Histone acetylation and GAF occupancy are important covariates in predicting HSF binding intensity.

<p>Plotted are the relative values of the sums of the coefficients associated with all rules that reference each covariate in the rules ensemble <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002610#pgen.1002610-Friedman1" target="_blank">[20]</a>. Results are shown for (A) the histone variant and modification model and (B) the non-Histone factor model.</p

Genomic chromatin and PB–seq data accurately predict in vivo HSF binding intensity.

<p>A) The intensity of in vivo ChIP-seq peaks is not recapitulated by in vitro PB–seq data; however, genomic DNase I hypersensitivity data and histone modification ChIP-chip data can be used to accurately predict HSF binding intensity. B) The experimentally determined ratio between in vivo ChIP-seq HSF intensity and in vitro PB–seq intensity is plotted against the predicted in vivo/actual PB–seq ratio. The Pearson correlation for each model is shown.</p

In vitro binding reveals potential HSF binding sites.

<p>The blue box highlights strong differences in the usage of potential binding sites in vivo at the Cpr67B locus, while the green boxes highlight differences in the magnitude of binding to major heat shock genes promoters, despite comparable in vitro binding affinities.</p

In vitro and in vivo binding of HSF to genomic HSEs do not correlate.

<p>A) A scatter plot comparing the observed in vivo HSF binding intensity and in vitro binding intensity for each isolated HSE indicates that the vast majority of in vivo binding is suppressed (green) or abolished (blue), if we assume that the top seven most DNase I hypersensitive isolated HSE clusters provide the best estimates for sites that are minimally influenced by chromatin. After scaling, red points have similar in vivo and in vitro intensity, black points may be enhanced in vivo, while green and blue points are suppressed and abolished, respectively. B) The points from panel A were categorized, and the resulting bar chart shows the relative frequencies of each category.</p

Pentamers within the HSEs are dependent upon their consensus match and also their position relative to the other pentamers.

<p>A) The mixture model defines each pentamer within the HSE as strict or relaxed depending upon how well it conforms to the canonical HSE. Note that the position of relaxed pentamers strongly influences their composition. B) A probabilistic sequence model reveals that the presence of two strict (red) and one relaxed (blue) pentamer provides the best explanation of the data.</p

DNase I hypersensitivity can be inferred using histone marks and MNase data.

<p>A) The intensity of DNase I hypersensitivity landscape is inferred by models (colors) that use histone modification profiles, non-histone factor profiles, DNase I data and MNase-seq data. B) The experimentally determined DNase I hypersensitivity data is plotted against inferred intensity for the various models. The Pearson correlation for each model is shown.</p

Recombinant HSF binds HSEs with picomolar affinity in vitro.

<p>A and B) The mobility of the constant 200 attomole HSE probe shifts into a trimeric-HSF:HSE complex as increasing HSF is added. There is no HSF in the left-most lane, the right-most lane contains 3 nM HSF (1 nM trimeric HSF), and the intervening lanes contain two-fold serial dilutions of HSF. C) A hyperbolic curve based on the Kd equation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002610#s4" target="_blank">Methods</a>) was modeled using the band shift data, indicating a Kd of 42.6 pM (95% confidence interval of 36.8–49.4 pM). D) A hyperbolic curve based on the Kd equation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002610#s4" target="_blank">Methods</a>) was modeled using the band shift data, indicating a Kd of 224 pM (95% confidence interval of 181–276 pM). E) The intensity of each isolated HSE in the <i>Drosophila</i> genome is transformed to an absolute Kd using the absolute Kds calculated from band shift data in panels A and B. The Kd values range from 40–400 pM.</p

Genes with reduced pausing in GAF-RNAi are enriched for GAF-bound promoters.

<p><b>(A)</b> Fraction of all genes or genes with significantly reduced promoter GRO-seq that are paused, have GAF-bound promoter, or high-confidence GAF peaks within their promoter. <b>(B)</b> The average GAF ChIP-seq reads from untreated (black and grey lines) or GAF-RNAi (maroon and red lines) cells between 500bp upstream to 500bp downstream for the TSS of paused genes with GAF-bound promoters separated into genes with significantly reduced promoter GRO-seq reads (Pause reduced) and all other paused genes with GAF-bound promoters (Paused unchanged).</p

GAF knock-down reduces promoter-proximal polymerase on many genes.

<p><b>(A)</b> The average GRO-seq reads (per million mapped reads) between 500bp upstream to 1000bp downstream for the TSS of all genes binned by 10bp. The reads from the sense strand are plotted above zero and the reads from the anti-sense strand are plotted below zero. <b>(B)</b> Promoter-proximal GRO-seq reads (100bp window with the most reads within 250bp of the TSS) of each gene for LacZ-RNAi and GAF-RNAi libraries plotted as the log₂ ratio of GAF-RNAi to LacZ-RNAi reads is plotted on the y-axis and log₂ of the average of LacZ-RNAi and GAF-RNAi reads on the x-axis. The regions with significant changes between the LacZ-RNAi and GAF-RNAi as determined by edgeR are colored red. <b>(C)</b> Gene body GRO-seq reads (500bp downstream of the TSS to the polyadenylation site) of each gene for LacZ-RNAi and GAF-RNAi libraries are plotted as in B. The regions with significant changes between the LacZ-RNAi and GAF-RNAi as determined by edgeR are colored orange. <b>(D)</b> The change in promoter-proximal and gene body reads represented as log₂ of the GAF-RNAi to LacZ-RNAi ratio. The promoter regions with significant changes between the LacZ-RNAi and GAF-RNAi as determined by edgeR are colored red.</p

Promoters with reduced pausing fill-in with nucleosomes.

<p><b>(A)</b> The average profile of LacZ-RNAi and GAF-RNAi MNase-seq reads 500bp upstream and downstream of the TSS of paused genes with GAF-bound promoters separated into genes with significantly reduced promoter GRO-seq reads (Pause reduced) and all other paused genes with GAF-bound promoters (Pause unchanged). <b>(B)</b> Heatmaps showing the LacZ-RNAi MNase-seq read level, GAF-RNAi MNase-seq read level, and the change in MNase-seq reads (GAF-RNAi subtracted from LacZ-RNAi) 500bp upstream and downstream from each TSS of paused genes with GAF-bound promoters arranged based on the significance of GRO-seq promoter read reduction in 10bp bins, as indicated by the left heatmap. The Pause reduced genes are indicated by the red bar at the bottom of the left heatmap. The p-values for increased MNase-seq reads from 100bp upstream to 50bp downstream of each TSS are indicated in the right heatmap. <b>(C)</b> The average profile of LacZ-RNAi and GAF-RNAi H2AvD reads 500bp upstream and downstream of the TSS of paused genes with GAF-bound promoters separated into Pause reduced genes and Pause unchanged genes. <b>(D)</b> The average profile of LacZ-RNAi and GAF-RNAi MNase-seq reads 500bp upstream and downstream of high confidence intergenic GAF peaks.</p