Repeat elements and the Arabidopsis DNA methylation landscape.

DNA methylation is an epigenetic mark that has key roles in the control of genome activity in plants and mammals. It is critical for the stable silencing of repeat elements and is also involved in the epigenetic regulation of some genes. Despite similarities in the controlling functions of DNA methylation, its dynamics and deposition patterns differ in several respects between plants and mammals. One of the most striking differences is that plants tend to propagate pre-existing DNA methylation states across generations, whereas mammals re-establish them genome wide at every generation. Here, we review our current understanding of DNA methylation in the flowering plant Arabidopsis. We discuss in particular the role of RNAi in the incremental methylation and silencing of repeat elements over successive generations. We argue that paramutation, an epigenetic phenomenon first described in maize, is an extreme manifestation of this RNAi-dependent pathway.

DNA methylation and epigenetic inheritance in plants and filamentous fungi.

Plants and filamentous fungi share with mammals enzymes responsible for DNA methylation. In these organisms, DNA methylation is associated with gene silencing and transposon control. However, plants and fungi differ from mammals in the genomic distribution, sequence specificity, and heritability of methylation. We consider the role that transposons play in establishing methylation patterns and the epigenetic consequences of their perturbation.

New approaches for the analysis of Arabidopsis thaliana small RNAs.

We are interested in the study of small RNAs (sRNAs) that over-accumulate during the plant hypersensitive response (HR), a form of programmed cell death that occurs in and around the site of infection when plants are challenged by pathogens. For this purpose, we have constructed, by subtractive hybridization, a cDNA library of Arabidopsis sRNAs that are enriched during HR. Sequencing of randomly chosen clones provided evidence for the specific accumulation of several microRNAs as well as previously unidentified sRNAs. In a second approach, we have tested the possibility to hybridize labelled cDNAs derived from sRNAs to a DNA tiling array. We could reproducibly hybridize a custom-made tiling array covering Arabidopsis chromosome 4, with small cDNAs as targets. Furthermore, we have found that the distribution of hybridized fragments with sRNAs extracted from control leaves is in good agreement with the abundance of Arabidopsis sRNAs that correspond to this chromosome as determined by massive parallel sequence signature (MPSS).

Extensive, nonrandom diversity of excision footprints generated by Ds-like transposon Ascot-1 suggests new parallels with V(D)J recombination.

Upon insertion, transposable elements can disrupt or alter gene function in various ways. Transposons moving through a cut-and-paste mechanism are in addition often mutagenic when excising because repair of the empty site seldom restores the original sequence. The characterization of numerous excision events in many eukaryotes indicates that transposon excision from a given site can generate a high degree of DNA sequence and phenotypic variation. Whether such variation is generated randomly remains largely to be determined. To this end, we have exploited a well-characterized system of genetic instability in the fungus Ascobolus immersus to perform an extensive study of excision events. We show that this system, which produces many phenotypically and genetically distinct derivatives, results from the excision of a novel Ds-like transposon, Ascot-1, from the spore color gene b2. A unique set of 48 molecularly distinct excision products were readily identified from a representative sample of excision derivatives. Products varied in their frequency of occurrence over 4 orders of magnitude, yet most showed small palindromic nucleotide additions. Based on these and other observations, compelling evidence was obtained for intermediate hairpin formation during the excision reaction and for strong biases in the subsequent processing steps at the empty site. Factors likely to be involved in these biases suggest new parallels between the excision reaction performed by transposons of the hAT family and V(D)J recombination. An evaluation of the contribution of small palindromic nucleotide additions produced by transposon excision to the spectrum of spontaneous mutations is also presented.

Isolation of the Ascobolus immersus spore color gene b2 and study in single cells of gene silencing by methylation induced premeiotically.

The ascomycete Ascobolus immersus has been extensively used as a model system for the genetic study of meiotic recombination. More recently, an epigenetic process, known as methylation induced premeiotically (MIP), that acts on duplicated sequences has been discovered in A. immersus and has raised a new interest in this fungus. To try and extend these studies, we have now cloned the A. immersus spore color gene b2, a well characterized recombination hot-spot. Isolation of the whole gene was verified by physical mapping of four large b2 alterations, followed by transformation and mutant rescue of a null b2 allele. Transformation was also used to duplicate b2 and subject it to MIP. As a result, we were able for the first time to observe gene silencing as early as just after meiosis and in single cells. Furthermore, we have found evidence for a modulating effect of MIP on b2 expression, depending on the region of the gene that is duplicated and hence subjected to MIP.

