Interaction of CCR4–NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis

Abstract

Transcription factor EBF1 (early B-cell factor 1) regulates early B-cell differentiation by poising or activating lineage-specific genes and repressing genes associated with alternative cell fates. To identify proteins that regulate the diverse functions of EBF1, we used SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry of proteins associated with endogenous EBF1 in pro-B cells. This analysis identified most components of the multifunctional CCR4–NOT complex, which regulates transcription and mRNA degradation. CNOT3 interacts with EBF1, and we identified histidine 240 in EBF1 as a critical residue for this interaction. Complementation of Ebf1−/− progenitors with EBF1H240A revealed a partial block of pro-B-cell differentiation and altered expression of specific EBF1 target genes that show either reduced transcription or increased mRNA stability. Most deregulated EBF1 target genes show normal occupancy by EBF1H240A, but we also detected genes with altered occupancy, suggesting that the CCR4–NOT complex affects multiple activities of EBF1. Mice with conditional Cnot3 inactivation recapitulate the block of early B-cell differentiation, which we found to be associated with an impaired autoregulation of Ebf1 and reduced expression of pre-B-cell receptor components. Thus, the interaction of the CCR4–NOT complex with EBF1 diversifies the function of EBF1 in a context-dependent manner and may coordinate transcriptional and post-transcriptional gene regulation.