Abstract
Significant insight into the signaling pathways leading to activation of the Rel transcription factor family, collectively termed NF-κB, has been gained. Less well understood is how subsets of NF-κB-dependent genes are regulated in a signal specific manner. The SIMPL protein (signaling molecule that interacts with mouse pelle-like kinase) is required for full Tumor Necrosis Factor-α (TNFα) induced NF-κB activity. We show that SIMPL is required for steady-state hematopoiesis and the expression of a subset of TNFα induced genes whose products regulate hematopoietic cell activity. To gain insight into the mechanism through which SIMPL modulates gene expression we focused on the Tnf gene, an immune response regulator required for steady-state hematopoiesis. In response to TNFα SIMPL localizes to the Tnf gene promoter where it modulates the initiation of Tnf gene transcription. SIMPL binding partners identified by mass spectrometry include proteins involved in transcription and the interaction between SIMPL and MED1 was characterized in more detail. In response to TNFα, SIMPL is found in p65-MED1 complexes where SIMPL enhances p65/MED1/SIMPL complex formation. Together our results indicate that SIMPL functions as a TNFα-dependent p65 co-activator by facilitating the recruitment of MED1 to p65 containing transcriptional complexes to control the expression of a subset of TNFα-induced genes.
Highlights
NF-kB controlled genes are a defining feature of the immune system, regulating both immune cell development and function
SIMPL is a key regulator of Tumor Necrosis Factor-a (TNFa), but not IL-1 induced NF-kB activity [20], [21], identifying SIMPL as a unique target to probe for signal specific NF-kB regulation
We performed a microarray analysis on RNA isolated from TNFa treated mouse embryo fibroblasts (MEFs) derived from littermate control or SIMPL2/2 mice (Fig. 1)
Summary
NF-kB controlled genes are a defining feature of the immune system, regulating both immune cell development and function. Analysis of endogenous gene expression in mouse embryo fibroblasts (MEFs) derived from mice lacking individual Rel family members has revealed that DNA binding site specificity of the c-rel/NF-kB family members is not merely regulated by the binding site sequence [1]. KB, involves activation of a preformed complex containing IkB kinase-a (IKKa), IkB kinase-b (IKKb) and IKKc/NEMO, a nonenzymatic scaffold protein. Activated IKKa and IKKb phosphorylate two key serine residues located amino terminal to the ankyrin repeat region in IkBa and IkBb [4]–[8]. Phospho-IkB is ubiquitylated on lysine residues near the phosphorylated residues, leading to removal of IkB from the NF-kB (p65/p50) containing complex via proteolysis. Analysis of mouse embryo fibroblasts derived from IKKa2/2 and IKKb2/2 mice suggest that IKKa controls basal NF-kB activity whereas IKKb controls cytokine induced NF-kB activity [9]–[14]
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