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. 2014 Feb;11(2):203-9.
doi: 10.1038/nmeth.2766. Epub 2013 Dec 15.

High-resolution mapping of transcription factor binding sites on native chromatin

Sivakanthan Kasinathan  1 , Guillermo A Orsi  2 , Gabriel E Zentner  3 , Kami Ahmad  4 , Steven Henikoff  5
Affiliations

High-resolution mapping of transcription factor binding sites on native chromatin

Sivakanthan Kasinathan et al. Nat Methods. 2014 Feb.

Abstract

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. TF profiling is commonly carried out by formaldehyde cross-linking and sonication followed by chromatin immunoprecipitation (X-ChIP). We describe a method to profile TF binding at high resolution without cross-linking. We begin with micrococcal nuclease-digested non-cross-linked chromatin and then perform affinity purification of TFs and paired-end sequencing. The resulting occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide high-resolution maps that are accurate, as defined by the presence of known TF consensus motifs in identified binding sites, that are not biased toward accessible chromatin and that do not require input normalization. We profiled Drosophila melanogaster GAGA factor and Pipsqueak to test ORGANIC performance on larger genomes. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.

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Conflict of interest statement

Competing Financial Interests

None.

Figures

Figure 1
Figure 1. Robust identification of Reb1 binding sites on native chromatin
(a) ORGANIC profiling scheme. (b) Representative example of a region showing ORGANIC profiling ChIP Reb1 occupancy and input tracks for chromatin extracted at 80, 150, and 600mM salt, scaled by normalized counts. The locations and relative occupancies of ChIP-exo calls and location of X-ChIP-chip calls are shown in the lower tracks. Note the different scales in input and IP tracks.
Figure 2
Figure 2. ORGANIC TF binding sites have characteristic binding site motifs
(a, b) Number of sites associated with MEME-discovered Reb1 (a) and Abf1 (b) motifs. Sequence logos displaying representative MEME-derived motifs are included as insets. Note that all of the ChIP-chip TF binding sites have characteristic motifs because of strict motif criteria imposed in determining high-quality binding sites. (c, d) Venn diagrams show degree of peak overlap between Reb1 (c) and Abf1 (d) datasets. Peaks called for each Abf1 and Reb1 ORGANIC dataset and position-specific log-odds matrices corresponding to MEME-discovered motifs are included (Supplementary Tables 1 and 2).
Figure 3
Figure 3. High sensitivity and specificity of ORGANIC profiling applied to TF binding sites
Histograms of motif scores determined using MEME-derived position-specific log-odds scoring matrices are shown for Reb1 (a) and Abf1 (b) binding sites. MEME-ChIP-derived motifs corresponding to each 1,000-unit log-odds motif score cohort are included above each histogram. Bins that contained either too few sites or sequences that did not produce a MEME-ChIP-derived motif are designated ‘N/A.’
Figure 4
Figure 4. ORGANIC sites are stably bound in vivo and are conserved throughout Saccharomyces evolution
(a–c) DNase I–seq profiles at ORGANIC Reb1 (a), ORGANIC Abf1 (b) and ChIP-exo Reb1 (c) sites. For c, both primary and secondary ChIP-exo sites are shown (see supplementary Fig. 5 for a separate analysis). (d) Paired-end sequencing fragment length distributions of reads from input (left) and IP (right) libraries mapped to fly and yeast genomes in a mixing experiment in which yeast Reb1 ORGANIC profiling was done in the presence of Drosophila nuclei. (e) Correlation between occupancy and log-odds motif score for Reb1 (left) and Abf1 (right) ORGANIC sites. (f,g) Average phastCons scores in 200-bp windows centered at ‘new sites’ (sites detected by ORGANIC but not by other methods) and sites detected by other methods for Reb1 (f) and Abf1 (g).
Figure 5
Figure 5. ORGANIC profiling identifies TF binding sites in inaccessible chromatin and does not require input normalization
(a, b) Nucleosome (Nucl.) occupancy for the top and bottom 200 Reb1 (a) and Abf1 (b) sites ranked by 80 mM ORGANIC occupancy. Motifs discovered by MEME for sites that are in nucleosome-depleted and –occluded DNA are included as insets. (c, d) Average Sono-Seq normalized counts in a 2 kb window centered Reb1 (c) and Abf1 (d) sites. Normalized counts are computed such that the genome-wide average is equal to 1 (see Online Methods).
Figure 6
Figure 6. ORGANIC profiling of Drosophila GAGA Factor and Pipsqueak DNA-binding proteins
(a) Representative GAF and Psq motifs found in ORGANIC GAF and Psq sites and Venn diagram showing overlap between GAF X-ChIP-chip (‘all sizes’ size class). (b) Representative TF hotspot-containing regions showing ORGANIC GAF profile and modENCODE X-ChIP-Seq tracks. Hotspots are occupied by GAF in the X-ChIP-Seq tracks, but not in the ORGANIC track. A common peak detected by both X-ChIP-Seq and ORGANIC profiling with high-scoring motifs is to the left of the HOT regions. Len25 GAF and Psq ORGANIC peaks are included (Supplementary Table 1).
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