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.     

Cloning and characterization of centromeric DNA from Neurospora crassa.

The centromere locus from linkage group VII of Neurospora crassa has been cloned, characterized, and physically mapped. The centromeric DNA is contained within a 450-kb region that is recombination deficient, A+T-rich, and contains repetitive sequences. Repetitive sequences from within this region hybridize to a family of repeats located at or near centromeres in all seven linkage groups of N. crassa. Genomic Southern blots and sequence analysis of these repeats revealed a unique centromere structure containing a divergent family of centromere-specific repeats. The predominantly transitional differences between copies of the centromere-specific sequence repeats and their high A+T content suggest that their divergence was mediated by repeat-induced point (RIP) mutations.     

DNA methylation associated with repeat-induced point mutation in Neurospora crassa.

Repeat-induced point mutation (RIP) is a process that efficiently detects DNA duplications prior to meiosis in Neurospora crassa and peppers them with G:C to A:T mutations. Cytosine methylation is typically associated with sequences affected by RIP, and methylated cytosines are not limited to CpG dinucleotides. We generated and characterized a collection of methylated and unmethylated amRIP alleles to investigate the connection(s) between DNA methylation and mutations by RIP. Alleles of am harboring 84 to 158 mutations in the 2.6-kb region that was duplicated were heavily methylated and triggered de novo methylation when reintroduced into vegetative N. crassa cells. Alleles containing 45 and 56 mutations were methylated in the strains originally isolated but did not become methylated when reintroduced into vegetative cells. This provides the first evidence for de novo methylation in the sexual cycle and for a maintenance methylation system in Neurospora cells. No methylation was detected in am alleles containing 8 and 21 mutations. All mutations in the eight primary alleles studied were either G to A or C to T, with respect to the coding strand of the am gene, suggesting that RIP results in only one type of mutation. We consider possibilities for how DNA methylation is triggered by some sequences altered by RIP.     

The A.T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure.

We have determined the domains of the mammalian high mobility group (HMG)I chromosomal proteins necessary and sufficient for binding to the narrow minor groove of stretches of A.T-rich DNA. Three highly conserved regions within each of the known HMG-I proteins is closely related to the consensus sequence T-P-K-R-P-R-G-R-P-K-K. A synthetic oligopeptide corresponding to this consensus "binding domain" (BD) sequence specifically binds to substrate DNA in a manner similar to the intact HMG-I proteins. Molecular Corey-Pauling-Koltun model building and computer simulations employing energy minimization programs to predict structure suggest that the consensus BD peptide has a secondary structure similar to the antitumor and antiviral drugs netropsin and distamycin and to the dye Hoechst 33258. In vitro these ligands, which also preferentially bind to A.T-rich DNA, have been demonstrated to effectively compete with both the BD peptide and the HMG-I proteins for DNA binding. The BD peptide also contains novel structural features such as a predicted Asx bend or "hook" at its amino-terminal end and laterally projecting cationic Arg/Lys side chains or "bristles" which may contribute to the binding properties of the HMG-I proteins. The predicted BD peptide structure, which we refer to as the "A.T-hook," represents a previously undescribed DNA-binding motif capable of binding to the minor groove of stretches of A.T base pairs.     

Increased temperature and 2-methyl-2,4-pentanediol change the DNA structure of both curved and uncurved adenine/thymine-rich sequences

DNA curvature is affected by elevated temperature and dehydrating agents such as 2-methyl-2,4-pentanediol (MPD) (used in crystallization). This effect of MPD has been ascribed to a specific distortion of the structure of adenine tracts (A-tracts), probably through a deformation of the characteristic narrow minor groove. Uranyl photoprobing indicates that a narrowed minor groove is present in all A/T regions containing four or more A/T base pairs. Consequently, this technique may be employed to study conformational changes in other A/T-rich sequences than pure A-tracts. In this study we use uranyl photoprobing to demonstrate that the effect of elevated temperature and MPD is analogous on both "normal" and curve-inducing A/T-rich sequences. The results therefore indicate that under these conditions the minor groove is widened in all A/T sequences and not only in pure A-tracts as previously suggested. Thus, the rather subtle structural difference of AT regions and A-tracts in nonbent DNA versus A-tracts in bent DNA may be quantitative rather than qualitative; i.e., the structure is more persistent and/or rigid in bent DNA.     