Interaction of a tryptophan-containing peptide with chromatin core particles. A fluorescence study.

The binding of a tetrapeptide lysyltryptophylglycyllysine to nucleosome core particles has been investigated using UV absorption and fluorescence spectroscopy. Modifications of the absorption spectra and fluorescence quenching of the tryptophyl residue are consistent with stacking between the indole ring and nucleic acid bases. Therefore DNA interactions with histones do not prevent stacking of the tryptophyl residue with nucleic acid bases in the peptide-core particle complexes. The number of peptide binding sites is reduced to half that of naked DNA.

Eukaryotic DNA methylation as an evolutionary device.

DNA methylation is catalyzed by a family of conserved DNA methyltransferases and is widespread among protists, plants, fungi and animals. It is however absent in some species and its genomic distribution varies among organisms. Sequence comparisons suggest that known and putative eukaryotic DNA methyltransferases fall into at least five structurally distinct subfamilies. Furthermore, it is now clear that DNA methylation can be involved in several functions, some of which may coexist within the same organism. It can inhibit transcription initiation, arrest transcript elongation, act as an imprinting signal, and suppress homologous recombination. On the basis of these observations, we argue that DNA methylation has been conserved during evolution because it provides unique possibilities for setting up functions of various types.

Molecular characterization of an active wheat LMW glutenin gene and its relation to other wheat and barley prolamin genes.

The isolation and characterisation by DNA sequencing of a low molecular weight (LMW) glutenin gene from wheat is described. The deduced protein contains a signal peptide, a central repetitive region rich in proline and glutamine and N and C terminal non-repetitive domains, similar to other prolamins. A detailed comparison of the C terminal domain of 20 prolamin genes enabled us to divide them into 4 families. The LMW glutenin family is distinct from the alpha, beta- and gamma-gliadin families of wheat and is closest to the B hordein genes of barley. This and other comparisons were also used to assess the pattern of genetic variation among prolamin sequences and to provide a molecular basis for the interpretation of prolamin size polymorphism. The 5' flanking fragment of the isolated gene was previously shown to direct endosperm-specific expression of a reporter gene in transgenic tobacco. Evidence is provided that the isolated gene is also active in wheat and its transcription initiation site was determined. Features of the gene which may be relevant to its activity are discussed.

Interchromosomal transfer of epigenetic states in Ascobolus: transfer of DNA methylation is mechanistically related to homologous recombination.

The transfer of methylation between alleles represents a plausible epigenetic mutational mechanism to explain loss of imprinting in mammals and paramutation in plants. Here, we have exploited advantages unique to the fungus Ascobolus immersus to obtain direct experimental evidence that methylation transfer can occur between homologous chromosomes. A methylated allele and an unmethylated allele of the Ascobolus b2 spore color gene were brought together in individual meiotic cells. Frequent transfer of methylation to the unmethylated allele was observed. This transfer was polarized 5' to 3' along the b2 gene, as is gene conversion, and always accompanied the latter process when tested in the same cross. These and other observations strongly suggest that methylation transfer and recombination are mechanistically related.

Translational misreading: a tRNA modification counteracts a +2 ribosomal frameshift.

Errors during gene expression from DNA to proteins via transcription and translation may be deleterious for the functional maintenance of cells. In this paper, extensive genetic studies of the misreading of a GA repeat introduced into the lacZ gene of Escherichia coli indicate that in this bacteria, errors occur predominantly by a +2 translational frameshift, which is controlled by a tRNA modification involving the MnmE and GidA proteins. This ribosomal frameshift results from the coincidence of three events: (1) decreased codon-anticodon affinity at the P-site, which is caused by tRNA hypomodification in mnmE(-) and gidA(-) strains; (2) a repetitive mRNA sequence predisposing to slippage; and (3) increased translational pausing attributable to the presence of a rare codon at the A-site. Based on genetic analysis, we propose that GidA and MnmE act in the same pathway of tRNA modification, the absence of which is responsible for the +2 translational frameshift. The difference in the impact of the mutant gene on cell growth, however, indicates that GidA has at least one other function.

[DNA repeats and homologous recombination: a probable role for DNA methylation in genome stability of eukaryotic cells]. / ADN répété et recombinaison homologue: un rôle probable de la méthylation de l'ADN dans la stabilité des génomes des cellules eucaryotes.

