Properties of Unpaired DNA Required for Efficient Silencing In

Properties of Unpaired DNA Required For Efﬁcient Silencing in Neurospora crassa

Dong W. Lee,*,1 Kye-Yong Seong,* Robert J. Pratt,* Kevin Baker* and Rodolfo Aramayo*,1,2 *Department of Biology, College of Science, Texas A&M University, College Station, Texas 77843-3258

ABSTRACT The presence of unpaired copies of a gene during meiosis triggers silencing of all copies of the gene in the diploid ascus cell of Neurospora. This phenomenon is called meiotic silencing and on the basis of genetic studies appears to be a post-transcriptional gene silencing (PTGS) mechanism. Previously, meiotic silencing was defined to be induced by the presence of a DNA region lacking an identical segment in the homologous chromosome. However, the determinants of unpaired DNA remained a mystery. Using the Ascospore maturation-1 (Asm-1) gene, we defined what needs to be “unpaired” to silence a gene. For efficient silencing, an unpaired region of DNA needs to be of a sufficient size and contain homology to the reporter transcript. The greater the size of the loop and the larger the homology to the reporter transcript, the better the resulting meiotic silencing is. Conversely, regions not containing homology to the transcript, e.g., intergenic regions, did not silence the reporter. Surprisingly, unpaired fragments lacking a canonical promoter silenced the reporter. Additionally, we detected the unpairing-dependent loss of a transcript during meiotic silencing. Our observations further support a PTGS mechanism for meiotic silencing and offer insight into the evolutionary consequences resulting from this novel meiotic checkpoint.

EIOTIC cells are unique in that their develop- diploid zygotic cell of Neurospora immediately after M ment entails profound changes that activate so- karyogamy, in the narrow window of time at the onset of phisticated cellular mechanisms to ensure precise chro- meiosis during which homologous chromosomes align mosome duplication, repair, and recombination. Thus, and “sense” each other, but before the first meiotic these cells must undergo internal morphological devel- division occurs (Aramayo and Metzenberg 1996b). opment. In Neurospora, meiosis occurs inside the zy- The zygote is the only known diploid cell of Neurospora gote, which forms by fusion of two haploid nuclei of and thus the only cell in Neurospora in which the trans- opposite mating type. The zygote undergoes karyogamy, sensing first seen in Drosophila (Lewis 1954) can be meiosis, and postmeiotic mitosis within the perithe- observed. When a gene fails to sense its partner in the cium—the complex multicellular sexual reproductive homologous chromosome, the resulting unpaired DNA apparatus of Neurospora (Raju 1992)—resulting in an triggers meiotic silencing, an RNA-mediated post-tran- ascus that contains eight haploid spores arrayed in an scriptional gene-silencing mechanism that, once acti- order that reflects their lineage (Raju 1980, 1992). Im- vated, persists during the subsequent meiotic divisions mediately following karyogamy, meiotic chromosomes (Aramayo and Metzenberg 1996b; Shiu et al. 2001), align, compact, pair, and recombine to produce progeny but is reset at some point prior to germination. that carry sets of newly shuffled DNA (Raju 1980; Kleck- Meiotic transvection was discovered through a com- ner 1996; Cook 1997; Zickler and Kleckner 1998, bination of design and serendipity during our studies 1999; Roeder and Bailis 2000). Inside the perithecium, of the complex Ascospore maturation-1 gene (Asm-1)in the developing asci are immersed in maternal tissue and Neurospora (Aramayo and Metzenberg 1996b). ASM-1 cannot be isolated in a pure form, especially at early stages (the product of asm-1ϩ) is an abundant nuclear protein in spore formation. Thus, any study of the molecular essential for the formation of aerial hyphae, the develop- mechanisms of meiosis in Neurospora is a challenge. ment of protoperithecia (the haploid female sexual Neurospora meiotic cells have developed mechanisms structures), and the maturation of the ascospores (the that control the integrity of the genomes that participate haploid sexual spores; Aramayo and Metzenberg 1996b; in the process. Meiotic transvection and meiotic silenc- Aramayo et al. 1996). Recessive, loss-of-function muta- ing are two such mechanisms. They are the two faces of tions in Asm-1 are spore autonomous within the ascus. the same coin. Meiotic transvection is initiated in the That is, spores carrying the mutant allele fail to develop, whereas spores with the asm-1ϩ allele mature normally. Surprisingly, we discovered that deletion alleles of Asm-1 1 These authors contributed equally to this work. are ascus dominant; all spores within the ascus fail to 2Corresponding author: Department of Biology, College of Science, ϩ Texas A&M University, Room 414A, Bldg. BSBW, College Station, TX develop, including the ones carrying the asm-1 allele. 77843-3258. E-mail: [email protected] Several models were proposed to explain this odd ge-

Genetics 167: 131–150 (May 2004) 132 D. W. Lee et al. netic behavior (Aramayo and Metzenberg 1996b). unpaired. The efficiency of silencing increased with the The favored interpretation was that pairing over the length of the unpaired DNA and with the size of the entire length of the Asm-1 gene is an essential step for unpaired loop. Additionally, we found that canonical normal development. This conclusion was based on the promoter elements did not have to be present in the observation that crosses between two deletion mutants, unpaired loop for meiotic silencing to occur. This work each one complemented by an identically placed ec- thus defined the primary qualitative and quantitative topic copy of asm-1ϩ, produced abundant, viable black properties of unpaired DNA in meiotic silencing and spores. These results are consistent with a model for offers further support to meiotic silencing being a post- transvection in which the presence of genes at identical transcriptional gene silencing (PTGS)-like mechanism. positions in homologous chromosomes alters the developmental outcome. Genes sense each other during chromosome pairing (i.e., undergo meiotic transvection). If MATERIALS AND METHODS a given gene fails to pair with (i.e., does not sense) its Procedures for DNA extraction from Neurospora crassa, South- partner, the silencing of all paired and unpaired copies ern blot analysis, and other nucleic acid manipulations were of the gene will follow. performed as described (Pratt and Aramayo 2002). Similarly, Shiu et al. (2001) demonstrated that mutations in the growth conditions, conidial spheroplast preparation, fungal Suppressor of ascus dominance-1 (Sad-1) gene eliminate the transformation, homokaryon purification, female fertility/ste- ascus dominance of paired and unpaired copies of Asm-1. rility determinations, and genetic crosses were performed as ϩ described (Pratt and Aramayo 2002). The formulas for Vo- The sad-1 gene encodes an RNA-dependent RNA poly- gel’s medium N, Westergaard’s medium, and the sugar mix- merase (RdRP; Shiu et al. 2001; Shiu and Metzenberg ture of Brockman and de Serres have been described by Davis 2002). This finding suggested that the mechanism be- and de Serres (1970). hind ascus dominance involves RNA silencing and that Scoring of genetic crosses: Generally the partners were co- it probably operates through the production of an RNA- inoculated in a petri dish and incubated at room temperature for 6 days. The bulk of the conidia were removed, and the based diffusible signal (Shiu et al. 2001). It also sug- remaining conidia were spread around the lawns with 1 ml of gested that in addition to the vegetative RNA-silencing water. Crosses made in this way started shooting ascospores -days after inoculation. The degree of silencing was deter 20ف -pathway, quelling, a second, meiosis-specific RNA-silenc ing pathway might exist in Neurospora (Galagan et al. mined by taking several pictures of each petri dish lid. Pictures 2003). This prediction was confirmed by our demonstra- were printed and spores counted. The percentage of black (viable) or white (inviable) ascospores shot was determined tion of the involvement of two genes in meiotic silenc- from the total number of ascospores present on each picture. ing: one coding for an Argonaute-like protein, Suppressor The strength of the observed silencing was defined as described of meiotic silencing-2 (Sms-2; Lee et al. 2003b), and the in the Figure 2 legend. other coding for a Dicer-like protein, Suppressor of meiotic Plasmid construction: The genome sequence of the Asm-1 lo- silencing-3 (Sms-3;M.Mclaughlin,D.W.Lee,R.Pratt cus is contained on contig 3.56 (Release 3, Whitehead Institute, http://www-genome.wi.mit.edu/annotation/fungi/neurospora/). and R. Aramayo, unpublished results). We arbitrarily defined as position 1 the HindIII site located Shiu et al. (2001) interpreted their results on meiotic 6128 bp upstream of the translational initiation signal (ATG) silencing of Asm-1 in a general context (unpaired DNA) for ASM-1 (Aramayo et al. 1996). Following this convention and predicted that the silencing mechanism works at the the HindIII fragment contained in pRAUW44 (Aramayo et al. post-transcriptional level. However, the authors did not 1996) maps from coordinates 1 to 12425. A detailed description of the plasmids used in this study address whether silencing could be achieved to the is presented in supplementary material online (Methods at same extent by unpairing different functional or non- http://www.genetics.org/supplemental/). Oligonucleotides functional fragments of the gene. For example, the used in this study are described in supplementary material synthesis of the first strand of RNA (the predicted tem- online (Table S1 at http://www.genetics.org/supplemental/). plate for the RdRP) could depend on a canonical pro- Total RNA isolation: Total RNA was extracted from 6-day-old perithecia using TRIzol reagent (GIBCO-BRL, Grand Island, moter recognized by a conventional RNA polymerase NY). Perithecia were harvested by removing them from Wes- II-directed transcription complex, as would be the case tergaard’s medium, solidified with 1.5% Bacto-Agar, with a for a retrotransposon undergoing activation (Sand- sterile razor blade, and were next ground with a mortar and meyer and Menees 1996). The use of Asm-1 as a reporter pestle under liquid nitrogen. One milliter of TRIzol reagent gene allowed us to ask these questions because ASM-1 (GIBCO-BRL) was added to the ground perithecia, and total is essential for normal ascospore development (Ara- RNA was extracted following the manufacturer’s protocol. The resulting RNA was subjected to an additional LiCl precipi- mayo et al. 1996). We therefore first mapped the pro- tation purification step. moter of the gene during both asexual and sexual devel- Rapid amplification of cDNA ends PCR: First strand was syn- opment and identified an Asm-1 transcript whose loss thesized by first mixing 3 ␮g of total RNA with 10 pmol of each correlates with the induction of meiotic silencing. We ODLAM018 (coordinates 6761–6742, Table S1) and SMART II then tested how the unpairing of different functional Oligo [SMART rapid amplification of cDNA ends (RACE) cDNA amplification kit; CLONTECH, Palo Alto, CA] in a and nonfunctional fragments of the gene affects meiotic 10-␮l reaction volume containing a final concentration of silencing. Silencing was observed only when regions of 50 mm Tris-HCl, pH 8.3, 10 mm dithiothreitol, 75 mm KCl, the gene with homology to the reporter transcript were 6mm MgCl2,and1mm dNTPs. The mixture was then dena- Properties of Unpaired DNA 133

