Use of forward genetic screens to identify genes required for RNA-directed DNA methylation in Arabidopsis thaliana.

RNA-directed DNA methylation is a small RNA-mediated epigenetic modification that contributes to transcriptional silencing of transposons and repetitive sequences in plants. We have conducted several forward genetic screens to identify factors required for RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis thaliana. Here, we review the findings from these screens and report on two new mutants, dms12 and dms13, that are defective in Pol V-specific subunits NRPE5 and NRPE9b. Cumulative results from genetic screens performed in our laboratory and those of other investigators have revealed that RNA-directed DNA methylation requires a complex transcriptional machinery comprising a number of plant-specific factors, many of which were functionally uncharacterized before being implicated in this pathway. Future challenges include unraveling the detailed mechanism and full range of functions of RNA-directed DNA methylation.

Involvement of CD44v6 in InlB-dependent Listeria invasion.

Listeria monocytogenes, a Gram-positive bacterium, is the causative agent for the disease called listeriosis. This pathogen utilizes host cell surface proteins such as E-cadherin or c-Met in order to invade eukaryotic cells. The invasion via c-Met depends on the bacterial protein InlB that activates c-Met phosphorylation and internalization mimicking in many regards HGF, the authentic c-Met ligand. In this paper, we demonstrate that the activation of c-Met induced by InlB is dependent on CD44v6, a member of the CD44 family of transmembrane glycoproteins. Inhibiting CD44v6 by means of a blocking peptide, a CD44v6 antibody or CD44v6-specific siRNA prevents the activation of c-Met induced by InlB. Subsequently, signalling, scattering and the entry of InlB-coated beads into host cells are also impaired by CD44v6 blocking reagents. For the entry process, ezrin, a protein that links the CD44v6 cytoplasmic domain to the cytoskeleton, is required as well. Most importantly, this collaboration between c-Met and CD44v6 contributes to the invasion of L. monocytogenes into target cells as demonstrated by a drastic decrease in bacterial invasion in the presence of blocking agents such as the CD44v6 peptide or antibody.

RNA-directed DNA methylation and Pol IVb in Arabidopsis.

Recent work in Arabidopsis has revealed a plant-specific RNA polymerase, pol IV, that is specialized for RNA interference (RNAi)-mediated, chromatin-based gene silencing. Two functionally diversified pol IV complexes have been identified: pol IVa is required to produce or amplify the small RNA trigger, whereas pol IVb, together with the plant-specific SWI/SNF-like chromatin remodeling factor DRD1, acts downstream from small RNA formation to induce de novo cytosine methylation of homologous DNA by an unknown mechanism. Retrotransposon long terminal repeats (LTRs) and other unannotated sequences that encode small RNAs are prime targets for DRD1/pol IVb-mediated cytosine methylation. In drd1 and pol IVb mutants, silent LTRs in euchromatin can be derepressed, resulting in enhanced transcription of adjacent genes or intergenic regions. In addition to mediating de novo methylation, some evidence suggests that DRD1 and pol IVb are also involved in a reciprocal process of active demethylation, perhaps in conjunction with DNA glycosylase domain-containing proteins such as ROS1. We speculate that DRD1/pol IV-dependent methylation/demethylation evolved in the plant kingdom as a means to facilitate rapid, reversible changes in gene expression, which might have adaptive significance for immobile plants growing in unpredictable environments.

Host defenses to transposable elements and the evolution of genomic imprinting.

Genomic imprinting is the differential expression of maternally and paternally inherited alleles of specific genes. Several organismic level hypotheses have been offered to explain the evolution of genomic imprinting. We argue that evolutionary explanations of the origin of imprinting that focus exclusively on the organismic level are incomplete. We propose that the complex molecular mechanisms that underlie genomic imprinting originally evolved as an adaptive response to the mutagenic potential of transposable elements (TEs). We also present a model of how these mechanisms may have been co-opted by natural selection to evolve molecular features characteristic of genomic imprinting.

