first step, we set up a transient expression system for leaf protoplasts of Petunia hybrida and used as a model system the inhibition of beta-glucuronidase (uidA) expression by uidA antisense gene segments. Both GUS enzyme activities and uidA RNA levels were measured. Co-introducing equal amounts of a full-length uidA antisense gene and a uidA sense gene reduced GUS activity by 60-70%. Various uidA antisense fragments also inhibited expression although with different efficiencies and we show that strong antisense fragments can be retrieved from weak antisense gene fragments. A promoter-less antisense gene did not reduce uidA expression indicating that the inhibition is mediated by antisense transcripts. Using quantitative PCR on first-strand cDNA we show that expression of functional antisense genes lead to reduced levels of uidA mRNA. This suggests that the mechanism of antisense RNA inhibition in protoplasts is similar to that in transgenic plants and that the protoplast system in combination with PCR can be used to preselect antisense fragments of any gene.

The petunia homologue of the Antirrhinum majus candi and Zea mays A2 flavonoid genes; homology to flavanone 3-hydroxylase and ethylene-forming enzyme.

The synthesis of anthocyanins in higher plants involves many enzymatic steps. Here we describe the isolation and characterization of a cDNA, ant17, which encodes a protein that has 73% amino acid sequence identity with the candi gene product of Antirrhinum majus and 48% with that of the maize a2 gene. This protein may therefore be involved in the synthesis of anthocyanins in the steps after the action of dihydroflavonol 4-reductase. This is consistent with the absence of ant17 expression in the regulatory anthocyanin mutants of petunia an1, an2 and an11. Furthermore, ant17 is predominantly expressed in corollas and anthers and is induced by gibberellic acid.

Gibberellic Acid Regulates Chalcone Synthase Gene Transcription in the Corolla of Petunia hybrida.

The pigmentation of Petunia hybrida corollas is regulated by gibberellic acid (GA(3)). It controls the increase of flavonoid enzyme levels and their corresponding mRNAs. We have used an in vitro culture system for corollas to study the regulatory role of GA(3) in the expression of flavonoid genes. By determining steady-state mRNA levels, we show that the accumulation of chalcone synthase (chs) mRNA in young corollas is dependent on the presence of both sucrose and GA(3) in the culture medium. Whereas sucrose had a general metabolic effect on gene expression, the stimulatory role of GA(3) was specific. Analysis of nascent transcripts in isolated corolla nuclei showed that changes in steady-state chs mRNA levels correlated very well with changes in the transcription rate. We therefore conclude that GA(3) controls the expression of chs at the transcriptional level. Preculturing the corollas in sucrose medium without GA(3) resulted in a lower chs mRNA level. The expression could be reinduced by the addition of GA(3). The hormone is thus required for the induction but also for the maintenance of chs transcription. The delayed reinduction of chs expression, the lag time in the kinetics of chs mRNA accumulation, and the inhibitory effect of cycloheximide on the action of GA(3) suggest that GA(3) controls chs transcription in an indirect manner. Our data support a model in which GA(3) induces the production of a regulatory protein such as a receptor or a trans-acting factor that is directly involved in chs transcription.

Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1.

Bean nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor, designated SBF-1, that specifically interacts with regulatory sequences in the promoter of the bean defense gene CHS15, which encodes the flavonoid biosynthetic enzyme chalcone synthase. SBF-1 binds to three short sequences designated boxes 1, 2 and 3 in the region -326 to - 173. This cis-element, which is involved in organ-specific expression in plant development, functions as a transcriptional silencer in electroporated protoplasts derived from undifferentiated suspension-cultured soybean cells. The silencer element activates in trans a co-electroporated CHS15-chloramphenicol acetyl-transferase gene fusion, indicating that the factor acts as a repressor in these cells. SBF-1 binding in vitro is rapid, reversible and sensitive to prior heat or protease treatment. Competitive binding assays show that boxes 1, 2 and 3 interact cooperatively, but that each box can bind the factor independently, with box 3 showing the strongest binding and box 2 the weakest binding. GGTTAA(A/T)(A/T)(A/T), which forms a consensus sequence common to all three boxes, resembles the binding site for the GT-1 factor in light-responsive elements of the pea rbcS-3A gene, which encodes the small subunit of ribulose bisphosphate carboxylase. Binding to the CHS15 -326 to -173 element, and to boxes 1, 2 or 3 individually, is competed by the GT-1 binding sequence of rbcS-3A, but not by a functionally inactive form, and likewise the CHS sequences can compete with authentic GT-1 sites from the rbcS-3A promoter for binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Boundaries of telomere conversion in Trypanosoma brucei.

Active genes for variant-specific surface glycoproteins (VSGs) reside in telomeric expression sites and may be replaced by other VSG genes via telomere conversions. The availability of a complete map of expression site 221 in variant 221a made it possible to determine the boundaries of such conversions and the sequences that are involved. We have analysed five trypanosome populations that arose from variant 221a through replacement of the 221 gene by another VSG gene. In each of these relapsed populations the telomere conversion ends at a different position in the expression site. In the relapsed population, 221aR3, the boundary was found in the coding region of an expression-site-associated gene (ESAG). This ESAG-2 codes for a potential 368-aa protein of unknown function; it contains a N-terminal signal peptide for mediating transfer to the endoplasmic reticulum and six potential N-glycosylation sites. It shares these structural features with the ESAG-1 protein encoded in the same expression site. ESAG-2 is a member of a large gene family which includes non-functional genes. In 221aR3, the partial conversion of ESAG-2 by an ESAG-2-like sequence has disrupted the open reading frame. The two ESAG-2 sequences are similar (92% identity) suggesting that sequence homology between telomeres provides the opportunity for gene conversion.

