title: Small interfering RNAs that trigger posttranscriptional gene silencing are not required for the histone H3 Lys9 methylation necessary for transgenic tandem repeat stabilization in Neurospora crassa.
abstract: In Neurospora crassa, the introduction of a transgene can lead to small interfering RNA (siRNA)-mediated posttranscriptional gene silencing (PTGS) of homologous genes. siRNAs can also guide locus-specific methylation of Lys9 of histone H3 (Lys9H3) in Schizosaccharomyces pombe. Here we tested the hypothesis that transgenically derived siRNAs may contemporaneously both activate the PTGS mechanism and induce chromatin modifications at the transgene and the homologous endogenous gene. We carried out chromatin immunoprecipitation using a previously characterized albino-1 (al-1) silenced strain but detected no alterations in the pattern of histone modifications at the endogenous al-1 locus, suggesting that siRNAs produced from the transgenic locus do not trigger modifications in trans of those histones tested. Instead, we found that the transgenic locus was hypermethylated at Lys9H3 in our silenced strain and remained hypermethylated in the quelling defective mutants (qde), further demonstrating that the PTGS machinery is dispensable for Lys9H3 methylation. However, we found that a mutant in the histone Lys9H3 methyltransferase dim-5 was unable to maintain PTGS, with transgenic copies being rapidly lost, resulting in reversion of the silenced phenotype. These results indicate that the defect in PTGS of the Deltadim-5 strain is due to the inability to maintain the transgene in tandem, suggesting a role for DIM-5 in stabilizing such repeated sequences. We conclude that in Neurospora, siRNAs produced from the transgenic locus are used in the RNA-induced silencing complex-mediated PTGS pathway and do not communicate with an RNAi-induced initiation of transcriptional gene silencing complex to effect chromatin-based silencing.
authors.affiliation: Istituto Pasteur e Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Universita di Roma La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy.
authors.forename: Agustin
authors.initials: A
authors.lastname: Chicas
authors.forename: Emma C
authors.initials: EC
authors.lastname: Forrest
authors.forename: Silvia
authors.initials: S
authors.lastname: Sepich
authors.forename: Carlo
authors.initials: C
authors.lastname: Cogoni
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Macino
mesh_terms: D047369:Chromatin Immunoprecipitation; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000502:physiology*; D006657:Histones / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D009154:Mutation / Q000235:genetics; D009492:Neurospora crassa / Q000235:genetics* / Q000378:metabolism; D011496:Protein Methyltransferases; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000502:physiology*; D020080:Tandem Repeat Sequences / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D019076:Transgenes
chemical_list: D006657:Histones; D034741:RNA, Small Interfering; D000076983:Histone Methyltransferases; D011496:Protein Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine