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Background   N  6 -methyldeoxyadenosine (6mA or m 6 dA) was shown more than 40 years ago in simple eukaryotes. Recent studies revealed the presence of 6mA in more prevalent eukaryotes, even in vertebrates. However, functional characterizations have been limited.    Results  We use  Tetrahymena thermophila  as a model organism to examine the effects of 6mA on nucleosome positioning. Independent methods reveal the enrichment of 6mA near and after transcription start sites with a periodic pattern and anti-correlation relationship with the positions of nucleosomes. The distribution pattern can be recapitulated by in vitro nucleosome assembly on native  Tetrahymena  genomic DNA but not on DNA without 6mA. Model DNA containing artificially installed 6mA resists nucleosome assembling compared to unmodified DNA in vitro. Computational simulation indicates that 6mA increases dsDNA rigidity, which disfavors nucleosome wrapping. Knockout of a potential 6mA methyltransferase leads to a transcriptome-wide change of gene expression.    Conclusions  These findings uncover a mechanism by which DNA 6mA assists to shape the nucleosome positioning and potentially affects gene expression.    Electronic supplementary material  The online version of this article (10.1186/s13059-018-1573-3) contains supplementary material, which is available to authorized users.

N6-methyldeoxyadenosine directs nucleosome positioning in Tetrahymena DNA