Jan M. Kooter

Listening to the silent genes: transgene silencing, gene regulation and pathogen control

By capitalizing on the initially puzzling observations of unpredictable transgene silencing and variable expression, plant scientists have pioneered research into a novel type of epigenetic regulation, termed homology-dependent gene silencing. This silencing process has implications for natural mechanisms of gene expression in plants and other eukaryotes, and has branched out into studies of reversible DNA modifications; RNA metabolism, transport and processing; and host responses to plant viruses, viroids and transposable elements. The analysis of transgene silencing systems has enriched our understanding of other epigenetic phenomena, including paramutation, as well as heterosis and genome evolution. This research is also highly relevant to the biotechnology industry, which is interested in avoiding unwanted transgene silencing in genetically engineered lines and in exploiting various types of silencing to inactivate specific genes. Homology-dependent gene silencing can also be used in high-throughput approaches for functional genomics.

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 PTGS is induced by deliberate [1-4] or fortuitous production (R.v.B., unpublished data) of double-stranded RNA (dsRNA).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.

Position-Dependent Methylation and Transcriptional Silencing of Transgenes in Inverted T-DNA Repeats: Implications for Posttranscriptional Silencing of Homologous Host Genes in Plants

ABSTRACT Posttranscriptional silencing of chalcone synthase (Chs) genes in petunia transformants occurs by introducing T-DNAs that contain a promoter-driven or promoterless Chstransgene. 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 promoterlessChs 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.

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.

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.

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.

Trans-inactivation of gene expression in plants

Abstract Gene silencing induced by antisense RNA has been extremely helpful in characterizing the cellular functions of a number of plant gene products. The introduction of extra sense gene copies may lead not only to protein overproduction but also, surprisingly, to gene silencing. Elucidation of the mechanisms responsible for both types of gene silencing may reveal novel mechanisms by which gene expression is controlled.

hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis

BackgroundActivation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells.MethodsUsing luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter.ResultsWe found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity.By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls.ConclusionsMethylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these repressive regions may provide an attractive biomarker for early detection of cervical cancer.

A single transgene locus triggers both transcriptional and post-transcriptional silencing through double-stranded RNA production

Silencing of a target locus by an unlinked silencing locus can result from transcription inhibition (transcriptional gene silencing; TGS) or mRNA degradation (post-transcriptional gene silencing; PTGS), owing to the production of double-stranded RNA (dsRNA) corresponding to promoter or transcribed sequences, respectively. The involvement of distinct cellular components in each process suggests that dsRNA-induced TGS and PTGS likely result from the diversification of an ancient common mechanism. However, a strict comparison of TGS and PTGS has been difficult to achieve because it generally relies on the analysis of distinct silencing loci. We describe a single transgene locus that triggers both TGS and PTGS, owing to the production of dsRNA corresponding to promoter and transcribed sequences of different target genes. We describe mutants and epigenetic variants derived from this locus and propose a model for the production of dsRNA. Also, we show that PTGS, but not TGS, is graft-transmissible, which together with the sensitivity of PTGS, but not TGS, to RNA viruses that replicate in the cytoplasm, suggest that the nuclear compartmentalization of TGS is responsible for cell-autonomy. In contrast, we contribute local and systemic trafficking of silencing signals and sensitivity to viruses to the cytoplasmic steps of PTGS and to amplification steps that require high levels of target mRNAs.