PU.1 regulates many of the same genes and pathways in immature erythroid cells and other hematopoietic cells Since PU.1 is an established transcriptional regulator in myeloid cells, we sought to determine if genes that we observed to be regulated by PU.1 in erythroid cells are also regulated by PU.1 in myeloid cells. We performed qChIP analysis in a myeloid cell line (32D) at 15 gene targets bound by PU.1 in erythroid cells. We found PU.1 bound 100% of these genes in myeloid cells (Figure 7A), consistent with a recent study in which 80% of PU.1 peaks lying close to TSS were found to overlap in macrophages and B cells [17]. Since we also observed that PU.1 regulates many genes in the PI3K/Akt and ERK/MAPK pathways in erythroid cells (Figure 5), we compared our findings with a published gene expression analysis of PU.1 cells undergoing differentiation to macrophages in response to restoration of PU.1 [49]. We found that, just as we observed in erythroid cells, many of the genes in these two pathways are upregulated by PU.1 in macrophages (Figure S6). Additionally, we also compared all of the genes regulated by PU.1 in early erythroid committed (R1) cells with the macrophage gene expression data, as well as with a gene expression analysis of HSC from PU.1 knockdown mice [33]. Of the genes that we found to be occupied and upregulated by PU.1 at least 1.2-fold in R1 cells, and also annotated in the two other data sets, 58% (94/162) are also upregulated in a PU.1 dependent-manner during macrophage differentiation and 70% (113/162) are similarly regulated in HSC (Figure 7B left). Similarly, of the genes that we found to be bound and repressed by PU.1 in erythroid cells and also represented in the other data sets, 57% (150/265) are also downregulated by PU.1 in macrophages and 51% (134/265) are repressed in a PU.1- dependent manner in HSC (Figure 7B right). We conclude that PU.1 regulates many of the same genes and pathways in immature erythroid cells, myeloid cells and HSC (see Discussion). Discussion The different roles of PU.1 in immature erythroid cells PU.1 is a master regulatory transcription factor that plays an essential role in the development of the myeloid and B-cell lineages [4,5]. However, several lines of evidence suggest that PU.1 also plays important roles in erythropoiesis (see Introduction and Figure 6). Some of its effects are likely attributable to its ability to bind to and repress GATA-1 [3,7,50], but it is also very important to understand the global gene expression program controlled by PU.1 in immature erythroid cells. To investigate this program, we first determined the genome-wide binding patterns of PU.1 in normal erythroid progenitors and leukemic erythroblasts. We found that PU.1 occupancy is highly similar in both cell types and that PU.1 binds in close proximity to a large number of genes (Figure 1). Unexpectedly, PU.1 appears to occupy more sites (.16,000) in immature erythroid cells than three essential red blood cell transcription factors, including GATA-1 (reported to occupy between 4,000–14,400 sites) [51–54], SCL (,4,500 sites) [55], and Klf1 (,2,000 sites) [56]. Although some of these differences may be due to the use of different peak calling methods or other technical issues such as the use of different antibodies and different cells, our unpublished results on GATA-1 occupancy using the same methods and the same cells lead to a similar conclusion (unpublished data). Interestingly, the genomic distri- butions of PU.1 in erythroid cells are also dramatically different from that reported for GATA-1 and Klf1. Whereas we found ,40% of PU.1 bound sites are within 2 kb of TSS (Figure 2), only ,16% of Klf1 bound sites lie within 1 kb of TSS [56], and only ,13% of GATA-1 sites are found within 210 kb of TSS [51], PU.1-Directed Gene Networks in Erythroid Cells consistent with our unpublished results indicating that only 9% of GATA-1 bound sites are within 2 kb of TSS in ES-EP (unpublished data). These results suggest that PU.1 may act independently of these factors, at least at a subset of genes in erythroid progenitors. Recent work demonstrates that PU.1 directly regulates certain genes that can themselves affect erythroid differentiation. For example, we reported that the CDK6 gene is directly upregulated by PU.1 [11]. Furthermore, we also showed that CDK6, like PU.1, is rapidly downregulated as erythroid cells enter terminal differentiation and that constitutive expression of CDK6 blocks erythroid differentiation [57]. Likewise, Elf-1, another member of the Ets transcription factor family, was shown to be directly upregulated by PU.1 and to negatively regulate erythroid differentiation [22]. Here we report that PU.1 binds in close proximity to the genes that encode Fli1, c-myb, and PU.1 itself (Figure 4). We also showed that PU.1 upregulates the expression of each of these genes (Figure 3C). Like PU.1, Fli1 and c-myb have been shown to block erythroid differentiation [34,36,37,58–60]. On the other hand, we also found that PU.1 binds in close proximity to and inhibits expression of genes that promote erythroid differentiation, such as Gfi-1b, EpoR, and Klf1 (Figure 3C and Figure 4) [38–41]. Thus, our results show that PU.1 regulates the expression of genes that both inhibit and promote erythroid differentiation. However, the results reported here also reveal that PU.1 has a much broader role in these cells than simply regulating several factors that control erythroid differentiation. Indeed, the ChIP-Seq and transcriptome analyses show that PU.1 regulates many genes involved in several signaling pathways that are critical for erythropoiesis. These pathways include the PI3K/Akt, ERK/ MAPK, and Jak/Stat pathways (Figure 5 and data not shown), all of which have been implicated in survival and proliferation of erythroid progenitors [61–64]. Interestingly, the PI3K/Akt and Jak/Stat pathways regulate PU.1 levels in erythroid cells [65,66]. Taken together, these findings suggest the existence of a feedback loop in which PU.1 controls the expression of many genes in these crucial signaling pathways and these pathways upregulate the expression of PU.1. Our finding that mice with ,30% of normal PU.1 levels exhibit a marked reduction in the number of the earliest committed fetal liver erythroid progenitors (Figure 6) is consistent with an important role for PU.1 in regulating these pathways in vivo. Thus, our studies further advance the concept of master regulatory transcription factors regulating both lineage- specific genes and more widely expressed genes. Overlapping functions of PU.1 in several hematopoietic lineages Several recent papers reported the genome-wide binding patterns of PU.1 in macrophages [17,18,20], neutrophilic precursors [19], B-cells [17], and multipotent progenitors cells [29]. Interestingly, the genomic distributions of PU.1 in these cells are quite different from what we observed in erythroid cells. For example, whereas we found that ,40% of PU.1 occupied sites map within 2 kb of TSS (the overwhelming majority of these being bound within 500 bp of TSS) (Figure 2), one study found that only 12% and 18% of PU.1 binding sites in macrophages and B-cells, respectively, were within 500 bp of TSS [17], and another study in macrophages found only 20% of such sites to be within 2.5 kb of TSS [18]. Similarly, only 23% of PU.1 peaks mapped within 1 kb of TSS in multipotential hematopoietic progenitor cells [29]. It is likely that part of the reason for these differences is the larger number of PU.1 occupied sites in the other cell types compared with erythroid cells. In fact, comparison of ChIP-Seq data for PLoS Genetics | www.plosgenetics.org 10 June 2011 | Volume 7 | Issue 6 | e1001392