Neurospora crassa, a model system for epigenetics research

Abstract

The filamentous fungus Neurospora crassa has provided a rich source of knowledge on epigenetic phenomena that would have been difficult or impossible to gain from other systems. Neurospora sports features found in higher eukaryotes but absent in both budding and fission yeast, including DNA methylation and H3K27 methylation, and also has distinct RNA interference (RNAi)-based silencing mechanisms operating in mitotic and meiotic cells. This has provided an unexpected wealth of information on gene silencing systems. One silencing mechanism, named repeat-induced point mutation (RIP), has both epigenetic and genetic aspects and provided the first example of a homology-based genome defense system. A second silencing mechanism, named quelling, is an RNAi-based mechanism that results in silencing of transgenes and their native homologs. A third, named meiotic silencing, is also RNAi-based but is distinct from quelling in its time of action, targets, and apparent purpose.

Figure 1.

Images of Neurospora crassa. (A) Vegetative growth in the wild on sugarcane (photo by D. Jacobson, Stanford University). (B) Slants of vegetative cultures of N. crassa in the laboratory (photo by N.B. Raju, Stanford University). (C) Hyphae of N. crassa stained with 4′6-diamidino-2-phenylindole (DAPI) to show abundant nuclei (photo by M. Springer, Stanford University). (D) A rosette of maturing asci showing ascospores patterns (photo by N.B. Raju; reprinted, with permission, from Raju 1980, © Elsevier).

Figure 2.

Life cycle of N. crassa. Half of the sexual spores (ascospores) are mating type A (red) and half are mating type a (blue). Sexual spores (ascospores) and vegetative spores (conidia) germinate and form mycelia, from which asexual fruiting bodies (conidiophores) emerge. Conidiophores form conidia, which are typically multinucleate. In response to nitrogen starvation, mycelia of either mating type form specialized female structures called protoperithecia. Vegetative tissue (e.g., a conidium) of the opposite mating type serves as the “male” to fertilize and initiate development of fruiting bodies (perithecia). After fertilization, male- and female-derived nuclei coexist in the same cytoplasm, where they undergo mitoses and eventually become organized into a dikaryotic tissue in which each cell has one nucleus of each mating type. The nuclei then pair and undergo a series of synchronous mitoses until the tip of the hyphal cell in which they reside bends to form a hook-shaped cell called a crozier. Fusion of haploid nuclei is immediately followed by meiosis and a mitotic division such that one crozier gives rise to one ascus containing eight ascospores. The approximate stages in which the epigenetic processes described in the text occur are indicated.

Figure 3.

Repeat-induced point mutation (RIP). For clarity, only two chromosomes are illustrated. The open box represents a gene or chromosomal segment that was duplicated in one strain (top, right). Duplications are subject to RIP (symbolized by lightning bolt) between fertilization and karyogamy. Results of genetic experiments revealed that duplications can be repeatedly subjected to volleys of C to T transitions (symbolized by filled boxes) during this period of approximately 10 mitoses, right up to the final premeiotic DNA synthesis (Selker et al. 1987; Watters et al. 1999). The four possible combinations of chromosomes in progeny are indicated. Pink “Me” represents DNA methylation, which is frequently (although not always) associated with products of RIP.

Figure 4.

Mutations from RIP and methylation status of eight different am alleles (adapted from Singer et al. 1995). Vertical bars indicate mutations. Alleles shown in black were not methylated. Alleles in blue were initially methylated, but after loss of methylation induced by 5-azacytidine, or by cloning and gene replacement, did not become remethylated. Alleles shown in red were not only initially methylated, but also triggered methylation de novo.

Figure 5.

Basic components of the DNA methylation machine of Neurospora. Chromatin associated with DNA substantially mutated by RIP (orange spiral decorated with pink mC moieties) is subjected to methylation of H3K9 by the histone methyltransferase DIM-5, whose localization and action depends on a multiprotein complex, DCDC (DIM-5/-7/-9, CUL4/DDB1 complex; Lewis et al. 2010a,b). The CUL4 subunit of the DCDC complex is associated with the small protein Nedd (N), which resembles the E3 ubiquitin ligase complex. Trimethylated H3K9 (K9me3) is recognized by HP1, which is involved in at least three heterochromatin-associated complexes: (1) It recruits the DNA methyltransferase DIM-2 (Honda and Selker 2008); (2) it is required for localization and function of the HCHC silencing complex, which contains HP1, the chromodomain protein CDP-2, the histone deacetylase HDA-1, and a CDP-2/HDA-1-associated protein, CHAP (Honda et al. 2012); and (3) it is required to guide the DMM complex, which serves to block the spreading of heterochromatin into neighboring transcribed regions (Honda et al. 2010).

