A component of the TOR (Target Of Rapamycin) nutrient-sensing pathway plays a role in circadian rhythmicity in Neurospora crassa.

The TOR (Target of Rapamycin) pathway is a highly-conserved signaling pathway in eukaryotes that regulates cellular growth and stress responses. The cellular response to amino acids or carbon sources such as glucose requires anchoring of the TOR kinase complex to the lysosomal/vacuolar membrane by the Ragulator (mammals) or EGO (yeast) protein complex. Here we report a connection between the TOR pathway and circadian (daily) rhythmicity. The molecular mechanism of circadian rhythmicity in all eukaryotes has long been thought to be transcription/translation feedback loops (TTFLs). In the model eukaryote Neurospora crassa, a TTFL including FRQ (frequency) and WCC (white collar complex) has been intensively studied. However, it is also well-known that rhythmicity can be seen in the absence of TTFL functioning. We previously isolated uv90 as a mutation that compromises FRQ-less rhythms and also damps the circadian oscillator when FRQ is present. We have now mapped the uv90 gene and identified it as NCU05950, homologous to the TOR pathway proteins EGO1 (yeast) and LAMTOR1 (mammals), and we have named the N. crassa protein VTA (vacuolar TOR-associated protein). The protein is anchored to the outer vacuolar membrane and deletion of putative acylation sites destroys this localization as well as the protein's function in rhythmicity. A deletion of VTA is compromised in its growth responses to amino acids and glucose. We conclude that a key protein in the complex that anchors TOR to the vacuole plays a role in maintaining circadian (daily) rhythmicity. Our results establish a connection between the TOR pathway and circadian rhythms and point towards a network integrating metabolism and the circadian system.

Neurospora importin α is required for normal heterochromatic formation and DNA methylation.

Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6(dim-3) allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport.

The cullin-4 complex DCDC does not require E3 ubiquitin ligase elements to control heterochromatin in Neurospora crassa.

The cullin-4 (CUL4) complex DCDC (DIM-5/-7/-9/CUL4/DDB1 complex) is essential for DNA methylation and heterochromatin formation in Neurospora crassa. Cullins form the scaffold of cullin-RING E3 ubiquitin ligases (CRLs) and are modified by the covalent attachment of NEDD8, a ubiquitin-like protein that regulates the stability and activity of CRLs. We report that neddylation is not required for CUL4-dependent DNA methylation or heterochromatin formation but is required for the DNA repair functions. Moreover, the RING domain protein RBX1 and a segment of the CUL4 C terminus that normally interacts with RBX1, the E2 ligase, CAND1, and CSN are dispensable for DNA methylation and heterochromatin formation by DCDC. Our study provides evidence for the noncanonical functions of core CRL components.

Substitutions in the amino-terminal tail of neurospora histone H3 have varied effects on DNA methylation.

Eukaryotic genomes are partitioned into active and inactive domains called euchromatin and heterochromatin, respectively. In Neurospora crassa, heterochromatin formation requires methylation of histone H3 at lysine 9 (H3K9) by the SET domain protein DIM-5. Heterochromatin protein 1 (HP1) reads this mark and directly recruits the DNA methyltransferase, DIM-2. An ectopic H3 gene carrying a substitution at K9 (hH3(K9L) or hH3(K9R)) causes global loss of DNA methylation in the presence of wild-type hH3 (hH3(WT)). We investigated whether other residues in the N-terminal tail of H3 are important for methylation of DNA and of H3K9. Mutations in the N-terminal tail of H3 were generated and tested for effects in vitro and in vivo, in the presence or absence of the wild-type allele. Substitutions at K4, K9, T11, G12, G13, K14, K27, S28, and K36 were lethal in the absence of a wild-type allele. In contrast, mutants bearing substitutions of R2, A7, R8, S10, A15, P16, R17, K18, and K23 were viable. The effect of substitutions on DNA methylation were variable; some were recessive and others caused a semi-dominant loss of DNA methylation. Substitutions of R2, A7, R8, S10, T11, G12, G13, K14, and P16 caused partial or complete loss of DNA methylation in vivo. Only residues R8-G12 were required for DIM-5 activity in vitro. DIM-5 activity was inhibited by dimethylation of H3K4 and by phosphorylation of H3S10, but not by acetylation of H3K14. We conclude that the H3 tail acts as an integrating platform for signals that influence DNA methylation, in part through methylation of H3K9.