Phosphorylation by cdc2 kinase modulates DNA binding activity of high mobility group I nonhistone chromatin protein

Chromatin high mobility group protein I (HMG-I) is a mammalian nonhistone protein that has been demonstrated both in vitro and in vivo to preferentially bind to A.T-rich sequences of DNA. Recently the DNA-binding domain peptide that specifically mediates the in vitro interaction of high mobility group protein (HMG)-I with the narrow minor groove of A.T-DNA has been experimentally determined. Because of its predicted secondary structure, the binding domain peptide has been called "the A.T hook" motif. Previously we demonstrated that the A.T hook of murine HMG-I protein is specifically phosphorylated by purified mammalian cdc2 kinase in vitro and that the same site(s) are also phosphorylated in vivo in metaphase-arrested cells. We also found that the DNA binding affinity of short synthetic binding domain peptides phosphorylated in vitro by cdc2 kinase was significantly reduced compared with unphosphorylated peptides. Here we extend these findings to intact natural and recombinant HMG-I proteins. We report that the affinity of binding of full-length HMG-I proteins to A.T-rich sequences is highly dependent on ionic conditions and that phosphorylation of intact proteins by cdc2 kinase reduces their affinity of in vitro binding to A.T-DNA by about 20-fold when assayed near normal mammalian physiological salt concentrations. Furthermore, in cell synchronization studies, we demonstrated that murine HMG-I proteins are phosphorylated in vivo in a cell cycle-dependent manner on the same amino acid residues modified by purified cdc2 kinase in vitro. Together these results strongly support the assertion that HMG-I proteins are natural substrates for mammalian cdc2 kinase in vivo and that their cell cycle-dependent phosphorylation by this enzyme(s) significantly modulates their DNA binding affinity, thereby possibly altering their biological function(s).     

Effects of DNA sequence and histone-histone interactions on nucleosome placement

Using competitive reconstitution, we have refined the parameters for the binding of histone octamers to artificial nucleosome-positioning sequences of the form: (A/T3nn(G/C)3nn. We find that the optimal period between flexible segments is approximately 10.1 base-pairs, supporting the view that the DNA on the nucleosome surface is overwound. The strongest requirement for flexible DNA is near the protein dyad. However, we see no indication of changes in DNA helical repeat in this region. Using a series of repetitive sequences, we confirm that neither all A/T-rich nor all G/C-rich regions are identical in promoting nucleosome formation. Surprisingly, A/T-rich segments containing the TpA step, subject to purine-purine clash in the minor groove, favor nucleosome formation over sequences lacking this step. Short tracts of adenine residues are found to position on the histone surface like other A/T-rich regions, in the manner predicted by the direction of their sequence-directed bends as determined by electrophoretic methods. Tracts containing five adenine residues are extremely aniostropic in their flexibility and are strongly detrimental to nucleosome formation when positioned for major groove compression. Longer adenine tracts are found to position near the ends of the nucleosomal DNA. However, other positions may be occupied by an A12 tract, with only a minor penalty in the free energy of nucleosome formation. Overall, reconstituted nucleosome positions are translationally degenerate, suggesting a weak dependence on DNA flexibility for nucleosome positioning. Dinucleosomal reconstitutions on tandem dimers of the 5 S RNA gene of Lytechinus variegatus demonstrate a weak phasing dependence for the interaction between nucleosomes. This interaction is maximal for the 202 base-pair repeat and suggests a co-operative mechanism for the formation of ordered nucleosomal arrays based on a combination of DNA flexibility and nucleosome-nucleosome interactions.     

High mobility group proteins HMG-1 and HMG-I/Y bind to a positive regulatory region of the pea plastocyanin gene promoter

A 268 bp region (P268) of the pea plastocyanin gene promoter responsible for high-level expression has been shown to interact with the high mobility group proteins HMG-1 and HMG-I/Y isolated from pea shoot chromatin. cDNAs encoding an HMG-1 protein of 154 amino acid residues containing a single HMG-box and a C-terminal acidic tail and an HMG-I/Y-like protein of 197 amino acid residues containing four AT-hooks have been isolated and expressed in Escherichia coli to provide large amounts of full-length proteins. DNase I footprinting identified eight binding sites for HMG-I/Y and six binding sites for HMG-1 in P268. Inhibition of binding by the antibiotic distamycin, which binds in the minor groove of A/T-rich DNA, revealed that HMG-I/Y binding was 400-fold more sensitive than HMG-1 binding. Binding-site selection from a pool of random oligonucleotides indicated that HMG-I/Y binds to oligonucleotides containing stretches of five or more A/T bp and HMG-1 binds preferentially to oligonucleotides enriched in dinucleotides such as TpT and TpG.     

Crystal structure of a complex of DNA with one AT-hook of HMGA1

We present here for the first time the crystal structure of an AT-hook domain. We show the structure of an AT-hook of the ubiquitous nuclear protein HMGA1, combined with the oligonucleotide d(CGAATTAATTCG)(2), which has two potential AATT interacting groups. Interaction with only one of them is found. The structure presents analogies and significant differences with previous NMR studies: the AT-hook forms hydrogen bonds between main-chain NH groups and thymines in the minor groove, DNA is bent and the minor groove is widened.     

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:     

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.     

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.     

HMG-1 enhances HMG-I/Y binding to an A/T-rich enhancer element from the pea plastocyanin gene.

