The insulator binding protein CTCF positions 20 nucleosomes around its binding sites across the human genome - PubMed (original) (raw)

The insulator binding protein CTCF positions 20 nucleosomes around its binding sites across the human genome

Yutao Fu et al. PLoS Genet. 2008.

Abstract

Chromatin structure plays an important role in modulating the accessibility of genomic DNA to regulatory proteins in eukaryotic cells. We performed an integrative analysis on dozens of recent datasets generated by deep-sequencing and high-density tiling arrays, and we discovered an array of well-positioned nucleosomes flanking sites occupied by the insulator binding protein CTCF across the human genome. These nucleosomes are highly enriched for the histone variant H2A.Z and 11 histone modifications. The distances between the center positions of the neighboring nucleosomes are largely invariant, and we estimate them to be 185 bp on average. Surprisingly, subsets of nucleosomes that are enriched in different histone modifications vary greatly in the lengths of DNA protected from micrococcal nuclease cleavage (106-164 bp). The nucleosomes enriched in those histone modifications previously implicated to be correlated with active transcription tend to contain less protected DNA, indicating that these modifications are correlated with greater DNA accessibility. Another striking result obtained from our analysis is that nucleosomes flanking CTCF sites are much better positioned than those downstream of transcription start sites, the only genomic feature previously known to position nucleosomes genome-wide. This nucleosome-positioning phenomenon is not observed for other transcriptional factors for which we had genome-wide binding data. We suggest that binding of CTCF provides an anchor point for positioning nucleosomes, and chromatin remodeling is an important component of CTCF function.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Aggregation of genomic signals around CTCF sites.

The coordinate origin is set to the 5′-end position of the 20-bp-long CTCF site. Panel A shows the schematic arrangement of nucleosomes (blue ovals) around a CTCF binding site (orange rectangle). Blue arrows indicate sequence tags on the same strand as the CTCF site (plus strand), and orange arrows indicate opposite-strand (minus strand) tags. Panels B, C and D show the mean coverage of sequence tags for all mononucleosomes, CTCF ChIP-Seq and DNase-Seq around CTCF sites, respectively. The curves for occupied CTCF sites are colored blue for the plus strand tags and orange for minus strand. The green and purple curves in panel B represent plus strand and minus strand curves for unoccupied CTCF sites. Panel E shows the mean phastCons scores around occupied (green) and unoccupied (purple) CTCF sites. Note that the ChIP-Seq peaks are 12 bp inside the nucleosome boundaries as explained in Methods. The locations of the major peaks in all the panels are labeled.

Figure 2. Aggregated ChIP-Seq tag coverage of 20 histone modifications and H2A.Z around CTCF binding sites.

The blue and orange curves are for plus- and minus-strand tags around occupied CTCF sites, and green and purple curves are for plus- and minus-strand tags around unoccupied CTCF sites, respectively. A. Histone modifications enriched around occupied CTCF binding sites; B. Un-enriched histone modifications. Figures from top-left to bottom-right are sorted by descending level of enrichment over +/−2 kb.

Figure 3. Nucleosomes flanking occupied CTCF sites are highly enriched in H2A.Z.

The figure shows aggregated H2A.Z ChIP-Seq tag coverage on the plus and minus strands of occupied (blue and orange curves) or unoccupied (green and purple) CTCF sites. Note that the blue and orange curves are much higher than the green and purple curves.

Figure 4. Enrichment of individual nucleosomes in H2A.Z or histone modifications.

The columns index the nucleosomes flanking the anchoring CTCF site as in Figure 1A. Each row indicates one histone variant or modification, in descending order of overall enrichment. The numbers in parentheses indicate logarithm of average enrichment over 20 nucleosomes. Each cell shows log(enrichment) for a particular nucleosome, in a red to green color spectrum. Enrichment is defined as the ratio of the area under the curves for occupied and the area under the curves for unoccupied CTCF sites. The heatmap was created using matrix2png (

http://www.bioinformatics.ubc.ca/pavlidis/lab/cgi-bin/matrix2png.cgi

Figure 5. Analysis of in vitro nucleosome positioning surrounding CTCF binding sites.

A 5S rDNA control fragment or insulator fragments that contain CTCF binding sites (insulator 23 and 24) were reconstituted into nucleosomal arrays with recombinant histone octamers. Reconstituted nucleosomal arrays were digested with increasing amounts of MNase and purified products were separated by agarose gel electrophoresis followed by Southern hybridization using radio-labeled oligonucleotides that anneal to one end of each array. Ovals denote positioned nucleosomes assembled on the head-to-tail repeats of the 5S rDNA nucleosome positioning sequences. Bars denote locations of CTCF binding sites. Note that CTCF sites are located between MNase cleavage sites and are likely to be occluded by nucleosomes.

Figure 6. Great variation in DNA accessibility among H2A.Z and nucleosomes enriched in different histone modifications.

Panel A: definition of terms. L-Protect, the length of DNA protected from MNase digestion; L-Center, the length of DNA digested by MNase between the −1 and +1 nucleosomes; L-Digest, the length of digested DNA between other neighboring nucleosomes; Unit+, distances between neighboring plus-strand peaks; Unit−, distances between neighboring minus-strand peaks; L-CTCF, length of CTCF micrococcal nuclease protection footprint. Panel B: bar graphs of distance measures. The datasets are arranged in the descending order of L-Protect, except for the mononucleosome mapping data (labeled nucleosome). Histone modifications colored in orange are enriched in occupied CTCF sites and the blue ones are not enriched; note the mixing of the datasets in the two colors. Panel C: negative correlation between L-Digest and L-Protect. Panel D: negative correlation between L-CTCF and L-Center. Panel E: negative correlation between FP-ratio and L-Center.

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The insulator binding protein CTCF positions 20 nucleosomes around its binding sites across the human genome - PubMed (original) (raw)