SRA

Functional genomic states are maintained by reinforcing chromatin interactions that exclude the components of other states. Plant heterochromatin features methylation of histone H3 at lysine 9 (H3K9me) and extensive DNA methylation. However, DNA methylation is also catalyzed by a mostly euchromatic small RNA-directed pathway (RdDM) thought to seek H3K9me. How RdDM is excluded from H3K9me-rich heterochromatin is unclear. Here we show that without histone H1, RdDM enters heterochromatin, 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 required for small RNA biogenesis, and without H1 small RNA 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 small RNA-generating branch of RdDM from non-CG methylated heterochromatin. Overall design: Bisulfite and RNA sequencing was performed using h1cmt2, h1cmt2shh1, h1cmt2cmt3 plants (1-2 replicates) to examine the effect of H1, non-CG DNA methylation and SHH1 on RNA-directed DNA methylation activity. sRNA-seq was performed using wt, h1, ddm1, h1ddm1, cmt2cmt3, h1cmt2cmt3, met1, and h1met1 plants (3 replicates, except for met1 and h1met1) to examine the effect of H1, non-CG DNA methylation on 24 nt sRNA expression. To explore H3K9me1 and H3K9me2 distribution in wt, h1, ddm1, h1ddm1, cmt2cmt3, and h1cmt2cmt3, antibody against H3K9me1 and H3K9me2 were used to enrich chromatin bound to modified histones.