Abstract
Canonically, heterochromatin formation in fission yeast and metazoans is initiated by di/trimethylation of histone H3 at lysine 9 position by the histone methyltransferase Suv39H1/Clr4, followed by binding of Swi6/HP1 to H3-K9- me2/me3 via its chromodomain. Subsequent self-association of Swi6/HP1 on adjacent nucleosomes or a cooperative interaction between Clr4 and Swi6/HP1 leads to folded heterochromatin structure. HP1 binding to RNA is shown to facilitate its localization at and assembly of heterochromatin in metazoans. Likewise, recruitment of Swi6/HP1 to centromere depends on the RNAi pathway in fission yeast; paradoxically, Swi6/HP1 is also thought to play a role in RNA turnover. Here we provide evidence in support of RNAi-independent recruitment of Swi6. We show that, apart from the low affinity binding to RNAs through its hinge domain, as already reported, Swi6/HP1 displays a hierarchy of increasing binding affinity through its chromodomain to the siRNAs corresponding to specific dg-dh repeats and even stronger binding to the cognate siRNA-DNA hybrids than to the siRNA precursors or general RNAs. Our results support a mechanism of recruitment of Swi6, which is dependent on its specific and high affinity binding to siRNA-DNA hybrid at the dg-dh repeats. This binding, which is independent of, albeit augmented by binding to H3- K9-Me2, leads to heterochromatin formation and silencing. We suggest that the net role of Swi6 in RNA physiology may be regulated by a balance between abundance and affinity of Swi6 towards heterochromatic and euchromatic RNAs and siRNAs.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
To support our claims, we provide additional evidence from our work and other publications. For example, we clearly show that while the binding of Swi6 to siRNAs and siRNA-DNA is independent of its property of binding to H3-K9-me2, the RNA and RNA-DNA hybrid binding facilitates the binding of Swi6 to H3-K9-Me2. We also stress that the binding affinity of Swi6 to siRNA and siRNA-DNA hybrid is much stronger than that for H3-K9-Me2, which justifies our conclusions mentioned above. Further, we provide supporting evidence, like the loss of Swi6 localization to heterochromatin regions by overexpression of RNaseH. Most tellingly, contrary to published claims showing partial reduction of heterochromatin levels of H3-K9-Me levels in the exosome mutant, we show a drastic reduction in the level of Swi6. Supporting this finding we show that the level of the centromere-linked euchromatic transcript associated with Swi6 is enhanced in the swi63K>3A mutant. We cite published work and our data to propose a model wherein the balance between the two fates of Swi6 is governed by the relative binding affinity to siRNA and mRNAs and their abundance.
