Upstream Regulatory Element (URE) at the Sfpi1 locus encoding PU.1, resulting in reduced PU.1 expression [30]. mRNA profiling was performed on very early committed erythroid progenitors (CD71med TER119low) isolated by flow cytometry [31]. The PU.1 mRNA level was found to be reduced by about 70% in the PU.1 low erythroid progenitors relative to wild-type progenitors (Figure 3C), similar to reports in other cell types [30,32,33]. The gene expression analysis revealed that 617 genes are up regulated and 836 genes are down regulated by PU.1 at least 1.5 fold in these cells. Of these 1453 genes exhibiting PU.1 dependent expression in erythroid progenitors, 504 genes (35%) have PU.1 bound within 2 kb of their TSS. Therefore, 7.4% (504/6826) of PU.1 bound genes are regulated by PU.1. Interestingly, when we correlated the gene expression changes in erythroid cells to the level of PU.1 occupancy, we found that genes exhibiting the strongest PU.1-dependent gene expression changes have a lower level of PU.1 binding (Figure 3C), a phenomenon that needs to be further explored. These findings reveal that a large number of genes are bound and regulated in a PU.1-dependent manner in erythroid progenitors. PU.1 regulates genes involved in controlling erythroid differentiation Besides PU.1, a number of other factors have been shown to be involved in regulating erythroid differentiation and erythroid- specific gene expression. For example, the Ets protein Fli-1 [34,35] and c-Myb [36,37], like PU.1, inhibit erythroid differentiation, whereas Gfi-1b [38], the erythropoietin receptor (EpoR) [39], and the erythroid kruppel-like factor Klf1 [40,41] promote erythroid differentiation. Comparative gene expression analysis in wild-type and PU.1 low erythroid progenitors revealed that c-myb and Fli-1 are upregulated by PU.1, whereas Gfi-1b, Klf1, and EpoR are significantly downregulated (Figure 3C). Furthermore, our ChIP- Seq data shows PU.1 occupancy either very near to the TSS and/ or within the transcribed region of each of these genes in both normal ES-EP and MEL cells (Figure 4A and 4B). These data were confirmed by qChIP in both cell types (Figure 4C and 4D). Interestingly, we observed very high levels of PU.1 occupancy in erythroid cells at the Upstream Regulatory Element (URE) lying ,14 kb upstream of the PU.1 (Sfpi1) gene itself (Figure 4AIII and 4C). The URE has been shown to have a strong positive effect on PU.1 expression in myeloid cells [30,42]. Indeed, deletion of the URE element resulted in about a 70% reduction of PU.1 in early fetal liver erythroid progenitors, similar to other cell types, suggesting PU.1 upregulates its own expression in immature erythroid cells (Figure 3C, [30,32,33]. The direct positive and negative effects of PU.1 on expression of the aforementioned genes fit well with their observed roles in erythroid differentiation, further strengthening the idea that PU.1 controls the differenti- ation decision in erythroid cells. PU.1 regulates several pathways involved in controlling the growth and survival of immature erythroid cells To further understand the categories of genes and biological pathways regulated by PU.1 in erythroid cells, we analyzed the ChIP-Seq PU.1 targets with Ingenuity Pathway Analysis (IPA) software. Table S1 shows the ten most significantly over- represented categories of molecular and cellular functions that are represented in the PU.1 target genes in both ES-EP and MEL cells. Interestingly, the two categories at the top are gene expression and cell cycle. To validate that PU.1 does indeed bind within the proximal promoter of genes in these two categories, we carried out qChIP experiments on a total of 32 genes, in both PU.1-Directed Gene Networks in Erythroid Cells ES-EP and MEL cells. 84% and 94% of the 32 genes in each cell type, respectively, were validated for PU.1 occupancy by qChIP (Figure 4, Figure S1, and Figure S4). IPA analysis also revealed several important cellular pathways that are regulated by PU.1 in erythroid cells. For example, 58% (79/137) of genes involved in the PI3K/Akt signaling pathway are occupied by PU.1 in ES-EP and MEL cells (Figure 5A). Gene expression analysis of wild-type and PU.1 low erythroid progenitors indicates that many genes that stimulate this pathway are positively regulated by PU.1 (Figure 5A). Another pathway found to be regulated by PU.1 in erythroid cells is the ERK/MAPK signaling pathway. 48% (92/192) of the genes in this IPA pathway are occupied by PU.1 (Figure 5B). As with the PI3K signaling pathway, gene expression analysis shows that PU.1 upregulates many genes that stimulate ERK/MAPK signaling (Figure 5B). IPA analysis also showed that PU.1 regulates the Jak/Stat signaling pathway. 67% (43/64) of the genes in this pathway are occupied by PU.1, and many of these genes are upregulated by PU.1 (data not shown). As discussed below, the PI3K/Akt, ERK/MAPK, and Jak/Stat sig- naling pathways have all been shown to play important roles in erythroid cell proliferation, survival and differentiation. Therefore, these results indicate that PU.1 regulates many genes and pathways that are crucial for erythroid cell function. PU.1 levels regulate the number of early erythroid committed cells in vivo The foregoing results indicate that PU.1 regulates several pathways that control the proliferation, survival and differentiation of erythroid cells. Previous studies reported that ex vivo cultures of PU.1-depleted fetal liver erythroblasts [10] or preleukemic erythroblasts [43] exhibit defects in proliferation and increased cell death. To determine whether PU.1 levels regulate the number of erythroid progenitors in vivo and the precise stage of its effects, we analyzed the distribution of five distinct populations of erythroid cells in fetal livers of mutant PU.1 low embryos and wild-type littermate embryos [30]. The five populations were identified by flow cytometry analysis for erythroid-specific TER119 and the transferrin receptor (CD71), representing populations of progressively more mature stages of erythroid cells from the earliest erythroid committed cells (R1 - CD71med TER119low) to late orthochromatophilic erythroblasts and reticulo- cytes (R5 - CD71med TER119high), as described previously [31]. We observed a dose-dependent reduction in the percentage of R1 cells from fetal livers of heterozygous and homozygous mutant embryos compared to wild-type littermate embryos (Figure 6A). Whereas, on average 8.6% of the fetal-liver of wild-type embryos are comprised of R1 cells, this compartment constitutes only 6.1% (t-test p-value 0.045 compared to wild-type) and 2.7% (t-test p- value 0.009 compared to wild-type) in heterozygous and homozygous mutant embryos, respectively. Given that PU.1 promotes several critical pathways involved in promoting survival, we hypothesized that the reduction of the R1 population may be due to increased apoptosis of these cells. Indeed, Annexin-V staining showed that the reduction in R1 cells is due, at least in part, to increased cell death in this population (Figure 6B). These results are consistent with a role for PU.1 in promoting widely utilized pathways for maintaining cell survival in very early erythroid committed cells in vivo. PU.1 may cooperate with E2Fs and other transcription factors in erythroid cells Lineage determining transcription factors, like PU.1, are thought to function together with other general and cell type- PLoS Genetics | www.plosgenetics.org 6 June 2011 | Volume 7 | Issue 6 | e1001392