Biomedical subjects
Publications and source records attributed to G Faugeron.
The experimental introduction of DNA in fungi has revealed a variety of gene inactivation strategies triggered in response to the presence of homologous nucleic acid sequences. These homology effects reveal distinct classes of gene inactivation resulting from hypermutation or relying on regulatory mechanisms that operate at either the transcriptional or the post-transcriptional level.
A gene encoding a protein that shows sequence similarity with the histone H1 family only was cloned in Ascobolus immersus. The deduced peptide sequence presents the characteristic three-domain structure of metazoan linker histones, with a central globular region, an N-terminal tail, and a long positively charged C-terminal tail. By constructing an artificial duplication of this gene, named H1, it was possible to methylate and silence it by the MIP (methylation induced premeiotically) process. This resulted in the complete loss of the Ascobolus H1 histone. Mutant strains lacking H1 displayed normal methylation-associated gene silencing, underwent MIP, and showed the same methylation-associated chromatin modifications as did wild-type strains. However, they displayed an increased accessibility of micrococcal nuclease to chromatin, whether DNA was methylated or not, and exhibited a hypermethylation of the methylated genome compartment. These features are taken to imply that Ascobolus H1 histone is a ubiquitous component of chromatin which plays no role in methylation-associated gene silencing. Mutant strains lacking histone H1 reproduced normally through sexual crosses and displayed normal early vegetative growth. However, between 6 and 13 days after germination, they abruptly and consistently stopped growing, indicating that Ascobolus H1 histone is necessary for long life span. This constitutes the first observation of a physiologically important phenotype associated with the loss of H1.
We have shown previously that masc1, a gene encoding a putative C5-DNA-methyltransferase (MTase), was necessary for the de novo 'Methylation Induced Premeiotically' (MIP) process and sexual reproduction in Ascobolus, whereas it was dispensable for maintenance methylation. A second MTase gene from Ascobolus, masc2, encodes a protein, Masc2, which possesses the large amino-terminal part characteristic of eukaryotic maintenance MTases. In vitro assays have shown that Masc2 displays a methylation activity, suggesting that it might be the MTase responsible for maintenance methylation. To check its function in vivo, we engineered a disruption of the masc2 gene. The resulting mutant strains did not exhibit any particular phenotype during either vegetative growth or sexual reproduction. Neither the masc2 mutation nor the double masc1 masc2 mutation had any detectable effect upon the maintenance of the pre-existing methylation of single gene copies previously subjected to MIP, natural retroelement-like repeats and tandemly repeated rDNA. The masc2 mutation did not alter either MIP or the other de novo methylation process that operates in vegetatives cells. Nor did it impair the meiotic process of methylation transfer. These results suggest that at least a third MTase gene responsible for maintenance and vegetative de novo methylation is present in Ascobolus.
Molecular mechanisms determining methylation patterns in eukaryotic genomes still remain unresolved. We have characterized, in Ascobolus, a gene for de novo methylation. This novel eukaryotic gene, masc1, encodes a protein that has all motifs of the catalytic domain of eukaryotic C5-DNA-methyltransferases but is unique in that it lacks a regulatory N-terminal domain. The disruption of masc1 has no effect on viability or methylation maintenance but prevents the de novo methylation of DNA repeats, which takes place after fertilization, through the methylation induced premeiotically (MIP) process. Crosses between parents harboring the masc1 disruption are arrested at an early stage of sexual reproduction, indicating that the activity of Masc1, the product of the gene, is crucial in this developmental process.
We identified two classes of native dispersed DNA repeats in the Ascobolus genome. The first class consisted of several kilobase long, methylated repeats. These repeats, named Mars (methylated Ascobolus repeated sequences), fell in one family of LINE-like elements and in three families of LTR-containing retrotransposable elements. The methylation features of Mars elements were those expected if they were natural targets for the MIP (methylation induced premeiotically) previously discovered in Ascobolus. The second class consisted of short repeats, approximately 100 bp long, corresponding to 5S rRNA and tRNA genes. As expected from their size, which was too small to allow MIP to occur, they were unmethylated, as were 26 kb of unique sequences tested. These observations are consistent with the hypothesis that MIP is targeted at natural DNA repeats and constitutes a defensive process against the detrimental consequences of the spreading of mobile elements throughout the genome. The 9 kb tandem repeats harbouring the 28S, 18S and 5.8S rRNA genes displayed methylation features suggesting that rDNA methylation proceeds through a process other than MIP.
