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Transcription factors (TFs) are DNA-binding proteins that regulate gene expression. Sequence-specific TFs recognize DNA via specific amino acid-base hydrogen bonds and contacts that read local DNA shape1. Studying base and shape readout modes of TFs in vivo has been challenging due to technical issues associated with current approaches for mapping TF-binding sites (TFBSs). We recently introduced Chromatin Endogenous Cleavage with sequencing (ChEC-seq), an in vivo mapping method based on fusing Micrococcal Nuclease (MNase) to a TF (ref. 2). Upon addition of calcium to permeabilized cells, tethered MNase cuts DNA adjacent to the bound TF and the released fragments are sequenced to provide a high-resolution genomewide TFBS map. We used ChEC-seq to map the budding yeast TFs Abf1, Reb1 and Rap1 and obtained data similar to highresolution ChIP-seq without the need for cross-linking, chromatin solubilization or antibodies. When cells were collected o1 min after calcium addition, most TFBSs contained a TF-specific sequence motif (‘fast’ sites). We also reported ‘slow’ sites with low motif scores that appeared after B10 min. We found that DNA shape features of high-scoring (mostly fast) and low-scoring (mostly slow) TFBSs corresponded closely, but differed from randomly chosen sites not overlapping highor low-scoring sites. In our study, DNA shape features of fast and slow sites were centred on the best match to the TF consensus motif; however, randomly chosen genomic intervals were not similarly centred on the best motif match. Rossi, Lai and Pugh now find that when random sites are motif-centred, the shape features correspond closely to slow site features3, which might suggest that DNA shape is insufficient to explain binding site selection by the TFs Abf1, Reb1 and Rap1. However, given that sequence and shape features covary4, it is problematic to rely on motif-dependent analyses to draw conclusions about whether a TF recognizes DNA shape5. To address this problem, we aligned DNA shape feature vectors for unique fast and slow ChEC-seq sites for each TF using a procedure that relied only on shape data and was not directly informed by sequence alignment. Given the possibility for overlap between nearby TFBSs, we identified unique sites that do not intersect with any other ChEC-seq sites within intervals ranging from 100 to 500 bp surrounding ChEC-seq peak maxima, with larger windows associated with increasing stringency. For Abf1 and Reb1, we found that average fast and slow site shape features were well correlated at a range of interval widths (Poo0.001; Fig. 1a–c). We also searched sites using a ‘shape profile’ defined using the average fast site features and found that score distributions for fast and slow sites only slightly differed (P40.03), but were very different from random and free MNase sites (Poo10 10) for Abf1 (Fig. 1b) and Reb1 (not shown). The major shape feature proximal to Abf1 motifs is a deformation to the helix indicative of motif-proximal poly(dA:dT) tracts (Fig. 1a), a sequence feature we observed at slow sites in our original study2. Consistent with the recognition of a preferred shape signature by Abf1 and Reb1 at fast and slow sites, random sites and free MNase sites were not well correlated with fast and slow sites (Fig. 1a–c). We do not observe shape features enriched for poly(dA:dT) tracts at free MNase sites (Fig. 1a,b), suggesting that the detection of this shared shape feature at fast and slow ChEC-seq sites is not simply due to the higher prevalence of these features within nucleosome-depleted regions. Shape features at Rap1 fast and slow sites were not well correlated (Po0.1; Fig. 1c,d). The robustness of the correlation between average fast and slow shape features for Abf1 and Reb1 across a range of interval widths (Fig. 1d) suggests that sampling of similar shapes by TFs may explain binding events, even within promoters where fast and slow sites co-occur. From these motif-independent analyses, we conclude that fast and slow binding sites for Abf1 and Reb1 have similar shape features. We next queried a TF-gene regulatory association database6, and asked whether TF-slow site associations had been previously observed in mapping or gene expression studies orthogonal to ChEC-seq. Consistent with our previous demonstration that slow sites were recovered as sites without the canonical motif in other studies2, the proportion of fast and slow sites documented or proposed to regulate proximal genes in previous studies (Fig. 1e) was similar across a range of interval widths. This suggests that slow sites with shape features similar to fast sites are likely true DOI: 10.1038/ncomms15644 OPEN

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