Recurrence of Repeat-Induced Point Mutation (Rip) in Neurospora Crassa

Duplicate DNA sequences in the genome of Neurospora crassa can be detected and mutated in the sexual phase of the life cycle by a process termed RIP (repeat-induced point mutation). RIP occurs in the haploid nuclei of fertilized, premeiotic cells before fusion of the parental nuclei. Both copies of duplications of gene-sized sequences are affected in the first generation at frequencies of approximately 50-100%. We investigated the extent to which sequences altered by RIP remain susceptible to this process in subsequent generations. Duplications continued to be sensitive to RIP, even after six generations. The fraction of progeny showing evidence of RIP decreased rapidly, however, apparently as a function of the extent of divergence of the duplicated sequences. Analysis of the stability of heteroduplexes of DNA altered by RIP and their native counterpart indicated that linked duplications diverged further than did unlinked duplications. DNA methylation, a common feature of sequences altered by RIP, did not seem to inhibit the process. A sequence that had become resistant to RIP was cloned and reintroduced into Neurospora in one or more copies to investigate the basis of the resistance. The altered sequence regained its methylation in vegetative cells, indicating that the methylation of sequences altered by RIP observed in vegetative cells is a consequence of the mutations. Duplication of the sequence restored its sensitivity to RIP suggesting that resistance to the process was due to loss of similarity between the duplicated sequences. Consistent with this, we found that the resistant sequence did not trigger RIP of the native homologous sequences of the host, even when no other partner was available. High frequency intrachromatid recombination, which is temporally associated with RIP, was more sensitive than RIP to alterations in the interacting sequences.     

In vivo levels of S-adenosylmethionine modulate C:G to T:A mutations associated with repeat-induced point mutation in Neurospora crassa

In Neurospora crassa, the mutagenic process termed repeat-induced point mutation (RIP) inactivates duplicated DNA sequences during the sexual cycle by the introduction of C:G to T:A transition mutations. In this work, we have used a collection of N. crassa strains exhibiting a wide range of cellular levels of S-adenosylmethionine (AdoMet), the universal donor of methyl groups, to explore whether frequencies of RIP are dependent on the cellular levels of this metabolite. Mutant strains met-7 and eth-1 carry mutations in genes of the AdoMet pathway and have low levels of AdoMet. Wild type strains with high levels of AdoMet were constructed by introducing a chimeric transgene of the AdoMet synthetase (AdoMet-S) gene fused to the constitutive promoter trpC from Aspergillus nidulans. Crosses of these strains against tester duplications of the pan-2 and am genes showed that frequencies of RIP, as well as the total number of C:G to T:A transition mutations found in randomly selected am(RIP) alleles, are inversely correlated to the cellular level of AdoMet. These results indicate that AdoMet modulates the biochemical pathway leading to RIP.     

Synthesis of signals for de novo DNA methylation in Neurospora crassa

Most 5-methylcytosine in Neurospora crassa occurs in A:T-rich sequences high in TpA dinucleotides, hallmarks of repeat-induced point mutation. To investigate how such sequences induce methylation, we developed a sensitive in vivo system. Tests of various 25- to 100-bp synthetic DNA sequences revealed that both T and A residues were required on a given strand to induce appreciable methylation. Segments composed of (TAAA)(n) or (TTAA)(n) were the most potent signals; 25-mers induced robust methylation at the special test site, and a 75-mer induced methylation elsewhere. G:C base pairs inhibited methylation, and cytosines 5' of ApT dinucleotides were particularly inhibitory. Weak signals could be strengthened by extending their lengths. A:T tracts as short as two were found to cooperate to induce methylation. Distamycin, which, like the AT-hook DNA binding motif found in proteins such as mammalian HMG-I, binds to the minor groove of A:T-rich sequences, suppressed DNA methylation and gene silencing. We also found a correlation between the strength of methylation signals and their binding to an AT-hook protein (HMG-I) and to activities in a Neurospora extract. We propose that de novo DNA methylation in Neurospora cells is triggered by cooperative recognition of the minor groove of multiple short A:T tracts. Similarities between sequences subjected to repeat-induced point mutation in Neurospora crassa and A:T-rich repeated sequences in heterochromatin in other organisms suggest that related mechanisms control silent chromatin in fungi, plants, and animals.     