Identification of DIM-7, a protein required to target the DIM-5 H3 methyltransferase to chromatin.

Functionally distinct chromatin domains are delineated by distinct posttranslational modifications of histones, and in some organisms by differences in DNA methylation. Proper establishment and maintenance of chromatin domains is critical but not well understood. We previously demonstrated that heterochromatin in the filamentous fungus Neurospora crassa is marked by cytosine methylation directed by trimethylated Lysine 9 on histone H3 (H3K9me3). H3K9me3 is the product of the DIM-5 Lysine methyltransferase and is recognized by a protein complex containing heterochromatin protein-1 and the DIM-2 DNA methyltransferase. To identify additional components that control the establishment and function of DNA methylation and heterochromatin, we built a strain harboring two selectable reporter genes that are silenced by DNA methylation and employed this strain to select for mutants that are defective in DNA methylation (dim). We report a previously unidentified gene (dim-7) that is essential for H3K9me3 and DNA methylation. DIM-7 homologs are found only in fungi and are highly divergent. We found that DIM-7 interacts with DIM-5 in vivo and demonstrated that a conserved domain near the N terminus of DIM-7 is required for its stability. In addition, we found that DIM-7 is essential for recruitment of DIM-5 to form heterochromatin.

DNA methylation and normal chromosome behavior in Neurospora depend on five components of a histone methyltransferase complex, DCDC.

Methylation of DNA and of Lysine 9 on histone H3 (H3K9) is associated with gene silencing in many animals, plants, and fungi. In Neurospora crassa, methylation of H3K9 by DIM-5 directs cytosine methylation by recruiting a complex containing Heterochromatin Protein-1 (HP1) and the DIM-2 DNA methyltransferase. We report genetic, proteomic, and biochemical investigations into how DIM-5 is controlled. These studies revealed DCDC, a previously unknown protein complex including DIM-5, DIM-7, DIM-9, CUL4, and DDB1. Components of DCDC are required for H3K9me3, proper chromosome segregation, and DNA methylation. DCDC-defective strains, but not HP1-defective strains, are hypersensitive to MMS, revealing an HP1-independent function of H3K9 methylation. In addition to DDB1, DIM-7, and the WD40 domain protein DIM-9, other presumptive DCAFs (DDB1/CUL4 associated factors) co-purified with CUL4, suggesting that CUL4/DDB1 forms multiple complexes with distinct functions. This conclusion was supported by results of drug sensitivity tests. CUL4, DDB1, and DIM-9 are not required for localization of DIM-5 to incipient heterochromatin domains, indicating that recruitment of DIM-5 to chromatin is not sufficient to direct H3K9me3. DIM-7 is required for DIM-5 localization and mediates interaction of DIM-5 with DDB1/CUL4 through DIM-9. These data support a two-step mechanism for H3K9 methylation in Neurospora.

Mapping of lysine methylation and acetylation in core histones of Neurospora crassa.

Core histones are susceptible to a variety of post-translational modifications (PTMs), among which methylation and acetylation play critical roles in various chromatin-dependent processes. The nature and biological functions of these PTMs have been extensively studied in plants, animals, and yeasts. In contrast, the histone modifications in Neurospora crassa, a convenient model organism for multicellular eukaryotes, remained largely undefined. In this study, we used several mass spectrometric techniques, coupled with HPLC separation and multiple-protease digestion, to identify the methylation and acetylation sites in core histones isolated from Neurospora. Electron transfer dissociation (ETD) was employed to fragment the heavily modified long N-terminal peptides. In addition, accurate mass measurement of fragment ions allowed for unambiguous differentiation of acetylation from trimethylation. Many modification sites conserved in other organisms were identified in Neurospora. In addition, some unique modification sites in histone H2B, including N-terminal alpha methylation, methylation at K3, and acetylation at K19, K28, and K29, were observed. Our analysis provides a potentially comprehensive picture of methylation and acetylation of core histones in Neurospora, which should serve as a foundation for future studies of the function of histone PTMs in this model organism.