The antiproliferative alkylphospholipid S-1-O-phosphocholine-2-N-acetyl-octadecane induces apoptosis in leukemia cell lines.

Lipids are involved in a multitude of important cellular functions. They act as signaling molecules and can even provoke apoptosis. In this context we investigated the efficacy of synthetic alkylphosphocholines (APCs) as potential anti-cancer membrane-affecting drugs. Leading to novel therapeutic strategies for cancer treatment, the new agents interact with the cell membrane and do not affect the DNA. The data presented here show a cell death-inducing capacity for 1-O-phosphocholine-2[S]-O-acetyl-octadecane and 1-O-phosphocholin-2[S]-N-acetyl-octadecane in Jurkat T cells as well as in BJAB cells. The activation of caspases is generally required for the induction of apoptosis as shown by experiments with specific caspase inhibitors. The results point on the one hand to the formation of a functional DISC after APC-treatment as indicated by the clustering of receptor molecules and on the other hand to the dependency on the instrinsic apoptotic machinery and the downstream of mitochondria-activated apoptosome.

Endogenous viral sequences and their potential contribution to heritable virus resistance in plants.

Tobacco endogenous pararetroviruses (TEPRVs) represent the first virus-derived repetitive sequence family found in plants. The sequence conservation of TEPRVs and the lack of an exogenous form of the virus suggest that TEPRVs serve a beneficial function, perhaps by furnishing virus resistance via homologous sequence interactions. This hypothesis is supported by the observation that TEPRVs are methylated and negligibly transcribed. Moreover, transgenes driven by the TEPRV enhancer are silenced and methylated when introduced into tobacco, but remain active and unmethylated in non-host species devoid of sequences homologous to TEPRVs. In transgenic Arabidopsis, the TEPRV enhancer is active primarily in shoot meristems. This suggests that the virus giving rise to TEPRVs could infect germ cell precursors, a prerequisite for meiotically heritable insertions into host chromosomes. The copy number, organization and methylation of TEPRVs in tetraploid tobacco and one of its diploid ancestors, Nicotiana sylvestris, the presumed original host for the virus, have remained constant since polyploid formation. The remarkable conservation of these features in two independently evolving species further supports a role for TEPRVs in viral immunity.

Transposition of IS10 from the host Escherichia coli genome to a plasmid may lead to cloning artefacts.

During recloning of Nicotiana tabacum L. repetitive sequence R8.3 in Escherichia coli, a modified clone that differed from the original by the insertion of an IS10 sequence was unintentionally produced. The insert was flanked by a 9-bp direct repeat derived from the R8.3 sequence, the 9-bp duplication of acceptor DNA in the site of insertion being a characteristic of IS10 transposition events. A database search using the FASTA program showed IS10 and other prokaryotic IS elements inserted into numerous eukaryotic clones. Unexpectedly, the IS10, which is not a natural component of the E. coli genome, appeared to be by far the most frequent contaminant of DNA databases among several IS sequences tested. In the GenEMBL database, the IS10 query sequence yielded positive scores with more than 500 eukaryotic clones. Insertions of shortened IS10 sequences having only one intact terminal inverted repeat were commonly found. Most full-length IS10 insertions (32 out of 40 analyzed) were flanked by 9-bp direct repeats having the consensus 5'-NPuCNN-NGPyN-3' with a strong preference for 5'-TGCTNA-GNN-3'. One insertion was flanked by an inverted repeat of more than 400 bp in length. PCR amplification and Southern analysis revealed the presence of IS10 sequences in E. coli strains commonly used for DNA cloning, including some reported to be Tn10-free. No IS10-specific PCR product was obtained with N. tabacum or human DNA. Our data suggest that transposition of IS10 elements may accompany cloning steps, particularly into large BAC vectors. This might lead to the relatively frequent contamination of DNA databases by this bacterial sequence. It is estimated that one in approximately every thousand eukaryotic clone in the databases is contaminated by IS-derived sequences. We recommend checking submitted sequences for the presence of IS10 and other IS elements. In addition, DNA databases should be corrected by removing contaminating IS sequences.

