separately for peaks shared by the two cell types (Overlap) or enriched in either MEL cells or ES-EP, as described in Materials and Methods. (B) The derived position-weighted matrices from MEME analyses of PU.1 ChIP-Seq peaks enriched in MEL cells (top) and ES-EP (bottom) are shown. The thymidine [T] and adenosine [A] residues that differ relative to matrices derived from MEME analyses of all PU.1 ChIP-Seq peaks in each cell type (Figure 2B) are highlighted. Found at: doi:10.1371/journal.pgen.1001392.s003 (0.27 MB PDF) Figure S3 High correlation of gene expression in MEL cells and ES-EP. Gene expression levels were obtained separately for MEL cells and ES-EP with the Affymetrix microarray platform. After data processing and normalization, the relative expression of genes in the two samples are shown here as a scatter plot with larger units representing higher expression. The Pearson’s correlation coefficient and its statistical significance are shown at the top of the figure. Found at: doi:10.1371/journal.pgen.1001392.s004 (0.77 MB PDF) Figure S4 qChIP validation of PU.1 occupancy near genes involved in gene regulation and cell cycle regulation. (A) and (B) qChIP was performed as described in Materials and Methods with chromatin from MEL cells (top) and ES-EP (bottom) with primers described in Table S2. The genes analyzed represent examples of genes from the IPA gene expression (A) and cell cycle (B) categories that have PU.1 ChIP-Seq peaks within +/22 kb of their TSS. Myogenin and b-HS2 serve as negative and positive controls, respectively. A HA antibody was used as an isotype control. Standard deviations were calculated from triplicate PCR reactions. Similar results were obtained with at least two inde- pendent chromatin preparations. Found at: doi:10.1371/journal.pgen.1001392.s005 (0.16 MB PDF) Figure S5 E2F factors occupy PU.1 target genes in MEL cells. (A) DNA sequences from PU.1 ChIP-Seq peaks within the proximal promoter in both ES-EP and MEL cells were analyzed using the TRANSFAC database as described in Materials and Methods. The names of the ten most significant observed transcription factor motifs (p-value,1025) that were found in at least 10% of peaks and $2 fold enriched over the expected frequency are shown. The ratio between observed frequency and expected frequency is represented by a log2ratio. (B) and (C) qChIP analysis of E2F2 (B) and E2F4 (C) occupancy was References 1. Orkin SH, Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132: 631–644. 2. Graf T, Enver T (2009) Forcing cells to change lineages. Nature 462: 587–594. 3. Stopka T, Amanatullah DF, Papetti M, Skoultchi AI (2005) PU.1 inhibits the erythroid program by binding to GATA-1 on DNA and creating a repressive chromatin structure. EMBO J 24: 3712–3723. 4. McKercher SR, Torbett BE, Anderson KL, Henkel GW, Vestal DJ, et al. (1996) Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J 15: 5647–5658. 5. Scott EW, Simon MC, Anastasi J, Singh H (1994) Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265: 1573–1577. 6. Pevny L, Simon MC, Robertson E, Klein WH, Tsai SF, et al. (1991) Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349: 257–260. 7. Rekhtman N, Radparvar F, Evans T, Skoultchi AI (1999) Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells. Genes Dev 13: 1398–1411. 8. Zhang P, Behre G, Pan J, Iwama A, Wara-Aswapati N, et al. (1999) Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1. Proc Natl Acad Sci U S A 96: 8705–8710. 9. Nerlov C, Querfurth E, Kulessa H, Graf T (2000) GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription. Blood 95: 2543–2551. PU.1-Directed Gene Networks in Erythroid Cells performed as described in Materials and Methods at the indicated PU.1 target genes in MEL cells. A HA antibody was used as an isotype control. Standard deviations were calculated from triplicate PCR reactions. Similar results were obtained with at least two independent chromatin preparations. The myogenin locus serves as a negative control. Found at: doi:10.1371/journal.pgen.1001392.s006 (0.16 MB PDF) Figure S6 PU.1 promotes PI3K/Akt and ERK/MAPK signal- ing in macrophages. A previously published [49] gene expression analysis of PU.12/2 cells induced to differentiate into macro- phages was used to generate heatmaps depicting the response of the indicated genes in the PI3K/Akt and ERK/MAPK signaling pathways to the activation of PU.1. The genes shown in this analysis are the same as the genes found to be regulated by PU.1 in MEL cells (Figure 5). Found at: doi:10.1371/journal.pgen.1001392.s007 (0.11 MB PDF) Table S1 Results of Ingenuity Pathway Analysis of PU.1 Target Genes. Ingenuity Pathway Analysis was performed on genes with one or more PU.1 ChIP-Seq peaks within 2 kb of TSS in both MEL and ES-EP. The ten most significant molecular and cellular functions identified are listed in descending order of significance. The number of genes associated with a given function and bound by PU.1 is shown. p-values have been corrected for multiple testing using the Holm-Bonferroni method. Found at: doi:10.1371/journal.pgen.1001392.s008 (0.01 MB DOCX) Table S2 Primers used in this study. Found at: doi:10.1371/journal.pgen.1001392.s009 (0.02 DOCX) Text S1 Supplemental Materials and Methods. Found at: doi:10.1371/journal.pgen.1001392.s010 (0.06 DOC) MB MB Author Contributions Conceived and designed the experiments: AI Skoultchi, SN Wontakal. Performed the experiments: SN Wontakal, B Will. Analyzed the data: AI Skoultchi, SN Wontakal, X Guo, D Zheng. Contributed reagents/ materials/analysis tools: M Shi, D Raha, MC Mahajan, S Weissman, M Snyder, U Steidl. Wrote the paper: AI Skoultchi, SN Wontakal, X Guo, D Zheng. 10. Back J, Dierich A, Bronn C, Kastner P, Chan S (2004) PU.1 determines the self- renewal capacity of erythroid progenitor cells. Blood 103: 3615–3623. 11. Choe KS, Ujhelly O, Wontakal SN, Skoultchi AI (2010) PU.1 directly regulates cdk6 gene expression, linking the cell proliferation and differentiation programs in erythroid cells. J Biol Chem 285: 3044–3052. 12. Pop R, Shearstone JR, Shen Q, Liu Y, Hallstrom K, et al. (2010) A key commitment step in erythropoiesis is synchronized with the cell cycle clock through mutual inhibition between PU.1 and S-phase progression. PLoS Biol 8: e1000484. doi:10.1371/journal.pbio.1000484. 13. Rao G, Rekhtman N, Cheng G, Krasikov T, Skoultchi AI (1997) Deregulated expression of the PU.1 transcription factor blocks murine erythroleukemia cell terminal differentiation. Oncogene 14: 123–131. 14. Rhodes J, Hagen A, Hsu K, Deng M, Liu TX, et al. (2005) Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish. Dev Cell 8: 97–108. 15. Galloway JL, Wingert RA, Thisse C, Thisse B, Zon LI (2005) Loss of gata1 but not gata2 converts erythropoiesis to myelopoiesis in zebrafish embryos. Dev Cell 8: 109–116. 16. Moreau-Gachelin F, Tavitian A, Tambourin P (1988) Spi-1 is a putative oncogene in virally induced murine erythroleukaemias. Nature 331: 277–280. 17. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, et al. (2010) Simple combinations of lineage-determining transcription factors prime cis-regula- tory elements required for macrophage and B cell identities. Mol Cell 38: 576–589. PLoS Genetics | www.plosgenetics.org 13 June 2011 | Volume 7 | Issue 6 | e1001392