Nucleosome Positioning by an Evolutionarily Conserved Chromatin Remodeler Prevents Aberrant DNA Methylation in Neurospora.

In the filamentous fungus Neurospora crassa, constitutive heterochromatin is marked by tri-methylation of histone H3 lysine 9 (H3K9me3) and DNA methylation. We identified mutations in the Neurospora defective in methylation-1 (dim-1) gene that cause defects in cytosine methylation and implicate a putative AAA-ATPase chromatin remodeler. Although it was well-established that chromatin remodelers can affect transcription by influencing DNA accessibility with nucleosomes, little was known about the role of remodelers on chromatin that is normally not transcribed, including regions of constitutive heterochromatin. We found that dim-1 mutants display both reduced DNA methylation in heterochromatic regions as well as increased DNA methylation and H3K9me3 in some intergenic regions associated with highly expressed genes. Deletion of dim-1 leads to atypically spaced nucleosomes throughout the genome and numerous changes in gene expression. DIM-1 localizes to both heterochromatin and intergenic regions that become hyper-methylated in dim-1 strains. Our findings indicate that DIM-1 normally positions nucleosomes in both heterochromatin and euchromatin and that the standard arrangement and density of nucleosomes is required for the proper function of heterochromatin machinery.

ASH1-catalyzed H3K36 methylation drives gene repression and marks H3K27me2/3-competent chromatin.

Methylation of histone H3 at lysine 36 (H3K36me), a widely-distributed chromatin mark, largely results from association of the lysine methyltransferase (KMT) SET-2 with RNA polymerase II (RNAPII), but most eukaryotes also have additional H3K36me KMTs that act independently of RNAPII. These include the orthologs of ASH1, which are conserved in animals, plants, and fungi but whose function and control are poorly understood. We found that Neurospora crassa has just two H3K36 KMTs, ASH1 and SET-2, and were able to explore the function and distribution of each enzyme independently. While H3K36me deposited by SET-2 marks active genes, inactive genes are modified by ASH1 and its activity is critical for their repression. ASH1-marked chromatin can be further modified by methylation of H3K27, and ASH1 catalytic activity modulates the accumulation of H3K27me2/3 both positively and negatively. These findings provide new insight into ASH1 function, H3K27me2/3 establishment, and repression in facultative heterochromatin.

PRD-1, a Component of the Circadian System of Neurospora crassa, Is a Member of the DEAD-box RNA Helicase Family.

The circadian rhythms found in almost all organisms are driven by molecular oscillators, including transcription/translation feedback loops (TTFLs). However, TTFL-independent oscillators can drive rhythms in both eukaryotes and prokaryotes. The fungus Neurospora crassa is a model organism for studying the molecular mechanism of the circadian clock. Although a circadian TTFL involving the proteins FRQ, WC-1, and WC-2 is well-characterized in N. crassa, rhythms can still be observed in the absence of this feedback loop. These rhythms are said to be driven by 1 or more FRQ-less oscillator(s) (FLOs). The prd-1 mutation lengthens the period in frq wild type and was previously shown to severely disrupt FRQ-less rhythms in frq null mutants under several different conditions; therefore, the prd-1 gene product is a candidate for a component of a FLO. We report here that prd-1 also disrupts free-running rhythms in wc-1 null mutants, confirming its effects on FRQ-less rhythms. We have now mapped and identified the prd-1 gene as NCU07839, a DEAD-box RNA helicase dbp-2 Complementation with the wild-type gene corrects the rhythm defects of the prd-1 mutant in the complete circadian system (when the FRQ-based TTFL is intact) and also the free-running FRQ-less rhythm on low choline. A PRD-1-GFP fusion protein localizes to the nucleus. The prd-1 mutant has a single base pair change in the first base of an intron that results in abnormally spliced transcripts. FRQ-less rhythms on low choline, or entrained to heat pulses, were only marginally affected in strains carrying deletions of 2 other RNA helicases (prd-6 and msp-8). We conclude that PRD-1 is a member of an RNA helicase family that may be specifically involved in regulating rhythmicity in N. crassa in both the complete circadian system and FLO(s).

