PU.1-Directed Gene Networks in Erythroid Cells Figure 6. Reduction in early erythroid progenitors in PU.1 depleted embryos. (A) Fetal livers were isolated from E13.5–14 day old embryos from WT (URE+/+), heterozygous (URE+/2), and homozygous (URE2/2) animals. Distinct erythroid populations were identified by staining with TER119 and CD71, with representative stainings shown. The percent of R1 cells present from two animals from each genotype was averaged and is displayed as a bar graph. * t-test,0.05, ** t-test,0.01 compared to URE+/+. (B) Apoptosis was analyzed by Annexin-V staining of R1 cells. The percentage of Annexin-V positive cells is shown as an average of two animals from each genotype. *t-test,0.05 compared to URE+/+. doi:10.1371/journal.pgen.1001392.g006 that there is a non-random, statistically significant (p-value,1025) association of PU.1 occupied sites with consensus binding-site sequences for a number of well-studied and less well-studied transcription factors (Figure S5A). For example, consistent with a potential role for PU.1 in cell cycle regulation, consensus binding- site sequences for E2F factors are found within the PU.1 ChIP-Seq peaks. Consensus binding site sequences for another factor, ETF, which has been implicated in control of organ size via effects on cell proliferation [44], are also enriched within the PU.1 ChIP-Seq peaks. To determine if E2F factors are indeed associated with PU.1- occupied genes in erythroid cells, we performed qChIP studies of E2F2 and E2F4, two factors that have been reported to be involved in erythropoiesis [45–48]. One or both factors were found to bind near the sites occupied by PU.1 in the promoters of a number of genes in MEL cells (Figure S5B, S5C, and Figure S4). Occupancy of PU.1 and E2F2 was more prevalent than PU.1 and E2F4. Interestingly, both E2F2 and E2F4 are present, along with PU.1, at the PU.1 URE itself, while E2F4 and PU.1 are bound to the PU.1 promoter region. Although further studies are needed to understand how E2Fs may cooperate with PU.1 to regulate gene expression in erythroid and possibly other hematopoietic cells, these results support the view that lineage-specific transcription factors work in concert with widely expressed factors. PLoS Genetics | www.plosgenetics.org 9 June 2011 | Volume 7 | Issue 6 | e1001392