Homologous recombination between DNA repeats directly threatens the intact transmission of repeat-rich eukaryotic genomes through mitotic and meiotic cell divisions. Besides several other factors already known, DNA methylation might contribute, in some eukaryotes, to the limitation of crossover events between repeats. A strong inhibitory effect of DNA methylation has now been directly demonstrated, in the filamentous fungus Ascobolus. This therefore reinforces the question of the biological impact of this DNA modification on the recombinational stability of repeat-rich genomes, such as those of mammals.

Localization of sequences in wheat endosperm protein genes which confer tissue-specific expression in tobacco.

The developing cereal grain accumulates large quantities of proteins which are unique to the endosperm tissue. The DNA sequences which determine their endosperm-specific expression have not yet been identified. In the absence of a suitable transformation-regeneration system for cereals, we have investigated whether chimaeric genes consisting of low mol. wt (LMW) and high mol. wt (HMW) glutenin gene upstream sequences coupled to the coding region of the bacterial chloramphenicol acetyl transferase (CAT) gene could be specifically expressed in transgenic tobacco. The fusions, made in a Ti-derived binary vector, were introduced into tobacco via Agrobacterium tumefaciens-mediated transformation and their activity assayed. Both the LMW and HMW glutenin chimaeric genes exhibited endosperm-specific CAT activity in the transformed plants. In addition, a deletion series of the LMW glutenin sequence indicated that sequences present between 326 bp and 160 bp upstream of the transcription start point are necessary to confer endosperm-specific CAT activity.

The wheat transcriptional activator SPA: a seed-specific bZIP protein that recognizes the GCN4-like motif in the bifactorial endosperm box of prolamin genes.

The conserved bifactorial endosperm box found in the promoter of wheat storage protein genes comprises two different cis elements that are thought to be involved in regulating endosperm-specific gene expression. Endosperm nuclear extracts contain binding activities. One is called ESBF-I, which binds to the endosperm motif (EM), and the other is called ESBF-II, which binds to the GCN4-like motif(GLM). Here, we present a functional analysis of the endosperm box of a low-molecular-weight glutenin gene found on the 1D1 chromosome of hexaploid wheat (LMWG-1D1) in transgenic tobacco plants. Our analysis demonstrates the necessity of the EM and GLM for endosperm-specific gene expression and suggests the presence in tobacco of functional counterparts of wheat ESBF-I and ESBF-II. Furthermore, we describe the isolation and characterization of cDNA clones encoding SPA, a seed-specific basic leucine zipper protein from wheat that can activate transcription from the GLMs of the -326-bp LMWG-1D1 promoter in both maize and tobacco leaf protoplasts. This activation is also partially dependent on the presence of functional EMs, suggesting interactions between SPA with ESBF-I-like activities.

The maize transcription factor Opaque-2 activates a wheat glutenin promoter in plant and yeast cells.

The promoter of the wheat low-molecular-weight glutenin (LMWG1D1) gene contains a cis element called the GCN4 like motif (GLM) which has low homology to one class of binding site for the maize endosperm-specific b-ZIP transcription factor Opaque-2 (O2). Previous work has shown that the GLM element interacts with the nuclear factor ESBFII during wheat endosperm development at the time of maximum transcription of the LMWG1D1 gene. In this paper we demonstrate that O2 binds to the GLM element and can activate high levels of transcription from the wheat GLM in transient assays in plant protoplasts and in yeast cells. Lower levels of O2 activation through the GLM element in yeast containing a defective GCN4 gene showed that GCN4 was necessary for high levels of O2 transcriptional activation, indicating that O2 may need to heterodimerise with GCN4 to activate transcription in yeast. These observations provide evidence that the GLM represents a new type of O2 DNA-binding site, and support a postulate that an O2 homologue may activate endosperm-specific expression of wheat storage protein genes.

Transcriptional control of plant storage protein genes.

The accumulation of plant storage proteins is controlled primarily by the transcriptional activation of their genes. Two classes of storage proteins, the zygotic or seed-specific, and the somatic, such as tuber proteins, have been studied. Gene expression analysis in transgenic plants has defined small regions of the promoters of such genes that are able to confer the appropriate patterns of expression. Protein-DNA interactions, both in vivo and in vitro, have revealed proteins that bind to regions implicated in expression, and these may be transcription factors. Promoter deletion analysis has determined the role of some of these DNA-binding proteins, such as in determining tissue-specificity or levels of expression. A common theme linking the expression of both classes of storage proteins is the involvement of metabolite levels in directly controlling gene expression.