Biophysical investigation of Forkhead box protein O4 – CREB binding protein complex formation

Abstract

The transcription machinery assembles through cooperative interactions between DNA-bound activators and coactivators. During RNA polymerase II complex assembly, CREB-binding protein (CBP) functions as a general coactivator, linking transcription factors to the core transcriptional apparatus via multiple domains, including the KIX domain. The forkhead box protein O4 (FOXO4), a transcription factor involved in DNA damage-induced senescence, interacts with CBP using its intrinsically disordered transactivation domains (TADs). Although CBP-KIX–FOXO-TAD interactions have been characterized in FOXO3a, the details of FOXO4 interactions with CBP-KIX remain uninvestigated. To understand the formation of the transcription initiation complex for anti-apoptotic gene expression, CBP–FOXO4 interactions should be investigated. This thesis presents detailed information on the interactions of the intrinsically disordered conserved region 3 (CR3) and the conserved region 2C (CR2C) domain of FOXO4 with the CBP-KIX, primarily using solution NMR spectroscopy. Chapter 1 focuses on CR3, which contains tandem ΦXXΦΦ motifs, and adopts an α- helical conformation upon the MLL site of KIX binding. The binding driving energy at the MLL site appears to be entropy-driven, whereas at the c-Myb site it is enthalpy-driven. Chapter 2 explores the CR2C, which contains a single ΦXXΦΦ motif and a proline-rich segment that maintains heterogeneous conformation even in the bound state. It is well established that KIX-binding proteins share a common ΦXXΦΦ motif, and the importance of this motif was confirmed in both CR2C and CR3. Despite the distinct conformational characters of each region, both CR3 and CR2C preferentially interact with the MLL-binding site. Our experimental results and structural modeling collectively suggest the promiscuous binding of CR3 and CR2C to the KIX domain. This study highlights how a compact folded KIX can recognize multiple intrinsically disordered regions through their structural plasticity. The molecular distinctions between FOXO3a and FOXO4-TADs emphasize the diversity of FOXO–CBP regulatory mechanisms and provide broader insight into how disordered transactivation domains modulate coactivator recruitment and transcription.