Short TpA-rich segments of the zeta-eta region induce DNA methylation in Neurospora crassa

The mechanisms that establish DNA methylation in eukaryotes are poorly understood. In principle, methylation in a particular chromosomal region may reflect the presence of a "signal" that recruits methylation, the absence of a signal that prevents methylation, or both. Experiments were carried out to address these possibilities for the 1.6 kb zeta-eta (zeta-eta) region, a relict of repeat-induced point mutation (RIP) in the fungus Neurospora crassa. The zeta-eta region directs its own de novo methylation at a variety of chromosomal locations. We tested the methylation potential of a nested set of fragments with deletions from one end of the zeta-eta region, various internal fragments of this region, chimeras of eta and the homologous unmutated allele, theta (theta), and various synthetic variants, integrated precisely in single copy at the am locus on linkage group (LG) VR or the his-3 locus on LG IR. We found that: (1) the zeta-eta region contains at least two non-overlapping methylation signals; (2) different fragments of the region can induce different levels of methylation; (3) methylation induced by zeta-eta sequences can spread far into flanking sequences; (4) fragments as small as 171 bp can trigger methylation; (5) methylation signals behave similarly, but not identically, at different chromosomal sites; (6) mutation density, per se, does not determine whether sequences become methylated; and (7) neither A:T-richness nor high densities of TpA dinucleotides, typical attributes of methylated sequences in Neurospora, are essential features of methylation signals, but both promote de novo methylation. We conclude that de novo methylation of zeta-eta sequences does not simply reflect the absence of signals that prevent methylation; rather, the region contains multiple, positive signals that trigger methylation. These findings conflict with earlier models for the control of DNA methylation, including the simplest version of the collapsed chromatin model.     

A targeted-replacement system for identification of signals for de novo methylation in Neurospora crassa.

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.     

Cytosine Methylation Associated with Repeat-Induced Point Mutation Causes Epigenetic Gene Silencing in Neurospora Crassa

Repeated DNA sequences are frequently mutated during the sexual cycle in Neurospora crassa by a process named repeat-induced point mutation (RIP). RIP is often associated with methylation of cytosine residues in and around the mutated sequences. Here we demonstrate that this methylation can silence a gene located in nearby, unique sequences. A large proportion of strains that had undergone RIP of a linked duplication flanking a single-copy transgene, hph (hygromycin B phosphotransferase), showed partial silencing of hph. These strains were all heavily methylated throughout the single-copy hph sequences and the flanking sequences. Silencing was alleviated by preventing methylation, either by 5-azacytidine (5AC) treatment or by introduction of a mutation (eth-1) known to reduce intracellular levels of S-adenosylmethionine. Silenced strains exhibited spontaneous reactivation of hph at frequencies of 10(-4) to 0.5. Reactivated strains, as well as cells that were treated with 5AC, gave rise to cultures that were hypomethylated and partially hygromycin resistant, indicating that some of the original methylation was propagated by a maintenance mechanism. Gene expression levels were found to be variable within a population of clonally related cells, and this variation was correlated with epigenetically propagated differences in methylation patterns.     

RIP: the evolutionary cost of genome defense

Repeat-induced point mutation (RIP) is a homology-based process that mutates repetitive DNA and frequently leads to epigenetic silencing of the mutated sequences through DNA methylation. Consistent with the hypothesis that RIP serves to control selfish DNA, an analysis of the Neurospora crassa genome sequence reveals a complete absence of intact mobile elements. As in most eukaryotes, the centromeric regions of N. crassa are rich in sequences that are related to transposable elements; however, in N crassa these sequences have been heavily mutated. The analysis of the N. crassa genome sequence also reveals that RIP has impacted genome evolution significantly through gene duplication, which is considered to be crucial for the evolution of new functions. Most if not all paralogs in N. crassa duplicated and diverged before the emergence of RIP. Thus, RIP illustrates the extraordinary extent to which genomes will go to defend themselves against mobile genetic elements.     