In Ascobolus immersus, DNA duplications are subject to the process of methylation induced premeiotically (MIP), which methylates the cytosine residues within the repeats and results in reversible gene silencing. The triggering of MIP requires pairing of the repeats, and its detection requires maintenance of the resulting methylation. MIP of kilobase-size duplications occurs frequently and leads to the methylation of all C residues in the repeats, including those belonging to non-CpG sequences. Using duplications of decreasing sizes, we observed that tandem repeats never escaped MIP when larger than 630 bp and showed a sudden and drastic drop in MIP frequencies when their sizes decreased from 630 to 317 bp. This contrasted with the progressive decrease of MIP frequencies observed with ectopic repeats, in which apparently the search for homology influences the MIP triggering efficiency. The minimal size actually required for a repeat to undergo detectable MIP was found to be close to 300 bp. Genomic sequencing and Southern hybridization analyses using restriction enzymes sensitive to C methylation showed a loss of methylation at non-CpG sites in short DNA segments, methylation being restricted to a limited number of CpG dinucleotides. Our data suggest the existence of two distinct mechanisms underlying methylation maintenance, one responsible for methylation at CpG sites and the other responsible for methylation at non-CpG sites.
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In the ascomycete Ascobolus immersus, duplicated DNA segments are subject to the methylation induced premeiotically (MIP) process. Affected sequences are heavily methylated at their cytosine residues. We used the bisulphite genomic sequencing method to determine the methylation status of every cytosine residue in a gene which had undergone MIP. Several individual DNA molecules, all issued from the replication of a single molecule initially subject to MIP, were sequenced. In each molecule, methylation extended over almost the whole length of the previously duplicated segment. The methylation extent was precisely delimited and constant in each of the molecules, leaving unmethylated a nearly 100-nucleotide region next to each end. In none of the molecules did methylation resulting from MIP extend beyond the ends. Although the DNA molecules were not all methylated with the same intensity, all cytosine residues in the methylated portion could be methylated, most of them belonging to non-symmetrical sequences. This finding contrasts with the situation in higher eukaryotes in which most, if not all, methylation is at short symmetrical sequences such as CpG or CpNpG, ensuring perpetuation of methylation. Methylation at non-symmetrical sequences implies that in A. immersus maintenance involves a novel sequence-non-specific methyltransferase.
This chapter focuses on phenomena of gene inactivation resulting from the presence of repeated gene copies within the genome of plants and fungi, and on their possible relationships to homologous DNA-DNA interactions. Emphasis is given to two related premeiotic processes: Methylation Induced Premeiotically (MIP) and Repeat-Induced Point mutation (RIP) which take place in the fungi Ascobolus immersus and Neurospora crassa, respectively. The relationships between these processes and genetic recombination are discussed.
A large proportion of Ascobolus immersus transformants are highly unstable in crosses: the phenotype conferred by the transgene is not transmitted to the progeny, irrespective of the endogenous or foreign origin of the transgene. They all have integrated multiple transgene copies, clustered at a single chromosomal site or at tightly-linked sites. Clustered non-homologous integrations are always rearranged. Yet they never escape the "methylation induced premeiotically" (MIP) process. This always results in gene silencing, even when the transgene is partially repeated, accounting for the high instability of these transformants.
The effect of duplications of gene fragments on the triggering of DNA methylation induced premeiotically (MIP) was studied in the sexual progeny of strains harboring, in addition to the resident met2 gene, a fragment of this gene inserted at an ectopic position. Cytosine methylation of the resident gene was checked for each of the eight duplications tested. Methylation was always found and it was coextensive with the length of the duplications. Silencing of the resident gene was triggered by duplications of segments corresponding to the region 5' to the open reading frame, to only the open reading frame, or to segments beginning 0.87-1.2 kb downstream from the transcription start. Silencing was accompanied by either the absence of transcripts or the presence of truncated transcripts, which suggests that methylation acts on transcript elongation.
Ascobolus immersus artificial gene repeats were shown previously to be subject premeiotically to both cytosine methylation and inactivation. We studied sexual progenies of strains harbouring two wild type copies of the endogenous met2 gene lying either in tandem array or at ectopic unlinked positions, by (i) investigating the methylation status, (ii) searching for mutations and (iii) analysing the inheritance of inactivation both in mitotic and sexual offspring. 100% of the 'tandem' progeny and 64% of the 'ectopic' progeny had methylated repeats and displayed gene inactivation. Similar methylation patterns involving all or most of the cytosine residues within the repeats were observed in both arrangements. The inactivated met2 copies were totally devoid of mutation, as deduced from: (i) extensive restriction site analysis and DNA sequencing; (ii) the finding that all the Met- derivatives tested reverted to prototrophy in selective conditions; and (iii) the finding that an inactivated copy of met2 stripped of its methylation through amplification in Escherichia coli regained activity when reintroduced in A.immersus. In the absence of selection, gene silencing and methylation were faithfully maintained through mitotic divisions and through five successive sexual cycles. Altogether, these data show the epimutational nature of this methylation induced premeiotically (MIP) process.
