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DNA Methylation Is Independent of RNA Interference in Neurospora

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Science  25 Jun 2004:
Vol. 304, Issue 5679, pp. 1939
DOI: 10.1126/science.1099709

RNA has emerged as an important regulatory substance. Studies with worms, plants, and the filamentous fungus Neurospora crassa have demonstrated that double-stranded RNA (dsRNA) can cause degradation of matching mRNA, resulting in RNA interference (RNAi) (1). In fission yeast [reviewed in (2)] and Drosophila (3), components of the RNAi machinery are also involved in the formation of heterochromatin, including localization of a key heterochromatin protein, HP1. In addition, ties have been found between RNAi and DNA methylation, a common feature of heterochromatin in many organisms including mammals and Neurospora (but absent in fission yeast and scant in Drosophila). In tobacco and Arabidopsis, dsRNA can induce DNA methylation, and Arabidopsis mutants with defects in components of the RNAi machinery show defects in de novo methylation (2, 4). Here we show that DNA methylation and HP1 localization occur normally in Neurospora in the absence of key elements of the RNA-silencing machinery.

Neurospora has genes for three RNA-dependent RNA polymerase (RdRP), two dicer, two argonaute, and two RecQ helicase homologs (5). Genetic experiments demonstrated that most of these are components of one or both of two Neurospora RNA-silencing pathways (table S1): quelling (which silences genes homologous to transgenes in vegetative cells) and meiotic silencing (which reversibly silences unpaired genes and any homologs during meiosis). To determine whether Neurospora relies on RNA silencing to induce DNA methylation, we analyzed DNA of mutants defective in components of the RNAi machinery. Southern blots showed full DNA methylation in all chromosomal regions tested, in every mutant and with every combination of mutations (e.g., Sms-3 dcl-2 double dicer and Sad-1 qde-1 rrp-3 triple RdRP mutants) (Fig. 1A). Moreover, upon transformation of RNA-silencing mutants, unmethylated test segments (hph and amRIP8) reliably signaled de novo DNA methylation (Fig. 1, B and C). Thus, establishment and maintenance of DNA methylation in Neurospora are independent of both quelling and meiotic silencing. They also do not depend on RRP-3, an RdRP of unknown function.

Fig. 1.

(A) DNA methylation is maintained in silencing mutants. DNA of the wild type (WT), the DNA methyltransferase DIM-2 mutant (DIM-2), a Sms-3 dcl-2 double dicer mutant (Dicer), and a Sad-1 qde-1 rrp-3 triple RdRP mutant (RdRP) was digested with 5-methylcytosine (5mC)–sensitive Sau 3AI (S) and its 5mC-insensitive isoschizomer, Dpn II (D), and probed for methylated regions (7). Eight additional methylated regions of varying copy number also retained methylation (8). Positions of the origin (Ori) and size standards are shown. (B and C) De novo DNA methylation is normal in silencing mutants. (B) The Sad-1 qde-1 rrp-3 his-3 mutant (H) was cotransformed with hph and his-3+ (+ hph/his-3+). (C) The Sms-3 dcl-2 mutant (H) was cotransformed with amRIP8 and a dominant β-tubulin (Bml) allele that confers resistance to benomyl (+ amRIP8/Bml). DNA of independent transformants (identified by number) was digested as in (A) and probed for am or hph. (D) HP1-GFP is localized to heterochromatin in Sms-3 dcl-2 mutants. Foci observed in mutants (Dicer) and the wild type represent heterochromatin, as we demonstrated by counterstaining DNA with Hoechst 33258 (8). In contrast, HP1-GFP is dispersed in nuclei of dim-5Y178V, a strain without DIM-5 activity (Dim-5). Equivalent results were obtained with other RNAi mutants (table S1).

Localization of Neurospora HP1 is controlled by the histone H3 Lys9 methyltransferase DIM-5 and depends on Lys9 methylation (6). We used green fluorescent protein–tagged HP1 (HP1-GFP) to test HP1 localization in RNA-silencing mutants (Fig. 1D). Localization was normal in all silencing mutants, indicative of normal methylation of Lys9, in contrast to a control strain without DIM-5 activity.

We conclude that RNA silencing is not universally involved in formation and maintenance of heterochromatin and that RNA-mediated DNA methylation, although prevalent in plants, is not “a general means of targeting de novo methylation” (4). At least in Neurospora, RNA-mediated silencing and silencing by DNA methylation are independent genome defense systems. It remains to be seen whether DNA methylation in mammals depends, at least in part, on components of the RNAi machinery, as in plants, or if it is independent of RNAi, as in Neurospora.

Supporting Online Material

www.sciencemag.org/cgi/content/full/304/5679/1939/DC1

Table S1

References and Notes

References and Notes

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