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 lineagespecific 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.

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells

“…EBF1 can bind DNA as a homodimer [60], but can further interact and cooperate with other transcription factors like MEF2C [61], the deoxygenase TET2, an enzyme involved in the DNA demethylation process [62], or the histone acetyltransferase CBP [63]. EBF1 also binds to CNOT3, a subunit of the CCR4-NOT complex [64] which regulates multiple steps in RNA metabolism including transcription, nuclear RNA export and RNA decay [65], and thereby also modulates target gene profiles of EBF. In addition, two multi-zinc finger proteins, ZNF423 and ZNF521, antagonize the biological activity of EBF1 and thereby might promote tumorigenesis [66].…”

Polypyrimidine Tract Binding Proteins are essential for B cell development

“…To understand the roles of PTBP1 in B cell development we first inspected mRNA expression of genes important for B cell lymphopoiesis. We found similar mRNA abundance in P1P2dKO compared to control and P1sKO pro-B cells in most of these genes including Cnot3 (Inoue et al, 2015;Yang et al, 2016) and Pax5 (Urbánek et al, 1994) ( Figure S4E). E2A (encoded by Tcf3) and IKAROS are two transcription factors with AS (Schjerven et al, 2013;Sun & Baltimore, 1991) important for B cell ontogeny.…”

Interplay of primary bovine lymphocytes and Mycobacterium avium subsp. paratuberculosis shows distinctly different proteome changes and immune pathways in host-pathogen interaction

“…Poor MHC II expression can cause autoimmune or infectious diseases, since MHCII is indispensable for adequate immune responses (Rodríguez-Gil et al, 2017). Additionally, CNOT proteins also contribute to the downregulation of MHC class I gene expression by influencing transcription and mRNA degradation (Yang et al, 2016). Enriched network analyses of secreted proteins from ID PBL revealed further major different functions of the 38 differentially abundant proteins (Figure 4B).…”