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. 2005 May;25(9):3793-801.
doi: 10.1128/MCB.25.9.3793-3801.2005.

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

Agustin Chicas  1 , Emma C Forrest, Silvia Sepich, Carlo Cogoni, Giuseppe Macino
Affiliations

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

Agustin Chicas et al. Mol Cell Biol. 2005 May.

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.

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Figures

FIG. 1.
FIG. 1.
Lys9H3 hypermethylation of the transgenic but not the endogenous al-1 loci. ChIP analysis showed that (A) there was no enrichment in immunoprecipitated endogenous al-1 DNA using the following antibodies: H3, nonacetylated H3; AK9/K14H3, histone H3 acetylated in Lys9/Lys14; 3mK4H3, trimethylated in Lys4; Tetra AH4, tetra-acetylated H4; AK8H4, histone H4 acetylated in Lys8; 3mK9H3, trimethylated in Lys9; 3mK27H3, trimethylated in Lys27. (B) Instead, the transgenic al-1 locus shows a sixfold enrichment of Lys9H3 methylation. The error bars represent the standard deviation of one IP analyzed in triplicate. (C) RT-PCR analysis to detect transgenic transcripts. Reverse transcription was carried out with either the RTSS or RTAS primers, which are immediately upstream of P1 and P2, respectively. One-tenth of the RT reaction volume was used for the PCRs, which were done using the P1-P2 primer pair. The PCR products were run on a 2% agarose gel and visualized by ethidium bromide staining. (D) Schematic diagram of the al-1 transgenic locus compared to the endogenous al-1 gene depicting the position of the different primers used for quantitative PCR. Thick shadow regions represent the al-1 coding regions, the clear areas represent the introns of the al-1 gene, and the solid lines represent the vector sequences. A primer derived from the bacterial vector sequence (P2) and the other from the al-1 gene (P1) were used to detect al-1 transgene-specific amplicons of 89 bp.
FIG. 2.
FIG. 2.
Analysis of Lys9H3 methylation at both transgenic (A) and endogenous (B) loci in the different PTGS-defective (qde) mutants. 6xw is the stably silenced parental strain (10). The genes involved in the PTGS pathway were found through mutagenesis and are as follows: qde-1 is the RNA-dependent RNA polymerase mutant (9), qde-2 is the PPD protein mutant (3), and qde-3 is the putative RecQ helicase mutant (11). The dim-5 mutant knocked out in the 6xw background (see Materials and Methods) and was included as a control. The revertant strain is one that is no longer silenced due to loss of 80% of transgenic copies (10). The error bars represent the standard deviation for two different immunoprecipitations analyzed in duplicate. We used three different antibodies for H3 trimethylated in Lys9; one was a gift from Prim Singh, one was purchased from AbCAM, and the third was a gift from Thomas Jenuwein. All antibodies gave similar results.
FIG. 3.
FIG. 3.
Characterization of the Δdim-5 strain. (A) Western blot analysis showing a reduction in Lys9H3 methylation in the Δdim-5 strain. (B) Southern blot showing loss of transgene methylation in the Δdim-5 strain. Genomic DNA from the parental 6xw strain or the Δdim-5 strain was digested with either Sau3AI (S), which is inhibited by cytosine methylation, or the cytosine methylation-insensitive isoschizomer DpnII (D). The probe used detects an endogenous al-1 product of 521 bases (lane 2) and a transgene product of 583 bases (lane 1). The restriction site that gives the transgene product of 583 bases is methylated in the 6xw strain and hence is not cut efficiently by Sau3AI. This is shown by the reduction in the 583-base band and an increase in higher-molecular-weight bands.
FIG. 4.
FIG. 4.
Southern blots showing the instability of the transgene in the Δdim-5 strain. (A) Revertants show loss of copies of the transgene. Genomic DNAs of 10 revertants (lanes 1 to 10), 2 silenced (S) (12 to 13), and the parental (P) strain (11) were digested with SmaI and HindIII, which cut in the endogenous al-1 locus to give a 3.1-kb band using the appropriate probe. Using this same probe, the transgenic locus gives a band of 5.5 kb because it is only cut by SmaI. In some strains, the transgenic bands are weaker than the endogenous band, since in this analysis the strains were not purified by microconidia (see Materials and Methods), so that some nuclei have completely lost transgenic copies whereas others maintain them. (B) Remaining silenced transformants progressively lost the al-1 transgene in the Δdim-5 strain. Genomic DNA was extracted from mycelia grown for 20 (II), 30 (III), 40 (IV), and 50 (V) generations from both the Δdim-5 strain and the parental strain (6xw), respectively, and digested as indicated in panel A. Loss of transgenic copies are represented in graphic form, using the PhosphorImager to measure the intensity of the bands normalized against the endogenous 3.1-kb band. (C) When Δdim-5 was transformed with the fragment of al-1 (pX16), PTGS occurred in 6% of transformants, represented in this blot as D5 al-1 S (silenced). The transformants were analyzed for copy number and found to contain transgenes in tandem. WT al-1 S (silenced) transformants are shown as a control. Transformants marked 1 or 2 were analyzed for stability in panel D. (D) In the Δdim-5 mutant strains (D5-1, D5-2), transgenic copies are lost in subsequent passages, whereas the WT al-1 silenced (WT-1, WT-2) control maintained the transgenic tandem. II and IV represent about 20 and 40 generations, respectively.
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