Fig. 2: Lola-I establishes paused Pol II throughout the embryo.
From: Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development
a Immunostaining using the Lola-I antibodies (yellow) shows that Lola-I is expressed ubiquitously in the late (14–17 h AEL) Drosophila embryo. As control, Lamin is shown (pink), which is also ubiquitous and nuclear. The brightness and contrast settings of the lookup table are linearly adjusted for clarity. The settings in the individual panels are the same as in the merge. scale bar: left - 100 µm and right - 10 µm. b ChIP-seq data shown as normalized reads per million (RPM) from isolated embryonic tissues of late-stage embryos (14–17 h AEL) using either Lola-I antibodies (turquoise) or Pol II antibodies (dark blue) reveal that Lola-I binding and paused Pol II are found in all examined tissues at Lola-I targets, even when the target gene is expressed only in a specific tissue (Gip is shown as example). Ubiquitous Pol II is not found for all genes, e.g., for the control gene Osi20, Pol II binding and expression is restricted to tracheal cells. The tissue-specific expression of Gip in crystal cells and Osi20 in tracheal cells are known from in situ hybridization shown below, courtesy of Berkeley Drosophila Genome Project93,94. c Average Pol II occupancy, Lola-I occupancy, and scRNA-seq levels across tissues confirm that Lola-I target genes show paused Pol II broadly across tissues but show tissue-specific gene expression, similar to the expression of previously described control genes that have restricted Pol II occupancy across tissues18. The enrichment of the Pol II ChIP-seq signal was calculated for each promoter over input, and values of <2 fold were set to 0 (min). For each tissue, the values were then sorted from low to high and normalized to the highest value (max). These sorted values were then averaged across tissues for either the Lola-I targets or the control, showing that the values extend much broader across tissues for the Lola-I targets than for the control. The same procedure was used to depict the scRNA-seq expression values. The Lola-I binding signal was calculated in the same way, except that the values from all genes were normalized to the same maximum signal in order to show that the control genes have lower Lola-I binding.