Repeated genes, artificially introduced in Ascobolus immersus by integrative transformation, are frequently inactivated during the sexual phase. Inactivation is observed in about 50% of meioses if duplicated genes are at ectopic chromosomal locations, and in 90% of meioses if genes are tandemly repeated. Inactivation is associated with extensive methylation of the cytosine residues of the duplicated sequences and is induced in the still haploid nuclei of the dikaryotic cell which will undergo karyogamy and subsequent meiosis. Only repeated sequences become methylated. This raises the intriguing question of how the premeiotic inactivation machinery is informed that a nucleus contains multiple copies of a gene. By using in crosses recombinant strains of A. immersus in which either one, two or three genetically independent copies of the exogenous amdS gene from Aspergillus nidulans had been introduced, we could follow the premeiotic inactivation of each one of the ectopic amdS copies. This led us to propose that a prerequisite for inactivation is a premeiotic pairing of repeated sequences and that each copy can undergo successive cycles of pairing. In fact, once methylated, a copy can pair with a still unmethylated copy, so that an uneven number of copies can be subject to inactivation.
An homologous transformation system for the filamentous fungus Ascobolus immersus has been developed, based on the complementation of a met2 mutation by the wild-type (wt) allele gene encoding homoserine O-transacetylase. Transformation of A. immersus met2 mutants occurs with moderate frequencies (about 50 transformants per microgram input DNA). Analysis of the DNA of the met2+ transformants showed that transformation resulted either in a single integration of the donor DNA into the genome by many different nonhomologous recombination events or in the substitution of the endogenous met2 mutation by the wt transforming allele. The relative frequencies of both events depended on the vector sequences carrying the cloned met2 gene. Whereas the substitution event led, as expected, to genetically stable transformants, the non-homologous integration was always associated with a strong instability when transformants were crossed and underwent meiosis.
To develop a method to modify genomic sequences in Ascobolus immersus by precisely reintroducing defined DNA segments previously manipulated in vitro, we investigated the effect of transforming DNA conformation on recombination with chromosomal sequences. Circular single-stranded DNA carrying the met2 gene and double-stranded DNA linearized by cutting within the met2 gene both transformed protoplasts of a met2 mutant strain of A. immersus to prototrophy. In contrast to the equivalent circular double-stranded DNA, which chiefly integrated at nonhomologous chromosomal sites, single-stranded and double-stranded cut DNAs recombined primarily with the homologous chromosomal met2 sequence. Of the single-stranded DNA transformants, 65% resulted from replacement of the resident met2 mutation by the exogenous wild-type allele. In 70% of the double-stranded-cut DNA transformants, one or more copies of the transforming DNA had integrated at the met2 locus, leading to tandem duplications of the met2 target region separated by plasmid DNA. These duplicated sequences could recombine, leading to progeny containing only one copy of the met2 region. This resulted in a precise gene replacement if the wild-type allele had been retained. In addition, we show that newly duplicated sequences were most often de novo methylated at the cytosine residues during the sexual phase. Cytosine methylation was associated with inactivation of the integrated met2 gene(s) in segregants of crosses. However, methylation was not accurately maintained at each DNA replication cycle, so that Met- segregants recovered a wild-type phenotype through successive mitotic divisions. This finding indicated that met2 genes were silenced by methylation alone.
We have cloned the met2 gene from Ascobolus immersus by heterologous hybridization with the MET2 gene of Saccharomyces cerevisiae. This gene codes for the homoserine O-transacetylase, one of the methionine biosynthetic enzymes. The complete nucleotide sequence of a 2910-bp DNA fragment carrying the met2 gene has been determined. The gene contains a 165-bp intron which is similar in structure to other fungal introns. The deduced amino acid (aa) sequence (518 aa residues; Mr of 57726) shows three domains with a significant level of homology with the corresponding yeast protein. Northern-blot analysis reveals at least two transcripts (2.4 and 2.1 kb) probably due to transcription termination heterogeneity, as suggested by S1-mapping experiments. Polymorphism has been observed in the met2 gene flanking regions of Ascobolus strains from two different stocks.
A 5.1-kb DNA fragment from Saccharomyces cerevisiae, which complements a yeast met2 mutant strain, has been cloned. This fragment contains the wild-type MET2 gene which codes for the homoserine O-transacetylase, one of the methionine biosynthetic enzymes. The presence of the MET2 gene has been shown by integrative transformation experiments and genetic analyses of the resulting transformants. The complete nucleotide sequence of a 2826-bp DNA fragment carrying the MET2 gene has been determined. The sequence contains one major open reading frame of 438 codons, giving a calculated Mr of 48,370 for the encoded protein. We have identified the transcriptional product of the MET2 gene and estimated its size at 1650 nucleotides.