DNA methylation analysis of genetic subregions.

<p>(A) Histograms showing the distribution of promoter methylation in several tissue samples, as indicated. Average methylation levels were determined for all promoters (≥3 CpGs, coverage ≥3 reads) and then distributed into bins with increasing methylation ratios. (B) Histograms showing the distribution of CpG island-associated promoters. Average methylation levels were determined (>5 CpGs, coverage ≥3 reads) and then distributed into bins with increasing methylation ratios. (C) Average DNA methylation ratios of promoters, gene bodies and intergenic regions.</p

Characterization of hypomethylated domains in Dnmt3a^wt tumors.

<p>All tracks show data for the entire mouse chromosome 4, the distance between the vertical black lines corresponds to 10 Mb of DNA sequence. (A) Methylation differences between two independent sets of Dnmt3a^wt tumors (small and large) and normal lung. Conserved partially methylated domains (PMDs) are indicated as red bars. (B) PMDs coincide with lamina-associated domains (LADs). Lamin B1 binding profiles are shown for mouse neural precursors (NPCs, blue), astrocytes (ACs, magenta), and embryonic fibroblasts (MEFs, green).</p

Sequencing data.

<p>Coverage indicates the average genome coverage. Dnmt3a ratio indicates the coverage ratios of the targeted region of Dnmt3a (exons 17–19) and the complete Dnmt3a locus. Differences in the Dnmt3a ratio illustrate the different rates of Cre-mediated recombination. Bisulfite conversion rates were determined by analyzing the conversion of non-CpG dinucleotides (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003146#s4" target="_blank">Materials and Methods</a> for details).</p

Analysis of large-scale DNA methylation patterns.

<p>(A) Density plots of average DNA ratios for 100-kb windows covering the entire mouse genome. Numbers indicate the number of windows with a methylation loss >0.15 (dotted line). (B) Methylation tracks consisting of 100-kb windows, covering the entire mouse chromosome 4. (C) Methylation changes of 100-kb windows covering the entire mouse chromosome 4. Methylation differences are plotted for Dnmt3a^wt lung tumors vs. normal lung and for Dnmt3a^KO lung tumors vs. normal lung, as indicated.</p

Overview of the experimental system used for this study.

<p>(A) Representative photographs of lungs from Dnmt3a wildtype and Dnmt3a deficient mice. Dnmt3a deficient mice have more advanced lung tumors. (B) Histophathology of two large Dnmt3a wildtype (wt) and two large Dnmt3a deficient tumors (KO). wt1: intermediate grade, solid growth pattern; wt2: intermediate to high grade, solid growth pattern; KO1: intermediate grade, papillary growth pattern; KO2: high grade, papillary growth pattern. Scale bars: 100 µM. (C) Schematic illustration of relevant comparisons for the characterization of methylation changes.</p

Methylation levels of CpGs and repetitive elements.

<p>(A) Methylation levels of individual CpGs in several tissue samples. Average methylation levels were determined for all covered CpG dinucleotides and then distributed into bins with increasing methylation ratios. Red bars bars indicate unmethylated CpG dinucleotides, orange bars partially methylated CpG dinucleotides and yellow bars completely methylated CpG dinucleotides. Percentages indicate the fractions of unmethylated (red), partially methylated (orange) and completely methylated (black) CpGs, respectively. (B) Color-coded histograms showing the distribution of repetitive element methylation in several tissue samples, as indicated. Average methylation levels were determined for all covered repeat elements and then distributed into bins with increasing methylation ratios.</p

BRN2 co-occupies distal enhancers with SOX2 in NPCs.

<p>(A) <i>de novo</i> MEME motif analysis of SOX2 bound regions in ESCs and NPCs revealed canonical SOX2 motif. (B) TRANSFAC BRN2 motifs enriched in SOX2 target regions in NPCs. p-values represent significance of enrichment based on Mann-Whitney Wilcoxon ranked sum test with Benjamini-Hochberg multiple hypothesis testing correction. (C) Heat maps of SOX2 and OCT4 at SOX2 bound promoters and enhancers centered on peaks of SOX2 enrichment and extended 4 kb in each direction. (D) Gene plots showing SOX2, OCT4, H3K4me1, and H3K27Ac density at the <i>Fbxo15</i> promoter and at poised enhancers of <i>Pax6</i> in ESCs. y-axis corresponds to reads per million. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8). Gray boxes indicate regions co-occupied by SOX2 and OCT4. (E) Heatmaps of SOX2 and BRN2 enrichment at SOX2-bound promoters and enhancers in NPCs centered on peaks of SOX2 enrichment and extended 4 kb in each direction. (F) Gene plots showing SOX2, BRN2, H3K4me1, and H3K27Ac density at <i>Olig1</i> and <i>Ascl1</i> loci in NPCs. y-axis corresponds to reads per million. Due to the high enrichment of H3K27Ac at active promoters, y-axis was cut off to show full dynamic range of enhancer-associated H3K27Ac density. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8). Gray boxes indicate regions of SOX2-BRN2 co-occupancy. * indicates known enhancer. (G) Breakdown of number of SOX2-BRN2 target enhancers that are H3K4me1+, H3K27Ac− (poised) or H3K4me1+/−, H3K27Ac+ (active). (H) Box and Violin plots representing expression data from Affymetrix arrays of genes linked to poised and active SOX2-BRN2 target enhancers in NPCs. y-axis corresponds to percentile expression rank, * denotes p-value<0.01, Student's T-test, two tailed. (I, J) GREAT analysis of genes linked to poised and active SOX2-BRN2 target enhancers in NPCs.</p

Additional transcription factor motifs are enriched in SOX2-POU-bound regions.