tured at 70Њ for 5 min. Two hundred units of Superscript II mycin phosphotransferase (hphϩ) marker, to generate the (Invitrogen, Carlsbad, CA) was added and the reaction was Asm-1⌬(3426–9336) deletion allele (Figure 1). The resulting incubated at 42Њ for 90 min. Reaction was stopped by adding ␮ mutant strains had the same complex phenotype as that 100 l of tricine-EDTA buffer (10 mm tricine-KOH, pH 8.5, 1 mm ⌬ EDTA). PCR was performed by mixing 2.5 ␮l of the first-strand of the Asm-1 mutants described before (Aramayo et al. synthesis reaction product (cDNA) with 10 pmol of ODLAM067 1996). They were female sterile (i.e., they did not form (coordinates 5952–5929, Table S1) and 1ϫ Universal Primer protoperithecia) but male fertile (i.e., they could fer- ␮ Mix (UPM; CLONTECH) in a 50- l reaction volume containing tilize strains of the opposite mating type). The aerial a final concentration of 40 mm tricine-KOH, pH 8.7, 15 mm potassium acetate, 3.5 mm magnesium acetate, 3.75 ␮g/ml BSA, hyphae of the mutants were stunted, compared to those 0.005% (v/v) Tween-20, 0.005% Nonidet-P40, 0.2 mm dNTPs, of wild type; consequently, they conidiated very close and 1ϫ Advantage 2 polymerase mix (CLONTECH). Reaction to the agar surface. was incubated as follows: 1 cycle, 94Њ for 2 min; followed by We then transformed one of these strains either with 5 cycles of 94Њ for 5 sec, 72Њ for 3 min; followed by 5 cycles Њ Њ Њ the identical 5910-bp DNA fragment that was deleted of 94 for 5 sec, 70 for 10 sec, 72 for 3 min; followed by 25 ⌬(3426-9336) cycles of 94Њ for 5 sec, 68Њ for 10 sec, and 72Њ for 3 min. in Asm-1 (Figure 1, construct A) or with one of a Strain description: Strains of N. crassa are described in Ta- series of 5Ј-truncated Asm-1 fragments (constructs B–G). ble 1. The following strains have been previously described: All these fragments were inserted ectopically at the his- DLNCR83A and DLNCT62A (Lee et al. 2003a), FGSC 4564 3 locus in LG I. We then tested these transformants for (Griffiths and DeLange 1978), and RPNCR4A (Pratt and their ability to undergo asexual and sexual develop- Aramayo 2002). The allele hphϩ::tkϩ consists of the hygromycin B phosphotransferase fused in frame to the herpes simplex virus ment. Asexual complementation was scored by the abil- thymidine kinase gene, as described by Lupton et al. (1991). ity of strains containing different Asm-1 fragments to Escherichia coli K12 XL1-Blue MR (Stratagene, La Jolla, CA) form aerial hyphae. Sexual complementation was scored was the host for all our bacterial manipulations. When non- by their ability to form protoperithecia and by their methylated DNA was needed for enzyme digestions, either ability to form viable ascospores in a cross with an Asm- GM2163—an E. coli K12 derivative containing, among other ⌬(3426-9336) markers, a dam13::Tn9 (CamR) and a dcm-6 mutation (New 1 deletion mutant of the opposite mating type England BioLabs, Beverly, MA)—or JM110—an E. coli K12 complemented with the 5910-bp Asm-1 fragment in- derivative containing, among others, dam and dcm mutations serted at the his-3 locus in LG I. In the absence of meiotic (Yanisch-Perron et al. 1985)—was used. silencing, successful ascospore maturation complemen- ؉ (3430-9336)⌬ A note about strains containing the Asm-1 ::hph :: tation should result in eight mature ascospores per as- mcl-1 deletion allele of Asm-1: In these strains, the region deleted encompassed a region predicted to direct the tran- cus. Failure to complement should produce four mature scription of a gene that we call myosin chain-like-1 (mcl-1ϩ), on ascospores and four immature ones. No meiotic silenc- the basis of its weak homology to myosin-chain-like genes in ing was observed in cross 1 and crosses 3–7 (Figure 2). other organisms (data not shown). Strains containing a disrup- The meiotic silencing observed in cross 8 precluded us tion in this predicted gene are viable and do not have any from determining the ascospore maturation comple- detectable developmental or metabolic phenotypes. A note about the integration at the his-3 locus: Due to the mentation phenotype of construct G (Figures 1 and 2). nature of the histidine-3 (his-3) integration vectors used in this As expected, deletion mutants carrying an ectopic copy study (Aramayo and Metzenberg 1996a), the lysophospholi- of the entire Asm-1 region complemented both the asex- pase (lpl) gene, located downstream of the his-3 gene, was ual and sexual developmental defects associated with deleted during the integration of our constructs. When we Asm-1⌬(3426–9336) (Figure 1, construct A). In contrast, strains arbitrarily define the HindIII restriction site present in the coding region of his-3 as position 1, the region deleted dur- carrying truncated ectopic alleles of Asm-1 lacking the ing the gene replacement spans from position 5192 to posi- promoter or the promoter plus transcription-initiation tion 6046. We include this deletion [lpl⌬(5192–6046)] as part of sites formed neither normal aerial hyphae nor proto- the genotype of all our strains containing integrations at the perithecia (constructs B–G), with the exception of con- his-3 locus (Table 1), to distinguish them from strains construct B, which could form protoperithecia when pres- taining integrations at the his-3 locus obtained with a third generation of his-3-integration vectors (Margolin et al. 1997). ent in mating type a (mat a) strains (data not shown). The genome sequence of the his-3ϩ locus can be found on The B, C, D, and E constructs did, however, support contig 3.165 (Release 3, Whitehead Institute, http://www- normal ascospore development, whereas strains car- genome.wi.mit.edu/annotation/fungi/neurospora/). rying segment F did not. These results indicate that the region between nucleotides 5437 and 5714 can drive the expression of Asm-1 in the absence of its canonical RESULTS promoter during ascospore development but not dur- The leader of Asm-1 contains a promoter element: To ing vegetative growth (compare construct E with con- determine the regions capable of driving Asm-1 expres- structs F and G). This “transcriptionally active element” sion, we performed a deletion-scanning analysis of the mapped to a region that encodes the leader of the Asm-1 Asm-1 promoter (Figure 1). First, we constructed two transcript that is expressed during asexual development strains, one of each mating type, that contain a deletion (Aramayo et al. 1996). of the 5910-bp chromosomal region spanning the pro- To confirm this result, we constructed a strain con- moter, coding, and downstream regions of Asm-1 in link- taining an 854-bp deletion at the normal Asm-1 locus age group V (LG V), which was replaced with the hygro- in LG V. This deletion removed most of the promoter 134 D. W. Lee et al.

