Properties of Unpaired DNA Required For Efficient Silencing in Neurospora crassa

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.

Neurospora crassa, a Model System for Epigenetics Research

“…Silencing was observed, implying that meiotic silencing results from the presence of unpaired alleles rather than from absence of paired ones [ Fig. 8F; asm-1 + × asm-1 + , asm-1 +(ectopic) ] (Shiu et al 2001;Kutil et al 2003;Lee et al 2003;Lee et al 2004). …”

“…Some of the quantitative and qualitative aspects of the "sensing" threshold have been addressed, however . The findings can be summarized as follows: (1) Given one small and one large loop of unpaired DNA, both carrying the same length of DNA homologous to a set of paired reporter genes, the large loop will silence more efficiently than the smaller one; (2) given two loops of identical size, but one carrying twice as much DNA homologous to a set of paired reporter genes, the loop carrying more homologous DNA will silence more efficiently than the one carrying less homologous DNA; (3) the silencing signal produced by an unpaired loop is confined to the unpaired region and does not "spread" to neighboring regions (e.g., paired reporter genes can be located next to a region of unpaired DNA without being significantly perturbed); (4) the canonical promoter of a gene need not be present in the loop of unpaired DNA for a gene to be silenced; and (5) meiotic silencing does not affect the ability of a promoter to direct transcription at a later developmental time (Kutil et al 2003;Lee et al 2003Lee et al , 2004Pratt et al 2004).…”

Efficient Detection of Unpaired DNA Requires a Member of the Rad54-Like Family of Homologous Recombination Proteins

“…It has the ability to identify and silence an unpaired segment of DNA as small as 700 bp between homologous chromosomes that are millions of base pairs long (Lee et al 2004). Unfortunately, the identities of the proteins that mediate unpaired DNA detection have remained elusive.…”

Relationship between allelic state of T-DNA and DNA methylation of chromosomal integration region in transformed Arabidopsis thaliana plants

“…Homozygous seeds produced by a hemizygous parent contain heavily methylated cytosines, at symmetrical as well as nonsymmetrical positions, in the region between the T-DNA insertion and the ATG codon. In the haploid Neurospora crassa, a gene not paired with its homologue in prophase I of meiosis forms a DNA loop, initiating Meiotic Silencing by Unpaired DNA (MSUD) (Shiu et al, 2001;Lee et al, 2004). This loop is detected and leads to a transient silencing by a posttranscriptional mechanism of all homologous sequences, including those that are themselves paired.…”