Functional domain analysis of SOX18 transcription factor using a single-chain variable fragment-based approach

Abstract

Antibodies are routinely used to study the activity of transcription factors, using various in vitro and in vivo approaches such as electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, genome-wide method analysis coupled with next generation sequencing, or mass spectrometry. More recently, a new application for antibodies has emerged as crystallisation scaffolds for difficult to crystallise proteins, such as transcription factors. Only in a few rare cases, antibodies have been used to modulate the activity of transcription factors, and there is a real gap in our knowledge on how to efficiently design antibodies to interfere with transcription. The molecular function of transcription factors is underpinned by complex networks of protein-protein interaction and in theory, setting aside intra-cellular delivery challenges, developing antibody-based approaches to modulate transcription factor activity appears a viable option. Here, we demonstrate that antibodies or an antibody single-chain variable region fragments are powerful molecular tools to unravel complex protein-DNA and protein-protein binding mechanisms. In this study, we focus on the molecular mode of action of the transcription factor SOX18, a key modulator of endothelial cell fate during development, as well as an attractive target in certain pathophysiological conditions such as solid cancer metastasis. The engineered antibody we designed inhibits SOX18 transcriptional activity, by interfering specifically with an 8-amino-acid motif in the C-terminal region directly adjacent to α-Helix 3 of SOX18 HMG domain, thereby disrupting protein-protein interaction. This new approach establishes a framework to guide the study of transcription factors interactomes using antibodies as molecular handles.

Biophysical Techniques for Target Validation and Drug Discovery in Transcription-Targeted Therapy

“…In addition, they found that chemical modifications to SM4 dramatically changed its PPI disrupting capacity, opening the door to fine-tuning of SOX18 activity [130]. In line with this, they reported that antibodies and antibody fragments could be used to modulate transcriptional output [131].…”

Recombinant Antibody Engineering Enables Reversible Binding for Continuous Protein Biosensing

“…Phage particles were purified from the culture supernatant by two rounds of PEG/NaCl precipitation as described elsewhere. 55 The phage titer was determined by infecting an exponentially growing E. coli XL1-blue culture with serial dilutions of purified library phage. Infected bacteria were plated on 2YT-AmpGlu plates and colonies were counted following overnight incubation at 37 °C.…”

“…One round of biopanning was performed against EGFR-His as described previously. 55 In brief, immunotubes (Nunc Maxisorp, Thermo Fischer Scientific) were coated overnight with 2 × 10 μg interleukin 22-His (negative antigen) and 10 μg EGFR-His (positive antigen) followed by a series of washes with PBS. The tubes were blocked with 1% casein in PBS (CPBS) for 1 h at room temperature (RT).…”

“…Phages were amplified overnight at 30 °C, 200 rpm and separated from bacterial cells by centrifugation on the following day. Phage particles were purified from the culture supernatant by two rounds of PEG/NaCl precipitation as described elsewhere . The phage titer was determined by infecting an exponentially growing E.…”

Homodimerization regulates an endothelial specific signature of the SOX18 transcription factor

“…The HMG-box is present in all groups of SOX proteins (A-H, 20 SOX) and is classically used as a reference to align and compare these proteins since this region is highly conserved ( 7 ). It is made up of 3 α-helixes, whereby α1 and α2 are involved with DNA binding while α3 is involved in protein-protein interactions ( 13 ). The HMG-box recognizes a heptameric consensus sequence (5-A/TA/TCAAA/TG-3) on the DNA.…”