Figure 6.

Epigenomic features of N. crassa genome. The genomic distributions of H3K9me3 (orange), HP1 (yellow), 5-methylcytosine (green), H3K27me3 (medium blue), and H3K4me3 (dark blue) are displayed for each of N. crassa’s seven linkage groups (OR74A NC10 sequence assembly, http://www.broadinstitute.org/annotation/genome/neurospora/MultiHome.html) using the Integrative Genomics Viewer (http://www.broadinstitute.org/igv) (Jamieson et al. 2013; MR Rountree and EU Selker, unpubl.). Base composition is shown at the top of each linkage group as the moving average of %GC (red) calculated for 500-bp windows in 100-bp steps, whereas the positions of predicted genes (purple) and repeats (black) are indicated below. The predicted gene file was downloaded from The Broad Institute (http://www.broadinstitute.org/annotation/genome/neurospora) and repeats were determined using the RepeatMasker program (http://www.repeatmasker.org).

Figure 7.

Quelling. For simplicity, only two of the seven chromosomes are diagrammed (straight line segments in gray circles representing nuclei). The native albino gene (al) is indicated by the dark orange rectangle on the top chromosome; rectangles on the lower chromosome (dark orange or yellow) represent ectopic al sequences introduced by transformation. Because transformed cells are often multinucleate, transformants are often heterokaryotic, as illustrated. Whether or not the transforming DNA includes the entire coding region, in some transformants it silences (“quells”) the native al⁺ gene in both transformed and nontransformed nuclei through an undefined trans-acting molecule (red lines emanating from the transforming DNA indicated by the yellow rectangle). This results in poorly pigmented or albino (Al^–) tissue in some transformants, as shown.

Figure 8.

Discovery and characterization of meiotic silencing. Key genetic experiments are illustrated using the Ascospore maturation-1 (Asm-1) gene, as a reporter. For each cross, the relevant genotype of the haploid parents of mating type A (red boxes) or mating type a (blue boxes) is shown on the left, and cartoons showing the predicted chromosome pairing in the diploid cell (violet boxes) is shown on the right. The phenotypes of resulting asci are presented on the far right. Black represents mature (typically viable) ascospores and white represents immature (inviable) ascospores. (A) Wild-type cross. (B) 4:4 segregation of ascospores from a heterozygous cross of wild type and a frameshift mutant in which alleles can pair and no meiotic silencing occurs. (C) Cross of strains with wild-type and deletion alleles triggers meiotic silencing. (D) Meiotic silencing is not rescued by ectopic wild-type allele, indicating that the developmental defect is not due to haploinsufficiency. (E) Allelic (pairable) ectopic copies asm-1⁺ in crossing partners rescue Asm-1⁻ defect. (F) Presence of an unpaired allele triggers silencing of all asm-1⁺ alleles (paired and unpaired) in meiosis. (G) Silencing of the suppressor of ascospore dominance (Sad-1), because of a Sad-1 deletion in one parent, suppresses meiotic silencing.

Figure 9.

A model for meiotic silencing. An image of a developing ascus from a cross between parents engineered to contain paired copies of sad-1⁺ fused to a reporter gene gfp⁺(i.e., sad-1⁺::gfp⁺) at the Pachytene stage of meiosis I (left; DW Lee and R Aramayo, unpubl.). Inside this cell, the meiotic nucleus, delineated by its nuclear membrane, is surrounded by a perinuclear structure that supports the attachment of components of the meiotic silencing apparatus. Predicted nuclear and perinuclear steps in meiotic silencing are diagrammed (right). It is hypothesized that trans-sensing, a mechanism preceding silencing, identifies heterologous regions of interacting chromosomes. The degree of heterology determines the strength of the induction step, which presumably involves the synthesis of aRNA and its conversion to double-stranded RNA (dsRNA) by the SAD-1 RdRP, a perinuclear event. The presence of dsRNA triggers the initiation of the silencing process, which involves the conversion of the dsRNA trigger into siRNAs via the DCL-1/SMS-3 Dicer (initiation step), and use of these siRNAs primers and normal RNAs as templates, by SAD-1 RdRP to generate dsRNA (amplification cycle). The incorporation of the siRNAs, generated by both the initiation step and the amplification cycles, into the RNA-inducing silencing complex (RISC) directs the endonucleolytic cleavage of mRNA or ssRNA (single-stranded RNA).