Fig. 5: Lola-I is a promoter pioneer factor.

a Heatmap of normalized MNase-seq data shows specific loss of nucleosome occupancy at the Lola-I target promoters during the late stages of Drosophila embryogenesis (Wilcoxon two-sided test, *P wt-early vs wt-late: 3.4e−9, wt-late vs mut-late 14–17 h: 3.4e−8). This does not occur in lola-I mutant embryos, as there is no statistically significant difference between wild-type early embryos and late lola-I mutant embryos. b Box plots of the MNase signal (centered on the Lola-I peaks) are shown for promoter-proximal regions and distal Lola-I-bound regions (Supplementary Data 2), which both show a significant decrease in late wild-type embryos (Wilcoxon two-sided test, *P distal, wt-early vs wt-late: *P = 4.5e−09, promoter wt-early vs wt-late: *P < 2e−16, distal wt-late vs mut-late: *P = 1.1e−08, promoter wt-late vs mut-late: *P = 4.1e−10). c Pol II recruitment, shown as change in normalized Pol II enrichment from the early to the late stage, only occurs when the Lola-I peak is found in close proximity to the TSS, showing that Lola-I depletes nucleosomes independently of Pol II recruitment (Wilcoxon two-sided test, *P = 1.2e−8). d Lola-I binds its motif preferentially at the edge of a nucleosome in vitro. Purified full-length Lola-I protein expressed in insect cells by baculovirus is incubated in vitro at different concentrations with nucleosomes reconstituted with Lola-I binding sites embedded in the 601 Widom sequence at different positions. e The relative binding of the Lola-I protein to the 601-templates containing the Lola-I motif vs the control 601-template without the motif is measured as the loss of signal in the nucleosomal band or the gain of signal in the super-shifted band (Supplementary Fig. 6) (relative to the no Lola-I lane - see methods). The results show that Lola-I binds most strongly when the motif is located along the nucleosomal edge. Significant cooperativity with additional motifs is not observed. f Illustration of the preferred position of the Lola-I motif with regard to the nucleosome structure. The nucleosome structure is from the RSCB protein data bank (5NL0). Box plots in the figure show the median (central line), the first and the third quartiles (box), and the largest/smallest value within 1.5 times of the interquartile range as whiskers.