Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells

<div><p>The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. One of the reasons for this dichotomy is that many other factors, such as nucleosomes, compete for binding. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights a complex interplay between Oct4 and Sox2 transcription factors and nucleosomes among different target genes, which may result in distinct patterns of stem cell gene regulation.</p></div

ABI SOLiD sequencing results of in vivo and in vitro nucleosome occupancy maps.

<p>We analyzed the results of the sequencing run in the following manner. First, we defined paired reads for pairs where each end aligned to the same chromosome. From that subset, we removed alignment scores that were not unique as assigned by bowtie. Finally we estimated the sequencing coverage (Reads/bp) by multiplying the number of reads by the average length in bp of each read, then dividing by the number of bps each BAC pool contained.</p><p>ABI SOLiD sequencing results of in vivo and in vitro nucleosome occupancy maps.</p

BEM-seq generated nucleosome occupancies are reproducible and reveal correlation between In vivo and in vitro occupancy.

<p>(a) Tracks of nucleosome occupancy are displayed across the Pou51f (Oct4) gene. In vivo nucleosome occupancy is represented in blue, and in vitro nucleosome occupancy is represented in green. Occupancy is center-weighted but not normalized, representing the amount of sequencing reads present in the experiment. Predicted in vitro occupancy, calculated as described in Kaplan <i>et al</i>, is also shown. Predicted in vitro data is uniformly weighted and displays the probability of in vitro nucleosome occupancy for a given bp from 0 to 1. (b) The sequence read coverage of each BAC is shown. The percentage of on target reads is calculated by taking the actual number of reads per BAC and dividing by the expected number of reads per BAC (The number of total sequencing reads multiplied by the ratio of the BAC length in bp to the total experiment size). Each BAC shows similar levels of enrichment from in vivo and in vitro experiments. (c) A density scatter dot plot of in vivo versus in vitro normalized occupancy scores is shown. For each sample, the log₂ of the normalized occupancy is taken, and the genome average is subtracted to set the average plotted value to zero. In vivo and in vitro nucleosome occupancy tightly correlate with each other. Spearman-rank correlation analysis for all base pairs in the datasets confirms the similarity between the two datasets (0.73, p <2.2x10¹⁶). This reflects the influence of intrinsic nucleosome preferences on in vivo occupancy.</p

In vivo and in vitro nucleosome occupancy tracks at functional and non functional transcription factor binding sites.

<p>(a) A 10kb region containing the Oct4 (Pou5f1) gene locus is shown with tracks for Oct4 occupancy and Sox2 occupancy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127214#pone.0127214.ref037" target="_blank">37</a>], as well as normalized, GC-adjusted in vitro nucleosome occupancy and in vivo nucleosome occupancy. The TFBS track contains predicted binding sites, generated by the Oct4:Sox2 binding matrix from JASPAR database, with functional sites in black and nonfunctional sites in red. The horizontal scale from Fig 3a is maintained for all subsequent figures. For each gene in our experiment, we chose example regions to examine the patterns of nucleosome occupancy over functional and nonfunctional sites. We highlight the selections for Oct4 to be shown in detail in Fig 3b. (b) Two regions from the Oct4 promoter region are shown. The region in the left panel contains two functional TFBSs. The overlap between the TFBS and the occupancy tracks is displayed with a grey vertical bar across all tracks. In the case of the Oct4 gene, in vivo and in vitro occupancy overlap at both functional and nonfunctional binding sites. (c) Two regions from the Sox2 promoter are shown in detail. Like Oct4, in vivo and in vitro occupancy are colocalized. (d-e) Unlike Oct4 and Sox2, Nes and Pax6 are poised, and show low in vivo occupancy at functional sites, compared to in vitro occupancy. (f-g) Sox1 and Olig2 are not regulated by Sox2 or Oct4, and consequently do not contain any functional TFBS. In vivo and in vitro occupancy seem similar at these nonfunctional sites.</p

Correlations between functional transcription factor binding and nucleosome occupancies.

<p>Using the average transcription factor (TF) occupancy (Oct4 and Sox2, from Whyte <i>et al</i>.) across the 15bp predicted binding sites for Oct4 and Sox2 (n = 58), we identified each site as functional or nonfunctional using a TF occupancy score of > = 20 as a cutoff. We also separated each set of factors by the type of downstream gene regulation (Class 1 Active: functional (n = 10) and nonfunctional (n = 12), Class 2 Poised: functional (n = 4) and nonfunctional (n = 13), Class 3 Repressed: nonfunctional (n = 19)). We calculated the in vitro and in vivo occupancy over the 15bp of each binding site and found the average of each gene type and binding site type. We also calculated the log₂ of the ratio of in vivo to in vitro occupancy at each binding site and found the median for each gene and site type. The standard error of the mean is displayed in the error bars. Paired Z-scores between different classes were calculated and p-values < 0.05 are marked with an asterisk. (a) In vitro occupancy is not significantly different at functional versus nonfunctional sites for both class 1 and class 2 genes. (b) At class 1 genes, in vivo occupancy is not significantly different at functional TFBS. At class 2 genes however, in vivo occupancy is significantly lower at functional TFBS. (c) For class 1 genes’ functional sites, the median fold-change is small and positive, while at class 2 the median fold-change is large and negative. Nonfunctional sites across all gene classes were positive.</p

Mapping in vivo and in vitro nucleosome occupancy using BAC-based enrichment.

<p>(a) Protocols were modified so that permeabilization and micrococcal nuclease digestion occur while embryonic stem cells are attached to the tissue culture surface to improve recovery and digestion reproducibility. The amount of micrococcal nuclease (MNase) in the digestion was titrated so that the mononucleosome band at 147bp is the primary band, without overdigestion. Digests were measured in Worthington Units of MNase * Time of digestion / volume of cell culture (U*min/mL). Lanes 1 and 8: 50bp ladder. Lanes 2–7 range from 2500 U*min/mL to 25,000 U*min/mL of MNase, with ideal digestion in the third condition, 10,000 U*min/mL. This amount of digestion was used in all future experiments. (b) Genomic DNA was purified from embryonic stem cell cultures and combined with histone octamer purified from chicken erythrocytes in a ratio of 100μg:30μg under high salt (2M NaCl) conditions. Removal of salt via dialysis results in reconstituted chromatin, representing histone proteins’ preferred DNA sequences. Reconstituted chromatin was digested with 5 Worthington Units of micrococcal nuclease per 10μg of genomic DNA present, for a digestion of 5 minutes at 37°C (Lanes 1 and 7: 50 bp ladder; Lanes 2–6: digested chromatin). (c) Bacterial Artificial Chromosome (BAC) DNA, which was nicked with biotin-dUTP, was blocked with Cot-1 DNA at a ratio of 100ng:10μg. 1μg of library-adapted mononucleosome DNA was denatured and mixed with BAC DNA. Mononucleosome DNA was hybridized to the corresponding BAC region and was isolated by removing BACs from solution with streptavadin beads, stringently washing the beads, and eluting single stranded DNA from the beads. Double stranded products were amplified using PCR and sent for paired-end sequencing.</p