TABLE 1 Fungal strains used in this study

Namea Genotypeb Originc DLNCR83A (his-3::lpl ⌬(5192-6046)::hphϩ::tkϩ; inl a) Progeny from DLNCT62A ϫ RPNCR4Ad DLNCT150A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[8082-6018]; inl A) Transformation of RANCR05A with pDLAM112 DLNCT151A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[8082-6018]; inl a) Transformation of RANCR06A with pDLAM112 DLNCT152A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[8442-7035]; inl A) Transformation of RANCR05A with pDLAM113 DLNCT153A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[8442-7035]; inl a) Transformation of RANCR06A with pDLAM113 DLNCT44A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[7758-6320]; inl A) Transformation of RANCR05A with pDLAM075 DLNCT45A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[7758-6320]; inl a) Transformation of RANCR06A with pDLAM075 DLNCT46A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[9336-8387]; inl A) Transformation of RANCR05A with pDLAM076 DLNCT47A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[9336-8387]; inl a) Transformation of RANCR06A with pDLAM076 DLNCT54A (his-3ϩ::lpl ⌬(5192-6046)::␭[26104-21226]::Asm-1[7758-6320]; inl A) Transformation of RANCR05A with pDLAM082 DLNCT55A (his-3ϩ::lpl ⌬(5192-6046)::␭[26104-21226]::Asm-1[7758-6320]; inl a) Transformation of RANCR06A with pDLAM082 DLNCT56A (his-3ϩ::lpl ⌬(5192-6046)::␭[31747-39168]::Asm-1[7758-6320]; inl A) Transformation of RANCR05A with pDLAM083 DLNCT57A (his-3ϩ::lpl ⌬(5192-6046)::␭[31747-39168]::Asm-1[7758-6320]; inl a) Transformation of RANCR06A with pDLAM083 DLNCT62A (his-3::lpl ⌬(5192-6046)::hphϩ::tkϩ; inl A) Transformation of RANCR06A with pDLAM073d FGSC 4564 (ad-3B cyh-1 R am1) FGSCe KBNCT04A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[6690-5465]; inl a) Transformation of DLNCR83A with pKYAM199 KYNCT01A (his-3; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl A) Transformation of RANCR05A with pKYAM052 KYNCT02A (his-3; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl a) Transformation of RANCR06A with pKYAM055 KYNCT03A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-3426]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl A) Transformation of KYNCT01A with pKYAM011 KYNCT04A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-4615]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl A) Transformation of KYNCT01A with pKYAM005 KYNCT05A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-3426]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl a) Transformation of KYNCT02A with pKYAM011 KYNCT06A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-3827]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl a) Transformation of KYNCT02A with pKYAM016 KYNCT07A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-4615]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl a) Transformation of KYNCT02A with pKYAM005 KYNCT08A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-4999]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl a) Transformation of KYNCT02A with pKYAM041 KYNCT09A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-5272]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl a) Transformation of KYNCT02A with pKYAM032 KYNCT10A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-5437]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl a) Transformation of KYNCT02A with pKYAM034 KYNCT11A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[9336-5714]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl a) Transformation of KYNCT02A with pKYAM046 KYNCT20A (his-3; asm-1ϩ[⌬(4615-5469)]::hphϩ, inl a) Transformation of RANCR06A with pKYAM116 KYNCT21A (his-3; asm-1⌬(4615-5977)::hphϩ, inl a) Transformation of RANCR06A with pKYAM114 KYNCT23A (his-3; Asm-1⌬(4615-6442)::hphϩ, inl a) Transformation of RANCR06A with pKYAM135 KYNCT24A (his-3; Asm-1⌬(4615-6690)::hphϩ, inl a) Transformation of RANCR06A with pKYAM086 KYNCT25A (his-3; Asm-1⌬(4615-7012)::hphϩ[←], inl a) Transformation of RANCR06A with pKYAM180 KYNCT30A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[9336-6690]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl A) Transformation of KYNCT01A with pKYAM030 KYNCT31B (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[9336-6107]; Asm-1⌬(3426-9336)::hphϩ::mcl-1, inl a) Transformation of KYNCT02A with pKYAM080 KYNCT34A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-3426]; inl A) Transformation of RANCR05A with pKYAM011 KYNCT35A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1ϩ[9336-3426]; inl a) Transformation of RANCR06A with pKYAM011 KYNCT46A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[7012-4615]; inl A) Transformation of RANCR05A with pKYAM156 KYNCT47A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[7012-4615]; inl a) Transformation of RANCR06A with pKYAM156 KYNCT53A (his-3; Asm-1⌬(5414-6690)::hphϩ, inl a) Transformation of RANCR06A with pKYAM198 KYNCT54A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[9336-6690]; inl A) Transformation of RANCR05A with pKYAM030 KYNCT55A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[9336-6690]; inl a) Transformation of RANCR06A with pKYAM030 KYNCT57A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[6118-4615]; inl A) Transformation of DLNCT62A with pKYAM203 KYNCT58A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[7758-6320][6118-4615]; inl A) Transformation of DLNCT62A with pKYAM204 KYNCT60A (his-3ϩ::lpl ⌬(5192-6046)::trpC[890-1245]::Asm-1[6324-7764]; inl A) Transformation of DLNCT62A with pKYAM206 KYNCT64A (his-3ϩ::lpl ⌬(5192-6046)::␭[5818-7229]::Asm-1[6118-4615]; inl A) Transformation of DLNCT62A with pKYAM210 KYNCT68A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[7758-6320][5467-4615]; inl A) Transformation of DLNCT62A with pKYAM219 KYNCT76A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[7758-6320][5467-4615]; inl a) Transformation of DLNCR83A with pKYAM219 KYNCT77A (his-3ϩ::lpl ⌬(5192-6046)::trpC[890-1245]::Asm-1[6324-7764]; inl a) Transformation of DLNCR83A with pKYAM206 KYNCT80A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[6118-4615]; inl a) Transformation of DLNCR83A with pKYAM203 KYNCT86A (his-3ϩ::lpl ⌬(5192-6046)::Asm-1[7758-6320][6118-4615]; inl a) Transformation of DLNCR83A with pKYAM204 KYNCT88A (his-3ϩ::lpl ⌬(5192-6046)::␭[5818-7229]::Asm-1[6118-4615]; inl a) Transformation of DLNCR83A with pKYAM210 RANCR05A (his-3; inl A) RANC collectionf RANCR06A (his-3; inl a) RANC collectionf RANCR49A (flA) RANC collectionf RANCR50A (fla) RANC collectionf a DLNC, KBNC, KYNC, and RANC indicate strains constructed or provided for this study by Dong W. Lee, Kevin D. Baker, Kye- Yong Seong, and Rodolfo Aramayo, respectively. b Allele numbers or designations are: A, mating type A; a, mating type a; ad-3B, adenine-3B (12-17-114); a m1, a mutant allele of the a idiomorph; Asm-1, Ascospore maturation-1; cyh-1R, cycloheximide resistant-1 (KH52r); fl, fluffy (P); his-3, histidine-3 (1-234-723); hphϩ, hygromycin B phosphotransferase; hphϩ::tkϩ, hygromycin B phosphotransferase fused in frame to the herpes simplex virus thymidine kinase gene; inl, inositol (89601); ␭, ␭DNA; lpl ⌬(5192-6046), lysophospholipase; mcl-1, myosin chain-like-1; trpC, tryptophan-C.Seematerials and methods for details. c Construction of the different plasmids is described in Methods in supplementary material at http://www.genetics.org/supplemental/. d See materials and methods (Strain description). e FGSC indicates strains acquired from the Fungal Genetics Stock Center, University of Kansas Medical Center, Kansas City. f Rodolfo Aramayo Neurospora crassa collection. Properties of Unpaired DNA 135

Figure 1.—Promoter scanning of Asm-1. (Top) A diagram of the 12,425- bp Asm-1 chromosomal region shows the relative positions of the ␣-transcript (shaded area) and the coding region of the gene. (Bottom) A detailed diagram depicts the 5910-bp region (3426–9336) containing the Asm-1 haploid promoter (3426–5118); the ␣ (5370), ␤ (5163), and ␥ (5118) transcription-start sites; the leader region (5370–6128); an intron (5238–5345, open box); a micro-open reading frame (␮ORF, 5600–5821; short open arrow); the transcriptionally active element (5469–5714); and the asm-1ϩ open reading frame (6128–8059; Ara- mayo et al. 1996). The horizontal bars labeled A–G represent DNA fragments inserted at the his-3 locus (LG I) in strains carrying the Asm-1⌬(3430–9336) deletion in LG V. The ability of each fragment to complement the aerial hyphal formation, protoperithecial development, and ascospore maturation defects associated with Asm-1⌬(3430–9336) is indicated. The asterisk indicates that construct B can direct the formation of protoperithecia in mat a strains only. The ascospore maturation of strains carrying construct G could not be determined (n.d.). The bar labeled H shows the extent of the Asm-1⌬(4615–5469) deletion introduced into the Asm-1 locus at its native position in LG V and the behavior of the resulting strain.

