The blood vasculature instructs lymphatic patterning in a SOX7-dependent manner

Despite a growing catalog of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in a non-cell-autonomous manner by modulating the transcription of angiocrine signals to pattern lymphatic vessels. While SOX7 is not expressed in lymphatic endothelial cells (LECs), the conditional loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signaling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium.Peer reviewe

Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice.

Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics

Exploring the interactome of SOX18 transcription factor and its druggability

An understanding of how genes are regulated in humans and higher eukaryotes is essential for the understanding of normal development and disease. Therefore genetic regulation is a crucial area of research, encompassing a diverse network of scientific disciplines. Early research was based mainly on bacterial systems of genetic regulation and the important findings of the basic mechanisms were extended by more recent advances in eukaryotic gene regulation. My PhD aims to contribute to the field by focusing on a single transcription factor and uncovering its network of interactions.The aim of my research was to explore the interactome of the SOX18 transcription factor and its druggability allowing us to understand the crucial role of protein interactions in fine tuning gene expression. Through utilising a combination of cell-free expression coupled to single molecule techniques, I developed a new platform to study transcriptions factors physical interactions and the ability of small molecules to disrupt these interactions. Deciphering how transcription factors (TFs) coordinate genes expression is fundamental to understanding biological processes and further developing new therapeutic approaches. A single transcription factor has multiple transcriptional effects that are context-dependent. This versatility of activity is thought to be mediated by different protein-protein interactions (PPIs). These PPIs offer a new avenue for the selective pharmacological modulation of transcription factor activity, which has proven to be a challenging endeavour using standard drug discovery approaches centred on blocking protein/DNA binding or interfering with post-translational modifications. In this study we show that SOX18, a transcription factor that acts as a molecular switch of embryonic lymphatic vascular development and neo-lymphangiogenesis during cancer metastasis, interacts with itself (homodimer) and multiple others protein partners specific to endothelial cells. Using a systematic truncation analysis of SOX18 domains, we propose a comprehensive map of the protein binding domains, revealing the existence of different interacting regions specific to subsets of SOX18 interactors. Using mutant forms of SOX18 protein associated with the rare human disease Hypotrichosis-Lymphedema-Telangiectasia (HLTRS) we validate domains essential to drive specific SOX18-dependent PPIs and correlate these variations with phenotypic variations observed in HLTRS.Using a focused library of small molecules, we are able to show that distinct SOX18 protein partner recruitment can be disrupted. On this basis we propose an initial Structure-Activity Relationship study, paving the way to the development of specific inhibitors to SOX18-dependent protein-protein interactions. This approach has enabled us to better understand the mode of action of a compound currently used in clinics to manage the HLTRS condition. Finally, we show that one of our newly identified SOX18 small molecule inhibitors is able to prevent metastasis in a pre-clinical model of breast cancer, further validating the value of targeting SOX18-dependent transcription factor complexes. This work opens up a new molecular strategy to target transcription factor activity and reposition this class of protein as viable a drug target

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

In the post-genome era, pathologies become associated with specific gene expression profiles and defined molecular lesions can be identified. The traditional therapeutic strategy is to block the identified aberrant biochemical activity. However, an attractive alternative could aim at antagonizing key transcriptional events underlying the pathogenesis, thereby blocking the consequences of a disorder, irrespective of the original biochemical nature. This approach, called transcription therapy, is now rendered possible by major advances in biophysical technologies. In the last two decades, techniques have evolved to become key components of drug discovery platforms, within pharmaceutical companies as well as academic laboratories. This review outlines the current biophysical strategies for transcription manipulation and provides examples of successful applications. It also provides insights into the future development of biophysical methods in drug discovery and personalized medicine

A split-luciferase reporter recognizing GFP and mCherry tags to facilitate studies of protein–protein interactions

The use of fluorescently-tagged proteins in microscopy has become routine, and anti-GFP (Green fluorescent protein) affinity matrices are increasingly used in proteomics protocols. However, some protein–protein interactions assays, such as protein complementation assays (PCA), require recloning of each protein as a fusion with the different parts of the complementation system. Here we describe a generic system where the complementation is separated from the proteins and can be directly used with fluorescently-tagged proteins. By using nanobodies and performing tests in cell-free expression systems, we accelerated the development of multiple reporters, detecting heterodimers and homodimers or oligomers tagged with GFP or mCherry. We demonstrate that the system can detect interactions at a broad range of concentrations, from low nanomolar up to micromolar

Homodimerization\ua0regulates an endothelial specific signature of the SOX18 transcription factor

During embryogenesis, vascular development relies on a handful of transcription factors that instruct cell fate in a distinct sub-population of the endothelium (1). The SOXF proteins that comprise SOX7, 17 and 18, are molecular switches modulating arterio-venous and lymphatic endothelial differentiation (2,3). Here, we show that, in the SOX-F family, only SOX18 has the ability to switch between a monomeric and a dimeric form. We characterized the SOX18 dimer in binding assays in vitro, and using a split-GFP reporter assay in a zebrafish model system in vivo. We show that SOX18 dimerization is driven by a novel motif located in the vicinity of the C-terminus of the DNA binding region. Insertion of this motif in a SOX7 monomer forced its assembly into a dimer. Genome-wide analysis of SOX18 binding locations on the chromatin revealed enrichment for a SOX dimer binding motif, correlating with genes with a strong endothelial signature. Using a SOX18 small molecule inhibitor that disrupts dimerization, we revealed that dimerization is important for transcription. Overall, we show that dimerization is a specific feature of SOX18 that enables the recruitment of key endothelial transcription factors, and refines the selectivity of the binding to discrete genomic locations assigned to endothelial specific genes

Rapid mapping of interactions between human SNX-BAR proteins measured in vitro by AlphaScreen and single-molecule spectroscopy

Protein dimerization and oligomerization is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to transfer information in signaling, transcriptional, and membrane trafficking events. Here we show that a combination of cell-free protein expression, a proximity based interaction assay (AlphaScreen), and single-molecule fluorescence allow rapid mapping of homo- and hetero-oligomerization of proteins. We have applied this approach to the family of BAR domain-containing sorting nexin (SNX-BAR) proteins, which are essential regulators of membrane trafficking and remodeling in all eukaryotes. Dimerization of BAR domains is essential for creating a concave structure capable of sensing and inducing membrane curvature. We have systematically mapped 144 pair-wise interactions between the human SNX-BAR proteins and generated an interaction matrix of preferred dimerization partners for each family member. We find that while nine SNX-BAR proteins are able to form homo-dimers, several including the retromer-associated SNX1, SNX2, and SNX5 require heteromeric interactions for dimerization. SNX2, SNX4, SNX6, and SNX8 show a promiscuous ability to bind other SNX-BAR proteins and we also observe a novel interaction with the SNX3 protein which lacks the BAR domain structure