1 DDM 1 and Lsh remodelers allow methylation of DNA wrapped in nucleosomes 1 2

9 Cytosine methylation regulates essential genome functions across eukaryotes, but the 10 fundamental question of whether nucleosomal or naked DNA is the preferred substrate of plant 11 and animal methyltransferases remains unresolved. Here, we show that genetic inactivation of a 12 single DDM1/Lsh family nucleosome remodeler biases methylation toward inter-nucleosomal 13 linker DNA in Arabidopsis thaliana and mouse. We find that DDM1 enables methylation of 14 DNA bound to the nucleosome, suggesting that nucleosome-free DNA is the preferred substrate 15 of eukaryotic methyltransferases in vivo. Furthermore, we show that simultaneous mutation of 16 DDM1 and linker histone H1 in Arabidopsis reproduces the strong linker-specific methylation 17 patterns of species that diverged from flowering plants and animals over a billion years ago. Our 18 results indicate that in the absence of remodeling, nucleosomes are strong barriers to DNA 19 methyltransferases. Linker-specific methylation can evolve simply by breaking the connection 20 between nucleosome remodeling and DNA methylation. 21