PU.1-Directed Gene Networks in Erythroid Cells Figure 3. PU.1-dependent gene expression in erythroid cells. (A) PU.1 target genes with a 2-fold expression difference (as measured by Affymetrix gene array) between MEL and ES-EP cells were identified. The log2 of the number of PU.1 ChIP-Seq reads within peaks of these genes’ promoters are computed and plotted here. Blue circles represent genes for which expression is higher in MEL cells, while red circles represent genes for which expression is higher in ES-EP. The diameter of each circle is proportional to the degree of difference in gene expression between the two cell types. (B) The expression levels of genes with and without PU.1 bound at proximal promoters in MEL and ES-EP are shown as boxplots, with the lower and upper sides of the box representing the lower and upper quartiles, respectively, and the line in the box representing the median expression value. (C) The log2ratio of the fold change in gene expression from early erythroid progenitors derived from wild-type embryos compared with PU.1 low embryos for genes bound by PU.1 in MEL (left) or ES-EP (right) are plotted as a function of the number of reads associated with the peak(s) corresponding to each gene. The positions of the values for Klf1, EpoR, Gfi1b, Myb, Fli1, and Sfpi1 in the plots are labeled. doi:10.1371/journal.pgen.1001392.g003 activator. Consistent with this view, we also find in both ES-EP and MEL cells that the average level of expression of PU.1 target genes is significantly higher than that of non-PU.1 target genes (Figure 3B). To determine how PU.1 affects the expression of its target genes, we combined the ChIP-Seq data with comparative gene expression analysis of fetal liver erythroid progenitors from wild- type E13.5–14.0 embryos and embryos that have a deletion in an PLoS Genetics | www.plosgenetics.org 5 June 2011 | Volume 7 | Issue 6 | e1001392