and all of the known transcription start sites of Asm-1 cloned and sequenced. This analysis identified cDNAs (Figure 1, coordinates 4615–5469, construct H). The corresponding to the ␣-, ␤-, and ␥-messages and also re- resulting strain produced stunted aerial hyphae and was vealed a 107-bp intron (coordinates 5238–5345) down- unable to form protoperithecia. This phenotype was stream of the ␤- and ␥-transcription start sites (Figure 1). similar to those strains carrying the ectopically located Taken together, these experiments identified the predom- constructs B–G. However, this strain supported normal inant transcription start sites detected for Asm-1 in wild- ascospore development (compare constructs E and H type cells during sexual development. with F). These results confirm that a DNA element con- To determine which transcripts are used during as- tained within nucleotides 5469–5714 (Figure 1) can cospore development, we performed 5Ј-RACE-PCR on drive the expression of Asm-1 during ascospore develop- mRNA extracted from 6-day-old perithecial tissues of ment. The canonical Asm-1 open reading frame (ORF) wild-type crosses and crosses where meiotic silencing must be transcribed and translated, because mutation was induced. Given that ASM-1 is essential for ascospore of the ATG translation initiation codon (located at posi- development, we predicted that silencing of the gene tion 6128–6130) inactivates Asm-1 function during asco- would eliminate the transcript(s) specific to ascospore spore development (Kutil et al. 2003). development. We crossed wild-type strains with each The ␣-transcript of Asm-1 is a target for meiotic silenc- other (Figure 3, lanes 2 and 5) and with strains carrying ing: To map the 5Ј-end(s) of the Asm-1 transcripts during the Asm-1⌬(3426–9336) deletion allele (lanes 3 and 6) or car- sexual development, we conducted two experiments: rying the Asm-1⌬(3426–9336) deletion allele complemented First, we performed 5Ј-RACE-PCR on mRNA extracted with the 5910-bp DNA fragment of Asm-1 inserted at from tissues undergoing sexual development (6 days the his-3 locus in LG I (lanes 4 and 7). DNA fragments after fertilization) and cloned and sequenced the ampli- corresponding to the ␤- and ␥-transcripts were present fied DNAs. This experiment confirmed the presence of in all crosses (lanes 2–7). In contrast, DNA fragments the previously determined transcription start site of the corresponding to the ␣-transcript could be detected gene [henceforth called ␣-start (coordinate 5370); Fig- only when the Asm-1 region was paired, i.e., when silenc- ure 1] (Aramayo et al. 1996) and also identified two ing was not induced (lanes 2 and 5). The unpairing- additional transcription start sites [henceforth called dependent loss of the ␣-message strongly suggests that ␤- (coordinate 5163) and ␥-start (coordinate 5118); Fig- this transcript is a target for meiotic silencing. The ure 1]. Second, a sexual-stage-specific cDNA library was ␤- and ␥-transcripts were not sensitive to meiotic silenc- screened with a probe spanning the entire Asm-1 region ing. Given the strong sequence similarity between the (coordinates 2992–8387). The cross-reacting cDNAs were ␣-, ␤-, and ␥-transcripts and that meiotic silencing is 136 D. W. Lee et al.

Figure 2.—Testing cis-silencing in linkage group I. (A) A diagram of a diploid zygote cell shows the pairing of the LG V and LG I chromosomes. For sim- plicity, only one of the two sister chroma- tids is indicated. Both LG V chromosomes carry the Asm-1⌬(3430–9336)-deletion allele. Unless otherwise indicated, the LG I Dad chromosome contains a DNA fragment corresponding to the asm-1ϩ region from coordinates 3426 to 9336 inserted at the his-3 locus, whereas the Mom chromosome carries a series of Asm-1- deletion alleles inserted at the same position. (B) A diagram of the Asm-1 region from coordinates 1 to 12425 shows the ␣-transcript and the coding region of asm-1ϩ. In crosses 1–10 (Table 2), a series of pairing Dad (solid bar) and Mom (open bar) chromosomes are presented. The numbers to the left and right of each DNA fragment indicate the positions of the left and right borders of the fragment, respectively. Whenever present, the size of the loop represents the length of unpaired DNA. The DNA region with homology to the ␣-transcript present in the unpaired DNA is indicated by open bars. To the right, the length of total unpaired DNA [DNA (bp)], the length of unpaired DNA corresponding to the ␣-transcript [Tran- script (bp)], and the extent of meiotic silencing (Silencing?) for each cross are presented. The strength of the observed silencing is indicated as the proportion of immature (white) ascospores: ϩ,0– 25%; ϩϩ, 25–45%; ϩϩϩ, 45–65%; ϩϩϩϩ, 65–85%; and ϩϩϩϩϩ, 85– 100%. The percentage of mature (black) ascospores produced by a cross showing a 4:4 segregation was multiplied by 2 to calculate the proportion of immature (white) ascospores.

ascus autonomous (Aramayo and Metzenberg 1996b; both complemented with the 5910-bp DNA fragment Shiu and Metzenberg 2002), the simplest explanation inserted at his-3, produced many mature, black, viable for their persistence is that these transcripts reside in the ascospores (Figure 2, cross 1). This result obeyed the surrounding maternal tissue and are, therefore, protected rules for meiotic transvection and indicated that pairing from degradation. However, these results do not rule of the ectopic copies of Asm-1 was both necessary and out that ␤- and ␥-transcripts are present inside the devel- sufﬁcient for normal sporulation. oping asci or that the ␣-transcript might be present in We reasoned that, if silencing of Asm-1 requires un- maternal tissue in undetectable amounts, but they do pairing of a speciﬁc DNA region or regions present in strongly suggest that the ␣-transcript is being selectively the unpaired DNA fragment (e.g., a promoter element), used during ascospore development. these silencing elements could be mapped by the pro- Only unpaired DNA regions with homology to the gressive-deletion method, according to the following transcript can silence Asm-1: To determine what proper- logic: Pairing of a “Dad” chromosome carrying the en- ties unpaired DNA must have to silence Asm-1, we per- tire 5910-bp Asm-1 fragment at the his-3 locus with differ- formed crosses of the same type that were used to test ent “Mom” chromosomes, each carrying a different de- sexual complementation and determined the percent- letion at the his-3 locus, results in the unpairing of the age of black ascospores shot from these crosses (Figure 2). region in the Dad chromosome that corresponds to Crosses between Asm-1⌬(3426–9336) deletion parent strains, the deletion in the Mom chromosome (Figure 2). If Properties of Unpaired DNA 137

