Article Figures & Data
Figures
- Figure S1. Defining conserved and species-specific T-bet binding in human and mouse.
(A) Numbers of T-bet binding sites (q < 0.01) in each human and mouse ChIP-seq replicate and the number of these sites present in all replicates. The number of sites present in all replicates are split into those that liftOver to the other species and those that do not. Sites that liftOver are in turn divided into those that liftOver to a region that overlaps a high-confidence T-bet binding site in the other species (q < 0.01 in all reps), that exhibit no overlap with any binding site in the other species (q > 0.1 in all reps), or which are indeterminant (all others). (B, C, D, E) Further examples of T-bet binding at genes with (B) conserved, (C) alternative, (D) Hs-specific and (E) Mm-specific T-bet binding. Details as for Fig 1C.
- Figure 1. Conserved and specific-specific T-bet binding in human and mouse Th1 cells.
(A) Cartoon depicting four different classes of T-bet target genes and the numbers and proportions that fall into each category. Conserved target genes are defined as orthologous genes associated with one or more high-confidence T-bet binding sites at an equivalent location (defined by liftOver) in both species. Alternatively bound genes are bound by T-bet in both species but at different locations (no conserved binding sites). Hs-specific and Mm-specific target genes are only bound by T-bet in human or mouse, respectively. (A, B) Heat maps showing T-bet occupancy at the sets of sites described in (A). Sequence reads (per million total reads) at each position are represented by colour, according to the scale on the right (numbers of sites: Hs conserved = 2,625, Hs alternative = 611, Mm alternative liftOver to Hs = 633, Hs-specific = 222, Mm-specific liftOver to Hs = 229, Mm conserved = 2,311, Hs alternative liftOver to Mm = 648, Mm Alternative = 628, Hs-specific liftOver to Mm = 223, Mm-specific = 234). (C) T-bet binding at example genes with conserved, alternative, Hs-specific and Mm-specific T-bet binding. The red dashed lines show the equivalent locations of T-bet binding sites in the other species, as defined by liftOver.
- Figure 2. Species-specific T-bet binding is associated with species-specific recruitment of P-TEFb, the super elongation complex and mediator.
Average number of ChIP-seq reads (per million total reads) for T-bet and its co-factors P-TEFb, the super elongation complex subunit AFF4 and the Mediator subunit MED1 across conserved, alternative, human-specific and mouse-specific T-bet binding sites in human and mouse Th1 cells. Numbers of sites as in Fig 1B.
- Figure S2. Occupancy of transcriptional co-activators at T-bet binding sites.
(A, B, C) Heat maps showing (A) P-TEFb, (B) AFF4, and (C) MED1 occupancy at the sets of sites at which T-bet binding is shown in Fig 1B. For each factor, sequence reads (per million total reads) at each position are represented by colour, according to the scales on the right. The number of sites in each heatmap is the same as Fig 1B.
- Figure S3. Differential gene expression between human and mouse Th1 cells.
(A) Relative expression of orthologous genes (n = 16,672) between human and mouse Th1 cells. Genes differentially expressed between species (Welch’s t test, unadjusted P < 0.01 or <0.05) and genes shown in Fig 3B are marked. (B) Distribution of absolute expression levels of the gene sets defined in Fig 1A or at other genes. Median values are marked by a dot.
- Figure 3. Species-specific T-bet binding is associated with species-specific Th1 gene expression.
