A picture of a colorectal adenocarcinoma with a cartoon illustrating three possible growth models: rapid growth, gradual growth, asymmetric growth.

<p>A picture of a colorectal adenocarcinoma with a cartoon illustrating three possible growth models: rapid growth, gradual growth, asymmetric growth.</p

The number of glands in a tumor as a function of the number of generations of cell division for Gompertzian growth models with inflection times of 49 and 320 generations and a maximum of 524,288 glands.

<p>The number of glands in a tumor as a function of the number of generations of cell division for Gompertzian growth models with inflection times of 49 and 320 generations and a maximum of 524,288 glands.</p

The relationship between physical proximity in the tumor and epigenetic distance.

<p>(a) The relationship between the fraction of tumor sampled and time to the most recent common ancestor. (b) Mean estimates of average Hamming distance for three physical distances: between tumor half (BH), within tumor half (WH) and within gland (WG) under different growth curve inflection times (inflection time is 49 for figure on left and 320 for figure on right) (N = 10,000 replicates).</p

Tumor sampling scheme with bisulfite sequence data generated from one colorectal tumor.

<p>Grey circles denote cancer glands. Six to seven glands are sampled from each tumor half and multiple clones are sequenced for each tumor gland (e.g. 7–8). Black circles denote methylated CpGs and white circles unmethylated CpGs. Three physical distances: within a cancer gland, between cancer glands from the same tumor half, and between cancer glands from opposite tumor halves.</p

Schematic of sample genealogy.

<p>(a) Example genealogy. Black circles denote cancer stem cells and grey circles denote cancer glands. (b) Two models for cell division. Black circles denote CSCs and white circles denote non-CSCs.</p

Mean estimate of average Hamming distance between-half (BH), within-half (WH), and within gland (WG) from a simulation study (N = 10,000 replicates).

<p>Growth curve inflection time is 49 generations of cell division for fast growing tumors and 320 generations for slow-growing tumors.</p

Average Hamming distance by tumor half for the 12 tumors published by Siegmund et al.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012002#pone.0012002-Siegmund2" target="_blank">[3]</a> The x-axis represents the distance in the right tumor half and the y-axis the distance in the left tumor half. Within-gland distances are denoted by G and within-half by H. Lines connect the pairs (G,H) from the same tumor. The line type indicates differences in distance between-half and within-half (BH-WH). Solid lines denote small BH-WH differences (difference <0.1, 8 “flat” tumors) and broken lines denote larger differences (BH-WH difference >0.4, 4 tumors). The type of broken line indicates WH differences between left and right tumor half; dotted lines indicate tumors with large WH differences (>0.6, 3 tumors); the dashed line indicates one tumor with small WH difference (0.14).</p

Chromatin changes during AEC differentiation.

<p>A) Manhattan plot of differential chromatin changes. X-axis = chromosomal location, Y-axis = number of cell type-specific chromatin changes within 2 MB region. Upper panel = H3K9/14^Ac changes, blue = AT2 cell-specific acetylation, purple = AT1 cell-specific acetylation. Lower panel = H3K27^me3 changes, orange = AT2 cell-specific methylation, grey = AT1 cell-specific methylation. B) 135 TFBS enrichment in domains of chromatin change from HOMER. X-axis = H3K9/14^Ac, Y-axis = H3K27^me3 enrichment. AT2 enrichment is shown as the log₁₀ TFBS p-value, AT1 enrichment is shown as the −log₁₀ TFBS p-value. C) Example of chromatin changes at an upregulated gene, <i>FZD2</i>, using IGV to visualize chromatin tracks. Blue = H3K9/14^Ac raw reads and SICER peaks called, green = predicted RXR binding site from HOMER analysis. D) Example of downregulated gene expression at the <i>PGC</i> gene locus. Lavender = predicted FOXA1 binding sites from HOMER analysis. AT2 = AEC chromatin signature (D0), AT1 = AEC chromatin signature (D8).</p

Transcriptomic profiling of human AEC differentiation.

<p>A) Heatmap of top 5% variant-VSN normalized gene expression probes. Blue = low expression, red = high expression. DAY = number of days AT2 cells were allowed to differentiate. “Prep” = donor lung origin by color (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003513#pgen.1003513.s001" target="_blank">Figure S1</a>). B) Principal component analysis of normalized hAEC samples. Samples color coded by donor lung as in (A). C) Significant changes in hAEC gene expression. Black line = BH-adjusted cutoff (FDR adjusted p≤0.05) calculated between D0 and D8. 20 genes show both significant up and downregulation for probes in different locations of the gene. D) Manhattan plot of differentially expressed genes. X-axis = chromosomal location, Y-axis = number of genes in each 2 MB region. E) qRT-PCR validation of microarray, data expressed in log₂-fold change of differences between D0 and D8. Circles = top 10 up- and down-regulated genes, triangles = known AT1 cell differentiation markers (<i>AQP5, PDPN, CAV1</i>). F) IPA of significantly up- or down-regulated genes. Bars expressed as log₁₀-BH corrected p-values of enrichment for pathway members in significant list against RefSeq db38 background. Whole figure: Red = upregulated, green = downregulated.</p

Functional validation of a transcription factor signaling pathway predicted from bioinformatics analysis.

<p>A) Western blots examining AT2 and AT1 cell markers during differentiation in the presence or absence of RXR antagonist UVI-3003. LAMIN A/C is the loading control. B) Transepithelial resistance as measured in kΩ-cm² over the course of differentiation. Error bars represent technical duplicates for each plating. C) Rat <i>Aqp5</i>-luciferase 4.3 kb promoter construct. Grey lines = 34 putative PPARA:RXR binding sites (Explain3.0). No sites were predicted from −900 to +6 bp due to lack of rat sequence information in the Explain v3.0 database. The asterisk marks the approximate location in the promoter of the ChIPed RXR site in E, below. The average number of PPARA:RXR sites per kilobase in the listed human/rat/mouse promoters is given in the table, with consensus site listed at the top. D) MLE-15 cells were transiently transfected with the <i>Aqp5</i>-luciferase construct and treated for 48 hours with vehicle (DMSO) or 7.5 µM UVI-3003. UV1-3003 treatment reduced <i>Aqp5</i>-luc activity by 48%±0.06. Values were normalized to vehicle control and represent the mean, error bars represent SEM, N = 3. All experiments represent 3 biological replicates. E) ChIP was performed on primary cultured rat AEC at day 0 (AT2, D0, n = 2) and day 8 (AT1-like, D8, n = 3). A region ∼4 kb upstream of the transcription start site specifically precipitated with RXR in day 8 samples. ChIP of GAPDH with RXR was performed as a control, and POL2 (POLR2A) binding to the GAPDH promoter was included as a positive control for the quality of day 0 DNA.</p