STRUCTURAL ANALYSES OF TRANSCRIPTION FACTOR/DNA COMPLEXES: NFI-X AND NF-Y

Binding of transcription factors (TFs) to discrete sequences in gene promoters and enhancers is crucial to the process by which genetic information is transferred to biological functions. TF structural analysis is the key to understanding their DNA-binding mode and for the design of specific inhibitors. In this context, the present PhD project focuses on two TFs: (1) NFI-X, a TF with unknown structure that binds to the palindromic motif TTGGC(n5)GCCAA and plays an essential role in skeletal muscle development; and (2) NF-Y, a histone-like TF that binds the CCAAT box in promoters of cell cycle genes. (1) The crystal structure of NFI-X was obtained in our lab a few years ago. However, no structural information regarding the DNA-binding mode of NFI-X was available until now. We were able to obtain a functional truncated DNA-binding domain (DBD) construct of NFI-X and purify it for cryo-EM analysis. This allowed us to solve a cryo-EM structure at 3.86 Å resolution, revealing for the first time the architecture of the NFI-X/DNA complex. Surprisingly, the NFI-X/DNA sample formed filaments in the grid after vitrification, which allowed to solve the structure by helical processing. This structure provides critical insights into how NFI-X recognizes its DNA target and mediates dimerization. (2) A new crystal structure of an extended NF-Y construct, termed the NF-Y long-minimal domain (NF-Yl-md), were obtained, which included additional regions of the NF-YC subunit not present in previous structures. These additional regions could potentially influence the DNA-binding and cooperative interactions of NF-Y with other TFs. In parallel, two cryo-EM structures of NF-Y in complex with ATF6 and two cryo-EM maps of NF-Y in complex with USF1 have been elucidated, demonstrating how NF-Y cooperates with these transcription factors to recognize composite DNA elements. These structures provide unprecedented insights into the cooperative assembly of multi-TF complexes on DNA. These findings contribute to the broader understanding of TF-DNA interactions, offering potential avenues for the design of targeted inhibitors that could modulate TF activity in various biological processes.