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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.
Highlights
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
The resulting values for area under the receiver operating characteristic (AUROC) showed that sequence is a better discriminator between high-scoring and low-scoring sites than shape (Supplementary Fig. 9), indicating that DNA shape is more similar than sequence between these two classes of sites. These results suggest that ChEC-seq kinetics separates TF binding sites (TFBSs) on the basis of their recognition mode
We have shown that ChEC-seq robustly identifies global protein– DNA interactions with high spatial and temporal resolution
Summary
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. While ChIP-seq is most frequently used to map genome-wide protein–DNA interactions, a number of orthogonal methods, each involving fusion of chromatin proteins to DNA-modifying enzymes, have been implemented In one such method, DNA adenine methyltransferase identification (DamID)[14], a protein of interest is fused to the Dam methyltransferase, resulting in methylation at regions bound by the protein and containing GATC sequences. A second enzymatic method is Calling Card-seq, in which a chromatin protein of interest is fused to a transposase to facilitate targeted integration of transposons into the genome[20] This method offers advantages similar to DamID with the added benefit of somewhat higher resolution, though it may be limited by transposase sequence preferences and depends on the presence of restriction sites an appropriate distance from the inserted transposon to create templates for circularization and inverse PCR. ChEC is controllable, as robust MNase activity depends on the addition of calcium to millimolar concentrations, several orders of magnitude greater than the 50–300 nM free calcium observed in unstimulated yeast[24] and mammalian cells[25,26,27,28,29]
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