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.

Resistance of RNA-mediated TGS to HC-Pro, a viral suppressor of PTGS, suggests alternative pathways for dsRNA processing.

In plants, double-stranded (ds) RNA that is degraded to small (sm) RNAs that are approximately 23 nucleotides in length can trigger the degradation of homologous RNAs in the cytoplasm (posttranscriptional gene silencing or PTGS) and de novo methylation of homologous DNA in the nucleus [1]. PTGS is similar to quelling in fungi [2] and RNAi in animals [3]. RNA-directed DNA methylation (RdDM) can lead to transcriptional gene silencing (TGS) and the methylation of homologous target promoters if dsRNAs containing promoter sequences are involved [4]. HC-Pro is a plant viral suppressor of PTGS that acts by preventing the accumulation of smRNAs [5, 6] that provide the specificity determinant for homologous RNA degradation [7-10]. Here, we show that HC-Pro does not suppress TGS induced by promoter dsRNA. Moreover, the amount of promoter smRNAs is elevated 5-fold in the presence of HC-Pro, and target promoter methylation is slightly increased without a concomitant rise in the level of promoter dsRNA. The promoter dsRNA, which is not polyadenylated, failed to trigger substantial degradation of polyadenylated, single-stranded promoter RNA. The differential effects of HC-Pro on smRNA accumulation associated with dsRNA-mediated TGS and at least some cases of PTGS suggest that dsRNA processing can occur by alternative pathways, and they support the idea that RdDM is triggered by smRNAs.

A test for transvection in plants: DNA pairing may lead to trans-activation or silencing of complex heteroalleles in tobacco.

To study whether DNA pairing that influences gene expression can take place in somatic plant cells, a system designed to mimic transvection was established in transgenic tobacco. Pairing was evaluated by testing whether an enhancerless GUS gene on one allele could be activated in trans by an enhancer on the second allele. The required heteroalleles were obtained at four genomic locations using Cre-lox-mediated recombination. In one transgenic line, elevated GUS activity was observed with the heteroallelic combination, suggesting that trans-activation occurred. Conversely, when the unaltered allele was homozygous, GUS activity dropped to hemizygous levels in a silencing phenomenon resembling dosage compensation. Double-stranded GUS RNAs or small GUS RNAs indicative of RNA-based silencing mechanisms were not detected in plants displaying reduced GUS activity. These results suggested that a transgene locus capable of pairing, as revealed by trans-activation, could also become silenced in an RNA-independent manner, thus linking DNA pairing and gene silencing. The transgene locus was complex and comprised an inverted repeat, which possibly potentiated allelic interactions. The locus was unable to trans-activate transgenes at ectopic sites, further implicating allelic pairing in the transvection effects.

RNA-based silencing strategies in plants.

In plants, double-stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms, which require common proteins in diverse organisms, and are assessing the role of methylation in transcriptional and posttranscriptional gene silencing.

Killing tumour cells by alkylphosphocholines: evidence for involvement of CD95.

Many lipids act as cellular messengers and lead to a variety of different cellular responses. Out of the group of these compounds the ceramides are able to induce apoptosis, and some synthetic lipids can mimic this effect. Apoptosis is an important mechanism whereby chemotherapeutics exhibit their anti-oncogenic activity. Although, some lipid analogues were used in clinical trials, they exert severe side effects and their mechanism of action is widely unknown. We present here a new class of synthetic alkylphosphocholines (APC) that induce programmed cell death in leukaemia cells. The signs of apoptosis arise after 1 h of incubation with these compounds as shown by phosphatidylserine externalisation followed by caspase activation and DNA fragmentation. We demonstrate that the molecular target of these lipids is upstream of caspases and Bcl-2. Experiments with FADD dominant negative cells reveal that induction of apoptosis occurs on the level of CD95 and that these compounds can now be optimised for their capacity to activate the apoptosis-inducing receptor CD95.

