A Large Gene Network in Immature Erythroid Cells Is Controlled by the Myeloid and B Cell Transcriptional Regulator PU.1 Sandeep N. Wontakal1., Xingyi Guo2., Britta Will1, Minyi Shi3,4, Debasish Raha3, Milind C. Mahajan5, Sherman Weissman5, Michael Snyder3,4, Ulrich Steidl1, Deyou Zheng2,6*, Arthur I. Skoultchi1* 1 Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 The Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3 Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, United States of America, 4 Department of Genetics, Stanford University, Stanford, California, United States of America, 5 Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, United States of America, 6 Departments of Genetics and Neuroscience, Albert Einstein College of Medicine, Bronx, New York, United States of America Abstract PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages. Citation: Wontakal SN, Guo X, Will B, Shi M, Raha D, et al. (2011) A Large Gene Network in Immature Erythroid Cells Is Controlled by the Myeloid and B Cell Transcriptional Regulator PU.1. PLoS Genet 7(6): e1001392. doi:10.1371/journal.pgen.1001392 Editor: Mitchell J. Weiss, Children’s Hosptial of Philadelphia, United States of America Received July 27, 2010; Accepted May 10, 2011; Published June 9, 2011 Copyright: 2011 Wontakal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grants HL078381/CA154239 (AI Skoultchi), NEI grant EY012200 (D Zheng), and NIH/MSTP Grant 5T32GM07288 (SN Wontakal). AI Skoultchi also receives support from National Cancer Institute Cancer Center Grant 2P30CA13330. This work was also partially supported by startup funds from Albert Einstein College of Medicine of Yeshiva University (D Zheng). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: arthur.skoultchi@einstein.yu.edu (AI Skoultchi); deyou.zheng@einstein.yu.edu (D Zheng) . These authors contributed equally to this work. Introduction Cellular identities are established through the actions of master regulatory transcription factors. In addition to promoting their lineage-specific gene expression programs, these factors may also inhibit the transcriptional programs of alternative cell lineages [1]. These inhibitory functions may serve to ensure that genes of closely related lineages are not mis-expressed [2,3]. The mecha- nisms used by such transcription factors to inhibit alternative lineage-specific gene expression are not well understood. The mutual antagonism between hematopoietic master regulators PU.1 and GATA-1 has served as an important paradigm, both for understanding lineage specification as well as these types of inhibitory interactions. PU.1 is an Ets family transcription factor that is required for the development of myeloid and B-cells [4,5]. GATA-1 is a Zn-finger DNA binding protein that is required for the development of erythrocytes and megakaryocytes [6]. These two factors have a particularly close developmental relationship because they direct lineage commitment from common multipotential progenitors. Indeed, PU.1 and GATA-1 physically interact and repress each other’s transcriptional activation and lineage specification func- tions [7–9]. Although PU.1 is highly expressed in myeloid and B-cells, it is also normally present in immature erythroid cells [10–12]. Down regulation of PU.1 is required for erythroid terminal differentia- tion [11–13]. This property has been conserved throughout vertebrate evolution [7,14,15]. The Sfpi1 locus encoding PU.1 is also a frequent target for integration by the spleen-focus-forming virus during Friend leukemia virus-induced murine erythroleuke- mia (MEL) [16]. The ability of PU.1 to bind to and repress GATA-1 transcriptional activity accounts for some of its functions in erythroid progenitors. However, PU.1 is also a DNA binding protein, with a number of well-established gene targets that it regulates in myeloid cells and B-cells [17–20]. Therefore, PU.1 might also regulate important genes in erythroid progenitors. Several examples of PU.1 gene targets with effects on erythroid cells have been described recently [11,21,22]. However, it is not known how many genes PU.1 controls in erythroid cells. Here we report that PU.1 does indeed direct an extensive transcriptional network in immature erythroid cells, a network consisting of pathways that are important for the growth and survival of PLoS Genetics | www.plosgenetics.org 1 June 2011 | Volume 7 | Issue 6 | e1001392