Histone H1 prevents non-CG methylation-mediated small RNA biogenesis in Arabidopsis heterochromatin

eLife digest Cells adapt to different roles by turning different groups of genes on and off. One way cells control which genes are on or off is by creating regions of active and inactive DNA, which are created and maintained by different groups of proteins. Genes in active DNA regions can be turned on, while genes in inactive regions are switched off or silenced. Silenced DNA regions also turn off 'transposable elements': pieces of DNA that can copy themselves and move to other regions of the genome if they become active. Transposons can be dangerous if they are activated, because they can disrupt genes or regulatory sequences when they move. There are different types of active and inactive DNA, but it is not always clear why these differences exist, or how they are maintained over time. In plants, such as the commonly-studied weed Arabidopsis thaliana, there are two types of inactive DNA, called E and H, that can silence transposons. In both types, DNA has small chemicals called methyl groups attached to it, which help inactivate the DNA. Type E DNA is methylated by a process called RNA-directed DNA methylation (RdDM), but RdDM is rarely seen in type H DNA. Choi, Lyons and Zilberman showed that RdDM is attracted to E and H regions by previously existing methylated DNA. However, in the H regions, a protein called histone H1 blocks RdDM from attaching methyl groups. This helps focus RdDM onto E regions where it is most needed, because E regions contain the types of transposons RdDM is best suited to silence. When Choi, Lyons and Zilberman examined genetically modified A. thaliana plants that do not produce histone H1, they found that RdDM happened in both E and H regions. There are many more H regions than E regions, so stretching RdDM across both made it less effective at silencing DNA. This work shows how different DNA silencing processes are focused onto specific genetic regions, helping explain why there are different types of active and inactive DNA within cells. RdDM has been studied as a way to affect crop growth and yield by altering DNA methylation. These results may help such studies by explaining how RdDM is naturally targeted. Flowering plants utilize small RNA (sRNA) molecules to guide DNA methyltransferases to genomic sequences. This RNA-directed DNA methylation (RdDM) pathway preferentially targets euchromatic transposable elements. However, RdDM is thought to be recruited by methylation of histone H3 at lysine 9 (H3K9me), a hallmark of heterochromatin. How RdDM is targeted to euchromatin despite an affinity for H3K9me is unclear. Here, we show that loss of histone H1 enhances heterochromatic RdDM, preferentially at nucleosome linker DNA. Surprisingly, this does not require SHH1, the RdDM component that binds H3K9me. Furthermore, H3K9me is dispensable for RdDM, as is CG DNA methylation. Instead, we find that non-CG methylation is specifically associated with sRNA biogenesis, and without H1 sRNA production quantitatively expands to non-CG-methylated loci. Our results demonstrate that H1 enforces the separation of euchromatic and heterochromatic DNA methylation pathways by excluding the sRNA-generating branch of RdDM from non-CG-methylated heterochromatin.