Transcriptional silencing and promoter methylation triggered by double-stranded RNA.

Double-stranded RNA induces a post-transcriptional gene silencing process, termed RNAi, in diverse organisms. It is shown here that transcriptional gene silencing accompanied by de novo methylation of a target promoter in plants can be triggered by a double-stranded RNA containing promoter sequences. Similar to the double-stranded RNA involved in RNAi, this promoter double-stranded RNA, which is synthesized in the nucleus, is partially cleaved into small RNAs approximately 23 nucleotides in length. Both transcriptional and post-transcriptional gene silencing can thus be initiated by double-stranded RNAs that enter the same degradation pathway. The results also implicate double-stranded RNA in directing DNA methylation. Different constructs designed to produce double-stranded promoter RNA in various ways were evaluated for their ability to induce gene silencing in tobacco and Arabidopsis. RNA hairpins transcribed from inverted DNA repeats were the most effective trans-acting silencing signals. This strategy could be useful for transcriptionally downregulating genes in a variety of plants.

Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates.

Increasing evidence supports the idea that various transgene silencing phenomena reflect the activity of diverse host defense responses that act ordinarily on natural foreign or parasitic sequences such as transposable elements, viroids, RNA and DNA viruses, and bacterial DNA. Transgenes or their transcripts can resemble these cellular invaders in a number of ways, thus making them targets of host protective reactions. At least two distinct host defense systems operate to silence transgenes. One acts at the genome level and is associated with de novo DNA methylation. A second line of defense operates post-transcriptionally and involves sequence-specific RNA degradation in the cytoplasm. Transgenes that are silenced as a consequence of the genome defense are revealing that de novo methylation can be cued by DNA-DNA or RNA-DNA interactions. These methylation signals can be interpreted in the context of transposable elements or their transcripts. During evolution, as transposable elements accumulated in plant and vertebrate genomes and as they invaded flanking regions of genes, the genome defense was possibly recruited to establish global epigenetic mechanisms to regulate gene expression. Transposons integrated into promoters of host genes could conceivably change expression patterns and attract methylation, thus imposing on endogenous genes the type of epigenetic regulation associated with the genome defense. This recruitment process might have been particularly effective in the polyploid genomes of plants and early vertebrates. Duplication of the entire genome in polyploids buffers against insertional mutagenesis by transposable elements and permits their infiltration into individual copies of duplicated genes.

Integrated pararetroviral sequences define a unique class of dispersed repetitive DNA in plants.

Although integration of viral DNA into host chromosomes occurs regularly in bacteria and animals, there are few reported cases in plants, and these involve insertion at only one or a few sites. Here, we report that pararetrovirus-like sequences have integrated repeatedly into tobacco chromosomes, attaining a copy number of approximately 10(3). Insertion apparently occurred by illegitimate recombination. From the sequences of 22 independent insertions recovered from a healthy plant, an 8-kilobase genome encoding a previously uncharacterized pararetrovirus that does not contain an integrase function could be assembled. Preferred boundaries of the viral inserts may correspond to recombinogenic gaps in open circular viral DNA. An unusual feature of the integrated viral sequences is a variable tandem repeat cluster, which might reflect defective genomes that preferentially recombine into plant DNA. The recurrent invasion of pararetroviral DNA into tobacco chromosomes demonstrates that viral sequences can contribute significantly to plant genome evolution.

Rapid structural and epigenetic changes in polyploid and aneuploid genomes.

Recent work with plants has demonstrated that genome instability can be triggered by a change in chromosome number arising from either whole genome duplications (polyploidy) or loss/gain of individual chromosomes (aneuploidy). This genome instability is manifested as rapid structural and epigenetic alterations that can occur somatically or meiotically within a few generations after heteroploid formation. The intrinsic instability of newly formed polyploid and aneuploid genomes has relevance for genome evolution and human carcinogenesis, and points toward recombinational and epigenetic mechanisms that sense and respond to chromosome numerical changes.