<p>(A) Heatmaps display the relationship between expression changes in transcription factors and the enrichment of their motifs in SOX2-BRN2 bound regions compared to SOX2-OCT4-bound regions. The full set of TRANSFAC motifs were ranked by statistical significance of enrichment in SOX2-BRN2-bound regions and in SOX2-OCT4-bound regions using the Mann-Whitney Wilcoxon test. The heat map on the right displays the change in rank of 108 TRANSFAC motifs between the two datasets. Only motifs that were ranked in the top 200 in either dataset are shown. The heat map on the left shows the fold change in gene expression between ESCs and NPCs of a transcription factor that recognizes the corresponding motif on the right. Scale bars: Left, fold gene expression change of transcription factors between ESCs (blue) and NPCs (yellow); Right, change in rank of TRANSFAC motifs between SOX2-OCT4 bound regions (blue) and SOX2-BRN2 bound regions (yellow). p-value reflects correlation of motif enrichment and gene expression of transcription factors which can recognize the motifs by a Monte-Carlo analysis. (B, C) Ingenuity Pathway Analysis to visualize the functional interconnection among genes associated with SOX2-BRN2 regions that also contain either an NF-I or RFX motif.</p

Motif configuration affects binding by SOX2 and cell-type-specific POUs.

<p>(A) Frequency distribution of distances in 25 base pair bins between peaks of OCT4 (top) and BRN2 (bottom) from SOX2 bound peaks. (B) Gene plots at the <i>Wwc1</i> locus. Direction of transcription (5′-3′) is left to right. Hashed line represents position of peaks of SOX2 and BRN2 enrichment separated by 100 bp. y-axis corresponds to reads per million. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8). (C) Distribution of orientation of and distance in base pairs between of SOX and POU motifs within SOX2-OCT4 (top), SOX2-BRN2 (bottom) bound regions. Negative values on x-axis reflect instances where SOX and OCT TRANSFAC motifs overlap. y-axis reflects fraction of occurrences of indicated spacing and orientation of all bound regions which contain a SOX and OCT motif. (D) Gene plots 3′ of the <i>Chd6</i> locus, which contains a SOX-OCT motif in the canonical orientation with a −1 bp spacer. Hashed line represents sequence under the peaks of enrichment, and boxed sequence represents the canonical SOX-OCT motif with a −1 bp spacer at this locus. y-axis corresponds to reads per million. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8).</p

Brn2 biases ES cells towards neural differentiation.

<p>(A) Staining with DAPI (blue) and immunocytochemistry of NESTIN (green) in ESCs induced to differentiate in adherent cultures with or without ectopic <i>Brn2</i>. (B) qRT-PCR of the indicated genes in ESCs with (black lines) and without (gray lines) ectopic <i>Brn2</i> expression through differentiation. y-axis represents relative expression normalized to <i>Gapdh</i> in 3 biological replicates, measured in triplicate. ESC time point is ESCs without doxycycline, and d1–d9 time points represent time in differentiation medium. * denotes p-value<0.05, ** denotes p-value<0.01 ANOVA with Bonferroni correction (C) Heatmap of OCT4 and SOX2 enrichment in ESCs and ectopic BRN2 and SOX2 in TetO-Brn2 cells of 701 genomic regions occupied by only ectopic BRN2 and SOX2. (D) GREAT GO biological processes enriched in 701 regions in (C). x-axis reflects negative log base 10 of raw binomial p-value for enrichment versus a whole genome background. (E) Gene plots depicting peaks of enrichment in indicated datasets at a locus distal to <i>Lrrn1</i>. y-axis corresponds to reads per million. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8). (F) qRT-PCR of genes associated with SOX2-BRN2 binding in TetO-Brn2 cells and NPCs, in ESCs with (black lines) and without (gray lines) ectopic <i>Brn2</i> expression through differentiation. y-axis represents relative expression normalized to <i>Gapdh</i> in 3 biological replicates, measured in triplicate. ESC time point is ESCs without doxycycline, and d1–d9 time points represent time in differentiation medium. * denotes p-value<0.05, ** denotes p-value<0.01 ANOVA with Bonferroni correction. (G) Pie chart reflecting overlap between SOX2-BRN2 regions and enhancer chromatin marks in TetO-Brn2 cells. Percentages in legend reflect fraction of 1,533 SOX2-BRN2 regions in each category. (H) Example region that was occupied by ectopic BRN2 in TetO-Brn2 cells, leading to recruitment of endogenous SOX2 and the deposition of H3K4me1 and H3K27Ac. y-axis corresponds to reads per million. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8). Gray box indicates region of SOX2-BRN2 co-occupancy in TetO-Brn2 cells which was not occupied by SOX2 in control cells. (I) Example poised enhancer occupied by ectopic BRN2 in induced cells, leading to recruitment of endogenous SOX2, and deposition of H3K27Ac. y-axis corresponds to reads per million. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8). Gray box indicates region of SOX2-BRN2 co-occupancy in TetO-Brn2 cells which was not occupied by SOX2 in control cells.</p