Fig. 4: Single-molecule FISH reveals that Lola-I lowers the gene activation barrier.
From: Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development
a Example of single-molecule FISH images of Gip (yellow), and PPO1 and PPO2 genes (pink) in wild-type embryos and lola-I −/− mutant embryos (DAPI - blue). Data were acquired with ×40 magnification, scale bar - 20 µm, with the same brightness and contrast settings. Top insets show the nascent Gip signal in the nuclei; bottom insets show the single Gip RNAs in the cytoplasm. The brightness and contrast in each inset was adjusted linearly for clarity. b The percentage of crystal cells (defined by high expression of PPO1 and PPO2) that express high levels of Gip is strongly reduced in lola-I mutant embryos compared to wild-type. Barplots show the mean, and the whiskers show the standard error of the mean, with the number of embryos (n): wild-type: 10–12 h n = 7, 12–14 h n = 8, 14–16 h n = 4, lola-I mutant: 10–12 h n = 7, 12–14 h n = 7, 14–16 h n = 7. Reduced Gip expression is observed for multiple time points showing that it is not due to a developmental delay and that Gip expression only mildly recovers over time. c The percentage of PPO1 and PPO2 positive crystal cells with a visible Gip nascent site of transcription (indicative of a transcriptional burst) is also strongly reduced in lola-I mutants (12–14 h AEL). The bar represents the mean, and the whiskers show the standard error of the mean (wild-type n = 7 and lola-I mutant n = 14, where n is the number of embryos). d Two-state model that was fitted to the data. e Histograms of mRNA/cell (dots), calculated from fluorescent intensities at ×100 magnification (see Methods), and fitted lines. The ratio of Koff and Kon was fixed to the ratio of crystal cells with visible nascent transcripts between wild-type and mutants as shown in (c).
