Studies of penta-decameric binding proteins: AP-4 and CBF-A

Abstract: Each immunoglobulin (Ig) V gene segment contains an upstream promoter region. The octamer element and the TATA box are the only elements that are conserved in all Ig promoters. The presence of the octamer element is necessary, but not sufficient to support high levels of transcription. Therefore, the activity of other co-stimulatory elements is required. This investigation has focused on the study of one such regulatory region: the penta-decameric (pd) element. The pd element is an integral part of the SP6 V-kappa promoter and shows functional synergism with the octamer element at the late stages in B cell differentiation. The pd element is comprised of an E2A type of E-box (5’-CAGCTG-3-) and an AT-rich region. Previous work had identified CArG box-binding factor-A (CBF-A) as the protein interacting with the AT-rich moiety. However, the ligand for the E-box was an unknown. Our work has now identified the bHLH-Zip protein AP-4 as the E-box binding factor. In addition, AP-4 has been found to bind to the second E-box (5’-CAGCTG-3’) found 3’ from the octamer site. E-boxes are conserved within human V-kappa promoter subgroups I, III, IV, V and VII, and related families, hence reinforcing the idea that the natural ligand for Ig-kappa promoter E-boxes is AP-4. Lastly, we pursued the study of CBF-A by searching for interacting partners by using a yeast Two-Hybrid screen. We have now characterised two important proteins interacting with CBF-A, the nucleolar phosphoprotein nucleophosmin(NPM/B23) and hnRNP H. Furthermore, CBF-A shows selectivity in its interactions, since CBF-A/NPM complexes are detected in the nucleus only, as opposed to the cytoplasmic only CBF-A/hnRNP H heterotypic complexes. Interestingly, we have shown that CBF-A undergoes self-oligomerisation in both the nuclear and cytoplasmic compartments. In addition, we have evidence to suggest that CBF-A might be shuttling between the nucleus and the cytoplasm. Specifically, we show that mitogenic stimulation of resting B cells promotes CBF-A accumulation in the nucleus. We have mapped the region responsible for the import/export activity of CBF-A to the most carboxyl-terminal 13 amino acid residues.