High-mobility-group proteins HMG-1 and HMG-I/Y bind at overlapping sites within the A/T-rich enhancer element of the pea plastocyanin gene. Competition binding experiments revealed that HMG-1 enhanced the binding of HMG-I/Y to a 31-bp region (P31) of the enhancer. Circularization assays showed that HMG-1, but not HMG-I/Y, was able to bend a linear 100-bp DNA containing P31 so that the ends could be ligated. HMG-1, but not HMG-I/Y, showed preferential binding to the circular 100-bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG-1 was not responsible for enhanced binding of HMG-I/Y. Direct interaction of HMG-I/Y and HMG-1 in the absence of DNA was demonstrated by binding of 35S-labeled proteins to immobilized histidine-tagged proteins, and this was due to an interaction of the N-terminal HMG-box-containing region of HMG-1 and the C-terminal AT-hook region of HMG-I/Y. Kinetic analysis using the IAsys biosensor revealed that HMG-1 had an affinity for immobilized HMG-I/Y (Kd = 28 nM) similar to that for immobilized P31 DNA. HMG-1-enhanced binding of HMG-I/Y to the enhancer element appears to be mediated by the formation of an HMG-1-HMG-I/Y complex, which binds to DNA with the rapid loss of HMG-1.     

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.     

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.     

Binding and catalytic contributions to site recognition by flp recombinase

Flp catalyzes site-specific recombination in a highly sequence-specific manner despite making few direct contacts to the bases within its binding site. Sequence discrimination could take place in the binding and/or the catalytic steps. In this study, we independently measure the binding affinity and initial cleavage rate of Flp recombinase with approximately 20 designed alternate target DNA sequences. Our results show that Flp specificity is largely, although not entirely, imparted at the binding step and is the result of a combination of direct and indirect readout. The Flp binding site includes an A/T-rich region that displays a characteristically narrow minor groove. We find that many A --> T changes are tolerated at the binding step, whereas C or G substitutions tend to decrease binding affinity. The effects of the latter can be alleviated by replacing guanine with inosine, which removes the N2 amino group that protrudes into the minor groove. Some A --> T changes reduce binding affinity, due to clashing with nearby residues, reinforcing that specificity requires avoiding negative contacts as well as creating positive ones. A tracts, which can lead to unusually rigid DNA structure, are tolerated during the binding step when placed within the region where the minor groove is already narrow. However, most A tracts slow catalysis more than C or G substitutions. Understanding what kind of sequence variation is tolerated in the binding and catalytic steps helps us understand how the target DNA is recognized by Flp and will be useful in guiding the design of Flp variants with altered specificities.     

Enhancement and alteration of bleomycin-catalyzed site-specific DNA cleavage by distamycin A and some minor groove binders.

The effects of compounds which bind in the DNA minor groove of A.T rich sequences, on bleomycin-catalyzed site-specific DNA cleavage were investigated by a DNA sequencing technique. Distamycin A enhanced bleomycin-catalyzed DNA cleavage in G.C rich sequences such as 5'-GGGGC-3' (under scoring; the cleaved nucleotide). The cleavage in such a sequence in the presence of distamycin A was greater than that in the absence of distamycin A by as much as about 100 times. Neither Hoechst 33258, 4',6-diamidino-2-phenylindole (DAPI) nor berenil caused extensive enhancement. The results suggest that the distamycin-induced conformational changes of DNA through interactions other than the DNA minor groove binding in A.T-rich sequences are specifically suitable for the bleomycin action.     

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.     

Molecular dynamics simulations of A. T-rich oligomers: sequence-specific binding of Na+ in the minor groove of B-DNA

Molecular dynamics simulations have been employed to probe the sequence-specific binding of sodium ions to the minor groove of B-DNA of three A. T-rich oligomers having identical compositions but different orders of the base pairs: C(AT)(4)G, CA(4)T(4)G, and CT(4)A(4)G. Recent experimental investigations, either in crystals or in solution, have shown that monovalent cations bind to DNA in a sequence-specific mode, preferentially in the narrow minor groove regions of uninterrupted sequences of four or more adenines (A-tracts), replacing a water molecule of the ordered hydration structure, the hydration spine. Following this evidence, it has been hypothesized that in A-tracts these events may be responsible for structural peculiarities such as a narrow minor groove and a curvature of the helix axis. The present simulations confirm a sequence specificity of the binding of sodium ions: Na(+) intrusions in the first layer of hydration of the minor groove, with long residence times, up to approximately 3 ns, are observed only in the minor groove of A-tracts but not in the alternating sequence. The effects of these intrusions on the structure of DNA depend on the ion coordination: when the ion replaces a water molecule of the spine, the minor groove becomes narrower. Ion intrusions may also disrupt the hydration spine modifying the oligomer structure to a large extent. However, in no case intrusions were observed to locally bend the axis toward the minor groove. The simulations also show that ions may reside for long time periods in the second layer of hydration, particularly in the wider regions of the groove, often leading to an opening of the groove.