The Neurospora Transposon Tad Is Sensitive to Repeat-Induced Point Mutation (Rip)

RIP (repeat-induced point mutation) efficiently mutates repeated sequences in the sexual phase of the Neurospora crassa life cycle. Nevertheless, an active LINE-like retrotransposon, Tad, was found in a N. crassa strain from Adiopodoume. The possibility was tested that Tad might be resistant to RIP, or that the Adiopodoume strain might be incompetent for RIP. Tad elements derived from the Adiopodoume strain were found to be susceptible to RIP. In addition, strains lacking active Tad elements, including common laboratory strains and strains representing seven species of Neurospora, were found to have sequences closely related to Tad but with numerous mutations of the type resulting from RIP (G:C to A:T). Even the Adiopodoume strain showed Tad-like elements with mutations characteristic of RIP. Results of crossing of an Adiopodoume transformant with progeny of Adiopodoume suggest that the Adiopodoume strain is proficient at RIP. We conclude that Tad is an old transposable element that has been inactivated by RIP in most strains. Finding relics of RIP in both heterothallic and homothallic species of Neurospora implicates RIP across the genus.     

A natural case of RIP: degeneration of the DNA sequence in an ancestral tandem duplication.

5S rRNA genes of Neurospora crassa are generally dispersed in the genome and are unmethylated. The xi-eta region of Oak Ridge strains represents an informative exception. Most of the cytosines in this region, which consists of a diverged tandem duplication of a 0.8-kilobase-pair segment including a 5S rRNA gene, appear to be methylated (E. U. Selker and J. N. Stevens, Proc. Natl. Acad. Sci. USA 82:8114-8118, 1985). Previous work demonstrated that the xi-eta region functions as a portable signal for de novo DNA methylation (E. U. Selker and J. N. Stevens, Mol. Cell. Biol. 7:1032-1038, 1987; E. U. Selker, B. C. Jensen, and G. A. Richardson, Science 238:48-53, 1987). To identify the structural basis of this property, we have isolated and characterized an unmethylated allele of the xi-eta region from N. crassa Abbott 4. The Abbott 4 allele includes a single 5S rRNA gene, theta, which is different from all previously identified Neurospora 5S rRNA genes. Sequence analysis suggests that the xi-eta region arose from the theta region by duplication of a 794-base-pair segment followed by 267 G.C to A.T mutations in the duplicated DNA. The distribution of these mutations is not random. We propose that the RIP process of N. crassa (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987; E. U. Selker, and P. W. Garrett, Proc. Natl. Acad. Sci. USA 85:6870-6874, 1988; E. B. Cambareri, B. C. Jensen, E. Schabtach, and E. U. Selker, Science 244:1571-1575, 1989) is responsible for the numerous transition mutations and DNA methylation in the xi-eta region. A long homopurine-homopyrimidine stretch immediately following the duplicated segment is 9 base pairs longer in the Oak Ridge allele than in the Abbott 4 allele. Triplex DNA, known to occur in homopurine-homopyrimidine sequences, may have mediated the tandem duplication.     

De Novo Methylation of Repeated Sequences in Coprinus Cinereus

We have examined the stability of duplicated DNA sequences in the sexual phase of the life cycle of the basidiomycete fungus, Coprinus cinereus. We observed premeiotic de novo methylation in haploid nuclei containing either a triplication, a tandem duplication, or an ectopic duplication. Methylation changes were not observed in unique sequences. Repeated sequences underwent methylation changes during the dikaryotic stage. In one cross, 27% of the segregants exhibited methylation-directed gene inactivation. However, all auxotrophs eventually reverted to prototrophy. C to T transition mutations were not observed in this study. Our studies also revealed one inversion that occurred in 50% of the segregants in a single triplication cross, and a single pop-out event that occurred during vegetative growth. These alterations were similar to changes reported in experiments with duplicated sequences in Neurospora crassa and Ascobolus immersus. However, significant differences were also noted. First, the extent of methylation was much less in C. cinereus than in the other two fungi. Second, CpG sequences appeared to be the preferred targets of methylation.     

