Distinct Promoters Mediate the Regulation of Ebf1 Gene Expression by Interleukin-7 and Pax5

Abstract

Early differentiation of B lymphocytes requires the function of multiple transcription factors that regulate the specification and commitment of the lineage. Loss- and gain-of-function experiments have provided important insight into the transcriptional control of B lymphopoiesis, whereby E2A was suggested to act upstream of EBF1 and Pax5 downstream of EBF1. However, this simple hierarchy cannot account for all observations, and our understanding of a presumed regulatory network, in which transcription factors and signaling pathways operate, is limited. Here, we show that the expression of the Ebf1 gene involves two promoters that are differentially regulated and generate distinct protein isoforms. We find that interleukin-7 signaling, E2A, and EBF1 activate the distal Ebf1 promoter, whereas Pax5, together with Ets1 and Pu.1, regulates the stronger proximal promoter. In the absence of Pax5, the function of the proximal Ebf1 promoter and accumulation of EBF1 protein are impaired and the replication timing and subcellular localization of the Ebf1 locus are altered. Taken together, these data suggest that the regulation of Ebf1 via distinct promoters allows for the generation of several feedback loops and the coordination of multiple determinants of B lymphopoiesis in a regulatory network.

SUPPLEMENTAL MATERIAL

We are grateful to E. Kremmer (GSF) for the preparation of the monoclonal anti-EBF antibody and Ingrid Falk and Andreas Wurch for FACS sorting. We thank Veronique Azuara for help and advice with the replication timing analyses. Bacterial expression plasmids for the DNA-binding domains of Pax5 and Ets1 proteins have been a kind gift of Cynthia Wolberger. We thank Yuzuru Kanakura for the STAT5A-CA and cyclin D1-luciferase plasmids.

This work was supported by a grant from the German Research Foundation (DFG).