Inhaltszusammenfassung

The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) play a central role in circadian transcriptional regulation and chromatin modification. The interactions of BMAL1’s C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with CRY1 (repressing) as well as the BMAL1 G-region preceding the TAD regulate the circadian oscillations. The repressive BMAL1-TAD-CRY1 interactio...The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) play a central role in circadian transcriptional regulation and chromatin modification. The interactions of BMAL1’s C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with CRY1 (repressing) as well as the BMAL1 G-region preceding the TAD regulate the circadian oscillations. The repressive BMAL1-TAD-CRY1 interactions are enhanced by the circadian acetylation of Lys-537 within the BMAL1 G-region. The CBP-KIX domain interacts with a plethora of transcription regulators via its two distinct pockets referred to as MLL- and CREB-pKID/c-Myb-binding pockets, typically targeting the intrinsically disordered regions within the TAD, which often attain folding upon binding to the KIX domain. In this thesis, we characterized the interaction of the CBP-KIX domain with BMAL1 proteins including the BMAL1-TAD, parts of the G-region, and Lys-537. Tethering the small compound 1-10 in the MLL-binding pocket of the CBP-KIX domain weakened BMAL1 binding and MLL1-bound KIX did not form a ternary complex with BMAL1, indicating that the MLL-binding pocket is important for KIX-BMAL1 interactions. Additionally, mutations in the second pKID/c-Myb-binding pocket of the KIX domain moderately impacted BMAL1 binding. The BMAL1(K537Q) mutation mimicking Lys-537 acetylation, however, did not affect the KIX-binding affinity, in contrast to its enhancing effect on CRY1 binding. Moreover, KIX binding does not induce the formation of extended regular secondary structures in BMAL1. SAXS models of BMAL1 and BMAL1:KIX complexes revealed that the N-terminal BMAL1 G-region including Lys-537 forms elongated extensions emerging from the bulkier BMAL1-TAD:KIX core complex. Fitting high-resolution KIX domain structures into the SAXS-derived envelopes suggested that the G-region emerges near the MLL-binding pocket, further supporting a role of this pocket in BMAL1 binding. This study significantly advances the mechanistic understanding of the roles of the BMAL1-TAD-CBP-KIX interaction and its interplay with other KIX ligands and with the BMAL1-CRY1 interaction in circadian gene regulation.» weiterlesen» einklappen

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DDC Sachgruppe:
Biowissenschaften, Biologie