C/EBP alpha induces Ebf1 gene expression in common lymphoid progenitors

C/EBP alpha is required for formation of granulocyte-monocyte progenitors (GMP) and also participates in B lymphopoiesis. The common lymphoid progenitor (CLP) and preproB populations but not proB cells express Cebpa, and pan-hematopoietic deletion of the +37 kb Cebpa enhancer using Mx1-Cre leads not only to reduced GMP but also to 2-fold reduced marrow preproB and >15-fold reduced proB and preB cells. We now show that IL7R alpha-Cre-mediated deletion of the +37 kb Cebpa enhancer, which occurs in 89% of Ly6D(+) and 65% of upstream Ly6D(-) CLP, leads to a 2-fold reduction of both preproB and proB cells, and a 3-fold reduction in preB cells, with no impact on GMP numbers. These data support a direct role for C/EBP alpha during B lineage development, with reduced enhancer deletion in Ly6D(-) CLP mediated by IL7R alpha-Cre diminishing the effect on B lymphopoiesis compared to that seen with Mx1-Cre. Amongst mRNAs encoding key transcriptional regulators that initiate B lymphoid specification (PU.1, E2A, IKAROS, EBF1, FOXO1, and BACH2), only Ebf1 levels are altered in CLP upon Mx1-Cre-mediated Cebpa enhancer deletion, with Ebf1 reduced similar to 40-fold in Flt3(+)Sca-1(int)c-kit(int)IL7R alpha(+) CLP. In addition, Cebpa and Ebf1 RNAs were 4- and 14-fold higher in hCD4(+) versus hCD4(-) CLP from Cebpa-hCD4 transgenic mice. Histone modification ChIP-Seq data for CLP indicate the presence of active, intronic Ebf1 enhancers located 270 and 280 kb upstream of the transcription start sites. We identified a cis element in this region that strongly binds C/EBP alpha using the electrophoretic mobility shift assay. Mutation of this C/EBP alpha-binding site in an Ebf1 enhancer-TK-luciferase reporter leads to a 4-fold reduction in C/EBP alpha-mediated trans-activation. These findings support a model of B lymphopoiesis in which induction of Ebf1 by C/EBP alpha in a subset of CLP contributes to initiation of B lymphopoiesis.