Protein phosphatase PP1 is required for normal DNA methylation in Neurospora.

Covalent modifications of histones integrate intracellular and extracellular cues to regulate the genome. H3 Lys 9 methylation (H3K9me) can direct heterochromatin formation and DNA methylation, while phosphorylation of H3 Ser 10 (H3S10p) drives gene activation and chromosome condensation. To examine the relationship between H3S10p, H3K9me, and DNA methylation in Neurospora crassa, we built and tested mutants of the putative H3S10 phosphatase, PP1. A PP1-impaired mutant showed increased H3S10p and selective reduction of methylation of H3K9 and DNA. Similarly, amino acid substitutions of H3S10 abolished methylation of H3K9 and DNA. Thus, H3S10 dephosphorylation by PP1 is required for DNA methylation of some loci.

The fungus Neurospora crassa displays telomeric silencing mediated by multiple sirtuins and by methylation of histone H3 lysine 9.

BACKGROUND: Silencing of genes inserted near telomeres provides a model to investigate the function of heterochromatin. We initiated a study of telomeric silencing in Neurospora crassa, a fungus that sports DNA methylation, unlike most other organisms in which telomeric silencing has been characterized. RESULTS: The selectable marker, hph, was inserted at the subtelomere of Linkage Group VR in an nst-1 (neurospora sir two-1) mutant and was silenced when nst-1 function was restored. We show that NST-1 is an H4-specific histone deacetylase. A second marker, bar, tested at two other subtelomeres, was similarly sensitive to nst-1 function. Mutation of three additional SIR2 homologues, nst-2, nst-3 and nst-5, partially relieved silencing. Two genes showed stronger effects: dim-5, which encodes a histone H3 K9 methyltransferase and hpo, which encodes heterochromatin protein-1. Subtelomeres showed variable, but generally low, levels of DNA methylation. Elimination of DNA methylation caused partial derepression of one telomeric marker. Characterization of histone modifications at subtelomeric regions revealed H3 trimethyl-K9, H3 trimethyl-K27, and H4 trimethyl-K20 enrichment. These modifications were slightly reduced when telomeric silencing was compromised. In contrast, acetylation of histones H3 and H4 increased. CONCLUSION: We demonstrate the presence of telomeric silencing in Neurospora and show a dependence on histone deacetylases and methylation of histone H3 lysine 9. Our studies also reveal silencing functions for DIM-5 and HP1 that appear independent of their role in de novo DNA methylation.

Methylation of histone H3 lysine 36 is required for normal development in Neurospora crassa.

The SET domain is an evolutionarily conserved domain found predominantly in histone methyltransferases (HMTs). The Neurospora crassa genome includes nine SET domain genes (set-1 through set-9) in addition to dim-5, which encodes a histone H3 lysine 9 HMT required for DNA methylation. We demonstrate that Neurospora set-2 encodes a histone H3 lysine 36 (K36) methyltransferase and that it is essential for normal growth and development. We used repeat induced point mutation to make a set-2 mutant (set-2(RIP1)) with multiple nonsense mutations. Western analyses revealed that the mutant lacks SET-2 protein and K36 methylation. An amino-terminal fragment that includes the AWS, SET, and post-SET domains of SET-2 proved sufficient for K36 HMT activity in vitro. Nucleosomes were better substrates than free histones. The set-2(RIP1) mutant grows slowly, conidiates poorly, and is female sterile. Introducing the wild-type gene into the mutant complemented the defects, confirming that they resulted from loss of set-2 function. We replaced the wild-type histone H3 gene (hH3) with an allele producing a Lys to Leu substitution at position 36 and found that this hH3(K36L) mutant phenocopied the set-2(RIP1) mutant, confirming that the observed defects in growth and development result from inability to methylate K36 of H3. Finally, we used chromatin immunoprecipitation to demonstrate that actively transcribed genes in Neurospora crassa are enriched for H3 methylated at lysines 4 and 36. Taken together, our results suggest that methylation of K36 in Neurospora crassa is essential for normal growth and development.