ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo

Abstract

Chromatin endogenous cleavage (ChEC) uses fusion of a protein of interest to micrococcal nuclease (MNase) to target calcium-dependent cleavage to specific genomic loci in vivo. Here we report the combination of ChEC with high-throughput sequencing (ChEC-seq) to map budding yeast transcription factor (TF) binding. Temporal analysis of ChEC-seq data reveals two classes of sites for TFs, one displaying rapid cleavage at sites with robust consensus motifs and the second showing slow cleavage at largely unique sites with low-scoring motifs. Sites with high-scoring motifs also display asymmetric cleavage, indicating that ChEC-seq provides information on the directionality of TF-DNA interactions. Strikingly, similar DNA shape patterns are observed regardless of motif strength, indicating that the kinetics of ChEC-seq discriminates DNA recognition through sequence and/or shape. We propose that time-resolved ChEC-seq detects both high-affinity interactions of TFs with consensus motifs and sites preferentially sampled by TFs during diffusion and sliding.

Figure 1. Phenotypic characterization of strains bearing MNase-tagged TFs.

(a) Growth of free MNase and TF–MNase strains on YPD. (b) Western blot analysis of H2A serine 129 phosphorylation (γH2A) in free MNase and TF–MNase strains. The γH2A/total H2A ratio is indicated under each lane. (c) ChEC-seq workflow. Yeast cells expressing a chromatin protein (CP) of interest genetically fused to MNase are permeabilized with digitonin and calcium is added to induce cleavage by CP-MNase. Digested DNA is then purified and prepared for high-throughput sequencing.

Figure 2. Genome-wide mapping of TF binding with ChEC-seq.

Tracks of ChEC-seq signal for (a) Abf1, (b) Rap1, and (c) Reb1 along the indicated 200-bp segments of the genome. The positions of Abf1 and Reb1 motifs detected within ORGANIC peaks and a Rap1 motif detected within a ChIP-exo peak are indicated. Tracks of free MNase ChEC-seq signal at these genomic regions are also shown.

Figure 3. Temporal analysis of ChEC-seq data reveals distinct classes of TFBSs.

Heatmaps of raw Z-scored, clustered ChEC-seq signals and motif scores for (a) Abf1, (b) Rap1 and (c) Reb1 sites. The sequence logos of the highest scoring motif discovered by MEME-ChIP and plotted with LogOddsLogo in fast and slow sites are displayed below the heatmap for the corresponding factor.

Figure 4. ChEC-seq peaks display enrichment in ChIP experiments.

Average plots of Abf1 X-ChIP-seq and ORGANIC signal around high-scoring, low-scoring and random sites. Shaded areas represent 95% confidence intervals.

Figure 5. ChEC-seq sites display distinct cleavage profiles based on motif strength.

(a) Average plots of Abf1 cleavage around high-scoring and low-scoring Abf1 sites. The boundaries of the motif match are indicated by vertical dotted lines. (b) Same as a but for Rap1 sites. (c) Same as a but for Reb1 sites. Sites are oriented such that the best match to the consensus motif used for scoring is left to right on the top strand. The y axis scale has been expanded in the lower panels to reveal details in the low-scoring site profiles.

Figure 6. TF–MNase linker alteration modulates cleavage patterns.

(a) Average plots of Abf1-SL cleavage around high-scoring and low-scoring Abf1 sites. The boundaries of the motif match are indicated by vertical dotted lines. (b) Same as a but for Reb1 sites. (c) Average plots of N-terminal Reb1 cleavage around high-scoring and low-scoring Reb1 sites. The y axis scale has been expanded in the lower panels.

Figure 7. ChEC-seq-derived sites display distinctive DNA shape profiles for each TF regardless of the strength of a consensus motif.

Average profiles of minor groove width (MGW), Roll, propeller twist (ProT) and helix twist (HelT) at high-scoring, low-scoring and random (a) Abf1, (b) Rap1 and (c) Reb1 sites. Plots were centered on the middle of the best match to the consensus motif used for scoring within each peak. Schematic representations of shape features are shown to the left of the row of plots for the corresponding feature.

Figure 8. Characteristics of high-scoring and low-scoring ChEC-seq sites.

A schematic representation of two TFBSs with differing affinity for the cognate TF. On the left, at a high-scoring site, a TF binds its cognate motif with high affinity, likely forming hydrogen bonds and other base-specific contacts, thus rapidly generating high levels of cleavage at specific sites. On the right, at a low-scoring site, a TF scans along the genome by sliding and sampling shape-preferred low-scoring sites, likely without forming contacts with the functional groups of the bases, thus generating dispersed low-level cleavages around these sites.