Rotational settings quantize nucleosome movement by chromatin regulators.

Proper nucleosome positioning is essential for gene regulation and genomic integrity. Current models posit that chromatin regulators override histones' intrinsic sequence preferences to establish nucleosome landscapes observed in vivo, implying a minimal role for DNA sequence in guiding nucleosomal structure. In contrast, we show that DNA sequence intrinsically guides nucleosome structure and remodeling from yeast to mammals. Nucleosomes with weak translational settings in vitro, in yeast, and in mammalian cells display strong rotational positioning that favors inward-facing A/T and outward-facing G/C dinucleotides at 10 bp spacings. Foreign DNA sequences heterologously inserted into the yeast genome obey the same rules, indicating that DNA sequence itself is causal. Remodelers and transcription elongation shift the preference among translational positions 10 bp apart, which maintains the same rotational setting. We propose that DNA sequence creates an energy landscape with preferred rotational settings, and that chromatin regulators navigate within, rather than override, these preferences.