increasing size of the unpaired DNA loop. Therefore, these results could be explained in three ways. First, the extent of silencing could simply correlate with the length of unpaired DNA (i.e., larger deletions cause silencing and smaller ones do not). If this is true, unpaired DNA would have to exceed some threshold length between 2288 and 2681 bp (crosses 7 and 8) to trigger silencing. Second, silencing might occur only when the unpaired DNA contains a discrete region required for silencing (e.g., homology to the canonical Asm-1 ␣-transcript of the gene). Third, silencing could be the combined outcome of these two effects. To discriminate among these possibilities, we decreased the length of the unpaired DNA loop from 2681 to Figure 3.—The ␣-transcript of Asm-1 is a target for meiotic silencing. Ethidium bromide agarose gel [1% (w/v)] is shown. 1492 bp, which is below the predicted threshold, by Lane 1, marker consisting of ␭DNA digested with EcoRI and constructing a new Dad chromosome with a truncation HindIII (␭/EcoRI/HindIII). Lanes 2–7: Reverse-transcribed at the 5Ј end of the original 5910-bp insert. The length PCR products were generated from total RNA as described of the unpaired transcript was held constant (cross 10). in materials and methods. RNA was extracted from the ϩ If only the size of the loop is important, then the smaller following crosses (Table 2): lane 2, cross 11 [asm-1 A (female) ϫ asm-1ϩ a (male)] (no silencing); lane 3, cross 12 [asm-1ϩ loop should not silence. If unpairing of the transcribed A (female) ϫ asm-1⌬(3426–9336) a (male)] (silencing induced by region is important, then the smaller loop should si- unpairing one copy of Asm-1); lane 4, cross 13 [asm-1ϩ A lence as efficiently as the larger one. If both factors are ϩ ϩ ⌬ (female) ϫ his-3 ::Asm-1 ; asm-1 (3426-9336) a (male)] (silencing important, then the smaller loop should silence less ef- induced by unpairing two copies of Asm-1); lane 5, cross 14 ϩ ϩ ficiently than the larger loop but to a greater extent than [asm-1 A (male) ϫ asm-1 a (female)] (no silencing); lane 6, cross 15 [asm-1⌬(3426–9336) A (male) ϫ asm-1ϩ a (female)] a loop of equal size that does not contain the transcribed (silencing induced by unpairing one copy of Asm-1); and lane region. The result was consistent with the third possi- 7, cross 16 [his-3ϩ::Asm-1ϩ; asm-1⌬(3426–9336) A (male) ϫ asm-1ϩ bility (compare crosses 8 and 10), suggesting that both a (female)] (silencing induced by unpairing two copies of qualitative (compare crosses 4–7 to cross 10) and quanti- Asm-1). The gel was loaded with 10% of each PCR reaction. tative (compare crosses 8 and 10) properties determine Note that the ␣-transcript is present only in crosses in which meiotic silencing was not induced. the silencing potential of unpaired DNA. To ensure that the his-3 locus was not introducing an unknown artifact, we next tested if unpaired Asm-1 DNA at the canonical chromosomal position in LG V behaves the unpaired DNA thus generated does not induce si- in the same manner. We constructed a series of strains lencing, the deletion will be recessive. In this situation, carrying progressively larger deletions of the Asm-1 deletion mutations that do not affect expression of gene. The deleted fragment was replaced by the hphϩ Asm-1 during sexual development will produce eight marker gene (Figure 4). Although the presence of the viable ascospores per ascus (e.g., crosses 1–6). In con- hphϩ marker gene at the site of deletion makes it difficult trast, any mutation that blocks expression of Asm-1 dur- to compare the results in Figure 2 and Figure 4 directly, ing ascospore development (i.e., that prevents ascospore the trends were similar. Crosses 17, 18, and 19 did not maturation) will affect the development of the haploid result in silencing, whereas crosses 20 to 24 did. Consis- ascospore containing it. In this case, even in the total tent with the results shown in Figure 2, unpaired DNA bp or more of homology to the canonical 700ف absence of silencing, half of the spores will be black and with half white (e.g., cross 7). If the unpaired DNA induces transcript was required for silencing (compare cross 7 silencing, the deletion will be dominant and no viable with 8 and cross 19 with 20). To confirm that the main spores will form (e.g., cross 8). Also, since Mom chromo- determinant of silencing was unpaired DNA correspond- somes carried progressively larger deletions of Asm-1, ing to the canonical transcript rather than the length of the unpaired loops in the Dad chromosomes will be- the unpaired DNA, we deleted a fragment of Asm-1 that come progressively larger. We can thus test the effect is completely within the region of homology to the of the size of the unpaired loop on meiotic silencing ␣-transcript (Figure 4, cross 25, coordinates 5414–6690). (Figure 2). Thus, the lower the percentage of black The silencing was not as strong as in crosses 20–24, but ascospores, the stronger the silencing will be (Table 2). since the total length of the unpaired DNA was shorter The results of the crosses are summarized in Figure 2. than that in cross 19, the enhancement of silencing was The unpaired DNA in crosses 1–7 did not cause silenc- significant (compare cross 25 with cross 19). ing, whereas it did in crosses 8 and 9. The ability to si- These results suggest that unpairing the promoter of lence thus correlated both with encroachment into re- Asm-1 is not sufficient to induce silencing (Figure 2, gions containing homology to the transcript and with crosses 2–7 and Figure 4, crosses 18 and 19) and thus 138 D. W. Lee et al. argue against the presence of a mechanism that tran- fragment of Asm-1 (coordinates 6320–7758) to the trpC scriptionally inactivates the loop of unpaired DNA dur- promoter of Aspergillus nidulans (Mullaney et al. 1985; ing meiosis and subsequent cell divisions. In addition, Hamer and Timberlake 1987). Strains carrying this they show that the signal produced by the unpaired fusion inserted at the his-3 locus in LG I were crossed kb 1.8ف DNA loop does not propagate in cis to other paired to wild-type strains, leaving an unpaired loop of 1.4ف segments of the gene. This result is in agreement with carrying a region of homology to the transcript of our own previous observations (Kutil et al. 2003). Fi- kb. Silencing was not observed (crosses 35 and 36). bp Next, we repeated the scanning experiment using larger 700ف nally, it seems that a DNA region with at least of homology to the canonical transcript is required for unpaired fragments. Fragments of 2397 bp (crosses 37 silencing the gene, which reinforces an RNA-mediated and 38), 2064 bp (crosses 39 and 40), 2646 bp (crosses model for meiotic silencing. 41 and 42), and 5910 bp (crosses 43 and 44), again span- Loops of unpaired DNA need not carry known pro- ning the entire Asm-1 genomic region, were tested. With- moter elements to silence: The loop of unpaired DNA out exception, silencing was observed. tested in our previous experiments was always linked to These results demonstrate that, under the conditions its normal flanking DNA and always contained segments used here, trans-silencing was not as efficient as cis-silenc- required for transcription of Asm-1. To dissociate the ing when the length of unpaired DNA is short. They unpaired loop from its surrounding sequences in the also demonstrate that promoter elements in the un- chromosome and to test whether promoter elements paired DNA are not required for the production of the are necessary for meiotic silencing we unpaired differ- diffusible silencing signal (e.g., small RNAs). Thus, the ent Asm-1 segments of similar sizes at his-3 and tested synthesis of the first strand of RNA for meiotic silencing their abilities to silence paired wild-type copies of the (the predicted template for the RdRP) does not obey gene. We call this test trans-silencing. If promoter ele- the known rules for transcription (i.e., the absolute re- ments are required, segments containing them will si- quirement for promoter elements observed for meta- lence in trans, whereas those lacking them will not. bolic genes in haploid tissues, for example). Similarly, any silencing-active fragment of Asm-1 DNA Bigger is better: We consistently found a positive cor- unpaired ectopically—where it is dissociated from its relation between the length of the unpaired DNA region normal flanking DNA in the chromosome—should and the strength of silencing. This observation suggested silence, unless its silencing activity depends on sur- that larger unpaired loops of DNA silence more effi- rounding cis-linked sequences that are normally present ciently than smaller ones. We tested this hypothesis fol- at its wild-type chromosomal position. lowing the same experimental design used to test trans- We performed this analysis by carrying out crosses silencing. between two sets of strains. All strains contained the First, we selected a fragment of DNA that did not si- wild-type asm-1ϩ allele at its normal position in LG V lence Asm-1 in trans (Figures 5 and 6, coordinates 4615– (Figure 5). At the his-3 locus in LG I, Dad chromosomes 6118, crosses 26 and 27). This fragment was selected contained no insert. Mom chromosomes each contained because, when unpaired, it exposes a region of homol- a different Asm-1 insert. We then examined whether ogy to the Asm-1 transcript of only 748 bp, near the these segments of Asm-1 DNA would activate meiotic minimal length required for cis-silencing (Figure 2, trans-silencing when unpaired. crosses 7 and 8). We predicted that if the size of the The Asm-1 region was scanned by unpairing fragments loop matters (i.e., bigger is better), silencing should of 1503 bp (crosses 26 and 27), 1225 bp (cross 28), be enhanced by the addition of “neutral” DNA. The 1438 bp (crosses 29 and 30), 1407 bp (crosses 31 and 32), 1503-bp fragment was therefore fused to 1411 bp of ␭ and 949 bp (crosses 33 and 34). Together, they spanned the DNA, and the fusion construct was integrated at the entire Asm-1 region of the genome (Figure 5). Only his-3 locus. When the resulting strain was crossed to wild the fragment spanning coordinates 7035–8442 induced type, Asm-1 was silenced (Figure 6, crosses 45 and 46). silencing (crosses 31 and 32). This result contrasted Since the only difference between crosses 26, 27 and with what was observed in cis-silencing (Figures 1–4). 45, 46 is the total length of unpaired DNA (i.e., the kb carrying regions of length of the region of homology to the transcript was 1.4ف In cis, unpaired loops of kb induced kept constant), this result implies that efficient silencing 0.7ف homology to the transcript as small as silencing (Figure 2, cross 10), whereas under the experi- is indeed correlated with the size of the loop. We also mental conditions tested here, an unpaired region of predicted that holding the length of unpaired DNA kb) while increasing the extent of 2.9ف ,.kb belonging entirely to the transcript could not constant (i.e 1.4ف induce silencing in trans (Figure 5, crosses 29 and 30). homology to the transcript should enhance silencing. It seemed conceivable that silencing was not observed The same 1503-bp Asm-1 fragment tested before was in some cases because the ectopic fragments lack expres- therefore fused to a 1438-bp fragment internal to the sion signals that are normally present. To ensure that Asm-1 coding region (coordinates 6320–7758). Crosses an absence of transcription was not responsible for the between wild-type strains and strains carrying this fusion failure to silence, we constructed a fusion of an internal construct at the his-3 locus resulted in very efficient Properties of Unpaired DNA 139

