Fig. 1: Lola-I is required for the loading of paused Pol II to target genes in the late Drosophila embryo.

a At promoters that have no Pol II occupancy in the early (2–4 h AEL) embryo but acquire paused Pol II in the late (14–17 h AEL) embryo (opening set), the Lola-I motif was identified de novo within 200 bp upstream of the TSS by MEME analysis (e-value = 8.1e−091). The Lola-I motif was enriched 6.6-fold in the opening set (n = 492) vs the constant set (n = 843) (*P = 8.75e−34, chi-squared test with multiple-testing correction). b A Western blot with antibodies specific for the Lola-I isoform shows that Lola-I increases in expression during embryogenesis. Tubulin is shown as control. Results shown are representative of at least comparable biological experiments and are consistent. Source data is provided as a Source Data file. c Single-gene example of the ChIP-seq data showing that Lola-I binding is found at the promoter of Tpi, a gene that acquires paused Pol II over time. d Heatmaps showing that the ~60% of Lola-I-bound regions found at promoters (n = 329) is associated with an increase in Pol II occupancy, RNA levels from the early (2–4 h AEL) to the late (14–17 h AEL) embryo, and DNAse hypersensitivity from the early (stage 5/3 h AEL) to the late (stage 14/11 h AEL) embryo⁹². A random sample of 250 promoters from the constant set¹⁹ is used as control. The star denotes significance (*P < 2e−16) using a two-sided Wilcoxon test. e Mutant line lola-I ^{ORC4 32} has a premature stop codon before the C2H2 zinc-finger region that codes for the DNA-binding domain. A Western blot confirms that lola-I ^ORC4 homozygous embryos produce a low amount of truncated Lola-I product (white arrow). The Rpb3 subunit of Pol II is shown as control below. The wt and lola ^−/− lanes were not run adjacently in the original gel. Results shown are representative of at least two biological replicates and are consistent. Source data is provided as a Source Data file. f Pol II ChIP-seq signal at the Lola-I target gene Tpi is strongly reduced in homozygous lola ^ORC4 mutant embryos, while the control gene Dl remains unchanged. In the rescue line, which expresses lola-I cDNA in the lola-I ^ORC4 mutant background, Pol II occupancy is rescued. g Heatmap showing that the Pol II ChIP-seq signal and ATAC-seq accessibility is specifically reduced at the Lola-I target promoters in lola-I ^ORC4 mutant embryos. In the rescue line, Pol II occupancy and accessibility are rescued to levels comparable to wild-type. The star denotes significance (Pol II – wt-mutant: *P = 6.0e−15, mutant-rescue: *P = 2.3e−4; ATAC– wt-mutant: *P = 1.6e−10, mutant-rescue: *P = 2.8e−08) using a two-sided Wilcoxon test. RPM: normalized reads per million.