PU.1 in macrophages and B-cells that we have generated, which resembles published data, shows that most peaks observed in the erythroid cells are also present in macrophages and B-cells (unpublished data). Consistent with this observation, we found that 100% (15/15) of genes occupied by PU.1 in erythroid cells are also occupied by PU.1 in 32D cells (Figure 7). Furthermore, most of the genes targeted for regulation by PU.1 in erythroid cells are not lineage specific; rather they are genes that are widely expressed in many cell types. This observation begs the question: Does PU.1 control common sets of genes in the several hematopoietic lineages in which it is expressed? To answer this question, we made several different types of comparisons of PU.1 gene targets in erythroid cells, myeloid cells and HSC. For example, we observed that many PU.1 gene targets in the PI3K/Akt and ERK/MAPK pathways that we identified in erythroid cells are similarly regulated by PU.1 in macrophages (Figure 5 and Figure S6). We also found that the majority of genes that are occupied and either positively or negatively regulated in erythroid cells are similarly regulated in macrophages and HSC (Figure 7B). Taken together, these comparative analyses indicate a very significant overlap in PU.1- dependent gene regulation in erythroid cells and other hemato- poietic cells. Much evidence supports the view that PU.1 plays a major role in establishing cellular identity in B-cells and myeloid cells. Finding such an extensive overlap among the PU.1 gene targets in several related but quite distinct hematopoietic cell types raises the question as to how cellular identity is established by PU.1. We suggest that the answer lies, at least in part, in the differences in the levels of PU.1 in different hematopoietic cells. Myeloid cells have the highest level of PU.1, B-cells have an intermediate level [67], and immature erythroid cells have a much lower level [68,69]. Overexpression of PU.1 in B-cells reprograms the cells to ma- crophages [70], similar to what has been reported in MEL cells [71,72]. These observations, taken together with our finding that many widely expressed genes are occupied and similarly regulated by PU.1 in immature erythroid cells, myeloid cells and HSC (Figure 7 and Figure S6), suggest that PU.1 has both shared and lineage-specific functions in these lineages, depending on the level of the factor in each lineage. We suggest that even at low concentrations, PU.1 can promote ubiquitous cellular functions such as proliferation and survival. Indeed, PU.1 has been reported to promote proliferation of both erythroid progenitors [10,43] and bone marrow derived macrophages [73]. However, higher con- centrations of PU.1 are required to promote its lineage-specific functions [67,70–72]. It will be interesting to determine whether this principle of overlapping gene networks regulated by a transcription factor in several related lineages, proposed here for PU.1, is applicable to master transcriptional regulators in other developmental systems. Materials and Methods Cell culture MEL cells (clone DS19) stably expressing a GATA-1-estrogen receptor ligand-binding domain (ER) fusion protein (GATA-1/ ER) was described previously [74]. ES-EP cells were cultured as previously described [23]. Further details can be found in Text S1. qChIP and ChIP-Seq qChIP was performed as previously described [11]. ChIP for ChIP-Seq analysis was performed similarly but using 56107 cells and 60 mg of anti-PU.1 antiserum. A list of antibodies used in this study, along with further information on processing of ChIP-Seq PU.1-Directed Gene Networks in Erythroid Cells samples can be found in Text S1. A summary of PU.1 ChIP-Seq data can be found in Dataset S1. ChIP-Seq data analysis Uniquely mapped reads from ChIP-Seq data were mapped to the mouse genome (mm9). Details on peak calling and subsequent statistical analysis can be found in Text S1. ChIP-Seq data is deposited on GEO database under the accession number GSE21953. Gene expression data analysis Total RNA was isolated from duplicate cultures of proliferating ES-EP and MEL cells or R1 cells sorted from two wild-type and URE2/2 animals using the RNeasy Kit (Qiagen) following the manufacturer’s instructions. Total RNA was further processed by the AECOM microarray facility using the standard Affymetrix pipeline and hybridized to GeneChip Mouse Gene 1.0 ST (Affymetrix). This expression data can be accessed from the GEO database under the accession number GSE21953. Subsequent statistical analysis is described in Text S1. Motif enrichment analysis For each PU.1 peak, we extracted a 500-bp sequence around the peak center (i.e., +/2250 bp) and used it for searching de novo motifs with the MEME software [26] and also known motifs in the TRANSFAC software suit (release 11) [75]. For motif analysis with TRANSFAC, we utilized the program MATCH in the TRANSFAC software package and applied the default parameters for minimizing the false positive rate (i.e., minFP option). As our background control, we ran MATCH against a randomly chosen set of 10,000 sequences (500-bp each). The enrichment of a known motif in PU.1 peaks was then calculated as the ratio of the motif occurrence frequency in PU.1 peaks and its corresponding frequency in background sequences. P-value was derived with a binomial test of the difference in motif frequency. Supporting Information Dataset S1 PU.1 ChIP-Seq Summary. Summary of PU.1 ChIP- Seq data. File includes all peaks identified in ES-EP and MEL cells. Peaks that are within 2 kb of TSS are assigned with a RefSeq gene. Found at: doi:10.1371/journal.pgen.1001392.s001 (2.05 MB XLS) Figure S1 Comparisons of ChIP-Seq and qChIP data for loci differentially occupied by PU.1 in MEL cells and ES-EP. (A) Comparisons for loci exhibiting enriched occupancy of PU.1 in MEL cells. Panels 1–3 display signal tracks corresponding to 1) a gene-poor region on chromosome 15, 2) the region near the Mxi1 gene, 3) the region near the Dnmt3a gene. Panel 4 shows qChIP analyses near the peaks denoted with arrows in panels 1–3. A HA antibody was used as an isotype control. Sequences of qChIP primers are shown in Table S2. Standard deviations represent the errors from triplicate PCR reactions. Similar results were obtained with at least 2 independent chromatin preps. (B) As in (A) for loci exhibiting enriched occupancy of PU.1 in ES-EP. Panels 1–3 display signal tracks corresponding to 1) a gene-poor region on chromosome 8, 2) the region near the Mta3 gene, 3) the region near the Gpr149 gene. Found at: doi:10.1371/journal.pgen.1001392.s002 (0.41 MB PDF) Figure S2 Properties of PU.1 ChIP-Seq peaks enriched in MEL cells or ES-EP. (A) The percentage of PU.1 ChIP-Seq peaks lying within the indicated distance from the closest TSS was calculated PLoS Genetics | www.plosgenetics.org 12 June 2011 | Volume 7 | Issue 6 | e1001392