dim-2 encodes a DNA methyltransferase responsible for all known cytosine methylation in Neurospora

To understand better the control of DNA methylation, we cloned and characterized the dim-2 gene of Neurospora crassa, the only eukaryotic gene currently known in which mutations appear to eliminate DNA methylation. The dim-2 gene is responsible for methylation in both symmetrical and asymmetrical sites. We mapped dim-2 between wc-1 and un-10 on linkage group (LG) VIIR and identified the gene by RFLP mapping and genetic complementation. Dim-2 encodes a 1454 amino acid protein including a C-terminal domain homologous to known DNA methyltransferases (MTases) and a novel N-terminal domain. Neither a deletion that removed the first 186 amino acids of the protein nor a mutation in a putative nucleotide binding site abolished function, but a single amino acid substitution in the predicted catalytic site did. Tests for repeat-induced point mutation (RIP) indicated that dim-2 does not play a role in this process, i.e. duplicated sequences are mutated in dim-2 strains, as usual, but the mutated sequences are not methylated, unlike the situation in dim-2+ strains. We conclude that dim-2 encodes an MTase that is responsible for all DNA methylation in vegetative tissues of NEUROSPORA:     

Rearrangement of duplicated DNA in specialized cells of Neurospora

Introduction of DNA into Neurospora crassa can lead to sequence instability in the sexual phase of the life cycle. Sequence instability was investigated by using a set of strains transformed with single copies of a plasmid including host sequences, Neurospora sequences deleted from the host genome, and foreign sequences. The sequences already represented in the host were rearranged at high frequency in a cross. In general, both elements of the duplication, that from the plasmid and that from the host, became rearranged, whether or not they were linked. Unique sequences were left unaltered. Cytosine residues in the rearranged sequences typically became methylated de novo. Results from tetrad analyses indicated that the rearrangements occur before meiosis, during a stage between fertilization and karyogamy. We suggest that this previously unrecognized genetic process, RIP (rearrangement induced premeiotically), may contribute diversity for evolution and also maintain the gross organization of the genome.     

DNA methylation inhibits expression and transposition of the Neurospora Tad retrotransposon

Tad is a LINE-like retrotransposon of the filamentous fungus Neurospora crassa. We have analyzed both expression and transposition of this element using strains with a single copy of Tad located in the 5' noncoding sequences of the am (glutamate dehydrogenase) gene. Tad in this position has been shown to carry a de novo cytosine methylation signal which causes reversible methylation of both Tad and am upstream sequences. Here we find that methylation of the Tad sequences inhibits both Tad expression and transposition. This inhibition can be relieved by the use of 5-azacytidine, a drug which reduces cytosine methylation, or by placing the Tad/am sequences in a dim-2 genetic background.     

Eth-1, the Neurospora Crassa Locus Encoding S-Adenosylmethionine Synthetase: Molecular Cloning, Sequence Analysis and in Vivo Overexpression

Intense biochemical and genetic research on the eth-1(r) mutant of Neurospora crassa suggested that this locus might encode S-adenosylmethionine synthetase (S-Adomet synthetase). We have used protoplast transformation and phenotypic rescue of a thermosensitive phenotype associated with the eth-1(r) mutation to clone the locus. Nucleotide sequence analysis demonstrated that it encodes S-Adomet synthetase. Homology analyses of prokaryotic, fungal and higher eukaryotic S-Adomet synthetase polypeptide sequences show a remarkable evolutionary conservation of the enzyme. N. crassa strains carrying S-Adomet synthetase coding sequences fused to a strong heterologous promoter were constructed to assess the phenotypic consequences of in vivo S-Adomet synthetase overexpression. Studies of growth rates and microscopic examination of vegetative development revealed that normal growth and morphogenesis take place in N. crassa even at abnormally high levels of cellular S-Adomet. The degree of cytosine methylation of a naturally methylated genomic region was dependent on the cellular levels of S-Adomet. We conclude that variation in S-Adomet levels in N. crassa cells, which in addition to the status of genomic DNA methylation could modify the flux of other S-Adomet-dependent metabolic pathways, does not affect growth rate or morphogenesis.     