(A) Violin plot of the distribution of log2 human versus mouse Th1 cell expression ratios for gene sets defined in Fig 1A or at other genes. Median values are marked by a dot. Mean log2 Hs/Mm ratio for Hs-specific genes = 1.95, P = 4.4e−13 (t test versus Conserved). Mean log2 Hs/Mm for Mm-specific genes = −1.55, P = 0.0011 (t test versus Conserved). Numbers of genes: Conserved 1,518, Alternative 349, Hs-specific 169, Mm-specific 150, Other 13,282. (B) Heat map showing expression (log2 human versus mouse expression ratio) of Hs-specific and Mm-specific genes that are significantly differentially expressed between human and mouse Th1 cells (Welch’s t test: unadjusted p < 1e−4). The study from which each dataset was taken is indicated by the coloured bar at the top and the key to the right hand side. (C) Loess regression fit of the relation between the log2 difference in human and mouse Th1 gene expression and the difference between the number of human and mouse T-bet binding sites for genes bound by T-bet in both species (grey area = 95% confidence interval). Genes with a greater number of T-bet binding sites in human tend to be more highly expressed in human, and vice versa. (D) Examples of genes with more T-bet binding sites and which are significantly more highly expressed in human than mouse Th1 cells. (E) Examples of genes with more T-bet binding sites and which are significantly more highly expressed in mouse than human Th1 cells.
- Figure 4. Species-specific T-bet binding is associated with enrichment of DNA sequence motifs.
(A) DNA binding motifs matching a previously identified consensus T-bet DNA binding motif (12) enriched in the set of T-bet binding sites in human (top) and mouse (bottom) Th1 cells. (B) Proportion of all T-bet binding sites, conserved T-bet binding sites, Hs-specific T-bet binding sites and Mm-specific T-bet binding sites in human and mouse that contain a sequence matching the consensus T-bet DNA binding sequence in that species (error bars = 95% confidence interval of the binomial test). (C) Enrichment of transcription factor binding motifs (−log10 P-value) within Conserved versus Hs-specific T-bet binding sites in the human genome (left) or within Conserved versus Mm-specific binding sites in the mouse genome (right). The class of the most highly enriched motifs are labelled. The dashed line shows the linear regression line for all motifs excluding AP-1 bZIP motifs.
- Figure S4. Transcription factor binding motif enrichment at species-specific T-bet binding sites.
(A) De novo motifs enriched within different sets of T-bet binding sites that were identified by HOMER to most closely resemble a T-box motif. Sites are divided into those bound by T-bet in both human and mouse (conserved), in humans only (Hs-specific) or mouse only (Mm-specific). For species-specific binding sites, the equivalent coordinates of the site in the other species’ genome was identified by liftOver. The P-values for enrichment are shown to the top right of each motif. The known motifs matched to each de novo motif are shown below, leading with the position of the motif in the ranked list of all matched known motifs, the motif itself, and then the matching score. (B) Enrichment of transcription factor binding motifs (−log10 P-value) within Hs-specific T-bet binding sites versus the liftOver coordinates of Mm-specific binding sites in the human genome (left) or within the liftOver coordinates of Hs-specific binding sites versus Mm-specific binding sites in the mouse genome (right). The classes of the most highly enriched motifs are labelled.
- Figure 5. Species-specific T-bet binding sites overlap transposable elements (TEs).
(A) Permutation test of the association between binding site types and TEs. In both human and mouse, species-specific binding sites are more likely to overlap a TE than conserved binding sites. The red bars show the observed overall χ2 of the inset table. The histograms show a χ2 null-distribution based on 10,000 permutations of the data. (B) Heat plot of the chi-square overlaps of the different classes of T-bet binding sites with different classes of TEs. The numbers show the raw table data, colour represents the standardised residuals according to the scale on the right, and circle size represents the absolute standardised residual value.
- Figure S5. Transposable element (TE) genomic distribution and enriched transcription factor binding motifs.
(A) Left: Stacked bar plots showing the proportion of the different classes of T-bet binding sites located at varying distances from the closest gene TSS (− upstream, + downstream). Right: Stacked bar plots showing the proportion of different classes of TEs located at varying distances from the closest gene TSS (− upstream, + downstream). (B) Enrichment of motifs within sets of human (left) and mouse (right) T-bet binding sites separated according to the specific TE classes with which they overlap. Motifs are filtered to only show those enriched (q < 0.05) in at least one set of T-bet binding sites in each species. To adjust for differences in significance scores due to differences in the number of binding sites that overlap each TE class, −log10 P-values were normalised within each set of T-bet binding sites and are indicated by colour, according to the scale on the left. Grey indicates lack of enrichment of the motif in that subset of T-bet binding sites (q > 0.05).
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