Author Summary Cellular identities are established by master regulatory transcription factors that promote cell type–specific gene expression. In some instances such factors also inhibit differentiation of alternative, closely related lineages. PU.1 is an Ets family transcription factor that is required for myeloid and B cell development. PU.1 is also expressed in red blood cell progenitors where it blocks erythroid terminal differentiation. One mechanism used by PU.1 to block red blood cell differentiation is by binding to and inhibiting the erythroid master regulator GATA-1. Here, we describe another mechanism utilized by PU.1 in erythroid progenitors. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) combined with gene expression profiling of erythroid progenitors, we show that PU.1 controls a large gene network in immature erythroid cells, including genes in pathways involved in regulating growth, survival, and differentiation of these cells. We also find that PU.1 controls many of the same genes and pathways in other blood cells. The results indicate that, in addition to activating lineage-specific genes, master regulatory transcription factors, like PU.1, also control numerous, widely expressed genes in multiple cell lineages. erythroid progenitors. Moreover, we find that several of these pathways are also regulated by PU.1 in other hematopoietic lineages, suggesting that PU.1 has overlapping functions in several hematopoietic lineages. Results Genome-wide occupancy of PU.1 is highly similar in normal and leukemic erythroid cells As a first step in identifying the transcriptional network controlled by PU.1 in immature erythroid cells, we performed ChIP-Seq in normal proliferating erythroid progenitors derived from embryonic stem cells (ES-EP) [23] and leukemic erythro- blasts (MEL cells). We obtained a total of 13,416,531 and 12,710,420 uniquely mapped reads in ES-EP and MEL cells, respectively. Using two peak calling programs, cisGenome and spp [24,25], we identified a total of 16,241 peaks of PU.1 occupancy in ES-EP and 16,599 peaks in MEL cells. We also compared the number of reads in a given peak in MEL cells and ES-EP and then assigned a peak as present in both cell types (#5 fold difference in the number of reads) or enriched in one cell type (.5 fold difference) (Figure 1A). With this classification scheme, we identified 16,011 peaks that are shared between the two cell types and 230 and 588 peaks that are enriched in ES-EP and MEL cells, respectively (Figure 1B left). Strikingly, more than 95% of the peaks are shared between the two cell types (Figure 1B left). Statistical analysis of the data presented in Figure 1A also revealed a strong similarity between PU.1 binding in the two cell types as evidenced by a correlation coefficient of 0.800 (p-value ,2.2610216). This similarity is even visually evident from examination of the signal tracks of 500 kb windows, such as the one displayed in Figure 2A. We used qChIP to verify that some of the rare loci enriched in one cell type are indeed differentially occupied in the two cell types (Figure S1). Overall, our results indicate that the binding patterns of PU.1 are highly similar in normal and leukemic erythroid cells. Since PU.1 occupies a surprisingly large number (.16,000) of sites in ES-EP and MEL cells (Figure 1B), it was of interest to PU.1-Directed Gene Networks in Erythroid Cells determine whether association of PU.1 with such sites is directed by a PU.1 consensus binding sequence or whether many of these sites do not contain such a sequence, which would suggest that PU.1 occupancy is likely due to protein-protein interactions with other transcription factors. Analysis of the ChIP-Seq peaks in ES- EP and MEL cells with the MEME program [26] generated the position-weighted matrices shown in Figure 2B. The matrices, which are nearly identical for the two cell types, contain a purine- rich core sequence, GGAA, corresponding to the previously described core sequence present in PU.1 binding sites [27,28] and matching the known binding sequence of the Ets family of transcription factors [19]. However, our analysis also identified a bias for an AAAGA sequence upstream of the purine-rich core, consistent with a recent report [17]. Of note, 13 of 14 positions in the matrices have a strong bias for purine residues. We found that 74% of peaks shared in ES-EP and MEL cells contain the consensus motif shown in Figure 2B, indicating that occupancy by PU.1 at the majority of observed sites is due to direct interaction between PU.1 and DNA. MEME analysis of the 26% of peaks that did not contain a PU.1 consensus motif showed enrichment for highly repetitive sequences that did not match any known transcription factor binding sites in the TRANSFAC database (data not shown). PU.1 binds near transcription start sites and regulates a large number of genes in immature erythroid cells To better understand the network of genes that are potentially regulated by PU.1 in immature erythroid cells, we sought to associate peaks with genes. We determined that 6,826 of the PU.1 occupied sites shared by ES-EP and MEL cells lie within 2 kb of the transcription start site (TSS) of known genes (referred to as the proximal promoter) (Figure 1B right). Moreover, we find that the greatest concentration of peaks within the proximal promoter is found at the TSS in both cell types (Figure 2C left). Further analysis of the genome-wide distribution of peaks showed that ,70% of peaks are found either within a gene structure or in the flanking 2 kb region, whereas ,30% of peaks are located in intergenic regions, suggesting the main mechanism of PU.1- mediated transcriptional regulation is through short-range inter- actions with the transcriptional machinery (Figure 2C right). This is in contrast to recent reports on the distribution of PU.1 in other hematopoietic cell types [17,18,29] and our unpublished data in macrophages and B-cells (see Discussion). Interestingly, a larger percentage (,40%) of shared peaks lie within proximal promoters, whereas a much smaller percentage (,10%) of the peaks enriched in either ES-EP or MEL cells exhibit this characteristic (Figure S2A). Moreover, MEME analysis showed that the weighted matrices of the enriched peaks are slightly different from the weighted matrices from all peaks (compare Figure 2B and Figure S2B). Since PU.1 was found to bind to many genes, it was important to understand the functional consequences of PU.1 occupancy in immature erythroid cells. Therefore, we correlated the PU.1 ChIP-Seq data with two types of mRNA profiling in these cells. First, we compared the transcriptomes of ES-EP and MEL cells. We found that the mRNA profiles of the two cell types are quite similar, despite the differences in their phenotypic properties (Figure S3). Nevertheless, we identified 758 genes that are occupied by PU.1 near their TSS (PU.1 target genes) and that are differentially expressed by 2-fold or more between the two cell types (440 genes upregulated in ES-EP and 318 genes upregulated in MEL). For these genes, we compared their PU.1 occupancy (represented as the number of reads in the PU.1 peak(s) near their TSS) with their relative levels of expression in the two cell types. PLoS Genetics | www.plosgenetics.org 2 June 2011 | Volume 7 | Issue 6 | e1001392