Genome quality control: RIP (repeat-induced point mutation) comes to Podospora

RIP (repeat-induced point mutation) is a silencing process discovered in Neurospora crassa and so far clearly established only in this species as a currently occurring process. RIP acts premeiotically on duplicated sequences, resulting in C-G to T-A mutations, with a striking preference for CpA/TpG dinucleotides. In Podospora anserina, an RIP-like event was observed after several rounds of sexual reproduction in a strain with a 40 kb tandem duplication resulting from homologous integration of a cosmid in the mating-type region. The 9 kb sequenced show 106 C-G to T-A transitions, with 80% of the replaced cytosines located in CpA dinucleotides. This led to the alteration of at least six genes, two of which were unidentified. This RIP-like event extended to single-copy genes between the two members of the repeat. The overall data show that the silencing process is strikingly similar to a light form of RIP, unaccompanied by C-methylation. Interestingly, the N. crassa zeta-eta sequence, which acts as a potent de novo C-methylation RIP signal in this species, is weakly methylated when introduced into P. anserina. These results demonstrate that RIP, at least in light forms, can occur beyond N. crassa.     

Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing

Proper DNA methylation patterning requires the complementary processes of de novo methylation (the initial methylation of unmethylated DNA sequences) and maintenance methylation (the faithful replication of preexisting methylation). Arabidopsis has two types of methyltransferases with demonstrated maintenance activity: MET1, which maintains CpG methylation and is homologous to mammalian DNMT1, and CHROMOMETHYLASE 3 (CMT3), which maintains CpNpG (N = A, T, C, or G) methylation and is unique to the plant kingdom. Here we describe loss-of-function mutations in the Arabidopsis DOMAINS REARRANGED METHYLASE (DRM) genes and provide evidence that they encode de novo methyltransferases. drm1 drm2 double mutants retained preexisting CpG methylation at the endogenous FWA locus but blocked de novo CpG methylation that is normally associated with FWA transgene silencing. Furthermore, drm1 drm2 double mutants blocked de novo CpNpG and asymmetric methylation and gene silencing of the endogenous SUPERMAN (SUP) gene, which is normally triggered by an inverted SUP repeat. However, drm1 drm2 double mutants did not show reactivation of previously established SUPERMAN epigenetic silenced alleles. Thus, drm mutants prevent the establishment but not the maintenance of gene silencing at FWA and SUP, suggesting that the DRMs encode the major de novo methylation enzymes affecting these genes.     

Novel pattern of DNA methylation in Neurospora crassa transgenic for the foreign gene hph.

It has previously been reported that multiple copies of the hph gene integrated into the genome of Neurospora crassa are methylated at Hpa II sites (CCGG) during the vegetative life cycle of the fungus, while hph genes integrated as single copies are not methylated. Furthermore, methylation is correlated with silencing of the gene. We report here the methylation state of cytosine residues of the major part of the promoter region of the hph gene integrated into the genome of the multiple copy strain HTA5.7 during the vegetative stage of the life cycle. Cytosine methylation is sequence dependent, but the sequence specificity is complex and is different from the sequence specificity known for mammals and plants (CpG and CpNpG). The pattern of DNA methylation reported here is very different from that measured after meiosis in Neurospora or in Ascobulus . After the sexual cycle in those two fungi all the cytosines of multiple stretches of DNA are heavily methylated. This indicates that the still unknown methyltransferase in Neurospora has a different specificity in the sexual and the vegetative stages of the life cycle or that there are different methyltransferases. The pattern of methylation reported here is also different from the pattern of cytosine methylation of transgenes of Petunia , the only pattern published until now in plants that has DNA methylation at cytosines which are not in the canonical sequences CpG and CpNpG.