Amplification of HMG-CoA reductase production enhances carotenoid accumulation in Neurospora crassa

Neurospora crassa, a filamentous fungus, naturally produces the carotenoids lycopene and neurosporaxanthin. To increase the carbon flux through the carotenoid biosynthetic pathway, the 1658-bp region of the HMG1 gene encoding the catalytic domain (cHMG1) of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Saccharomyces cerevisiae was expressed in N. crassa under control of the strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and the inducible alcohol dehydrogenase (alcA) promoter. Overexpressing cHMG1 under control of the GPD promoter increased lycopene and neurosporaxanthin production 6- and 1.5-fold, respectively, relative to the wild-type strain. Over-expression of cHMG1 under control of the alcA promoter increased production of lycopene and neurosporaxanthin 3- and 2-fold, respectively.     

Effect of the cyclic nucleotide level on the degree of carotenoid pigment formation in the mycelial cells of Neurospora crassa

The relation between the content of cyclic nucleotides and the rate of formation of carotenoid pigments in the Neurospora crassa mycelium cells was investigated. Light derepression of the carotenoid synthesis during the photoinduction lag-period induced a transient decrease of the cAMP content. The intracellular cAMP content was in negative correlation with the constitutive level of carotenoid pigments. The cGMP content remained unchanged during the photoinduction lag-period and showed no correlation with the constitutive level of carotenoids in N. crassa cells.     

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.     

Effect of 5-azacytidine on the light-sensitive formation of sexual and asexual reproductive structures in wc-1 and wc-2 mutants of Neurospora crassa

Under the conditions of nitrogen starvation, illumination by blue light of wc-1 and wc-2 mutants of the ascomycete Neurospora crassa failed to stimulate the formation of protoperithecia and inhibit conidiation (contrary to what was observed in the mycelium of the wild-type fungus). The data obtained indicate that wc-1 and wc-2 genes of N. crassa are involved in light-dependent formation of protoperithecia and conidia. The effects of 5-azacytidine (an inhibitor of DNA methylation) under the same experimental conditions suggest that the balance between the formation of sexual and asexual reproductive structures, maintained in N. crassa, depends on genome methylation processes sensitive to the action of light, which is mediated by the photoreceptor complex of WC proteins.     

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.     

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.     

Signal for DNA methylation associated with tandem duplication in Neurospora crassa.

Most cytosine residues are subject to methylation in the zeta-eta (zeta-eta) region of Neurospora crassa. The region consists of a tandem direct duplication of a 0.8-kilobase-pair element including a 5S rRNA gene. The repeated elements have diverged about 15% by the occurrence of numerous CG to TA mutations, which probably resulted from deamination of methylated cytosines. Most but not all common laboratory strains of N. crassa have methylated duplicated DNA at the zeta-eta locus. However, many strains of N. crassa and strains of N. tetrasperma, N. sitophila, and N. intermedia have one instead of two copies of the homologous DNA and it is not methylated. A cross of strains differing at the zeta-eta locus produced progeny which all had duplicated, methylated, or unique, unmethylated DNA, like the parental strains. We conclude that a signal causing unprecedented heavy DNA methylation is present in the zeta-eta region.     

Mutations in the dim-1 gene of Neurospora crassa reduce the level of DNA methylation

Mutants that show reduced DNA methylation were identified in a mutant screen based on the assumptions that (i) the nucleoside analog 5-azacytidine (5-azaC) promotes the formation of potentially lethal DNA-methyltransferase adducts; (ii) reduction in DNA methyltransferase will decrease the sensitivity of cells to 5-azaC; and (iii) this potential selective advantage will be enhanced in mutants that are deficient in the repair of 5-azaC-induced DNA damage. Of fifteen potential repair mutants screened for sensitivity to 5-azaC, five (mus-9, mus-10, mus-11, mus-18, and uvs-3) showed moderately increased sensitivity and two (mus-20, mei-3) showed highly increased sensitivity. A mus-20 mutation was used to isolate three non-complementing methylation mutants. The mutations, named dim-1 (defective in methylation), reduced female fertility, reduced methylation by 40–50%, and altered patterns of methylation. In wild-type strains hypomethylation perse fails to alter methylation specificity. We demonstrate a growth-phase-dependent change in methylation patterns, detectable only in hypomethylated DNA from dim ⁺ cultures. This may represent a growth-phase-dependent change in the relative amounts of distinct species of methyltransferase, one of which may be encoded by the dim-1 gene.     