TABLE 2 Genetic crosses

Relevant genotypeb LG I Total Mature LG V: ascospores ascospores Cross no.a mat his-3 Asm-1 Parents examinedc (%)c Observationsa,b Silencing?d

1 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT03A [9336–3426]; ϫϫ664 94.4 No a his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT05A [9336–3426];

2 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT03A [9336–3426]; ϫϫ897 94.4 No a his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT06A [9336–3827];

3 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT03A [9336–3426]; ϫϫ653 94.6 No a his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT07A [9336–4615];

4 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT03A [9336–3426]; ϫϫ1148 95.2 No a his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT08A [9336–4999];

5 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT03A [9336–3426]; ϫϫ1011 94.6 No a his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT09A [9336–5272];

6 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT03A [9336–3426]; ϫϫ825 91.3 No a his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT10A [9336–5437];

7 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT03A [9336–3426]; ϫϫ612 51.9 4:4 segregation No a his-3ϩ::Asm-1 Asm-1⌬(3426–9336) KYNCT11A [9336–5714];

8 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT03A [9336–3426]; ϫϫ652 3.7 4:4 segregation Yes (ϩϩϩϩϩ) a his-3ϩ::Asm-1 Asm-1⌬(3426–9336) KYNCT31B [9336–6107];

9 A his-3ϩ::Asm-1 Asm-1⌬(3426–9336) KYNCT30A [9336–6690]; ϫϫHundreds Ͻ1 4:4 segregation Yes (ϩϩϩϩϩ) a his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT05A [9336–3426]; (continued) 140 D. W. Lee et al.

TABLE 2 (Continued)

Relevant genotypeb LG I Total Mature LG V: ascospores ascospores Cross no.a mat his-3 Asm-1 Parents examinedc (%)c Observationsa,b Silencing?d

10 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT04A [9336–4615]; ϫϫ1042 9.7 4:4 segregation Yes (ϩϩϩϩ) a his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT31B [9336–6107];

11 A his-3ϩ; asm-1ϩ RANCR49A ϫϫThousands Ͼ99 Perithecial tissue No was harvested 6 days after fertilization a his-3; asm-1ϩ RANCR06A

12 A his-3ϩ; asm-1ϩ RANCR49A ϫϫThousands Ͻ1 Perithecial tissue Yes was harvested (ϩϩϩϩϩ) 6 days after fertilization a his-3; Asm-1⌬(3426–9336) KYNCT02A

13 A his-3ϩ; asm-1ϩ RANCR49A ϫϫThousands Ͻ1 Perithecial tissue Yes was harvested (ϩϩϩϩϩ) 6 days after fertilization a his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT05A [9336–3426];

14 A his-3; asm-1ϩ RANCR05A ϫϫThousands Ͼ99 Perithecial tissue No was harvested 6 days after fertilization a his-3ϩ; asm-1ϩ RANCR50A

15 A his-3; Asm-1⌬(3426–9336) KYNCT01A ϫϫThousands Ͻ1 Perithecial tissue Yes was harvested (ϩϩϩϩϩ) 6 days after fertilization a his-3ϩ; asm-1ϩ RANCR50A

16 A his-3ϩ::Asm-1ϩ Asm-1⌬(3426–9336) KYNCT03A [9336–3426]; ϫϫThousands Ͻ1 Perithecial tissue Yes was harvested (ϩϩϩϩϩ) 6 days after fertilization a his-3ϩ; asm-1ϩ RANCR50A

17 A his-3; asm-1ϩ RANCR05A ϫϫ794 Ͼ97 No a his-3; asm-1ϩ RANCR06A (continued) Properties of Unpaired DNA 141

TABLE 2 (Continued)

Relevant genotypeb LG I Total Mature LG V: ascospores ascospores Cross no.a mat his-3 Asm-1 Parents examinedc (%)c Observationsa,b Silencing?d

18 A his-3; asm-1ϩ RANCR05A ϫϫThousands Ͼ99 No a his-3; asm-1ϩ[⌬(4615–5469)] KYNCT20A

19 A his-3; asm-1ϩ RANCR05A ϫϫ774 47.5 4:4 segregation No a his-3; asm-1⌬(4615–5977) KYNCT21A

20 A his-3; asm-1ϩ RANCR05A ϫϫ1048 3 4:4 segregation Yes (ϩϩϩϩϩ) a his-3; Asm-1⌬(4615–6442) KYNCT23A

21 A his-3; asm-1ϩ RANCR05A ϫϫ895 5.4 4:4 segregation Yes (ϩϩϩϩϩ) a his-3; Asm-1⌬(4615–6690) KYNCT24A

22 A his-3; asm-1ϩ RANCR05A ϫϫ650 5.5 4:4 segregation Yes (ϩϩϩϩϩ) a his-3; Asm-1⌬(4615–7012) KYNCT25A

23 A his-3; Asm-1⌬(3426–9336) KYNCT01A ϫϫ596 0 4:4 segregation Yes (ϩϩϩϩϩ) a his-3; asm-1ϩ RANCR06A

24 A his-3; asm-1ϩ RANCR05A ϫϫ315 0 4:4 segregation Yes (ϩϩϩϩϩ) a his-3; Asm-1⌬(3426–9336) KYNCT02A

25 A his-3; asm-1ϩ RANCR05A ϫϫ361 34.6 4:4 segregation Yes (ϩϩ) a his-3; Asm-1⌬(5414–6690) KYNCT53A

26 A his-3; asm-1ϩ RANCR05A ϫϫ3381 84.5 Ϯ 2.3 RIPe No a his-3ϩ::Asm-1 asm-1ϩ KYNCT80A [6118–4615];

26B A his-3; asm-1ϩ RANCR05A ϫϫ1249 84.5 Ϯ 2.3 RIPe, both strains No heterokaryons with FGSC 4564 a his-3ϩ::Asm-1 asm-1ϩ KYNCT80A [6118–4615];

27 A his-3ϩ::Asm-1 asm-1ϩ KYNCT57A [6118–4615]; ϫϫ5221 84.5 Ϯ 2.3 RIPe No a his-3; asm-1ϩ RANCR06A

27B A his-3ϩ::Asm-1 asm-1ϩ KYNCT57A [6118–4615]; (continued) 142 D. W. Lee et al.

TABLE 2 (Continued)

Relevant genotypeb LG I Total Mature LG V: ascospores ascospores Cross no.a mat his-3 Asm-1 Parents examinedc (%)c Observationsa,b Silencing?d

ϫϫ1257 84.5 Ϯ 2.3 RIPe, both strains No heterokaryons with FGSC 4564 a his-3; asm-1ϩ RANCR06A

28 A his-3; asm-1ϩ RANCR05A ϫϫ1622 92 RIPe No a his-3ϩ::Asm-1 asm-1ϩ KBNCT04A [6690–5465];

29 A his-3; asm-1ϩ RANCR05A ϫϫ7592 81.5 Ϯ 1.6 RIPe No a his-3ϩ::Asm-1 asm-1ϩ DLNCT45A [7758–6320];

29B A his-3ϩ; asm-1ϩ RANCR49A ϫϫ1330 81.5 Ϯ 1.6 RIPe No a his-3ϩ::Asm-1 asm-1ϩ DLNCT45A [7758–6320];

30 A his-3ϩ::Asm-1 asm-1ϩ DLNCT44A [7758–6320]; ϫϫ5997 81.5 Ϯ 1.6 RIPe No a his-3; asm-1ϩ RANCR06A

30B A his-3ϩ::Asm-1 asm-1ϩ DLNCT44A [7758–6320]; ϫϫ1345 81.5 Ϯ 1.6 RIPe No a his-3ϩ; asm-1ϩ RANCR50A

31 A his-3; asm-1ϩ RANCR05A ϫϫ1867 56.5 Ϯ 3.3 RIPe Yes (ϩϩ) a his-3ϩ::Asm-1 asm-1ϩ DLNCT153A [8442–7035];

32 A his-3ϩ::Asm-1 asm-1ϩ DLNCT152A [8442–7035]; ϫϫ1153 56.5 Ϯ 3.3 RIPe Yes (ϩϩ) a his-3; asm-1ϩ RANCR06A

33 A his-3; asm-1ϩ RANCR05A ϫϫ200 90.7 Ϯ 2.3 RIPe No a his-3ϩ::Asm-1 asm-1ϩ DLNCT47A [9336–8387];

34 A his-3ϩ::Asm-1 asm-1ϩ DLNCT46A [9336–8387]; ϫϫ169 90.7 Ϯ 2.3 RIPe No a his-3; asm-1ϩ RANCR06A

35 A his-3; asm-1ϩ RANCR05A ϫϫ5208 80.3 Ϯ 7.3 RIPe No (continued) Properties of Unpaired DNA 143

TABLE 2 (Continued)

