br Introduction Hematopoiesis is thought to begin

Introduction Hematopoiesis is thought to begin from stem Madecassic acid that progress through consecutive precursor cell stages in a hierarchical fashion whereby lineage commitment precludes alternative cell differentiation. However, there has been increasing evidence of hematopoietic plasticity and cell lineage conversion, particularly during leukemogenesis (Cobaleda and Busslinger, 2008; Graf, 2008; Greaves et al., 1986; Regalo and Leutz, 2013). The transcription factors C/EBPα and C/EBPβ are potent inducers of myelomonocytic genes in heterologous cell types (Ness et al., 1993), and the experimental conversion of lymphoid cells to myeloid cells modulated by both C/EBPs has highlighted their lympho-myeloid transdifferentiation potential (Graf and Enver, 2009). The C/EBP family members C/EBPα, C/EBPβ, C/EBPδ, and C/EBPε are expressed in myeloid cells (Cloutier et al., 2009; Scott et al., 1992). Loss-of-function studies in genetically modified mice suggested combinatorial and partially redundant functions of C/EBPs in myelopoiesis (Tsukada et al., 2011). Knockout studies showed that deletion of Cebpa has the strongest impact on myelopoiesis, resulting in an almost complete loss of neutrophils and impaired development of granulocyte-macrophage progenitor (GMP) cells (Zhang et al., 1997, 2004). However, cytokines could compensate for the lack of Cebpa by the concomitant activation of Cebpb, and genetic replacement of Cebpa with Cebpb in the Cebpa locus compensates for the Cebpa requirement in hematopoiesis and liver functions (Chen et al., 2000; Hirai et al., 2006; Jones et al., 2002). Individual deletions of C/EBPβ, δ, and ε evoke milder and gene-specific phenotypes, such as susceptibility to infections, failure of emergency granulopoiesis, impaired cytokine production, and partial granulocyte deficiency that is intensified by compound C/EBP gene deletions. For example, compound Cebpb/Cebpe deletion mutants display impaired granulopoiesis, defective macrophage functions, and a disrupted innate immune regulatory gene expression network, confirming the compensatory and redundant functions of the C/EBPs (Akagi et al., 2010; Hirai et al., 2006; Litvak et al., 2009; Tanaka et al., 1995; Yamanaka et al., 1997). C/EBPα can stimulate the transdifferentiation of B and T cells and, together with PU.1, even fibroblasts into macrophages (Bussmann et al., 2009; Feng et al., 2008; Ness et al., 1993; Xie et al., 2004). Conversion of B cells into inflammatory-type macrophages occurs rapidly after C/EBP expression, with high efficiency and through a direct route (Bussmann et al., 2009; Di Tullio et al., 2011; Xie et al., 2004). An experimental transdifferentiation system based on an estrogen-responsive, conditional C/EBPα protein in the v-H-ras-transformed pre-B cell line HAFTL1 (Holmes et al., 1986) has served as a tool to examine the mechanistic aspects of lympho-myeloid lineage conversion, including alterations of chromatin occupancy, gene expression kinetics, non-coding RNA expression, and DNA methylation (Barneda-Zahonero et al., 2013; Di Tullio et al., 2011; Kallin et al., 2012; Krijger et al., 2016; Rodriguez-Ubreva et al., 2012, 2014; van Oevelen et al., 2015). We recently found that structural alterations and post-translational modification sites of C/EBPβ may determine the path of transdifferentiation of primary progenitor B cells toward distinct myeloid cell fates (including granulocytes and dendritic cells), suggesting that epigenetic instructions beyond the inflammatory macrophage cell fate are encoded in the C/EBP structure and could account for cell-type specification (Stoilova et al., 2013). This observation has prompted us to compare the lineage conversion capacity of all transactivator C/EBP family members and to develop a lympho-myeloid transdifferentiation system that is amenable to targeted mouse genetics and cell-culture manipulation. In this study, we generated murine v-Abl-immortalized B cells from wild-type and genetically altered mice to compare the lympho-myeloid transdifferentiation potential of the C/EBP family members C/EBPα, C/EBPβ, C/EBPδ, and C/EBPε. Our data showed that C/EBPβ and C/EBPε readily induce a granulocytic fate in addition to macrophage formation. Granulocytic conversion largely depended on transgene dosage. In addition, efficient transdifferentiation required endogenous Cebpa/Cebpb. Importantly, applying selective pressure on immortalized B cells expressing C/EBPβ by depriving β-mercaptoethanol resulted in the rapid extinction of B cells and massive expansion of stable myeloid cells. These myeloid progenitors displayed bipotential GMP-like properties and continuously produced macrophages and granulocytes. This process could suggest a link between C/EBP-induced lympho-myeloid lineage switch and a B cell-derived leukemic myelomonocytic GMP-like phenotype (Slamova et al., 2013).