Intrinsic DNA codes govern distinct modes of nucleosome-transcription factor interactions

The interplay between transcription factors (TFs) and nucleosomes is central to gene regulation, yet most studies adopt a protein-centric perspective that largely overlooks DNA sequence contributions. Here, we identify four distinct nucleosomal DNA classes, including the canonical WW/SS pattern (W = A/T, S = G/C). All four nucleosome types are widespread across the human genome both in vivo and in vitro. Nucleosomes sharing the same pattern are rotationally in phase, whereas different patterns exhibit specific rotational offsets, revealing distinct DNA codes for nucleosome positioning. Analysis of 744 TFs across eukaryotes shows that the WW/SS and anti-WW/SS patterns are associated with TF binding in chromatin. Differences in the proportions of these patterns in a region, quantified as ΔNPS values, correlate closely with in vitro nucleosome occupancy. Integrating ΔNPS with in vivo nucleosome occupancy identifies four distinct modes of nucleosome–TF interaction and links nucleosomal DNA patterns to nucleosome-depleted regions around TF binding sites. Here, the authors describe four distinct DNA sequence patterns in nucleosomes that shape how transcription factors interact with chromatin, revealing previously hidden DNA-encoded rules governing nucleosome positioning and gene regulation.