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Differential Nuclear Localization of SOX18 Variants in Transiently Transfected Epithelial Cells
Author(s) -
Cvammen William,
Prokop Jeremy W.,
Qutob Dinah,
Freeland Thomas,
Underwood Adam
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.717.3
Subject(s) - transfection , biology , microbiology and biotechnology , population , genetics , mutagenesis , gene , mutation , demography , sociology
Sry‐related box (SOX) proteins are transcription factors containing a conserved High Mobility Group‐box (HMG‐box) DNA‐binding domain that regulate embryogenesis and tissue maintenance. SOX mutations result in cancer, developmental abnormalities, sex reversal, and circulatory dysfunction. Genomic analysis of the 20 human SOX genes identified 3,049 variants from gnomAD and 1,566 from COSMIC with an enrichment of variants that falls within the HMG box. The most impactful variant identified occurred in the third alpha helix of the SOX18 HMG‐box that replaces a glutamic acid (E) for a lysine (K) (rs201931544). Since other variants occurring within the HMG box of SOX18 are linked to vascular and lymphatic disfunction, we predict SOX18 E137K will also derange protein function through altered DNA interaction and localization to the nucleus. No literature exists regarding E137K, although it is present in 0.8% of Latino population. The purpose of this project was to produce native and mutated SOX18 expression constructs to determine if nuclear accumulation is impacted. Native human SOX18 was first cloned into pHTCHaloTag® CMV‐neo vector (Promega), followed by site‐directed mutagenesis to synthesize pHTCHalo/SOX18‐E137K. After sequence confirmation, human dermal lymphatic endothelial cells, HeLa and A375 were transiently transfected with either the native or mutated construct, or a control vector (pHTCHaloTag® CMV‐neo vector with no insert), in glass chamber slides. After 24hr, live cells were incubated with HaloTag® Oregon Green® Ligand (Promega) and micrographs were captured on an Olympus iX51 with DP71 digital camera. Controls showing cytochemistry specificity include cells transfected with SOX18 incubated with PBS in place of Oregon Green® Ligand. From these trials, it was determined that E137K reduces nuclear localization of SOX18 in all cell lines and may account for divergent phenotypic consequences when compared to native SOX18. Support or Funding Information Support for this study was from NIH‐K01ES025435 (to JWP) and Walsh University This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .