Nodal/Activin Signaling in ES Cell Fate Decisions Figure 5. Target Gene Expression Increases with Phospho-Smad2 Binding, Which Demonstrates Dose-Dependent Affinity for Specific DNA Motifs. (A) Plot of relationship between pSmad2 binding affinity and expression levels of associated target genes in the Activin, DMSO and SB conditions. Horizontal axis shows ,8000 RefSeq microarray targets ranked based on normalized log2 expression with a cutoff of 5.5 to exclude non-expressing genes and non-functional probes. The largest pSmad2 ChIP-Seq peaks occurring within +/-50 kb of the RefSeq genes were identified for each condition, normalized to average DMSO enrichments and subjected to a 500 gene moving average calculation on the vertical axis. (B) Plot showing frequency distribution of basic CAGA Smad-binding DNA element (SBE) sequence in 100 base pair (bp) intervals up- and downstream of ChIP-Seq peak positions. Horizontal axis shows distance in bp from the center (broken red line) of ChIP-Seq peaks and vertical axis shows frequency of CAGA motifs in each interval. The frequency of CAGA sequences in 10,000 random mouse genome locations was computed as a control for comparison (black line). (C) Plot of frequency distribution for the canonical Smad CAGAC sequence in intervals around ChIP-Seq peak PLoS Genetics | www.plosgenetics.org 10 June 2011 | Volume 7 | Issue 6 | e1002130