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.
Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. While the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity (https://research.cchmc.org/Ciliome_Gene_Expression/). Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by utilizing Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore long-standing questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.
The SOX transcription factor family is pivotal in controlling aspects of development. To identify genotype–phenotype relationships of SOX proteins, we performed a non-biased study of SOX using 1890 open-reading frame and 6667 amino acid sequences in combination with structural dynamics to interpret 3999 gnomAD, 485 ClinVar, 1174 Geno2MP, and 4313 COSMIC human variants. We identified, within the HMG(High Mobility Group)- box, twenty-seven amino acids with changes in multiple SOX proteins annotated to clinical pathologies. These sites were screened through Geno2MP medical phenotypes, revealing novel SOX15 R104G associated with musculature abnormality and SOX8 R159G with intellectual disability. Within gnomAD, SOX18 E137K (rs201931544), found within the HMG box of ~0.8% of Latinx individuals, is associated with seizures and neurological complications, potentially through blood–brain barrier alterations. A total of 56 highly conserved variants were found at sites outside the HMG-box, including several within the SOX2 HMG-box-flanking region with neurological associations, several in the SOX9 dimerization region associated with Campomelic Dysplasia, SOX14 K88R (rs199932938) flanking the HMG box
Our group and others have previously linked SOX (SRY‐related HMG‐box) genes to cardiovascular regulation and cancer. SOX proteins are transcription factors containing a highly conserved HMG (high mobility group) box DNA binding domain that enables sequence specific DNA interaction, bending, and gene regulation. Sequence variation in the HMG box of SOX genes has not been fully explored in humans; therefore, we have evaluated the variants within the 20 human SOX genes from ~60,000 exomes/genomes of gnomAD and also the cancer COSMIC database for somatic variations. Each variant was analyzed using codon selection and molecular modeling. A total of 3,049 variants were identified from gnomAD and 1,566 from COSMIC, with an enrichment of variants that falls within the HMG domain in cancer patients. Uniquely we highlighted an enrichment of HMG box variants in cancers of the large intestine within SOX genes with the highest enrichment in the SOX9 gene. Our most conserved and potentially impactful variant was identified as SOX18 E137K, found within 0.8% of Latino individuals. By comparing stability differences of the E137K variant against wild type SOX18 structures, using molecular dynamics simulations, SNP analysis at amino acid 137 (Glutamic acid to Lysine) was shown to disrupt the structure of the third α‐helix of the HMG box. Since other variants that fall within the HMG box of SOX18 are associated with disorders of the vasculature and lymphatics, we suspect SOX18 E137K to potentially affect vascular function through the alteration of its interaction with DNA. Work is underway to begin to assess the physiologic implications of this novel variant.Support or Funding InformationSupport for this study was from NIH‐K01ES025435 (to JWP) and Walsh UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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