Oxygen and hydrogen peroxide enhance light-induced carotenoid synthesis in Neurospora crassa

Previously, we found that intracellular reactive oxygen species (ROS) affect photomorphogenesis in Neurospora crassa. In this study, we investigated the physiological roles of ROS in the response to light and found that the exposure of mycelia to air was important for the light-induced carotenogenesis. Mycelia treated with a high concentration of O(2) gas and H(2)O(2) to release ROS showed an enhancement of light-induced carotenoid accumulation and the expression of gene related to light-inducible carotenogenesis. These results suggested that stimuli caused by the exposure of the mycelia to air containing O(2) gas triggered the light-induced carotenoid synthesis.     

Mutations affecting the biosynthesis of S-adenosylmethionine cause reduction of DNA methylation in Neurospora crassa.

A temperature-sensitive methionine auxotroph of Neurospora crassa was found in a collection of conditional mutants and shown to be deficient in DNA methylation when grown under semipermissive conditions. The defective gene was identified as met-3, which encodes cystathionine-gamma-synthase. We explored the possibility that the methylation defect results from deficiency of S-adenosylmethionine (SAM), the presumptive methyl group donor. Methionine starvation of mutants from each of nine complementation groups in the methionine (met) pathway (met-1, met-2, met-3, met-5, met-6, met-8, met-9, met-10 and for) resulted in decreased DNA methylation while amino acid starvation, per se, did not. In most of the strains, including wild-type, intracellular SAM peaked during rapid growth (12-18 h after inoculation), whereas DNA methylation continued to increase. In met mutants starved for methionine, SAM levels were most reduced (3-11-fold) during rapid growth while the greatest reduction in DNA methylation levels occurred later. Addition of 3 mM methionine to cultures of met or cysteine-requiring (cys) mutants resulted in 5-28-fold increases in SAM, compared with wild-type, at a time when DNA methylation was reduced approximately 40%, suggesting that the decreased methylation during rapid growth in Neurospora is not due to limiting SAM. DNA methylation continued to increase in a cys-3 mutant that had stopped growing due to methionine starvation, suggesting that methylation is not obligatorily coupled to DNA replication in Neurospora.     

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.     

Direct interaction between DNA methyltransferase DIM-2 and HP1 is required for DNA methylation in Neurospora crassa

DNA methylation is involved in gene silencing and genomic stability in mammals, plants, and fungi. Genetics studies of Neurospora crassa have revealed that a DNA methyltransferase (DIM-2), a histone H3K9 methyltransferase (DIM-5), and heterochromatin protein 1 (HP1) are required for DNA methylation. We explored the interrelationships of these components of the methylation machinery. A yeast two-hybrid screen revealed that HP1 interacts with DIM-2. We confirmed the interaction in vivo and demonstrated that it involves a pair of PXVXL-related motifs in the N-terminal region of DIM-2 and the chromo shadow domain of HP1. Both regions are essential for proper DNA methylation. We also determined that DIM-2 and HP1 form a stable complex independently of the trimethylation of histone H3K9, although the association of DIM-2 with its substrate sequences depends on trimethyl-H3K9. The DIM-2/HP1 complex does not include DIM-5. We conclude that DNA methylation in Neurospora is largely or exclusively the result of a unidirectional pathway in which DIM-5 methylates histone H3K9 and then the DIM-2/HP1 complex recognizes the resulting trimethyl-H3K9 mark via the chromo domain of HP1.