Optogenetic Regulation of Transcription Factor Dynamics and Downstream Gene Expression in Single Cells

Transcription factor p65, a subunit of the NFκB family, is involved in the regulation of cell immune response, inflammation, and carcinogenesis. The downstream gene expression from p65 activation is regulated by localization of the transcription factor into the nucleus of mammalian cells. Localization of p65 is triggered by external stimuli in the cell and is regulated by IκB proteins and the endogenous Nuclear Exporting Signal (NES) and Nuclear Localization Signal (NLS) within p65. For precise manipulation of the translocation of p65, a synthetic approach utilizing an optogenetic system was employed to deliver the stimuli. The endogenous NLS and NES were mutated, allowing for the localization of the transcription factor to be completely dependent on the synthetic, optogenetic signal, and to be unaffected by any naturally occurring signal in the cell. Variations of the mutant p65 plasmid were cloned to compare how deviations of the NLS and NES sequence affect the localization of p65 and the corresponding gene expression output. Using a fluorescent tag, quantification of the abundance of p65 within the nucleus can be achieved through microscopy. By implementing this synthetic approach, precise control over gene expression can be employed, and naturally induced and synthetically induced gene expression can be compared to better understand p65-implicated disease progression.