Inactivation of transcription by UV irradiation of T. brucei provides evidence for a multicistronic transcription unit including a VSG gene.

We have used inactivation of transcription by UV irradiation to map transcription units in trypanosomes. The relative inactivation rate of the transcription of mini-exon, 5S, and rRNA genes was inversely proportional to the previously estimated lengths of these transcription units. The telomeric transcription unit containing the gene for variant-specific surface glycoprotein (VSG) 221 was inactivated as a single unit of 60 kb. This long transcription unit comprises at least one other protein-coding gene and yields seven other stable mRNAs. These data thus provide evidence for a multicistronic transcription unit for cellular genes in a eukaryote.

The anatomy and transcription of a telomeric expression site for variant-specific surface antigens in T. brucei.

The variant specific surface glycoprotein (VSG) genes of T. brucei are expressed in telomeric expression sites. We have determined the structure of the active site in trypanosome variant 221a, which contains VSG gene 221, by analysis of cloned DNA segments that represent 65 kb of the 5'-flanking region of the VSG gene. In nuclear run-on experiments, 57 kb of adjacent sequences are cotranscribed with the VSG gene at approximately similar rates and in the alpha-amanitin-resistant manner characteristic of VSG genes. Besides the VSG mRNA, this expression site yields at least seven stable RNAs, suggesting that it is a multicistronic transcription unit. Our results also show that insertion of a transcriptional terminator is not the general mechanism of switching off expression sites.

Coincident multiple activations of the same surface antigen gene in Trypanosoma brucei.

Trypanosomes with a coat of variant surface glycoprotein (VSG) 118, consistently appear around day 20 when a rabbit is infected with Trypanosoma brucei strain 427. There is a single chromosome-internal gene for VSG 118 and this is activated by duplicative transposition to a telomeric expression site. We show here that the expression-linked extra copy of VSG gene 118 in a day 18 population of a chronic infection is heterogeneous, and we infer that the population is not monoclonal but is the result of multiple independent activations of the 118 gene. We show that the heterogeneity of expression-linked extra copies is also present in other trypanosome populations expressing chromosome-internal VSG genes. We present a model for the timing of VSG gene activation during chronic infection that emphasizes two features: the relative activation and inactivation frequencies of different expression sites, and the degree of homology of the sequences flanking VSG genes with expression sites.

Pulsed field gradient electrophoresis of DNA digested in agarose allows the sizing of the large duplication unit of a surface antigen gene in trypanosomes.

Intact chromosome-sized DNA molecules from eukaryotes may be prepared by performing lysis and enzymic deproteinization on cells embedded in agarose [Schwartz and Cantor, Cell 37 (1984), 67-75]. Here we show that DNA prepared by this method may be cut with restriction enzymes, or modified with site-specific methylases and cut by DpnI. As the DNA remains incorporated in the gel matrix, shear degradation of large fragments is avoided. The fragments can then be sized by conventional or pulsed field gradient gel electrophoresis. Phage lambda genomic oligomers are used as size markers, allowing the estimation of fragment sizes up to about 1200 kb. We apply these techniques to show that activation of the telomeric gene encoding variant surface antigen 1.3 in Trypanosoma brucei strain 427, involves the duplication of a DNA segment that starts between 29 and 42 kb upstream of the gene and to assign a chromosomal fragment into which the duplicated 1.3 gene may have transposed.

Transcription of a transposed trypanosome surface antigen gene starts upstream of the transposed segment.

The non-telomeric variant surface glycoprotein (VSG) genes in Trypanosoma brucei are activated by a duplicative transposition to a telomeric expression site. We have determined the 5' end of the transposed segment of the gene for VSG 117 and infer from comparison with similar data obtained by others that the crossover can occur at variable positions within short repeats present upstream of this gene and in the expression site. We have analysed nascent and steady state transcripts of the transposed gene and its neighbouring expression site DNA. The results indicate that transcription starts upstream of the transposed gene segment in the expression site and that transcripts are rapidly processed at specific points identified by protection of DNA-RNA hybrids against digestion by nuclease S1 or Exo VII. Hence, this gene appears to be activated by a process akin to promoter addition.

Two simultaneously active VSG gene transcription units in a single Trypanosoma brucei variant.

Trypanosomes can change their surface coat either by slotting a different surface antigen gene copy into an active (telomeric) expression site or by activating a new VSG gene expression site and inactivating the old one. How expression sites are activated or inactivated is not clear. We report an exceptional trypanosome variant in which the inactivation of a surface antigen gene is accompanied by a 30 kb DNA insertion 5' of the gene. Transcription of the region upstream of the insertion continues unaltered and retains the characteristic insensitivity to alpha-amanitin of VSG gene transcription units, showing that the expression site is still active. The expressed VSG gene in this trypanosome variant resides in another telomere. Hence, two VSG gene transcription units can be simultaneously active. This argues against a single mobile activating element controlling VSG gene transcription and favors a stochastic model of telomere activation/inactivation.