Overlap of Cell Cycle Groups

<p>Venn Diagram for the total number of genes cycling in each of the three synchronization methods after our filtering and normalization.</p

Confusion Array Display for the aobANN versus Membership in EM MoDG Expression Class

<p>Expression class predictions from the aobANN (based on ChIPchip factor binding data) are displayed in a confusion array against the starting expression classes from EMDoG clustering. Each of the 40 contributing “best” ANNs were trained on 80% of the data and tested on the remaining 20% to evaluate performance. They were selected as the best performing network out ten networks trained on the same data split, but initialized with differing random seeds. These two classifications have a similarity of .86 by linear assignment [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-b009" target="_blank">9</a>]: an LA value of 1.0 would indicate perfect classification success by the ANNs.</p

Transcription Factor Rankings by aobANN Weights for Alpha Factor Arrest Data

<div><p>(A) ANN weights are sorted by the SOS metric described in the text and in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-g003" target="_blank">Figure 3</a>B.</p><p>(B) ANN weights from the aobANN network, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-g004" target="_blank">Figure 4</a>, for ANNs trained to predict RNA expression clusters derived from yeast cultures synchronized using alpha factor arrest to syncrhonize cells [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-b001" target="_blank">1</a>].</p></div

Enrichment and Depletion of Binding Sites in Individual Cell Cycle Phase Classes for Transcription Factors Highly Ranked in aobANNs in Budding Yeast Genomes

<div><p>For several regulators highlighted by strong positive or negative association with particular expression classes in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-g004" target="_blank">Figure 4</a> (denoted parenthetically), site enrichment <i>p</i>-values were calculated for each EM MoDG expression cluster. Each <i>p</i>-value was calculated using only the cell cycle identified genes that were also used as input genes to the ANN. Each block of bars along the <i>x</i>-axis represent log <i>p</i>-values (<i>y</i>-axis) for an EM MoDG cluster. Each bar within these blocks represents the log <i>p</i>-value measurements for a different <i>Saccharomyces</i> species as indicated by the color legend. Enrichment is shown as positive values (−log <i>p</i>-value), and depletion is shown as negative values (log <i>p</i>-value). The species have been arranged by to reflect evolutionary distance from S. cerevisiae. From left to right: <i>S. cerevisiae, S. paradoxus, S. mikatae, S. bayanus</i>. A dashed line along the graphs at <i>p</i>-value = .05 has been drawn to help visualize the scale difference between the plots.</p><p>(A–D) Enrichment bar charts for the specified binding sites. If the binding site is referred to by a standard name other than that of the regulator that binds to it, the regulator name is in parentheses. The color map key for each specie is at the bottom.</p></div

The Artificial Neural Network Architecture

<div><p>(A) Shown is the simple single layer network we trained to predict expression behavior based on the in vivo binding activity of ∼75% of the transcription regulators in yeast. A 204-dimension vector containing the measured transcription factor binding data from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-b021" target="_blank">21</a>] was used as the input vector. Given this binding vector, the ANN was trained to predict which of the five cell cycle expression classes (clusters) each gene belongs to. These expression classes were determined using EM MoDG.</p><p>(B) Matrix representation of the ANN. Each matrix cell, <i>W_c,r,</i> represents the real-valued connection strength, or weight, between a regulator (<i>r</i>) and an expression class (<i>c</i>) and is shown in (A) as an edge between a regulator and an expression class. These weights represent the importance of binding activity or inactivity for each transcription factor in associating a member gene with its expression class (cluster) under the ANN model.</p></div

Weight Matrix Analysis for the aobANN

<div><p>(A) Regulators were sorted based on the SOS metric (Methods and text), and the resulting total SOS rank for each regulator is plotted as a bar.</p><p>(B) The top 20 regulators are shown, ordered by importance in predicting expression behavior using the sum-of-squared weights metric. The top panel reproduces a zoomed-in view of the top 20 regulators as in (A). The bar representing each regulator is split to display positive (red) and negative (blue) contributions. The left-hand column shows a trajectory summary for each expression cluster as classified by EM MoDG. The right-hand side color map represents the weight matrix where expression classes are displayed along the rows corresponding to the drawn trajectory summaries. Regulators are sorted along the columns in rank order. Each cell is colored according to its value in the weights matrix.</p></div

Neural Network Rank Order Stability

<div><p>(A) Regulators are sorted by their SOS rank order (see text and Methods). The line indicates the mean rank for each regulator across each of 40 best ANNs, with variance of each ranking indicated by the error bar.</p><p>(B) Top 20 regulators show high stability across ANNs.</p></div

In Silico Network Mutations

<div><p>Shown are results from training ANNs missing one or more regulators as indicated on the left margin of each heatmap. Within each heatmap, each cell represents a regulator, the position of the cell along the <i>x</i>-axis of the plot is determined by the mutated network, but the color is indicative of the regulator's rank in the unperturbed network (as shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-g003" target="_blank">Figure 3</a>). The lowest strip shows the rank order color spectrum for the wild-type network.</p><p>(A) An overview showing the overall rank stability of the regulators across all mutant networks generated.</p><p>(B) A higher resolution view of the top-ranked regulators for each mutant network. Only the top 50 regulators are shown, and the color spectrum is adjusted to only span 1–50. Any regulator that was ranked within the top 50 regulators in a mutant network, but not in the wild-type network, is shown as white. The position of Swi4 in each network is denoted by *.</p><p>(C) A zoomed-in version of our mutant network analysis focusing only on networks generated by the top G1 regulators (Swi6, Mbp1, Stb1, Ace2, Swi5, Swi4).</p></div

Binding Site Enrichment and Depletion for <i>S. Pombe</i>

<p>MCB consensus binding site enrichment <i>p</i>-values are shown for <i>S. pombe,</i> based an EM MoDG clustering of expression data from ([<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-b003" target="_blank">3</a>]. Cluster trajectory summaries as a function of timepoint in the cell cycle are shown for each expression cluster in the top panels; red lines highlight the mean expression trajectory, and cluster gene number is given in the upper left corner. Below is a bar chart of <i>p</i>-values. <i>p</i>-Values are normalized against only cycling genes (blue), or are normalized against all genes (red).</p

Transcription Factor Rankings by aobANN Weights for Cdc15 Syncrhonized Data

<div><p>(A) ANN weights are sorted by the SOS metric as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-g003" target="_blank">Figure 3</a>B.</p><p>(B) ANN weights from the aob network as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-g004" target="_blank">Figure 4</a> for ANNs trained to predict RNA expression clusters derived from yeast cultures synchronized using Cdc15 TS mutant [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020169#pcbi-0020169-b001" target="_blank">1</a>].</p></div