Abstract
Transcription factor CCAAT/enhancer-binding protein (C/EBP)-beta is crucial for regulating transcription of genes involved in a number of diverse cellular processes, including those involved in some cytokine-induced responses. However, the mechanisms that contribute to its diverse transcriptional activity are not yet fully understood. To gain an understanding into its mechanisms of action, we took a proteomic approach and identified cellular proteins that associate with C/EBP-beta in an interferon (IFN)-gamma-dependent manner. Transcriptional mediator (Mediator) is a multisubunit protein complex that regulates signal-induced cellular gene transcription from enhancer-bound transcription factor(s). Here, we report that the Med1 subunit of the Mediator as a C/EBP-beta-interacting protein. Using gene knock-out cells and mutational and RNA interference approaches, we show that Med1 is critical for IFN-induced expression of certain genes. Med1 associates with C/EBP-beta through a domain located between amino acids 125 and 155 of its N terminus. We also show that the MAPK, ERK1/2, and an ERK phosphorylation site within regulatory domain 2, more specifically the Thr(189) residue, of C/EBP-beta are essential for it to bind to Med1. Last, an ERK-regulated site in Med1 protein is also essential for up-regulating IFN-induced transcription although not critical for binding to C/EBP-beta.
Highlights
The CCAAT/enhancer-binding proteins (C/EBPs)3 are a group of structurally similar, but functionally and genetically distinct, transcription factors, which participate in a number of physiological activities
Using IFN signaling as a model, we searched for cellular factors that associate with C/EBP- using a proteomic approach
To identify the proteins that associated with C/EBP-, mouse macrophage cell line RAW264.7 was stimulated with mouse IFN-␥ for 2, 4, 6, 12, 16, and 24 h
Summary
The CCAAT/enhancer-binding proteins (C/EBPs) are a group of structurally similar, but functionally and genetically distinct, transcription factors, which participate in a number of physiological activities. Using IFN signaling as a model, we searched for cellular factors that associate with C/EBP- using a proteomic approach These studies identified Med ( known as TRAP220/PBP/DRIP220/CRSP220), a subunit of the transcriptional Mediator as an IFN-induced C/EBP--binding protein [15,16,17]. Med is subject to regulation by ERKs. Mutations in the recently reported ERK1/2-regulated motifs of Med did not inhibit its interaction with C/EBP- but suppressed IFN-induced transcription. Mutations in the recently reported ERK1/2-regulated motifs of Med did not inhibit its interaction with C/EBP- but suppressed IFN-induced transcription These studies, identify a novel regulatory aspect of C/EBP--driven transcription in response to IFN-␥
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