Modulation of DNA Binding Properties of CCAAT/Enhancer Binding Protein Epsilon by Heterodimer Formation and Interactions with NFkappaB Pathway.

Specific regulation of gene expression is achieved through interaction of multiple transcriptional activators and repressors with their cognate DNA sites. This activity is subject to several levels of control, including post-translational modification by phosphorylation, acetylation, selective degradation and interaction with co-activators and co-repressors.

Recent studies directed at discovering C/EBP- responsive genes resulted in identification of a diverse and degenerate collection of putative C/EBP binding sites. Identification of binding sites for each individual member of the C/EBP family is complicated by common co-expression of several of these proteins in the same cell and their ability to form hetero-dimeric complexes with each other and structurally-related ATF/CREB proteins. Activity of C/EBP proteins is regulated by phosphorylation mediated through several major kinase pathways, that affect DNA-binding, subcellular localization in the nucleus, and interaction with cell cycle proteins.

Our studies of C/EBP-e phosphorylation identified several sites, including one for p38 MAPK within the transactivation domain of C/EBP-e. As a result of phosphorylation at Threonine-75 (T75), C/EBP-e DNA binding and specific transcriptional activity was significantly enhanced.

In this study, we identified consensus binding sequences for C/EBP-e and for heterodimers of C/EBP-e with its most common dimerization partner ATF4. Furthermore, we find that C/EBP-e- DNA interaction is highly upregulated by interaction with activated NFkappaB pathway protein p65-RelA. This interaction depends on the T75 phosphorylation status of C/EBP-e and can be enhanced by introduction of phosphomimetic Thr to Asp mutation. By using selective removal of p65-RelA and siRNA mediated p65 knock-down, we demonstrate that activated NFkappaB pathway is required for high level expression of genes with C/EBP-e responsive promoter. This novel interaction may play an important role in regulation of a C/EBP-e - dependent genes in myeloid cells, for example at sites of infection or other inflammations.