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Single molecule tracking raw data: SOX18 and its dominant-negative mutant SOX18RaOp

Citation

McCann, Alex et al. (2021), Single molecule tracking raw data: SOX18 and its dominant-negative mutant SOX18RaOp, Dryad, Dataset, https://doi.org/10.5061/dryad.xsj3tx9fp

Abstract

Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-Lymphedema-Telangiectasia-Renal Syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.

Methods

Single Molecule Tracking – Imaging

Immediately prior to imaging cells were washed twice and replaced with Fluorobrite DMEM (Glibco) imaging media. JF549 dye was a gift from Dr. Luke Lavis (Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, United States). 2 drops/mL of NucBlue Live ReadyProbes Reagent (Hoechst 33342) was added directly to the media and cells were incubated for 5 min at 37 oC with 5 % CO2, prior to adding 2 nM of JF549 Halo-tag dye directly to the media and cells and incubation for a further 15 min at 37 oC with 5 % of CO2. Following incubation, cells were washed twice and replaced with Fluorobrite DMEM (Glibco).

Images were acquired on an Elyra single molecule imaging (PALM/STORM) total internal reflection fluorescence (TIRF) microscope, with an Andor 897 EMCCD camera, SR Cube 05 RL – BP 420-480 / BP 570-640 / LP 740 filter set and 100 X oil 1.46 NA TIRF objective using ZEISS ZEN blue software.

Cells were imaged using a 561 nm excitation laser (power = 11.6 µW oblique illumination, 1.96 mW epi illumination; power density = 0.728 W/cm2 oblique illumination, 123.24 W/cm2 epi illumination) with a high-power TIRF filter (TIRF_HP). Using these parameters, we performed two different acquisition techniques; fast SMT which uses a 20 ms acquisition speed to acquire 6000 frames without intervals, and slow SMT which uses a 500 ms acquisition speed to acquire 500 frames without intervals. A low laser power was used to achieve a good signal-to-noise ratio with minimal photobleaching during imaging. Target and surrounding cells were prebleached for approximately 3 minutes (total across cells) prior to imaging to reduce the density of HALO-tagged molecules, background fluorescence, and the fluorescence interference from surrounding cells.

Funding

National Health and Medical Research Council, Award: GNT1120381

National Health and Medical Research Council, Award: GNT1164000

National Health and Medical Research Council, Award: GNT1111169

National Health and Medical Research Council, Award: GNT1155794

National Health and Medical Research Council, Award: GNT1104461

Australian Research Council, Award: LE130100078

National Health and Medical Research Council, Award: GNT1124762

Jacob Haimson Beverly Mecklenburg Lectureship

Biological Optical Microscopy Platform (BOMP)

Jacob Haimson Beverly Mecklenburg Lectureship

Biological Optical Microscopy Platform (BOMP)