Relevant genotypeb LG I Total Mature LG V: ascospores ascospores Cross no.a mat his-3 Asm-1 Parents examinedc (%)c Observationsa,b Silencing?d

a his-3ϩ::trpC asm-1ϩ KYNCT77A [890–1245]:: Asm-1 [6324–7764];

35B A his-3; asm-1ϩ RANCR05A ϫϫ757 80.3 Ϯ 7.3 RIPe, both strains No heterokaryons with FGSC 4564 a his-3ϩ::trpC asm-1ϩ KYNCT77A [890–1245]:: Asm-1 [6324–7764];

36 A his-3ϩ::trpC asm-1ϩ KYNCT60A [890–1245]:: Asm-1 [6324–7764]; ϫϫ3169 80.3 Ϯ 7.3 RIPe No a his-3; asm-1ϩ RANCR06A

36B A his-3ϩ::trpC asm-1ϩ KYNCT60A [890–1245]:: Asm-1 [6324–7764]; ϫϫ1084 80.3 Ϯ 7.3 RIPe, both strains No heterokaryons with FGSC 4564 a his-3; asm-1ϩ RANCR06A

37 A his-3; asm-1ϩ RANCR05A ϫϫ1736 43.4 Ϯ 7.5 RIPe Yes (ϩϩϩ) a his-3ϩ::Asm-1 asm-1ϩ KYNCT47A [7012–4615];

38 A his-3ϩ::Asm-1 asm-1ϩ KYNCT46A [7012–4615]; ϫϫ1978 43.4 Ϯ 7.5 RIPe Yes (ϩϩϩ) a his-3; asm-1ϩ RANCR06A

39 A his-3; asm-1ϩ RANCR05A ϫϫ1485 33.5 Ϯ 8.1 RIPe Yes (ϩϩϩϩ) a his-3ϩ::Asm-1 asm-1ϩ DLNCT151A [8082–6018];

40 A his-3ϩ::Asm-1 asm-1ϩ DLNCT150A [8082–6018]; ϫϫ1738 33.5 Ϯ 8.1 RIPe Yes (ϩϩϩϩ) a his-3; asm-1ϩ RANCR06A (continued) 144 D. W. Lee et al.

TABLE 2 (Continued)

Relevant genotypeb LG I Total Mature LG V: ascospores ascospores Cross no.a mat his-3 Asm-1 Parents examinedc (%)c Observationsa,b Silencing?d

41 A his-3; asm-1ϩ RANCR05A ϫϫ570 0.5 Ϯ 0.6 RIPe Yes (ϩϩϩϩϩ) a his-3ϩ::Asm-1 asm-1ϩ KYNCT55A [9336–6690];

42 A his-3ϩ::Asm-1 asm-1ϩ KYNCT54A [9336–6690]; ϫϫ636 0.5 Ϯ 0.6 RIPe Yes (ϩϩϩϩϩ) a his-3; asm-1ϩ RANCR06A

43 A his-3; asm-1ϩ RANCR05A ϫϫ555 0.1 RIPe Yes (ϩϩϩϩϩ) a his-3ϩ::Asm-1 asm-1ϩ KYNCT35A [9336–3426];

44 A his-3ϩ::Asm-1 asm-1ϩ KYNCT34A [9336–3426]; ϫϫ643 0.03 RIPe Yes (ϩϩϩϩϩ) a his-3; asm-1ϩ RANCR06A

45 A his-3; asm-1ϩ RANCR05A ϫϫ3229 52.2 Ϯ 7.9 RIPe Yes (ϩϩϩ) a his-3ϩ::␭ asm-1ϩ KYNCT88A [5818–7229]:: Asm-1 [6118–4615];

45B A his-3; asm-1ϩ RANCR05A ϫϫ1011 52.2 Ϯ 7.9 RIPe, both strains Yes heterokaryons (ϩϩϩ) with FGSC 4564 a his-3ϩ::␭ asm-1ϩ KYNCT88A [5818–7229]:: Asm-1 [6118–4615];

46 A his-3ϩ::␭ asm-1ϩ KYNCT64A [5818–7229]:: Asm-1 [6118–4615]; ϫϫ2944 52.2 Ϯ 7.9 RIPe Yes (ϩϩϩ) a his-3; asm-1ϩ RANCR06A

46B A his-3ϩ::␭ asm-1ϩ KYNCT64A [5818–7229]:: Asm-1 [6118–4615]; ϫϫ1221 52.2 Ϯ 7.9 RIPe, both strains Yes heterokaryons (ϩϩϩ) with FGSC 4564 (continued) Properties of Unpaired DNA 145

TABLE 2 (Continued)

Relevant genotypeb LG I Total Mature LG V: ascospores ascospores Cross no.a mat his-3 Asm-1 Parents examinedc (%)c Observationsa,b Silencing?d

a his-3; asm-1ϩ RANCR06A

47 A his-3; asm-1ϩ RANCR05A ϫϫ4045 21.7 Ϯ 7.8 RIPe Yes (ϩϩϩϩ) a his-3ϩ::Asm-1 asm-1ϩ KYNCT86A [7758–6320] [6118–4615];

47B A his-3; asm-1ϩ RANCR05A ϫϫ1148 21.7 Ϯ 7.8 RIPe, both strains Yes heterokaryons (ϩϩϩϩ) with FGSC 4564 a his-3ϩ::Asm-1 asm-1ϩ KYNCT86A [7758–6320] [6118–4615];

48 A his-3ϩ::Asm-1 asm-1ϩ KYNCT58A [7758–6320] [6118–4615]; ϫϫ5166 21.7 Ϯ 7.8 RIPe Yes (ϩϩϩϩ) a his-3; asm-1ϩ RANCR06A

48B A his-3ϩ::Asm-1 asm-1ϩ KYNCT58A [7758–6320] [6118–4615]; ϫϫ1158 21.7 Ϯ 7.8 RIPe, both strains Yes heterokaryons (ϩϩϩϩ) with FGSC 4564 a his-3; asm-1ϩ RANCR06A

49 A his-3; asm-1ϩ RANCR05A ϫϫ4176 61.9 Ϯ 5.5 RIPe Yes (ϩϩ) a his-3ϩ::Asm-1 asm-1ϩ KYNCT76A [7758–6320] [5467–4615];

49B A his-3; asm-1ϩ RANCR05A ϫϫ1269 61.9 Ϯ 5.5 RIPe, both strains Yes heterokaryons (ϩϩ) with FGSC 4564 a his-3ϩ::Asm-1 asm-1ϩ KYNCT76A [7758–6320] [5467–4615];

50 A his-3ϩ::Asm-1 asm-1ϩ KYNCT68A [7758–6320] [5467–4615]; ϫϫ4437 61.9 Ϯ 5.5 RIPe Yes (ϩϩ) a his-3; asm-1ϩ RANCR06A (continued) 146 D. W. Lee et al.

TABLE 2 (Continued)

Relevant genotypeb LG I Total Mature LG V: ascospores ascospores Cross no.a mat his-3 Asm-1 Parents examinedc (%)c Observationsa,b Silencing?d

50B A his-3ϩ::Asm-1 asm-1ϩ KYNCT68A [7758–6320] [5467–4615]; ϫϫ1179 61.9 Ϯ 5.5 RIPe, both strains Yes heterokaryons (ϩϩ) with FGSC 4564 a his-3; asm-1ϩ RANCR06A

51 A his-3; asm-1ϩ RANCR05A ϫϫ9645 60.3 Ϯ 3.8 RIPe Yes (ϩϩ) a his-3ϩ::␭ asm-1ϩ DLNCT55A [26104–21226]:: Asm-1 [7758–6320];

52 A his-3ϩ::␭ asm-1ϩ DLNCT54A [26104–21226]:: Asm-1 [7758–6320]; ϫϫ6174 60.3 Ϯ 3.8 RIPe Yes (ϩϩ) a his-3; asm-1ϩ RANCR06A

53 A his-3; asm-1ϩ RANCR05A ϫϫ175 30.4 RIPe Yes (ϩϩϩϩ) a his-3ϩ::␭ asm-1ϩ DLNCT57A [31747–39168]:: Asm-1 [7758–6320];

54 A his-3ϩ::␭ asm-1ϩ DLNCT56A [31747–39168]:: Asm-1 [7758–6320]; ϫϫ155 30.4 RIPe Yes (ϩϩϩϩ) a his-3; asm-1ϩ RANCR06A a When necessary, heterokaryons were constructed between the Griffiths sterile helper strain [Fungal Genetics Stock Center (FGSC) 4564] and the required strains. On those occasions, sexual crosses were set up with both parents homokaryotic and also with both parents heterokaryotic. The results of the homokaryotic and of the heterokaryotic crosses were equivalent. b Complete genotypes are described in Table 1. c Scoring was done either by inspecting the lid of the petri dish or by counting the indicated number of spores as described in materials and methods. d The strength of the observed silencing is defined in the Figure 2 legend. e Reduced percentage of black ascospores in these crosses reflects the vulnerability of one or both parents to repeat-induced point mutation of the Asm-1 gene. silencing (Figure 6, compare the results from crosses coordinates 6320–7758, crosses 29, 30, 35, and 36) was 45 and 46 with crosses 47 and 48). fused to an additional 852-bp fragment of Asm-1 DNA In a further test, the fragment that did not silence (Figure 6). The resulting 2290-bp construct, which has Asm-1 even when coupled to the trpC promoter (Figure 5, 1535 bp of homology to the transcript, was integrated Properties of Unpaired DNA 147

Figure 4.—Testing cis- silencing in linkage group V. The presentation is as in Figure 2, except that the diagram of the diploid zygote cell represents the pairing of a wild-type LG V chromosome (Dad) with a Mom chromosome carrying a deletion allele. The coordinates indicate the unpaired region in the Dad chromosome corresponding to the region that was deleted and replaced by the hphϩ gene in the Mom chromosome.

at the his-3 locus. When this strain was crossed to wild quence homology have evolved in N. crassa. During hap- type, weak silencing was observed (Figure 6, compare the loid development (i.e., vegetative growth), the presence results of crosses 29 and 30 with crosses 49 and 50). of repeated elements can activate a vegetative silencing Finally, we tested whether increasing the length of un- mechanism called quelling (Cogoni and Macino 2000; paired DNA while keeping the length of homology to Cogoni 2001, 2002). If cells containing repeated DNA the transcript constant (i.e., 1438 bp) would result in elements overcome quelling and enter the sexual phase stronger silencing. First, the 6320–7758 fragment of of their life cycle, the sequences are subjected to another Asm-1 DNA was fused to 4878 bp of ␭ DNA. The result- unique silencing mechanism called repeat-induced point ing 6316-bp construct was integrated at the his-3 locus. mutation (RIP; Selker 1990, 1997). In this process, a When strains carrying this construct were crossed to series of GC-to-AT transition mutations are introduced wild type, the result was mild silencing (crosses 51 and into the duplicated sequence. Most of the remaining 52). We next increased the length of the unpaired DNA nonmutated bases are methylated. Both of these mecha- from 6316 to 8859 bp by adding additional ␭ DNA while nisms belong to the repertoire of strategies that Neuro- keeping the length of homology to the transcript con- spora uses to maintain the integrity of its genome. Mei- stant at 1438 bp. When the resulting strain was crossed otic silencing is another such process. to wild type, stronger silencing was observed (Figure 6, This work was aimed at understanding which regions crosses 53 and 54). We therefore conclude that a longer of a gene need to be unpaired during meiosis to signal stretch of unpaired DNA silences more efﬁciently than silencing. Detecting gene silencing during meiosis is not a shorter one. trivial. It requires choosing reporter genes whose products are essential and constantly required for the com- pletion of meiosis or ascospore development or that DISCUSSION can be easily detected (e.g., green ﬂuorescent protein). In summary, a number of elegant molecular mecha- It also requires protein products produced before the nisms to control gene expression based on DNA se- induction of meiotic silencing to be unstable. We used 148 D. W. Lee et al.

Figure 5.—Loops of unpaired DNA need not carry known promoter elements to silence. The presentation is as in Figure 2, with the following alterations. Both LG V chromosomes carry the asm-1ϩ wild-type allele. On LG I, Dad chromosomes have no Asm-1 DNA at his-3, whereas Mom chromosomes carry different Asm-1 alleles inserted at his-3 (open bars). The hatched region indicates the position of the canonical promoter and the transcriptionally active element of Asm-1. The shaded area indicates the position of the region corresponding to the ␣-transcript of the gene.

Asm-1 because its gene product (ASM-1) fulﬁlls both these ﬁciency by the unpaired DNA, (2) the presence of ca- requirements (Aramayo and Metzenberg 1996b). nonical promoter elements in the unpaired loop is not In this report we established that: (1) The size of the required for silencing, (3) the unpairing of promoter loop and the degree of homology to the reporter tran- elements does not affect their transcriptional compe- script (i.e., Asm-1) directly correlate with the silencing ef- tence later, during spore development, (4) the ␣-tran-

Figure 6.—Bigger is better. The presentation is as in Figure 5. In crosses 45 and 46, the unpaired Asm-1 fragment corresponding to coordinates 4615–6118 was augmented by the addition of 1411 bp of DNA from bacteriophage ␭. In crosses 47 and 48, the same fragment was fused to a 1438-bp fragment internal to the Asm-1 coding region (coordinates 6320–7758). Simi- larly, the 1438-bp fragment internal to the Asm-1 coding region (coordinates 6320– 7758) was fused to a 852-bp upstream fragment (coordinates 4615–5467). In crosses 51–54, the loop of unpaired DNA of the Asm-1 fragment corresponding to coordinates 6320–7758 was augmented by the addition of 4878 bp (crosses 51 and 52) and 7421 bp (crosses 53 and 54), respectively, of DNA from bacteriophage ␭. Properties of Unpaired DNA 149

with the size of the loop. The complex is more active in larger loops than in smaller ones. In model II, unpaired DNA is the target of multiple transcription complexes, all of them with similar transcriptional activities. Larger loops silence more efficiently because they accommodate more transcription complexes than smaller ones do. The net result is the same. The larger the loop, the higher is the concentration of postulated aRNAs and small interfering RNAs (siRNAs) that would be produced by the meiotic silencing pathway. Paired genes do not misbehave. Unpaired genes do, regardless of their genomic location. If pairing occurs, meiosis proceeds normally. If a gene is unpaired, then the unpaired DNA will be detected by the meiotic silenc- Figure 7.—Models for transcription of unpaired DNA. In ing machinery. If the unpaired region is small, it poses model I, the transcriptional activity of the single complex present in each loop is represented by the number of concen- no danger and elicits little or no response. If it is large, tric circles. The transcription complex is more active in larger it activates meiotic silencing. In this regard, the meiotic loops than in smaller ones. In model II, each complex has machinery involved in chromosome pairing is extremely an equivalent transcriptional activity, but larger loops silence sensitive. Even the largest loop tested in this work (i.e., more efficiently because they accommodate more transcrip- 8859 bp) corresponds to Ͻ0.09% of LG I. A loop this size tion complexes than do smaller ones. The outcome is the same: the larger the loop, the higher the concentration of should have negligible effect on chromosome pairing. siRNAs predicted to be produced by the unpaired DNA. Clearly, the combined activities of meiotic transvection and meiotic silencing perform a critical check of the sequence composition of the genomes participating in meiosis. For example, an infection by retrotrans- cript of Asm-1 is a target for meiotic silencing, and (5) posable elements in one of the participating genomes the leader of Asm-1 contains a DNA element capable of would reveal itself through the presence of unpaired driving its expression during sexual development. DNA corresponding to the invading elements. Their Our results provide strong confirmatory evidence that active presence at this particular developmental stage meiotic silencing is post-transcriptional, because silenc- constitutes a grave danger to the integrity of the ge- ing of Asm-1 can be achieved only by unpairing regions nomes because of the highly recombinogenic nature of with homology to the transcript. They also raise several meiotic cells. From an evolutionary perspective, meiotic important questions. How is the unpaired DNA tran- silencing does not impose a constraint on rearrange- scribed? And why do long stretches of unpaired DNA ments in intergenic regions or regions of genes whose silence more efficiently than smaller ones? products are not essential for meiosis or ascospore devel- Promoter elements need not be present in the un- opment. It does, however, limit the rate of evolution of paired DNA to induce silencing, suggesting that un- critical components of meiosis and ascospore develop- paired DNA is the target of a modified form of a known ment. In this scenario, two rapidly evolving but related RNA polymerase. Alternatively, meiotic chromosomes genomes could accumulate a number of noncritical si- might be the target of an undescribed form of transcrip- lent rearrangements without losing their interbreeding tion that originates in paired regions and extends into ability, up to a point, where a rearrangement in one unpaired DNA, whenever present. Whatever the mecha- critical gene could result in their reproductive isolation. nism of transcription of unpaired DNA, the postulated It is thus appropriate to consider meiotic silencing not aberrant RNAs (aRNAs) produced likely do not need only as another checkpoint for meiosis, but also as a poly(A) tails to play their part in meiotic silencing, as critical mechanism of reproductive isolation. evidenced by the strong silencing activity of truncated fragments of the gene. Moreover, these “truncated” We thank Michael D. Manson for critical review of the manuscript. This work was supported by U.S. Public Health Service grant GM58770 transcripts either must be immediately converted into to R.A. double-strand RNAs by the SAD-1 RdRP or somehow escape the RNA-quality-control mechanisms that oper- ate in eukaryotic cells (Moore 2002), unless, of course, LITERATURE CITED those mechanisms are inactive during early meiosis. Aramayo, R., and R. L. 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