RNA Interference Regulates Histone Methylation in Eukaryotic Heterochromatin

While the experiments in Selker's laboratory (see p. 157) were in progress, Andrew Fire and Craig Mello and colleagues66 discovered that double-stranded RNA (dsRNA) was much more potent than sense or antisense single strands of RNA at silencing gene expression in Caenorhabditis elegans. They found that only a few molecules of dsRNA were required to cause silencing. They suggested that a catalytic or amplification step might be involved, and named this phenomenon RNA interference (RNAi).

In a study using fission yeast (S. pombe) as a model, Shiv Grewal and Robert Martiennssen and colleagues67 obtained evidence suggesting histone modifications were guided by RNAi by deleting several genes (argonaute, dicer, and RNA-dependent RNA polymerase) that encode part of the molecular machinery of RNAi. Deletion resulted in aberrant accumulation of complementary transcripts from centromeric heterochromatic repeats, as well as transcriptional derepression of transgenes located at the centromere, loss of histone H3 Lys9 methylation, and loss of centromere function. For intact cells, they explained their findings by the following mechanism: dsRNA derived from repeat sequences in heterochromatin would trigger RNAi that would initiate histone 3 lysine9 methylation. The covalently modified histone would then signal DNA methylation. This mechanism could guide eukaryotic methyltransferases to specific regions of the genome, such as retroposons and other parasitic elements. They believed this arrangement could be reinforced by maintenance methyltransferase activity as well as by histone deacetylation guided by CpG-methyl-binding proteins.

RNAi is still a comparatively new model in regulatory biology,68 and the mechanistic complexity of the process and its biological ramifications are only beginning to be appreciated. The technique has been harnessed for the analysis of gene function in several diverse organisms and systems including plants, fungi, and metazoans, but its use in mammalian systems has lagged behind somewhat.68 The first indication that RNAi could induce gene silencing in mammals came from observations in early mouse embryos and numerous mammalian cell lines, but silencing in these systems was transient. By utilizing long, hairpin dsRNAs, Paddison and colleagues69 have recently succeeded in creating stable gene silencing in mouse cell lines substantially increasing the power of RNAi as a genetic tool. The ability to create permanent cell lines with stable "knockdown" phenotypes extends the utility of RNAi in several ways, one of which will be its application to epigenetics research.

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