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) NFIX, 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) There is a lack of structural information on NFIX and relative TF family members because of the challenge expression and purification of soluble protein constructs in the amount required for structural characterization. We were able to obtain functional NFIX constructs using E. coli cells, and to purify them with a high yield. NFIX constructs were tested for correct folding, stability, and DNA-binding through a series of biochemical and biophysical methods. Furthermore, we managed to produce well-diffracting NFIX crystals, which were used for Single Anomalous Diffraction (SAD) phasing. We collected two different datasets of same NFIX construct at 2.7 Å and 3.5 Å resolution, in two different space groups. Structural analysis of this NFIX construct shed first light on this class of TFs and put the bases for the understanding of its DNA-binding mode. (2) Genomic data of NF-Y locations at gene promoters indicate that NF-Y plays a key role in oncogenic activation. The knowledge of the 3D structure of NF-Y in complex with its CCAAT box provided the rationale for developing inhibitors able to interfere with DNA-binding. The pipeline used to search for NF-Y inhibitors consisted of in silico screenings of compounds that interfere with NF-Y functional trimerization and/or with CCAAT box interaction, followed by in vitro biochemical/biophysical confirmation of inhibition and X-ray crystallography validation. The selected compound from the initial screening was suramin, which proved to bind to NF-Y and to functionally inhibit the binding of DNA. We obtained suramin/protein complex co-crystals, which diffracted up to 2.3 Å resolution. The crystal structure of the suramin/NF-Y provides the first evidence of NF-Y inhibition by a small molecule.

STRUCTURAL ANALYSIS OF TRANSCRIPTION FACTOR/DNA COMPLEXES / M. Lapi ; scientific tutor: M. Nardini ; coordinator: A. Chaves Sanjuan. - : . Dipartimento di Bioscienze, 2021 Apr 22. ((33. ciclo, Anno Accademico 2020.

STRUCTURAL ANALYSIS OF TRANSCRIPTION FACTOR/DNA COMPLEXES

M. Lapi
2021

Abstract

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) NFIX, 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) There is a lack of structural information on NFIX and relative TF family members because of the challenge expression and purification of soluble protein constructs in the amount required for structural characterization. We were able to obtain functional NFIX constructs using E. coli cells, and to purify them with a high yield. NFIX constructs were tested for correct folding, stability, and DNA-binding through a series of biochemical and biophysical methods. Furthermore, we managed to produce well-diffracting NFIX crystals, which were used for Single Anomalous Diffraction (SAD) phasing. We collected two different datasets of same NFIX construct at 2.7 Å and 3.5 Å resolution, in two different space groups. Structural analysis of this NFIX construct shed first light on this class of TFs and put the bases for the understanding of its DNA-binding mode. (2) Genomic data of NF-Y locations at gene promoters indicate that NF-Y plays a key role in oncogenic activation. The knowledge of the 3D structure of NF-Y in complex with its CCAAT box provided the rationale for developing inhibitors able to interfere with DNA-binding. The pipeline used to search for NF-Y inhibitors consisted of in silico screenings of compounds that interfere with NF-Y functional trimerization and/or with CCAAT box interaction, followed by in vitro biochemical/biophysical confirmation of inhibition and X-ray crystallography validation. The selected compound from the initial screening was suramin, which proved to bind to NF-Y and to functionally inhibit the binding of DNA. We obtained suramin/protein complex co-crystals, which diffracted up to 2.3 Å resolution. The crystal structure of the suramin/NF-Y provides the first evidence of NF-Y inhibition by a small molecule.
NARDINI, MARCO
CHAVES SANJUAN, ANTONIO
Settore BIO/10 - Biochimica
Settore BIO/18 - Genetica
STRUCTURAL ANALYSIS OF TRANSCRIPTION FACTOR/DNA COMPLEXES / M. Lapi ; scientific tutor: M. Nardini ; coordinator: A. Chaves Sanjuan. - : . Dipartimento di Bioscienze, 2021 Apr 22. ((33. ciclo, Anno Accademico 2020.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/834212
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